Correcting nova-associated human neurological disorders

Abstract

The invention relates to methods of altering expression or activity of NOVA 1 or NOVA2. The invention provides anti-microRNA molecules directed against miRNA binding sites on untranslated regions that serve to redirect RNA translation, including acting as a competitor for miRNA binding on targets, including NOVA1 and NOVA2. The invention provides methods for inhibiting microRNAs directed to NOVA1 or NOVA 1 in an animal by administering one of more anti-microRNA molecule specific therefore. Methods of alleviating one or more disease or condition, including altering or modulating vocalization, language dysfunction, autism (including non-verbal autism), microencephaly, seizures, developmental delay or cancer are provided. In accordance with such methods, NOVA1 or NOVA2 expression or activity is modulated to result in alleviation of one or more disease or condition.

Description

[0001] CORRECTING NOVA-ASSOCIATED HUMAN NEUROLOGICAL DISORDERS

[0002] STATEMENT OF GOVERNMENT RIGHTS

[0003]

[0001] This invention was made with government support under R35NS097404, R01DC018691, and R35GM127070, awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] INCORPORATION OF SEQUENCE LISTING

[0005]

[0002] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document encoded as XML in UTF-8 text. The electronic document, created on February 1, 2026, is entitled “1119-85_PCT_ST26.xml”, and is 87,928 bytes in size.

[0006] FIELD OF THE INVENTION

[0007]

[0003] The present invention relates to methods of altering expression or activity of NOVA 1 or NOVA2. The invention provides anti-microRNA molecules directed against miRNA binding sites on untranslated regions that serve to redirect RNA translation, including acting as a competitor for miRNA binding on targets, including NOVAI and NOV A2. The invention provides anti-microRNA molecules directed to and complementary to microRNA binding sites on the NOVAI transcript or on the NOVA2 transcript. The invention provides methods for inhibiting microRNAs directed to NOVAI or NOVAI in an animal by administering one of more anti-microRNA molecule specific therefore. Methods of alleviating one or more disease or condition, including altering or modulating vocalization, language dysfunction, autism (including non-verbal autism), microencephaly, seizures, developmental delay or cancer are provided. In accordance with such methods, NOVAI or NOVA2 expression or activity is modulated to result in alleviation of one or more disease or condition.

[0008] BACKGROUND OF THE INVENTION

[0009]

[0004] Humans differ significantly from their closest living relatives, the great apes, and from archaic humans such as Neanderthals and Denisovans. This is particularly evident in comparing the ability of great apes and humans to communicate through complex learned vocal communication, a necessary component of spoken language. This complexity is driven by some anatomical adaptions of the vocal tract and intricate neural networks linking various brain regions

[0010]

[0011] .

[0012]

[0005] The transcription factor forkhead box P2 (FOXP2) is a potential driver of human language function, as it harbors two amino acid substitutions present in human but not in chimpanzee and many other mammal genomes, although it is not human specific, as these substitutions are also present in otherhominids (e.g. Neanderthal and Denisovan; Ref 8). Families with FOXP2 mutations exhibit severe speech defects^’^ while FOXP2 disruption in mice may lead to vocalization abnormalities^^ suggesting a role in spoken language function. Studies on mice with the two amino acids substituted to the human version have reported vocal changes both in the neonatal and adult stages

[0013]

[0014] ’. While Hammerschmidt et al. observed minimal vocal changes, von Merten et al. reported qualitative changes under a more natural vocalization paradigm

[0015]

[0016] , suggesting the potential involvement of these two amino acids in vocalization.

[0017]

[0006] Similarly, the TKTL1 gene contains what may be a human-specific amino acid thought to influence greater neurogenesis in human than Neanderthal frontal cortex, though this finding is based only on European ancestry genome datasets

[0018]

[0019] . Broader analyses of modern human genomes reveal that 0.03-0.2% of individuals possess the ‘putative Neanderthal variant’, indicating its presence in a significant portion of the population14.

[0020]

[0007] Genomic comparisons between archaic humans, ape genomes, and the broader human population have identified 61 human-specific nonsynonymous coding variants that are fixed or nearly fixed in modem humans 17. One of the genes includes an isoleucine to valine substitution at position 197 (I197V) in the RNA binding protein neuro-oncological ventral antigenl (NOVAI). NOVAI is highly expressed in neurons of the central nervous system (CNS) in both mice and humans"^, and its expression has also been observed in cultured human and rat cells

[0021]

[0022] . NOVAI was first identified as an autoantigen targeted in the paraneoplastic neurologic disorder (PND) opsoclonus-myoclonus ataxia (POMA)"^. PNDs develop when tumor cells ectopically express proteins normally restricted to the nervous system, triggering an antitumor immune response that breaches the blood-brain barrier, leading to autoimmune neurologic disease ’. In POMA, a robust immune response is mounted against NOVAI and its paralog,

[0023] n

[0024] NOVA2 and the autoimmune disorder is characterized by motor dysfunction due to failure of inhibition of midbrain neurons, which results in the hyperactivity associated with opsoclonus-myoclonus ataxia"^ Nova I is required for the survival of neurons in the spinal cord and brainstem after birth. In mice, homozygous deletion of the Nova! gene results in an early postnatal lethal phenotype due to abnormal motor function. Therefore, N0VA1 plays a crucial role in neural development and neuromuscular control in mammals.

[0025]

[0008] NOVA proteins directly bind RNA in the mouse brain5"’55to regulate pre-mRNA

[0026] 71 7 A 7 z 7

[0027] processing-51A' ’55, translation50and neurophysiology5'. Genetic studies mapping NOVA target RNAs in mice and humans have also linked it to autism. A human patient with a heterozygous deletion ofNO VAI presented with delay of language development, learning disabilities, motor hyperactivity and behavioral dysregulation^^. A number of neurological disorders feature or present with motor vocalization disability — including development disorders (non-verbal autism and others) and degenerative disorders (e.g. frontal dementias).

[0028]

[0009] Several human clinical studies have reported that abnormalities in chromosomal regions including NOVAI cause severe psychiatric disorders and motor developmental abnormalities (Fryssira, H et al (2016) Mol. Cytogenet. 9, 55.; Qi M et al (2013) Gene 531, 457-461). NOVAI expression appears to be associated with Autism and the progression and prognosis of cancers such as gastric cancer, hepatocellular carcinoma, and lymphoma, as well as glioblastoma and astrocytoma and oligodendroglioma, making NOVAI function relevant and clinically applicable from multiple perspectives (Zhang C et al (2010) Science 239, 439-443; Parikshak, N. N. et al (2016) Nature 540, 423-427; Gimenez, M. et al (2015) BMC Cancer 15, 481; Zhi, F. et al (2014) PLoS One 9, eiO9124; Yoon, S. O. et al (2016) Oncotarget 7, 2475—2495; Kim, E. K. et al (2017) Gastric Cancer 20, 43 M-47; Shen, B. et al (2015) FEBS Lett. 589, 3205-3211; Zhang, Y.-A. et al (2014) PLoS One 9, e90955; Kim, E. K. et al (2016) J. Pathol. Transl. Med. 50, 104-112). In mice, NOVAI deficiency leads to a lethal phenotype within a few days after birth, with marked motor dysfunction (weakness and action-induced tremors) and neuronal apoptosis in the spinal cord and brain stem (Jensen KB et al (2000) Neuron 25,359-371).

[0029]

[0010] Further, a human with very specific deletion in the NOVAI gene, leading to haploinsufficiency, was recently described. This patient presented with a series of neuro-behavioral deficits, including motor dysfunction and developmental delay (Tajima et al (2023) Cell Reports 42, 112050). This human also demonstrated language dysfunction and speech delay. Despite the implied importance of NOVA 1 in maintaining neural activity and function in the brain, other than a role in Agrin splicing and development of the neuromuscular junction, the specific physiological functions of NOVAI remain largely unknown (Ruggiu M et al (2009) PNAS USA 106, 3513-3518).

[0030] [OH] Correction ofNoval deficiency and / or altered Nova l expression, such as inhibition of or blocking expression of a variant or abnormal NOVA! or interference with NOVAI suppressing microRNAs, is provided herein to impact and alleviate neural, neuro-behavioral, motor, and verbal deficiencies or dysfunctions.

[0031]

[0012] The citation of references herein shall not be construed as an admission that such is prior art to the present invention.

[0032] SUMMARY OF THE INVENTION

[0033]

[0013] NOVAI is a neuronal RNA-binding protein expressed in the central nervous system and is essential for survival in mice and normal development in humans. Modern humans specifically share and carry a single amino acid change (I197V) inNOVAl’s second RNA binding domain that is notpresent in other ancient hominids or other mammals. In an aspect of the invention, mice were generated carrying the human-specific I197V variant (denoted

[0034]

[0035] and the molecular and behavioral consequences were assessed and evaluated. The II 97V substitution had minimal impact onNOVAl’sRNA binding capacity. The I197V substitution led to specific effects on alternative splicing, andmultiple binding peaks in mouse brain transcripts involved in vocalization were revealed. Gene ontology analysis of these transcripts revealed that the highest ranked behavioral function of these genes was in vocalization. Experimentally, introducing the II 97V substitution into mice was associated with behavioral differences in vocalization patterns inNoval^u / ^umice as pups and adults. Thus, this human-specific NOVAI substitution may have been part of an ancient evolutionary' selective sweep in a common ancestral population of Homo sapiens, linked to the development of spoken language via differential RNA regulation during brain development.

[0036]

[0014] In accordance with aspects of the invention, gene-editing is implemented to substitute the NOVAI isoleucine (I) isoform present in most mammals and archaic hominids (Neanderthals and Denisovans) with the human-specific valine (V) variant at position 197 in a non-human mammal (mice). Comparison of humanized NOVAI mice

[0037]

[0038] with wild-type mice carrying the ancestral Noval gene

[0039]

[0040] reveals specific transcriptomic and behavior differences related to vocalization. The humanspecific NOVAI amino acid 197 variant confers vocalization changes in humanized mice.

[0041]

[0015] The invention provides molecules and methods for altering expression or activity of NOVAI or NOVA2. In an aspect, anti-microRNA molecules directed against rniRNA binding sites on untranslated regions are provided that serve to redirect RNA translation, including acting as a competitor for miRNA binding on targets, including NOVAI and N0VA2. The invention provides anti-microRNA molecules directed to and complementary to microRNA binding sites on the NOVAI transcript. The invention provides methods for inhibiting microRNAs directed to NOVAI in an animal by administering one of more anti-microRNA molecule specific therefore. The invention provides anti-microRNA molecules directed to and complementary to microRNA binding sites on the NOVA2 transcript. The invention provides methods for inhibiting microRNAs directed to NOVA2 in an animal by administering one of more anti-microRNA molecule specific therefore. In aspects, the anti-microRNA molecules are directed to 3’ untranslatedregion sequence.

[0042]

[0016] The invention provides an anti-microRNA molecule comprising at least 8 moieties or nucleotides and having complementarity to one or more micro-RN A binding sites present in and normally targeting NOVAI, particularly NOVAI 3’ untranslated region (UTR). In an embodiment the anti-microRNA molecule comprises at least 8 moieties or nucleotides forming a complementary base pair with nucleotides of the NOVAI mRNA, particularly the 3’UTR. The invention provides an anti-microRNA molecule comprising at least 8 moieties or nucleotides and having complementarity to one or more micro-RNAbinding sites present in and normally targeting NOVA2, particularly NOVA23’ untranslated region (UTR). In an embodiment the anti-microRNA molecule comprises at least 8 moieties or nucleotides forming a complementary base pair with nucleotides of the N0VA2 mRNA. particularly the 3 ’UTR.

[0043]

[0017] In some embodiments, the anti-microRNA molecule acts as a competitor for miRNA binding. In one such embodiment, the anti-microRNA molecule relieves / corrects / blocks existing miRNA mediated RNA translational inhibition and degradation. One such aspect is described in Tajima Y et al (2023) Cell Reports 42, 112050, which details that NOVAI ordinarily stabilizes mRNA through 3 UTR binding, antagonizing miRNA binding, resulting in translational controls and effects on intracellular and systemic energy homeostasis. Thus, N0VA1 stabilizes Impact mRNA by binding its 3’ UTR, antagonizing the actions of miR-138 and miR-124. In an aspect of the invention, the anti-microRNA molecule acts to antagonize the actions of miRNAs and miR molecules by binding to the 3 ’ UTR they target, in aspects the 3’ UTR of NOVA 1 or of NOVA2.

[0044]

[0018] In another aspect, the invention provides an anti-microRNA molecule comprising at least 8 moieties or nucleotides and having complementarity to a micro-RNA targeting NOVAI, particularly NOVAI 3’ untranslated region (UTR). In an embodiment the anti-microRNA molecule comprises at least 8 moieties or nucleotides forming a complementary base pair with nucleotides of a micro-RNA targeting NOVAI, particularly the 3’UTR. In an embodiment, the micro-RNA targets human NOVAI. In an embodiment, the micro-RNA targets the 3’ UTR of NOVAI. In an embodiment, the micro-RNA targets human NOVA 1. In an embodiment, the micro-RNA targets the 5 ’ UTR of NOVA I.

[0045]

[0019] In some embodiments, the ant-microRNA molecule is an antagomir or antisense oligonucleotide (ASO).

[0046]

[0020] In an embodiment, the anti-microRNA molecule comprises a minimum of eight moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base fonning a base pair with a complementary base wherein at least eight contiguous bases form a complementary base pair with a or one or more micro-RNA’s target sequence of NOVAI, particularly NOVAI 3’ untranslated region (UTR). In an embodiment, the anti-microRNA molecule comprises a minimum of eight moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a base pair with a complementary base wherein at least eight contiguous bases form a complementary base pair with a or one or more micro-RNA’s target sequence ofNOVA2, particularly NOV A23’ untranslatedregion (UTR). In such an aspect, the anti-microRNA molecule specifically blocks microRNA activity directed at NOVAI or NOVA2 by blocking microRNA binding at the UTR target sequence itself by binding to and blocking the target.

[0047]

[0021] In one embodiment, the invention provides an isolated single stranded anti-microRNA molecule, particularly an antagomir or antisense oligonucleotide (ASO), comprising a minimum of eight moieties anda maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a base pair with a complementary base wherein at least eight contiguous bases form a complementary base pair with a N0VA1 sequence that is targeted by a microRNA, particularly NOVA! 3’ untranslated region (UTR). In one embodiment, the invention provides an isolated single stranded anti-microRNA molecule, particularly an antagomir or antisense oligonucleotide (ASO), comprising a minimum of eight moieties and a maximum of fifty' moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a base pair with a complementary base wherein at least eight contiguous bases form a complementary' base pair with a N0VA2 sequence that is targeted by a microRNA, particularly N0VA23’ untranslated region (UTR).

[0048]

[0022] In an alternative embodiment, the anti-microRNA molecule comprises a minimum of eight moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a base pair with a complementary' base wherein at least eight contiguous bases form a complementary base pair with a microRNA targeting NOVA1, particularly NOVA! 3’ untranslated region (UTR). In an alternative embodiment, the anti-microRNA molecule comprises a minimum of eight moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a base pair with a complementary' base wherein at least eight contiguous bases form a complementary base pair with a micro-RNA targeting NOVA2, particularly NOVA2 3'' untranslated region (UTR).

[0049]

[0023] In one embodiment, the invention provides an isolated single stranded anti-microRNA molecule, particularly an antagomir or antisense oligonucleotide (ASO), comprising a minimum of eight moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a base pair with a complementary base wherein at least eight contiguous bases form a complementary base pair with a microRNA targeting NO VAI, particularly NOVAI 3:untranslated region (UTR).

[0050]

[0024] In another embodiment, the invention provides a method for inhibiting a microRNA molecule targeting NOVAI, particularly NOVAI 3’ untranslated region (UTR), the microRNA molecule comprising a sequences of bases complementary of the sequence of bases in a single stranded anti-microRNA molecule, the method comprising introducing into the cell the single-stranded anti-microRNA molecule comprising a sequence of a minimum of eight moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a base pair with a complementary base wherein at least eight contiguous bases form a complementary base pair with a micro-RNA targeting NOVAI.

[0025] In yet a further embodiment, the invention provides a method for inhibiting a microRNA molecule targeting NO VA 1, particularly NOVA 1 3 ’ untranslated region (UTR), the microRNA molecule comprising a sequences of bases complementary of the sequence of bases in a single stranded anti-microRNA molecule, the method comprising introducing into the cell the single-stranded anti-microRNA molecule comprising a sequence of a minimum of eight moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a base pair with a complementary base wherein at least eight contiguous bases of the anti-microRNA molecule are complementary to the microRNA, except that up to thirty percent of the bases may be substituted by wobble base pairs, and up to ten percent of the at least eight moieties may be additions, deletions, mismatches, or combinations thereof. In yet a further embodiment, the invention provides a method for inhibiting a microRNA molecule targeting NOVA2, particularly NOVA2 3’ untranslated region (UTR), the microRNA molecule comprising a sequences of bases complementary of the sequence of bases in a single stranded anti-microRNA molecule, the method comprising introducing into the cell the single-stranded anti-microRNA molecule comprising a sequence of a minimum of eight moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a base pair with a complementary' base wherein at least eight contiguous bases of the anti-microRNA molecule are complementary to the microRNA, except that up to thirty percent of the bases may be substituted by wobble base pairs, and up to ten percent of the at least eight moieties may be additions, deletions, mismatches, or combinations thereof. In an embodiment, no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units.

[0051]

[0026] The invention relates to an isolated single stranded anti-microRNA molecule. The molecule comprises a minimum number of eight moieties, preferably a minimum of ten, preferably a minimum of thirteen, preferably a minimum of fifteen, preferably a minimum of 18, or preferably a minimum of 21 moieties.

[0052]

[0027] Each moiety comprises a base bonded to a backbone unit. A base refers to any one of the nucleic acid bases present in DNA or RNA. The base can be a purine or pyrimidine. Examples of purine bases include adenine (A) and guanine (G). Examples of pyrimidine bases include thymine (T), cytosine (C) and uracil (U). Each base of the moiety forms a Watson-Crick base pair with a complementary base. The adenine can be replaced with 2,6-diaminopurine without compromising base-pairing. The backbone unit may be any molecular unit that is able stably to bind to a base and to form an oligomeric chain.

[0053]

[0028] Suitable backbone units are well known to those in the art. For example, suitable backbone units include sugar-phosphate groups, such as the sugar-phosphate groups present in ribonucleotides, deoxyribonucleotides, phosphorothioate deoxyribose groups, N’3-N’5 phosphoroamidate deoxyribose groups, 2’0-alkyl-ribose phosphate groups, 2’-O-alkyl-alkoxy ribose phosphate groups, ribose phosphategroup containing a methylene bridge, 2’-Fluororibose phosphate groups, morpholino phosphoroamidate groups, cyclohexene groups, tricyclo phosphate groups, and amino acid molecules.

[0054]

[0029] In one embodiment, the anti-microRNA molecule comprises at least one moiety which is a ribonucleotide moiety or a deoxyribonucleotide moiety.

[0055]

[0030] In another embodiment, the anti-microRNA molecule comprises at least one moiety which confers increased nuclease resistance. The nuclease can be an exonuclease, an endonuclease, or both. The exonuclease can be a 3’— 5’ exonuclease or a 5’— >3’ exonuclease. Examples of 3’— >5’ human exonuclease include PNPT1, Werner syndrome helicase, RRP40, RRP4I, RRP42, RRP45, and RRP46. Examples of 5’— >3’ exonuclease include XRN2, and FEN1. Examples of endonucleases include Dicer, Drosha, RNase4, Ribonuclease P, Ribonuclease Hl, DHP1, ERCC-I and OGGI. Examples of nucleases which function as both an exonuclease and an endonuclease include APE1 and EXO1.

[0056]

[0031] An anti-microRNA molecule comprising at least one moiety which confers increased nuclease resistance means a sequence of moieties wherein at least one moiety is not recognized by a nuclease. Therefore, the nuclease resistance of the molecule is increased compared to a sequence containing only unmodified ribonucleotide, unmodified deoxyribonucleotide or both. Such modified moieties are well known in the art.

[0057]

[0032] A modified moiety can occur at any position in the anti-microRNA molecule. For example, to protect the anti-microRNA molecule against 3’— >5’ exonucleases, the molecule can have at least one modified moiety at the 3’ end of the molecule and preferably at least two modified moieties at the 3’ end. If it is desirable to protect the molecule against 5’— 3’ exonuclease, the anti-microRNA molecule can have at least one modified moiety and preferably at least two modified moieties at the 5’ end of the molecule. The anti-microRNA molecule can also have at least one and preferably at least two modified moieties between the 5’ and 3’ end of the molecule to increase resistance of the molecule to endonucleases. In one embodiment, all of the moieties are nuclease resistant.

[0058]

[0033] In another embodiment, the anti-microRNA molecule comprises at least one modified deoxyribonucleotide moiety. Suitable modified deoxyribonucleotide moieties are known in the art. An example of a modified deoxyribonucleotide moiety is a phosphorothioate deoxyribonucleotide moiety. Another suitable example of a modified deoxyribonucleotide moiety is an N’3-N’5 phosphoroamidate deoxyribonucleotide moiety. An oligonucleotide molecule comprising more than one phosphoroamidate deoxyribonucleotide moiety is referred to as phosphoroamidate (NP) DNA.

[0059]

[0034] In another embodiment, the molecule comprises at least one modified ribonucleotide moiety. Suitable modified ribonucleotide moieties are known in the art. A suitable example of a modified ribonucleotide moiety is a ribonucleotide moiety that is substituted at the 2’ position. The substituents at the 2’ position may, for example, be a C1 to C4 alkyl group. The C1 to C4 alkyl group may be saturated or unsaturated, and unbranched or branched. Some examples of C1 to C4 alkyl groups include ethyl, isopropyl,and allyl. An oligoribonucleotide molecule comprising more than one ribonucleotide moiety that is substituted at the 2’ position with a Ci to C4 alkyl group is referred to as a 2'-O -(C1-C4 alkyl) RNA, e.g.,2’-O-methyl RNA (OMe RNA). Another suitable example of a substituent at the 2’ position of a modified ribonucleotide moiety is a Ci to C4 alkoxy - Ci to C4 alkyl group. The Ci to C4 alkoxy (alkyloxy) and Ci to C alkyl group may comprise any of the alkyl groups described above. An oligonucleotide molecule comprising more than one ribonucleotide moiety that is substituted at the 2’ position with a C 1 to C4 alkoxy-Ci to C4 alkyl group is referred to as a 2’-O-(Cj to C4 alkoxy - Ci to C4 alkyl) RNA, e.g., 2’-O-methoxyethyl RNA (MOE RNA). Another suitable example of a modified ribonucleotide moiety is a ribonucleotide that has a methylene bridge between the 2’-oxygen atom and the 4’-carbon atom. An oligoribonucleotide molecule comprising more than one ribonucleotide moiety that has a methylene bridge between the 2’-oxygen atom and the 4’-carbon atom is referred to as locked nucleic acid (LNA). Locked nucleic acids are commercially available.

[0060]

[0035] Another suitable example of a modified ribonucleotide moiety is a ribonucleotide that is substituted at the 2’ position with fluoro group. A modified ribonucleotide moiety having a fluoro group at the 2' position is a 2′-fluororibonucleotide moiety. Such moieties are known in the art. Molecules comprising more than one 2 ’-fluororibonucleotide moiety are referred to herein as 2’-fluororibo nucleic acids (FANA).

[0061]

[0036] In another embodiment, the anti-microRNA molecule comprises at least one base bonded to an amino acid residue. Moieties that have at least one base bonded to an amino acid residue will be referred to herein as peptide nucleic acid (PNA) moieties. Such moieties are nuclease resistance, and are known in the art. Molecules having more than one PNA moiety are referred to as peptide nucleic acids.

[0062]

[0037] The anti-microRNA molecules of the present invention are capable of inhibiting microRNA activity, preferable in a cell. Inhibiting microRNA activity refers to the inhibition of cleavage of the microRNA’s target sequence or the repression of translation of the microRNA’ s target sequence. The method comprises introducing into the cell a single-stranded microRNA molecule.

[0063]

[0038] The anti-microRNA molecules of the present invention may be used as a modulator of the expression of genes which are at least partially complementary to the anti-microRNA molecules and microRNA, particularly the gene encoding NOVAI protein. For example, if a particular microRNA is beneficial for the survival of a cell, an appropriate isolated microRNA of the present invention may be introduced into the cell to promote survival. Alternatively, if a particular microRNA is harmful (e.g., induces apoptosis, induces cancer, etc.), an appropriate anti-microRNA molecule can be introduced into the cell in order to inhibit the activity of the microRNA and reduce the harm.

[0064]

[0039] The cell can be any cell which expresses microRNA molecules, including the microRNA molecules listed herein. Alternatively, the cell can be any cell transfected with an expression vector containing the nucleotide sequence of a microRNA.

[0040] The anti-microRNA molecules or microRNAs can be introduced into a cell by any method known to those skilled in the art. Useful delivery systems, include for example, liposomes and charged lipids. Other methods for introducing an anti-microRNA molecule or a microRNA into a cell include use of delivery vehicles, such as dendrimers, biodegradable polymers, polymers of amino acids, polymers of sugars, and oligonucleotide-binding nanoparticles. In addition, pluoronic gel as a depot reservoir can be used to deliver the anti-microRNA oligonucleotide molecules over a prolonged period. Targeting of an anti-microRNA molecule or a microRNA to a particular cell can be performed by any method known to those skilled in the art. For example, the anti-microRNA molecule or microRNA can be conjugated to an antibody or ligand specifically recognized by receptors on the cell.

[0065]

[0041] An exemplary mouse NOVAI protein is a follows:

[0066] Mouse NOVAI protein - 197 Isoleucine (I) in bold. (507 amino acids)

[0067] 1 MtLAAAFTQQNGTHTGVPIDLDFPDSRKRPLEAPFEAGSTKRTNTGEDGQYFLKVLIFSYA

[0068] 61 AGSI IGKGGQTIVQLQKETGATIKLSKSKDFYPGTTERVCLIQGTIEALNAVHGFIAEKI

[0069] 121 REMPQNVAKTEPVSILQPQTTVNPDRIKQTLPSSPTTTKSSPSDPMTTSRANQVKIIVPN

[0070] 181 STAGLIIGKGGATVKAIMEQSGAWVQLSQKPDGINLQERVVTVSGEPEQNRKAVELI IQK

[0071] 241 IQEDPQSGSCLNISYANVTGPVANSNPTGSPYANTAEVLPTAAAAAGLLGHANLAGVAAF

[0072] 301 PAVLSGFTGNDLVAITSALNTLASYGYNLNTLGLGLSQAAATGALAAAAASANPAAAAAN

[0073] 361 LLATYASEASASG3TAGGTAGTFALGSLAAATAATNGYFGAASPLAASAILGTEKSTDGS

[0074] 421 KDVVEIAVPENLVGAILGKGGKTLVEYQELTGARIQISKKGEFVPGTRNRKVTITGTPAA

[0075] 481 TQAAQYLITQRITYEQGVRAANPQKVG (SEQ ID NO: 1 )

[0076]

[0042] An exemplary human NOVAI protein is a follows:

[0077] Human NOVAI protein - human specific 197 (V) Valine in bold (507 amino acids) 1 iAAAPIQQNGTHTGVPIDLDPPDSRKRPLEAPPEAGSTKRTNTGEDGQYFLKVLIFSYA

[0078] 61 AGSI IGKGGQ IVQLQKETGATIKLSKSKDFYPGTTERVCLIQGTVEALNAVHGFIAEKI

[0079] 121 REMPQNVAKTEPV3ILQFQTTVNPDRIKQTLPSSPTTTKSSPSDPMTTSRANQVKIIVPN

[0080] 181 STAGLIIGKGGATVKAVMEQSGAWQLSQKPDGINLQERVVTVSGEPEQNRKAVELI IQK

[0081] 241 IQEDPQSGSCLNISYANVTGPVANSNPTGSPYANTAEVLPTAAAAAGLLGHANLAGVAAF

[0082] 301 PAVLSGFTGNDLVAITSALNTLASYGY LNTLGLGLSQAAATGALAAAAASANPAAAAAN

[0083] 361 LLATYASEA3ASGSTA, GGTAGTFALGSLAAATAATNGYFGAASPLAASAILGTEKSTDGS

[0084] 421 KDVVEIAVPENLVGAILGKGGKTLVEYQELTGARIQISKKGEFVPGTRNRKVTITGTPAA

[0085] 481 TQAAQYLITQRITYEQGVRAANPQKVG (SEQ ID NO: 2 )

[0086]

[0043] Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

[0087] BRIEF DESCRIPTION OF THE DRAWINGS

[0088]

[0044] Figure 1A-1E: Evolutionarily conserved NOVAI harbors a modern human-specific amino acid at position 197. (a) Conservation analysis of NOVAI and NOVA2 protein conservation across speciesusing high-quality genome assemblies from NCBI. The analysis was performed using NCBI's Constraintbased Multiple Alignment Tool (Cobalt). “Conserved Region” is defined by the relative entropy threshold of the amino acid residue. Green bars indicate highly conserved positions. Gray-red bars indicate the Column Quality score: scores for amino acid residues based on agreement within the column / position. Rare residues are highlighted in darker red, while positions with any mismatch are anchored, (b) Sequence comparison of NOVA 1 CDS in 4 ancient humans (3 Neanderthals and 1 Denisovan; blue bar) and 8 modern humans; red bar. The upper panel shows the 50 bases around the 197thamino acid and the lower shows the entire NOVAI CDS. (c) SNP frequency analysis of NOVAI gene in modem humans. Mean allele frequencies (MAFs) on the NOVAI gene detected in the genome analysis of 121,412 modem humans are indicated by gray dots; the upper bound of 95% confidence interval for SNPs in the NO VA 1 gene is indicated by red dotted lines, and for comparison, those for NOVA2 or all genes on chromosome 14 (where NOVA1 gene is located) are indicated by blue- or black- dotted lines, respectively. The red star indicates the V197I variant: rs762662114, chrl 4:26448894 / GRCh38.pl4. (d) Comparison of normalized Tajima’s D values. The first gene set includes NOVAI and NOVA2, and NOVA 1 -neighboring genes (FOXG1 and STXBP6 on chrl4. The second gene set includes all genes on chr14 where NOVAI is located, (e) A model of the evolutionary timing of the 197thamino acid change that occurred in the NOVAI gene, noting the Nova1hu / humice generated in this study. Nova1hu / humice have the modern human specific amino acid in NOVAI protein. The bottom panel shows the corresponding location within the KH2 domain of the NOVAI protein. Amino acids structurally proximal (<5 A) to the 197thamino acid predicted by AlphaFold2 are boxed in. The KH2 domain sequence of Nova1wt / wtANQVKI IVPNSTAGLI IGKGGATVKAIMEQSGAWVQLSQKPDGINLQERVVTVSGEPEQNRKAVELI IQK IQE (SEQ ID NO:3) and of Noval ANQVKI IVPNSTAGLI IGKGGATVKAVMEQSGAWVQLSQKPDGINLQERVVTVSGEPEQNRKAVELI IQK IQE (SEQ ID NO:4) are depicted.

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[0045] Figure 2A and 2B: (a) The SNP report from dbSNP database. The single nucleotide variation (rs762662114) responsible for modern human specific amino acid in NOVAI (197^ Vai). Alternate allele frequencies in the total number of samples for each genome project are shown, (b) MAF analysis in the NOVAI gene across human ancestries. SNPs detected in the genome analysis of 121,410 modem humans on the NOVAI CDS, along with the NOVAI protein structure. Each SNP is color- coded by ethnicity. SNP corresponding to the 197thamino acid (ancient human-type variant) is indicated by a star.

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[0046] Figure 3: Comparison of Normalized Tajima’s D values. The closer the normalized Tajima’s D value approaches -1, the greater the likelihood that the gene has undergone strong purifying selection The first gene set includes NOVAI and NOVA2, and closest protein-coding NOVA 1 -neighboring genes (FOXG1 and STXBP6) on chr14. Each gray dot indicates a gene in each gene list. The normalized Tajima's D value for NOVAI (red) is -0.9993. The second gene set includes all genes on chr.14 where NOVAI is located. These gene sets arc also shown in Figure Id. The third and fourth gene sets arc based on Meyer's reporfr, with genes related to the 260 human-specific single- nucleotide changes (SNCs) that cause fixed amino acid substitutions in well-defined human coding sequence, or to the subset of these genes (eight among the 260 SNCs) whose function is associated with brain function or nervous system development. The fifth gene set is based on Trujillo’s report, with genes associated with 61 autosomal fixed derived mutations in all humans compared to Neanderthal genomes and Denisovan genome. The sixth and seventh gene sets include human RNA binding proteins (RBPs) with KH domain or all annotated RBPs.

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[0047] Figure 4: Recent burst of coalescence of a sweep for NOVA 1 197V. The left tree shows the NOVA 1 SNP, with the shortened branches and burst of relatively recent coalescence events leading to the modem humans (reflecting a rise in frequency of the derived allele). The right tree shows FRMD8 for contrast, with longer branches and delayed coalescence: more typical of what would be expected in the absence of a selective sweep. The sampled ARGs included two Yoruba (HGDP00927, SS6004475), two Mbuti (SS6004471, HGDP0456), and two San (HGDP01029, SS6004473) individuals, as well as the Altai Neanderthal and Denisovan sequences and a chimpanzee outgroup (panTro4). The numbers following the samples represent the separate haplotypes from each individual. Red lines indicate derived allele.

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[0048] Figure 5A-5E: Generation of humanized Nova1 mice {Nova1hu / hu). (a) Overview of the strategy to generate mice with a modem human-specific amino acid substitution in NOVAI protein. Using CRISPR / Cas9, nucleotide substitutions (that lead to a single amino acid change from Isoleucine to Valine) are introduced. Two silent mutations were introduced for genotyping, (b) DNA sequencing of Noval allele in wild-type mice and in mice in which correct knock-in was introduced {Nova1hu. (c) Genotyping of Noval^u'^umice using restriction enzymes. The introduction of silent mutations creates a BtsIα recognition site. Noval^uallele is distinguished from wild-type Nova! allele by restriction enzyme treatment after PCR. (d) The gRNA sequence TGCTACTGTGAAGGCTATAATGG (SEQ ID NO:5) and predicted off-target site information predicted by CRISPR direct (crispr.dbcls.jp). There are 10 potential off target loci with mismatches (chr2 TGAAGGCTATAACGG (SEQ ID NO:7); chr5 TGAAGGCTATAATGG (SEQ ID NO: 8); chr6 TGAAGGCTATAAAGG (SEQ ID NO: 9); chr7 TGAAGGCTATAAAGG (SEQ ID NO:10); chr8 TGAAGGCTATAAAGG (SEQ ID NO: 11); chr9 TGAAGGCTATAAGGG (SEQ ID NO: 12); chr 17 TGAAGGCTATAAGGG (SEQ ID NO: 13); chrl CCTTTATAGCCTTCA (SEQ ID NO: 14); chr3 CCTTTATAGCCTTCA (SEQ ID NO: 15); chrl 8 CCTTTATAGCCTTCA (SEQ ID NO: 16) outside of the PAM+12mer core sequences chr12 CCATTATAGCCTTCA (SEQ ID NO: 6). (e) The genomic sequencing of the potential off target (POT)loci. Alignment of each genotype and reference genome for the genomic sequence of 100 bases around the POTs are shown. Asterisks indicate identical nucleotides. All POT sites were identical between genotypes and the reference genome, with the target site (responsible for II 97V substitution) being the only detectable edits. The mouse reference and wt target site sequence NOVAI sequence corresponds to AGTTCCCAAC AGCACAGCAGGTCTGATAATAGGGAAGGGAGGTGCTACTGTGAAGGCTATAATGGAGCAGT CAGGGGCTTGGGTGCAGCTTTCCCAGAAACCCGATGGGATCAAC (SEQ ID NO:21) (mouse sequence regions around the Novallle amino acid 197 sequence underlined). The human sequence at the target site is AGTTCCCAACAGCACAGCAGGTCTGATAATAGGGAAGGGAGGTGCTACTGTGAAGGCAGTG ATGGAGCAGTCAGGGGCTTGGGTGCAGCTTTCCCAGAAACCCGATGGGATCAAC (SEQ ID NO:22) (mouse sequence regions around the Noval amino acid 197 sequence underlined and the valine VI 97 is shown in bold).

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[0049] Figure 6A-6J: (a) Body weight comparison of wild type (Nova1wt / wt, N=19), heterozygous (Noval, N=12), and homozygous (Noval^u'^u, N=14) mice, measured from 2-12 weeks postnatal, (b) Brain-to-body weight ratio comparison in 3-week-old mice (N=8 per group), (c) Gene expression correlations between Nova1wt / wtand Noval^u^ in midbrain at E18.5 and P21. Scatterplots show gene expression in average TPMs (log2 scale). The red dot marks one differentially expressed gene (Gkn3 J<0.05, FDR<0.1), and the yellow dot indicates Noval. Pearson correlations are noted above the plots. E18.5 midbrain: Novalwt,wtN=6, Nova1hu / huN=6; P21 midbrain: Nova1wt / wtN=4, Noval^u / ^uN.

[0094] (d) Autoradiography images from NOVAI- CLIP of 3-week-old midbrain in Nova1wt / wtand Noval^u / ^umice. The yellow line marks the NOVAI protein size, and the red outline highlights RNA extraction by sectioning, (e) Distribution of NOVA 1 CLIP peaks on the mouse genome, (f) The most enriched binding sequence from NOVAI CLIP peaks (top) and frequency of that sequence (UCAU) present around the binding site (lower part), (g) Scatterplot of CLIP tag number per peak between Nova1wt / wtand Nova1hu / humice atP21 midbrain. The axes are shown in log2 scales. Sample sizes are Noval"“'!mN=3, Novarn'wt=3.

[0095] (h) Gene annotation analysis52of N0VA1 bound transcripts. Transcripts with the top 1% peak height (read count) for each genotype were analyzed. Genes expressed in the midbrain of P21 mice were used as background for analysis. The term ‘behavior’ is indicated with a black arrowhead, (i) Representative data from gel shift assay using each purified NOVAI protein and -’■^P-Iabeled UCAU RNA oligo probe. Leftmost lane is probe only, no purified protein. The bottom band represents the free probe, and the top shifted band represents the purified protein bound to the RNA probe, (j) The bands were quantified from gel images of the gel shift assay and the amount of binding per protein concentration were plotted. Thedissociation constants (Kd values) for each purified NOVAI protein are shown in the graph. N=5 for each point.

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[0050] Figure 7A-7F: Comprehensive gene expression analysis in the brain, (a) Global correlation matrix of gene expression levels between brain samples: midbrain at E18.5, cortex, midbrain, cerebellum at P21 in Novalh" / h" and Nova1wt / wtmice. Heatmap showing correlation coefficients for log2 (TPM+1), color intensity and the size of the circle are proportional to the correlation coefficients, (b) Principal component analysis of gene expression levels between samples. The X axis is the first principal component, and the Y axis is the second principal component, with a percentage of variances explained by each component approximately 53% and 29%, respectively. The ellipses indicate confidence ellipses around group mean points (large dot), (c) Principal component analysis of gene expression levels in each corresponding sample (age and brain region), (d) Gene expression correlations between Novalwt / Wtand

[0097]

[0098] in corresponding brain regions and age. Scatterplots of gene expressions measured in average TPMs are shown. The axes are shown in log2 scales. The red dot indicates a differentially expressed gene between genotypes (p<0.05, FDR<0.1). The yellow dot indicates Nova J gene. Pearson correlation is reported on top of the plots. The upper two plots (midbrain) are identical with Figure 2c. (e) Principal component analysis of gene expression levels between Nova1ko / koand Nova1wt / wtmidbrain at E18.5. The RNA sequencing data are from GEO (GSE69711). The first principal component is 37.5%, and the second principal component is 18.6%, respectively. The ellipses indicate confidence ellipses around group mean points (large markers), (f) Gene expression correlations. Scatterplots of gene expressions measured in average TPMs are shown. The axes are shown in log2 scales. The yellow dot indicates Noval gene. Pearson correlation is reported on top of the plots, (left) Novalwt'wtand Noval^°'^° at E18.5 midbrain. The red dots indicate differentially expressed genes between genotypes (FDR<0.05) (see Supplemental Table 18). (right) Novalwt'wtand Nova1hu / huat E18.5 midbrain (the same with top left panel in d). The green dots indicate differentially expressed genes in the comparison between Nova1wt / wtand Nova1ko / ko(left panel, corresponding to the red dot). The midbrain sample of E18.5, N

[0099]

[0100] oval^0'^0N=3, Novalwt^vtN=3, Nova1hu^111N=6, Novalwt'wtN=6. The cortex, midbrain, and cerebellum samples of P21, Nova1hu / huN=4, Nova1wt / wtN=4, respectively.

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[0051] Figure 8A and 8B: Comparison of NOVAI protein expression between the brains of Nova1wt / wtand Nova1hu / humice, (a) Comparison of N0VA1 protein expression in dissected 3-week-old mouse brain tissues. Expression of NOVA proteins were analyzed by immunoblotted on cortex (ex), midbrain (mid), and cerebellum (cb) by panNOVA antibody, NOVAI antibody recognizing the N-terminus and the C-terminus, respectively. The predicted NOVA1 / 2 protein isoforms are listed in the notes, (b) NOVA 1 proteinexpression in Nova1wt / wtand Nova1hu / humice. Immunostaining for N0VA1 protein in sagittal sections of the brain at 3 and 12 weeks of age, respectively.

[0102]

[0052] Figure 9: KH domain sequence alignment. Figure adapted from Lewis et al., 2000 with modifications^. Each RNA binding protein (Nova-2, Nova-1, FMR-1, hnRNP El, hnRNP E2, hnRNP K, GLD-1, Bicaudal-C, PNP and NusA) and comprising KH domain number (KH1, KH2, KH3) are listed on the left. Secondary structural elements were based on the X-ray structure. Color coding scheme: yellow, invariant GXXG motif; purple, hydrophobic core (aliphatic a / p platform). Functional classifications: A, aliphatic stacking interaction; S, side chain-base hydrogen bond, including water-mediated contacts; M, protein backbone-base hydrogen bond; *, van der Waals contact. Amino acids on the red background are those for which loss of protein function was reported due to substitution in the hydrophobic core. Amino acids on the green background indicate the 197^ valine of NOVAI, which is unique to modern humans.

[0103]

[0053] Figure 10A-10J: NOVAI -CLIP analysis in 3-week-old mouse cortex and cerebellum, (a-e) cortex samples, (f-j) cerebellum samples, (a, f) Representative images of autoradiography in NOVA 1 -CLIP of 3-week- old Nova1wt / wtand Nova1hu / humice. The yellow line indicates NOVAI protein size, and the red enclosing line indicates where RNA was extracted by sectioning, (b, g) Distribution of NOVAI CLIP peaks on the genome, (c, h) The most enriched binding sequence from NOVAI CLIP peak (upper part) and frequency of that sequence (UCAU) present around the binding site (lower part), (d, i) Scatterplot of CLIP tag number per peak between Novalwt'wtand Noval^u'^u. The axes are shown in log2 scales. R square value is shown, (e) Gene annotation analysis of NOVA 1 bound transcripts. Transcripts with the top 100 peak height (read count) in each genotype were analyzed. Genes expressed in the P21 cortex were used as background for analysis. The term “behavior” is indicated with a black arrowhead, (j) Gene annotation analysis of NOVA 1 bound transcripts. Transcripts with the top 1% peak height (read count) in each genotype were analyzed. Genes expressed in the P21 cerebellum were used as background for analysis. The term “behavior” is indicated with a black arrowhead. The cortex and cerebellum samples of P21, Nova1hu / huN=3, Nova1wt / wtN=3, respectively.

[0104]

[0054] Figure 11A-11D: Predicted structural model caused by I197V substitution in the KH2 domain of NOVA1 (a) 3D structure prediction by AlphaFold2 (alphafold.ebi.ac.uk / ), showing the expanded KH2 domain of NOVA1 The 197thamino acid is centered, and its proximal amino acids (<5 k) are colored in pink. In this model, the change from isoleucine to valine results in the loss of contact with the amino acid residues at H 1 and H3 due to the loss of one carbon chain of the amino acid side chain, (b) Illustration of the amino acids surrounding 1197V relative to the secondary structure sequence of KH2. Amino acids structurally proximal to the 197thamino acid (<5 A) predicted by AlphaFold2 are colored in pink. Corresponding to Fig. le. Amino acids structurally proximal (<5 A) to the 197thamino acid predicted byAlphaFold2 are boxed in. The KH2 domain sequence of NovalKt'"rANQ> VKI IVENSTAGLI IGKGGATVKAIMEQSGAWVQLSQKPDGINLQERVVTVSGEPEQNRKAVELI IQK IQE (SEQ ID NO:3) and of Novalh“'1mANQVKI IVPNSTAGLI IGKGGATVKAVMEQSGAIWQLSQKPDGINLQERVVTVSGEPEQNRKAVELI IQK IQE (SEQ ID NO:4) are depicted, (c-d) Structures of models based on crystallographic data, in which the 197thamino acid is adjacent to amino acids involved in KH- domain-RNA interactions (Q32 in c, equivalent to the relative position of amino acid 198 in KH 1 ) or KH-domain-protein interactions (M32 in d, equivalent to amino acid 198 in KH2). The models are from Figure 9 of Teplova et al., 2011.

[0105]

[0055] Figure 12A-12F: Nova1hu / humice exhibit alternative splicing changes in specific neuronal genes, (a) NOVAI immunostaining in P21 mouse brain. Scale bars represent 500pm. Abbreviations: CTX, cortex; HIP, hippocampus; CP, Caudate putamen; TH, thalamus; HY, hypothalamus; MB, midbrain; CB, cerebellum; SN, substantia nigra; PAG. periaqueductal gray, (b) Western blotting analysis of NOVAI in dissected mouse brain regions. The corresponding isoforms for each band is listed on the right. Each lane’s NOVAI band is quantified and normalized to the ACTB band signal. The cortex value is set at 1. (c) Alternative splicing (AS) changes in the midbrain of 3- week-old Nova1hu / humice. Differential AS events with p value and delta PSI (di), di >0.05 aNova1hu / huvs. Nova1wt / wt) events are shown in light green, those having NOVA 1 -CLIP peaks on the transcript are shown in dark green, di > -0.05 (in Nova1hu / huvs. Nova1wt / wt) events are shown in light orange, those having NOVAI -CLIP peaks on the transcript are shown in magenta. Representative differential AS events are labeled with each gene name. Differential AS events in vocal behavior related genes are labeled in each gene name with a yellow box (Fig. 15). Novalwt / wtN=4, Noval^u / ^uN=4. (d) Examples of transcripts showing significant AS changes and having NOVA1-CLIP peaks on its transcript. Differential AS exons are colored in yellow. Information of each AS event is described below the IGV snapshots; Gene name, AS exon number, AS type, percent splice-in value (PSI, the percent of transcripts that include a specific AS exon), percent change (Al, APSI; Nova1hu / huvs. Nova1wt / wtand p-value. AS splicing events are classified into the following types: Cassette exon (cass), alternative 5’ splice site (alt5), alternative 3’ splice site (alt3), tandem cassette (taca), mutually exclusive exons (mutx). (e) Gene annotation analysis for the transcripts with differential AS events. The expressed genes in the P21 midbrain were set as background for the analysis, (f) Percentage of genes with differential AS occupying each behavior-related gene ontology category. The number of transcripts with differential AS event in Nova1hu / hurelative to the total number of genes comprising each category are shown.

[0106]

[0056] Figure 13: NOVAI immunostaining in the mouse brain. Immunostaining for NOVAI (green) and DAPI (blue) in postnatal day 21 (P21) and 0 (P0) mouse brain. The scale bars indicate 500pm. Thecorresponding brain regions are indicated in the orange characters. CTX: cortex, HIP: hippocampus, CP: Caudate putamen, TH: thalamus, HY: hypothalamus, MB: midbrain, CB: cerebellum. The images of N0VA1 staining of P21 mouse brain are corresponding to Figure 12a.

[0107]

[0057] Figure 14: N0VA1 protein expression in dissected mouse brain. Western blotting for N0VA1, panNOVA and ACTB proteins in dissected brain regions of adult mouse brain. Proteins and isoforms corresponding to each band are listed on the right. The highly expressed NOVAI in the hypothalamus, substantia nigra and periaqueductal gray is Exon4 minus isofom Three biological replicates are shown. The N0VA1 and ACTB plots in replicate 2 are corresponding to Fig 3b.

[0108]

[0058] Figure 15: V ocal behavior related transcripts showing differential AS in Noval^uj,^umice. AS exons are colored in yellow. Information of each AS event is described below the IGV snapshots; Gene name, AS exon number, AS type, percent spliced-in value (PSI, the percent of transcripts that include a specific AS exon), percent change (Al, APSI; Nova1hu / huvs. Nova1wt / wt) and / 7-value. AS splicing events are classified into the following types: Cassette exon (cass), alternative 5’ splice site (alt5), alternative 3’ splice site (alt3 ), tandem cassette (taca), mutually exclusive exons (mutx).

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[0059] Figure 16A and 16B: The resampling analysis for differential AS events in Nova1hu'^1mice, (a) The 650 random resampling were repeated 1000 times from a list of transcripts detected in the RNAseq dataset to calculate the number of transcripts those annotated in the behavior category in gene ontology database (https: / / geneontology.org / ). The histogram shows the density of the number of transcripts detected for each resampling, the boxplots at the top show the distribution features (median and quartiles (box) and maximum minimum (whiskers) and outliers (dots)). The green triangle indicates mean value (21.6) of the resampling. The red triangle indicates the number of transcripts detected in this study (27). The number of trials that exceeded the number of 27 was 99, 9.9% probability, indicating the number of transcripts detected in this study is higher than the average number of transcripts detected by chance, (b) The 27 random resampling were repeated 1000 times from the 843 transcripts annotated as the behavior category in the gene ontology database to calculate the number of transcripts those annotated in the vocalization category. The green triangle indicates the mean value (0.711) of the resampling. The red triangle indicates the number of transcripts detected in this study (4). Four trials detected the same number of transcripts as the observed number of transcripts 4, and zero trials exceeded 4, the probability is less than 4%, indicating the number of transcripts detected in this study is higher than the number of transcripts detected by chance with the 5% level of significance.

[0110]

[0060] Figure 17A and 17B: NOVA 1 -CLIP binding peaks in vocalization related transcripts, (a) A list of genes classified to be involved in vocal behavior in gene ontology analysis. Genes for which N0VA1 binding was detected on the transcript in CLIP analysis were marked (check mark). The threshold for NOVA 1 binding was a peak detected in all three biological replicates with peak height greater than 10. ForP21 samples (cortex, midbrain, cerebellum), the gene was marked if it meets the above criteria in either Novalhu / huor Novalwt / wt. (b) Examples of vocalization-related genes with N0VA1 CLIP peaks on their transcripts.

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[0061] Figure 18A-18I: Nova1hu / humice show altered vocal patterns, (a) Isolation induced ultrasonic vocalization (USV) test for pups, (b) USV parameters and syllable classification, (c) Fqmax distribution and two Gaussian fit in pup USVs. Ashman’s D score (a measure of separation of two distributions, above 2 means good separation) are shown. Each Gaussian center and weight are labeled. The intercept of the two Gaussian distributions (black triangle) were used as the cutoff between high and low Fqmax USVs. The dot plots at the bottom of the density plots show the mean (black dots) and standard deviation (whiskers) by peak for each genotype. There are no significant differences between genotypes, (d) Ratio of high or low Fqmax in syllables “d” and “m". The ratio of syllables that belong to each distribution (high or low) are calculated for the total number of each syllable type, (e) Courtship induced USV test for adult mice, (f) Duration distribution and two Gaussian fit for syllable “s” in adult USVs. Ashman’s D score, each Gaussian center and weight are shown. The intercept of the two Gaussian distributions (black triangle) was used as the cutoff between long and short duration. The dot plot at the bottom of the density plot shows the mean (black dots) and standard deviation (whiskers) by peak for each genotype. There are no significant differences between genotypes. Examples for short and long “s” are shown at the top of the plot, (g) Peak frequency parameters in long duration “s”. (h) Fqmax distribution and two Gaussian fit in adult USVs. Ashman’s D score, Gaussian center and weight are labeled. The black star indicates 100kHz cutoff of high Fqmax and low Fqmax. The dot plot at the bottom of the density plot shows the mean (black dots) and standard deviation (whiskers) in each USVs for genotype. There arc no significant differences between genotypes. Examples for low' and high Fqmax syllable are shown at the top of the plot, (i) Frequency variance (Fq variance) in high Fqmax in adult USVs. Data are represented as boxplots that include the minimum score, first (lower) quartile, median, third (upper) quartile, and maximum score, / ^-values were calculated by Wilcoxon rank sum test and corrected with Bonferroni method. * <0.05, **p<0.01. For pup (c-d), each circle indicates data from a single pup. Noval^u / ^uN=41, Nova1hu / wtN=23, Nova1wt / wtN=40 pups. For adults (f-i), experiments were conducted three times in consecutive weeks, and the average of the three experiments was plotted as the value for the mouse (white circle). N

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[0113] Nova1hu / huN=13, Nova1wt / wtN=14, Nova1wt / wtN=13 adults.

[0114]

[0062] Figure 19A-19E: USV characteristics in pups and adults, (a) Syllable composition (left) and amplitude (right) for each syllabic in pup USVs. Data is represented as boxplots. Each open circle indicates data from a single pup. p-values were calculated by Wilcoxon rank sum test and corrected with Bonferroni method. * p value <0.05. Nova1hu / huN=41, Nova1hu / wtN=23, Novalwt / wtN=40 pups, (b) Example ofbimodal distributions in peak frequency (Fq) in pup USVs. Density plots of start, minimum, mean, and maximum Fq for syllable “d” observed in each genotype, (c) Density plots of minimal and maximum Fq for jump syllables (“d”, “u”, “nr”) observed in each genotype. Arrows indicate high Fqmax syllables above 100kHz. (d) Density plots of Fqmin and Fqmax in adult USV s. Two Gaussians (blue and orange lines) fitted are overlaid. The Fqmax plot is corresponding to Figure 4h. The Gaussian centers (green and red circles) and weights are labeled. The black star indicates 100kHz cutoff of high and low Fqmax USVs. (e) Examples of mouse USVs spanning the high- frequency regions from previous studies. Figures from Vogel et al., 20197. and Grimsley et al., 20118. USVs with signals above 100 kHz are indicated by orange arrows.

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[0063] Figure 20A-20E: Playback behavioral experiments for pup USVs. (a) Apparatus used for neonatal mouse vocal selection tests with musing mother mice. The box consists of three rooms connected by a passageway through which the mouse can pass, and a speaker is attached to each room at each end. (b) Overview of the neonatal mouse vocal selection test with mother mice. Nursing mother mouse of Nova1wt / wtor Nova1hu^11was placed in the center of the room and their behavior was recorded while vocal recordings of neonatal mice were played. Vocal recordings of Novalwt / wtor Nova1hu / huneonatal mice were randomly played from speakers at both ends, respectively. To exclude direction preference, the recordings played from each speaker were switched after a one- minute break, (c) Vocal recordings of neonatal mice used in the experiments. The recordings were arranged from the data of the pup-USV test to reflect the overall parameters of each genotype (Nova1wt / wtor Nova1hu / hu. (d) Comparison of the time the mother mouse stayed in the room where the recording of each neonatal mouse w as played. Bars indicate mean ± standard error; dots indicate values for each individual. The time spent in each room by the same individual is connected by a line, (e) Comparison of the number of times the mother mouse entered the room where each neonatal mouse recording was played. Bars indicate mean ± standard error; dots indicate values for each individual. The time spent in each room by the same individual is connected by a line. Nova1wt / wtmother: N=32, Noval^u / ^umother: N=29.

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[0064] Figure 21A and 21B: (a) Rotarod performance test, (left) illustration of the test. To measure the locomotor performance (motor coordination), mice were placed on an elevated revolving rod that accelerates at a constant rate (4 to 40rpm in 300sec). The time it took the animals to fall were recorded. Tests were performed three times and the average value were calculated, (middle) Results in NOVAI deficiency mouse model (cKO mice: Tajima et al., 2023 (right) Results in humanized NOVAI mice. The bar graph represent mean ± standard deviation. The dot indicates average time for one mouse. Gad2CreNoval^ / wt^=4, Gad2CreNova1fl / flN=5, Novalwt / wt^=3, Nova1hu / huN=3. (b) Y-maze test, (left) illustration of the test and calculation for the alternation rate. Mice were allowed to freely explore a Y -shaped maze for 8 minutes. The number of entries into the arms and the number of triads were recordedto calculate the percentage of alternation. Alternations are consecutive entries into each arm of the Y-maze without any repeats (e.g., arm 1 -> 2 ->3). (middle) results in N0VA1 deficiency mouse model (cKO mice) The data is from Tajima et al., 20239. (right) results in humanized NOVAI mice. The alteration rates and total number of entries into arms during the tests are represented as boxplots with the minimum score, first quartile, median, third quartile, and maximum score. Each dot indicates data from a single mouse. Gad2CreNova1fl / wtN=7, Gad2CreNova1fl / flN=8, Novalwt / wtN=18, Nova1hu / wtN=23, Nova1hu / huN=18.

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[0065] Figure 22 depicts miRNAs that target Noval 3’UTR. Upon inspection (right panel) the miRNAs include miR-27a, miR-103. let-7a and miR-132.

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[0066] Figure 23. miRNAs that target Noval 3’ UTR. miRNA binding sites in Noval 3’ UTR mapped by Ago2 CLIP. Antagomirs (e.g. antisense to sequences surrounding miR-103) can block miRNA binding, offering the possibility of increasing Noval mRNA levels and translation of Noval protein (see Tajima et al., 2023 and related refs).

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[0067] Figure 24 depicts miRNAs that target Nova2 3’UTR. Upon inspection (right panel) the miRNAs include miR-26a, miR-484, miR-148a, miR-19a, miR-221, miR-124, miR-30b, miR-296-5p, miR-340-5p, miR-30b, miR-30e, miR-9, miR-288-5p, miR-124 let-7c.

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[0068] Corresponding reference characters indicate corresponding parts throughout the several views. The examples set out herein illustrate several embodiments of the invention but should not be construed as limiting the scope of the invention in any manner.

[0121] DETAILED DESCRIPTION

[0122] Definitions:

[0123]

[0069] As used herein the term “about” refers to ± 10 %,

[0124]

[0070] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”. It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of’ are also provided.

[0125]

[0071] The term “consisting of’ means “including and limited to”.

[0126]

[0072] The term “consisting essentially of’ means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0127]

[0073] As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

[0074] Throughout this application, various embodiments of this disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0128]

[0075] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0129]

[0076] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0130]

[0077] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0131]

[0078] The term “antibody” describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. CDR grafted antibodies are also contemplated by this term. An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U. S. Patent Nos. 4,816,397 and 4,816,567. The term “antibody(ies)” includes a wild type immunoglobulin (Ig) molecule, generally comprising four full length polypeptide chains, two heavy (H) chains and two light (L) chains, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain); including full length functional mutants, variants, or derivatives thereof, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain antibodies; Immunoglobulin molecules can be of anyclass (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2). Also included within the meaning of the term “antibody” are any “antibody fragment”.

[0132]

[0079] An “antibody fragment” means a molecule comprising at least one polypeptide chain that is not full length, including (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CHI) domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of an Fab (Fd) fragment, which consists of the VH and CHI domains; (iv) a variable fragment (Fv), which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain (Ward, E. S. et al., Nature 341, 544-546 (1989)); (vi) a camelid antibody; (vii) an isolated complementarity determining region (CDR); (viii) a Single Chain Fv Fragment wherein a VH domain and a VL domain are linked by a peptide I inker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (ix) a diabody, which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites (WO94 / 13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)); and (x) a linear antibody, which comprises a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; (xi) multivalent antibody fragments (scFv dimers, trimers and / or tetramers (Power and Hudson, J Immunol. Methods 242: 193-204 9 (2000)); (xii) a minibody, which is a bivalent molecule comprised of scFv fused to constant immunoglobulin domains, CH3 or CH4, wherein the constant CH3 or CH4 domains serve as dimerization domains (Olafsen T et al (2004) Prot Eng Des Sei 17(4):315-323; Hollinger P and Hudson PJ (2005) Nature Biotech 23(9): 1126-1136); and (xiii) other non-full length portions of heavy and / or light chains, or mutants, variants, or derivatives thereof, alone or in any combination. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are included.

[0133]

[0080] As antibodies can be modified in a number of ways, the term "antibody" should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP -A-0120694 and EP-A-0125023 and U. S. Patent Nos. 4,816,397 and 4,816,567.

[0081] An "antibody combining site" is that structural portion of an antibody molecule comprised of light chain or heavy and light chain variable and hypervariable regions that specifically binds antigen.

[0134]

[0082] The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule. Exemplary' antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab’, Ffab')? and F(v), which portions are preferred for use in the therapeutic methods described herein.

[0135]

[0083] The term “specific” may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partners). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.

[0136]

[0084] The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

[0137]

[0085] The term “consisting essentially of’ refers to a product, particularly a peptide sequence, of a defined number of residues which is not covalently attached to a larger product. In the case of the peptide of the invention referred to above, those of skill in the art will appreciate that minor modifications to the N- or C-terminal of the peptide may however be contemplated, such as the chemical modification of the terminal to add a protecting group or the like, e.g. the amidation of the C-terminus.

[0138]

[0086] The term “isolated” refers to the state in which specific binding members of the invention, or nucleic acid encoding such binding members will be, in accordance with the present invention. Members and nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo. Members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.

[0139]

[0087] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "pg" mean microgram, "mg" means milligram, "ul" or "pl" mean microliter, "ml" means milliliter, "1" means liter.

[0140]

[0088] The amino acid residues described herein are preferred to be in the " L" isomeric form. However, residues in the " D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin-binding is retained by the polypeptide. NH2refers to the free aminogroup present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.

[0141]

[0089] It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.

[0142]

[0090] A "replicon" is any genetic element

[0143]

[0144] plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo i.e., capable of replication under its own control.

[0145]

[0091] A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

[0146]

[0092] A " DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary' forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3’ direction along the nontranscribed strand of DNA i.e., the strand having a sequence homologous to the mRNA).

[0147]

[0093] An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.

[0148]

[0094] A DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3’ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0149]

[0095] Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

[0150]

[0096] A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5’ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein bindingdomains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain " TATA" boxes and " CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

[0151]

[0097] An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under tire control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

[0152]

[0098] A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

[0153]

[0099] The term "oligonucleotide," as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.

[0154]

[0100] The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

[0155]

[0101] The primers are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary' to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

[0102] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

[0156]

[0103] A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In rokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0157]

[0104] Two DNA sequences are "substantially homologous” when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.

[0158]

[0105] By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid.

[0159]

[0106] Mutations can be made in the sequences encoding the amino acids, such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein.

[0160]

[0107] The present invention includes sequences containing amino acid changes and substitutions, including conservative changes, which do not significantly alter the activity or binding characteristics of the resulting protein. In the instant invention, human NOVAI particularly and specifically has a valine amino acid at residue 197. Non-human N0VA1 protein does not have a valine at this position. Mouse NOVAI has an isoleucine. Isoleucine is encoded by ATA for example. Valine is encoded by any of GTA,GTT, GTC or GTG. In an aspect of the invention, the mouse ATA is mutated to GTA so that a valine is encoded and a human N0VA1 with I197V is expressed, generated, encoded.

[0161]

[0108] Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site (z.c., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces (l-tums in the protein's structure.

[0162]

[0109] Two amino acid sequences are "highly homologous" or "substantially homologous” when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90% or 95% or 98% or 99%) are identical, or represent conservative substitutions.

[0163] [HO] A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature

[0164]

[0165] a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

[0166] [Hl] A DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

[0167]

[0112] The term "agent” means any molecule, including polypeptides, antibodies, polynucleotides, chemical compounds and small molecules. In particular the term agent includes compounds such as test compounds or drug candidate compounds.

[0168]

[0113] The term "agonist" refers to a ligand that stimulates the receptor the ligand binds to in the broadest sense.

[0169]

[0114] The term "assay" means any process used to measure a specific property of a compound. A "screening assay" means a process used to characterize or select compounds based upon their activity' from a collection of compounds.

[0170]

[0115] The term "preventing” or "prevention" refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop) in asubject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.

[0171]

[0116] The term "prophylaxis" is related to and encompassed in the term ‘prevention’, and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non-limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.

[0172]

[0117] " Therapeutically effective amount" means that amount of a drug, compound, antimicrobial, antibody, or pharmaceutical agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician. In particular, with regard to gram-positive bacterial infections and growth of gram-positive bacteria, tire term “effective amount” is intended to include an effective amount of a compound or agent that will bring about a biologically meaningfid decrease in the amount of or extent of tumor regression and or increase in length of a subject’s survival or period disease-free or in remission. The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the growth or amount of tumor size, or enhanced survival or disease-free period by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent.

[0173]

[0118] The term "treating" or "treatment” of any disease or infection refers, in one embodiment, to ameliorating the disease or infection (i.e., arresting the disease or growth of the infectious agent or bacteria or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or infection, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, "treating” or "treatment" relates to slowing the progression of a disease or reducing an infection.

[0174]

[0119] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

[0175]

[0120] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "pg" mean microgram, "mg" means milligram, "ul" or "pl" mean microliter, "ml" means milliliter, "1" means liter.

[0176]

[0121] “Therapeutically effective amount” or “effective amount” as used herein refers to an amount that is effective to elicit the desired biological or medical response, including the amount of a compound that, when administered to a subject for treating a disease, is sufficient to affect such treatment for the disease.The effective amount will vary depending on the compound, the disease, and its severity and the age, weight, etc., of the subject to be treated. The effective amount can include a range of amounts. As is understood in the art, an effective amount may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable or beneficial result may be or is achieved. Suitable doses of any co-administered compounds may optionally be lowered due to the combined action (e.g., additive or synergistic effects) of the compounds.

[0177]

[0122] The term “subject” or “animal” is meant any subject, particularly a mammalian subject, in need of treatment with a peptide or polypeptide provided herein. Mammalian subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows, apes, monkeys, orangutans, and chimpanzees, and so on. In one aspect, the subject or animal is a non-human subject or animal. In one aspect, the non-human subject or animal is a mouse. In an aspect, the non-human subject or animal is a domesticated animal. In an aspect, the non-human subject or animal is a cat or a dog. Detailed Disclosure:

[0178]

[0123] N0VA1 is a neuronal RNA-binding protein expressed in the central nervous system and is essential for survival in mice and normal development in humans. A single amino acid change (1197V) in N0VA1 ’s second RNA binding domain is present specifically in humans. As described herein, non-human animals (mice) carrying the human-specific 1197V variant (denoted Novalh‘'hi) have specific alterations in alternative splicing, including multiple binding peaks in mouse brain transcripts involved in vocalization. In particular, the human 1197V substitution results in behavioral differences in vocalization patterns in Naval1”1" non-human animals (mice) as pups and adults. This human-specific NOVAI substitution presents as being linked to the development of spoken language via differential RNA regulation during brain development.

[0179]

[0124] MicroRNAs (miRNAs)

[0180]

[0125] Most of the mammalian genome is untranslated and these untranslated genomic regions contain numerous sequences that are transcribed into “noncoding RNAs” (ncRNAs). NcRNAs are classified based on length into small RNAs [18-200 bp; including “microRNAs” or “miRNAs”, piRNAs, snoRNAs, tRNA-derived fragments ( tRFs), and others] and long RNAs (>200 bp; “long non-coding RNAs” or “IncRNAs”). MiRNAs are primarily involved in the posttranscriptional regulation of protein-coding genes. Consistent with their roles in regulating critical biological processes, expression of individual miRNAs is tightly regulated in tissue- and developmental stage-specific patterns.

[0181]

[0126] microRNAs (miRNAs) are recognized as key players in the pathophysiology of multiple human diseases. miRNAs regulate the development, homeostasis and pathologies of virtually all vertebrate tissues. Many miRNAs have specific or enriched expression in the central nervous system, regulating such diverseprocesses as neuronal differentiation, excitation, synaptogenesis and plasticity (Tan, C. L. et al (2013) Science 342, 1254-1258). Accordingly, miRNA dysregulation is implicated in neurological disorders and many cancers including glioma and liver cancer (Im, H. I. & Kenny, P. J. (2012) Trends Neurosci. 35, 325-334; Mizoguchi, M. et al. (2012) J. Oncol. 2012, 732874; Setty, M. et al. (2012) Mol. Syst. Biol. 8, 605).

[0182]

[0127] As such, miRNAs have emerged as promising therapeutic targets for preventing the onset and / or progression of several diseases. In one approach, anti-miRNA antisense oligonucleotides or “antagomirs” precisely block the activity' of specific miRNAs and are therefore a promising therapeutic strategy to repress pathological miRNAs (Saenz-Pipaon, G and Dichek DA (2022) Atherosclerosis 374:44-54). Canonical miRNA binding involves base pairing of the miRNA seed region (nucleotides 2-8) to complementary target sites (Friedman, R. C. et al (2009) Genome Res. 19, 92-105; Nielsen, C. B. et al. (2007) RNA 13, 1894-1910).

[0183]

[0128] Some microRNAs have been reported as candidates that target NOVAI. Further, the involvement of miRNAs downregulating NOVAI in relation to some diseases, including gastric cancer and astrocytoma, has been reported (Zhi, F. et al (2014) PLoS One 9, e!09124; Yoon, S. O. et al (2016) Oncotarget 7, 2475-2495; Shen, B. et al (2015) FEBS Lett. 589, 3205-3211).

[0184]

[0129] In an aspect of the present invention, the presence of Argonaute-miRNA binding elements in Noval and the specific sites they are bound to on Nova 3’ UTRs have been identified and characterized. In one such aspect, mir-103 has been identified. The miR-103 site is conserved across numerous animal species, including humans, mice, rats, rabbits, chimps, rhesus monkeys, etc.

[0185]

[0130] The miR-103 8mer target sequence is AUGCUGCA (listed 5’ to 3’) (SEQ ID NO:23).

[0186]

[0131] An antagomir or antisense oligonucleotide (ASO) relevant and specific for the target sequence microRNA or miR is provided in an aspect of the invention. The antagomir or ASO comprises sequence complementary to the mir target sequence miR-103, particularly the sequence UGCAGCAU (listed 5’-3’) (SEQ ID NO:61). The antagomir or ASO can include nucleotides complementary to additional adjacent sequence, such as up to 5, 10 or more nucleotides adjacent, including 5’ to, or 3’ to, or both 5’ and 3’ to the target 8mer sequence. For example, the antagomir or ASO may further comprise nucleotides complementary to adjacent sequence, such as AUA (SEQ ID NO:62), AAUA (SEQ ID NO:63), AAAUA (SEQ ID NO:64), GAAAUA (SEQ ID NO:65), GGAAAUA (SEQ ID NO:66), UGGAAAUA (SEQ ID NO:67), AUGGAAAUA (SEQ ID NO:68) or AAUGGAAAUA (SEQ IDNO:69). Thus, the antagomir may further comprise sequence selected from TAT, TATT, TATTT, TATTTC, TATTTCC, TATTTCCA.

[0187]

[0132] Therapies that target miRNAs aim either to increase the expression / abundance of miRNAs that prevent disease or to reduce the activity of miRNAs that cause disease. These therapies employ miRNA mimics or miRNA inhibitors, respectively. MiRNA mimics (or agomiRs) are synthetic double-stranded small RNA molecules that specifically match the sequence of endogenous mature miRNAs. MiRNA inhibitors are classified as either antisense oligonucleotides (ASOs) or miRNA sponges based on theirmolecular structure (Diener C et al (2022) Trends Genet 38(6):613-26.). MiRNA-targeting ASOs or antagomirs are single-stranded small RNAs that bind complementarity to their miRNA targets, blocking interaction of these targets with their corresponding endogenous mRNA targets, thereby increasing (or “de¬ repressing”) expression of these mRNA targets. Similarly, miRNA sponges possess multiple miRNA binding sequences that can sequester multiple copies of either a single miRNA species or several different miRNA species. Accordingly, sponges are potentially a more potent approach for reducing levels of target miRNA. miRNA mimics (i.e., agomiRs) have shown potential in preclinical studies and are being tested clinically (Hong DS et al (2020) British Journal of Cancer122(11):1630–7; Gallant-Behm CL et al (2019), Journal of Investigative Dermatology139(5):1073–81)..

[0188]

[0133] Antagomirs or antisense oligonucleotides (ASOs) of the invention may be chemically modified to improve stability, increase cellular uptake, and enhance affinity for target miRNAs. These modifications can be applied to the backbone as well as the ribose and / or nucleobase of ASOs and include: (1) changes in the phosphodiester bonds such as incorporating a phosphorothioate internucleotide linkage to confer resistance to nuclease degradation and increase bioavailability; (2) modification of ribose to enhance ASO stability and affinity for target miRNA (e.g., 2'-O-methyl, 2'-O-methoxyethyl, and 2′-fluoro modifications); (3) construction of a locked nucleic acid by placing a methyl bridge between the 2'-0 and 4' position of the ribose ring, thereby increasing the affinity of LNAs for their target miRNAs; and (4) substitution of the ribose-phosphate backbone with a pseudopeptide sequence [creating a peptide nucleic acid (PNA) molecule], or with a 6-membered morpholino ring and phosphorodiamidate linkages, generating a phosphorodiamidate morpholino oligomer (PMO), wherein the PNAs and PMOs are neutrally charged and resistant to cleavage by nucleases.

[0189]

[0134] In some embodiments, targeted delivery of antagomirs can make use of viral (i.e., adenovirus, adeno-associated virus, and lentivirus) and non-viral (i.e., liposomes, extracellular vesicles, and polymer nanoparticles) delivery systems.

[0190]

[0135] Antagomirs or antisense oligonucleotides (ASOs) of the invention can be delivered as nonencapsulated molecules, including via intravenous administration, subcutaneous administration or direct administration to the CNS. Alternatively, ASOs may be delivered via expression by a viral vector, such as one that has specificity or capability for relevant cell types, organ or body region. In another aspect, the ASOs are encapsulated in non-viral nanoparticles (NPs) constructed with a goal of targeting their ASO / antagomir cargo to a cell type, organ, or body region of interest. Viral vectors may be double stranded virus vectors or single stranded virus vectors. Viral vectors include adenovirus (double stranded)- based vectors and adeno-associated virus (AAV) (single stranded) -based vectors. Exemplary and suitable AAVs may be AA1, AA2, AA5, AA6, AA8 or AA9. Lentiviral vectors may also be utilized.

[0136] Use of antagomir-expressing viral vectors eliminates the need for resource-intensive synthesis and purification of clinical-grade antagomirs. A single viral vector injection could achieve long-term in vivo expression of an antagomir, leading to durable suppression of its miRNA target.

[0191]

[0137] In another aspect, the ASOs of the invention may be delivered as cargo in a delivery preparation designed for sustained or extended half-life. In one such aspect, liposomes which are synthetic lipid bilayer particles that can encapsulate molecular cargoes, may be utilized for delivery. The liposomes may be synthesized from cationic lipids and / or coated with a polymer such as polyethylene glycol (PEG). Alternatively, extracellular vesicles (EVs) may be utilized, which may be derived from the plasma membrane or from intracellular multivesicular bodies. In another aspect, polymer nanoparticles are utilized for delivery, wherein the ASOs are carried as cargo attached to a polymeric scaffold, such as poly(lactic-co-glycolic acid) (PGLA) or chitosan, a natural cationic polysaccharide.

[0192]

[0138] The examples herein demonstrate that correction of NO VAI encoding nucleic acid in a nonhuman animal to encode human N0VA1 protein, particularly N0VA1 having a valine at amino acid 197, results in altered vocalization in mice. Thus, expression of human NOVAI protein, particularly N0VA1 having a valine at amino acid 197, is associated with specific vocalizations and RNA processing and presents a particular neuronal transcriptome in an animal, even in a non-human animal. Alterations in N0VA1 protein and / or N0VA1 expression is associated with severe psychiatric disorders, motor developmental abnormalities, Autism (including non-verbal autism), neuro-behavioral deficits, including motor dysfunction and developmental delay, language dysfunction and speech delay. Further, alterations in N0VA1 protein and / or N0VA1 expression is associated with or linked to the progression and prognosis of cancers such as gastric cancer, hepatocellular carcinoma, and lymphoma, as well as glioblastoma and astrocytoma and oligodendroglioma. In aspects of the invention, correction of the NOVAI encoding nucleic acid and / or genetic manipulation to express unaltered and non-pathogenic NOVA1 is implemented including for alleviation of disorders or conditions associated with altered N0VA1 expression and / or expression and activity of pathogenic N0VA1 protein or N0VA1 variants. Such correction may include genetic correction techniques such as tire CRISPR / CAS type system and such methods implemented and described herein in humans. Correction may include vector expression and / or genome integration of unaltered and non-pathogenic N0VA1. Methods for altering or modifying vocalization in a human are also provided.

[0193]

[0139] In an embodiment, the invention provides a method for inhibiting a microRNA molecule targeting N0VA1, particularly N0VA1 3’ untranslated region (UTR), in a cell or animal, particularly a human, comprising introducing one or more microRNA molecule comprising introducing into the cell or animal, particularly human, one or more anti-micro RNA molecule directed to NOVA1 UTR which is ordinarily targeted by the microRNA. In one such embodiment, the anti-microRNA molecule acts a a competitive inhibitor for miRNA binding. Blocking the miRNA via an antimicroRNA molecule or ASO that binds tothe NOVA 3’UTR could only specifically block that one site of action (i.e. within the N0VA1 or within the N0VA23’UTR).

[0194]

[0140] In another further embodiment, the invention provides a method for inhibiting a microRNA molecule targeting NOVAI, particularly NOVAI 3’ untranslated region (UTR), in a cell or animal, particularly a human, comprising introducing one or more microRNA molecule comprising introducing into the cell or animal, particularly human, one or more anti-micro RNA molecule directed to microRNA targeting NOVAI. In an aspect of the method, a single-stranded anti-microRNA molecule directed against microRNA targeting NOVAI comprising a sequence of a minimum of eight moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a base pair with a complementary base wherein at least eight contiguous bases of the anti-microRNA molecule are complementary to the microRNA.

[0195]

[0141] In one aspect, up to thirty percent of the bases may be substituted by wobble base pairs, and up to ten percent of the at least eight moieties may be additions, deletions, mismatches, or combinations thereof. In an embodiment, no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units.

[0196]

[0142] Methods to alleviate or modulate or treat diseases or conditions associated with altered NOVAI activity, expression, or pathogenic forms of NOVAI are provided herein. Such disorders or conditions include psychiatric disorders, motor developmental abnormalities, Autism (including non-verbal autism), neuro-behavioral deficits, including motor dysfunction and developmental delay, language dysfunction, speech delay and cancers. Cancers include gastric cancer, hepatocellular carcinoma, and lymphoma, as well as glioblastoma and astrocytoma and oligodendroglioma.

[0197]

[0143] Methods to alleviate or modulate or treat diseases or conditions associated with altered NOVA2 activity, expression, or pathogenic forms ofNOVA2 are provided herein.

[0198]

[0144] Methods for altering or modifying vocalization in a human are also provided. Vocalization may be assessed, evaluated and tested in accordance with standards and methods known in the art, including as provided and described herein.

[0199]

[0145] In other aspects, facilitated by early diagnostic(s) to identify NOVA-related issues, upregulating NOVAI (or NOVA2) by intervention as provided herein provides methods, molecules, and approaches for correcting developmental (e.g. non-verbal autism) or degenerative (e.g. frontal dementias, characterized by motor language production defects, e.g. FTDP17) issues and diseases or conditions. Thus, in embodiments, congenital language deficiencies (such as non-verbal autism) could be alleviated, modulated, or treated by increasing Noval levels (or Nova2). Similar situations and disease targets include frontotemporal dementias, which present as motor-verbal problems (Broca’s area). Early diagnosis, such as in utero or through pre-implantation diagnosis could facilitate and allow correction inutero or selection during IVF, particularly in as much as studies show that Nova! gets turned on and is expressed and impactful early in development.

[0200]

[0146] In an aspect of the invention, the neuronal transcriptome may be altered or modified. Thus, methods are provided for altering or modifying the neuronal transcriptome in a human are further provided via the anti-RNA molecules or via the N0VA1 correction outlined herein.

[0201]

[0147] The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.

[0202]

[0148] While the present disclosure has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof to adapt to particular situations without departing from the scope of the present disclosure. Therefore, it is intended that the present disclosure not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out the present disclosure, but that the present disclosure will include all embodiments falling within the scope and spirit of the appended claims.

[0203] EXAMPLES EXAMPLE 1

[0204] Introduction

[0205]

[0149] Fossil records indicate that modem humans (Homo Sapiens)' emerged 200,000-300,000 years ago as the predominant species from several now-extinct hominid subclades •

[0206]

[0207] . Humans differ significantly from their closest living relatives, the great apes, particularly in their ability to communicate through complex learned vocal communication, a necessary component of spoken language. This complexity is driven by some anatomical adaptions of the vocal tract and intricate neural networks linking various brain regions3-'. However, the genetic basis underlying these specialized human traits remains to be fully identified.

[0208]

[0150] The closest evolutionary relatives of modern humans are two extinct lineages: Neanderthals and Denisovans. Genome sequencing from fossilized remains of these archaic humans has identified distinct genetic differences between them and modern humans, which may be relevant to recent human

[0209] Q _ 1 5

[0210] evolution. Additionally, the availability of extensive human genome data over the past few decades, initially focused on European populations, has significantly expanded the scope of evolutionary studies P-14.

[0151] The transcription factor forkhead box P2 (FOXP2) is of particular interest as a potential driver of human language function, as it harbors two amino acid substitutions present in human but not in chimpanzee and many other mammal genomes. Families with FOXP2 mutations exhibit severe speech defects ^46wj1iieFOXP2 disruption in mice leads to vocalization abnormalities^ ^ suggesting a role in spoken language function. Studies on mice with the two amino acids substituted to the human version have reported vocal changes both in the neonatal and adult stages

[0211]

[0212] ’. While Hammerschmidt et al. observed minimal vocal changes, von Merten et al. reported qualitative changes under a more natural vocalization paradigm 90 ’ 91, suggesting the involvement of these two amino acids in vocalization.

[0213] However, these substitutions are also present in archaic humans, and comprehensive analyses using diverse human genome datasets have found no evidence of recent selection. This suggests that the FOXP2 substitutions occurred earlier than initially thought12,22. Similarly, the TKTL1 gene contains a humanspecific amino acid thought to influence greater neurogenesis in human than Neanderthal frontal cortex, though this finding is based on European ancestry genome datasets23. Broader analyses of modem human genomes reveal that 0.03-0.2% of individuals possess the ‘putative Neanderthal variant’, indicating its presence in a significant portion of the population14. These findings underscore the importance of incorporating diverse human samples to identify and validate the genetic background of modern human traits through genomic comparisons.

[0214]

[0152] Genomic comparisons between archaic humans, ape genomes, and the broader human population have identified 61 human-specific nonsynonymous coding variants that arc fixed or nearly fixed in modern humans13. Notably, one of the genes includes an isoleucine to valine substitution at position 197 (I197V) in the RNA binding protein neuro-oncological ventral antigenl (N0VA1). NOVAl is highly expressed in neurons of the central nervous system (CNS) in both mice and humans24, and its expression has also been observed in cultured human and rat cells25–27. NOVAl was first identified as an autoantigen targeted in the paraneoplastic neurologic disorder (PND) opsoclonusmyoclonus ataxia (POMA)24. PNDs develop when tumor cells ectopically express proteins normally restricted to the nervous system, triggering an anti-tumor immune response that breaches the blood-brain barrier, leading to autoimmune neurologic

[0215]

[0216] disease" ’. In POMA, a robust immune response is mounted against NOVAl and its paralog, NOVA230The autoimmune disorder is characterized by motor dysfunction due to failure of inhibition of midbrain neurons, which results in the hyperactivity associated with opsoclonus-myoclonus ataxia24. In mice, homozygous deletion of the Noval gene results in an early postnatal lethal phenotype due to abnormal motor function31. Therefore, NOVAI plays a crucial role in neural development and neuromuscular control in mammals. NOVA proteins directly bind RNAO'") 72

[0217] in the mouse brain-’V-53to regulate pre-mRNA processing1’3^’33, translation30and 77

[0218] neurophysiology. Genetic studies mapping NOVA target RNAs in mice and humans have also 1 inked it to autism. Interestingly, a human patient with a heterozygous deletion of NOVAI presented with delay of language development, learning disabilities, motor hyperactivity and behavioral dysregulation36,

[0219]

[0153] Studies have explored the N0VA1 I197V variant by reverting the ancestral isoleucine 197 variant back into human iPSC-derived organoids, revealing morphological and electrophysiological changes in vitro. However, these effects were not observed in another study that reintroduced the same substitution an

[0220] in different iPSCs. This discrepancy underscores challenges of obtaining consistent results with varying experimental methods and materials in vitrei ’41.

[0221]

[0154] In this work, we used gene-editing to substitute the NOVA 1 isoleucine (I) isoform present in most mammals and archaic hominids (Neanderthals and Denisovans) with the human-specific valine (V) variant at position 197 in mice. Comparison of these humanized NOVAI mice {Nova1hu / hu) with wild-type mice carrying the ancestral Noval gene (Nova1wt / wt) revealed specific transcriptomic and behavior differences related to vocalization. Taken together, the unique role of N0VA1 in neurons, its association with human disease, and evidence that the human-specific amino acid 197 variant confers vocalization changes in humanized mice suggest a role for N0VA1 in the evolution of human-specific language.

[0222] RESULTS NOVAI I197V has characteristics of a variant that underwent a strong evolutionary selective sweep in modern humans

[0223]

[0155] NOVA proteins have three K homology (KH)-type RNA binding domains and have been shown to bind to YCAY repeat sequences on target transcripts36,42–46Comparison of the amino acid sequences of NOVA 1 and N0VA2 in various organisms showed that N0VA1 is extremely highly conserved across the whole protein sequence, whereas N0VA2 shows higher variability between species (Fig. la). We compared eight human genomes with three high-coverage Neanderthal genomes and one high-coverage Denisovan genome. The only change between modern and ancient humans was a nonsynonymous nucleotide substitution encoding the 197thamino acid of NOVAI, resulting in a valine in modern humans replacing an isoleucine in ancient humans (Fig. Ib). An expanded genomic analysis of the dbSNP database revealed that this substitution was present in all but six of 650,058 human sequences, five of which were from individuals of Asian descent (Figure 2a). The samples aredeidentified, so it is not possible to assess those individuals.

[0224]

[0156] We analyzed the mean allele frequencies (MAF) of NOVAI in 121,412 human genomes from the ExAC database (Fig. 1c, Fig. 2b). This analysis confirmed the extremely low frequency of variants encoding 197V in humans, consistent with strong selective pressure across the entire N0VA1 coding sequence. Specifically, the upper bound of 95% confidence interval for the MAF of NOVAI was 0.00071, significantly lower than that of NOVA2 (0.0099) or the average across all genes on chromosome 14, where NOVAI is located (0.0042; Fig. 1c). Moreover, evolutionary analysis on the NOVAI gene yielded a Tajima’s D statistic of -2.48 (Table 1). Normalizing Tajima’s D values between genes by the theoretical minimum 47, we found that NO VA 1 ’s normalized Tajima’s D statistic was exceedingly low. This suggests that NO VAI has undergone strong purifying selection, particularly in comparison to NOVA2, neighboring genes (FOXG1, STXBP6), and a set of genes associated with 61 human-specific nonsynonymous coding v

[0225]

[0226] ariants^ (Fig. Id, Fig. 2, Table 1).

[0227]

[0157] To further examine whether a selective sweep occurred at the NOVAI gene locus, we performed JO the DH test — a robust method for detecting selection that remains insensitive to other perturbations. Using human genetic data from the 1000 Genomes Project, we calculated the DH value for the NOVAI gene locus, which was 0.42643, reaching the 5% significance level among the genes on the same chromosome (chr. 14). In contrast, adjacent genes did notreach significance (FOXG1: 0.90390, STXBP6'.

[0228] 0.77477). It has been reported that the human-specific variant in NO VAI resides on the third-largest human-specific haplotype among the fixed human-specific sites, featuring two high-frequency haplotypes Our results, together with those of Trujillo et al, support amodel whereby the NOVAI 197V variant distinguishes NOVA 1 in modern humans from ancient humans, primates, or more distant species (Fig. la-c).

[0229]

[0158] To further evaluate the hypothesis that the NOVAI 197V variant may have been part of a selective sweep, we performed an analysis of the ancestral recombination graph (ARG) for modern and archaic hominins in the surrounding region, using ARGs previously inferred using ARGweaver-D49. These ARGs explicitly describe gene trees and accompanying recombination events throughout the region. Focusing on the NOVAI 197V variant, we analyzed the sampled ARGs using CLUES250, a method that estimates a selection coefficient to best explain observed changes in allele frequency over time, based on indirect information from the ARG.

[0230]

[0159] Our analysis revealed that selection at the NOVAI SNP is relatively strong and statistically significant, with an estimated selection coefficient of s = 0.00082 ip = 0.019 for the null hypothesis of no selection). While this estimate is an order of magnitude less than those observed at the strongest sweeps in the human genome (such as LCT, which has s ≅ 0.0151), it is still substantial, correspondingto a population- scaled coefficient of 5 = 2Nes = 19, indicating strong selection relative to nearly neutral evolution (where |5|<= 1) (Fig. 4). For comparison, we applied the same CLUES2 analysis

[0231] to 38 other SNPs for which informative ARGs were available, previously identified as potentially 1 -1

[0232] selected in the human genome. The results showed that the selection observed at the N0VA1 SNP is relatively strong compared to these other genes, with 33 of the 38 showing either non- significant results or smaller selection coefficients (Table 2).

[0233]

[0160] Taken together, the observation that the NOVAI 197V allele became nearly fixed and is shared across human population groups suggests that it arose and increased to high frequency before their divergence. Our analyses support the idea that the N0VA1 197V variant was part of an ancient selective sweep in modern humans, predating many other known sweeps in the human genome.

[0234] Humanized NO VAI mice are comparable to wild-type mice in development and gene expression in the brain

[0235]

[0161] To explore the physiologic and biological significance of the 1197 V amino acid substitution in NOVAI, we used CRISPR / Cas9-based gene editing to introduce nucleotide changes that result in humantype NOVAI knock-in mice (Nova1hu / humice; Fig. le. Fig. 5). Nova1hu / humice developed normally (Fig. 6a) and exhibited fertility similar to littermate controls Nova1wt / wt) mice. The brain to body weight ratio was comparable between Nova

[0236]

[0237] and Nova1wt / wtmice (Fig. 6b). Comprehensive gene expression analysis of the midbrain at embryonic day 18.5 (E18.5) and at postnatal day 21 (P21), when fundamental neural circuits and behaviors have been established, revealed that tire transcript levels, including Noval itself, were nearly identical between genotypes throughout development (Fig. 6c) and across different brain regions (Fig.7a-d). Similarly, the expression pattern and levels of NOVA1 protein were equivalent between genotypes in the brain (Fig. 8).

[0238]

[0162] The only gene that showed a significant steady-state difference in Noval^lu^’umice was Gkn3 atP21, a secreted protein involved in endothelial cell proliferation (Figure 6c; jJ-value<0.05, FDR<0.1). Gkn3 is thought to be involved in adaptive gene loss during recent human evolution

[0239]

[0240] , and showed down regulation in the P21 midbrain of Noval^u'^l{mice (average TPM 18.6 in Nova1wt / wt, 11.3 in Nova1hu / hu, log2FC = -0.71, p-value = 2.3×10-6, FDR = 0.037).

[0241] Modern human specific amino acid substitution does not affect sequence specific RNA- binding capacity of NOVAI

[0242]

[0163] NOVA proteins harbor three KH domains that are responsible for sequence-specific RNA-binding 2>45,46

[0243]

[0244] domain, found in many RNA binding proteins, includes common motifs: an invariant Gly-X-X-Gly motif, a hydrophobic core, and a variable loop. The 197thamino acid in NO VAI is a part of the hydrophobic core. Several studies have shown that a single amino acid substitution in thehydrophobic core cause loss of function of RNA binding proteins54–56ForNoval, in vitro binding assays have demonstrated that single amino acid substitutions (He— > Thr in KH1, Leu— »Asn in KH3) in the hydrophobic cores cause loss of RNA binding capability'^’57

[0245]

[0246] 9)

[0247]

[0164] To examine whether the I197V substitution affects NOVAl’s RNA binding ability', we performed CLIP (cross-linking and immunoprecipitation) to compare N0VA1 genome-wide binding maps in Noval^u'^l‘ and Nova1wt / wtmidbrain at P21 (Fig. 6d). Across three biological replicates for each genotype, we identified 26,155 binding peaks in Noval^u'^uand 27,720 in Novalwt'wtmice, respectively. NOVAI binding peaks were detected primarily on introns and 3’UTRs (Fig. 6e). The binding motifs were highly enriched for the known NOVAI binding sequences (UCAU), and the genomic distribution of NOVA 1 binding to this motif was highly similar between genotypes (Fig. 6f). The number of tags in the detected CLIP peaks were highly comparable (R2=0.982), with only minor differences in low-count peaks (a total of 250 peaks: average CLIP tags / peak 16.3 m. Nova1hu / hu, 13.1 in Nova1wt / wt;i-value<0.01, |log2FC|>l) (Fig. 6g, Table 3). In both genotypes, N0VA1 bound transcripts were strongly enriched for those encoding proteins involved in the synaptic signaling, transmission and secretion (Fig. 6h). The nearly identical characteristics between genotypes were also observed in the genomic distribution of NOVA 1 binding peaks, enriched motifs, and peak correlations in cortex and cerebellum at P21 (Fig. 10). Notably, NOVAI bound transcripts identified by CLIP analysis were enriched for behavior- and synapse-related categories across midbrain, cortex and cerebellum (Fig. 6h, Fig. lOe, lOj).

[0248]

[0165] We performed in vitro RNA binding assays to compare the RNA binding characteristics of the N0VA1 proteins. Full-length proteins were purified from E. coli, and RNA oligonucleotides with the N0VA1 binding site (UCAU repeat sequence with stem-loop structure42) were used for gel shift assays. Each NOVA1 protein bound32P-labeled UCAU-RNA and caused a dose-dependent shift in migration (Fig. 6i), with indistinguishable binding dissociation constants (Kd for NO VAI protein was 105.83 ± 8.3, and for NOVAlwtprotein was 104.8 ± 11.6; (Fig. 6j). Taken together, these in vivo and in vitro studies reveal not only the I197V variant’s resilience in maintaining the biophysical features of RNA binding with minimal global disruption but also its remarkable conservation of overall function. However, this variant exerts specific effects on alternative splicing (AS) as explored in the following section.

[0249] Humanized NO VAI mice exhibit alternative splicing changes of specific genes

[0250]

[0166] The role of RNA binding proteins in mRNA processing is often influenced by various factors beyond their RNA binding capability. Several studies suggest that competing or cooperating proteins.as well as non-protein factors like metals or ATP, play roles in determining the downstream effects on target mRNAs58–63. The function of NOVA proteins on specific mRNA targets, such as splicing or stabilization, also depends on their association with other proteinsJ>44,64 Although the I197V substitution has little effect on the RNA affinity and sequence specificity, X- ray crystallography analysis and protein structure prediction suggested subtle changes in amino acid interactions within the KH domain (Fig. 11, see Discussion). These changes may affect protein-protein interactions or NOVA dimerization65, leading us to investigate their impact on RNA regulation, particularly on AS.

[0251]

[0167] We first determined the brain regions for AS analysis based on the expression patterns of N0VA1. The immunostaining of mice brains at E18.5 and P21 shows that NOVAI is expressed throughout brain development across various regions (Fig. 12a, Fig. 13). N0VA1 is most highly expressed in midbrain, low-level across cortical layers, sparse in striatum and hippocampus, and intermediate level in the granular layer in cerebellum. Analysis of NOVA protein expression in P21 mouse brain using N0VA1 and panNOVA antibodies revealed that among the various isoforms of N0VA1 and N0VA2, a specific N0VA1 isoform (N0VA1 without Exon4^ ) is highly expressed in the midbrain, particularly in the periaqueductal gray (PAG) region (Fig. 12b, Fig. 14). The PAG is involved in a broad range of physiological and behavioral functions, including defense reaction, pain and anxiety, fear, micturition, and vocalization66,67. It is thought to integrate sensory signals from the periphery, acting as a control center for behavioral

[0252]

[0253] regulation66AS analysis was performed in the mouse midbrain, the region where NOVAI is most highly expressed.

[0254]

[0168] AS analysis in the P21 midbrain revealed that several AS events were specifically altered in Nova1hu / humice compared to Novalwt / vtcontrols (Fig. 12c). Specifically, 720 events showed significant changes (delta PSI (di) more than 5% (|<7Z|>0.05, <0.05)) (Table 4). Among the most intriguing changes were tandem cassette exons in Fnbp1l (formin binding protein I -like, exons 10 and 11), a gene implicated in human intelligence 7Q ’ ^0, and cassette exons in Itprl (inositol 1,4,5-trisphosphate receptor type 1, exon 41), a receptor that mediates calcium release from the endoplasmic reticulum (Fig. 12d). Although the effect of the 1197V substitution on AS was smaller than the effects observed in previous NOVA knockout studies34,64, these findings highlight the variant’s subtle but specific molecular impact. We further investigated the NOVAI I197V variant’s influence on AS by cross-referencing the CLIP and gene annotation datasets and assessing the statistical significance of the results using random resampling methods, as detailed in the section below.

[0255]

[0169] To assess the molecular impact and potential direct effect of the I197V substitution, we examined N0VA1 binding on AS transcripts by analyzing the CLIP dataset. Among the 720 differential AS events, 258 (41%) had N0VA1 binding peaks on their transcripts (Figure 12c). Gene annotationanalysis revealed that the 630 transcripts with differential AS events are enriched in processes related to cell projection organization or chromatin remodeling, and the transcripts with N0VA1 binding peaks show further enrichment in processes involving cell projection, morphogenesis, and synaptic function (Fig. 12e).

[0256]

[0170] To explore the potential effects on behavioral control in NOVA1^U / ^Umice, we examined genes associated with behavior among transcripts showing differential AS events. Of the 630 transcripts with differential AS events, 27 transcripts were associated with the behavior category (Table 5). Remarkably, the vocal behavior term showed the highest ratio of gene coverage, with four of the 22 genes annotated for vocal behavior (Auts2, Myh14, Nrxn2, Srpx2)^81–84^ being differentially spliced in Noval^u'^umice relative to Noval^'t,wtmice (Fig. 12f, Fig. 15). This number of vocalization-related genes was significantly higher than expected by chance, exceeding the 5% significance level in random resamplings tests (1000 resamplings, mean 0.71, median 1; Fig. 16).

[0257]

[0171] Interestingly, many genes involved in vocalization, including Foxp2, Celf6, Auts2, Nrxnl-3, and ShankI-3, showed reproducible NOVAI binding peaks on their transcripts across multiple brain regions (Fig. 17). Given that Pasilla, the fly ortholog of mammalian NOVA 1 / 2, regulates splicing of o

[0258] target transcript in an experience-dependent manner, it is plausible that these vocalization related transcripts are similarly affected in a context-dependent manner, such as in response to sensory cues from surrounding environment. Together, these findings indicate that Noval^u'^n>mice with I197V substitution exhibit changes in specific splicing events in the brain, including those in genes involved in animal behavior, particularly vocalization.

[0259] Humanized NOVAI mice pups have altered vocalization

[0260]

[0172] We next examined whether Noval^U / ^nimice with I197V substitution exhibit changes in vocalization behavior. Prior studies in birds, fish, and mammals have shown that all vocal species have a conserved midbrain / brainstem vocal motor pathway^^-^^. Of particular importance in the vocal circuit in the mammalian brain is the periaqueductal gray (PAG), which plays a central role in the neural basis of primate vocal production

[0261]

[0262] . The PAG projects to brainstem respiratory premotor and vocal motor nuclei, including the nucleus ambiguous (Amb), which directly innervates the

[0263]

[0264] larynx >. Inhibition of PAG or Amb function results in loss of innate v

[0265]

[0266] ocalization^ “93, while stimulation of the PAG induces vocalizations in both primates and mice^^ suggesting that the PAG and downstream brainstem circuits are essential for vocalization. We found that N0VA1 is highly expressed in the midbrain, including the PAG and Amb (Fig. 12a- b, Fig. 13-14). In addition to A'ova7^u / ^?w-specific actions on RNA splicing, we also found that many of the genes reported to be involved in vocal behavior(14 out of 22 transcripts) were NOVAI binding targets (Fig. 17). These data strengthen the possibility of a relationship between Nova1hu / huand vocalization, suggesting that vocalization studies in these mice would be valuable.

[0267]

[0173] We first compared vocalizations from pups of Nova1hu / hu, Nova1^hu / wt^ (heterozygous) and NovalHt / Vi 7mice. When pups are isolated from their mothers, they produce isolation induced ultrasonic vocalizations (USVs), which are distress calls that attract their mother^^-^^. We recorded USVs from 7-day-old mice pups for 5 minutes in a dark sound isolation chamber (Fig. 18a, Table 6, Table 7).

[0268] no

[0269] Following a previous protocol, we classified the syllables into four types (simple [s], upward [u], downward [d], multiple [m]), based on the direction and number of pitch jumps that separate notes within a syllable. Each syllable was analyzed for peak frequency measures (frequency value of start: Fqstart, minimum: Fqmin, maximum: Fqmax, end: Fqend, mean: Fqmean), variance (Fqvariance), bandwidth, duration, amplitude and purity (Fig. 18b).

[0270]

[0174] The average number of USVs were 77.9 ± 6.0 per minute in Novalwt'wtand 62.7 ± 6.4 per minute in Nova1hu / hu(mean ± standard error), with no significant difference between genotypes (Table 8). However, several changes in USV features which are thought to be important for mouse vocal communication

[0271]

[0272] were observed. The “s” syllable (without pitch jumps), the most abundant syllable type, showed a trend of increased percentage within the total syllables in Nova1hu / hupups, while its amplitude was significantly lower in Noval^lu / '^uthan that of Novalwt, vtpups. The amplitude of syllables containing pitch jumps (“u”, “d” and “m”) did not differ between genotypes, but these percentages were decreased in Noval^u''^upups, particularly for the “u” and “m” syllables (Fig. 19a, Table 8).

[0273]

[0175] Pup isolation-induced USVs in mice show a bimodal distribution in their peak frequency (Fq), especially at 5-9 days of age, consolidating to a single peak as they grow^. In our study, 7-day- old pups exhibited a clear bimodal distribution in the Fq parameter regardless of the genotypes (Fig. 19b). We assessed bimodality in Fqmax, showing clear separation using an Ashman's D score^ This analysis indicated a clear separation in two peaks for the “s”, “d” and “m” syllables (£»2.0), but not for the “u” syllable (79=1.603) (Fig. 18c). Inorder to separate these two peaks, we fitted two Gaussians

[0274]

[0275] for each syllable type showing bimodal distribution, comparing USV characteristics for each peak (Fig.

[0276] 18c, Table 9). Individual syllables were classified into those with Tow’ or ‘high’ Fqmax according to the cutoff value (the intersection point of each distribution). In the jump syllables of Noval^u'^upups, the proportion of low Fqmax decreased, while the proportion of high Fqmax increased (Fig. 18d, Table10). We also tested the bimodality and syllable ratio with Fqmin and confirmed the same trend (increased ratio in high Fq in Noval^l^mpups). Heterozygous Novalwt^upups showed intermediate values between Noval^l< / ^uand Novalwt^vtpups for these parameters, suggesting that the effect of the I197V substitution in N0VA1 protein on pup USVs is dosage dependent. These observations demonstrate distinct changes in the vocalizations of Novani'^upups.

[0277]

[0176] The vocalizations of pups isolated from the nest are known to be able to influence maternal behavior in mice^^’^^,103 -0 exp]oiethe potential significance of vocal motor changes in Nova1hu^11pups on their mothers, we tested whether mother mice were more attracted to recorded vocalizations from each genotype. We set up an experiment using a three-room box connected by passageways, with speakers placed in each of the two end rooms (Fig. 20a). Neonatal vocal recordings (from Noval^ni / ^umice or Novalw^'wtcontrol mice) were played from each speaker, and either a Noval^w'^uor a Novalw1 / wtmother mouse was placed in the center room (Fig. 20a-c). Regardless of the mother’s genotype or the neonatal vocal recordings, no significant differences were observed in the mother’s orientation toward the vocalizations of pups (Fig. 20d-e). Thus, changes in vocal quality in Nova1hu'^11pups had no impact on the behavior of the mother mice in this assay.

[0278] Humanized NOVAI adult mice have altered vocalization

[0279]

[0177] Adult mouse USVs are often produced in long, continuous sequences consisting of the same four major syllable types (Figure 18b), particularly during courtship J04,105 yeexplored whether changes in courtship vocalization behavior would occur in Noval^u'^umice, using a previously established paradigm*^’ ^5, 106

[0280]

[0281] elicited courtship USVs from adult male mice by exposing them to adult female mice in estrus (Fig. 18e, Table 11, Table 12). In this context, more than 90% of the vocalizations come from the male 107. The average number of courtship-induced USVs did not differ between genotypes: 144.8 ± 24.0 per minute in Noval ^1and 165.5 ± 27.0 per minute in Noval^u'^ni(mean ± standard error) (Table 13). We next examined short-duration and long-duration syllables

[0282]

[0283] ■, which has been previously shown to be bimodal especially for “s” syllables

[0284]

[0285] Two Gaussian distributions were fitted to characterize each class of USVs (Fig. 18f). The cutoff duration (the intersection point of each distribution) between short and long USVs was determined to be 44 ms, with 29% of “s” syllables categorized as long duration. The average duration of short- and long-“s” syllables was 22.6 ± 9.1 ms and 69.0 ± 40.1 ms, respectively. Noval^ni^umice tended to have a lower frequency characteristic for long- duration “s” syllables, where the differences for Fqstart, Fqend and Fqmean were significantly lower compared to controls (Fig. 18g, Table 14). These changes were not observed in pupisolation induced USVs, indicating that this effect is developmentally specific and / or context dependent.

[0286]

[0178] We next examined peak frequencies. In adult USVs, the Fqmax parameter exhibited a unique distribution. High Fqmax syllables were those that exceeded 100 kHz, and constituted a distinct population from the main population (Fig. 19c, d). Syllables with High Fqmax of wild type animals are often observed in harmonic syllables, containing pitch jumps and some simple syllables^ ^9 (fig 19e). Fitting two Gaussian distributions to the data, the High Fqmax was found to be 107.1 ± 2.1 kHz, whereas the main peak was 80.8 ± 11.5 kHz (mean± standard deviation) (Fig. 18h). Using this definition, the High Fqmax syllables accounted for roughly 8.5 % of total USVs observed. There were no significant differences in syllable composition with High Fqmax among genotypes (Table 15). However, the Fqvariance of High Fqmax syllables containing pitch jumps (“d”, “u” and “m”) was significantly greater in Nova1^hu / hu^ mice (Fig. 18i, Table 16). Fqvariance tends to increase with syllable complexity with simple syllables like “s” having lower values and more complex syllables like “m” with multiple jumps having higher values. This suggests that Noval^uj^umice produce more complex high frequency USVs than Nova1^wt / wt^ mice. These findings demonstrate that vocal behavior is altered in both pups and adults in Noval^u^nimice, resulting in unique vocal characteristics.

[0287] DISCUSSION

[0288]

[0179] In line with studies of genetic variants that have played a role in the evolution of modern humans 19,111,112weinvestigated the biological effect of a single amino acid substitution, Il 97V in NOVAI, which is unique to modem humans. By analyzing Noval^lu''^umice carrying this allele, we identified molecular changes in alternative splicing in the brain, including brain regions associated with vocal behavior, and identified changes in vocalization patterns in pups and adult mice. These findings suggest that during human evolution, the 1197V substitution in NOVAI protein may have contributed to the development of neural systems involved in more complex vocal communication.

[0289]

[0180] The importance of NOVAI in mammals is evident from the lethal phenotype of Noval knockout mice01and the neurological symptoms caused by NOVAI haploinsufficiency in humans00. This significance is further highlighted by the high conservation of the NOVAI protein in mammals. Interestingly, the NOVAI gene harbors an Ultra Conserved Element (UCE; uc.359) at the end of the 3’

[0290]

[0291] UTR113,114 additional high conservation extending upstream from the NOVAI UCE to most of the 3’ UTR and terminal exon encoding N0VA1 KH2 and KH3 domains. This underscores the unique nature of the 1197 V variant, which occurred within a region of the genome resistance to change.

[0292]

[0181] Previous studies have confirmed the evolutionary restriction of NOVA 1 variants, including theI197V variant (term I200V in one study^13^). We support this analysis and have expanded upon it with larger human sequence datasets across diverse ethnic groups, and from methods that infer selection coefficients from ancient samples^49,50^ These results confirm that N0VA1 has undergone strong positive selection, and that the 1197V variant is part of an evolutionary selective sweep in the emergence of Homo sapiens.

[0293]

[0182] The observation that the 1197V N0VA1 allele is nearly fixed across human populations (Figure 2a) suggests that it emerged and increased in frequency well before the divergence of ancient human lineages. The earliest divergence among modem human groups - the San divergence- is currently estimated to be around 250 - 300kya, prior to their migration to Eurasia

[0294]

[0295] Unlike more recent selective sweeps, such as the LCT locus, which is dated around 10kya, and is population-specific, the NOVAI variant is part of an older, more widespread sweep. These older sweeps, shared across modern human populations, may leave subtler genetic signatures that require novel detection methods. This suggests that the ancient NOVAI selective sweep may represent part of a broader set of undiscovered ancient sweeps.

[0296]

[0183] One possible explanation for the changes in vocal behavior observed in Novani^umice could be molecular changes in midbrain and brainstem vocal pathways, which express high levels of NOVA 1 and are involved in regulating innate vocalizations (USVs), including breath coordination, timing, and amplitude^116–118^

[0297]

[0298] alternative possibility is that changes occurred in more recently evolved cortical vocal regions, which control pitch, frequency modulation, and duration (the Kuypers / Jurgens hypothesis^86,119^ and the volitional articulatory motor network^89,120^

[0299]

[0300] Given that NOVA 1 is expressed in the mouse cortex, predominantly in inhibitory neurons^, it is plausible that the 1197V substitution affects cortical regulation of vocalization.

[0301]

[0184] Notably, Novalu'^umice exhibit qualitative changes in vocal characteristics compared to control mice both in pups and adults, despite producing a similar number of calls. These findings suggest that the vocalization changes in Nova1hu'^11mice are not simply the result of alterations in general motor performance. This idea is supported by other observations showing that Noval^111'^11mice perform similarly to control mice in motor function tests, such as the rotarod, and display comparable locomotion activity levels in the Y-maze test (Fig. 21). Additionally, the Y-maze test results indicated that Noval^l / ^umice had spatial working memory comparable to that of control mice.

[0302]

[0185] The changes in vocalization in Nova

[0303]

[0304] mice varied in a development- or context-dependent manner. It has been reported that high-frequency (Fq) USVs are emitted more frequently by male adult mice during social interactions

[0305]

[0306] , and that female mice are attracted to male mice that emit moreno

[0307] complex USVs. Given these reports, the increased the proportion of higher Fq USVs in pups, and the increased complexity of these USVs in adults, may potentially offer social advantages in mice. However, since auditory frequency resolution in mice has been reported to be limited

[0308]

[0309] and our preference experiment using humanized N0VA1 pup USVs showed no significant preferences in the mother's responses, it remains unclear to what extent other mice can recognize these vocalization changes. It should be noted that we were unable to address the effect of the 1197V substitution on the vocalizations of adult female mice, as our study focused on a courtship-induced vocalization paradigm that predominantly elicits USVs from male mice

[0310]

[0311] . However, recent studies suggest that female mice also vocalize under certain experimental conditions or social contexts^ ^4, 125 Future studies will be necessary to investigate the effects of the 1197 V substitution on USVs in female mice, as well as adult female preferences to USVs in adult Noval^111^11male mice.

[0312]

[0186] Interestingly, the vocalization changes observed in Noval^u'^umice share some similarities with those observed in humanized Foxp2 mice (with two human-specific substitutions). In both cases, the changes were developmental or context-dependent, and included a decrease in peak frequency in simple syllables and modulation of high frequency regions in complex syllables

[0313]

[0314] ^19–21^ (Table 17). Conversely, male mice with a humanized Foxp2 mutation produced simpler song bouts with more “s” syllables ’. These observations may indicate a common or related molecular alteration in the neural circuits involved in the USV production between humanized Nova1 mice and humanized Foxp2 mice. Future studies should aim to identify the molecular and neural basis of these alterations, as well as the physiological significance of these vocalization changes in the context of social behavior.

[0315]

[0187] Our molecular analysis showed that the sequence-specific RNA binding of NOVAI was unaffected by the human substitution and that steady-state gene expression levels in the brains of Nova1hu / humice were nearly identical to those of wild type mice. However, we detected alternative splicing changes in several transcripts associated with vocalization. The expression pattern of N0VA1 in the brain and the enrichment of its target transcripts to specific biological pathway support a link between N0VA1 function and vocal behavior. Uncovering the precise molecular mechanisms underlying the phenotypes in Noval^u'^ntmice will require further study of the neural circuits for vocalization, as well as on regulatory factors influencing NOVA protein function. This study sets the groundwork for understanding molecular mechanisms driving the evolution of human vocal communication.

[0316]

[0188] Biochemically, NOVA proteins harbor three KH domains responsible for sequence-specific RNA- b

[0317]

[0318] inding42>45,46, amino acid 197 located in the KH2 domain. Although the I197Vsubstitution in N0VA1 alters the hydrophobic core of the KH domain, it does not lead to a loss of RNA-binding capacity or functional attenuation, in contrast to other KH domain point mutations'^ This is supported by the unchanged global gene expression levels observed in the brains

[0319]

[0320] mice, while N0VA1 knockout mice (which exhibit postnatal lethality ) show significant expression changes of key neuronal genes in the midbrain at E18.5 (Fig. 7e, 7f, Table 18). KH domains harbor three alphahelices (H) and three beta-sheets (S) (S1-H1-H2-S2-S3-H3; Fig. l la-b); the first and second alpha helices (H1-H2) determine single-stranded RNA binding specificity’^-'^,a[1may also be involved in protein- protein dimerization^ (Fig. llc-d). Protein structure predictions suggest that the addition of a single carbon atom in valine 197 extends its ability to interact with several nearby amino acids (185Ile in Hl; 232Ala, 235Leu, 236Ile, 239Lys in H3), allowing the KH2 domain to gain contacts with Hl and 239Lys in H3. Thus, while the I197V substitution does not change sequence specificity or binding affinity of N0VA1 with RNA (Fig. 6i-j), it may affect KH domain dimerization, or may have undiscovered effects on protein-protein interactions^. Interestingly, the amino acid corresponding to N0VA1 amino acid 197 is also an isoleucine in the related proteins FMRI and hnRNP E1 / E2 / K, but is a valine in both human and mouse N0VA2^^ (Fig 9) Functional differences between N0VA1 and N0VA2 in mice^>64mayrcqCC[ structural and functional differences in their respective KH domains.

[0321]

[0189] In summary, we analyzed a single amino acid unique to modern humans in the RNA binding protein NOVA1 and examined its biological effects in vivo by introducing this amino acid in mice. N0VA1 is highly intolerant to changes in amino acid sequences during evolution with the exception of this single amino acid change in humans. We propose that this change was part of an evolutionary sweep associated with specific changes in the neuronal transcriptome and vocal communication.

[0322] Methods

[0323]

[0190] Animal experiments

[0324]

[0191] All procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) at Rockefeller University. C57BL / 6J (stock no. 000664) mice were obtained from the Jackson Lab. Noval^u'^umice generated in this study were backcrossed to C57BL / 6J strain at least 8 times. Mice were housed in a 12-h light / dark cycle, up to 5 mice per cage. Male or female mice aged 7 days to 20 weeks were used for animal experiments, as described. Littermates of the same-sex were randomly assigned to experimental groups.

[0325]

[0192] Generation of Nova1hu^ mice

[0326]

[0327]

[0193] Noval^u'^umice were generated by directly injecting the sgRNA / Cas9 RNP with a single-stranded repair template DNA (ssDNA) into C57BL6 zygote to substitute isoleucine to valine at aminoacid 197 of mouse NOVAI. gRNA and the ssDNA were designed as follows. gRNA (5’-TGCTACTGTGAAGGCTATAA-3’(SEQ ID NO: 17)): overlapping theDNA sequence (mmlO / chrl2: 46,700,902- 46,700,904) of the mouse Novai genomic locus encoding the 197thamino acid ofNOVAl. ssDNA: 140 nt length DNA homologous to the NOVAI locus with a nucleotide substitution (A to_G) to cause an amino acid change from isoleucine to valine at the 197^ position. Two silent mutations were also designed to create Btsal restriction enzyme recognition site for genotyping.

[0328]

[0194] Genomic DNA was extracted from the tail of the F0 animals, and the DNA corresponding to the area around the 197thamino acid was amplified by PCR and subsequently cloned into a plasmid for determining the sequence of the modified allele. Genomic sequence analysis revealed that among 13 F0 animals, 8 animals harbored the designed allele (with three nucleotide substitutions: one causing 1197V amino acid substitution, two for restriction enzyme recognition site for genotyping (not causing amino acid changes)). Animals carrying the designed humanized Novai allele were crossed to the wildtype C57 / BL6 mice, and this process was continuously repeated for subsequent generations to eliminate possible off-target mutations.

[0329]

[0195] Genome sequencing analyses were performed on the possible off-target sites of the gRNA used (10 potential off-target loci with mismatches outside of the PAM+12mer core sequences; predicted by CRISPR direct: https: / / crispr.dbcls.jp / ) to compare sequences of control (wild type) and humanized NOVAI mouse. For all the potential off-target sites, the sequence in the humanized NOVAI mice were identical to control mice and the reference genome, with 1197 V substitutions being the only detectable edits (Table 4d-e).

[0330]

[0196] For routine genotyping, sequences around the genomic DNA encoding the 197thamino acid were amplified by PCR subsequently digested with the Btsal restriction enzyme. Each Mouse genotype; wild type (Novalwt, / wt), humanized Novai homozygous (Noval^u'^'), heterozygous (

[0331]

[0332] Nova1hu^) was determined by band size obtained by electrophoresis. Siblings obtained by crossing heterozygous parents were used in the experiment. Primers used for typing are shown below.

[0333] NOVAlhu-Fwd: 5’- ccctcttttgacatgctggt -3’ (SEQ ID NO: 18)

[0334] NOVAlhu-Rvs: 5’- cataaggagatccggttgga -3‘ (SEQ ID NO:19)

[0335] DNA band size after restriction enzyme treatment: wild type (613 bp), homozygous (389 bp and 224 bp), heterozygous (613 bp / 389 bp + 224 bp) (see Fig. 4c).

[0336]

[0197] Antibodies

[0337]

[0198] Primary antibodies used for immunohistochemistry and western blotting were as follows; rabbit anti-NOVAl [EPR13847] (ab 183024, abeam), rabbit anti-NOVAl C-terminal [EPR13848] (ab183723, abeam), human anti-pan NOVA (anti-Nova paraneoplastic human serum) and rabbit anti-ATCB (ab8227, abeam).

[0199] Immunohistochemistry

[0338]

[0200] Postnatal day 0, three or twelve-week-old mice were perfused with PBS and 4% paraformaldehyde (PF A), and the brain was dissected. Dissected brain was further fixed. Overnight by 4% PFA at 4°C. The solution was sequentially replaced with 15% sucrose / PBS and 30% sucrose / PBS, embedded with OCT compound, and stored at -80°C until use. Frozen brains were sliced into 30-50 pm thick sections in a cryostat (CM3O5OS, LEICA). Slices were washed three times with PBS at room temperature (RT), incubated in 0.2% Triton X-100 / PBS for 15 min at RT, blocked in 1.5% normal donkey serum (NDS) / PBS for 1 h at RT, incubated overnight at 4°C with primary antibody in 1.5% NDS / PBS, then incubated in Alexa Incubated with 488, 555 or 647 conjugated donkey secondary antibody. The nuclei were stained using 4’,6-diamidino-2- phenylindole (DAPI) solution (1 pg / ml). Images of specimens were collected with a BZ-X700 (KEYENCE) microscope.

[0339]

[0201] Western blotting

[0340]

[0202] Each dissected brain region (cortex, midbrain and cerebellum) of P21 mouse brains was lysed in RIPA buffer (50mM Tris-HCl; 150mM NaCI; 0.1% SDS; 0.5% sodium deoxycholate; I%NP- 40). Extracts were separated by SDS-PAGE, and subjected to immunoblotting using the antibodies described above. Quantification of western blots were done with ImageJ (v1.53). Each band signal was quantified and normalized with ACTB signal to control for differences in loading.

[0341]

[0203] Electrophoretic Mobility Shift Assay (EMSA)

[0342]

[0204] Protein purification

[0343]

[0205] The genes encoding each N0VA1 protein (NOVAlwtand N0VA1 ^u) were cloned into the pGEX6pl vector and expressed in E. coli BL21 strain. N-terminally GST (Glutathione S- Transferase) fused NOVAI was induced by the addition of IPTG (final cone. O.lmM) for 4hr. Pelleted cells were sonicated, and then incubated in the presence of Triton-X (final cone. 1%, 30 min). Cleared supernatant was collected after centrifugation (12,000 xg 10 min 4°C). After incubating with Glutathione Sepharose beads (GE Healthcare Biosciences, 17075601) for 30 minutes, the mixture was washed three times with PBS. The GST tag was cleaved from the NOVAI protein by PreScission Protease treatment (GE Healthcare, 27-0843-01; 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.5, 4°C for 4hr) to obtain purified NOVA 1 protein. The concentration of each purified NOVAI protein was determined by SDS-PAGE followed by GelCode Blue staining (Thermo Fisher Scientific, PI24590) using BSA (Sigma- Aldrich, B8667) as standards.

[0344]

[0206] Single strand RNA probe preparation

[0345] 4^

[0346]

[0207] The single-stranded RNA probe was designed as previously. The following single strand RNA were synthesized by IDT:CCTTATCATGCTGACTCACGTCATTTCATCTCATCAAGGGAGTCAGTGGGATA (SEQ ID NO:20)

[0347]

[0208] Synthesized RNA was first incubated at 80°C for 10 min, then rapidly incubated on ice, mixed with [gamma-32P] ATP (3000 Ci / mmol, 10 mCi / ml) (Revvity, BLU502A), and labeled at the 5' end by T4 polynucleotide kinase treatment (New England BioLabs, M0201S). The labeled probes were purified by G-25 column (VWR, 95017-621) and diluted to the appropriate concentration with water.

[0348]

[0209] Binding assay

[0349]

[0210] Purified NOVAI protein (0.5-2 pmol / reaction) and labeled RNA probe (0.08 pmol / reaction) were mixed under the following buffer conditions: lOmM HEPES, 3mM MgC12, 150mM NaCl, 5% Glycerol, O.lmM DTT, 0. lU / pl RNase OUT (Thermo Fisher Scientific, 10777019)., lOmg / ml Yeast tRNA (Thermo Fisher Scientific, AM7119), Img / ml poly dl-dC (Thermo Fisher Scientific, 20148E). The binding reaction was performed at 22°C for 60 minutes, and the reaction solution was mixed with loading dye and run on an 8% acrylamide native gel at 150 V for 3 hours to separate RNA-protein binding. After the gel electrophoresis, the gels were dried by a gel dryer and autoradiographs were detected. Quantification of each signal was performed by ImageJ, and the Kd value of each NOVAI protein for the RNA probe was calculated using Prism software (https: / / www.graphpad.com / features).

[0350]

[0211] RNA-seq library preparation and analysis

[0351]

[0212] For RNA-seq, samples included dissected cortex, midbrain and cerebellum at P21, or dissected midbrain at E18.5 of Nova1hu / huand Novalwt,wtmice. The mRNA-seq library was prepared from RNA extracted with Trizol following the Illumina TruSeq protocol of polyA selection, fragmentation, and adapter ligation. Multiplex libraries were sequenced as 125 nt paired- end runs on the HiSeq-2500 platform at Rockefeller University Genomic Core. These raw datasets and processed data files have been deposited with Gene Expression Omnibus (GSE253297).

[0352]

[0213] Cross-linking Immunoprecipitation (CLIP)

[0353]

[0214] NOVA1-CLIP was performed in P21 dissected cortex, midbrain and cerebellum of Noval^u / ^uand Nova1^wt / wt^ using each three biological replicates. Tissues were dissected in PBS, triturated using 20G needle and crosslinked three times on ice for 400 mJ / cm2 using Stratalinker. Crosslinked material was collected by centrifugation, resuspended in wash buffer (IX PBS, 0.5% NP-40, 0.5% deoxycholate and 0.1 % SDS with protease inhibitor), and subjected to DNase (RQ1 DNase: Promega) and RNase (RNase A: Affymetrix) treatment at a final dilution of 1:20,000 for 5 min. The lysate was clarified by centrifugation at 20,000 x g for 20 min. The supernatant was used for immunoprecipitation with 200 pL of Protein A Dynabeads (Invitrogen) loaded with 18 pg anti-Noval antibody (abeam) for 2 hours at 4°C. The samples were washed as follows: twice with wash buffer, twice with Nelson stringent wash buffer(15mM Tris pH 7.4, 5mM EDTA, 2.5 mM EGTA, 1% Triton X-100. 1% Sodium deoxycholate, 0.1% SDS, 120 mM NaCl, 25mM KCI), twice with Nelson high salt buffer (15mM Tris pH 7.4, 5mM EDTA, 2.5 mM EGTA, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS, IM NaCl), twice with Nelson low salt buffer (15mM Tris pH 7.4, 5mM EDTA), and twice with PNK wash buffer (50mM Tris pH 7.4, 10 mM MgCl2, 0.5% NP-40). RNA fragments were dephosphorylated using FastAP Alkaline phosphatase (Thermo Fisher Scientific) and subjected to 3' ligation overnight at 16°C with a preadenylated linker (preA-L32) using truncated KQ T4 RNA Ligase2 (NEB). The RNA-protein complexes were labeled with32P-γ-ATP using T4 PNK (NEB) and subjected to SDS-PAGE and transfer to nitrocellulose membrane. Appropriate regions of the membrane were cut out and RNA was extracted according to the following conditions: lOOmM Tris PH7.5, 50mM NaCl, lOmM EDTA, 7M Urea with proteinase K. RNA was purified by phenol-chloroform extraction method. Cloning was performed using the BrdU-CLIP protocol. Briefly, the reverse transcription reaction was performed using Superscript III (Thermo Fisher Scientific), and the cDNA was BrdU-labeled by including BrdU in the reaction solution. Immunoprecipitation was performed with 5 μg anti-BrdU antibody (abeam) and 25 pg protein G Dynabeads per reaction (45 min at room temperature), followed by washing with the following solutions (including Denhardt’s solution): once with IP buffer (0.3x SSPE, ImM EDTA, 0.05% Tween 20), twice with Nelson low salt buffer, twice with Nelson stringent wash buffer, twice with IP buffer. After eluting the cDNA, BrdU- immunoprecipitation was performed again under the same conditions. cDNA was circularized on beads using CircLigase II (Epicentre) and PCR was performed using Accuprime Pfx supermix (Thermo Fisher Scientific) and Syber Green until RFU 250-500. PCR products were purified using Agencourt AMPure XP (Beckman Coulter) and concentrations were measured by TapeStation. High-throughput sequencing was performed at the Rockefeller University Genome Resource Center. These raw' datasets and processed data files have been deposited w ith Gene Expression Omnibus (GSE253296).

[0354]

[0215] Bioinformatics

[0355]

[0216] Paired-end reads from RNA sequencing were aligned to the mouse genome (mml 0) builds of the 1 mouse genome using OLego (v1.1.7) (https: / / zhanglab.c2b2.columbia.edu / index.php / OLego) Mapped reads were counted using gapless (for inference of transcript structure) and countit (for quantification of gene expression and alternative splicing) in Quantas (v1.0.9) (https: / / zhanglab.c2b2.columbia.edu / index.php / Quantas)127. All reads mapping to transcripts were included in the differential expression analysis using edgeR

[0356]

[0357] . The data set for Nova1 knockout mouse (E18.5 midbrain) was kindly provided by Dr. Yuhki Saito. Tire data are available from GEO submission GSE69711. Data visualizations were done using R (v4.2.0). Correlation matrix was visualized using corrplot package. PCA analysis was performed using FactoMineR and factoextrapackages and visualized using ggplot2 package. Sequencing tracks were visualized using Integrative Genomic Viewer (IGV, v2.13. O).

[0358]

[0217] Quantification of splicing for annotated cassette exons was performed using the Quantas pipeline (v1.0.9)127

[0359]

[0360] . In brief, the inclusion level of each cassette exon (percent-spliced-in: PSI) was calculated from the number of supporting exon junction reads for the inclusion and skipping isoforms. Only quantifications with > 20 supporting junction reads were used for downstream analysis. Gene annotation analysis was performed using Metascape (v3.5.20240101)^

[0361]

[0362] 52. The expressed genes in the P21 midbrain (filtering lowly expressed genes by edgeR) were set as background for the analysis. Attribution for genes was performed using Gene Ontology (GO) resource in MG1 (6.24) (https: / / www.informatics.jax.org / ).

[0363]

[0218] CLIP reads were processed using the CLIP Tool Kit (CTK, vl.1.3) as described previously129. Briefly, raw reads were filtered for quality and demultiplexed using indexes introduced during the reverse transcription reaction. PCR duplicates were collapsed and adapter sequences removed. Reads were mapped to the mmlO build of mouse genome using novoalign (v3.09.02) (www.novocraft.com). Mapped reads were further collapsed for potential PCR duplicates by coordinates and taking into consideration the degenerate barcodes introduced during the reverse transcription. Only unique CLIP tags were used for subsequent analyses. We performed three biological replicates per sample. All scripts used in the analysis including the peak finding algorithm and more information can be publicly obtained at (https: / / zhanglab.c2b2.columbia.edu / index.php / Standard / BrdU- CLIP data analysis using CTK). De novo motif analysis and motif density analysis were done using findMotifsGenome.pl and annotatePeaks.pl commands in HOMER (v4.11).

[0364]

[0219] Ultrasonic vocalization (USV) tests

[0365]

[0220] Isolation induced pup USV test

[0366]

[0221] To elicit isolation-induced USV, 7-day-old pups were isolated from their mother and littermates. Each pup was placed quietly on a small open-faced plastic plate in the sound attenuating chamber (15” x24” xl2” Igloo beach cooler with a tube for pumped air circulation input, no light). An ultrasonic microphone was suspended a small distance from the pup, and the USVs were recorded for 5 minutes. Between trials, the recording box was cleaned with 70% alcohol and distilled water, and allowed to fully dry before the next experiments. Vocalizations were recorded with UltraSoundGateCM16 / CMPA ultrasonic microphones connected to an Ultrasound Gate USGH amplifier. Recordings were saved using the AvisoftRecorderUSG software (Sampling frequency: 250 kHz; FFT-length: 1024 points; 16-bits). All acoustic hardware was obtained from Avisoft Bioacoustics®1(Berlin, Germany).

[0367]

[0222] Playback behavioral experiment

[0368]

[0223] Mothers rearing 7-day-old offspring were used in the playback experiment. We used a three-chamber box (12” x 23.5” x 15.5”) connected by a passageway through which a mouse could pass for the test. Each chamber at both ends was equipped with a speaker (Vifa ultrasonic speaker, Avisoft Bioacoustics), and a camera (Firefly S, FFY-U3-16S2C-S, FLIR) was placed on the ceiling of the chamber to record the behavior of the mouse. The speakers were connected to an UltraSoundGate Player 216H (Avisoft Bioacoustics), using Avisoft Recorder USGH and had a frequency range (±12 dB as the maximum deviation from the average sound volume) of 25-125 kHz. We adjusted the loudness between the channels by controlling the level of the peak power before the experiment. Using two microphones, we made sure that both songs were audible at the entrance of both rooms so that the mother can respond to the songs but not loud enough that the microphones could detect the song being played in the other room. After the 10 min habituation period, playbacks were triggered when the mouse broke an infrared sensor located in the center of the three-chamber box. One speaker on one side played one pup-USV recording and the other speaker simultaneously played another pup-USV recording both of which were previously recorded during the pup isolation induced USV test for 5 min.

[0369]

[0224] Pup-USV recording was prepared in Audacity® by stitching vocalizations from 4-5 pups for each genotype. These recording files contained an equivalent number of pup-USVs (Nova1wt / wt1689 USVs, Nova1hu / hu1561 USVs) and were confirmed to reflect the vocal characteristics of each genotype. After the first 5 min playback session, a second 5 min playback session was conducted after 1 min quiet period. In the second playback experiment, the two recordings playing from the speakers were switched to eliminate the possible preference by the location. During each 5 min period, the mother was allowed to explore freely in the box and the time she spent in each room was counted. The box was cleaned between experiments with 70% alcohol and distilled water and allowed to fully dry before the next experiments.

[0370]

[0225] Courtship induced adult USV test

[0371]

[0226] The protocol for the courtship-induced vocalization test in adult male mice has been performed according to previous studies98with minor modifications. Briefly, adult male mice (8-12 weeks old) were sexually socialized by spending one night with a sexually mature female to enhance the male's motivational state to exhibit courtship USV behavior110. On the next day, female mouse was removed from the cage, and the male mice housed in the same cage until the test day. On the day of the recording, the males were placed in a new cage and then singly habituated in the sound recording environment (as described for pup USV test) for 15 min. The males were then exposed to adult female mice for 5 min. We used the females (8-12 weeks old) in estrus (selected visually for wide vaginal opening and pink surrounding). The test was conducted three times per mouse, one week apart, and a different female mouse was used as the stimulus each time to avoid familiarity effects. The order of mice tested each time was shuffled to avoid the possible order effects. Between trials, the mouse cage was cleaned with 70%alcohol and distilled water, and allowed to fully dry before the next experiments.

[0372]

[0227] USV analysis

[0373]

[0228] Acoustic waveforms were processed using a custom Python program called “Mouse Song Analyzer 2” (MSA2): available on the website (https: Vgithub.com / Neurogenetics- Jarvis / MSA ^IOS, 110,130,anjanajySjs aspreviously described^’ 05,110 Biaef]y f]lcsoftware computed the sonograms from each waveform, threshold to eliminate the white noise component of the signal, and truncated for frequencies outside the USV song range (35-125 kHz). We used a criterion of 10 ms minimum to separate two syllables and 3 ms as the minimum duration of a syllable. The identified syllables were then classified by presence or absence of instantaneous “pitch jumps” separating notes within a syllable into four categories: (1) simple syllable without any pitch jumps (“s”); (2) complex syllables containing two notes separated by a single upward (“u”) or (3) downward (“d”) pitch jump; and (4) more complex syllables containing a series of multiple pitch jumps (type “m”). Any sounds the software could not classify were put into “not identified: notIDd” category. The following spectral features were calculated automatically by MSA2 from the sonograms of each of the classified syllable types: Syllable duration, inter-syllable interval, standard deviation of pitch distribution, pitch (mean frequency), frequency modulation, spectral purity, and bandwidth. The fitting of Gaussian in the duration and peak frequency maximum (Fqmax) distributions in pup isolation induced USVs and in adult courtship USVs were performed using R package mixtools: tools for analyzing finite mixture models101. The bimodality of the USV duration distributions were assessed using Ashman’s D Score100, where μ and σ are the center and standard deviation of each Gaussian, respectively. Cutoff Fqmax in pup USVs between low and high USVs were defined as the intercept of the two Gaussian fits to the distribution to the nearest frequency (kHz). Cutoff durations between short and long USVs were defined as the intercept of the two Gaussian fits to the USV duration distribution to the nearest millisecond. Statistical analysis was performed by pairwise Wilcoxon rank sum tests, with correction (Bonferroni) for multiple comparisons between genotypes. Correction was not applied for the parameters in call structure. This is because individual properties are assumed to be related to each other, which increases type 2 error caused by overcorrection.

[0374]

[0229] Rotarod test

[0375]

[0230] The tests were performed using the elevated revolving rod (Stoelting, Cat#. 57624). Mice were placed on the apparatus and habituated for few minutes. The rod accelerated at a constant rate (4 to 40rpm in 300sec) and the time it took the animals to fall was recorded. Tests were performed three times and the average value are calculated. Statistical analysis was performed by Wilcoxon rank sum test.

[0376]

[0231] Y-maze test

[0377]

[0232] The Y-maze tests were conducted according to the described procedure131. The tests wereperformed in a Y-maze with three arms of equal length at 120° angles to each other (Stoelting, Cat#. 60180). Mice were placed in the center of the maze and has free access to all three arms. If the animal chooses an arm different from the arm it arrived in, this choice is called an alteration. This is considered a correct response; conversely, returning to the previous arm is considered an error. The number of times and the order in which the animals entered the aims are recorded and used to calculate the alternation rate. The behavior of the mice was recorded for 8 min. Statistical analysis was performed by Wilcoxon rank sum test.

[0378]

[0233] Variant identification and annotations of Neanderthal and Denisovan

[0379]

[0234] The sequencing data of three Neanderthal genomes were obtained from The Draft Neanderthal Genome Project (https: / / www.ebi.ac.uk / ena / browser / view / PRJEB2065). The sequencing data of Denisovan genome accompanied with nine modem human genomes were obtained from Denisovan Genome Project (http: / / cdna.eva.mpg.de / denisova / ). The fastq files of each sample were aligned to the human genome (hg 19) by Burrows- Wheeler Aligner (BWA, v0.7.17-rl 188). The aligned SAM files were processed into BAM files by Picard (v2.18.7) and Genome Analysis Toolkit (GATK, v4.4.0.0). Variant calling for each sample was processed with Mutect2 of GATK4. The variants w’ere annotated by using ANNOVAR (v2).

[0380]

[0235] Variants in N0VA1 loci

[0381]

[0236] The variants in modern human populations w’ere obtained from ExAC database (vO.3.1) (https: / / gnomad.broadinstitute.org / downloads), which contains 60,706 exomes mapped to hg!9. Then, the variants in NOVA1 loci (chromosome 14: 26912296 -27067239) of Neanderthal, Denisovan, and modern human populations were subsetted by using bcftools (vl.19). The frequency of minor alleles for each position in NOVAI loci was calculated.

[0382]

[0237] Tajima’s D statistical analysis

[0383]

[0238] Corrected allelic frequencies of each SNP site from ExAC (v0.3.1) were extracted from the dbSNP database (version 2022-11-16). The sites with very low total allele count were filtered out (cutoff for total count: 200). pi, theta and Tajima’s D values were calculated for all genes examined132

[0384]

[0385] . The normalized Tajima’s D value were calculated as the ratio of Tajima’s D to its theoretical minimum value (D(min))47.

[0386]

[0239] Dll test

[0387]

[0240] Allelic frequency for each mutation site were extracted from refsnp files (version b 156) downloaded from NCBI (version of November 2022). In-house program was used to calculate the evolutionary statistic, Tajima’s D is calculated by according (Tajima, 1989), Fay and Wu’s / / -statistic is defined as 0a - 0 / / (Fay and Wu, 2000), we calculate normalized H (formula 11, 12), E-test (formula 13, 14), and DH-test (formula 15) (Zeng et al., 2006). Our in-house program is shared to public:https: / / g

[0388]

[0389] ithub.coi^^^

[0390]

[0241] Selection analysis on NOVA 1 197V

[0391]

[0242] The ancestral recombination graph (ARG) analysis was performed using ARGweaver-D49. These ARGs explicitly describe gene trees and accompanying recombination events throughout the region. They were sampled from an approximate posterior distribution using Markov chain Monte Carlo methods to capture uncertainty in the ARG, given the sequence data and evolutionary' parameters. The sampled ARGs included two Yoruba (HGDP00927, SS6004475), two Mbuti (SS6004471, HGDP0456), and two San (HGDP01029, SS6004473) individuals, all sequenced to high coverage, as well as the Altai Neanderthal and Denisovan sequences and a chimpanzee outgroup (panTro4). Because the N0VA1 197V variant is nearly fixed in modem humans and likely predates the separation of major continental population groups, we do not anticipate significant additional insights from including more modern human samples, as most would coalesce well after the allele reached high frequency. Based on information from the ARG, the sampled ARGs were analyzed using CLUES250.

[0392]

[0243] Statistical information

[0393]

[0244] Information of statistical methods and the number of biological replicates in the analysis are in the figure legends and methods section of each analysis as appropriate.

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[0535]

[0245] TABLE I

[0536] Tajima’s D and normalized Tajima’s D.

[0537] Tajima’s D values and their normalized values were calculated based on the ExAC data sets. The genes on datasets 3 and 4 are based on the reports of Meyer et al, and the genes on dataset 5 are based on Trujillo et al. See Methods for details on calculation of the Tajima’s D values. A subset of data is presented here for readability.

[0538] gene. chr i pi. i thetaW. i Tajima.s. D Tajima.s. D. I Gene set normalized.

[0539] NOVAI chr14 1.68E-06 0.00191534 [ -24807974 -0.999292729 NOVA1 and its related FOXG1 chr14 ] 1.00E-05 0.00267173 T -24017538 ■0.9963984 NOVA1 and its related NOVA2 chr19 5.26E-05 0.00129301 L2.3220406 -0.959435228 NOVA1 and its related STXBP6 chr14 i 9.66E-06 i 0.00127155 i -2.3828803 -0.992563164 i NOVA1 and its related ADSL chr22 3.81 E-05 i 0.00103031 i -2.2524394 -0.963218649 I Meyer2012_neuron. related ARHGAP32 chr11 i 9.87E-05; 0.00136933 i -2.2933292 -0.928104343 Meyer2012_neuron.related CNTNAP2 chr7 9.02E-05 0.00272893 -2.4524935 -0.967131933 i Meyer2012_neuron.related HTR2B chr2 i 3.68E-05 i 0.00097537 i -1.8752014 -0.96248593 Meyer2012_neuron.related KATNA1 chr6 0.00022786 0.00342952 -2.1938463 -0.93374683: Meyer2012_neuron.related LUZP1 chr1 i 0.00020082: 0.00015172 i 0.57781166 0.323680306 Meyer2012_neuron.related NOVA1 chr14 i 1.68E-06 i 0.00191534 i -2.4807974 -0.999292729 I Meyer2012_neuron.related SLITRK1 chr13 2.22E-07 1.57E-05 -0.5603988 -0.986039139 Meyer2012 neuron, related

[0540]

[0541]

[0246] TABLE 2

[0542] Selection analysis for human-specific SNPs using CLUES2.

[0543] The columns display the log-likelihood ratio (logLR), the negative lo lO-transformed p-value (-loglO(p-value)), and the selection coefficient (SelectionMLEl) for each SNP across a single epoch (0 to 200,000). The table is sorted by / j- alue, with SNPs highlighted in bold, including NOVAI (bold and underlined), showing stronger selection signals. Gene list and SNP information are from Trujillo et al., 20213.

[0544] Gene name Ensembl chr position logLR -Iog10 Epochl Epochl SelectionMLEl Gene ID (hg19) (hg19) (p-value) _start _end HERC5 ENSG00000138646 4 89410317 5.5077 3.04 0 200000 0.00135 AHR ENSG00000106546 7 17375392 6.2143 3.37 0 200000 0.00127 LAG3 ENSG00000089692 12 6883790 3.9207 2.29 0 200000 0.00097 C3 ENSG00000125730 19 6685111 3.8964 2.28 0 200000 0.00089 ZNF2 ENSG00000163067 2 95831534 3.8143 2.24 0 200000 0.00087 NOVA1 ENSG00000139910 14 26918100 2.7454 1.72 0 200000 0.00082 SSH2 ENSG00000141298 17 27959034 2.739 1.72 0 200000 0.00057 SSH2_2 ENSG00000141298 17 27959258 2.7372 1.71 0 200000 0.00057 IF144L ENSG00000137959 1 79106805 2.5711 1.63 0 200000 0.00054 KIAA1199 ENSG00000103888 15 81173308 2.0899 1.39 0 200000 0.00052 FAM166A ENSG00000188163 9 140139881 2.4931 1.59 0 200000 0.00051 KIF18A ENSG00000121621 11 28119295 2.3184 1.5 0 200000 0.00051 ZNF106 ENSG00000103994 15 42742312 2.2816 1.49 0 200000 0.00051 CDH16 ENSG00000166589 16 66947064 2.1992 1.44 0 200000 0.00051 KIF26B ENSG00000162849 1 245582905 1.6157 1.14 0 200000 0.00049 ITGB4 ENSG00000132470 17 73753035 1.7434 1.21 0 200000 0.00048 PRDM10 ENSG00000170325 11 129772293 1.8097 1.24 0 200000 0.00038 NLRP2 ENSG00000022556 19 55489189 1.8316 1.25 0 200000 0.00037 ADSL ENSG00000239900 22 40760978 1.6985 1.18 0 200000 0.00036 ADAM18 ENSG00000168619 8 39537618 1.6707 1.17 0 200000 0.00035 VCAN ENSG00000038427 5 82837946 1.6271 1.15 0 200000 0.00034 SCAP ENSG00000114650 3 47469149 1.5695 1.12 0 200000 0.00033 OR1K1 ENSG00000165204 9 125563200 1.5636 1.11 0 200000 0.00033 DNAJC11 ENSG00000007923 1 6694660 1.52 1.09 0 200000 0.00032 ADAM18_2 ENSG00000168619 8 39564352 1.2405 0.94 0 200000 0.00031 DCHS1 ENSG00000166341 11 6654769 1.3738 1.01 0 200000 0.0003 PIGZ ENSG00000119227 3 196674495 1.0181 0.81 0 200000 0.00024 ZNHIT2 ENSG00000174276 11 64884957 1.0153 0.81 0 200000 0.00021 MRPL49 ENSG00000149792 11 64893151 1.0113 0.81 0 200000 0.00021 PIEZO1 ENSG00000103335 16 88804443 0.9977 0.8 0 200000 0.00021 AC074212.3 ENSG00000237452 19 46265288 0.9664 0.78 0 200000 0.00021 CASC5_2 ENSG00000137812 15 40915640 0.9432 0.77 0 200000 0.00021 CASC5 ENSG00000137812 15 40912860 0.943 0.77 0 200000 0.00021 NOTO ENSG00000214513 2 73438011 0.9422 0.77 0 200000 0.00021

[0545]

[0546] NCOA6 ENSG00000198646 20 33337529 0.9277 0.76 0 200000 0.0002 ZNF726 ENSG00000213967 19 24116551 0.732 0.65 0 200000 0.00018 CCT5 ENSG00000150753 5 10250094 0.7118 0.63 0 200000 0.00013 TEX2 ENSG00000136478 17 62290457 0.6899 0.62 0 200000 0.00012 FRMD8 ENSG00000126391 11 65154602 0.5303 0.52 0 200000 0.00011

[0547]

[0548]

[0247] TABLE 3

[0549] Differential CLIP peaks between Novalwt / wt and Novalhu / hu.

[0550] Peaks are extracted that fulfill the criteria of being detected in all three biological replicates and having tags per peak at least 10 (peak height>10) for either Novalhu / hu or Novalwt / wt mice for NOVA1-CLIP analysis in P21 midbrain. Peaks on lowly expressed transcripts in P21 midbrain are excluded using RNA sequencing data. NOVAI binding peaks with -value less than 0.01 and absolute value of log2FC more than 1 were shown. The table contains the number of read counts for each genotype along with the genomic location of each peak: gene name, chromosome, start, end, strand, and annotation on the transcript and peak ID. The analysis was performed using edgeR. Noval wt / wt N=3, Novalhu / hu N=3.Gene_name chr start end strand annotation Noval Noval PValue peakJD

[0551] (wt / wt) (hu / hu)

[0552] Tln2 chr9 67375090 67375105 - intron 1 52 1.51E-08 218276[gene=chr9_r_c45452][PH=53][PH0=15.39][P=3.92e-12] Wfs1 chr5 36968335 36968337 - CDS) 13 45 6.81E-05 81327[gene=chr5_r_c18062] [PH =58] [PH0= 16,54][P=2.50e-13] downstream

[0553] 10K

[0554] Cog5 chr12 31782366 31782368 + intron 68 135 7.19E-05 63195[gene=chr12_f_c15280][PH=203][PH0=57.50][P=917e-14] TbHx chrX 77660566 77660573 + 3'UTR 24 4 8.81E-05 70789[gene=chrX_f_c12881][PH=28][PH0=12.95][P=2.92e-03] Usp25 chr16 77100491 77100495 + intron 3 20 0.00012122 347232[gene=chr16_f_c56828][PH=23][PH0=11,04][P=1,01e-02] Rhobtb2 chr14 69796341 69796343 - CDS 42 14 0.00012782 206997feene=chr14_r_c41627][PH=56][PHO=11.81][P=644e-15] Sgcz chr8 38483559 38483564 - intron 3 20 0.00022142 168310[gene=chr8_r_c28981][PH=23][PH0=6.24][P=1.37e-05] Celf2 chr2 6543160 6543166 - 3'UTR 17 2 0.00027482 9779[gene=chr2_r_c2189][PH=19][PH0=9.79][P=1.87e-01] Gript chrlO 119494678 119494680 + intron 6 27 0.00032527 266625[gene=chr10_f_c59146][PH=33][PH0= 10.02][P=9.87e-07] Nacc2 chr2 26056875 26056876 - 3'UTR 26 5 0.00032527 59469[gene=chr2_r_c13933][PH=31][PH0=10.03][P=6.53e-06] Opcml chr9 28827554 28827558 + intron 19 47 0.00037902 82659[gene=chr9_f_c16295][PH=66][PH0=18.33][P=1.63e-14] 8030462N17Rik chr18 77673267 77673277 - intron 0 12 0.00048846 164709[gene=chr18_r_c36578][PH=12][PH0=7.05][P=2.36e-01] Zbtb20 chr16 43176802 43176828 + intron 0 12 0.00048846 302113[gene=chr16_f_c47580][PH = 12] [PH0=5.81 ][P=1.13e-01 ] Cadps chr14 12435532 12435538 - intron 1 16 000051907 7826[gene=chr14_r_c2170][PH=17][PH0=4.20][P=1.18e-04] Fgf14 chr14 124326934 124326936 - intron 12 36 0.00053645 336266[gene=chr14_r_c64873][PH=48][PH0=9.19][P=6.55e-15] Fam 155a chr8 9238881 9238887 - intron 37 14 0.00053645 5870[gene=chr8_r_c1204] [PH=51 ][PH0=18.12][P=1.09e-08] Nudcdl chr15 44411046 44411054 - intron 1 15 0.00061696 43887[gene=chr15_r_c11355][PH=16][PH0=8.73][P=7.86e-02] Dmxl2 chr9 54418345 54418348 - intron 5 29 0.00065465 171082[gene=chr9_r_c34491][PH=34][PH0=14.60][P=1.14e-03] Rgs8 chr1 153693792 153693796 3'UTR 38 16 0.00071843 283660[gene=chr1_f_c62451][PH=54][PH0=13.66][P=5.87e-14] Rgs9 chr11 109234998 109234999 - downstream 2 19 0.00072905 394173[gene=chr11_r_c76058][PH=21][PH0=6.77][P=3.64e-04]

[0555] 10K|intron

[0556] Plxna2 chr1 194815094 194815101 + 3'UTR 32 11 0.00087261 388504[gene=chr1_f_c84665][PH=43][PH0=14.55][P=6.66e-08] 2210408121 Rik chr13 77207945 77207947 + intron 12 0 0.00097687 137388[gene=chr13_f_c30891][PH=12][PH0=4.83][P=4.22e-02] A330076H08Rik chr7 61941615 61941618 - non-coding 15 2 0.000977 155846[gene=chr7_r_c28589][PH=17][PH0=5.75][P=2.48e-03] Snx24 chr18 53311185 53311186 + intron 8 43 0.00099762 135693[gene=chr18_f_c28621][PH=51][PH0=14.58][P=936e-12]

[0557]

[0558] Ctnna2 chr6 77349600 77349611 - intron 2 14 0.00105834 128839[gene=chr6_r_c28203][PH=16][PH0=10.71][P=1.99e-01] Wwox chr8 115234745 115234746 + intron 11 0 0.00106582 263063[gene=chr8_f_c59865][PH=11][PH0=4.26][P=6.04e-02] Tmem64 chr4 15285827 15285831 + 3'UTR 9 30 0.00119457 17194[gene=chr4J_c4304][PH=39][PH0=17.82][P=5.20e-04] Gpr165 chrX 96718486 96718491 + 3'UTR 17 44 0.001215 90685[gene=chrX_f_c18065][PH=61][PH0=22.69][P=2.16e-09] Bmprlb chr3 141846816 141846821 - downstream 2 16 0.0012544 235944[gene=chr3_r_c49354][PH=18][PH0=9.26][P=5.49e-02]

[0559] 10K|intron

[0560] Tmem68 chr4 3552881 3552890 - downstream 35 15 0.00126174 224[gene=chr4_r_c18][PH=50][PH0=13.71][P=2.82e-12]

[0561] 10K|intran

[0562] Cstf2t chr19 31085227 31085229 + 3'UTR 34 14 000126174 81632[gene=chr19_f_c16468][PH=48][PH0= 19.34][P=1 51 e-06] Fam 155a chr8 9736285 9736292 - intron 16 3 0.00131294 40897[gene=chr8_r_c4373][PH=19][PH0=6.23][P=3.22e-03] Mbp chr18 82571209 82571218 + intron 5 21 0.00141581 203306[gene=chr18J_c44190][PH=26][PH0=12.91][P=1 10e-01] Ptprt chr2 162427558 162427559 - intron 9 38 0.00141669 403860[gene=chr2_r_c88859][PH=47][PH0=14.31][P=1.01e-09] Dab1 chr4 104734817 104734822 + downstream 3 19 0.00149054 187819[gene=chr4_f_c43783][PH=22][PH0=8.83][P=652e-03]

[0563] 10K|intron

[0564] Fgf9 chr14 58109986 58109988 + 3'UTR 41 20 0.0014987 114977[gene=chr14J_c28269][PH=61][PH0=16.01][P=2.34e-14] Tcf3 chr10 80412537 80412539 - intron 15 40 0.00151809 183602[gene=chr10_r_c42408][PH=54][PH0=14.15][P=144e-13] Bacel chr9 45863180 45863196 + 3'UTR 4 20 0.00154495 170783[gene=chr9_f_c27569][PH=23][PH0=10.19][P=4.45e-03] Gas5 chr1 161037408 161037414 + non-coding 4 21 0.00154495 319034 [gene=chr1_f_c68139][PH=25][PH0=15.49][P=7.28e-02] Foxn3 chr12 99301581 99301584 - intron 7 36 0.00159816 156172[gene=chr12_r_c33299][PH=43][PH0= 12.33][P=1,49e-09] Lingol chr9 56619908 56619909 - CDS 13 2 0.00183175 179080[gene=chr9_r_c36383][PH=15][PH0=5.89][P=2.49e-02] ipo11 chr13 106878281 106878293 - intron 2 14 000183175 216140[gene=chr13_r_c48769][PH=16][PH0=647][P=306e-02] Repsi chr10 18119017 18119032 + 3‘UTR|CDS|intron 13 2 0.00183175 26215[gene=chr10_f_c5525][PH=15][PH0=12.27][P=3.81e-01] Eif4e chr3 138555579 138555580 + 3'UTR 13 2 0.00183175 269987[gene=chr3_f_c59205][PH=15][PH0=9.33][P=4.15e-01] Zbtb20 chr16 43421583 43421585 + intron 2 15 0.00183175 304091 [gene=chr16_f_c48276][PH= 17][PH0=7.59][P=1,83e-02] Rasgrfl chr9 89931789 89931799 + intron 13 2 0.00183175 312891[gene=chr9_f_c54416][PH=15][PH0=6.14][P=2.41e-02] Phactr3 chr2 178219367 178219377 + intron 9 29 0.00188011 501468[gene=chr2_f_c104787][PH=38][PH0= 11.481[P=3.63e-08] Fam 120a chr13 48922185 48922188 - CDS 28 8 0.00188011 92863[gene=chr13_r_c21897][PH=36][PH0=22.72][P=5.48e-02] Fem 1c chr18 46504648 46504654 - 3'UTR 20 4 00019522 105803[gene=chr18_r_c22842][PH=24][PH0=11 98][P=1 67e-01] Nrxn3 chr12 89641569 89641574 + intron 0 11 0.00195362 230588[gene=chr12_f_c53385][PH=11][PH0=5.81][P=2.07e-01] Pls3 chrX 75786155 75786156 - 3'UTR 8 27 0.00210428 74940[gene=chrX_r_c15617][PH=35][PH0=1570][P=3.44e-03]

[0565]

[0566] Frmd6 chr12 70833161 70833167 + intron 35 12 0.00215546 137754[gene=chr12_f_c33175][PH=47][PH0=12.99][P= 1 92e-11] Scrtl chr15 76517795 76517799 - 3'UTR 11 31 0.00222392 145440[gene=chr15_r_c33056] [PH=42][PH0= 10.15)[P=6.17e-12] Shisa6 chr11 66479786 66479811 - intron 15 3 0.00235091 188045[gene=chr11_r_c40003][PH=18][PH0=10.15][P=8.56e-02] Sntgl chr1 9193556 9193578 - intron 3 15 0.00235091 22497[gene=chr1_r_c4951][PH=18][PH0=8.09][P=2.31e-02] Nrxn3 chr12 89835988 89836001 + intron 3 16 0.00235091 241655[gene=chr12_f_c54827][PH=19][PH0=9.62][P=3.87e-02] Smap2 chr4 120968800 120968801 - 3'UTR 15 3 000235091 254548[gene=chr4_r_c53732][PH=18][PH0=10 17][P=281 e-01 ] Kirrel chr3 87081635 87081644 - 3'UTR 22 7 0.00249566 131406[gene=chr3_r_c28517] [P H =29][PH 0=7.26][P=9.15e-08] Nudt11 chrX 6053068 6053074 + 3'UTR 22 7 0.00249566 47[gene=chrX_f_c11 ][PH=29][PH0=12.13][P= 1.30e-03] Arhgap44 chr11 65023153 65023155 - CDS 29 9 0.00256579 176033[gene=chr11_r_c37417][PH=37][PH0=22.80][P=3.11e-02] Sema4f chr6 82924400 82924405 - intron 18 4 0.00257819 147696[gene=chr6_r_c31467][PH=22][PH0=11.52][P=1.12e-01] Stxbp6 chr12 44854700 44854711 - 3'UTR 20 6 0.00260107 48761 [gene=chr12_r_c11800] [PH =26][PH0= 13.67][P=1.37e-02] Mapk8 chr14 33391156 33391165 - intron 5 20 0.00260107 98954[gene=chr14_r_c21513][PH=25][PH0=12.82][P=1.42e-02] Kirrel3 chr9 34697558 34697562 + intron 18 3 000262142 119765[gene=chr9_f_c19812][PH=21][PH0=925][P=1 93e-02] Numb chr12 83815750 83815772 - intron 6 22 0.00265304 136438[gene=chr12 r c29075] [PH =28][PH0= 17.42][P=5.06e-02] Auts2 chr5 131865939 131865958 - intron 2 13 0.00274509 356276[gene=chr5_r_c74056][PH=15][PH0=5.11][P=6.80e-03] Farpl chr14 121094837 121094842 + intron 2 20 0.00288105 234673[gene=chr14_f_c54729][PH=22][PH0=10.27][P=1.57e-02] Auts2 chr5 131615099 131615122 - intron 3 21 0.00297168 345719[gene=chr5_r_c72487][PH=24][PH0=14.14][P=4.85e-02] Sertad4 chr1 192845131 192845133 - 3'UTR 18 3 0.00311834 455652[gene=chr1_r_c99472][PH=21][PH0=8.61][P=3.53e-03] Phf20 chr2 156227793 156227808 + intron 7 23 0.00313858 435302[gene=chr2_f_c91425][PH=30][PH0=8.65][P=6.36e-07] Scamp5 chr9 57442182 57442183 - 3'UTR 45 21 0003202 184401 [gene=chr9_r_c37743][PH=66][PH0=2356][P=581e-10] Pcsk2 chr2 143616863 143616871 + intron 14 2 0.00328831 397207[gene=chr2_f_c82251][PH=16][PH0=8.83][P=8.39e-02] Plec chr15 76172043 76172062 - 3'UTR 10 31 0.00338126 142998[gene=chr15_r_c32635][PH=41][PH0=20.82][P=6.94e-04] Aqp4 chr18 15392136 15392137 - 3'UTR 12 30 0.00338126 24755[gene=chr18_r_c6655][PH=42][PH0=19.36][P=3.14e-04] Kcnip4 chr5 48800208 48800209 - intron 12 2 0.00341905 105704[gene=chr5_r_c24161][PH=14][PH0=3.77][P=1.17e-03] Kcnip4 chr5 49515481 49515486 - intron 12 2 0.00341905 146850[gene=chr5_r_c29186][PH=14][PH0=7.45][P=9.25e-02] Erbb4 chr1 68849423 68849428 - intron 1 13 0.00341905 152763[gene=chr1_r_c33551][PH=14][PH0=6.88][P=9.44e-02] 4933431E20Rik chr3 107891945 107891963 - NA 12 2 000341905 172994[gene=chr3_r_c36764][PH=14][PH0=439][P=4 14e-03] Hdlbp chr1 93413864 93413874 - CDS 12 2 0.00341905 203697[gene=chr1_r_c45482][PH=14][PH0=8.59][P=1.68e-01]

[0567]

[0568] Plekha6 chr1 133297020 133297041 + intron 12 2 0.00341905 252614[gene=chr1_f_c56079][PH=14][PH0=5.07][P=5.71e-02] Hecwl chr13 14427719 14427739 - intron 2 13 0.00341905 26548[gene=chr 13_r_c5850] [PH = 15][PH0=9.06] [P=1,58e-01 ] Paqr7 chr4 134508409 134508424 + 3'UTR 12 2 0.00341905 288802[gene=chr4_f_c67294][PH=14][PH0=6.19][P=1.58e-01] Vps33a chr5 123529952 123529966 - 3'UTR 12 1 0.00341905 307338[gene=chr5_r_c63396][PH=13][PH0=6.31][P=8.60e-02] Slc38a3 chr9 107651950 107651966 - CDS 12 2 0.00341905 314736[gene=chr9_r_c67228][PH=14][PH0=6.19][P=3.84e-02] St7 chr6 17916162 17916175 + downstream 13 2 000341905 36822[gene=chr6_f_c7347][PH=15][PH0=532][P=832e-03]

[0569] 10K|intron

[0570] Fam175b chr7 132883689 132883696 + 3'UTR 12 2 0.00341905 386934[gene=chr7_f_c81165][PH=14][PH0=4.69][P=7.92e-03] Actn4 chr7 28893487 28893492 - 3'UTR 12 2 0.00341905 39944[gene=chr7_r_c7021][PH=14][PH0=6.95][P=6.57e-02] Camtai chr4 151801066 151801069 - intron 12 2 0.00341905 407690[gene=chr4_r_c82368][PH=14][PH0=4.24][P=6.64e-03] Arhgap18 chr10 26786392 26786418 + intron 12 1 000341905 43473[gene=chr10_f_c9557][PH=13][PH 0=4.87] [P=1.84e-02] Zfp266 chr9 20499284 20499285 - CDS 13 2 0.00341905 44383[gene=chr9_r_c10451][PH=15][PH0=6.65][P=6.27e-02] Lingo2 chr4 36151380 36151389 - intron 1 12 0.00341905 49162[gene=chr4_r_c10673][PH=13][PH0=4.47][P=1.44e-02] Gridl chr14 35375525 35375530 + intron 1 13 0.00341905 91769[gene=chr14_f_c22328][PH=14][PH0=5.87][P=3.27e-02] Nlgn3 chrX 101324631 101324639 + 3'UTR 12 2 0.00341905 98346[gene=chrX_f_c19340][PH=13][PH0=4.91][P=2.31e-02] Malatl chr19 5802563 5802564 - non-coding 22 49 0.00355118 18052[gene=chr19_r_c2896j[PH=71][PH0=33.56][P=330e-06] Ank3 chr10 69988359 69988364 + CDSjintron 23 6 0.00372178 121240[gene=chr10_f_c27580][PH=29][PH0=10.48][P=329e-04] Lnx1 chr5 74671385 74671393 - intron 7 23 000372178 200642[gene=chr5_r_c41295][PH=30][PH0=18 15][P=326e-02] Rab6b chr9 103183763 103183767 + 3'UTR 54 26 0.00375134 338504[gene=chr9_f_c60832][PH=79][PH0=28.47][P=4.08e-13] Pcdh9 chr14 93886975 93886977 - CDS 10 28 0.00393673 277078[gene=chr14_r_c51949][PH=38][PH0=11.81][P=8.19e-08] Pcdh7 chr5 58029071 58029094 + intron 3 19 0.00401138 199109[gene=chr5_f_c39064][PH=22][PH0=14.29][P= 1.12e-01 ] Gpr83 chr9 14861784 14861797 + intron 5 20 0.00407982 22298[gene=chr9_f_c4710][PH=25][PH0=9.32][P=3.15e-04] Osbpl6 chr2 76428749 76428750 intron 24 55 0.00413442 216993[gene=chr2_f_c43929][PH=79][PH0=30.78][P=1.56e-11] Mobp chr9 120156451 120156452 + intron 22 52 0.00416529 381075[gene=chr9_f_c70113][PH=74][PH0=15.09][P=1.62e-14] Clmn chr12 104863094 104863116 - intron 3 15 00041825 178069[gene=chr 12_r_c38617] [PH = 18][PH0=982] [P =596e-02] Prepl chr17 85086906 85086920 - intron 3 15 0.0041825 179404[gene=chr17_r_c37901][PH=18][PH0=6.94][P=4.48e-03] Rnf152 chr1 105278568 105278571 - 3'UTR 2 14 0.0041825 212352[gene=chr 1 _r_c47602) [PH= 16][PH 0=9.75][P=4.67e-01 ] Dlg2 chr7 91644519 91644525 + intron 15 3 0.0041825 215217[gene=chr7_f_c43677][PH=18][PH0=7.43][P=7.63e-03] Cntn5 chr9 10496870 10496871 - intron 2 14 0.0041825 22971[gene=chr9_r_c5639][PH=16][PH0=5.91][P=6.04e-03]

[0571]

[0572] D17Wsu92e chr17 27764200 27764208 - downstream 3 16 0.0041825 48244[gene=chr17_r_c 10255] [PH= 19][PH0=10.90][P=7.15e-02]

[0573] 10K|intron

[0574] Cacnb2 chr2 14973623 14973637 + downstream 2 14 0.0041825 60322[gene=chr2_f_c8199][PH=16][PH0=8.73][P=7.86e-02]

[0575] 10K|intron

[0576] Lyrm4 chr13 35979561 35979563 - 3‘UTR 2 15 0.0041825 63572[gene=chr13_r_c 15403] [PH= 17][PH 0=7,22][P=3.22e-02] Fam 189a 1 chr7 64954199 64954211 - intron 12 36 0.00418453 166946[gene=chr7_r_c30976][PH=48][PH0=13.08][P=8.64e-12] Azi2 chr9 118050716 118050718 + intron 51 110 0.00432514 374034[gene=chr9_f_c68395][PH=160][PH0=19.50][P=391e-14] Grm7 chr6 111566704 111566712 + 3'UTR 31 13 0.00432839 357967[gene=chr6_f_c73851][PH=44][PH0=13.25][P=1.53e-09] Ctnna2 chr6 77359325 77359333 - intron 4 19 000434582 129354[gene=chr6_r_c28274][PH=23][PH0=7.41][P=1.02e-04] HiatH chr13 65066382 65066390 - 3'UTR 4 18 0.00434582 136248[gene=chr13 r c31593] [PH=22][PH0=9.15][P=5.15e-03] CoblH chr2 65106073 65106080 - intron 19 4 0.00434582 186842[gene=chr2_r_c42177][PH=23][PH0=9.32][P=1.53e-03] Grlf1 chr7 16561768 16561774 - CDS 19 6 0.00434582 20382[gene=chr7_r_c3277][PH=25][PH0=629][P=1.12e-06] Sec24c chr14 20684727 20684733 + intron 4 18 0.00434582 29427[gene=chr14_f_c7604][PH=22][PH0=9.55][P=4.88e-03] Tm9sf3 chr19 41223167 41223169 - CDS 18 6 0.00434582 89658[gene=chr19_r_c20096][PH=24][PH0=11.21][P=9.64e-03] Syn3 chr10 86293222 86293244 - 3'UTRjintron 1 12 0.00436048 201864[gene=chr10_r_c46311][PH=13][PH0=6.06][P=6.43e-02] RapgefB chr11 54676088 54676103 + intron 3 18 000442706 104111[gene=chr11_f_c22599][PH=21][PH0=1271l[P=800e-02] Atg3 chr16 45176241 45176246 + intron 3 17 0.00442706 312386[gene=chr16_f_c49796][PH=20][PHO=7.49][P=2.80e-03] Tex2 chr11 106502353 106502364 - 3'UTR 17 4 0.00442706 373752[gene=chr11_r_c71697][PH=21][PH0=6.85][P=5.16e-04] Atxn7l1 chr12 33250212 33250213 + 3'UTR|intron 16 4 0.00442706 67938[gene=chr12_f_c16456][PH=20][PH0=9.27][P=4.67e-02] Stx18 chr5 38056112 38056123 + intron 12 2 0.00444495 176536[gene=chr5_f_c33679][PH=14][PH0=8.03][P=1.29e-01] Msi2 chr11 88449510 88449511 - intron 13 40 0.00459909 321418[gene=chr11_r_c61284][PH=53][PH0=10.94][P=1.00e-100] Pcdh7 chr5 57994529 57994562 + intron 3 16 0.00489756 198369[gene=chr5_f_c38908][PH=19][PH0=16.46][P=4.05e-01] Ank2 chr3 126968902 126968907 - intron 11 30 000510227 209256[gene=chr3_r_c43622][PH=41][PH0=1274][P=1 99e-08] Ltn1 chr16 87377669 87377671 - 3'UTR 50 23 0.00519474 204946[gene=chr16_r_c42303][PH=73][PH0=21.43][P=1.00e-100] Grial chr11 57186931 57186933 + intron 8 25 0.00540195 121657[gene=chr11_f_c25669][PH=33][PH0=11,32][P=4.91 e-06 j Fam155a chr8 9587638 9587640 - intron 14 35 0.00544983 25500[gene=chr8_r_c3385][PH=48][PH0=18.26][P=2.47e-07] Cacnb4 chr2 52430329 52430330 - 3'UTR 39 18 0.00549324 161975[gene=chr2_r_c35753][PH=57][PH0=22.65][P=1.89e-07] Dab1 chr4 103991619 103991624 + intron 15 36 0.00552146 173200[gene=ch r4_f_c40210][PH=51 ] [P H0= 12.29][P=3.12e- 14] Gria4 chr9 4534702 4534716 - intron 6 21 0.00555689 5520[gene=chr9_r_c737][PH=27][PH0=893][P=3.47e-05] Rap2a chr14 120482218 120482236 + intron 13 1 000557665 233224[gene=chr14_f_c54321][PH=14][PH0=754][P=978e-02]

[0577]

[0578] Ubr4 chr4 139425322 139425323 + CDSjintron 22 6 0.00592828 305380[gene=chr4_f_c70955][PH=28][PH0=13.17][P=3.65e-03] Nlgnl chr3 26028563 26028565 - intron 7 23 0.00592828 42304[gene=chr3_r_c9019][PH=30][PH0=13.83][P=1.77e-03] Plbd2 chr5 120484878 120484879 - 3'UTR 122 232 0.00603636 288717[gene=chr5_r_c59873][PH=354][PH0=50.08][P=4.03e-13] Ildr2 chr1 166302739 166302763 + intron 3 15 0.00615219 325630[gene=chr1_f_c69530][PH=18][PH0=12.62][P=2.11e-01] Csnklal chr18 61589749 61589750 + 3'UTR 21 44 0.00622624 151980[gene=chr18_f_c32696][PH=65][PH0=20.45][P=9.85e-13] Ptprs chr17 56440551 56440553 - intron 1 11 0.0063493 100879[gene=chr17_r_c21306][PH=12][PH0=4.09][P=2.62e-02] Rbfoxl chr16 7190257 7190264 + intron 1 12 0.0063493 109859[gene=chr16_f_c12377][PH=13][PHO=4.95][P=2.03e-02] Nrg3 chr14 38658892 38658905 - intron 1 11 0.0063493 110477[gene=chr14_r_c23874][PH=12][PH0=4.75][P=3.48e-02] Ank3 chr10 69885184 69885188 + CDSjintron 12 1 0.0063493 116735[gene=chr10_f_c27021 ][PH=13][PH0=5.52][P=3.52e-02] Kcnn2 chr18 45431465 45431479 + intron 1 11 0.0063493 121451[gene=chr18_f_c24885][PH=12][PH0=6.31][P=1.18e-01] Megf9 chr4 70491002 70491012 - intron 2 11 0.0063493 134622[gene=chr4_r_c28731][PH=13][PH0=8.07][P=2.73e-01] Epha6 chr16 60393163 60393166 - intron 1 12 0.0063493 135852[gene=chr16_r_c28511][PH=13][PH0=6.68][P=9.17e-02] Foxn3 chr12 99266900 99266907 - intron 2 11 0.0063493 154841[gene=chr12_r_c33073][PH=13][PH0=6.65][P=1.00e-01] Kcnj3 chr2 55535452 55535464 + intron 11 1 0.0063493 162751 [gene=chr2_f_c31996][PH=12][PH0=6.39][P=1.24e-01] Atg9a chr1 75186254 75186265 - CDS 11 2 0.0063493 166510[gene=chr1_r_c36915][PH=13][PH0=6.61][P=1.71e-01] Chst11 chr1O 83098086 83098111 + intron 1 11 0.0063493 167378[gene=chr10_f_c38091 ][PH=12] [PH0=7,68][P=2.31 e-01 ] Trio chr15 27779612 27779616 - intron 2 11 0.0063493 18083[gene=chr15_r_c5239][PH=13][PH0=3.93][P=6.92e-03] Scn2a1 chr2 65724305 65724322 + intron 11 2 0.0063493 182203[gene=chr2_f_c36629][PH=13][PH0=9.73][P=3.30e-01] Pde4d chr13 109143085 109143091 + intron 2 11 0.0063493 182350[gene=chr13_f_c42168][PH=13][PH0=6.85][P=2.85e-01] Dlgapl chr17 70184422 70184424 + intron 1 11 00063493 183681[gene=chr17_f_c42440][PH=12][PH0=673][P=324e-01] Rgs6 chr12 82949776 82949778 + intron 2 11 0.0063493 184798[gene=chr12_f_c43840][PH=13][PH0=5.15][P=2.61e-02] Usp46 chr5 74000749 74000754 - 3'UTR 11 2 0.0063493 197455[gene=chr5_r_c40249][PH=13][PH0=4.56][P=2.12e-02] Gpr56 chr8 95011791 95011805 CDS 12 2 0.0063493 204796[gene=chr8_f_c45814][PH=14][PH0=6.38][P=4.12e-02] Pafahfbl chr11 74720203 74720207 - intron 12 2 0.0063493 209928[gene=chr11_r_c44123] [PH=14][PH0=7.62][P=1.13e-01] Abr chr11 76463642 76463643 - CDS 11 2 0.0063493 217102[gene=chr11_r_c45929][PH=13][PH0=5.18][P=2.94e-02] Carlo chr11 93268234 93268252 + intron 1 12 0.0063493 239264[gene=chr11_f_c50822][PH=13][PH0=5.89][P=5.54e-02] Rora chr9 68757549 68757550 + intron 11 2 00063493 242264[gene=chr9_f_c42091][PH=13][PH0=468][P=1 70e-02] Svop chr5 114026972 114026985 - 3'UTR 12 2 0.0063493 273107[gene=chr5_r_c56412][PH=14][PH0=4.93][P=2.05e-02]

[0579]

[0580] Nfasc chr1 132624330 132624341 - intron 1 11 0.0063493 284961 [gene=chr1_r_c64083][PH=12][PH0=6.31][P=1.18e-01] Parva chr7 112588604 112588615 + CDS 11 1 0.0063493 305980[gene=chr7_f_c64662][PH=12][PH0=5.42][P=6.83e-02] Spen chr4 141512222 141512235 - intron 1 11 0.0063493 316763[gene=chr4_r_c67497][PH=12][PH0=5.05][P=4.32e-02] Vamp4 chr1 162597669 162597670 + 3'UTR 12 2 0.0063493 320164[gene=chr1_f_c68357][PH=14][PH0=6.87][P=1.99e-01] Prune2 chr19 17221018 17221028 + 3'UTR 1 11 0.0063493 32655[gene=chr19_f_c6658][PH=12][PH0=7.07][P=2.57e-01] Pou2f1 chr1 165866880 165866885 - 3'UTR 11 1 0.0063493 369368[gene=chr1_r_c82165][PH=12][PH0=4.10][P=1.62e-02] Slc35c2 chr2 165282831 165282850 - CDS|downstream 11 2 0.0063493 414986[gene=chr2_r_c91440][PH=13][PH0=6.31][P=7.06e-02]

[0581] 10K

[0582] Nsg2 chr11 32037173 32037177 + downstream 12 2 0.0063493 44784[gene=chr11_f_c10024][PH=14][PH0=7.49][P=1.43e-01]

[0583] 10K|intron

[0584] Mcf2 chrX 60056436 60056445 - 3'UTR 1 11 0.0063493 53094[gene=chrX_r_c12000] [PH=12][PH0=5.62][P=1.46e-01] Bini chr18 32425420 32425422 intron 1 11 0.0063493 55756[gene=chr18_f_c13157][PH=12][PH0=4.39][P=3.38e-02] Ntm chr9 29932789 29932816 - intron 1 11 0.0063493 96131[gene=chr9_r_c19286][PH=12][PH0=6.97][P=1.71e-01] Ptprg chr14 11794267 11794272 intron 11 2 0.0063493 9921[gene=chr14_f_c2352][PH=13][PH0=4.50][P=1.40e-02] Scn8a chr15 100948358 100948361 + downstream 12 31 0.00643244 243072[gene=chr15_f_c53079][PH=43][PH0=11,25][P=5.52e-11]

[0585] 10K|intron

[0586] AW549877 chr15 3983814 3983821 - 3'UTR 25 8 0.00652143 1863[gene=chr15_r_c536][PH=33][PH0=12.19][P=3.08e-05] Gria2 chr3 80688704 80688706 - 3'UTR 17 37 0.00660797 116522[gene=chr3_r_c25424][PH=54][PH0=8.27][P=3.87e-14] Cdh23 chr10 60323354 60323357 - CDS 20 7 0.00661429 139347[gene=chr10_r_c32807][PH=27][PH0= 11,30][P=1.00e-03] Ppp1r13b chr12 111833650 111833654 - CDS 20 7 0.00661429 186042[gene=chr12_r_c40978][PH=26][PH0=8.32][P=2.31e-05] Gpr158 chr2 21532663 21532669 + intron 6 21 0.00661429 82137[gene=chr2_f_c13433][PH=27][PH0=12.42][P=2.54e-03] Plcxd2 chr16 45962796 45962807 - 3'UTR 34 14 0.00661558 115904[gene=chr16 _r c23848] [PH=48][PH0=23.97] [P=2.79e-03] Slc9a6 chrX 56662332 56662338 + 3'UTR 15 33 0.00661558 36506[gene=chrX_f_c7704][PH=48][PH0=20.98][P=2.33e-05] Nrg3 chr14 39347080 39347081 - intron 6 21 0.00662165 137104 [gene=chr14_r_c28013][PH=27][PH0=13.13][P=1.00e-02] Asxl2 chr12 3465161 3465179 + downstream 1 11 0.00674359 2824[gene=chr12_f_c352][PH=12][PH0=6.88][P=1.63e-01]

[0587] 10K|intron

[0588] Tacd chr8 25157244 25157245 - 3'UTR 30 14 0.00677474 96870[gene=chr8_r_c14876][PH=44][PH0=15.65][P=1.14e-06] Pcdh17 chr14 84535997 84536000 + 3'UTR 9 25 0.00700535 179904[gene=chr14_f_c40472][PH=34][PH0=21.49][P=4.65e-01] Plekhm2 chr4 141626063 141626064 - 3'UTR 25 9 0.00700535 316985[gene=chr4_r_c67569][PH=34][PH0=15.33][P=5.32e-04] Bai3 chr1 25639840 25639841 - intron 25 10 0.00700535 74076[gene=chr1_r_c16357][PH=35][PH0=13.91][P=3.84e-05] Prexl chr2 166673382 166673384 - intron 16 41 0.00718109 422880[gene=chr2_r_c93126][PH=57][PH0=11.64][P=2.30e-14]

[0589]

[0590] Rab11fip2 chr19 59904994 59905002 - 3‘UTR 18 6 0.00720067 131541[gene=chr19_r_c30851][PH=24][PH0=10.26][P=1.17e-02] Prkce chr17 86189466 86189468 + intron 18 6 0.00720067 215446[gene=chr17_f_c50287][PH=23][PH0=7.79][P=3.89e-04] MapkIO chr5 102911709 102911719 - 3‘UTR 18 5 0.00720067 235805[gene=chr5_r_c48873] [PH=23][PH0=10.96][P=1.36e-02] Snx5 chr2 144250967 144250972 - 3'UTR 4 18 0.00720067 351896[gene=chr2_r_c77937][PH=22][PH0=6.94][P=3.44e-04] Grial chr11 57100290 57100293 + intron 14 3 0.00738761 118324[gene=chr11_f_c25112][PH=17][PH0=6.18][P=6.25e-03] Macradl chr19 7109426 7109433 + intron 3 13 0.00738761 13464[gene=chr19_f_c2895][PH=15][PH0=5.21][P=7.47e-03] Zfp458 chr13 67255954 67255964 - 3'UTR 3 13 0.00738761 138590[gene=chr13_r_c32167][PH=16][PH0=6.95][P=4.00e-02] Mtmr10 chr7 64317457 64317458 + downstream 2 13 0.00738761 142217[gene=chr7_f_c26384][PH=15][PH0=5.48][P=9.18e-03]

[0591] 10K|intron

[0592] Ubxn7 chr16 32385529 32385531 + 3‘UTR 13 3 0.00738761 184185[gene=chr16_f_c28439][PH=16][PH0=4.88][P=3.35e-03] Pum2 chr12 8750486 8750493 + 3'UTR 13 3 0.00738761 21380[gene=chr12_f_c5115][PH=16][PH0=5.73][P=7.27e-03] Sparcl1 chr5 104088440 104088454 - CDS 13 2 0.00738761 243556[gene=chr5_r_c50477][PH=15][PH0=9.18][P=1.66e-01] Cntn5 chr9 10821898 10821905 - intron 2 14 0.00738761 26787[gene=chr9_r_c6611][PH=16][PH0=10.29][P=1.86e-01] Lsamp chr16 41655904 41655910 + intron 3 13 0.00738761 273646[gene=chr16_f_c43757][PH=16][PH0=5.06][P=4.35e-03] Zfp280d chr9 72343292 72343308 + downstream 14 3 0.00738761 280799[gene=chr9_f_c47244][PH=17][PH0=5.63][P=2.49e-03]

[0593] 10K|intron

[0594] Ksr2 chr5 117657054 117657056 + intron 13 2 0.00738761 322715[gene=chr5_f_c67714][PH=15][PH0=5.95][P=2.05e-02] Fgf14 chr14 124004855 124004856 - intron 14 3 0.00738761 327855[gene=chr14_r_c63184][PH=17][PH0=8.31][P=5.00e-02] Ptprt chr2 162532839 162532844 - intron 2 14 0.00738761 406485[gene=chr2_r_c89439][PH=16][PH0=6.36][P=1.17e-02] Aatk chr11 120011690 120011693 - CDS 3 13 0.00738761 448858[gene=chr11_r_c86316][PH=16][PH0=4.06][P=3.66e-04] Csmdl chr8 16865361 16865362 - intron 3 14 0.00738761 65016[gene=chr8_r_c9802][PH=17][PH0=5.42][P=1.23e-03] Stard4 chr18 33201593 33201613 - 3‘UTR 3 14 0.00738761 68258[gene=chr18_r_c14133j[PH=17j[PH0=4.53][P=2.48e-04] Ralgpsl chr2 33151900 33151920 - intron 2 14 0.00738761 84741[gene=chr2_r_c18587][PH=16][PH0=6.83][P=1.84e-02] 1810013L24Rik chr16 8843517 8843523 + intron 15 4 0.00754075 120910[gene=chr16_f_c14078][PH=19][PHO=6.00][P=1.34e-03] Arhgef12 chr9 42965228 42965231 - 3'UTR 15 4 0.00754075 126616[gene=chr9_r_c26635][PH=19][PH0=6.78][P=2.22e-03] Bail chr15 74553164 74553174 + intron 4 16 0.00754075 142801 [gene=chr15_f_c33450][PH=20][PH0=6.61][P=1.28e-03] Nrxnl chr17 90702013 90702019 - intron 3 16 0.00754075 204180[gene=chr 17_r_c42399] [PH = 19][PH0=8.83] [P = 1.87e-02] Ralgps2 chr1 156806788 156806794 - 3'UTR 16 4 0.00754075 339859[gene=chr1_r_c75429][PH=20][PH0=6.67][P=5.76e-04] Mapk8ip3 chr17 24897630 24897639 - 3'UTR|intron 3 16 0.00754075 34751 [gene=chr17_r_c7362][PH=19][PH0=7.69] [P=7.86e-03] Rab11fip3 chr17 25990143 25990149 - 3'UTR 15 4 0.00754075 41880[gene=chr17_r_c8747][PH=19][PH0=5.52][P=2.74e-04]

[0595]

[0596] Arhgap5 chr12 52559290 52559308 + intron 15 4 0.00754075 99670[gene=chr12_f_c24798][PH=19][PH0=6.82][P=1.92e-03] Dpysl3 chr18 43323323 43323339 - 3'UTR 13 3 0.00763313 97858[gene=chr18_r_c20773][PH=16][PH0=8.10][P=4.27e-01] Ctnna2 chr6 77507171 77507177 - intron 28 13 0.00763827 137154[gene=chr6_r_c29339][PH=41][PH0=6.72][P=1.67e-14] Ptprd chr4 76059952 76059955 - intron 11 27 0.00763827 139529[gene=chr4_r_c29682][PH=38][PH0=10.17][P=1.71e-09] Akt3 chr1 177022933 177022934 - 3'UTR|intron 28 10 0.00763827 419681[gene=chr1_r_c91076][PH=38][PH0=12.53][P=6.49e-07] Trpm3 chr19 22989168 22989171 + 3'UTR 11 28 0.00763827 62303[gene=chr19_f_c12516][PH=39][PH0=19.40][P=1.16e-02] Specc1 chr10 75276900 75276917 + CDS 15 3 0.00778858 149493[gene=chr10_f_c34045][PH=18][PH0=10.33][P=8.05e-02] Nrxn3 chr12 89679467 89679470 + intron 13 31 0.00792315 233329[gene=chr12_f_c53675][PH=44][PH0=14.79][P=502e-08] Pcdh10 chr3 45422808 45422814 + 3'UTR|intron 13 32 0.00792315 79265[gene=chr3_f_c17900][PH=45][PH0=28.22][P=1.27e-02] Plekha6 chr1 133272277 133272284 + CDS 36 15 0.00795088 251699[gene=chr1_f_c55952][PH=51][PH0=11.40][P=1.00e-100] Kcnip4 chr5 48667122 48667124 - intron 8 24 0.00813512 101305[gene=chr5_r_c23377][PH=32][PH0=15.96][P=4.20e-03] Kirrel3 chr9 34838356 34838357 + intron 7 24 0.00813512 131287[gene=chr9_f_c20770][PH=31][PH0=6.25][P=1.56e-10] Nrg3 chr14 39392839 39392846 - intron 23 9 0.00813512 140336[gene=chr14_r_c28343][PH=32][PH0=7.09][P=1.22e-09] Trp53bp1 chr2 121198311 121198313 - 3‘UTRjintron 23 8 0.00813512 303681 [gene=chr2_r_c67553][PH=31 ][PH0=7.88][P=3.85e-08] Pitpnd chr11 107212257 107212278 - 3’UTR 8 23 0.00813512 379186[gene=chr11_r_c72624][PH=31][PH0=9.97][P=3.63e-06] Mobp chr9 120160123 120160125 + intron 24 9 0.00813512 381850[gene=chr9_f_c70122][PH=33][PH0=13.57][P=1.72e-03] Fgf14 chr14 124574783 124574784 - intron 20 43 0.00865247 348422[gene=chr14_r_c66365][PH=63][PH0=17.75][P=3.87e-14] Nvl chr1 181103308 181103316 - intron 20 44 0.00865247 436813[gene=chr1_r_c94918][PH=64][PH0=26.03][P=9.24e-09] Slc25a12 chr2 71272613 71272614 - 3'UTR 26 10 0.00904766 201327[gene=chr2_r_c45292] [PH =36][PH 0= 15.57][P=3.06e-04] Grik2 chr10 49262892 49262893 - intron 17 38 0.00908375 119788[gene=chr10_r_c28184][PH=55][PH0=31.37][P=2.67e-03] Frmd3 chr4 74158541 74158545 + intron 2 14 0.00923366 99602[gene=chr4_f_c23253][PH=16][PH0=7.85][P=4.29e-02] Snx27 chr3 94526463 94526464 - CDSjdownstream 7 20 0.00936035 143799[gene=chr3_r_c30588][PH=27][PH0= 11,56][P=1,07e-03]

[0597] 10K|intron

[0598] Scamp5 chr9 57443829 57443830 - CDS 21 8 0.00936035 184996[gene=chr9_r_c37744][PH=29][PH0=9.47][P=1.78e-05] Pbx3 chr2 34172068 34172072 - 3'UTR 21 6 0.00936035 97894[gene=chr2_r_c21409][PH=27][PH0=12.29][P=4.19e-03] Hspa12a chr19 58840035 58840041 - intron 12 1 0.0093801 125266[gene=chr19_r_c29378][PH=13][PH0=5.63][P=1.17e-01] Mycbp2 chr14 103295678 103295687 - intron 3 14 0.00939383 299560[gene=chr14_r_c56989][PH=17][PH0=10.60][P=1.49e-01] Ephb1 chr9 102163510 102163516 - intron 36 16 0.00940893 292238[gene=chr9_r_c62047][PH=52][PH0= 13.48][P=1,48e-13] Gria2 chr3 80691959 80691960 - intron 12 28 0.00948303 116867[gene=chr3_r_c25446][PH=40][PH0=10.25][P=2.96e-10]

[0599]

[0600] Negri chr3 156892225 156892228 + intron 11 30 0.00948303 310769[gene=chr3_f_c67188][PH=41 ][PH0=12.88][P=3.88e-08] Rgs9 chr11 109230439 109230441 - downstream 12 29 0.00948303 393390[gene=chr11_r_c76035][PH=41 ][PH0=21.08][P=1.87e-02]

[0601] 10K|intron

[0602] Purg chr8 33404377 33404378 + intron 32 14 0.00956867 61592[gene=chr8_f_c14691][PH=46][PH0=14.36][P=1.07e-08] Pgbd5 chr8 124369837 124369844 - 3‘UTR 20 8 0.00978145 355417[gene=chr8_r_c73946][PH=28][PH0=7.95][P=2.76e-06]

[0603]

[0604]

[0248] TABLE 4

[0605] Differential alternative splicing (AS) events in Novalhu / hu mice

[0606] List of events for which differences were detected in AS analysis in the midbrain of Novalhu / hu and Novalwt / wt mice ( -value<0.05, |rf7]>0.05). For each event, the gene name, chromosome number, genomic start and end position of the event, strand, splicing type, PSI values and its differences (APSI, di), p- value and ID information are listed. PSI: percent spliced-in value, the percent of transcripts that include a specific AS exon. APSI: percent change in Novalhu / hu vs. Novalwt / wt. AS splicing events are classified into the following types: Cassette exon (cass), alternative 5’ splice site (alt5), alternative 3’ splice site (alt3), tandem cassette (taca), mutually exclusive exons (mutx), intron retention (iret). Novalwt / wt N=4, Novalhu / hu N=4.Gene name chr start end strand AS type PSI NOVA1(wt / wt) PSI NOVA1(hu / hu) delta. PSI Fnbpl I chr3 122549155 122558105 - taca 0 152 0.361 -0.209 Ccp110 chr7 118732393 118737023 + cass 0.637 0.754 -0.117 Itprl chr6 108431460 108438431 + cass 0864 0986 -0123 Tle6 chr1O 81595344 81595554 alt3 0.895 0.360 0.535 Tle6 chr1O 81595322 81595554 - alt3 0.895 0.360 0.535 Ccdc163 chr4 116712722 116714148 + alt3 0.102 0.625 -0.523 Rev3l chr10 39732159 39783331 + cass 0.194 0.627 -0.434 Mrps18b chr17 35911418 35914401 - cass 0.924 0.995 -0.071 Rhbdd3 chr11 5105180 5105716 alt5 0.813 0.929 -0.117 Dvl1 chr4 155856613 155859302 + cass 0381 0289 0091 Impa2 chr18 67302175 67306774 alt3 0.020 0.164 -0.144 Tcf3 chr1O 80415364 80415925 - alt3 0.399 0.572 -0.173 AbhdIO chr16 45737523 45742890 - cass 0.972 0.866 0.106 Htr3a chr9 48899212 48900781 alt3 0.004 0.278 -0.274 Zfp655 chr5 145233440 145235862 + cass 0.715 1.000 -0.285 Col4a5 chrX 141656684 141666974 + taca 0.076 0.375 -0.299 Stab1 chr14 31142957 31143266 - iret 0295 0075 0220 Rpain chr11 70973014 70977801 + taca 0.707 0.882 -0.175 Ilkap chr1 91373860 91376398 - alt3 0.143 0.857 -0.714 Cenpa chr5 30666896 30672569 + taca 0.073 0.700 -0.627 Atpaf2 chr11 60405775 60407390 - alt3 0.110 0.167 -0.057 BC029214 chr2 25459774 25460073 - alt3 0.139 0.208 -0.069 Fam73b chr2 30381938 30383018 + iret 0.134 0.081 0.053 Snapc4 chr2 26363637 26366036 alt3 0219 0331 -0112 6720401G13Rik chrX 50608477 50608813 - alt3 0571 0.827 -0.255 Ctc1 chr11 69021029 69022619 + alt3 0.810 0.586 0.224

[0607]

[0608] Ctc1 chr11 69021029 69022613 + alt3 0.810 0.586 0.2242700062C07Rik chr18 24471061 24473148 alt5 0.021 0.098 -0.078 Pex5l chr3 33014964 33143276 taca 0.495 0.363 0.131 Setd2 chr9 110573880 110590435 cass 0086 0034 0052 Gnbll chr16 18540971 18544272 alt3 0.609 0.966 -0.357 Dlg1 chr16 31842769 31853883 mutx 0.250 0.174 0.076 Amd1 chr10 40290117 40290614 iret 0.130 0.364 -0.233 Lrrc16b chr14 55501458 55502432 iret 0.114 0.055 0.058 Fam21 chr6 116208206 116209117 alt5 0.976 0.925 0.051 Rtel1 chr2 181335892 181339093 cass 0.747 0.445 0.301 Abtb1 chr6 88839626 88840899 cass 0.642 0.776 -0.134 Rps6kl1 chr12 85147745 85149907 alt5 0.828 0.938 -0.109 Atrx chrX 105879908 105884419 alt3 0.933 0.718 0.215 Pfkfb3 chr2 11471421 11478057 cass 0.289 0.468 -0.179 Zfp445 chr9 122856698 122857160 alt5 0.298 0.370 -0.071 Camta1 chr4 151061378 151074061 cass 0.637 0.565 0.072 Rnf111 chr9 70475775 70503726 cass 0.422 0.791 -0.369 Zfp239 chr6 117863083 117869272 taca 0075 0200 -0125 Gphn chr12 78492037 78504750 cass 0.187 0.263 -0.076 Itprl chr6 108417888 108438431 taca 0.729 0.601 0.128 Trp63 chr16 25866074 25868248 alt5 0.667 0.192 0.474 Samp chr10 128821922 128833410 cass 0.586 0.776 -0.190 Hsf4 chr8 105269800 105270895 cass 0.868 0.989 -0.121 Cadml chr9 47818733 47848297 cass 0.909 0.825 0.084 Snapc3 chr4 83418705 83435294 cass 0965 0832 0133 Hsd3b7 chr7 127801833 127802112 iret 0345 0.540 -0.195 Mprip chr11 59771616 59772216 iret 0.092 0.146 -0.054 Crispld2 chr8 120018852 120023701 alt3 0.143 0.527 -0.384

[0609]

[0610] A830018L16Rik chr1 11414183 11518708 cass 1.000 0.899 0.101Dyrkla chr16 94659496 94663860 + alt3 0.404 0.541 -0.136 Hmgn3 chr9 83109941 83111105 - iret 0.583 0.688 -0.105 Vldlr chr19 27239621 27240636 + cass 0.994 0.927 0.067 Naa40 chr19 7230067 7230282 iret 0.431 0.584 -0.153 Naa40 chr19 7230023 7230282 - iret 0.431 0.584 -0.153 Naa40 chr19 7230073 7230282 - iret 0.431 0.584 -0.153 Mast2 chr4 116330390 116337496 - cass 0.774 0.870 -0.096 Gak chr5 108569418 108569955 - iret 0.866 0.917 -0.051 Gak chr5 108569422 108569955 - iret 0.866 0.917 -0.051 Ccdc50 chr16 27406650 27409413 + cass 0048 0122 -0074 Cwc22 chr2 77936592 77946181 cass 0.843 0.506 0.337 Ppp3cc chr14 70256359 70267505 cass 1.000 0.941 0.059 Ppan chr9 20889337 20889678 + iret 0.049 0.168 -0.119 Appl2 chr10 83610977 83611198 - iret 0.300 0.386 -0.086 Suv39h1 chrX 8063695 8064056 - iret 0.016 0.074 -0.057 Galnt7 chr8 57540019 57545407 - mutx 0.678 0.466 0.212 Wdr70 chr5 8093675 8099020 - alt3 0.794 0.994 -0.200 Cyp4f13 chr17 32932628 32941199 - cass 0.132 0.042 0.090 Cabin 1 chr10 75725258 75725754 - alt3 0.916 0.983 -0.067 Ptprd chr4 76091421 76099604 - cass 0.316 0.478 -0.162 Osbpl9 chr4 109087394 109098552 - cass 0.659 0.913 -0.254 Ccdc50 chr16 27435561 27436601 cass 0.925 0.990 -0.065 Phldbl chr9 44696036 44697841 - alt5 0.289 0.351 -0.063 Foxm1 chr15 128371967 128372602 alt3 0.417 0.000 0.417 Dnajc6 chr4 101508103 101597950 + cass 0449 0.376 0.073 Cacnb2 chr2 14963905 14971641 + mutx 0.397 0.485 -0.088 Banp chr8 122007752 122024145 + cass 0.653 0.494 0.160

[0611]

[0612] Thada chr17 84464356 84466207 - alt3 0.458 0.880 -0.422Btbd6 chr12 112976675 112977370 + cass 0.986 0.912 0.073 Rasgrp2 chr19 6400546 6401862 + alt3 0.977 0.577 0.400 Lig3 chr11 82785314 82787827 + alt3 0672 0842 -0171 Bdp1 chr13 100098952 100103877 alt3 0.847 1.000 -0.153 Prrc2c chr1 162674098 162676816 - cass 0.910 0.834 0.075 Bap1 chr14 31256660 31257766 + iret 0.923 0.974 -0.051 Mbnl1 chr3 60501264 60595763 + cass 0.716 0.905 -0.189 Ubr2 chr17 46975926 46981441 - mutx 0.378 0.299 0.079 Gas7 chr11 67629609 67652911 cass 0.907 0.848 0.060 Zfp7 chr15 76881111 76888346 + alt3 0705 0937 -0233 Nme6 chr9 109833195 109835413 alt3 0.724 0.976 -0.252 Wdr52 chr16 44413647 44416052 + cass 0.053 0.440 -0.388 Zfp12 chr5 143239953 143240414 + alt5 0.178 0.273 -0.095 Mtnr2 chr9 13782276 13785926 + cass 0.134 0.083 0.051 Cars chr7 143559648 143563040 - cass 0.920 0.974 -0.054 Cgn chr3 94777993 94778269 - iret 0.385 0.000 0.385 Fbxw9 chr8 85064383 85064671 + iret 0444 0286 0158 Chrd chr16 20735745 20736142 + iret 0.023 0.169 -0.146 Snrk chr9 122117319 122137600 + taca 0.625 0.738 -0.113 Rimsl chr1 22346292 22373509 - alt3 0.798 0.890 -0.092 Hr chr14 70552244 70555106 + cass 0.242 0.577 -0.334 Slc25a19 chr11 115624198 115628111 - cass 0.068 0.128 -0.060 Fam161b chr12 84345316 84346934 - iret 0.425 0.264 0.161 Vldlr chr19 27240465 27241452 alt3 0.968 0.894 0.075 Slmap chr14 26428241 26438737 - cass 0852 0.774 0.078 Cln3 chr7 126582999 126583446 - alt3 0.020 0.157 -0.136 Baz2b chr2 59933670 59936750 - cass 0.716 0.893 -0.178

[0613]

[0614] Spopl chr2 23537460 23543323 - cass 0.244 0.145 0.099Psma3 chr12 70983318 70984593 + alt3 0.971 0.915 0.056 Atg9a chr1 75190822 75191954 - alt3 0.398 0.491 -0.093 Dlg1 chr16 31846833 31853883 + cass 0536 0645 -0109 Zfp273 chr13 67813815 67822366 + alt3 1.000 0.118 0.882 Inpp5e chr2 26399232 26399573 - iret 0.200 0.293 -0.093 Saft>2 chr17 56562938 56563430 - iret 0.163 0.229 -0.066 Clybl chr14 122181716 122371428 + taca 0.642 0.861 -0.219 Myl6 chr10 128492601 128493352 - cass 0.151 0.242 -0.091 Nemf chr12 69353793 69356131 - cass 0.871 0.969 -0.098 Drosha chr15 12881622 12883264 + alt5 0859 0915 -0055 Brf1 chr12 112964183 112965991 alt3 0.908 0.811 0.097 Mtg2 chr2 180084450 180085901 + alt5 0.924 0.998 -0.074 Nfib chr4 82310302 82327912 - taca 0.751 0.965 -0.215 Zfp87 chr13 67515780 67520720 - cass 0.070 0.001 0.069 Gpatch2l chr12 86268975 86291369 + cass 0.740 0.854 -0.114 Med16 chr10 79907490 79908918 - alt3 0.839 0.974 -0.135 Naa60 chr16 3884695 3897379 + taca 0817 0904 -0086 Ppfibpl chr6 147012454 147016392 + cass 0.049 0.140 -0.091 Ube4a chr9 44959978 44965548 - cass 0.552 0.742 -0.190 N28178 chr4 42916603 42933666 + cass 0.812 0.698 0.113 Gm 16596 chr12 108536401 108539472 - cass 0.000 0.067 -0.067 Robo2 chr16 73897015 73904555 - cass 0.243 0.346 -0.103 Lrrc1 chr3 14550250 14551526 + alt3 0.046 0.200 -0.154 B3galnt2 chr13 13966309 13987462 mutx 0482 0364 0118 C2cd3 chr7 100374263 100380124 + alt3 0786 0.979 -0.193 Trmt11 chr10 30590842 30591101 - iret 0.000 0.110 -0.110 Ip6k1 chr9 108044724 108045538 + cass 0.253 0.193 0.059

[0615]

[0616] Slc35a4 chr18 36681527 36682053 + iret 0.453 0.533 -0.080Slc35a4 chr18 36681527 36683856 iret 0.453 0.533 -0.080 Slc35a4 chr18 36681527 36683863 iret 0.453 0.533 -0.080 Slc35a4 chr18 36681527 36682030 iret 0453 0533 -0080 Ptprq chr14 12166744 12190772 cass 0.559 0.388 0.171 Cadps chr14 12467022 12473514 cass 0.775 0.825 -0.050 Pak3 chrX 143712277 143715288 cass 0.861 0.971 -0.110 Pari chr16 20287856 20293401 alt5 0.017 0.073 -0.056 Tmem70 chr1 16665225 16667772 alt3 0.106 0.041 0.065 Tmem70 chr1 16665225 16667769 alt3 0.106 0.041 0.065 Cdk13 chr13 17719355 17721292 alt3 0367 0442 -0075 Myo7a chr7 98098186 98102701 cass 0.419 0.204 0.215 Kdm6a chrX 18246974 18248358 cass 0.443 0.699 -0.257 Tcergl chr18 42573259 42575554 cass 0.073 0.142 -0.069 Clipl chr5 123627293 123631168 alt5 0.555 0.794 -0.240 Mgl2 chr11 70134118 70135161 alt3 0.600 0.000 0.600 Suds3 chr5 117091679 117093030 iret 0.602 0.530 0.072 Suds3 chr5 117092682 117093030 iret 0602 0530 0072 Suds3 chr5 117092434 117093030 iret 0.602 0.530 0.072 Bicd2 chr13 49383042 49387024 iret 0.397 0.479 -0.083 Slc15a2 chr16 36771876 36774640 cass 0.938 0.816 0.122 Dock11 chrX 36009338 36011809 cass 0.712 0.333 0.378 Eftud1 chr7 82672809 82674581 alt3 0.969 0.825 0.145 Rtel1 chr2 181355923 181356615 iret 0.975 0.862 0.113 Rtel1 chr2 181355923 181356427 iret 0975 0862 0113 Cenpt chr8 105849625 105849922 iret 0.109 0.231 -0.122 Rtel1 chr2 181356157 181356615 alt3 0.333 0.014 0.319 Rtel1 chr2 181356157 181356614 alt3 0.333 0.014 0.319

[0617]

[0618] Hnrnpk chr13 58395573 58396898 cass 0.227 0.304 -0.077Stk30 chr12 110807798 110808404 alt3 0.998 0.885 0.114 Dcaf17 chr2 71078059 71082052 cass 0.689 0.881 -0.192 Snph chr2 151597108 151601073 cass 0.801 0860 -0060 Golga1 chr2 39047072 39047765 iret 0.164 0.280 -0.117 Golga1 chr2 39047022 39047765 iret 0.164 0.280 -0.117 Em1 chr11 106434914 106459027 cass 1.000 0.715 0.285 Trdmt1 chr2 13523425 13525693 cass 0.176 0.060 0.116 Wdr47 chr3 108618489 108623448 alt3 0.815 0.733 0.082 Ptpmt1 chr2 90916853 90917508 alt3 0.286 0.000 0.286 D3Ertd751e chr3 41756048 41758929 iret 0.925 1.000 -0.075 Nudt1 chr5 140331908 140334618 cass 0.735 0.941 -0.206 Trmt2a chr16 18250819 18251659 taca 0.815 0.912 -0.097 Mapk8ip3 chr17 24892151 24894222 taca 0.906 0.973 -0.067 Vtila chr19 55380941 55391867 cass 0.663 0.765 -0.102 Plxnb2 chr15 89170568 89180787 cass 0.928 1.000 -0.072 Smek1 chr12 101051530 101053583 alt5 0.134 0.200 -0.066 Arap3 chr18 37974364 37974680 iret 0000 0089 -0089 Kif22 chr7 127027728 127027989 iret 0.067 0.197 -0.130 Zcchc6 chr13 59771879 59782342 taca 0.048 0.106 -0.058 Apbb3 chr18 36676864 36677255 alt3 0.370 0.267 0.103 Myh11 chr16 14246726 14250623 cass 0.938 0.500 0.438 Med12l chr3 59037559 59042412 cass 0.776 0.560 0.216 Sec23ip chr7 128778428 128779224 alt3 0.940 0.871 0.069 Fance chr17 28317467 28318155 alt3 0368 0227 0141 Slc19a2 chr1 164256746 164261016 alt5 0887 0.975 -0.088 Trmt13 chr3 116590229 116592258 cass 0.000 0.083 -0.083 Fbxw11 chr11 32642795 32711996 cass 0.215 0.368 -0.152

[0619]

[0620] Pcgf5 chr19 36437154 36437361 alt3 0.931 0.999 -0.068Smox chr2 131520094 131522218 + taca 0.751 0.805 -0.054 Man2b1 chr8 85084400 85084813 + alt3 0.105 0.173 -0.068 Man2b1 chr8 85084400 85084735 + alt3 0.105 0173 -0068 Man2b1 chr8 85084400 85084724 + alt3 0.105 0.173 -0.068 Wrn chr8 33353294 33385526 - cass 0.000 0.158 -0.158 Chrd chr16 20738552 20739163 + cass 0.686 0.921 -0.235 Crif1 chr8 70503304 70503638 + alt3 0.467 0.077 0.390 Comtd1 chr14 21847872 21848119 - iret 0.092 0.148 -0.057 Med 15 chr16 17655677 17673397 - cass 0.887 0.779 0.108 Acini chr14 54653213 54653680 - alt3 0320 0731 -0411 Osbpl6 chr2 76555013 76560259 cass 0.932 0.993 -0.061 Pde9a chr17 31459900 31461726 + cass 0.912 0.990 -0.078 Zcchc12 chrX 36195985 36196577 + alt3 0.864 0.740 0.124 Fam120b chr17 15401744 15405934 + iret 0.962 0.721 0.241 Ccdc91 chr6 147475870 147507826 + cass 0.035 0.095 -0.060 Armcxl chrX 134718919 134719724 + alt5 0.769 0.691 0.079 Golga4 chr9 118572917 118577227 + cass 0033 0084 -0051 Pickl chr15 79247264 79248295 + iret 0.249 0.185 0.064 Alg8 chr7 97386801 97388587 + alt3 0.536 0.788 -0.252 2010107G23Rik chr10 62109883 62111012 - cass 0.762 0.864 -0.102 Phykpl chr11 51593603 51594140 + iret 0.687 0.450 0.236 Fra10ac1 chr19 38207262 38214456 - cass 0.924 0.987 -0.063 Gfra2 chr14 70890129 70966315 + cass 0.905 0.968 -0.063 Mms19 chr19 41955418 41955960 alt3 0.025 0.085 -0.060 Nr1h2 chr7 44552034 44552619 - alt3 0.743 0.656 0.087 Svil chr18 5059229 5062404 + cass 0.440 0.187 0.253 Klhl32 chr4 24649539 24682269 - alt3 0.988 0.895 0.093

[0621]

[0622] Wheel chr5 33864659 33867657 + alt3 0.886 0.810 0.076Tra2b chr16 22247189 22248494 - alt3 0.707 0.827 -0.120 Arfgapl chr2 180977319 180979226 + cass 0.719 0.789 -0.069 Tnfsf13b chr8 10014168 10031421 + cass 0600 0953 -0353 Golga3 chr5 110184337 110185897 + alt5 0.326 0.522 -0.196 Sft2d1 chr17 8321799 8323334 + alt5 0.000 0.143 -0.143 Rmad1 chr3 87924343 87925430 - iret 0.538 0.662 -0.124 Rnf215 chr11 4139738 4140077 + iret 0.219 0.149 0.070 Ankrd54 chr15 79057954 79061210 - alt3 0.964 0.853 0.111 Immt chr6 71851771 71857047 taca 0.871 0.821 0.050 Zc3h13 chr14 75331705 75336073 + alt3 0640 0539 0.102 Kif21a chr15 90943764 90952817 taca 0.485 0.546 -0.061 Sema4c chr1 36555931 36557533 alt3 1.000 0.000 1.000 Pmp22 chr11 63128981 63133243 + alt3 0.671 0.724 -0.052 Ap3s1 chr18 46780660 46790622 + cass 0.588 0.678 -0.090 Cpsf3l chr4 155872849 155885195 + cass 0.899 0.975 -0.076 Sh2b3 chr5 121818464 121818853 - iret 0.149 0.251 -0.101 Wiz chr17 32378839 32381155 - alt5 0356 0053 0303 Luc7l chr17 26252909 26255124 + cass 0.110 0.177 -0.067 Slc4a9 chr18 36531099 36531525 + iret 0.000 1.000 -1.000 Nnat chr2 157560447 157562106 + cass 0.769 0.569 0.200 Navi chr1 135464705 135467788 - cass 0.907 0.828 0.079 Arfip2 chr7 105637120 105638326 - cass 0.836 0.921 -0.085 Zfyve16 chr13 92508121 92509045 - alt3 0.786 0.935 -0.149 Ankrd10 chr8 11619061 11628529 cass 0.302 0.445 -0.143 Inip chr4 59775446 59783851 - cass 0.807 0.931 -0.123 Mtg2 chr2 180071793 180078822 + mutx 0.667 0.718 -0.051 Gak chr5 108613512 108623373 - taca 0.674 0.785 -0.111

[0623]

[0624] Pacrgl chr5 48374179 48374537 + alt3 0.001 0.114 -0.113Ift88 chr14 57434794 57437282 + alt3 0.075 0.149 -0.074 Ino80d chr1 63093284 63093729 - alt3 0.800 0.970 -0.170 Adcy3 chr12 4206912 4208660 + alt3 0774 0678 0096 Cbx5 chr15 103215021 103215351 alt3 0.333 0.241 0.093 Ogg1 chr6 113328364 113329407 + alt3 0.457 0.341 0.116 Erbb2ip chr13 103824719 103830330 - cass 0.750 0.831 -0.081 Acsm5 chr7 119534233 119534914 + alt5 0.100 0.667 -0.567 Mark2 chr19 7275395 7280011 - cass 0.937 0.877 0.060 Srpx2 chrX 133908425 133910730 cass 0.599 0.286 0.313 Edc4 chr8 105887781 105888044 + iret 0.097 0.047 0.050 Dcaf11 chr14 55561209 55563018 alt5 0.737 0.658 0.079 Dcaf11 chr14 55561074 55563018 + alt5 0.737 0.658 0.079 Slc44a2 chr9 21342742 21343046 + iret 0.091 0.167 -0.076 Srrm4 chr5 116453408 116467587 - cass 0.156 0.098 0.058 Ripk2 chr4 16129022 16132861 - cass 0.462 0.172 0.289 2700094K13Rik chr2 84669217 84670115 - alt5 0.772 0.846 -0.074 Ppip5k2 chr1 97740823 97741194 - alt3 0.813 0.921 -0.108 Doxr chr11 120726087 120726310 - iret 0.203 0.099 0.104 Htr4 chr18 62437382 62464641 + cass 0.000 0.400 -0.400 Enoxl chr14 77615438 77637799 + cass 0.113 0.251 -0.138 Tinf2 chr14 55680014 55680706 - iret 0.131 0.226 -0.096 Col11a1 chr3 114212072 114213285 cass 0.080 0.000 0.080 Cacna2d4 chr6 119345043 119347250 + cass 0.129 0.440 -0.311 Iqce chr5 140669021 140670566 alt3 0.477 0.848 -0.370 Gale chr4 135965565 135966115 + alt3 0.925 0.999 -0.075 Efcc1 chr6 87749131 87751904 + cass 0.699 0.490 0.208 Cenpt chr8 105846983 105848898 - alt3 0.941 1.000 -0.059

[0625]

[0626] Ap3s1 chr18 46754370 46758113 + cass 0.069 0.144 -0.075Cog1 chr11 113659268 113661051 alt3 0.956 0.901 0.055 Supv3l1 chr10 62430470 62432464 alt5 0.925 0.987 -0.063 Map3k5 chr10 20000565 20023800 cass 0803 0962 -0159 Miip chr4 147862896 147865269 cass 0.423 0.289 0.133 Mtr chr13 12247892 12250690 cass 0.628 0.925 -0.297 Fam69c chr18 84720155 84730688 cass 0.313 0.000 0.313 Acsl6 chr11 54314951 54315761 cass 0.426 0.286 0.140 Reps1 chr10 18104153 18114485 cass 0.815 0.733 0.083 Chd2 chr7 73454328 73455640 cass 0.072 0.123 -0.050 Ift57 chr16 49699347 49703267 cass 0979 0926 0053 Mterfd1 chr13 66928169 66930222 alt3 0.978 0.902 0.076 Pum1 chr4 130752575 130763020 cass 0.760 0.830 -0.070 Grina chr15 76246789 76247736 alt3 0.380 0.220 0.160 Ccdc60 chr5 116225268 116288984 alt3 1.000 0.060 0.940 Naa25 chr5 121426726 121430755 cass 0.068 0.004 0.065 Ctc1 chr11 69031099 69031603 alt3 0.668 0.523 0.144 Gnmt chr17 46726060 46726410 iret 0.440 0.110 0.330 Ablim2 chr5 35836947 35841400 cass 0.468 0.535 -0.067 C2cd5 chr6 143017882 143020373 cass 0.925 0.774 0.150 Ccdc137 chr11 120458611 120460110 alt3 0.999 0.941 0.058 Tctex1d2 chr16 32425240 32426920 iret 0.109 0.190 -0.081 Trim30a chr7 104412214 104429350 cass 0.238 0.810 -0.571 Arfgapl chr2 180967244 180971113 cass 0.659 0.758 -0.099 Ptpn4 chr1 119765877 119783588 taca 0.868 0.953 -0.086 Gigyf1 chr5 137525171 137525558 alt3 1.000 0.947 0.053 Aifm1 chrX 48499771 48513397 mutx 0.634 0.739 -0.105 Sybu chr15 44746245 44748397 alt3 0.885 0.966 -0.081

[0627]

[0628] Amt chr3 95491022 95493859 alt3 0.793 0.900 -0.107Pbrml chr14 31019148 31025649 mutx 0.274 0.116 0.158 Casp2 chr6 42276694 42279925 cass 0.292 0.484 -0.192 Shroom2 chrX 152623125 152657523 alt3 0859 0697 0.163 Ggtal chr2 35422179 35432631 cass 1.000 0.722 0.278 Zfp655 chr5 145235305 145238321 cass 1.000 0.808 0.192 Skil chr3 31113407 31117038 alt5 0.597 0.395 0.202 Shq1 chr6 100632238 100637121 alt5 0.773 1.000 -0.227 Papd5 chr8 88247508 88250852 cass 0.893 0.963 -0.069 Aars chr8 111033878 111037072 cass 0.609 0.673 -0.064 Dus3l chr17 56768347 56768627 iret 0408 0478 -0070 Gpsm3 chr17 34589805 34590940 cass 1.000 0.873 0.127 Ccser2 chr14 36874935 36896343 cass 0.878 0.767 0.111 Dock7 chr4 98991363 99001201 cass 0.810 1.000 -0.190 Asnsdl chr1 53346579 53348557 alt5 0.831 0.907 -0.076 Stx3 chr19 11789556 11791844 cass 0.521 0.695 -0.174 Lrrc16b chr14 55507684 55508263 alt3 0.759 0.689 0.069 Gemin5 chr11 58122281 58125417 alt3 0.446 0.613 -0.167 Wbp1 chr6 83120259 83120875 iret 0.129 0.185 -0.056 BC003331 chr1 150388522 150390355 alt3 0.813 0.733 0.080 Ppip5k2 chr1 97755885 97759363 cass 1.000 0.941 0.059 Fam188a chr2 12386608 12405919 taca 0.929 0.992 -0.063 Mettl25 chr9 105832850 105841377 cass 0.404 0.754 -0.351 Ip6k2 chr9 108796060 108797689 cass 0.109 0.029 0.079 Ptcd3 chr6 71905097 71907845 cass 0.897 0.962 -0.065 Gspt1 chr16 11238871 11240706 alt3 0.492 0.582 -0.090 Gsptl chr16 11239031 11240706 alt3 0.492 0.582 -0.090 Rbbp5 chr1 132497929 132505664 alt5 0.212 0.316 -0.104

[0629]

[0630] Tec chr5 72773830 72782174 cass 0.000 1.000 -1.000L3mbtl1 chr2 162959515 162961081 cass 0.760 0.914 -0.154 Zscan29 chr2 121165641 121170241 cass 0.167 0.320 -0.153 Tpd52l2 chr2 181508165 181511614 cass 0804 0861 -0057 Slc25a25 chr2 32420304 32421389 cass 0.844 0.792 0.051 Plekha5 chr6 140536647 140544186 cass 0.716 0.854 -0.139 Agfg1 chr1 82883180 82886186 alt3 0.748 0.817 -0.069 Pdcd11 chr19 47119792 47120423 iret 0.114 0.203 -0.089 Ccdc171 chr4 83604021 83635741 cass 0.280 0.846 -0.566 Bratl chr5 140710137 140711652 iret 0.876 0.467 0.409 Ccnd3 chr17 47505050 47578791 cass 0.154 0.000 0.154 Thnsl2 chr6 71141219 71144346 cass 0.200 0.000 0.200 Rbm39 chr2 156178558 156179239 iret 0.559 0.669 -0.110 Robo2 chr16 73928023 73933867 cass 0.387 0.281 0.107 Pcsk6 chr7 66025227 66031850 cass 0.678 0.543 0.136 2210018M11Rik chr7 98600671 98610864 cass 0.744 0.844 -0.100 Fn1 chr1 71597310 71598462 alt3 0.960 0.910 0.050 Phf21b chr15 84791353 84791940 iret 0.265 0.470 -0.205 Exocl chr5 76537697 76542237 cass 0.911 0.978 -0.067 Ss18 chr18 14636495 14640360 cass 0.615 0.517 0.098 Fam188a chr2 12403997 12405919 alt3 0.982 0.913 0.069 Usp1 chr4 98923970 98928353 cass 0.685 0.878 -0.192 Erall chr11 78074279 78074626 iret 0.292 0.214 0.078 Pxk chr14 8152080 8155344 alt3 0.562 0.491 0.070 Fbrsl1 chr5 110378037 110379113 cass 0.329 0.202 0.127 Atxn2 chr5 121811384 121814719 cass 0.529 0.479 0.050 Gtpbp2 chr17 46164193 46164832 iret 0.124 0.176 -0.052 Taz chrX 74288177 74288512 iret 0.937 0.838 0.099

[0631]

[0632] Slc25a10 chr11 120497013 120497460 iret 0.197 0.126 0.070Ube2e1 chr14 18330949 18331790 alt3 1.000 0.837 0.163 Txlng chrX 162786616 162787677 iret 0.495 0.304 0.191 Nek1 chr8 61049783 61054621 cass 0.202 0.316 -0.115 Asapl chr15 64152829 64159003 alt5 0.385 0.335 0.050 Mtbp chr15 55571294 55572967 alt3 0.000 0.357 -0.357 Tarbp2 chr15 102522848 102523673 iret 0.486 0.626 -0.141 Ezh2 chr6 47540676 47542405 cass 0.183 0.323 -0.139 Mrohl chr15 76432148 76433616 cass 0.917 0.968 -0.051 Hmgxb4 chr8 74993708 74999707 cass 1.000 0.698 0.302 Hnrnph3 chr10 63017523 63018224 alt5 0.677 0.752 -0.075 Qpctl chr7 19144659 19147150 cass 0.974 0.908 0.066 Dok1 chr6 83032766 83033429 alt3 0.667 1.000 -0.333 Dok1 chr6 83032804 83033429 alt3 0.667 1.000 -0.333 Sema4d chr13 51725223 51748788 cass 0.448 0.353 0.095 Wdr4 chr17 31503509 31509911 alt5 0.933 0.839 0.094 B130024G19Rik chr7 70365382 70388458 cass 1.000 0.231 0.769 Dcaf11 chr14 55560491 55561441 alt3 0.583 0.900 -0.317 Zfp12 chr5 143239953 143244318 cass 0.674 0.811 -0.136 Nmi chr2 51960570 51973003 cass 0.700 0.250 0.450 Atf6 chr1 170787345 170788674 alt3 0.929 0.824 0.104 Jmjd4 chr11 59453510 59454058 alt3 0.440 0.321 0.119 Myef2 chr2 125095911 125098074 cass 0.805 0.751 0.054 Trim26 chr17 36837177 36851127 cass 0.846 0.943 -0.097 Tcf3 chr10 80418765 80419598 alt3 0.120 0.186 -0.066 Wdr17 chr8 54690003 54696396 cass 0.858 1.000 -0.142 Brox chr1 183292474 183294449 alt3 0.175 0.014 0.161 Ddx26b chrX 56493038 56496733 cass 0.332 0.594 -0.262

[0633]

[0634] Medag chr5 149422133 149427505 cass 0.913 0.782 0.131Taf6l chr19 8773504 8774452 - alt3 0.925 0.776 0.148 Nrxn2 chr19 6443630 6450598 + cass 0.703 0.793 -0.090 Stk35 chr2 129801403 129827986 + cass 0765 0649 0.116 Eps15f1 chr8 72340998 72358444 cass 0.864 0.924 -0.060 Ml!t1 chr17 56899768 56905854 - cass 0.387 0.314 0.072 Ildr2 chr1 166270498 166294736 + cass 0.838 0.914 -0.075 Smpd4 chr16 17625719 17626537 + alt3 0.020 0.500 -0.480 Nr1h2 chr7 44551954 44552619 - iret 0.671 0.580 0.091 Casp9 chr4 141793842 141796833 alt3 0.982 0.857 0.125 Casp9 chr4 141793842 141796690 + alt3 0982 0857 0 125 Rapgefl chr2 29679132 29686282 cass 0.422 0.477 -0.055 Pin chr1O 53337704 53344271 4- cass 0.000 0.067 -0.067 Fdxr chr11 115271876 115272281 - alt3 0.587 0.728 -0.141 Fdxr chr11 115271920 115272281 - alt3 0.587 0.728 -0.141 Cops4 chr5 100518442 100528689 + taca 0.772 0.844 -0.072 Mutyh chr4 116807733 116814435 + cass 0.000 0.217 -0.217 Uty chrY 1168089 1170183 - cass 0366 0730 -0364 Zfp384 chr6 125030797 125033317 + cass 0.668 0.565 0.103 Slc12a7 chr13 73809905 73813671 + cass 0.385 0.765 -0.381 Hax1 chr3 89997737 89998665 - alt3 0.727 0.937 -0.210 Acd chr8 105700006 105700357 - alt3 0.142 0.221 -0.079 Tjapl chr17 46260094 46261201 - cass 0.849 0.933 -0.084 Ank3 chr1O 69980271 69982196 + cass 0.423 0.476 -0.053 Fam 179b chr12 64965939 64976850 cass 0022 0088 -0066 MfsdIO chr5 34636592 34637145 - alt3 0299 0.045 0.254 Abcblb chr5 8806007 8812845 + cass 1.000 0.773 0.227 Tnntl chr7 4512264 4513612 - alt3 0.276 0.164 0.112

[0635]

[0636] Cecr2 chr6 120756548 120757690 + alt5 0.450 0.875 -0.425Zfp346 chr13 55113677 55122514 + cass 0.912 0.982 -0.069 Dars2 chr1 161046776 161051437 - cass 0.777 0.878 -0.102 Pax6 chr2 105692736 105697363 + cass 0934 1 000 -0066 1110007C09Rik chr13 49203717 49205344 cass 0.864 0.921 -0.057 Lmbrll chr15 98908519 98908920 - iret 0.035 0.109 -0.074 Cux1 chr5 136312642 136314402 - alt3 0.962 0.787 0.174 Cenpt chr8 105849186 105849520 - iret 0.079 0.163 -0.083 Mlh1 chr9 111249219 111255715 - cass 1.000 0.906 0.094 Usp24 chr4 106370983 106372763 iret 0.103 0.035 0.068 Oprll chr2 181715689 181718731 + cass 0.251 0170 0082 Wbpll chr19 46599136 46644454 cass 0.480 0.362 0.118 Orc5 chr5 22526362 22526582 iret 0.064 0.117 -0.053 L3mbtl1 chr2 162966545 162967084 + iret 0.198 0.319 -0.121 Slc38a10 chr11 120106433 120109535 - alt3 0.762 0.868 -0.106 Zfp58 chr13 67494602 67500452 - cass 0.069 0.182 -0.113 BC053749 chr7 30549606 30552271 - cass 0.333 0.722 -0.389 Apitdl chr4 149132258 149137581 - alt3 0083 0029 0054 Fam149b chr14 20375509 20378522 + cass 0.604 0.726 -0.123 Kcnh6 chr11 106023724 106025877 + alt3 0.071 0.167 -0.095 Cadml chr9 47813772 47848297 + taca 0.299 0.363 -0.064 Atg2b chr12 105648988 105649631 - alt3 0.885 0.950 -0.065 Soatl chr1 156457945 156474239 - cass 0.855 1.000 -0.145 Akap13 chr7 75743964 75746809 + alt3 0.829 0.657 0.172 Trmt2a chr16 18252401 18253497 cass 0928 0991 -0063 Grikl chr16 87950027 87957596 - cass 0456 0.317 0.138 Nckap5 chr1 125913619 125981696 - cass 1.000 0.692 0.308 MrpslO chr17 47372426 47375108 + cass 0.842 0.752 0.090

[0637]

[0638] Vps54 chr11 21263198 21264870 + alt3 0.343 0.500 -0.157Stxbp5l chr16 37139843 37174361 - taca 0.836 0.655 0.182 Timeless chr10 128249967 128250461 alt3 0.889 0.589 0.300 Dlq3 chrX 100767726 100771664 cass 0260 0312 -0052 Exod chr5 76554099 76559166 cass 0.702 0.550 0.153 Cxcl16 chr11 70458744 70459166 iret 0.026 0.220 -0.194 Capn3 chr2 120502386 120502592 iret 0.077 0.228 -0.150 9030617O03Rik chr12 100779094 100829724 cass 0.000 0.115 -0.115 RapgefS chr11 54691237 54699285 cass 0.212 0.358 -0.146 Trpm3 chr19 22732999 22766807 cass 0.550 0.473 0.078 Unc13b chr4 43115102 43165977 cass 0532 0250 0282 Pdlim5 chr3 142304704 142312213 cass 0.719 0.446 0.273 Mynn chr3 30603503 30607829 alt5 0.675 0.879 -0.204 Rrp36 chr17 46670110 46674286 taca 0.702 0.816 -0.114 Smarca2 chr19 26749851 26752040 cass 0.094 0.175 -0.081 Plekha5 chr6 140552714 140569414 taca 0.840 0.673 0.166 Porcn chrX 8201443 8203288 mutx 0.606 0.518 0.088 Auts2 chr5 131439297 131445481 mutx 0229 0301 -0072 Ngb chr12 87097530 87100114 alt5 0.155 0.098 0.057 Hnrnpk chr13 58396826 58399211 cass 0.876 0.802 0.074 Rpi12 chr2 32962984 32963852 cass 0.932 0.992 -0.060 Pex2 chr3 5560499 5563271 cass 0.510 0.613 -0.103 Fance chr17 28316858 28317610 cass 0.500 0.333 0.167 Gphn chr12 78412340 78454850 cass 0.297 0.210 0.086 Usp37 chr1 74441531 74450554 cass 0907 1 000 -0093 QricM chr9 108517717 108528917 cass 0955 0.853 0.102 Sh2b2 chr5 136224154 136224495 alt3 0.982 0.868 0.114 Proml chr5 44000776 44001900 cass 0.924 1.000 -0.076

[0639]

[0640] Cacnala chr8 84638611 84640248 alt3 0.978 0.896 0.082Flna chrX 74230505 74233337 - cass 0.568 0.652 -0.085 Hax1 chr3 89997386 89998002 - iret 0.736 0.554 0.182 Chd8 chr14 52212562 52213022 - alt3 0320 0264 0056 lfi203 chr1 173936517 173942293 cass 0.500 1.000 -0.500 Pixna3 chrX 74335769 74336190 + iret 0.024 0.112 -0.088 Faxc chr4 21948691 21982501 + cass 0.760 0.869 -0.109 Gria2 chr3 80690403 80692531 - cass 0.411 0.471 -0.060 Ssh3 chr19 4267746 4268581 - iret 0.174 0.270 -0.096 Kbtbd3 chr9 4309898 4313805 alt3 0.000 0.107 -0.107 Slx4ip chr2 137000183 137044076 + cass 0737 0417 0320 Slc22a5 chr11 53876008 53891677 cass 0.828 0.938 -0.110 Pou2f1 chr1 165931661 166002633 taca 0.565 0.354 0.211 Nuak2 chr1 132324952 132327847 + cass 0.097 0.429 -0.332 Lca5 chr9 83426592 83441098 - cass 0.210 0.538 -0.328 Ccdc33 chr9 58033219 58033714 - alt3 0.308 0.857 -0.549 Sema6c chr3 95171557 95173652 + cass 0.215 0.130 0.085 Lrrfip1 chr1 91107297 91112296 + taca 0869 0930 -0061 Ncaph2 chr15 89370394 89370642 + alt3 0.227 0.176 0.051 Zfp821 chr8 109717761 109721342 + alt5 0.485 0.597 -0.112 Fopnl chr16 14311046 14317332 - cass 0.914 1.000 -0.086 Serac 1 chr17 6067528 6070888 - cass 0.937 0.697 0.240 Ermard chr17 15059343 15061181 cass 0.957 0.861 0.096 Rgs12 chr5 35020313 35021258 + alt3 0.999 0.946 0.053 Kankl chr19 25422918 25425983 cass 0538 0605 -0067 Zkscan17 chr11 59502919 59503809 - alt5 1 000 0.938 0.062 0rmdl2 chr10 128820260 128821597 - alt3 0.557 0.741 -0.184 2310022A10Rik chr7 27571563 27574739 + alt5 0.006 0.062 -0.056

[0641]

[0642] Gnbll chr16 18499045 18548190 + cass 0.742 1.000 -0.2581700086006Rik chr18 38238404 38250197 taca 0.500 0.214 0.286 Eps8 chr6 137539321 137591491 alt3 0.868 0.968 -0.100 Tmem191c chr16 17277668 17277890 iret 0327 0390 -0064 Arhgap4 chrX 73906668 73911270 taca 0.108 0.250 -0.142 Gripl chr10 119819511 119930035 cass 0.895 0.630 0.265 Lyplal chr1 4886743 4889609 alt3 0.780 0.703 0.077 Lyplal chr1 4886743 4889601 alt3 0.780 0.703 0.077 Lyplal chr1 4886743 4889559 alt3 0.780 0.703 0.077 Lyplal chr1 4886743 4889508 alt3 0.780 0.703 0.077 Ankrd27 chr7 35620542 35622348 alt3 0907 0964 -0057 Eva 1c chr16 90830858 90876190 taca 0.591 1.000 -0.409 Setdbl chr3 95339906 95340311 alt3 0.841 0.911 -0.070 Sfswap chr5 129543213 129549683 alt5 0.949 0.881 0.068 1700066M21 Rik chr1 57377643 57380144 cass 0.067 0.579 -0.512 Mettl23 chr11 116843556 116845944 alt3 0.991 0.500 0.491 Kdm5c chrX 152237632 152240195 cass 0.976 0.912 0.064 Tcf7 chr11 52257683 52260620 cass 0210 0077 0133 Pgap2 chr7 102235661 102236345 alt5 0.193 0.740 -0.547 Itgb4 chr11 115997949 115999954 alt5 0.876 0.939 -0.062 Fhodl chr8 105337317 105337768 alt3 0.429 0.000 0.429 Acads chr5 115111856 115112390 iret 0.073 0.167 -0.094 Ocrl chrX 47948124 47960568 cass 0.765 0.685 0.081 Gtf2ird2 chr5 134191179 134192790 alt5 0.182 0.304 -0.123 Zbtb20 chr16 43569680 43577192 cass 0446 0261 0186 Kcntl chr2 25909204 25909676 iret 0234 0.294 -0.060 Usp47 chr7 112077791 112082560 cass 0.358 0.451 -0.092 Zkscan2 chr7 123484939 123490261 cass 0.292 0.122 0.170

[0643]

[0644] Dph6 chr2 114519711 114535585 cass 0.556 0.725 -0.170Nsun5 chr5 135374926 135375484 + alt5 0.383 0.535 -0.152 Rft1 chr14 30676849 30677816 + alt5 0.241 0.148 0.093 St18 chr1 6730050 6752367 + cass 0000 0750 -0750 Staql chr9 100643622 100705253 + cass 0.022 0.081 -0.059 Ddx41 chr13 55535729 55536044 - iret 0.098 0.159 -0.061 Ddx41 chr13 55535762 55536044 - iret 0.098 0.159 -0.061 Clasrp chr7 19603189 19604460 - alt3 0.905 0.986 -0.081 Cdc14b chr13 64196642 64205416 - taca 1.000 0.804 0.196 Zdhhc12 chr2 30091666 30092064 - alt3 1.000 0.924 0.076 Nfatc2ip chr7 126382853 126390571 - taca 1 000 0835 0.165 Cdanl chr2 120825241 120850438 cass 0.333 0.157 0.177 Bicdl chr6 149518905 149556900 4- cass 0.794 0.880 -0.086 Ulk3 chr9 57593750 57594045 + iret 0.114 0.204 -0.090 Kif17 chr4 138254272 138255697 + cass 0.941 0.882 0.059 5530601H04Rik chrX 105044001 105066876 - cass 0.641 0.769 -0.127 Sh2d3c chr2 32737518 32744878 + cass 0.667 0.463 0.204 Cyba chr8 122426185 122427302 - cass 0988 0.916 0072 Fbxl6 chr15 76537089 76537419 - iret 0.078 0.134 -0.055 Rars2 chr4 34623410 34630569 + cass 0.965 0.889 0.076 Sitm chr9 70559036 70572175 + cass 0.934 0.986 -0.052 Izumo4 chr10 80704417 80704712 + alt3 0.018 0.118 -0.101 Fam 195a chr17 25864593 25868542 - cass 0.249 0.161 0.088 Plekha6 chr1 133273857 133280406 + cass 0.846 0.920 -0.074 2410004N09Rik chr18 33794891 33795988 cass 0962 0906 0056 Ccdc64 chr5 115648174 115651938 - cass 0914 0.855 0.059 Erp44 chr4 48219342 48279451 - taca 1.000 0.896 0.104 D430042009Rik chr7 125707922 125753048 + cass 0.467 0.805 -0.338

[0645]

[0646] Htra2 chrtS 83052697 83053069 - iret 0.286 0.214 0.072Polr3gl chr3 96579797 96580088 - iret 0.349 0.258 0.091 Ppip5k2 chr1 97719833 97723833 cass 0.392 0.282 0.110 Hpca chr4 129118363 129121666 cass 0169 0239 -0070 RteH chr2 181354352 181355551 cass 0.765 0.452 0.313 Ube2q2 chr9 55162967 55176246 cass 0.780 0.716 0.064 D930015E06Rik chr3 83900326 83901465 alt3 1.000 0.860 0.140 Mms19 chrl 9 41962949 41963469 alt3 0.932 1.000 -0.068 Smpd4 chr16 17625719 17626018 iret 0.057 0.121 -0.065 Slx4ip chr2 137043999 137066229 taca 1.000 0.647 0.353 Prrxl chrl 163248255 163257941 cass 0420 0645 -0225 Epb4 chr10 25495444 25501630 cass 0.816 0.665 0.151 Nktr chr9 121741597 121742786 alt3 0.626 0.693 -0.067 Otud5 chrX 7873839 7875283 alt3 0.254 0.204 0.050 Otud5 chrX 7873839 7874861 alt3 0.254 0.204 0.050 Dpp7 chr2 25353152 25353515 iret 0.227 0.304 -0.077 Rabepk chr2 34785566 34790654 alt3 0.868 0.788 0.080 Fam126b chrl 58557976 58565953 alt5 0035 0098 -0063 Ythdcl chr5 86804536 86815729 cass 0.506 0.284 0.222 Tbc1d19 chr5 53830454 53833044 cass 0.267 0.393 -0.126 Ascc2 chr11 4656268 4664302 taca 1.000 0.941 0.059 Nae1 chr8 104527082 104528216 cass 0.955 0.880 0.075 Sltm chr9 70572148 70574624 cass 0.943 0.880 0.063 Mthfd2l chr5 90974322 91021367 cass 0.830 0.926 -0.096 Tle1 chr4 72158212 72169195 alt5 0259 0 100 0159 Aasdh chr5 76904208 76905451 cass 0846 0.481 0.365 Sorbs 1 chr19 40318021 40324831 cass 0.589 0.518 0.072 Pla2g3 chr11 3491891 3492241 iret 0.138 0.269 -0.132

[0647]

[0648] Nemf chr12 69340977 69341480 alt3 0.963 0.904 0.059Nemf chr12 69340994 69341480 - alt3 0.963 0.904 0.059 Znf512b chr2 181589372 181590180 - iret 0.872 0.778 0.095 Med7 chr11 46436970 46442720 + cass 0671 0837 -0166 Rpain chr11 70973014 70974131 + cass 0.846 0.926 -0.080 Focad chr4 88185879 88229426 + cass 0.879 0.966 -0.086 Coro6 chr11 77462624 77464109 + cass 0.120 0.030 0.090 Tfdp2 chr9 96287600 96295126 + cass 0.619 0.752 -0.133 8430427H17Rik chr2 153417959 153420808 - alt5 0.710 0.590 0.120 Itsn2 chr12 4639654 4650118 cass 0.405 0.497 -0.092 Dpf1 chr7 29313081 29314401 + cass 0788 0694 0094 Chd6 chr2 160969338 160970205 alt3 0.982 0.932 0.050 Mtssl chr15 58941233 58945523 alt5 0.275 0.193 0.082 Traf7 chr17 24516503 24518798 - alt3 0.296 0.415 -0.119 Traf7 chr17 24516504 24518798 - alt3 0.296 0.415 -0.119 Traf7 chr17 24516510 24518798 - alt3 0.296 0.415 -0.119 Traf7 chr17 24516526 24518798 - alt3 0.296 0.415 -0.119 Traf7 chr17 24516550 24518798 - alt3 0296 0415 -0119 Traf7 chr17 24516562 24518798 - alt3 0.296 0.415 -0.119 Pisd chr5 32764772 32785625 - cass 0.894 0.970 -0.076 Toplmt chr15 75669286 75670183 - alt3 0.968 0.884 0.084 Dpp7 chr2 25355813 25356141 - iret 0.054 0.110 -0.057 Ecscr chr18 35713087 35715214 - cass 0.890 1.000 -0.110 Gpr137b chr13 13359125 13361421 - cass 0.168 0.103 0.065 Mars chr10 127296540 127296943 cass 0596 0462 0135 Zcchc2 chr1 106023663 106027530 + alt3 0 104 0.200 -0.096 Trub2 chr2 29776110 29779894 - cass 0.974 0.912 0.062 Art13b chr16 62827163 62846997 - cass 0.999 0.843 0.156

[0649]

[0650] Smtn chr11 3517526 3521967 - cass 0.983 0.906 0.077Zmym3 chrX 101416908 101417329 alt3 0.463 0.538 -0.075 Zfp62 chr11 49214222 49215492 alt5 0.889 0.981 -0.093 Exoc7 chr11 116295543 116300432 cass 0773 0698 0075 Crtc2 chr3 90262450 90263356 cass 0.474 0.346 0.128 Map3k12 chr15 102509289 102510004 alt3 0.130 0.077 0.053 Myo19 chr11 84892075 84894651 cass 0.440 0.720 -0.280 Ntmtl chr2 30807967 30819831 mutx 0.300 0.214 0.086 Cdkl3 chr11 52033513 52084494 cass 0.167 0.500 -0.333 Hdac7 chr15 97798223 97802124 mutx 0.147 0.210 -0.062 Porcn chrX 8201443 8203042 cass 0896 0956 -0060 Ccnk chr12 108179801 108186621 cass 0.960 0.903 0.057 Mpv17 chr5 31144699 31145775 mutx 0.605 0.681 -0.076 Zfp788 chr7 41633530 41647616 taca 0.312 0.425 -0.113 Cflar chr1 58711507 58713658 alt3 0.000 0.111 -0.111 Fam126a chr5 23965255 23979646 cass 0.129 0.039 0.090 Mybptf chr10 88518281 88523152 cass 0.250 0.802 -0.552 Ccdc103 chr11 102883073 102884522 alt3 0154 0063 0091 D11Wsu47e chr11 113687773 113692501 cass 0.980 0.863 0.117 Brcc3 chrX 75449968 75455700 alt5 0.714 0.881 -0.167 Sorbsl chr19 40364993 40373616 cass 0.501 0.564 -0.063 Fam229b chr10 39122176 39132377 alt3 0.765 0.865 -0.100 Cep72 chr13 74037631 74040161 iret 0.462 0.191 0.270 Kctd20 chr17 28952715 28961686 cass 0.278 0.440 -0.162 Map3k4 chr17 12239969 12243595 cass 0947 0892 0055 Mdm2 chr10 117705154 117710027 cass 0725 0.605 0.120 Acyl chr9 106434964 106435168 iret 0.071 0.130 -0.059 Atp9b chr18 80738652 80739803 alt3 0.967 0.917 0.050

[0651]

[0652] Atp2b4 chr1 133702673 133711821 alt5 0.387 0.273 0.114Zfp688 chr7 127419003 127421517 cass 0.403 0.557 -0.154 Lyplall chr1 186089443 186114362 cass 0.895 0.765 0.130 Abca7 chr10 80007135 80007450 alt3 0755 0896 -0141 Pbrml chr14 31107108 31114003 cass 0.470 0.335 0.135 Sp100 chr1 85679058 85692048 cass 1.000 0.591 0.409 Cggbp! chr16 64852798 64855895 alt3 0.655 0.724 -0.070 Cggbp-I chr16 64852798 64855872 alt3 0.655 0.724 -0.070 Cggbpl chr16 64852798 64855632 alt3 0.655 0.724 -0.070 Pnpti chr11 29148277 29153337 alt3 0.909 0.990 -0.081 Myh14 chr7 44634358 44637878 cass 0411 0535 -0 124 Pds5a chr5 65627998 65630054 alt3 0.917 0.978 -0.061 LairT chr7 4028706 4055952 alt5 0.643 0.397 0.245 Slc35f5 chr1 125587364 125589964 alt3 0.890 0.958 -0.068 Trp53inp1 chr4 11165089 11174376 cass 0.350 0.459 -0.110 Zscan21 chr5 138116951 138125689 taca 1.000 0.708 0.292 Sic7a3 chrX 101083870 101085371 cass 0.176 0.248 -0.073 Gyk chrX 85737313 85740360 cass 0745 0623 0123 Camk4 chr18 32939171 33107941 cass 0.866 0.948 -0.082 Ankzfl chr1 75195800 75196380 iret 0.449 0.311 0.138 Prrgl chrX 78449612 78483910 cass 0.141 0.294 -0.153 N28178 chr4 42917250 42933666 cass 0.934 0.866 0.068 Lrp6 chr6 134520395 134566964 cass 0.901 0.986 -0.085 Slc44a2 chr9 21352466 21355027 cass 0.672 0.600 0.072 Cep164 chr9 45823645 45828584 cass 0128 0000 0128 Dnm2 chr9 21505463 21507145 alt3 0642 0.565 0.077 Dnm2 chr9 21505463 21506688 alt3 0.642 0.565 0.077 Dnm2 chr9 21505463 21506389 alt3 0.642 0.565 0.077

[0653]

[0654] Sh3pxd2a chr19 47314034 47343467 cass 0.854 0.690 0.164Atxn3 chr12 101948003 101948438 - alt3 0.677 0.876 -0.199 Cd97 chr8 83734295 83741182 - alt3 0.000 0.114 -0.114 Dec chr18 71378654 71384222 - alt3 0935 0996 -0061 Srr chr11 74912961 74925676 cass 0.841 0.896 -0.055 Miefl chr15 80234079 80236152 + cass 0.143 0.267 -0.124 Zfp945 chr17 22861505 22865335 - cass 0.481 0.279 0.202 Nup214 chr2 31989072 31991424 + cass 0.919 0.838 0.080 Rbms3 chr9 116636368 116681226 - cass 0.116 0.174 -0.059 6330408A02Rik chr7 13258966 13269646 - alt5 0.837 0.957 -0.120 Cd33 chr7 43527455 43529931 - cass 0231 0074 0 157 Ccdc74a chr16 17648066 17650073 taca 0.767 0.591 0.175 Adamts6 chr13 104399942 104427067 4- cass 0.238 0.429 -0.190 Eda chrX 100395018 100400759 + cass 0.048 0.385 -0.337 Trpm3 chr19 22897669 22901334 + cass 0.828 0.886 -0.058 Fcld2 chr17 29360931 29363941 + alt3 0.300 0.002 0.298 Zdhhc3 chr9 123089031 123091131 - alt5 0.333 0.002 0.331 Repsi chr10 18104153 18107747 + alt3 0796 0727 0069 Pter chr2 12924040 12978616 + cass 0.529 0.852 -0.322 Agrn chr4 156167281 156168568 - alt5 0.683 0.597 0.086 Maltl chr18 65448921 65451572 + cass 0.077 0.000 0.077 Dnajc24 chr2 105966709 105981119 - cass 0.909 0.967 -0.058 Lrrfip1 chr1 91079030 91085004 cass 0.324 0.444 -0.121 Acapl chr11 69881566 69882030 - iret 1.000 0.111 0.889 Lilrb4 chr10 51493169 51494177 iret 0000 0600 -0600 1112a chr3 68695189 68695345 + iret 0667 0.000 0.667 Tmem232 chr17 65486471 65517236 - cass 1.000 0.200 0.800 Mtif2 chr11 29526407 29530153 + cass 0.918 1.000 -0.082

[0655]

[0656] Phf23 chr11 69997745 69999131 + cass 0.629 0.729 -0.100Usp43 chr11 67876372 67880143 - alt5 1.000 0.774 0.226 Zfp182 chrX 21060502 21062021 - alt3 0.250 1.000 -0.750 Baz2b chr2 59978542 59983969 - alt3 0510 0718 -0207 Ambral chr2 91772290 91810208 + cass 0.725 0.660 0.065 Ube2v2 chr16 15581058 15594500 - cass 0.000 0.086 -0.086 Agapl chr1 89743727 89789323 + cass 0.716 0.654 0.062 Plekha5 chr6 140556803 140569414 + taca 0.880 0.977 -0.097 Pld5 chr1 176044834 176074524 - alt5 0.958 0.865 0.093 Slc50a1 chr3 89269826 89270138 - iret 0.344 0.285 0.059 Cdk11b chr4 155625530 155626888 + alt5 0399 0464 -0065 Myolh chr5 114361042 114361323 iret 0.231 0.000 0.231 Hacel chr10 45618497 45648686 4- cass 0.913 0.974 -0.061 Gtf2ird1 chr5 134363901 134380019 - taca 0.921 0.980 -0.059 Usp37 chr1 74456078 74461729 - cass 0.584 0.726 -0.142 Stxbp5l chr16 37142275 37174361 - cass 0.284 0.159 0.126 Ybx3 chr6 131370322 131379479 - cass 0.431 0.355 0.076 Arhgap12 chr18 6069830 6135930 - cass 0133 0024 0109 Upf1 chr8 70339742 70340098 - alt5 0.194 0.278 -0.084 Pusl1 chr4 155889433 155889722 - iret 0.061 0.111 -0.050 Bak1 chr17 27021166 27022581 - cass 0.414 0.296 0.118

[0657]

[0658] Rapgef3 chr15 97757682 97758104 - alt3 0.081 0.027 0.054

[0249] TABLE 5

[0659] Classification in transcripts belonging to behavioral gene ontology categories.

[0660] Detailed classification of 27 transcripts with differential AS events in the midbrain of P21 Novalhu / hu mice. The 27 transcripts that were classified into behavioral categories in the gene ontology were divided according to the minor classification to which each transcript belongs. The major gene ontology term. ID, minor classification, and category are shown.

[0661] Gene_name Name Minor Classification GO term ID Category ATP-binding cassette,

[0662] memory learning or memory G0:0007611 Learning Abca7 sub-family A member

[0663] 7 visual learning learning or memory GG:0007611 Learning Adcy3 adenylate cyclase 3 olfactory learning learning or memory G0:0007611 Learning Atxn3 ataxin 3 exploration behavior exploration behavior GG:0035640 Exploration autism susceptibility innate vocalization vocalization

[0664] Auts2 candidate 2 behavior behavior GG:0071625 Vocalization calcium channel, adult walking

[0665] voltage-dependent, behavior locomotory behavior GG:0007626 Locomotor Cacnala

[0666] P / Qtype, alpha 1A behavioral response

[0667] subunit to pain Other calcium / calmodulin- dependent protein

[0668] Camk4 kinase IV long-term memory learning or memory G0:0007611 Learning chromodomain

[0669] social behavior social behavior G0:0035176 Sociability Chd8 helicase DNA binding

[0670] protein 8 long-term memory learning or memory G0:0007611 Learning exploration behavior exploration behavior G0:0035640 Exploration Chrd chordin

[0671] visual learning learning or memory G0:0007611 Learning CLN3

[0672] associative learning learning or memory G0:0007611 Learning lysosomal / endosomal

[0673] Cln3

[0674] transmembrane

[0675] protein, battenin learning or memory learning or memory GG:0007611 Learning dishevelled segment

[0676] Dvl1 polarity protein 1 social behavior social behavior G0:0035176 Sociability behavioral response

[0677] epidermal growth

[0678] to ethanol Other

[0679] Eps8 factor receptor

[0680] adult locomotory

[0681] pathway substrate 8

[0682] behavior locomotory behavior G0:0007626 Locomotor guanine nucleotide

[0683] binding protein (G

[0684] protein), beta

[0685] Gnbll polypeptide 1-like social behavior social behavior GG:0035176 Sociability behavioral response

[0686] glutamate receptor,

[0687] Grikl to pain Other ionotropic, kainate 1

[0688] adult behavior Other adult locomotory

[0689] HtrA serine peptidase behavior locomotory behavior G0:0007626 Locomotor Htra2

[0690] 2 adult walking

[0691] behavior locomotory behavior G0:0007626 Locomotor intraflagellar transport regulation of feeding

[0692] Ift88 88 behavior feeding behavior G0:0007631 Feeding mannosidase 2, alpha

[0693] Man2b1 B1 learning or memory learning or memory G0:0007611 Learning myosin, heavy vocalization vocalization

[0694]

[0695] Myh14 polypeptide 14 behavior behavior G0:0071625 Vocalizationadult behavior Other vocalization vocalization

[0696] Nrxn2 neurexin II behavior behavior G0:0071625 Vocalization social behavior social behavior G0:0035176 Sociability vocal learning learning or memory G0:0007611 Learning eating behavior feeding behavior G0:0007631 Feeding regulation of

[0697] locomotor rhythm locomotory behavior G0:0007626 Locomotor Oprll opioid receptor-like 1

[0698] conditioned place

[0699] preference Other behavior Other learned vocalization

[0700] behavior or vocal

[0701] Pax6 paired box 6 learning learning or memory G0:0007611 Learning adult locomotory

[0702] behavior locomotory behavior G0:0007626 Locomotor peripheral myelin

[0703] Pmp22

[0704] protein 22 motor behavior motor behavior G0:0061744 Motor adult walking

[0705] behavior locomotory behavior G0:0007626 Locomotor pumilio RNA-binding adult locomotory

[0706] Pum1 family member 1 behavior locomotory behavior G0:0007626 Locomotor Rap guanine

[0707] nucleotide exchange

[0708] Rapgef3 factor (GE F) 3 associative learning learning or memory G0:0007611 Learning solute carrier family 22

[0709] (organic cation

[0710] Slc22a5 transporter), member 5 locomotory behavior locomotory behavior G0:0007626 Locomotor sushi-repeat- containing protein, X- vocalization vocalization

[0711] Srpx2 linked 2 behavior behavior G0:0071625 Vocalization VPS54 GARP complex

[0712] Vps54 subunit motor behavior motor behavior G0:0061744 Motor locomotory behavior locomotory behavior G0:0007626 Locomotor Wdr47 WD repeat domain 47 motor behavior motor behavior G0:0061744 Motor

[0713] adult locomotory

[0714]

[0715] behavior locomotory behavior G0:0007626 Locomotor

[0716]

[0250] TABLE 6

[0717] Syllables detected in isolation induced USV test in pups.

[0718] The acoustic waveform data for each pup was processed by the Mouse Song Analyzer (from Erich lands lab) to obtain values for each syllable: syllable type, duration (time per syllable [sec]), ISI (intersyllable interval), fqVariance (degree of variance), purity, amplitude (magnitude of loudness), bandwidth (width of peak frequency) and peak frequency (Fq) variabilities; fqmin (minimum), fqmean (mean), fqmax (maximum), fqstart (start), fqend (end). notIDd: not identified, (partial exemplary data shown)1 lf<

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[0757] Si $ § <*• v' •t>

[0251] TABLE 7

[0758] USV features in each pup.

[0759] In isolation induced USV test for pup, the following USV features are calculated for each pup: number of USVs, call rate, percent. starting (percent of starting syllable type of sequence (continuous syllables)), percent. composition (percent of syllable), sequence length. For each syllable type (“s”, “u”, “d”, “m”), following parameter are calculated: Bw (bandwidth), Amp (amplitude), fqVar (Fq variance), Purity, Dur (duration), and Fq variabilities; fqMin (minimum), fqMean (mean), fqMax (maximum), fqStart (start), fqEnd (end), (partial exemplary data shown)

[0760]

[0761]

[0252] TABLE 8

[0762] USV features in each genotype of pup.

[0763] USV features inNovalhu / hu, Novaihu / wt and Novalwt / wt pups. Bw (bandwidth). Amp (amplitude), fqVar (Fq variance). Dur (duration). The values represent the mean value for each genotype. The standard error (se) values for each parameter are inserted in the adjacent mean columns. The

[0764] I l lparameters statistically different from control (Novalwt / wt are indicated with asterisk (*p < 0.05).

[0765] / r-values were calculated by Wilcoxon rank sum test and were corrected with Bonferroni method.

[0766] Novalwt / wt pup N=40, Novalhu / wt pup N=23, Novalhu / hu pup N=41.

[0767] S 4 slfis & ®< U4si

[0768] 40 563354 £ 4® i^^3

[0769] ] riC'fsto:}■& $ 23 <65522 W 77.134 w G'S 0.254 34.i05 IS®! 3.016 6.533 '$>5$; v.< ®5| 1334

[0770]

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[0777]

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[0780]

[0781] 035 0.346?? 6Oj 61® 28?324 0.05 OF; 602 3®® 3710 65G®?| OT®

[0782]

[0253] TABLE 9

[0783] Bimodal distribution parameters at Fqmax (maximum frequency) in pup USVs

[0784] For each syllable type, the bimodality in Fqmax was assessed by Ashman’s D test (Fig. 4c). Two Gaussians were fitted to calculate each distribution parameters: component (weight), Fq mean

[0785] [Hz], Sd (standard deviation) and cutoff value (intersection point) |kHz|. The distributions are

[0786] classified as Low Fqmax and High Fqmax by the cutoff.

[0787] Simu ai tiistri&.ition analysis a i twO Gaussians fit at maximum frequenc **y#** o**f** P**wu*p**l> U***S*«*^V******w**w********«***l>*********p**w*******«**w**********************#***«** Syiia bis type r-qmax ^component (peak wsightj Ms-an Sd _ Ashman's 0 (tors; cutoff (u-itarssctiurQlkHz)

[0788] Low 0,56 67275,73 9845.6Sj ISi<,

[0789] 9.44 925. S9.37

[0790] *"^ L-a*-**w*-*^ 'aso

[0791] _5 U7W82,5

[0792] High 0.70 "gsoijua 6622,211

[0793] LOW 0,771 87754.79 773C.24:

[0794] S7.5

[0795] High <1?3 10S1M.58'

[0796] Low 0.62 872PS.49 76S2 13 i

[0797] - 3 876983

[0798]

[0799] High 038 W1S33.27

[0800]

[0254] TABLE 10

[0801] Proportion of high / low Fqmax in pup-USVs in each genotype.

[0802] The ratio of low Fqmax and high Fqmax in each syllable type are shown for each genotype. The

[0803] values represent the mean value for each genotype. The standard deviation (sd) and the standard

[0804] error (se) values for each parameter are inserted in the adjacent mean columns. The parameters statistically different from control (Novalwt / wt) are indicated with asterisk (*p<0.05, ** <0.01). ^valueswere calculated by Wilcoxon rank sum test and were corrected with Bonferroni method. Novalwt / wt pup N=40, Novalhu / wt pup N=23, Novalhu / hu pup N=41.

[0805] (*) p=0.039; (**) p=0.0069

[0806] genotype Syl ratio.lowFqmax sd se ratio.highFqmax sd $e 4 N««o««v«e«<l«4 (<4w<«4t« / 4w««t4)<«««" 0361 0.250 O. O42 0.635 0.259 0.042 Novel (hu / wt) d 0.272 0.236 0.050 0.728 0.236 0.050 Noval (hu / hu) 0.254 0.269 0.044 0.745 0.268 0.044 Nowl (wt / wt) 0.676 0.292 0.049 0324 0.292 0.049 Novel (hu / wt) m *0,498 0.301 0.067 *0.502 0301 0.067 Novel (hu / hu) _ **0,407 0360 0.067 **0393 0360 0.067 Novel (wt / wt) 0.553 0.157 0.025 0.432 0.158 0.025 Novel (hu / wt) s 0.522 0.145 0.030 0.467 0.143 0.030

[0807]

[0808] Novel (hu / hu) 0.541 0,155 0.024 0.442 0.149 0.023

[0809]

[0255] TABLE 11

[0810] Syllables detected in courtship induced USV test in adults.

[0811] The acoustic waveform data for each adult mouse was processed by the Mouse Song Analyzer (from Erich Jarvis lab) to obtain values for each syllable: syllable type, duration (time per syllable [sec]), ISI (intersyllable interval), fqVariance (degree of var iance), purity, amplitude (magnitude of loudness), bandwidth (width of peak frequency) and peak frequency (Fq) variabilities; fqmin (minimum), fqmean (mean), fqmax (maximum), fqstart (start), fqend (end). Day: recording day 1-3. The test was conducted three times per mouse, one week apart. LF: live female for stimulation. notlDd: not identified, (partial exemplary data shown)*t s 3 s? £ ft 2 S £ i g $ 1 J i i § ft ■ Ifl. I S ft i

[0812]

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[0814]

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[0818]

[0819]

[0256] TABLE 12

[0820] USV features in each adult mouse.

[0821] In courtship induced USV test for adult mouse, the following USV features are calculated: number of USVs, call rate, percent. starting (percent of starting syllabic type of sequence (continuous syllables)), percent. composition (percent of syllable), sequence length. For each syllable type (“s”, ‘u”, “d”, “m”), following parameter are calculated: Bw (bandwidth). Amp (amplitude), fqVar (Fq variance), Purity, Dur (duration), and Fq variabilities: fqMin (minimum), fqMean (mean), fqMax (maximum), fqStart (start), fqEnd (end). The values were shown by each recording day per mouse (dl-d3). (partial exemplary data shown)

[0257] TABLE 13

[0822] USV features in each genotype of adult mouse.

[0823] USV features in Novalhu / hu, Novalhu / wt and Novalwt / wt pups. Bw (bandwidth), Amp (amplitude), fqVar (Fq variance), Dur (duration, sec). The values represent the mean value for each genotype. The standard error (se) values for each parameter are inserted in the adjacent mean columns. The parameters statistically different from control (Novalwt / wt) are indicated with asterisk (*p < 0.05, ** p < 0. 1). -values were calculated by Wilcoxon rank sum test and were corrected with Bonferroni method. Novalwt / wt N=13, Novalhu / wt N=14, Novalhu / hu N=13.sc alt®® se SeejuenctUrat

[0824] 13 723.5’4 liSUW 344,755 5.359

[0825] 14 73S.4QS 21.450 $..:535

[0826]

[0827] ttosl [hu / ha) 13 S23.71S 135.354 27.031 3.77C Prarwrt.cramt 3® PWWtXWtH

[0828] 77.240 3.3SS 13 ^8 1.408 7.018 0.773

[0829] 78.532 2,4$2 1 ’ 40’ 1171 §.753

[0830]

[0831] 7S 3S1 3.137 1C 13$ 1.5& S 7,7S7 M97 gem&ype S.to’v’3? SC d.fqWr sc s^Var 5«

[0832] Nwal iwiAvn 0 ”3253 c was 0,11053 Q.0OJ47 0.13844 0.80818

[0833] 0 W.32 0.00308 8.13058 808473 8.13137 080332

[0834]

[0835] [ hv / ha) OM3121 Q.0029S G. 77 0. W98 8.12530 fi8Q494 gEncstyps s. Byr se slDtrr 5C U!>.:■( SC

[0836] Kfevsl 8. G34S2 8.08278 8.85918 0.88422 G. PS392 aw43

[0837] 0. P327S WK13 CsOSGS? 0.00339 8.80353

[0838]

[0839] 8.82977 0. W58 8.05348 9,08272 9.80277 8.88479

[0840]

[0258] TABLE 14

[0841] USV characteristics in long / short duration “s” in adult-USVs in each genotype.

[0842] The “s” syllables were classified by the cutoff (44ms) into short or long duration (Fig. 4f). The values represent the mean value for each genotype. The standard error (se) values for each parameter are inserted in the adjacent mean columns. The parameters statistically different from control (Novalwt / wt) are indicated with asterisk (*p < 0.05, **p < 0. 1). / ^-values were calculated by Wilcoxon rank sum test and were corrected with Bonferroni method. Aw<r7w / / N=13, Novalhu / wt N=14, Novalhu / hu N=13. (partial exemplary data shown)

[0843] Long duration A ‘

[0844] genotype ^Duration [se< St! Variance [se Purity [se Amplitude ise Bandwidth se Koval t. Wwij j 0.07713 0.03371 0.0620SS 0.00243 0.76752’ O. OU19 595.76] 62.74 15342.45 780.31 Koval (hu / wt) | 0.07599 0.00245 0.05325; 0,00395 O.7?577[ 0.01380 526.17! 53.02 13595.66 939.81

[0845]

[0846] Kwai fhu / hu) ] 0.07439 0.00277 O. O5i®0| 0.00333 6.767341 0.01218 695.03! 46.81 14343.48 863.80

[0847]

[0848] Short duration “s"

[0849] geiw.ype i Duration js* se rqV.jiiance? ise Purity ise Amphtude ise Sandwidth se Moral (wt / wt) | 0.02183 C.(K©79 002455^ 0.00145 0.72049] 801768 508.92] 50.34 5221.21 339.24 Moral (WwtJ | 0.02186 0. OTS1 Q.02488| 0.00140 0.73150] 0.01181 443.63i 20.96 5210.26 330.85

[0850]

[0851] Koval (hu / hu) | 0.02131 0.08869 002398! 0.00131 0.72621] 0.01183 458,36] 24.01 4994.89 288.37 g«Wge IfSSS.’S.&lL se Pqmin |Ht] [se Fqmean [Hzjse Fgstart |HzJ ise (gefid jtfaj iise Noral (wt / wt) | 7955349 ““74332.27] 745‘17 “‘J‘76935^i 81842 ““7fr985.33T 798’91 7753652^ SSSil Moral (hu / wt[ | 77653.52 164243 72443.25] 1388.72 75017.34] 1493.08 74282.30 i 1460.41 75750.95] 156S.9O

[0852]

[0853] Moral (hy / hu) j 78867,67 81252 73872.78] 795.18 76329.87] 791.84 75618.92] 793.42 76836.02 ] 800.98

[0259] TAB E 15

[0854] Proportion of syllable composition in high / low Fqmax in adult-USVs in each genotype.

[0855] The syllables were classified by the cutoff (100 kHz) into low or high Fqmax (Fig. 4h). The ratio of low Fqmax and high Fqmax in each syllable type are shown for each genotype. notIDd: not identified. The values represent the mean value for each genotype. The standard error (se) for each parameter is inserted in the adjacent mean columns. The parameters statistically different from control (Novalwt / wt) are indicated with asterisk (*p < 0.05). / ?-values were calculated by Wilcoxon rank sum test and were corrected with Bonferroni method. Novalwt / wt N=13, Novalhu / wt N=14, Novalhu / hu N=13.

[0856] Syllable Composition in low / high Fqmax US Vs

[0857] genotype Syl high. Fqmax_component se low. Fqm ax ^component se Naval (wt / wt) s 6,416 0.032 0.772 0.021 Naval (hu / wt) s 0.339 6.035 0.755 6.62 a Naval (hu / hu) s 6,394 0.035 0.769 0.023 Naval (wt / wt) u 6139 6.616 0.064 6.066 Naval (hu / wt) u 0.143 0.014 0-067 6.00S Naval (hu / hu) u 6,162 6.617 0.074 6.664 Naval (wt / wt) d 0.229 0.012 0.101 6.611 Naval (hu / wt) d 6,212 6.617 6.116 6.614 Naval (hu / hu) d 0.236 6.616 0.107 6.616 Neva! (wt / wt) m 0.117 0.017 0.046 0.008 Noval (hu / wt) m 6,129 6.626 0.044 6.666 Naval (hu / hu) m 0.100 0.011 0.040 0.007 Naval (wt / wt) notIDd 0.126 6.615 0.029 6.066 Naval (hu / wt) notIDd 0.201 0.064 0.028 0.004

[0858]

[0859] Naval (hu / hu) notIDd 0.171 6.636 0.038 6.066

[0860]

[0260] TABLE 16

[0861] USV characteristics in low / high Fqmax in adult-USVs in each genotype.

[0862] The syllables were classified by the cutoff ( 100kHz) into low or high Fqmax (Fig. 4h). The values represent the mean value for each genotype. The standard error (se) values for each parameter are inserted in the adjacent mean columns. The parameters statistically different from control (Novalwt / wf) are indicated with asterisk (*p < 0.05, < 0.01). p- values were calculated by Wilcoxon rank sum test and were corrected with Bonferroni method. Novalwt / wt N=13, Novalhu / wt N= 14, Novalhu / hu N= 13.t?$V charaaeriate a high Fornax iaM®

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[0879]

[0880] «:

[0881]

[0261] TABLE 17

[0882] Comparison of vocalization tests between humanized mouse models.Comparison on USV tests between this study and three studies using humanized Foxp2 mice (Enard et al., 2009, Hammerschmidts et al., 2015, von Merten et al., 2021)10-12. The table contains experimental conditions (methods and analysis) and findings in each study. To avoid changing the nuance of the words in each report, the terms in each paper were quoted verbatim in the table (e.g., calls, elements, vocalizations).

[0883] Compsrisoo of USV ansteis for HiimonzK! mouse Stmly TSfeJDOj Expwsrt HOUSS; tfft& Slf«3J:0rt;:lAge ®:!»>:;

[0884] W

[0885] :rs«ct»n ffsrrosFrw retisrei''g$c%'<er4 «!>isfte feSrsj sigwrois Ssiitoisn: jteotwe mates for scotisiK- cjrawtero rseptmtesw pawter

[0886] Souse O rewrites tesatefa sate S(C»pfcB» p'esrpMser, reptrfetg 'W.;s» -werte tcKfeng ajjorrtfe' "sii ' ssruefe-e ws’yii eetneoon for e«ust® se-amettrs

[0887] Stars* mferfKssron. Ass $w ^M®t®r®®®i®®®®®®®s ooBrtstsp ■note! cenMeo fiteordiagsrfes rotwoirtwne Stesresestate- aawfcr

[0888] _ ta

[0889]

[0890] Siaty

[0891] : aw» a® Mb tn w t®VAt pitp'Pii irate ®emie

[0892] MM NOVA* {tWI& Ste; s sffisse aria sMte is Basse O'At irate iWihieasgj

[0893] MM sis sis

[0894] :; XSO teoioie pap SWieir M erst fest

[0895] JSBK 3-ate wte whh if ts:< i M tor rateteaiiai:, recoKiteg

[0896] Ending sKtag

[0897] CS8s05iO®®0;ii sisiO$f jOisSatassWsi iff sev' t W. U'tnswea Gate tiSGH aapte. AviseWasfeUSG Ai'isafi StsiCiSiStirs Mstse Sotsg MW r -JBA3 Mra MK m iWten. MM. auaifctfe Mfity. tewsy was. star- BMW saws errf-fr«toeocy

[0898] Paar atete W sio^-e s#aife -sj, ®e tewrai pS see spMKi sta mi (■tj, suffle pttefi js^s ®

[0899] As? &3M with escn peak jpaarrsfer «* twai Mb;

[0900]

[0901] |tfc stgofficert is the Mtn sfe&fes

[0902] Stoat tatiu prcauced taver err^iteie tor *s' syfesfe

[0903] pup tfcni M has less Mining pfch ws

[0904] ttati teifeasea pms® d r«w Ms o»? Wi

[0905] ffe -S8:®effl;t •Setettce- » te Mb of sates

[0906] ttar i Mt ftSi Mr stop!ie» SKI r & seat; ■feaixtecies tR • to tong “ syfeise

[0907] ftoo hii sM u<t tte >%» e » ^{extaUatitesA^F^s

[0908]

[0909]

[0262] TABLE 18

[0910]

[0263] Expression changes in Novalko / ko midbrain at E18.5.

[0911] Transcripts whose expression was affected in E18.5 midbrain in Noval knockout mice were shown Novalwt / wt vs. Novalko / ko). RNA sequencing data are from Saito et al., 201613. Corresponding values on the same transcripts in humanized NOVAI mice at E18.5 midbrain are shown on the right column (Novalwt / wt vs. Novalhu / hu). Transcripts Per Kilobase Million (tpm) values represent the average value for each genotype. The analysis was performed using edgeR. Novalwt / wtN=, Novalko / ko N=3 for Noval knockout mice comparison, Novalwt / wt N=6, Novalhu / hu N=6 for humanized Noval mice comparison._

[0912] _

[0913] w

[0914]

[0915] -’ - p <5I >

[0916] J

[0917] W

[0918] 1

[0919] _ — L

[0920] 1^9

[0921]

[0922] _..

[0923] oj 1

[0924] sos_

[0925] ^w!&w535« „<<*, \\ 0 ••.' ■;:.-i.

[0926] ; %? Z i -• <;?;\

[0927]

[0928] X

[0929]

[0930] _

[0931]

[0932]

[0933] W

[0934] TABLE REFERENCES

[0935] 1. Schaeffer, S. W. Molecular population genetics of sequence length diversity in the Adh region of Drosophila pseudoobscura. Genet. Res. 80, 163-175 (2002).

[0936] 2. Meyer, M. et al. A High-Coverage Genome Sequence from an Archaic Denisovan Individual. Science 338, 222-226 (2012).

[0937] 3. Trujillo, C. A. et al. Reintroduction of the archaic variant of NOVA 1 in cortical organoids alters neurodevelopment. Science 371, (2021).

[0938] 4. Lewis, H. A. et al. Sequence-specific RNA binding by a Nova KH domain: implications for paraneoplastic disease and the fragile X syndrome. Cell 100, 323-332 (2000).

[0939] 5. Teplova, M. et al. Protein-RNA and protein-protein recognition by dual KH1 / 2 domains of the neuronal splicing factor Nova-1. Structure 19, 930-944 ( 2011).

[0940] 6. Dredge, B. K., Stefani, G., Engelhard, C. C. & Darnell, R. B. Nova autoregulation reveals dual functions in neuronal splicing. EMBO J. 24, 1608-1620 (2005).

[0941] 7. Vogel, A. P., Tsanas, A. & Scattoni, M. L. Quantifying ultrasonic mouse vocalizations using acoustic analysis in a supervised statistical machine learning framework. Sci. Rep. 9, 1-10 (2019). 8. Grimsley, J. M. S., Monaghan, J. J. M. & Wenstrup, J. J. Development of social vocalizations in mice. PLoS One 6, e!7460 (2011).

[0942] 9. Tajima, Y. et al. NOVAI acts on Impact to regulate hypothalamic function and translation in inhibitory neurons. Cell Rep. 42, 112050 (2023).

[0943] 10. Enard, W. et al. A Humanized Version of Foxp2 Affects Cortico-Basal Ganglia Circuits in Mice. Cell 137, 961-971 (2009).

[0944] 11. Hammerschmidt, K. et al. A humanized version ofFoxp2 does not affect ultrasonic vocalization in adult mice. Genes Brain Behav. 14, 583-590 (2015).

[0945] 12. von Merten, S., Pfeifle, C., Kunzel, S., Hoier, S. & Tautz, D. A humanized version of Foxp2 affects ultrasonic vocalization in adult female and male mice. Genes Brain Behav. 20, el2764 (2021).

[0946] 13. Saito, Y. et al. NOVA2-mediated RNA regulation is required for axonal pathfinding during development. Elife 5, (2016).EXAMPLE 2

[0947] CORRECTING N0VA1 EXPRESSION

[0948]

[0264] Noval and Nova2 were discovered as neuron-specific RNA binding proteins targeted in autoimmune nemologic disorders manifest by failure to inhibit motor functions. We recently described the first patient with a Noval genetic haploinsufficiency (Tajima, 2023) (2). This patient had neurologic dysfunction, initially presenting with delayed language development and progressing to show manifestations of failure to inhibit motor functions reminiscent of Tourette’s syndrome.

[0949]

[0265] Moreover, our most recent studies (including as described in the prior Example) have now found that human Noval is associated with human language function. We have also identified additional patients with point mutations in Noval and the highly related gene Nova2. Clinical phenotypes of these patients are varied, but include early life language dysfunction as one common outcome.

[0950]

[0266] Modeling these defects in genetically modified neurons indicate that these mutations lead to loss of Nova function as an RNA regulator. Identification of dysregulated transcripts points to key pathways that are then dysregulated in these neurons. Collectively, these observations indicate that loss of Nova function leads to human neurologic developmental disorders, including but not restr icted to errors in language development. Increasing Nova levels is likely to correct these defects.

[0951]

[0267] In one approach, upregulation of Nova is possible by antagonizing suppressors of Nova RNA translation and stability. microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs (Moore et al., 2015). Specifically, we note the presence of Argonaute-miRNA binding elements in Noval and the specific sites they are bound to on Nova 3’ UTRs.

[0952]

[0268] Antagonizing this binding, for example by anti-sense oligonucleotides (ASOs), will increase Nova levels and thereby have the potential to ameliorate clinical neurologic phenotypes (see Tajima et al., 2023). The binding can be antagonized by designing ASOs that bind directly and specifically to the Nova untranslated region sequence that is the target of miRNAs. This would only block the Nova site of action (i.e. the Novalor Nova23’UTR) and would specifically control Nova expression.

[0953]

[0269] Figure 22 depicts miRNAs that target Noval 3’UTR. Upon inspection (right panel) the miRNAs include miR-27a, miR-103, let-7a and miR-132.

[0954]

[0270] Figure 23. miRNAs that target Noval 3’ UTR. miRNA binding sites in Noval 3’ UTR mapped by Ago2 CLIP. Antagomirs (e.g. antisense to sequences surrounding miR-103) can block miRNA binding, offering the possibility of increasing Noval mRNA levels and translation of Noval protein (see Tajima et al., 2023 and related refs).

[0271] Figure 24 depicts miRNAs that target Nova2 3 ’UTR. Upon inspection (right panel) the miRNAs include miR-26a, miR-484, miR-148a, miR-I9a, miR-221, miR-124, miR-30b, miR-296-5p, miR-340-5p, miR-30b, miR-30e, miR-9, miR-288-5p, miR-124 and let-7c,

[0955]

[0272] The miR-103 site is conserved across numerous animal species, including humans, mice, rats, rabbits, chimps, rhesus monkeys, etc. as depicted in Figure 23 and shown below.

[0956] Mouse AAAA-GCACCUGUUCUCC AUCUGAACUAGACAAUGGAAAUAAUGCUGCAU ( SEQ ID NO: 2 ) Human AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 25) Chimp AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 25) Rhesus AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 27 ) Squirrel AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 28) Rat AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 29) Rabbit AAAAAGCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 30 )

[0957] Pig AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 31)

[0958] Cow AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 32 )

[0959] Cat AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 33 )

[0960] Dog AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 34 ) BrwnBatAAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU ( SEQ ID NO: 35 ) ElephantAAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 36 ) OpossomAAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU ( SEQ ID NO: 37 ) Macaw AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU ( SEQ ID NO: 38) Chicken AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 39) Lizard AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 40 )

[0961] X. tropicalis

[0962] - G - UCUCCAU - AACAACCUGUAUAAUGAUGAAUA (SEQID NO: 1 ) Con (Consensus)

[0963] AAAA-GCACCUGUUCUCCAUCUGAACUAGACAAUGGAAAUAAUGCUGCAU (SEQ ID NO: 2 )

[0964]

[0273] The miR-103 8mcr target sequence is set out as AUGCUGCA (SEQ ID NO:23) (listed 5’-3’).

[0965] Below is a Table of the Ago2 CLIP tag results that reveal miRNAs targeting NOVAI 3" UTR (and

[0966] NOVA2 3’ UTR). It also illustrates where exactly AGO2 is binding on the 3’ UTR sequences of the

[0967] NOVAI gene.

[0968] TABLE 1

[0969] location miRNA name tag sequence

[0970] mouse mmu-miR-27a GCAATATAGTTGCATAAAAGCACTGTAAAATA (SEQ ID NO: 43) Noval_3 ’ UTR mmu-miR-103 GACAATGGAAATAATGCTGCATGCTGGCCAGGGCCCATTCTTCATCATTTG (SEQ ID NO: 44 )

[0971] mmu-let7a GACCCAACAACTGTCACCCTCTTCTGTACCTCTCCTGAGTGCCAACTGT CCAGGCCA (SEQ ID NO: 45)

[0972] mmu-miR-132 ATGAAAGAGAAAAGCTGTAGTAAACTGTGTTAATTGGTAATGACTATT.

[0973] TATCGTCGA (SEQ ID NO: 46)

[0974] location miRNA name tag sequence

[0975] mmu-miR-484 TATGGGAGGAGGGGAGGGAGCCTTCAGGTCACCTGGAATG

[0976]

[0977] mouse i ( SEQ ID NO: 47 )

[0978] Nova2_3 ' UTR TCAAAATGTGAAAATCCCTAGAAAGCAATAGCCTTCGAGGTACCTCG 148a / 342-5p CACTGACTCCCCCTCC (SEQ ID NO: 48 )

[0979] mmu-miR- AAAACAATTCTTGATACCACTTTGTACATATCTTCTACAGTTGGGGCT 19a / 190 / 23b GCTGAGATTGGAGGTGG (SEQ ID NO: 49 )

[0980] i mmu-miR-221 CGCCCGTAAAACCCTCAGGCTCCATGAGGA (SEQ ID NO: 50)

[0981] ; mmu-miR-124 ATAGTTAAGTGTTGCCTGCATTGAACTGTCTCATCTGCCCTCCTGCTT CCGATACCCCTG (SEQ ID NO: 51 )

[0982] i mmu-miR-30b GTATTCTTCTCCTCCTGAATTCTCGACTCTCATTTCCCCTTTAAAAGCA AA. AA. GTGC (SEQ ID NO: 52 )

[0983] ; mmu-miR- TTAGAGGGGGGGGGGCCTTAAGAGGCAAAGAA (SEQ ID NO: 53)

[0984] 296-5p

[0985]

[0986] ; mmu-miR- T C A CC C T GA. GT T T T T T C T T AG AAT C I T T AT AAGA. A AA AA AG AAA AA

[0987] ( SEQ ID NO: 54 ) TTATTACTTTATGGCTAGCTGGTCTCCAGCTATTTGAAAAAGGAGAGG AGGTTCTCCC (SEQ ID NO: 55) mmu-miR-30e CACCACAGTCAGCGGTCTCGATGCCCCAGCTGGTGTGTCCGTTCCATC GGGTCCCCA ( SEQ ID NO: 56 ) mmu-miR-9 CCCTATCCCCCCATCGCCATGCCTGGGCCATGCCCACATCTCCCACCCA CTCACCC ( SEQ ID NO: 57 ) i mmu-miR- C TC CC AC C AC ACC C C T C C C CC AC C C C T TC T C C C TC GGC G AC T T C AC C T T T; 188-5p TTTTGCCG (SEQ ID NO: 58 ) i mmu-miR-124 TCTAATGTTGTCTTTTTTGCTTGGCATCACACACGCCTTCTTAACCCA ( SEQ ID NO: 59 ) CATACACA, GA. ACCCACA, GACAGCTCCTGGACACCCGTGTCCA. CTGGGA

[0988]

[0989] GCAGCT (SEQ ID NO: 60 )

[0990]

[0274] An antagomir or antisense oligonucleotide (ASO) relevant and specific for the target sequence

[0991] microRNA or miR is designed. The antagomir or ASO comprises sequence complementary to the mir

[0992] target sequence miR-103 comprises the sequence UGCAGCAU (SEQ ID NO:61) (listed 5’-3’). Tire

[0993] antagomir or ASO can include nucleotides complementary to additional adjacent sequence, such as up to

[0994] 5, 10 or more nucleotides adjacent, including 5’ to or 3’ to or both 5’ and 3’ to the target 8mer sequence.

[0995] For example, the antagomir or ASO may further comprise nucleotides complementary' to adjacent

[0996] sequence, such as AUA (SEQ ID NO:62), AAUA (SEQ ID NO:63), AAAUA (SEQ ID NO:64).

[0997] GAAAUA (SEQ ID NO:65), GGAAAUA (SEQ ID NO:66), UGGAAAUA (SEQ ID NO:67),

[0998] AUGGAAAUA (SEQ ID NO:68) or AAUGGAAAUA (SEQ ID NO:69).

[0999]

[0275] In another approach, the binding can be antagonized by designing ASOs that bind to and block

[1000] the miRNAs targeting Nova sequence.

[1001]

[0276] Given our discovery of the relationship between Noval and language (as described above in

[1002] Example I), clinical conditions where the ability to regulate Noval may be especially relevant include

[1003] those involving motor vocalizati...

Claims

Claims:

1. An anti-microRNA molecule comprising at least 8 moieties or nucleotides and having complementarity to an untranslated region (UTR) sequence of N0VA1 or N0VA2 targeted by micro RNA, or having complementarity to a micro-RNA that targets N0VA1 or N0VA2.

2. The anti-microRNA molecule of claim 1, wherein the micro-RNA targeting N0VA1 or N0VA2 targets the N0VA1 or NOVA23’ UTR.

3. The anti-microRNA molecule of claim 1 or 2, wherein the molecule has complementarity to N0VA1 3 ’UTR sequence.

4. The anti-microRNA molecule of claim 1 or 2, wherein the molecule target sequence is selected from sequence set out in TABLE 19 (SEQ ID NO:43-SEQ ID NO:60).

5. The anti-microRNA molecule of claim 1 or 2, wherein the molecule has complementarity to one or more microRNA targeting N0VA1 selected from miR-27a, miR-103, let-7a and miR-132, or wherein the molecule has complementarity to one or more microRNA targeting N0VA2 selected from miR-26a, miR-484, miR-148a, miR-19a, miR-221, miR-124, miR-30b, miR-296-5p, miR-340-5p, miR-30b, miR-30e, miR-9, miR-288-5p, miR-124 and let-7c.

6. The anti-microRNA molecule of any of claims 1-5, wherein the anti-microRNA molecule comprises at least 8 moieties or nucleotides forming a complementary base pair with nucleotides of UTR sequence targeted by micro-RNA or with sequence of micro-RNA targeting NOVA17. The anti-microRNA molecule of any of claims 1-6, wherein the micro-RNA targets human NOVA18. The anti-microRNA molecule of claim 7, wherein the micro-RNA targets the 3 ’ UTR of NOVA 1.

9. The anti-microRNA molecule of any of claims 1-8, wherein the anti-microRNA molecule is an antagomir or antisense oligonucleotide ( ASO).

10. The anti-microRNA molecule of any of claims 1-8, wherein the anti-microRNA molecule comprises at least one moiety which is a ribonucleotide moiety or a deoxyribonucleotide moiety.

11. The anti-microRNA molecule of any of claims 1-8, wherein the anti-microRNA molecule comprises at least one moiety or nucleotide which is modified to improve stability, increase cellular uptake, and / or enhance affinity for its target miRNA.

12. The anti-microRNA molecule of claim 11, wherein the anti-microRNA molecule comprises at least one moiety which confers increased nuclease resistance.

13. The anti-microRNA molecule of any of claims 1-12, wherein the molecule has complementarity to sequence AUGCUGCA (SEQ ID NO:23) or to sequence as set out in TABLE 19 (SEQ ID NO:43-60).

14. A method for inhibiting a microRNA molecule targeting NOVAI or NOVA2 in a cell or animal comprising introducing, administering or expressing in said cell or animal the anti-microRNA molecule of any of claims 1-13.

15. The method of claim 12, wherein the anti-microRNA molecule targets NOVA 1 or NOVA2 3' UTR or 'herein the anti-micro RNA molecule targets microRNA that binds NOVAI or NOVA2 UTR.

16. The method of claim 14 or 15, wherein the anti-microRNA is delivered to the cell or animal via a viral or non-viral delivery system.

17. The method of claim 16, wherein the viral delivery system is an adenovirus, adeno-associated virus or lentivirus system.

18. The method of claim 16, wherein the non-viral delivery system uses a liposome, extracellular vesicle, or polymer nanoparticle based delivery system.

19. A method of alleviating or modulating a disease or condition associated with altered NOVAI or N0VA2 activity, expression, or pathogenic forms of NOVAI or N0VA2 in a human, comprising introducing, administering or expressing in the human, or in the cells of the human, the anti-microRNA molecule of any of claims 1-13.

20. The method of claim 19, wherein the disease or condition is selected from psychiatric disorders, motor developmental abnormalities. Autism (including non-verbal autism), neuro-behavioral deficits, including motor dysfunction and developmental delay, language dysfunction, speech delay and cancer.

21. The method of claim 20, wherein the cancer is selected from gastric cancer, hepatocellular carcinoma, and lymphoma, as well as glioblastoma and astrocytoma and oligodendroglioma.

22. A method of altering or modifying vocalization in a human comprising introducing, administering or expressing in the human, or in the cells of the human, the anti-microRNA molecule of any of claims 1 -13.

23. A method of alleviating or modulating a disease or condition associated with altered NOVAI or NOVA2 activity, expression, or pathogenic forms of NOVA 1 orNOVA2 in a human, comprising correcting the NOVAI encoding nucleic acid and / or genetically manipulating the human or cells of the human to express unaltered and non-pathogenic NOVAI or NOVA2.

24. The method of claim 23, wherein the disease or condition is selected from psychiatric disorders, motor developmental abnormalities, Autism (including non-verbal autism), neuro-behavioral deficits, including motor dysfunction and developmental delay, language dysfunction, speech delay and cancer.

25. The method of claim 24, wherein the cancer is selected from gastric cancer, hepatocellular carcinoma, and lymphoma, as well as glioblastoma and astrocytoma and oligodendroglioma.

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