Treatment of cerebrovascular diseases with neurogenic locus Notch homolog protein 3 (NOTCH3) agents

Abstract

The present disclosure provides methods for treating a subject having cerebrovascular disease by administering a neurogenic locus Notch homolog protein 3 (NOTCH3) agent, and methods for identifying a subject at increased risk for developing cerebrovascular disease.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically as a text file of 364 kilobytes in size under the title 18923806502SEQ, created on May 30, 2022. The Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates generally to methods of treating subjects with cerebrovascular disease with neurogenic locus Notch homolog protein 3 (NOTCH3) agents and methods of identifying subjects at increased risk for developing cerebrovascular disease. [Background technology]

[0003] Cerebrovascular disease includes any abnormality of the brain resulting from a pathological process of blood vessels, which may include any one or more of the following: occlusion of the vessel lumen by a thrombus or embolus, rupture of the vessel, changes in blood-vessel wall permeability, and increased viscosity or other changes in blood quality. Cerebrovascular disease is usually easily diagnosed by how it manifests. Cerebrovascular disease usually manifests as a stroke. A stroke can be characterized as an abrupt, non-convulsive focal neurological deficit. That is, a stroke can be characterized as the death of brain tissue resulting from insufficient blood flow and insufficient oxygen to the brain. In the United States, stroke is the leading cause of death after heart disease and cancer. There are approximately 500,000 cases of stroke in the United States annually. And these 500,000 cases lead to approximately 175,000 deaths.

[0004] Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) or CADASIL syndrome is the most common form of inherited stroke disorder. The most common clinical symptoms are migraines and transient ischemic attacks or strokes, which usually occur during the fourth to fifth decade of life, although MRI can detect signs of the disease several years before clinical symptoms of the disease become apparent. The underlying pathology of CADASIL is the progressive hypertrophy of smooth muscle cells in blood vessels. No specific treatment for CADASIL is available. Most treatments for symptoms in CADASIL patients (including migraines and strokes) are similar to those for symptoms in patients without CADASIL, but these treatments are almost exclusively experimental due to limited data on their effectiveness in CADASIL patients.

[0005] Neurogenic locus Notch homolog protein 3 (NOTCH3) is a receptor for the membrane-bound ligands Jagged1, Jagged2, and Delta1 that control cell fate decisions. Upon ligand activation via the shed notch intracellular domain (NICD), the ligand forms a transcriptional activator complex with RBPJ / RBPSUH and activates genes in the enhancer of the mitotic locus. NOTCH3 influences the implementation of differentiation, proliferation, and apoptosis programs. Summary of the Invention

[0006] The present disclosure provides a method of treating a subject having a cerebrovascular disease, the method comprising administering a neurogenic locus Notch homolog protein 3 (NOTCH3) agent to the subject.

[0007] The disclosure also provides a method of treating a subject having a subcortical stroke, the method comprising administering to the subject a NOTCH3 agent.

[0008] The disclosure also provides a method of treating a subject having an ischemic stroke, the method comprising administering to the subject a NOTCH3 agent.

[0009] The disclosure also provides a method of treating a subject having a hemorrhagic stroke, the method comprising administering to the subject a NOTCH3 agent.

[0010] The present disclosure also provides a method of treating a subject having a parenchymal stroke, the method comprising administering to the subject a NOTCH3 agent.

[0011] The present disclosure also provides a method of treating a subject having cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the method comprising administering to the subject a NOTCH3 agent.

[0012] The disclosure also provides a method of treating a subject with a therapeutic agent for treating or inhibiting cerebrovascular disease, the subject being afflicted with cerebrovascular disease, the method comprising obtaining or obtaining a biological sample from the subject, and performing or performing sequence analysis on the biological sample to determine whether the subject has a genotype that includes a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide, thereby determining whether the subject has a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide, and administering to the subject, who is a NOTCH3 reference type, a standard dose of the therapeutic agent. and administering or continuously administering a therapeutic agent for treating or inhibiting cerebrovascular disease and administering a NOTCH3 agent to a subject, and administering to a subject who is heterozygous or homozygous for a NOTCH3 missense variant nucleic acid molecule a therapeutic agent for treating or inhibiting cerebrovascular disease in an amount equal to or greater than a standard dosage, and administering to the subject a NOTCH3 agent, wherein the presence of a genotype having a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide indicates that the subject has an increased risk of developing cerebrovascular disease.

[0013] The present disclosure also provides a method of identifying a subject having an increased risk of developing cerebrovascular disease, the method comprising determining or having determined the presence or absence in a biological sample obtained from the subject of a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide, wherein if the subject is of the NOTCH3 reference type, the subject does not have an increased risk of developing cerebrovascular disease, and if the subject is heterozygous or homozygous for the NOTCH3 missense variant nucleic acid molecule, the subject has an increased risk of developing cerebrovascular disease.

[0014] The disclosure also provides a genomic nucleic acid molecule having a nucleotide sequence encoding a NOTCH3 polypeptide, the nucleotide sequence including a thymine or a complement thereof at a position corresponding to position 21,944 according to SEQ ID NO:2; an mRNA molecule having a nucleotide sequence encoding a NOTCH3 polypeptide, the nucleotide sequence including a uracil or a complement thereof at a position corresponding to position 3,781 according to SEQ ID NO:8; a uracil or a complement thereof at a position corresponding to position 3,767 according to SEQ ID NO:9; a uracil or a complement thereof at a position corresponding to position 3,532 according to SEQ ID NO:10; a uracil or a complement thereof at a position corresponding to position 3,769 according to SEQ ID NO:11; or a thymine or a complement thereof at a position corresponding to position 3,544 according to SEQ ID NO:12. or a cDNA molecule having a nucleotide sequence encoding a NOTCH3 polypeptide, the nucleotide sequence comprising: a thymine or its complement at a position corresponding to position 3,781 according to SEQ ID NO:18; a thymine or its complement at a position corresponding to position 3,767 according to SEQ ID NO:19; a thymine or its complement at a position corresponding to position 3,532 according to SEQ ID NO:20; a thymine or its complement at a position corresponding to position 3,769 according to SEQ ID NO:21; or a thymine or its complement at a position corresponding to position 3,544 according to SEQ ID NO:22.

[0015] The disclosure also provides a genomic nucleic acid molecule having a nucleotide sequence encoding a NOTCH3 polypeptide, the nucleotide sequence comprising a thymine or a complement thereof at a position corresponding to position 21,944 according to SEQ ID NO:2; an mRNA molecule having a nucleotide sequence encoding a NOTCH3 polypeptide, the nucleotide sequence comprising a uracil or a complement thereof at a position corresponding to position 3,781 according to SEQ ID NO:8; a uracil or a complement thereof at a position corresponding to position 3,767 according to SEQ ID NO:9; a uracil or a complement thereof at a position corresponding to position 3,532 according to SEQ ID NO:10; a uracil or a complement thereof at a position corresponding to position 3,769 according to SEQ ID NO:11; or a uracil or a complement thereof at a position corresponding to position 3,544 according to SEQ ID NO:12. or a cDNA molecule having a nucleotide sequence encoding a NOTCH3 polypeptide, the nucleotide sequence comprising: a thymine or its complement at a position corresponding to position 3,781 according to SEQ ID NO:18; a thymine or its complement at a position corresponding to position 3,767 according to SEQ ID NO:19; a thymine or its complement at a position corresponding to position 3,532 according to SEQ ID NO:20; a thymine or its complement at a position corresponding to position 3,769 according to SEQ ID NO:21; or a thymine or its complement at a position corresponding to position 3,544 according to SEQ ID NO:22.

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain features of the present disclosure. [Brief description of the drawings]

[0017] [Figure 1] The results of EXWAS for the NOTCH3 locus are shown. [Figure 2-1] 1 shows the occurrence of cases and controls in EXWAS, grouped by the presence of rs201680145. [Figure 2-2] Same as above. [Diagram 3]The results of PheWAS for NOTCH3 p.Arg1231Cys are shown. [Figure 4] A table summarizing the PheWAS results for NOTCH3 p.Arg1231Cys is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Various terms relating to the aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein.

[0019] Unless expressly stated otherwise, it is not intended that any method or aspect described herein be interpreted as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically states in the claim or description that the steps are to be limited to a particular order, no order is intended to be implied in any respect. This is true for all potentially unclear criteria for interpretation, including questions of logic regarding the arrangement of steps or operational flow, the plain meaning derived from grammatical construction or punctuation, or the number or type of aspects described herein.

[0020] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0021] As used herein, the term "about" means that the recited numerical values ​​are approximate and that small variations would not significantly affect the practice of the disclosed embodiments. When numerical values ​​are used, unless otherwise indicated by the context, the term "about" means that the numerical values ​​can vary by ±10% and remain within the scope of the disclosed embodiments.

[0022] As used herein, the term "comprising" may in certain embodiments be replaced with "consisting" or "consisting essentially of," as desired.

[0023] As used herein, with respect to a nucleic acid molecule or polypeptide, the term "isolated" means that the nucleic acid molecule or polypeptide is in a state other than its native environment, e.g., away from blood and / or animal tissue. In some embodiments, an isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule or polypeptide can be in a highly purified form, i.e., greater than 95% pure or greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternatively phosphorylated or derivatized forms.

[0024] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include polymeric forms of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multistranded. A strand of a nucleic acid also refers to its complement.

[0025] As used herein, the term "subject" encompasses any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs, etc.), pet animals (e.g., dogs, cats, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), and non-human primates (e.g., apes and monkeys, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a patient under the care of a physician.

[0026] According to the present disclosure, common variant SNP rs201680145 in human NOTCH3 gene is identified to be associated with increased risk of developing cerebrovascular disease, such as CADASIL, subcortical stroke, ischemic stroke, hemorrhagic stroke, or parenchymal stroke. For example, it has been observed that a genetic change in the NOTCH3 reference genomic nucleic acid molecule (see SEQ ID NO: 1) that changes a cytosine to a thymine at position 21,944 indicates that subjects with such a change may have increased risk of developing cerebrovascular disease. As shown by genome-wide analysis, it is believed that variants of NOTCH3 gene or protein do not have any known significant association with cerebrovascular disease, such as subcortical stroke, ischemic stroke, hemorrhagic stroke, or parenchymal stroke.

[0027] In addition, CADASIL was originally reported to be caused by autosomal dominant mutations in the NOTCH3 gene (Joutel et al., Nature, 1996, 383, 707-10), including rs201680145 (Singhal et al., Brain, 2004, 127, 2031-2038). These mutations were thought to result in abnormal accumulation of Notch3 in the plasma membrane of vascular smooth muscle cells in both cerebral and extracerebral blood vessels, as seen by electron microscopy as accumulation of osmiophilic granules (Ruchoux et al., Acta Neuropathol., 1995, 89, 500-12) (Joutel et al., J. Clin. Invest., 2000, 105, 597-605). In addition, in many cases, leukoencephalopathy follows. All CADASIL pathogenic mutations in NOTCH3 share the common property of adding a Cys residue in the NOTCH3 extracellular domain or deleting it from one of the 34 EGFR domains, where each EGFR typically has six Cys residues that are highly conserved in human, mouse, and zebrafish NOTCH3. Hundreds of such Cys-adding or deleting variants have been identified. However, the notion that the NOTCH3 p.Arg1231Cys mutation is the cause of CADASIL has been abandoned as inaccurate due to the very high occurrence of this mutation (rs201680145) in South Asians, including a significant excess over the frequency predicted based on the presence of CADASIL (Rutten et al., Ann. Clin. Transl. Neurol., 2016, 3, 844-853). Taken together, the genetic analyses described herein surprisingly show that the NOTCH3 gene, and in particular, gain-of-function variants in the NOTCH3 gene, are associated with an increased risk of developing cerebrovascular disease, such as CADASIL, subcortical stroke, ischemic stroke, hemorrhagic stroke, or parenchymal stroke.Thus, subjects having a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide at increased risk of developing cerebrovascular disease, such as CADASIL, subcortical stroke, ischemic stroke, hemorrhagic stroke, or parenchymal stroke, may be treated such that cerebrovascular disease is prevented, its symptoms are reduced, and / or the onset of symptoms is inhibited. Thus, the present disclosure provides methods of utilizing the identification of such NOTCH3 missense variant nucleic acid molecules in subjects to identify or stratify the risk of developing cerebrovascular disease, such as CADASIL, subcortical stroke, ischemic stroke, hemorrhagic stroke, or parenchymal stroke, or to diagnose a subject as having increased risk of developing cerebrovascular disease, such as CADASIL, subcortical stroke, ischemic stroke, hemorrhagic stroke, or parenchymal stroke, in such subjects, such that subjects at risk of having or having active disease may be treated accordingly.

[0028] For the purpose of this disclosure, any particular subject can be classified as having one of three NOTCH3 genotypes: i) NOTCH3 reference type, ii) heterozygous for NOTCH3 missense variant nucleic acid molecule encoding NOTCH3 predicted gain-of-function polypeptide, or iii) homozygous for NOTCH3 missense variant nucleic acid molecule encoding NOTCH3 predicted gain-of-function polypeptide.If the subject does not have a copy of NOTCH3 missense variant nucleic acid molecule encoding NOTCH3 predicted gain-of-function polypeptide, the subject is NOTCH3 reference type.If the subject has a single copy of NOTCH3 missense variant nucleic acid molecule encoding NOTCH3 predicted gain-of-function polypeptide, the subject is heterozygous for NOTCH3 missense variant nucleic acid molecule encoding NOTCH3 predicted gain-of-function polypeptide. As used herein, a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide is any NOTCH3 nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a NOTCH3 polypeptide having a partial gain-of-function, a complete gain-of-function, a predicted partial gain-of-function, or a predicted complete gain-of-function. A subject having a NOTCH3 polypeptide having a partial gain-of-function (or a predicted partial gain-of-function) is a hypomorphic for NOTCH3.

[0029] In any of the embodiments described herein, the NOTCH3 missense variant nucleic acid molecule encoding the NOTCH3 predicted gain-of-function polypeptide can be any nucleic acid molecule encoding NOTCH3 Arg1178Cys, Arg1231Cys, or Arg1182Cys.In some embodiments, the NOTCH3 missense variant nucleic acid molecule encodes NOTCH3 Arg1178Cys, Arg1231Cys, or Arg1182Cys.If a subject has two copies of the NOTCH3 missense variant nucleic acid molecule encoding the NOTCH3 predicted gain-of-function polypeptide, the subject is homozygous for the NOTCH3 missense variant nucleic acid molecule encoding the NOTCH3 predicted gain-of-function polypeptide.

[0030] In any of the embodiments described herein, the NOTCH3 variant nucleic acid molecule can be any Cys-change variant (which changes from or to a cysteine ​​residue). In some embodiments, the Cys-change variant is located within the 34 EGF-like repeats in the NOTCH3 extracellular domain (as defined by Uniprot; see the World Wide Web at "uniprot.org / blast / ?about=Q9UM47[1335-1373]&key=Domain"). In any of the embodiments described herein, the NOTCH3 variant nucleic acid molecule can be any of the variants listed in Table 1 (ENST00000263388). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10]

[0031] For subjects who are genotyped or determined to be heterozygous or homozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by NOTCH3, such subjects have an increased risk of developing cerebrovascular disease, such as CADASIL, subcortical stroke, ischemic stroke, hemorrhagic stroke, or parenchymal stroke. For subjects who are genotyped or determined to be heterozygous or homozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by NOTCH3, such subjects can be treated with an agent effective for treating cerebrovascular disease, such as CADASIL, subcortical stroke, ischemic stroke, hemorrhagic stroke, or parenchymal stroke. For subjects who are genotyped or determined to be heterozygous or homozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by NOTCH3, such subjects can also or independently be treated with a NOTCH3 agent.

[0032] In any of the embodiments described herein, the NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide can be any NOTCH3 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule, etc.) encoding a NOTCH3 polypeptide with partial gain-of-function, complete gain-of-function, predicted partial gain-of-function, or predicted complete gain-of-function. For example, the NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide can be any nucleic acid molecule encoding NOTCH3 Arg1178Cys, Arg1231Cys, or Arg1182Cys. In some embodiments, the NOTCH3 missense variant nucleic acid molecule encodes NOTCH3 Arg1178Cys. In some embodiments, the NOTCH3 missense variant nucleic acid molecule encodes NOTCH3 Arg1231Cys. In some embodiments, the NOTCH3 missense variant nucleic acid molecule encodes NOTCH3 Arg1182Cys.

[0033] In any of the embodiments described herein, the NOTCH3 predicted gain-of-function polypeptide can be any NOTCH3 polypeptide with partial gain-of-function, complete gain-of-function, predicted partial gain-of-function, or predicted complete gain-of-function. In any of the embodiments described herein, the NOTCH3 predicted gain-of-function polypeptide can be any of the NOTCH3 polypeptides described herein, including, for example, NOTCH3 Arg1178Cys, Arg1231Cys, or Arg1182Cys. In some embodiments, the NOTCH3 predicted gain-of-function polypeptide is NOTCH3 Arg1178Cys. In some embodiments, the NOTCH3 predicted gain-of-function polypeptide is NOTCH3 Arg1231Cys. In some embodiments, the NOTCH3 predicted gain-of-function polypeptide is NOTCH3 Arg1182Cys.

[0034] In any of the embodiments described herein, the cerebrovascular disease is CADASIL, subcortical stroke, ischemic stroke, hemorrhagic stroke, or parenchymal stroke.In any of the embodiments described herein, the cerebrovascular disease is CADASIL.In any of the embodiments described herein, the cerebrovascular disease is subcortical stroke.In any of the embodiments described herein, the cerebrovascular disease is ischemic stroke.In any of the embodiments described herein, the cerebrovascular disease is hemorrhagic stroke.In any of the embodiments described herein, the cerebrovascular disease is parenchymal stroke.

[0035] Symptoms of cerebrovascular disease include, but are not limited to, dizziness, nausea, or vomiting; unusually severe headaches; blurred consciousness, disorientation, or memory loss; numbness or weakness of an arm, leg, or face, especially on one side; abnormal or rapid speech; difficulty understanding; blindness or decreased vision; or loss of balance, coordination, or ability to walk.

[0036] The present disclosure provides a method of treating a subject having cerebrovascular disease, the method comprising administering a NOTCH3 agent to the subject.

[0037] The disclosure also provides a method of treating a subject having a subcortical stroke, the method comprising administering to the subject a NOTCH3 agent.

[0038] The disclosure also provides a method of treating a subject having an ischemic stroke, the method comprising administering to the subject a NOTCH3 agent.

[0039] The disclosure also provides a method of treating a subject having a hemorrhagic stroke, the method comprising administering to the subject a NOTCH3 agent.

[0040] The present disclosure also provides a method of treating a subject having a parenchymal stroke, the method comprising administering to the subject a NOTCH3 agent.

[0041] The disclosure also provides a method of treating a subject having CADASIL, the method comprising administering to the subject a NOTCH3 agent.

[0042] In some embodiments, the NOTCH3 agent comprises an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense molecule, a small interfering RNA (siRNA) molecule, or a short hairpin RNA (shRNA) molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an siRNA molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an shRNA molecule. Such inhibitory nucleic acid molecules can be designed to target any region of a NOTCH3 nucleic acid molecule, such as an mRNA molecule. In some embodiments, the inhibitory nucleic acid molecule hybridizes to a sequence within a NOTCH3 genomic nucleic acid molecule or an mRNA molecule, and reduces the expression of a NOTCH3 polypeptide in cells in a subject. In some embodiments, the NOTCH3 agent comprises an antisense RNA that hybridizes to a NOTCH3 genomic nucleic acid molecule or an mRNA molecule, and reduces the expression of a NOTCH3 polypeptide in cells in a subject. In some embodiments, the NOTCH3 agent comprises an siRNA that hybridizes to a NOTCH3 genomic nucleic acid molecule or an mRNA molecule, and reduces the expression of a NOTCH3 polypeptide in cells in a subject. In some embodiments, the NOTCH3 agent comprises an shRNA that hybridizes to a NOTCH3 genomic nucleic acid molecule or mRNA molecule and reduces expression of a NOTCH3 polypeptide in cells in the subject.

[0043] In some embodiments, representative methodologies that can be used to design inhibitory nucleic acid molecules against NOTCH3 are described, for example, in US Patent Application Publication No. 2021 / 0115444, which illustrates allele-specific suppression. Briefly, this technology is based on oligonucleotides with phosphorothioate backbone linkages, where the backbone linkages contain specific stereoisomers at specific positions. The inclusion of specific chiral structures at specific positions within the oligonucleotide can result in improved stability, activity, and cleavage specificity of the target nucleotide. This technology is based on chiral-controlled (and / or stereochemically pure) oligonucleotide compositions that include oligonucleotides defined by having 1) a common base sequence and length; 2) a common pattern of backbone linkages; 3) a common pattern of backbone chiral centers; and 4) a common pattern of backbone P modifications. The purity of the chiral-controlled oligonucleotide composition can be controlled by the stereoselectivity of each coupling step in its preparation process. The initial overall structure of the stereochemically pure oligonucleotide (e.g., positioning and chirality of the internucleotide bond) is developed by rational design rules developed in structural studies of the substrate-ligand complex of interest, followed by screening of candidate oligonucleotides. The stereochemically pure preparations are generated in a solid support synthesis process, where the solid support is treated with various reagents in several synthesis cycles to achieve stepwise extension of the growing oligonucleotide chain by individual nucleotide units. The steps of each cycle include treatment with an activating reagent, reaction with a chiral agent, stereospecific condensation, capping of unreacted -OH groups with protecting groups, a modification step used to introduce modified internucleotide bonds, and a deblocking step, for example, to deprotect the 5'-OH group for the subsequent cycle. The oligonucleotides can be antisense nucleic acid molecules, siRNAs, RNaseH guide sequences, etc., useful for controlled cleavage of target nucleic acid polymers, such as transcripts derived from alleles.Increased specificity for a particular sequence (e.g., an allele or SNP) is achieved by the overall reduction in cleavage sites targeted by the chirally controlled oligonucleotide compared to the unmodified oligonucleotide.

[0044] In some embodiments, the NOTCH3 agent comprises a nuclease agent that induces one or more nicks or double-strand breaks in the recognition sequence(s), or a DNA binding protein that binds to the recognition sequence in the NOTCH3 genomic nucleic acid molecule. The recognition sequence can be located in the coding region of the NOTCH3 gene, or in a control region that affects the expression of the gene. The recognition sequence of the DNA binding protein or nuclease agent can be located in an intron, exon, promoter, enhancer, regulatory region, or any protein non-coding region. The recognition sequence can include or be adjacent to the start codon of the NOTCH3 gene. For example, the recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents can be used, each of which targets a nuclease recognition sequence that includes or is adjacent to the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence that includes or is adjacent to a start codon and one targeting a nuclease recognition sequence that includes or is adjacent to a stop codon, and cleavage by the nuclease agents can result in the deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-stranded break in the desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA binding protein that binds to the desired recognition sequence can be used in the methods and compositions disclosed herein.

[0045] Nuclease agents and DNA binding proteins suitable for use herein include, but are not limited to, zinc finger proteins or zinc finger nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALEN), or clustered regularly interspersed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary, including recognition sequences that are about 30 to about 36 bp for zinc finger proteins or ZFN pairs, about 15 to about 18 bp for each ZFN, about 36 bp for TALE proteins or TALEN, and about 20 bp for CRISPR / Cas guide RNAs.

[0046] In some embodiments, a CRISPR / Cas system can be used to modify a NOTCH3 genomic nucleic acid molecule in a cell. The methods and compositions disclosed herein can use the CRISPR-Cas system by utilizing a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of a NOTCH3 nucleic acid molecule.

[0047] Cas proteins generally contain at least one RNA recognition domain or RNA binding domain that can interact with gRNA. Cas proteins may also contain nuclease domains (such as DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 proteins and wild-type Cpf1 proteins (such as FnCpf1). Cas proteins may have full cleavage activity to generate double-stranded breaks in NOTCH3 genomic nucleic acid molecules, or may be nickases that generate single-stranded breaks in NOTCH3 genomic nucleic acid molecules. Additional examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasF), Cse5 (CasF), Cse6 (CasF), Cse7 (CasF), Cse8 (CasF), Cse9 (CasF), Cse1 (CasF), Cse2 (CasF), Cse3 (CasF), Cse4 (CasF), Cse5 (CasF), Cse6 (CasF), Cse7 (CasF), Cse8 (CasF), Cse9 (CasF), Cse1 ...1 (CasF), Cse1 (CasF), Cse1 (CasF), Cse1 (CasF), Cse1 (CasF), Cse1 (CasF), Cse Cas proteins include, but are not limited to, se4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, as well as homologs or modified forms thereof. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, Cas proteins can be fused to a cleavage domain, an epigenetic modification domain, a transcription activation domain, or a transcription repressor domain. Cas proteins can be provided in any form. For example, the Cas protein may be provided in the form of a protein, such as a Cas protein complexed with a gRNA. Alternatively, the Cas protein may be provided in the form of a nucleic acid molecule, such as RNA or DNA, that encodes the Cas protein.

[0048] In some embodiments, targeted genetic modification of a NOTCH3 genomic nucleic acid molecule can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize to one or more gRNA recognition sequences within a target genomic locus in a NOTCH3 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located within a region of SEQ ID NO:1. The gRNA recognition sequence can also include or be adjacent to a position corresponding to position 21,944 according to SEQ ID NO:1. For example, the gRNA recognition sequence can be located about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides away from a position corresponding to position 21,944 according to SEQ ID NO:1. The gRNA recognition sequence can include or be adjacent to a start codon or a stop codon of a NOTCH3 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start or stop codon.

[0049] The gRNA recognition sequence in the target genomic locus of the NOTCH3 genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence, which is a 2-6 base pair DNA sequence that immediately follows the DNA sequence targeted by the Cas9 nuclease. A canonical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase, followed by two guanine ("G") nucleobases. The gRNA can guide Cas9 to any location in the genome for gene editing, but editing will not occur at any site other than the site where Cas9 recognizes the PAM. In addition, 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells. Generally, the PAM is about 2 to about 6 nucleotides downstream of the DNA sequence targeted by the gRNA. The PAM can be adjacent to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 3' end. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 5' end. For example, the cleavage site of the Cas protein can be about 1 to about 10 base pairs, about 2 to about 5 base pairs, or 3 base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when Cas9 from S. pyogenes or a closely related Cas9 is used), the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is immediately 3' to the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand will be 5'-CCN-3', where N is any DNA nucleotide and is immediately 5' to the gRNA recognition sequence of the complementary strand of the target DNA.

[0050] gRNA is an RNA molecule that binds to Cas protein and targets Cas protein to a specific position in NOTCH3 genomic nucleic acid molecule. An exemplary gRNA is an effective gRNA that induces Cas enzyme to bind to or cleave NOTCH3 genomic nucleic acid molecule, and the gRNA comprises a DNA targeting segment that hybridizes with a gRNA recognition sequence in NOTCH3 genomic nucleic acid molecule that includes or is adjacent to the position corresponding to position 21,944 according to SEQ ID NO:1. For example, the gRNA can be selected to hybridize with a gRNA recognition sequence located at about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the position corresponding to position 21,944 according to SEQ ID NO:1. Other exemplary gRNA comprises a DNA targeting segment that hybridizes with the gRNA recognition sequence present in the NOTCH3 genomic nucleic acid molecule that includes or is adjacent to the start codon or stop codon.For example, gRNA can be selected to hybridize with the gRNA recognition sequence that is located at about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon, or about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the stop codon. A suitable gRNA may comprise from about 17 to about 25 nucleotides, from about 17 to about 23 nucleotides, from about 18 to about 22 nucleotides, or from about 19 to about 21 nucleotides. In some embodiments, the gRNA comprises 20 nucleotides.

[0051] Examples of suitable gRNA recognition sequences located within the NOTCH3 reference gene are shown in Table 2 as SEQ ID NOs: 29-43. [Table 2]

[0052] The Cas protein and gRNA form a complex, and the Cas protein cleaves the target NOTCH3 genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at a site inside or outside the nucleic acid sequence present in the target NOTCH3 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds. For example, the formation of a CRISPR complex (including the gRNA hybridized to the gRNA recognition sequence and complexed with the Cas protein) can result in the cleavage of one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from) the nucleic acid sequence present in the NOTCH3 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds.

[0053] Such a method may result in a NOTCH3 genomic nucleic acid molecule in which, for example, the region of SEQ ID NO:2 is destroyed, the start codon is destroyed, the stop codon is destroyed, or the coding sequence is destroyed or deleted. Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize with additional gRNA recognition sequences in the target genomic locus of the NOTCH3 genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (such as a second gRNA that hybridizes with a second gRNA recognition sequence), the cleavage by Cas protein can generate two or more double-strand breaks or two or more single-strand breaks.

[0054] In some embodiments, the NOTCH3 agent is an anti-aggregating or agonistic antibody that increases processing. Such antibodies include, but are not limited to, the 5E1 antibody (or a humanized version thereof) (Ghezali et al., Ann. Neurol. 2018, 84, 246-259) and the A13 antibody (Machuca-Parra et al., J. Exp. Med., 2017, 214, 2271-2282).

[0055] In some embodiments, the treatment method further comprises detecting the presence or absence of a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide in a biological sample from the subject. As used throughout this disclosure, a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide is any NOTCH3 nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a NOTCH3 polypeptide having partial gain-of-function, complete gain-of-function, predicted partial gain-of-function, or predicted complete gain-of-function.

[0056] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits cerebrovascular disease. In some embodiments, the subject has cerebrovascular disease. In some embodiments, the method includes obtaining or obtaining a biological sample from the subject, and performing or performing a sequencing analysis on the biological sample to determine whether the subject has a genotype that includes a NOTCH3 missense variant nucleic acid molecule that encodes a NOTCH3 predicted gain-of-function polypeptide, thereby determining whether the subject has a NOTCH3 missense variant nucleic acid molecule that encodes a NOTCH3 predicted gain-of-function polypeptide. The method further includes administering or continuously administering a therapeutic agent that treats or inhibits cerebrovascular disease at a standard dose to a subject that is a NOTCH3 reference type, and administering a NOTCH3 agent to the subject. Alternatively, the method further includes administering or continuously administering a therapeutic agent that treats or inhibits cerebrovascular disease at an amount equal to or greater than the standard dose to a subject that is heterozygous or homozygous for a NOTCH3 missense variant nucleic acid molecule, and administering a NOTCH3 agent to the subject. The presence of a genotype having a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide indicates that the subject has an increased risk for developing cerebrovascular disease.

[0057] Detecting the presence or absence of a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide in a biological sample from a subject and / or determining whether a subject has a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from the subject.

[0058] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits cerebrovascular disease. In some embodiments, the subject has cerebrovascular disease. In some embodiments, the method includes determining whether the subject has a NOTCH3 predicted gain-of-function polypeptide by obtaining or obtaining a biological sample from the subject and performing or performing an assay on the biological sample to determine whether the subject has a NOTCH3 predicted gain-of-function polypeptide. If the subject does not have a NOTCH3 predicted gain-of-function polypeptide, the therapeutic agent that treats or inhibits cerebrovascular disease is administered or continues to be administered to the subject at a standard dosage. If the subject has a NOTCH3 predicted gain-of-function polypeptide, the therapeutic agent that treats or inhibits cerebrovascular disease is administered or continues to be administered to the subject at an amount equal to or greater than the standard dosage. The presence of a NOTCH3 predicted gain-of-function polypeptide indicates that the subject has an induced risk of developing cerebrovascular disease. In some embodiments, the subject has a NOTCH3 predicted gain-of-function polypeptide. In some embodiments, the subject does not have a NOTCH3 predicted gain-of-function polypeptide.

[0059] Detecting the presence or absence of a NOTCH3 predicted gain-of-function polypeptide in a biological sample from a subject and / or determining whether a subject has a NOTCH3 predicted gain-of-function polypeptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the polypeptide can be present in a cell obtained from the subject.

[0060] Examples of therapeutic agents that treat or inhibit cerebrovascular disease include tissue plasminogen activator (tPA); anticoagulants such as apixaban, dabigatran, edoxaban, rivaroxaban, and warfarin; diuretics (e.g., bumetanide, ethacrynic acid, furosemide, and torsemide), ACE inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril), beta blockers (e.g., acebutolol, atenolol, blood pressure medications such as statins (e.g., atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin), or any combination thereof.

[0061] In some embodiments, the dose of the therapeutic agent for treating or inhibiting cerebrovascular disease can be increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% (i.e., higher than the standard dose) for subjects who are heterozygous or homozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by NOTCH3, compared to subjects who are NOTCH3 reference types (which may receive a standard dose). In some embodiments, the dose of the therapeutic agent for treating or inhibiting cerebrovascular disease can be increased by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, the dose of the therapeutic agent for treating or inhibiting cerebrovascular disease can be administered more frequently to subjects who are heterozygous or homozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted by NOTCH3, compared to subjects who are NOTCH3 reference types.

[0062] In some embodiments, the dose of a therapeutic agent for treating or inhibiting cerebrovascular disease can be increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% for a subject homozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3, compared to a subject heterozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3. In some embodiments, the dose of a therapeutic agent for treating or inhibiting cerebrovascular disease can be increased by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, the dose of a therapeutic agent for treating or inhibiting cerebrovascular disease can be administered more frequently to a subject homozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3, compared to a subject heterozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3.

[0063] Administration of the therapeutic agent and / or NOTCH3 agent for treating or inhibiting cerebrovascular disease can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months. Repeated administration can be the same dose or different dose. Administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. For example, according to a certain dosing regimen, subject can receive therapy for a long period of time (for example, 6 months, 1 year, or more).

[0064] Administration of the therapeutic agent and / or NOTCH3 agent for treating or inhibiting cerebrovascular disease can be by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically and pharma- ceutical acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration chosen. The term "pharmaceutical acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially deleterious to the recipient thereof.

[0065] The terms "treat," "treating," and "treatment" as well as "prevent," "preventing," and "prevention," as used herein, refer to eliciting a desired biological response, such as a therapeutic effect and a prophylactic effect, respectively. In some embodiments, the therapeutic effect comprises one or more of the following following administration of the agent or a composition comprising the agent: reduction / reduction of cerebrovascular disease, reduction / reduction in the severity of cerebrovascular disease (e.g., reduction or inhibition of onset of cerebrovascular disease), reduction / reduction in symptoms and effects associated with cerebrovascular disease, delay in onset of symptoms and effects associated with cerebrovascular disease, reduction in the severity of symptoms of effects associated with cerebrovascular disease, reduction in the severity of acute episodes, reduction in the number of symptoms and effects associated with cerebrovascular disease, reduction in the latency period of symptoms and effects associated with cerebrovascular disease, amelioration of symptoms and effects associated with cerebrovascular disease, reduction in secondary symptoms, reduction in secondary infections, prevention of recurrence of cerebrovascular disease, reduction in the number or frequency of recurrent episodes, increase in the latency period between symptomatic episodes, increase in the time to sustained progression, promotion of remission, induction of remission, increase in remission, accelerated recovery, or increase in the effectiveness of or decrease in resistance to alternative therapies, and / or increase in the survival time of the affected host animal. A prophylactic effect may include complete or partial avoidance / inhibition, or delay (e.g., complete or partial avoidance / inhibition or delay, etc.) of the onset / progression of cerebrovascular disease following implementation of a treatment protocol, and prolongation of survival of the affected host animal. Treatment of cerebrovascular disease encompasses treatment of a subject already diagnosed as having any form of cerebrovascular disease at any clinical stage or symptom, delaying the onset or progression or aggravation or worsening of symptoms or signs of cerebrovascular disease, and / or preventing and / or reducing the severity of cerebrovascular disease.

[0066] The present disclosure also provides a method for identifying a subject with an increased risk of developing cerebrovascular disease. In some embodiments, the method includes determining or has determined the presence or absence of a NOTCH3 missense variant nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) encoding a NOTCH3 predicted gain-of-function polypeptide in a biological sample obtained from the subject. If the subject lacks a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide (i.e., the subject is classified as a NOTCH3 reference type by genotyping), the subject is not at an increased risk of developing cerebrovascular disease. If the subject has a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide (i.e., the subject is heterozygous or homozygous for a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide), the subject is at an increased risk of developing cerebrovascular disease.

[0067] Having two copies of a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3 may place a subject at greater risk of developing cerebrovascular disease than having a single copy of a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3. Without intending to be limited to any particular logic or mechanism of action, it is believed that a single copy of a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3 (i.e., heterozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3) confers a greater risk of developing cerebrovascular disease compared to subjects who are of the NOTCH3 reference type, and it is also believed that having two copies of a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3 (i.e., homozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3) may confers a greater risk of developing cerebrovascular disease compared to subjects having a single copy.

[0068] Determining whether a subject has a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide in a biological sample from the subject and / or determining whether a subject has a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide can be performed by any of the methods described herein. In some embodiments, these methods may be performed in vitro. In some embodiments, these methods may be performed in situ. In some embodiments, these methods may be performed in vivo. In any of these embodiments, the nucleic acid molecule may be present in a cell obtained from the subject.

[0069] In some embodiments, if a subject is identified as having an increased risk of developing cerebrovascular disease, the subject is further treated with a therapeutic agent and / or a NOTCH3 agent for treating or inhibiting cerebrovascular disease as described herein. For example, if a subject is heterozygous or homozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3, and therefore has an increased risk of developing cerebrovascular disease, the subject is administered a therapeutic agent and / or a NOTCH3 agent for treating or inhibiting cerebrovascular disease at a dose equal to or higher than the standard dose. In some embodiments, if a subject is homozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3, the subject is administered a therapeutic agent for treating or inhibiting cerebrovascular disease at a dose equal to or higher than the dose administered to a subject who is heterozygous for a NOTCH3 missense variant nucleic acid molecule encoding a gain-of-function polypeptide predicted in NOTCH3. In some embodiments, the subject is NOTCH3 reference type. In some embodiments, the subject is heterozygous for a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide. In some embodiments, the subject is homozygous for a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide.

[0070] The present disclosure also provides a method for detecting the presence or absence of a NOTCH3 missense variant genomic nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide in a biological sample from a subject, and / or a NOTCH3 missense variant mRNA molecule encoding a NOTCH3 predicted gain-of-function polypeptide in a biological sample from a subject, and / or a NOTCH3 missense variant cDNA molecule encoding a NOTCH3 predicted gain-of-function polypeptide produced from an mRNA molecule in a biological sample from a subject. It is understood that gene sequences within a population and the mRNA molecules encoded by such genes may vary due to polymorphisms (such as single nucleotide polymorphisms). The sequences of the NOTCH3 variant genomic nucleic acid molecules, NOTCH3 variant mRNA molecules, and NOTCH3 variant cDNA molecules provided herein are only exemplary sequences. Other sequences of the NOTCH3 variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules are also possible.

[0071] Biological samples can be derived from any cell, tissue, or biological fluid from a subject. Biological samples can include any clinically significant tissue, such as bone marrow samples, tumor biopsies, fine needle aspirates, or samples of bodily fluids (such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine). In some embodiments, the sample includes a buccal swab. The biological samples used in the methods disclosed herein can vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. Depending on the assay being used, the biological sample can be processed differently. For example, when detecting any NOTCH3 variant nucleic acid molecules, a pretreatment designed to isolate or enrich the biological sample for NOTCH3 variant nucleic acid molecules can be employed. A variety of techniques can be used for this purpose. When detecting the level of any NOTCH3 variant mRNA molecules, a variety of techniques can be used to enrich the biological sample containing mRNA molecules. A variety of methods can be used to detect the presence or level of mRNA molecules or the presence of specific variant genomic DNA loci.

[0072] In some embodiments, detecting a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide in a subject comprises assaying or analyzing a biological sample obtained from the subject to determine whether a NOTCH3 missense variant genomic nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide in the biological sample, a NOTCH3 missense variant mRNA molecule encoding a NOTCH3 predicted gain-of-function polypeptide in the biological sample, and / or a NOTCH3 missense variant cDNA molecule encoding a NOTCH3 predicted gain-of-function polypeptide produced from an mRNA molecule in the biological sample contains one or more mutations that result in a gain of function (partial or complete) or that are predicted to result in a gain of function (partial or complete).

[0073] In some embodiments, a method of detecting the presence or absence in a subject of a NOTCH3 missense variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule produced from an mRNA molecule) encoding a NOTCH3 predicted gain-of-function polypeptide comprises performing an assay on a biological sample obtained from the subject, wherein the assay determines whether a nucleic acid molecule in the biological sample comprises a particular nucleotide sequence.

[0074] In some embodiments, the nucleotide sequence of the NOTCH3 genomic nucleic acid molecule comprises a thymine or its complement at a position corresponding to position 21,944 according to SEQ ID NO:2.

[0075] In some embodiments, the nucleotide sequence of the NOTCH3 mRNA molecule comprises uracil or its complement at a position corresponding to position 3,781 according to SEQ ID NO:8; uracil or its complement at a position corresponding to position 3,767 according to SEQ ID NO:9; uracil or its complement at a position corresponding to position 3,532 according to SEQ ID NO:10; uracil or its complement at a position corresponding to position 3,769 according to SEQ ID NO:11; or uracil or its complement at a position corresponding to position 3,544 according to SEQ ID NO:12. In some embodiments, the nucleotide sequence of the NOTCH3 mRNA molecule comprises uracil or its complement at a position corresponding to position 3,781 according to SEQ ID NO:8. In some embodiments, the nucleotide sequence of the NOTCH3 mRNA molecule comprises uracil or its complement at a position corresponding to position 3,767 according to SEQ ID NO:9. In some embodiments, the nucleotide sequence of the NOTCH3 mRNA molecule comprises uracil or its complement at a position corresponding to position 3,532 according to SEQ ID NO:10. In some embodiments, the nucleotide sequence of the NOTCH3 mRNA molecule comprises uracil, or its complement, at a position corresponding to position 3,769 according to SEQ ID NO: 11. In some embodiments, the nucleotide sequence of the NOTCH3 mRNA molecule comprises uracil, or its complement, at a position corresponding to position 3,544 according to SEQ ID NO: 12.

[0076] In some embodiments, the nucleotide sequence of the NOTCH3 cDNA molecule comprises a thymine or its complement at a position corresponding to position 3,781 according to SEQ ID NO:18; a thymine or its complement at a position corresponding to position 3,767 according to SEQ ID NO:19; a thymine or its complement at a position corresponding to position 3,532 according to SEQ ID NO:20; a thymine or its complement at a position corresponding to position 3,769 according to SEQ ID NO:21; or a thymine or its complement at a position corresponding to position 3,544 according to SEQ ID NO:22. In some embodiments, the nucleotide sequence of the NOTCH3 cDNA molecule comprises a thymine or its complement at a position corresponding to position 3,781 according to SEQ ID NO:18. In some embodiments, the nucleotide sequence of the NOTCH3 cDNA molecule comprises a thymine or its complement at a position corresponding to position 3,767 according to SEQ ID NO:19. In some embodiments, the nucleotide sequence of the NOTCH3 cDNA molecule comprises a thymine or its complement at a position corresponding to position 3,532 according to SEQ ID NO:20. In some embodiments, the nucleotide sequence of the NOTCH3 cDNA molecule comprises a thymine, or its complement, at a position corresponding to position 3,769 according to SEQ ID NO: 21. In some embodiments, the nucleotide sequence of the NOTCH3 cDNA molecule comprises a thymine, or its complement, at a position corresponding to position 3,544 according to SEQ ID NO: 22.

[0077] In some embodiments, the biological sample comprises cells or cell lysates. Such methods can further comprise, for example, obtaining a biological sample from a subject that comprises NOTCH3 genomic nucleic acid molecules or mRNA molecules, and optionally reverse transcribing the mRNA into cDNA if it is mRNA. Such assays can comprise, for example, determining the uniqueness of these positions of a particular NOTCH3 nucleic acid molecule. In some embodiments, the methods are in vitro methods.

[0078] In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a NOTCH3 genomic nucleic acid molecule, a NOTCH3 mRNA molecule, or a NOTCH3 cDNA molecule produced from an mRNA molecule in a biological sample, wherein the sequenced portion contains one or more mutations that result in or are predicted to result in a gain of function (partial or complete).

[0079] In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a NOTCH3 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 21,944 according to SEQ ID NO: 2, or a complement thereof. If the sequenced portion of the NOTCH3 nucleic acid molecule in the biological sample comprises a thymine at a position corresponding to position 21,944 according to SEQ ID NO: 2, then the NOTCH3 nucleic acid molecule in the biological sample is a NOTCH3 missense variant nucleic acid molecule that encodes a NOTCH3 predicted gain-of-function polypeptide.

[0080] In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a NOTCH3 mRNA molecule in the biological sample, or a cDNA molecule produced therefrom, wherein the sequenced portion comprises a position corresponding to position 3,781 according to SEQ ID NO:8 or its complement; and / or a position corresponding to position 3,781 according to SEQ ID NO:18 or its complement. If the sequenced portion of the NOTCH3 nucleic acid molecule in the biological sample comprises a uracil at the position corresponding to position 3,781 according to SEQ ID NO:8, or a thymine at the position corresponding to position 3,781 according to SEQ ID NO:18, then the NOTCH3 nucleic acid molecule in the biological sample is a NOTCH3 missense variant nucleic acid molecule that encodes a NOTCH3 predicted gain-of-function polypeptide.

[0081] In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a NOTCH3 mRNA molecule in the biological sample, or a cDNA molecule produced therefrom, wherein the sequenced portion comprises a position corresponding to position 3,767 according to SEQ ID NO:9 or its complement; and / or a position corresponding to position 3,767 according to SEQ ID NO:19 or its complement. If the sequenced portion of the NOTCH3 nucleic acid molecule in the biological sample comprises a uracil at the position corresponding to position 3,767 according to SEQ ID NO:9, or a thymine at the position corresponding to position 3,767 according to SEQ ID NO:19, then the NOTCH3 nucleic acid molecule in the biological sample is a NOTCH3 missense variant nucleic acid molecule that encodes a NOTCH3 predicted gain-of-function polypeptide.

[0082] In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a NOTCH3 mRNA molecule in the biological sample, or a cDNA molecule produced therefrom, wherein the sequenced portion comprises a position corresponding to position 3,532 according to SEQ ID NO: 10, or its complement; and / or a position corresponding to position 3,532 according to SEQ ID NO: 20, or its complement. If the sequenced portion of the NOTCH3 nucleic acid molecule in the biological sample comprises a uracil at the position corresponding to position 3,532 according to SEQ ID NO: 10, or a thymine at the position corresponding to position 3,532 according to SEQ ID NO: 20, then the NOTCH3 nucleic acid molecule in the biological sample is a NOTCH3 missense variant nucleic acid molecule that encodes a NOTCH3 predicted gain-of-function polypeptide.

[0083] In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a NOTCH3 mRNA molecule in the biological sample, or a cDNA molecule produced therefrom, wherein the sequenced portion comprises a position corresponding to position 3,769 according to SEQ ID NO: 11, or its complement; and / or a position corresponding to position 3,769 according to SEQ ID NO: 21, or its complement. If the sequenced portion of the NOTCH3 nucleic acid molecule in the biological sample comprises a uracil at the position corresponding to position 3,769 according to SEQ ID NO: 11, or a thymine at the position corresponding to position 3,769 according to SEQ ID NO: 21, then the NOTCH3 nucleic acid molecule in the biological sample is a NOTCH3 missense variant nucleic acid molecule that encodes a NOTCH3 predicted gain-of-function polypeptide.

[0084] In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a NOTCH3 mRNA molecule in the biological sample, or a cDNA molecule produced therefrom, wherein the sequenced portion comprises a position corresponding to position 3,544 according to SEQ ID NO: 12, or its complement; and / or a position corresponding to position 3,544 according to SEQ ID NO: 22, or its complement. If the sequenced portion of the NOTCH3 nucleic acid molecule in the biological sample comprises a uracil at the position corresponding to position 3,544 according to SEQ ID NO: 12, or a thymine at the position corresponding to position 3,544 according to SEQ ID NO: 22, then the NOTCH3 nucleic acid molecule in the biological sample is a NOTCH3 missense variant nucleic acid molecule that encodes a NOTCH3 predicted gain-of-function polypeptide.

[0085] In some embodiments, the determining step, detecting step, or sequencing analysis comprises sequencing at least a portion of a nucleotide sequence of a NOTCH3 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 21,944 according to SEQ ID NO: 2, or a complement thereof. If the sequenced portion of the NOTCH3 nucleic acid molecule in the biological sample comprises a thymine at a position corresponding to position 21,944 according to SEQ ID NO: 2, then the NOTCH3 nucleic acid molecule in the biological sample is a NOTCH3 missense variant nucleic acid molecule that encodes a NOTCH3 predicted gain-of-function polypeptide.

[0086] In some embodiments, the determining step, detecting step, or sequencing analysis comprises sequencing at least a portion of the nucleotide sequence of a NOTCH3 mRNA molecule in a biological sample, wherein the sequenced portion comprises a position corresponding to position 3,781 according to SEQ ID NO:8 or its complement; a position corresponding to position 3,767 according to SEQ ID NO:9 or its complement; a position corresponding to position 3,532 according to SEQ ID NO:10 or its complement; a position corresponding to position 3,769 according to SEQ ID NO:11 or its complement; or a position corresponding to position 3,544 according to SEQ ID NO:12 or its complement. If a sequenced portion of a NOTCH3 nucleic acid molecule in a biological sample contains uracil at a position corresponding to position 3,781 according to SEQ ID NO:8, a position corresponding to position 3,767 according to SEQ ID NO:9, a position corresponding to position 3,532 according to SEQ ID NO:10, a position corresponding to position 3,769 according to SEQ ID NO:11, or a position corresponding to position 3,544 according to SEQ ID NO:12, then the NOTCH3 nucleic acid molecule in the biological sample is a NOTCH3 missense variant nucleic acid molecule that encodes a gain-of-function polypeptide predicted for NOTCH3.

[0087] In some embodiments, the determining step, detecting step, or sequencing analysis comprises sequencing at least a portion of the nucleotide sequence of a NOTCH3 cDNA molecule produced from an mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 3,781 according to SEQ ID NO: 18 or its complement; a position corresponding to position 3,767 according to SEQ ID NO: 19 or its complement; or a position corresponding to position 3,532 according to SEQ ID NO: 20 or its complement. If the sequenced portion of the NOTCH3 nucleic acid molecule in the biological sample comprises a thymine at a position corresponding to position 3,781 according to SEQ ID NO: 18, a position corresponding to position 3,767 according to SEQ ID NO: 19, a position corresponding to position 3,532 according to SEQ ID NO: 20, a position corresponding to position 3,769 according to SEQ ID NO: 21, or a position corresponding to position 3,544 according to SEQ ID NO: 22, then the NOTCH3 nucleic acid molecule in the biological sample is a NOTCH3 missense variant nucleic acid molecule encoding a predicted gain-of-function polypeptide for NOTCH3.

[0088] In some embodiments, the determining step, detecting step, or sequencing analysis comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a NOTCH3 genomic nucleic acid molecule proximate to a position corresponding to position 21,944 according to SEQ ID NO:2; b) extending the primer through at least the position in the nucleotide sequence of a NOTCH3 genomic nucleic acid molecule corresponding to position 21,944 according to SEQ ID NO:2; and c) determining whether the extension product of the primer comprises a thymine at the position corresponding to position 21,944 according to SEQ ID NO:2.

[0089] In some embodiments, the determining step, detecting step, or sequencing analysis comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a NOTCH3 mRNA molecule adjacent to a position corresponding to position 3,781 according to SEQ ID NO:8 and / or a cDNA molecule adjacent to a position corresponding to position 3,781 according to SEQ ID NO:18; b) extending the primer through at least the position of the nucleotide sequence of a NOTCH3 mRNA molecule corresponding to position 3,781 according to SEQ ID NO:8 and / or a cDNA molecule corresponding to position 3,781 according to SEQ ID NO:18; and c) determining whether the extension product of the primer comprises uracil at the position corresponding to position 3,781 according to SEQ ID NO:8 and / or thymine at the position corresponding to position 3,781 according to SEQ ID NO:18.

[0090] In some embodiments, the determining step, detecting step, or sequencing analysis comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a NOTCH3 mRNA molecule adjacent to a position corresponding to position 3,767 according to SEQ ID NO:9 and / or a cDNA molecule adjacent to a position corresponding to position 3,767 according to SEQ ID NO:19; b) extending the primer through at least the position of the nucleotide sequence of a NOTCH3 mRNA molecule corresponding to position 3,767 according to SEQ ID NO:9 and / or a cDNA molecule corresponding to position 3,767 according to SEQ ID NO:19; and c) determining whether the extension product of the primer comprises a uracil at the position corresponding to position 3,767 according to SEQ ID NO:9 and / or a thymine at the position corresponding to position 3,767 according to SEQ ID NO:19.

[0091] In some embodiments, the determining step, detecting step, or sequencing analysis comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a NOTCH3 mRNA molecule adjacent to a position corresponding to position 3,532 according to SEQ ID NO:10 and / or a cDNA molecule adjacent to a position corresponding to position 3,532 according to SEQ ID NO:20; b) extending the primer through at least the position of the nucleotide sequence of a NOTCH3 mRNA molecule corresponding to position 3,532 according to SEQ ID NO:10 and / or a cDNA molecule corresponding to position 3,532 according to SEQ ID NO:20; and c) determining whether the extension product of the primer comprises a uracil at the position corresponding to position 3,532 according to SEQ ID NO:10 and / or a thymine at the position corresponding to position 3,532 according to SEQ ID NO:20.

[0092] In some embodiments, the determining step, detecting step, or sequencing analysis comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a NOTCH3 mRNA molecule adjacent to a position corresponding to position 3,769 according to SEQ ID NO:11 and / or a cDNA molecule adjacent to a position corresponding to position 3,769 according to SEQ ID NO:21; b) extending the primer through at least the position of the nucleotide sequence of a NOTCH3 mRNA molecule corresponding to position 3,769 according to SEQ ID NO:11 and / or a cDNA molecule corresponding to position 3,769 according to SEQ ID NO:21; and c) determining whether the extension product of the primer comprises a uracil at the position corresponding to position 3,769 according to SEQ ID NO:11 and / or a thymine at the position corresponding to position 3,769 according to SEQ ID NO:21.

[0093] In some embodiments, the determining step, detecting step, or sequencing analysis comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a NOTCH3 mRNA molecule adjacent to a position corresponding to position 3,544 according to SEQ ID NO:12 and / or a cDNA molecule adjacent to a position corresponding to position 3,544 according to SEQ ID NO:22; b) extending the primer through at least the position of the nucleotide sequence of a NOTCH3 mRNA molecule corresponding to position 3,544 according to SEQ ID NO:12 and / or a cDNA molecule corresponding to position 3,544 according to SEQ ID NO:22; and c) determining whether the extension product of the primer comprises a uracil at the position corresponding to position 3,544 according to SEQ ID NO:12 and / or a thymine at the position corresponding to position 3,544 according to SEQ ID NO:22.

[0094] In some embodiments, the determining step, detecting step, or sequencing analysis comprises the steps of: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a NOTCH3 mRNA molecule proximate to a position corresponding to position 3,781 according to SEQ ID NO:8; a position corresponding to position 3,767 according to SEQ ID NO:9; a position corresponding to position 3,532 according to SEQ ID NO:10; a position corresponding to position 3,769 according to SEQ ID NO:11; or a position corresponding to position 3,544 according to SEQ ID NO:12; and b) hybridizing to at least a portion of the nucleotide sequence of a NOTCH3 mRNA molecule proximate to a position corresponding to position 3,781 according to SEQ ID NO:8; a position corresponding to position 3,767 according to SEQ ID NO:9; a position corresponding to position 3,532 according to SEQ ID NO:10, a position corresponding to position 3,769 according to SEQ ID NO:11; or a position corresponding to position 3,544 according to SEQ ID NO:12. extending the primer through a position in the nucleotide sequence of the mRNA molecule; and c) determining whether the extension product of the primer contains uracil at a position corresponding to position 3,781 according to SEQ ID NO:8, a position corresponding to position 3,767 according to SEQ ID NO:9, a position corresponding to position 3,532 according to SEQ ID NO:10, a position corresponding to position 3,769 according to SEQ ID NO:11, or a position corresponding to position 3,544 according to SEQ ID NO:12.

[0095] In some embodiments, the determining step, detecting step, or sequencing analysis comprises the steps of: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a NOTCH3 cDNA molecule proximate to a position corresponding to position 3,781 according to SEQ ID NO:18; a position corresponding to position 3,767 according to SEQ ID NO:19; a position corresponding to position 3,532 according to SEQ ID NO:20; a position corresponding to position 3,769 according to SEQ ID NO:21; or a position corresponding to position 3,544 according to SEQ ID NO:22; and b) hybridizing to at least a portion of the nucleotide sequence of a NOTCH3 cDNA molecule proximate to a position corresponding to position 3,781 according to SEQ ID NO:18; a position corresponding to position 3,767 according to SEQ ID NO:19; a position corresponding to position 3,532 according to SEQ ID NO:20, a position corresponding to position 3,769 according to SEQ ID NO:21; or a position corresponding to position 3,544 according to SEQ ID NO:22. and c) determining whether the extension product of the primer contains a thymine at a position corresponding to position 3,781 according to SEQ ID NO:18, a position corresponding to position 3,767 according to SEQ ID NO:19, a position corresponding to position 3,532 according to SEQ ID NO:20, a position corresponding to position 3,769 according to SEQ ID NO:21, or a position corresponding to position 3,544 according to SEQ ID NO:22.

[0096] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only NOTCH3 genomic nucleic acid molecules are analyzed. In some embodiments, only NOTCH3 mRNA is analyzed. In some embodiments, only NOTCH3 cDNA obtained from NOTCH3 mRNA is analyzed.

[0097] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a NOTCH3 polypeptide, wherein the amplified portion comprises a thymine or its complement at a position corresponding to position 21,944 according to SEQ ID NO:2; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, wherein the variation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising a thymine or its complement at a position corresponding to position 21,944 according to SEQ ID NO:2; and d) detecting the detectable label.

[0098] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a NOTCH3 polypeptide, wherein the amplified portion comprises uracil or its complement at a position corresponding to position 3,781 according to SEQ ID NO:8; and / or thymine or its complement at a position corresponding to position 3,781 according to SEQ ID NO:18; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, wherein the variation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 3,781 according to SEQ ID NO:8; and / or thymine or its complement at a position corresponding to position 3,781 according to SEQ ID NO:18; and d) detecting the detectable label.

[0099] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a NOTCH3 polypeptide, wherein the amplified portion comprises uracil or its complement at a position corresponding to position 3,767 according to SEQ ID NO:9; and / or thymine or its complement at a position corresponding to position 3,767 according to SEQ ID NO:19; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, wherein the variation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 3,767 according to SEQ ID NO:9; and / or thymine or its complement at a position corresponding to position 3,767 according to SEQ ID NO:19; and d) detecting the detectable label.

[0100] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a NOTCH3 polypeptide, wherein the amplified portion comprises uracil or its complement at a position corresponding to position 3,532 according to SEQ ID NO:10; and / or thymine or its complement at a position corresponding to position 3,532 according to SEQ ID NO:20; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, wherein the variation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 3,532 according to SEQ ID NO:10; and / or thymine or its complement at a position corresponding to position 3,532 according to SEQ ID NO:20; and d) detecting the detectable label.

[0101] In some embodiments, the determining step, detecting step, or sequencing analysis comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a NOTCH3 polypeptide, wherein the amplified portion comprises uracil or its complement at a position corresponding to position 3,769 according to SEQ ID NO:11; and / or thymine or its complement at a position corresponding to position 3,769 according to SEQ ID NO:21; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, wherein the variation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 3,769 according to SEQ ID NO:11; and / or thymine or its complement at a position corresponding to position 3,769 according to SEQ ID NO:21; and d) detecting the detectable label.

[0102] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a NOTCH3 polypeptide, wherein the amplified portion comprises uracil or its complement at a position corresponding to position 3,544 according to SEQ ID NO:12; and / or thymine or its complement at a position corresponding to position 3,544 according to SEQ ID NO:22; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variation-specific probe, wherein the variation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 3,544 according to SEQ ID NO:12; and / or thymine or its complement at a position corresponding to position 3,544 according to SEQ ID NO:22; and d) detecting the detectable label.

[0103] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a NOTCH3 polypeptide, wherein the amplified portion comprises uracil or its complement at a position corresponding to position 3,781 according to SEQ ID NO:8, uracil or its complement at a position corresponding to position 3,767 according to SEQ ID NO:9, uracil or its complement at a position corresponding to position 3,532 according to SEQ ID NO:10, uracil or its complement at a position corresponding to position 3,769 according to SEQ ID NO:11, or uracil or its complement at a position corresponding to position 3,544 according to SEQ ID NO:12; b) labeling the amplified nucleic acid molecule with a detectable label; and c) detecting the amplified nucleic acid molecule. ) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 3,781 according to SEQ ID NO:8, uracil or its complement at a position corresponding to position 3,767 according to SEQ ID NO:9, uracil or its complement at a position corresponding to position 3,532 according to SEQ ID NO:10, uracil or its complement at a position corresponding to position 3,769 according to SEQ ID NO:11, or uracil or its complement at a position corresponding to position 3,544 according to SEQ ID NO:12; and d) detecting the detectable label.

[0104] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a NOTCH3 polypeptide, wherein the amplified portion comprises a thymine or its complement at a position corresponding to position 3,781 according to SEQ ID NO:18, a thymine or its complement at a position corresponding to position 3,767 according to SEQ ID NO:19, a thymine or its complement at a position corresponding to position 3,532 according to SEQ ID NO:20, a thymine or its complement at a position corresponding to position 3,769 according to SEQ ID NO:21, or a thymine or its complement at a position corresponding to position 3,544 according to SEQ ID NO:22; b) labeling the amplified nucleic acid molecule with a detectable label; and c) detecting the amplified nucleic acid molecule. ) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising a thymine or its complement at a position corresponding to position 3,781 according to SEQ ID NO:18, a thymine or its complement at a position corresponding to position 3,767 according to SEQ ID NO:19, a thymine or its complement at a position corresponding to position 3,532 according to SEQ ID NO:20, a thymine or its complement at a position corresponding to position 3,769 according to SEQ ID NO:21, or a thymine or its complement at a position corresponding to position 3,544 according to SEQ ID NO:22; and d) detecting the detectable label.

[0105] In some embodiments, the nucleic acid molecule is mRNA and the determining step further comprises reverse transcribing the mRNA into cDNA prior to the amplifying step.

[0106] In some embodiments, the determining step, detecting step, or sequencing analysis comprises contacting a nucleic acid molecule in a biological sample with a variation-specific probe comprising a detectable label, the variation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising a thymine or its complement at a position corresponding to position 21,944 according to SEQ ID NO:2, and detecting the detectable label.

[0107] In some embodiments, the determining step, detecting step, or sequencing analysis comprises contacting a nucleic acid molecule in a biological sample with a variation-specific probe comprising a detectable label, wherein the variation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 3,781 according to SEQ ID NO:8 and / or thymine or its complement at a position corresponding to position 3,781 according to SEQ ID NO:18; and detecting the detectable label.

[0108] In some embodiments, the determining step, detecting step, or sequencing analysis comprises contacting a nucleic acid molecule in a biological sample with a variation-specific probe comprising a detectable label, wherein the variation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 3,767 according to SEQ ID NO:9, and / or thymine or its complement at a position corresponding to position 3,767 according to SEQ ID NO:19; and detecting the detectable label.

[0109] In some embodiments, the determining step, detecting step, or sequencing analysis comprises contacting a nucleic acid molecule in a biological sample with a variation-specific probe comprising a detectable label, wherein the variation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 3,532 according to SEQ ID NO:10, and / or thymine or its complement at a position corresponding to position 3,532 according to SEQ ID NO:20; and detecting the detectable label.

[0110] In some embodiments, the determining step, detecting step, or sequencing analysis comprises contacting a nucleic acid molecule in a biological sample with a variation-specific probe comprising a detectable label, wherein the variation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 3,769 according to SEQ ID NO:11, and / or thymine or its complement at a position corresponding to position 3,769 according to SEQ ID NO:21; and detecting the detectable label.

[0111] In some embodiments, the determining step, detecting step, or sequencing analysis comprises contacting a nucleic acid molecule in a biological sample with a variation-specific probe comprising a detectable label, wherein the variation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 3,544 according to SEQ ID NO:12 and / or thymine or its complement at a position corresponding to position 3,544 according to SEQ ID NO:22; and detecting the detectable label.

[0112] In some embodiments, the determining step, detecting step, or sequencing analysis comprises contacting a nucleic acid molecule in a biological sample with a variation-specific probe comprising a detectable label, the variation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising uracil or its complement at a position corresponding to position 3,781 according to SEQ ID NO:8, uracil or its complement at a position corresponding to position 3,767 according to SEQ ID NO:9, uracil or its complement at a position corresponding to position 3,532 according to SEQ ID NO:10, uracil or its complement at a position corresponding to position 3,769 according to SEQ ID NO:11, or uracil or its complement at a position corresponding to position 3,544 according to SEQ ID NO:12, and detecting the detectable label.

[0113] In some embodiments, the determining step, detecting step, or sequencing analysis comprises contacting a nucleic acid molecule in a biological sample with a variation-specific probe comprising a detectable label, the variation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising a thymine or its complement at a position corresponding to position 3,781 according to SEQ ID NO:18, a thymine or its complement at a position corresponding to position 3,767 according to SEQ ID NO:19, a thymine or its complement at a position corresponding to position 3,532 according to SEQ ID NO:20, a thymine or its complement at a position corresponding to position 3,769 according to SEQ ID NO:21, or a thymine or its complement at a position corresponding to position 3,544 according to SEQ ID NO:22, and detecting the detectable label.

[0114] Mutations (such as SNPs in nucleic acid sequences) can be detected using mutation-specific polymerase chain reaction techniques. Because DNA polymerase will not extend if there is a mismatch with the template, a mutation-specific primer can be used.

[0115] In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is present in a cell obtained from the subject.

[0116] In some embodiments, the assay involves contacting a biological sample under stringent conditions with a primer or probe, such as a mutation-specific primer or mutation-specific probe, that specifically hybridizes to a NOTCH3 variant genomic sequence, variant mRNA sequence, or variant cDNA sequence, but not to a corresponding NOTCH3 reference sequence, and determining whether hybridization occurs.

[0117] In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay also comprises reverse transcribing the mRNA into cDNA, such as by reverse transcription polymerase chain reaction (RT-PCR).

[0118] In some embodiments, the method utilizes probes and primers of sufficient nucleotide length to bind to a target nucleotide sequence and specifically detect and / or identify polynucleotides including NOTCH3 variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules. The operator can determine the hybridization or reaction conditions to achieve this result. The nucleotide length can be any length sufficient for use in the selected detection method, including any assay described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under high stringency hybridization conditions. The probes and primers can have perfect nucleotide sequence identity of consecutive nucleotides in the target nucleotide sequence, but probes that differ from the target nucleotide sequence and retain the ability to specifically detect and / or identify the target nucleotide sequence can be designed by conventional methods. The probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity to the nucleotide sequence of the target nucleic acid molecule.

[0119] In some embodiments, to determine whether a NOTCH3 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or a complement thereof, in a biological sample comprises a nucleotide sequence (genomic nucleic acid molecule) comprising a thymine at a position corresponding to position 21,944 according to SEQ ID NO:2, the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from the 5' flanking sequence adjacent to the thymine at a position corresponding to position 21,944 according to SEQ ID NO:2 and a second primer derived from the 3' flanking sequence adjacent to the thymine at a position corresponding to position 21,944 according to SEQ ID NO:2 to produce an amplicon indicative of the presence of a SNP at the position encoding the thymine at a position corresponding to position 21,944 according to SEQ ID NO:2. In some embodiments, the amplicon can range in length from the primer pair plus one nucleotide base pair to any length of an amplicon that can be produced by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a thymine at a position corresponding to position 21,944 according to SEQ ID NO:2 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides on each side of the position that includes a thymine at a position corresponding to position 21,944 according to SEQ ID NO:2.

[0120] In some embodiments, to determine whether a NOTCH3 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample comprises a nucleotide sequence comprising uracil at a position corresponding to position 3,781 according to SEQ ID NO:8 (mRNA molecule) or thymine at a position corresponding to position 3,781 according to SEQ ID NO:18 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5' flanking sequence adjacent to the uracil at a position corresponding to position 3,781 according to SEQ ID NO:8 or thymine at a position corresponding to position 3,781 according to SEQ ID NO:18 and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 3,781 according to SEQ ID NO:8 or thymine at a position corresponding to position 3,781 according to SEQ ID NO:18 to generate an amplicon indicative of the presence of a SNP at a position encoding the uracil at a position corresponding to position 3,781 according to SEQ ID NO:8 or thymine at a position corresponding to position 3,781 according to SEQ ID NO:18. In some embodiments, the amplicon can range in length from the primer pair plus one nucleotide base pair to any length of the amplicon that can be produced by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a uracil at a position corresponding to position 3,781 according to SEQ ID NO:8, or a thymine at a position corresponding to position 3,781 according to SEQ ID NO:18, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the uracil at a position corresponding to position 3,781 according to SEQ ID NO:8, or a thymine at a position corresponding to position 3,781 according to SEQ ID NO:18.

[0121] In some embodiments, to determine whether a NOTCH3 nucleic acid molecule (mRNA molecule, or cDNA molecule) or its complement in a biological sample comprises a nucleotide sequence comprising uracil at a position corresponding to position 3,767 according to SEQ ID NO:9 (mRNA molecule) or thymine at a position corresponding to position 3,767 according to SEQ ID NO:19 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5' flanking sequence adjacent to the uracil at a position corresponding to position 3,767 according to SEQ ID NO:9 or thymine at a position corresponding to position 3,767 according to SEQ ID NO:19 and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 3,767 according to SEQ ID NO:9 or thymine at a position corresponding to position 3,767 according to SEQ ID NO:19 to generate an amplicon indicative of the presence of a SNP at a position encoding the uracil at a position corresponding to position 3,767 according to SEQ ID NO:9 or thymine at a position corresponding to position 3,767 according to SEQ ID NO:19. In some embodiments, the amplicon can range in length from the primer pair plus one nucleotide base pair to any length of the amplicon that can be produced by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a uracil at a position corresponding to position 3,767 according to SEQ ID NO:9, or a thymine at a position corresponding to position 3,767 according to SEQ ID NO:19, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the uracil at a position corresponding to position 3,767 according to SEQ ID NO:9, or a thymine at a position corresponding to position 3,767 according to SEQ ID NO:19.

[0122] In some embodiments, to determine whether a NOTCH3 nucleic acid molecule (mRNA molecule, or cDNA molecule) or its complement in a biological sample comprises a nucleotide sequence comprising uracil at a position corresponding to position 3,532 according to SEQ ID NO: 10 (mRNA molecule) or thymine at a position corresponding to position 3,532 according to SEQ ID NO: 20 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5' flanking sequence adjacent to the uracil at a position corresponding to position 3,532 according to SEQ ID NO: 10 or thymine at a position corresponding to position 3,532 according to SEQ ID NO: 20 and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 3,532 according to SEQ ID NO: 10 or thymine at a position corresponding to position 3,532 according to SEQ ID NO: 20 to generate an amplicon indicative of the presence of a SNP at a position encoding the uracil at a position corresponding to position 3,532 according to SEQ ID NO: 10 or thymine at a position corresponding to position 3,532 according to SEQ ID NO: 20. In some embodiments, the amplicon may range in length from the primer pair plus one nucleotide base pair to any length of the amplicon that can be produced by the DNA amplification protocol. This distance may range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a position that includes uracil at a position corresponding to position 3,532 according to SEQ ID NO: 10, or a thymine at a position corresponding to position 3,532 according to SEQ ID NO: 20, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position that includes uracil at a position corresponding to position 3,532 according to SEQ ID NO: 10, or a thymine at a position corresponding to position 3,532 according to SEQ ID NO: 20.

[0123] In some embodiments, to determine whether a NOTCH3 nucleic acid molecule (mRNA molecule, or cDNA molecule) or its complement in a biological sample comprises a nucleotide sequence comprising uracil at a position corresponding to position 3,769 according to SEQ ID NO:11 (mRNA molecule) or thymine at a position corresponding to position 3,769 according to SEQ ID NO:21 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5' flanking sequence adjacent to the uracil at a position corresponding to position 3,769 according to SEQ ID NO:11 or thymine at a position corresponding to position 3,769 according to SEQ ID NO:21 and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 3,769 according to SEQ ID NO:11 or thymine at a position corresponding to position 3,769 according to SEQ ID NO:21 to generate an amplicon indicative of the presence of a SNP at a position encoding the uracil at a position corresponding to position 3,769 according to SEQ ID NO:11 or thymine at a position corresponding to position 3,769 according to SEQ ID NO:21. In some embodiments, the amplicon can range in length from the primer pair plus one nucleotide base pair to any length of the amplicon that can be produced by the DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a position that includes a uracil at a position corresponding to position 3,769 according to SEQ ID NO: 11, or a thymine at a position corresponding to position 3,769 according to SEQ ID NO: 21, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position that includes a uracil at a position corresponding to position 3,769 according to SEQ ID NO: 11, or a thymine at a position corresponding to position 3,769 according to SEQ ID NO: 21.

[0124] In some embodiments, to determine whether a NOTCH3 nucleic acid molecule (mRNA molecule, or cDNA molecule) or its complement in a biological sample comprises a nucleotide sequence comprising uracil at a position corresponding to position 3,544 according to SEQ ID NO: 12 (mRNA molecule) or thymine at a position corresponding to position 3,544 according to SEQ ID NO: 22 (cDNA molecule), the biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5' flanking sequence adjacent to the uracil at a position corresponding to position 3,544 according to SEQ ID NO: 12 or thymine at a position corresponding to position 3,544 according to SEQ ID NO: 22 and a second primer derived from a 3' flanking sequence adjacent to the uracil at a position corresponding to position 3,544 according to SEQ ID NO: 12 or thymine at a position corresponding to position 3,544 according to SEQ ID NO: 22 to generate an amplicon indicative of the presence of a SNP at a position encoding the uracil at a position corresponding to position 3,544 according to SEQ ID NO: 12 or thymine at a position corresponding to position 3,544 according to SEQ ID NO: 22. In some embodiments, the amplicon may range in length from the primer pair plus one nucleotide base pair to any length of the amplicon that can be produced by the DNA amplification protocol. This distance may range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a uracil at a position corresponding to position 3,544 according to SEQ ID NO: 12, or a thymine at a position corresponding to position 3,544 according to SEQ ID NO: 22, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the uracil at a position corresponding to position 3,544 according to SEQ ID NO: 12, or a thymine at a position corresponding to position 3,544 according to SEQ ID NO: 22.

[0125] Similar amplicons can be generated from mRNA and / or cDNA sequences. PCR primer pairs can be derived from known sequences, for example, using computer programs designed for that purpose, such as the PCR primer analysis tools in Vector NTI version 10 (Informax Inc., Bethesda Md.), PrimerSelect (DNASTAR Inc., Madison, Wis.), and Primer3 (Version 0.4.0.COPYRGT., 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Additionally, sequences can be visually scanned and primers manually identified using known guidelines.

[0126] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods include nucleic acid hybridization methods other than sequencing, including using labeled primers or probes on purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, the target nucleic acid molecule can be amplified prior to or simultaneously with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0127] Hybridization techniques can employ stringent conditions so that the probe or primer specifically hybridizes to its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other non-target sequences, for example, at least 2-fold, at least 3-fold, at least 4-fold or more, for example, more than 10-fold higher than background. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence at least 2-fold higher than other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence at least 3-fold higher than other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence at least 4-fold higher than other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence at more than 10-fold background to a detectably greater extent than to other nucleotide sequences. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0128] Suitable stringency conditions that promote DNA hybridization, such as 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by a 2x SSC wash at 50°C, are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection include conditions in which the salt concentration is less than about 1.5 M NaCl at pH 7.0-8.3. + ion, usually about 0.01 to 1.0 M Na + The conditions are: ionic concentration (or other salts) and temperature of at least about 30°C for short probes (e.g., 10-50 nucleotides) and at least about 60°C for longer probes (e.g., more than 50 nucleotides). Stringent conditions may be achieved by the addition of destabilizing agents such as formamide. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash will be at least long enough to reach equilibrium.

[0129] The present disclosure also provides a method for detecting the presence of a NOTCH3 predicted gain-of-function polypeptide, comprising performing an assay on a biological sample obtained from a subject to determine whether a NOTCH3 polypeptide in the subject contains one or more mutations that cause the polypeptide to have a gain-of-function (partial or complete) or a predicted gain-of-function (partial or complete). The NOTCH3 predicted gain-of-function polypeptide can be any of the NOTCH3 variant polypeptides described herein. In some embodiments, the method detects the presence of NOTCH3 Arg1178Cys, Arg1231Cys, or Arg1182Cys. In some embodiments, the method detects the presence of NOTCH3 Arg1178Cys. In some embodiments, the method detects the presence of NOTCH3 Arg1231Cys. In some embodiments, the method detects the presence of NOTCH3 Arg1182Cys.

[0130] In any of the embodiments described herein, the NOTCH3 variant polypeptide can be any Cys-altered variant (which is altered from or to a cysteine ​​residue). In some embodiments, the Cys-altered variant is located within the 34 EGF-like repeats in the NOTCH3 extracellular domain (as defined by Uniprot; see the World Wide Web at "uniprot.org / blast / ?about=Q9UM47[1335-1373]&key=Domain"). In any of the embodiments described herein, the NOTCH3 variant polypeptide can be any of the variants listed in Table 1 (ENST00000263388).

[0131] In some embodiments, the method comprises assaying a sample from the subject to determine whether a NOTCH3 polypeptide in the sample comprises a cysteine ​​at a position corresponding to position 1,178 according to SEQ ID NO: 26. In some embodiments, the method comprises assaying a sample from the subject to determine whether a NOTCH3 polypeptide in the sample comprises a cysteine ​​at a position corresponding to position 1,231 according to SEQ ID NO: 27. In some embodiments, the method comprises assaying a sample from the subject to determine whether a NOTCH3 polypeptide in the sample comprises a cysteine ​​at a position corresponding to position 1,182 according to SEQ ID NO: 28.

[0132] In some embodiments, the detecting step comprises sequencing at least a portion of the polypeptide that includes a position corresponding to position 1,178 according to SEQ ID NO:26 or SEQ ID NO:23. In some embodiments, the detecting step comprises sequencing at least a portion of the polypeptide that includes a position corresponding to position 1,231 according to SEQ ID NO:27 or SEQ ID NO:24. In some embodiments, the detecting step comprises sequencing at least a portion of the polypeptide that includes a position corresponding to position 1,182 according to SEQ ID NO:28 or SEQ ID NO:25.

[0133] In some embodiments, the detecting step comprises an immunoassay for detecting the presence of a polypeptide that includes a position corresponding to position 1,178 according to SEQ ID NO:26 or SEQ ID NO:23. In some embodiments, the detecting step comprises an immunoassay for detecting the presence of a polypeptide that includes a position corresponding to position 1,231 according to SEQ ID NO:27 or SEQ ID NO:24. In some embodiments, the detecting step comprises an immunoassay for detecting the presence of a polypeptide that includes a position corresponding to position 1,182 according to SEQ ID NO:28 or SEQ ID NO:25.

[0134] In some embodiments, if the subject does not have a NOTCH3 predicted gain-of-function polypeptide, the subject does not have an increased risk of developing cerebrovascular disease, or any of CADASIL, subcortical stroke, ischemic stroke, hemorrhagic stroke, or parenchymal stroke. In some embodiments, if the subject has a NOTCH3 predicted gain-of-function polypeptide, the subject has an increased risk of developing cerebrovascular disease, or any of CADASIL, subcortical stroke, ischemic stroke, hemorrhagic stroke, or parenchymal stroke.

[0135] The disclosure also provides isolated nucleic acid molecules that hybridize to a NOTCH3 variant genomic nucleic acid molecule, a NOTCH3 variant mRNA molecule, and / or a NOTCH3 variant cDNA molecule (such as any of the genomic variant nucleic acid molecules, mRNA variant molecules, and cDNA variant molecules disclosed herein). In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a NOTCH3 nucleic acid molecule that includes a position corresponding to position 21,944 according to SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a NOTCH3 nucleic acid molecule that includes a position corresponding to position 3,781 according to SEQ ID NO:8, or a position corresponding to position 3,781 according to SEQ ID NO:18. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a NOTCH3 nucleic acid molecule that includes a position corresponding to position 3,767 according to SEQ ID NO:9, or a position corresponding to position 3,767 according to SEQ ID NO:19. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a NOTCH3 nucleic acid molecule comprising a position corresponding to position 3,532 according to SEQ ID NO: 10, or a position corresponding to position 3,532 according to SEQ ID NO: 20. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a NOTCH3 nucleic acid molecule comprising a position corresponding to position 3,769 according to SEQ ID NO: 11, or a position corresponding to position 3,769 according to SEQ ID NO: 21. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a NOTCH3 nucleic acid molecule comprising a position corresponding to position 3,544 according to SEQ ID NO: 12, or a position corresponding to position 3,544 according to SEQ ID NO: 22.

[0136] In some embodiments, such isolated nucleic acid molecules comprise at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55 , at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecules comprise or consist of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecules comprise or consist of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 18 to about 30 nucleotides.In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

[0137] In some embodiments, such isolated nucleic acid molecules hybridize under stringent conditions to a NOTCH3 variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule). Such nucleic acid molecules can be used, for example, as probes, primers, variation-specific probes, or variation-specific primers as described or exemplified herein, including, but not limited to, primers, probes, antisense RNA, shRNA, and siRNA, each of which is described in more detail elsewhere herein and can be used in any of the methods described herein.

[0138] In some embodiments, the isolated nucleic acid molecule hybridizes to at least about 15 contiguous nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a NOTCH3 variant genomic nucleic acid molecule, a NOTCH3 variant mRNA molecule, and / or a NOTCH3 variant cDNA molecule. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 35 nucleotides.

[0139] In some embodiments, the isolated variation-specific probe or variation-specific primer comprises at least about 15 nucleotides, and the variation-specific probe or variation-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence encoding a NOTCH3 polypeptide, the portion including a position corresponding to position 21,944 according to SEQ ID NO: 2, or a complement thereof. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleotide sequence that is complementary to a portion of a nucleotide sequence including a position corresponding to positions 21,944 to 21,946 according to SEQ ID NO: 2, or a complement thereof.

[0140] In some embodiments, the isolated variation-specific probe or variation-specific primer comprises at least about 15 nucleotides, the variation-specific probe or variation-specific primer comprising a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a NOTCH3 polypeptide, the portion comprising a position corresponding to position 3,781 according to SEQ ID NO:8 or a complement thereof, or a position corresponding to position 3,781 according to SEQ ID NO:18 or a complement thereof. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence comprising a position corresponding to positions 3,781 to 3,783 according to SEQ ID NO:8 or a complement thereof, and / or a position corresponding to positions 3,781 to 3,783 according to SEQ ID NO:18 or a complement thereof.

[0141] In some embodiments, the isolated variation-specific probe or variation-specific primer comprises at least about 15 nucleotides, which variation-specific probe or variation-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a NOTCH3 polypeptide, the portion comprising a position corresponding to position 3,767 according to SEQ ID NO:9 or its complement, or a position corresponding to position 3,767 according to SEQ ID NO:19 or its complement. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence comprising a position corresponding to positions 3,767-3,769 according to SEQ ID NO:9 or its complement, and / or a position corresponding to positions 3,767-3,769 according to SEQ ID NO:19 or its complement.

[0142] In some embodiments, the isolated variation-specific probe or variation-specific primer comprises at least about 15 nucleotides, the variation-specific probe or variation-specific primer comprising a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a NOTCH3 polypeptide, the portion comprising a position corresponding to position 3,532 according to SEQ ID NO: 10 or its complement, or a position corresponding to position 3,532 according to SEQ ID NO: 20 or its complement. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence comprising a position corresponding to positions 3,532 to 3,534 according to SEQ ID NO: 10 or its complement, and / or a position corresponding to positions 3,532 to 3,534 according to SEQ ID NO: 20 or its complement.

[0143] In some embodiments, the isolated variation-specific probe or variation-specific primer comprises at least about 15 nucleotides, which variation-specific probe or variation-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a NOTCH3 polypeptide, the portion comprising a position corresponding to position 3,769 according to SEQ ID NO:11 or its complement, or a position corresponding to position 3,769 according to SEQ ID NO:21 or its complement. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence comprising a position corresponding to positions 3,769 to 3,771 according to SEQ ID NO:11 or its complement, and / or a position corresponding to positions 3,769 to 3,771 according to SEQ ID NO:21 or its complement.

[0144] In some embodiments, the isolated variation-specific probe or variation-specific primer comprises at least about 15 nucleotides, which variation-specific probe or variation-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a NOTCH3 polypeptide, the portion comprising a position corresponding to position 3,544 according to SEQ ID NO: 12 or its complement, or a position corresponding to position 3,544 according to SEQ ID NO: 22 or its complement. In some embodiments, the variation-specific probe or variation-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence comprising a position corresponding to positions 3,544 to 3,546 according to SEQ ID NO: 12 or its complement, and / or a position corresponding to positions 3,544 to 3,546 according to SEQ ID NO: 22 or its complement.

[0145] In some embodiments, the variation-specific probe and the variation-specific primer comprise DNA. In some embodiments, the variation-specific probe and the variation-specific primer comprise RNA.

[0146] In some embodiments, the probes and primers described herein (including mutation-specific probes and mutation-specific primers) have nucleotide sequences that specifically hybridize to any of the nucleic acid molecules disclosed herein or their complements. In some embodiments, the probe or primer specifically hybridizes to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0147] In some embodiments, the primers can be used in second generation or high throughput sequencing, including variation specific primers. Sometimes the primers can be modified, including variation specific primers. In particular, the primers can include various modifications used in various steps of, for example, Massive Parallel Signature Sequencing (MPSS), Polony sequencing, and 454 pyrosequencing. Modified primers can be used in several steps of the process, including biotinylated primers in the cloning step, and fluorescently labeled primers used in the bead loading and detection steps. Polony sequencing is typically performed using paired-end tag libraries, where each molecule of DNA template is about 135 bp in length. Biotinylated primers are used in the bead loading and emulsion PCR. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. The adapters can contain a 5'-biotin tag for immobilizing the DNA library on streptavidin-coated beads.

[0148] The probes and primers described herein can be used to detect nucleotide variations within any of the NOTCH3 variant genomic nucleic acid molecules, NOTCH3 variant mRNA molecules, and / or NOTCH3 variant cDNA molecules disclosed herein. The primers described herein can be used to amplify NOTCH3 variant genomic nucleic acid molecules, NOTCH3 variant mRNA molecules, or NOTCH3 variant cDNA molecules, or fragments thereof.

[0149] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than thymine) at a position corresponding to position 21,944 according to SEQ ID NO:1 in a particular NOTCH3 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of a NOTCH3 reference-type genomic nucleic acid molecule. Conversely, if one of the 3' ends of the primer hybridizes with thymine (rather than cytosine) at a position corresponding to position 21,944 according to SEQ ID NO:2 in a particular NOTCH3 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of a NOTCH3 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer that is complementary to the thymine at a position corresponding to position 21,944 according to SEQ ID NO:2 can be present at the 3' end of the primer.

[0150] The present disclosure also provides a pair of primers comprising any of the above-mentioned primers. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than uracil) at the position corresponding to position 3,781 according to SEQ ID NO:3 in a particular NOTCH3 nucleic acid molecule, the presence of the amplified fragment will indicate the presence of a NOTCH3 reference-type mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with uracil (rather than cytosine) at the position corresponding to position 3,781 according to SEQ ID NO:8 in a particular NOTCH3 mRNA molecule, the presence of the amplified fragment will indicate the presence of a NOTCH3 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at the position corresponding to position 3,781 according to SEQ ID NO:8 can be present at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 3,781 according to SEQ ID NO: 13 in a particular NOTCH3 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of a NOTCH3 reference-type cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 3,781 according to SEQ ID NO: 18 in a particular NOTCH3 cDNA molecule, the presence of an amplified fragment will indicate the presence of a NOTCH3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at the position corresponding to position 3,781 according to SEQ ID NO: 18 can be present at the 3' end of the primer.

[0151] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than uracil) at the position corresponding to position 3,767 according to SEQ ID NO: 4 in a particular NOTCH3 nucleic acid molecule, the presence of the amplified fragment will indicate the presence of a NOTCH3 reference-type mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with uracil (rather than cytosine) at the position corresponding to position 3,767 according to SEQ ID NO: 9 in a particular NOTCH3 mRNA molecule, the presence of the amplified fragment will indicate the presence of a NOTCH3 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at the position corresponding to position 3,767 according to SEQ ID NO: 9 can be present at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 3,767 according to SEQ ID NO: 14 in a particular NOTCH3 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of a NOTCH3 reference-type cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 3,767 according to SEQ ID NO: 19 in a particular NOTCH3 cDNA molecule, the presence of an amplified fragment will indicate the presence of a NOTCH3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at a position corresponding to position 3,767 according to SEQ ID NO: 19 can be present at the 3' end of the primer.

[0152] The present disclosure also provides a pair of primers comprising any of the above-mentioned primers. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than uracil) at the position corresponding to position 3,532 according to SEQ ID NO:5 in a particular NOTCH3 nucleic acid molecule, the presence of the amplified fragment will indicate the presence of a NOTCH3 reference-type mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with uracil (rather than cytosine) at the position corresponding to position 3,532 according to SEQ ID NO:10 in a particular NOTCH3 mRNA molecule, the presence of the amplified fragment will indicate the presence of a NOTCH3 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at the position corresponding to position 3,532 according to SEQ ID NO:10 can be present at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 3,532 according to SEQ ID NO: 15 in a particular NOTCH3 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of a NOTCH3 reference-type cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 3,532 according to SEQ ID NO: 20 in a particular NOTCH3 cDNA molecule, the presence of an amplified fragment will indicate the presence of a NOTCH3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at a position corresponding to position 3,532 according to SEQ ID NO: 20 can be present at the 3' end of the primer.

[0153] The present disclosure also provides a pair of primers comprising any of the above-mentioned primers. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than uracil) at the position corresponding to position 3,769 according to SEQ ID NO:6 in a particular NOTCH3 nucleic acid molecule, the presence of the amplified fragment will indicate the presence of a NOTCH3 reference-type mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with uracil (rather than cytosine) at the position corresponding to position 3,769 according to SEQ ID NO:11 in a particular NOTCH3 mRNA molecule, the presence of the amplified fragment will indicate the presence of a NOTCH3 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at the position corresponding to position 3,769 according to SEQ ID NO:11 can be present at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 3,769 according to SEQ ID NO: 16 in a particular NOTCH3 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of a NOTCH3 reference-type cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 3,769 according to SEQ ID NO: 21 in a particular NOTCH3 cDNA molecule, the presence of an amplified fragment will indicate the presence of a NOTCH3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 3,769 according to SEQ ID NO: 21 can be present at the 3' end of the primer.

[0154] The present disclosure also provides a pair of primers comprising any of the above-mentioned primers. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than uracil) at the position corresponding to position 3,544 according to SEQ ID NO: 7 in a particular NOTCH3 nucleic acid molecule, the presence of the amplified fragment will indicate the presence of a NOTCH3 reference-type mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with uracil (rather than cytosine) at the position corresponding to position 3,544 according to SEQ ID NO: 12 in a particular NOTCH3 mRNA molecule, the presence of the amplified fragment will indicate the presence of a NOTCH3 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the uracil at the position corresponding to position 3,544 according to SEQ ID NO: 12 can be present at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 3,544 according to SEQ ID NO: 17 in a particular NOTCH3 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of a NOTCH3 reference-type cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 3,544 according to SEQ ID NO: 22 in a particular NOTCH3 cDNA molecule, the presence of an amplified fragment will indicate the presence of a NOTCH3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at a position corresponding to position 3,544 according to SEQ ID NO: 22 can be present at the 3' end of the primer.

[0155] In the context of this disclosure, "specifically hybridizes" means that a probe or primer (e.g., a mutation-specific probe or mutation-specific primer) does not hybridize to a nucleic acid sequence encoding a NOTCH3 reference-type genomic nucleic acid molecule, a NOTCH3 reference-type mRNA molecule, and / or a NOTCH3 reference-type cDNA molecule.

[0156] In some embodiments, the probe (e.g., the mutation-specific probe) comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0157] The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are attached. A solid support is a solid-state substrate or support to which a molecule such as any of the probes disclosed herein can associate. A form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which a plurality of different probes are attached in an array, grid, or other organized pattern. A form of solid-state substrate is a microtiter dish, such as a standard 96-well format. In some embodiments, a multi-well glass slide can be used, which usually contains one array per well.

[0158] The nucleotide sequence of the NOTCH3 reference genomic nucleic acid molecule is set forth in SEQ ID NO: 1. With reference to SEQ ID NO: 1, position 21,944 is a cytosine.

[0159] There is a variant genomic nucleic acid molecule of NOTCH3 in which the cytosine at position 21,944 is replaced with a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:2.

[0160] The nucleotide sequence of the NOTCH3 reference-type mRNA molecule is set forth in SEQ ID NO: 3. With reference to SEQ ID NO: 3, position 3,781 is a cytosine.

[0161] The nucleotide sequence of another NOTCH3 reference-type mRNA molecule is set forth in SEQ ID NO: 4. With reference to SEQ ID NO: 4, position 3,767 is a cytosine.

[0162] The nucleotide sequence of another NOTCH3 reference-type mRNA molecule is set forth in SEQ ID NO: 5. With reference to SEQ ID NO: 5, position 3,532 is a cytosine.

[0163] The nucleotide sequence of another NOTCH3 reference-type mRNA molecule is set forth in SEQ ID NO: 6. With reference to SEQ ID NO: 6, position 3,769 is a cytosine.

[0164] The nucleotide sequence of another NOTCH3 reference-type mRNA molecule is set forth in SEQ ID NO: 7. With reference to SEQ ID NO: 7, position 3,544 is a cytosine.

[0165] There is a variant mRNA molecule of NOTCH3 in which the cytosine at position 3,781 is replaced by uracil, the nucleotide sequence of which is set forth in SEQ ID NO:8.

[0166] There is another variant mRNA molecule of NOTCH3 in which the cytosine at position 3,767 is replaced with a uracil. The nucleotide sequence of this variant mRNA molecule is set forth in SEQ ID NO:9.

[0167] There is another variant mRNA molecule of NOTCH3 in which the cytosine at position 3,532 is replaced by uracil. The nucleotide sequence of this variant mRNA molecule is set forth in SEQ ID NO:10.

[0168] There is another variant mRNA molecule of NOTCH3 in which the cytosine at position 3,769 is replaced with uracil. The nucleotide sequence of this variant mRNA molecule is set forth in SEQ ID NO:11.

[0169] There is another variant mRNA molecule of NOTCH3 in which the cytosine at position 3,544 is replaced by uracil. The nucleotide sequence of this variant mRNA molecule is set forth in SEQ ID NO:12.

[0170] The nucleotide sequence of the NOTCH3 reference type cDNA molecule is set forth in SEQ ID NO: 13. With reference to SEQ ID NO: 13, position 3,781 is a cytosine.

[0171] The nucleotide sequence of another NOTCH3 reference type cDNA molecule is set forth in SEQ ID NO: 14. With reference to SEQ ID NO: 14, position 3,767 is a cytosine.

[0172] The nucleotide sequence of another NOTCH3 reference type cDNA molecule is set forth in SEQ ID NO: 15. With reference to SEQ ID NO: 15, position 3,532 is a cytosine.

[0173] The nucleotide sequence of another NOTCH3 reference type cDNA molecule is set forth in SEQ ID NO: 16. With reference to SEQ ID NO: 16, position 3,769 is a cytosine.

[0174] The nucleotide sequence of another NOTCH3 reference type cDNA molecule is set forth in SEQ ID NO: 17. With reference to SEQ ID NO: 17, position 3,544 is a cytosine.

[0175] A variant cDNA molecule of NOTCH3 exists in which the cytosine at position 3,781 is replaced with a thymine, the nucleotide sequence of which is set forth in SEQ ID NO:18.

[0176] Another variant cDNA molecule of NOTCH3 exists in which the cytosine at position 3,767 is replaced with a thymine. The nucleotide sequence of this variant cDNA molecule is set forth in SEQ ID NO:19.

[0177] Another variant cDNA molecule of NOTCH3 exists in which the cytosine at position 3,532 is replaced by a thymine. The nucleotide sequence of this variant cDNA molecule is set forth in SEQ ID NO:20.

[0178] Another variant cDNA molecule of NOTCH3 exists in which the cytosine at position 3,769 is replaced with a thymine. The nucleotide sequence of this variant cDNA molecule is set forth in SEQ ID NO:21.

[0179] Another variant cDNA molecule of NOTCH3 exists in which the cytosine at position 3,544 is replaced by a thymine. The nucleotide sequence of this variant cDNA molecule is set forth in SEQ ID NO:22.

[0180] The genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be derived from any organism. For example, the genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be an ortholog from another organism, such as human or non-human mammal, rodent, mouse, or rat. It is understood that gene sequences within a population can differ due to polymorphisms, such as single nucleotide polymorphisms. The examples provided herein are merely exemplary sequences. Other sequences are possible.

[0181] Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Functional polynucleotides can act as effectors, inhibitors, modulators, and stimulators of the specific activity of target molecules, or functional polynucleotides can have de novo activity independent of any other molecules.

[0182] The isolated nucleic acid molecules disclosed herein can include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to heterologous nucleic acid sequences, for example in a vector, or heterologous labels. For example, the isolated nucleic acid molecules disclosed herein can be present in a vector containing the isolated nucleic acid molecule and a heterologous nucleic acid sequence, or as an exogenous donor sequence containing them. The isolated nucleic acid molecules can also be linked or fused to heterologous labels. The labels can be directly detectable (e.g., fluorophores, etc.) or indirectly detectable (e.g., haptens, enzymes, or fluorophore quenchers, etc.). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, dyes, dyes, chromogens, spin labels, and fluorescent labels. The labels can also be, for example, chemiluminescent substances; metal-containing substances; or enzymes (where enzyme-dependent secondary generation of signal occurs). The term "label" is used to indicate that the conjugated molecule produces a detectable signal when subsequently added together with a substrate. It may also refer to a "tag" or hapten that can selectively bind to a conjugated molecule, such as a conjugate of avidin or streptavidin. For example, biotin can be used as a tag together with a conjugate of horseradish peroxidase (HRP) with avidin or streptavidin to bind to the tag and detect the presence of HRP by using a colorimetric substrate (such as tetramethylbenzidine (TMB)) or a fluorogenic substrate. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3xFLAG, 6xHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes, and their colorimetric, fluorogenic, and chemiluminescent substrates, as well as other labels.

[0183] The disclosed nucleic acid molecules can include, for example, nucleotides, or non-natural or modified nucleotides, such as, for example, nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or nucleotides that incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0184] The nucleic acid molecules disclosed herein can also include one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide that contains a modification to either the base moiety, sugar moiety, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as different purine or pyrimidine bases, such as pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenine-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine, and 6-azothymine, 5-uracil, 6-azocytosine, and 6-azothymine, Examples of uracil and cytosines include, but are not limited to, uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo (such as 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0185] Nucleotide analogs can also include modifications to the sugar moiety, including, but not limited to, natural and synthetic modifications of ribose and deoxyribose. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1-10 Alkyl or C 2-10Alkenyl, and C 2-10 Exemplary 2' sugar modifications also include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON [(CH2) n Other modifications at the 2' position include, but are not limited to, C 1-10 These include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those containing modifications at the bridging ring oxygen (such as CH2 and S). Nucleotide sugar analogs can also have sugar mimetics (such as cyclobutyl moieties) in place of the pentofuranosyl sugar.

[0186] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. These phosphate bonds or modified phosphate bonds between two nucleotides can be via 3'-5' or 2'-5' bonds, and the bonds can contain reverse polarity (such as 3'-5' to 5'-3' or 2'-5' to 5'-2'). Various salts, mixed salts, and free acid forms are also included. Nucleotide alternatives also include peptide nucleic acids (PNAs).

[0187] The present disclosure also provides a vector comprising any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vector can be a viral vector or a non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or a cosmid (such as a circular double-stranded DNA to which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector to which additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses (such as cauliflower mosaic virus and tobacco mosaic virus), yeast artificial chromosomes (YACs), Epstein-Barr (EBV) derived episomes, and other expression vectors known in the art.

[0188] Desirable regulatory sequences for mammalian host cell expression may include, for example, viral elements directing high levels of polypeptide expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), Simian Virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma derived promoters and / or enhancers, as well as strong mammalian promoters (e.g., native immunoglobulin promoters and actin promoters). Methods for expressing polypeptides in bacterial or fungal cells (e.g., yeast cells) are also well known. The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter).

[0189] The percent identity (or percent complementarity) between particular stretches of nucleotide sequences in a nucleic acid molecule or amino acid sequences in a polypeptide can be routinely determined using the BLAST program (Basic Local Alignment Search Tool) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings that employ the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). As used herein, when percent sequence identity is referred to, a higher percentage of sequence identity is preferred over a lower one.

[0190] The present disclosure also provides a composition comprising any one or more of the isolated nucleic acid molecules, genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules disclosed herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution, such as PBS, HBSS, etc.

[0191] As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in the context of the numbering of a particular nucleotide or nucleotide sequence or position, refers to the numbering of the specified reference sequence when the particular nucleotide or nucleotide sequence is compared to a reference sequence (e.g., SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:13, etc.). In other words, the number of a residue (e.g., nucleotide or amino acid, etc.) of a particular polymer or the position of a residue (e.g., nucleotide or amino acid, etc.) is specified with reference to the reference sequence, not by the actual position number of that residue within the particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence can be aligned to a reference sequence by introducing gaps to optimize the residue matching between the two sequences. In these cases, although gaps exist, the numbering of the residues in the particular nucleotide or nucleotide sequence is done with reference to the reference sequence to which it is aligned.

[0192] For example, a nucleic acid molecule comprising a nucleotide sequence encoding a NOTCH3 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 21,944 according to SEQ ID NO:2, means that when the nucleotide sequence of a NOTCH3 genomic nucleic acid molecule is aligned against the sequence of SEQ ID NO:2, the NOTCH3 sequence has a thymine residue at a position corresponding to position 21,944 of SEQ ID NO:2. The same applies to an mRNA molecule comprising a nucleotide sequence encoding a NOTCH3 polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 3,781 according to SEQ ID NO:8, and to a cDNA molecule comprising a nucleotide sequence encoding a NOTCH3 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 3,781 according to SEQ ID NO:18. In other words, these terms refer to a nucleic acid molecule encoding a NOTCH3 polypeptide, wherein a genomic nucleic acid molecule has a nucleotide sequence that includes a thymine residue that is homologous to the thymine residue at position 21,944 of SEQ ID NO:2 (or an mRNA molecule has a nucleotide sequence that includes a uracil residue that is homologous to the uracil residue at position 3,781 of SEQ ID NO:8, or a cDNA molecule has a nucleotide sequence that includes a thymine residue that is homologous to the uracil residue at position 3,781 of SEQ ID NO:18).

[0193] As described herein, the position in the NOTCH3 genomic nucleic acid molecule corresponding to position 21,944 according to SEQ ID NO:2 can be identified, for example, by performing sequence alignment between the nucleotide sequence of a particular NOTCH3 nucleic acid molecule and the nucleotide sequence of SEQ ID NO:2. For example, there are various computer algorithms that can be used to perform sequence alignment to identify the nucleotide position corresponding to position 21,944 of SEQ ID NO:2. For example, sequence alignment can be performed by using the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res., 1997, 25, 3389-3402) or CLUSTALW software (Sievers and Higgins, Methods Mol. Biol., 2014, 1079, 105-116). However, sequences can also be aligned manually.

[0194] The amino acid sequence of the NOTCH3 reference polypeptide is set forth in SEQ ID NO: 23. With reference to SEQ ID NO: 23, the NOTCH3 reference polypeptide is 1,286 amino acids in length. With reference to SEQ ID NO: 23, position 1,178 is an arginine.

[0195] Another NOTCH3 reference polypeptide amino acid sequence is set forth in SEQ ID NO: 24. With reference to SEQ ID NO: 24, the NOTCH3 reference polypeptide is 2,321 amino acids in length. With reference to SEQ ID NO: 24, position 1,231 is an arginine.

[0196] Another NOTCH3 reference polypeptide amino acid sequence is set forth in SEQ ID NO: 25. With reference to SEQ ID NO: 25, the NOTCH3 reference polypeptide is 1,289 amino acids in length. With reference to SEQ ID NO: 25, position 1,182 is an arginine.

[0197] There is a variant polypeptide (Arg1178Cys) of NOTCH3, the amino acid sequence of which is set forth in SEQ ID NO: 26. With reference to SEQ ID NO: 26, the NOTCH3 variant polypeptide is 1,286 amino acids long. With reference to SEQ ID NO: 26, position 1,178 is a cysteine.

[0198] There is another variant polypeptide of NOTCH3 (Arg1231Cys), the amino acid sequence of which is set forth in SEQ ID NO: 27. With reference to SEQ ID NO: 27, the NOTCH3 variant polypeptide is 2,321 amino acids long. With reference to SEQ ID NO: 27, position 1,231 is a cysteine.

[0199] There is another variant polypeptide of NOTCH3 (Arg1182Cys), the amino acid sequence of which is set forth in SEQ ID NO: 28. With reference to SEQ ID NO: 28, the NOTCH3 variant polypeptide is 1,289 amino acids long. With reference to SEQ ID NO: 28, position 1,182 is a cysteine.

[0200] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for the nucleotide bases and three-letter code for the amino acids. The nucleotide sequences follow the standard convention of starting at the 5'-end of the sequence and proceeding forward to the 3'-end (i.e., from left to right on each line). Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be encompassed by any reference to the presented strand. The amino acid sequences follow the standard convention of starting at the amino terminus of the sequence and proceeding forward to the carboxy terminus (i.e., from left to right on each sequence).

[0201] The present disclosure also provides a therapeutic agent for treating or inhibiting cerebrovascular disease for use in treating cerebrovascular disease in a subject (or for use in preparing a medicament for treating cerebrovascular disease), wherein the subject has any of NOTCH3 variant genomic nucleic acid molecules, variant mRNA molecules, and / or variant cDNA molecules encoding a NOTCH3 polypeptide described herein. The therapeutic agent for treating or inhibiting cerebrovascular disease can be any of the therapeutic agents for treating or inhibiting cerebrovascular disease described herein.

[0202] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a NOTCH3 polypeptide, the nucleotide sequence including a thymine or its complement at a position corresponding to position 21,944 according to SEQ ID NO:2.

[0203] In some embodiments, the subject is identified as having an mRNA molecule having a nucleotide sequence encoding a NOTCH3 polypeptide, the nucleotide sequence including uracil or its complement at a position corresponding to position 3,781 according to SEQ ID NO:8; uracil or its complement at a position corresponding to position 3,767 according to SEQ ID NO:9; uracil or its complement at a position corresponding to position 3,532 according to SEQ ID NO:10; uracil or its complement at a position corresponding to position 3,769 according to SEQ ID NO:11; or uracil or its complement at a position corresponding to position 3,544 according to SEQ ID NO:12.

[0204] In some embodiments, the subject is identified as having a cDNA molecule having a nucleotide sequence encoding a NOTCH3 polypeptide, the nucleotide sequence including a thymine or its complement at a position corresponding to position 3,781 according to SEQ ID NO:18; a thymine or its complement at a position corresponding to position 3,767 according to SEQ ID NO:19; a thymine or its complement at a position corresponding to position 3,532 according to SEQ ID NO:20; a thymine or its complement at a position corresponding to position 3,769 according to SEQ ID NO:21; or a thymine or its complement at a position corresponding to position 3,544 according to SEQ ID NO:22.

[0205] The present disclosure also provides a NOTCH3 agent for use in treating cerebrovascular disease (or for use in preparing a medicament for treating cerebrovascular disease) in a subject, wherein the subject has any of a NOTCH3 variant genomic nucleic acid molecule, variant mRNA molecule, and / or variant cDNA molecule encoding a NOTCH3 polypeptide described herein. The NOTCH3 agent can be any of the NOTCH3 agents described herein.

[0206] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a NOTCH3 polypeptide, the nucleotide sequence including a thymine or its complement at a position corresponding to position 21,944 according to SEQ ID NO:2.

[0207] In some embodiments, the subject is identified as having an mRNA molecule having a nucleotide sequence encoding a NOTCH3 polypeptide, the nucleotide sequence including uracil or its complement at a position corresponding to position 3,781 according to SEQ ID NO:8; uracil or its complement at a position corresponding to position 3,767 according to SEQ ID NO:9; uracil or its complement at a position corresponding to position 3,532 according to SEQ ID NO:10; uracil or its complement at a position corresponding to position 3,769 according to SEQ ID NO:11; or uracil or its complement at a position corresponding to position 3,544 according to SEQ ID NO:12.

[0208] In some embodiments, the subject is identified as having a cDNA molecule having a nucleotide sequence encoding a NOTCH3 polypeptide, the nucleotide sequence including a thymine or its complement at a position corresponding to position 3,781 according to SEQ ID NO:18; a thymine or its complement at a position corresponding to position 3,767 according to SEQ ID NO:19; a thymine or its complement at a position corresponding to position 3,532 according to SEQ ID NO:20; a thymine or its complement at a position corresponding to position 3,769 according to SEQ ID NO:21; or a thymine or its complement at a position corresponding to position 3,544 according to SEQ ID NO:22.

[0209] All patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be so incorporated by reference. If various versions of a sequence are associated with accession numbers at various times, the version associated with the accession number at the effective filing date of this application is meant. Effective filing date means the earlier of the actual filing date or the filing date of the priority application that refers to the accession number, if applicable. Similarly, if different versions of publications, websites, etc. are published at different times, the version last published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure may be used in combination with any other feature, step, element, embodiment, or aspect, unless otherwise indicated. Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be made within the scope of the appended claims.

[0210] The following examples are provided to describe the embodiments in more detail. They are intended to illustrate, not limit, the claimed embodiments. The following examples provide those skilled in the art with disclosure and explanation of how the compounds, compositions, articles, devices and / or methods described herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of any claims. Attempts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some error and deviation can be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is ° C. or at ambient temperature, and pressure is at or near atmospheric pressure. EXAMPLES

[0211] Example 1: Association of NOTCH3 with increased risk of developing cerebrovascular disease Over 19,000 Pakistanis were recruited for the genetic study from 22 clinical centers in five cities, including 4,882 stroke cases and 6,904 controls classified using TOAST and Oxfordshire criteria. The demographics of stroke cases and controls are shown in Tables 3 and 4. [Table 3] [Table 4]

[0212] Exome sequencing and ExWAS were performed for 12 binary stroke traits and identified 36 hits, including 29 enriched in Pakistani compared to Europeans (p-value < 1e-7), of which 7 were missense or LoF (Table 5). [Table 5]

[0213] Read stack QC excluded 7 closely spaced associated variant pairs (PTPRD: 10bp, ZNF33A: 6bp, PTPRB: 3bp, MYCBP2: 5bp) with MAC=5, and 1 variant excluded due to unrelated biology (increased fertility) (GPR149 3:154429224:A:C, p.Phe131Cys, p-value 7.96E-08, effect 28.04, cases 2698|2693|5|0 controls 6871|6871|0|0, MAF 2.6E-04).

[0214] A missense SNV in NOTCH3 (p.Arg1231Cys, GRCh38:Chr19:15179052:G:A, rs201680145) was the only significant hit in the CNCD F2 subcortical stroke EXWAS (p-value 2.1e-8, effect size 2.97 (2.01,4.35), MAF 7.1e-3 (Table 6). The variant has an elevated MAF in cases, is predicted pathogenic by PolyPhen (score 0.83), and affects an epidermal growth factor (EGF) repeat in a gene that is highly conserved in mice (93%). The variant adds a Cys to number 31 of 34 EGF repeats (each of which normally has 6 Cys residues) in the NOTCH3 extracellular domain (ECD). Variants that add to or remove from the ECD are known to be pathogenic in CADASIL. [Table 6]

[0215] P.Arg1231cys is the only associated variant at the NOTCH3 locus (Figure 1). The variant has an elevated MAF in cases, suggesting a risk-enhancing effect. There was one homozygous control. This individual was 45 years old, younger than the mean age of stroke onset in Pakistan (50) and younger than the median age of subcortical stroke cases (56, the same as the recruitment age of this cohort). We hypothesize that this individual may be asymptomatic for stroke but may exhibit early symptoms of CADASIL, such as migraine headaches, GOM accumulation in the vasculature, and white matter hyperintensities, and may be at increased risk for stroke. Previous studies have suggested that there is a more severe CADASIL phenotype in homozygotes than heterozygotes for this variant. [Table 7]

[0216] PheWAS of NOTCH3 p.Arg1231Cys revealed nominal p-values ​​for multiple stroke phenotypes, including lacunar artifacts (LACI), small artery atherosclerosis (SAA), partial anterior circulation infarction (PACI), family history of stroke, and ischemic stroke (Figures 3 and 4).

[0217] The NOTCH3 Arg1231Cys variant was observed in RGC cohorts of African, European, mixed American, and South Asian ancestry. The highest prevalence was observed in South Asians (Table 8). One homozygote was observed in a mixed American (Mexico City cohort) (55-year-old male, blood pressure 80 / 116, BMI 25, HbA1c 5.2, started smoking at age 18, no history of major CNS bleeding or other diseases). Similar to the homozygote in CNCD F2, we hypothesize that this individual may have other symptoms of CADASIL, such as migraines, GOM, vascular pathology, and cerebral white matter loss, that were not collected in this cohort. [Table 8]

[0218] The arginine (R) residue is highly conserved across mammalian species, and the p.Arg1231Cys variant is predicted to be deleterious (PolyPhen2 score 0.843).

[0219] In addition to those described herein, various modifications of the described subject matter will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, etc.) is incorporated herein by reference in its entirety for all purposes.

Claims

1. 1. An in vitro method for identifying a subject's susceptibility to developing cerebrovascular disease, the method comprising: determining the presence or absence of a NOTCH3 missense variant nucleic acid molecule encoding a predicted gain-of-function polypeptide at neurogenic locus Notch homolog protein 3 (NOTCH3) in a biological sample obtained from the subject; the subject being a NOTCH3 reference type is an indication that the subject does not have an increased risk of developing the cerebrovascular disease; The method, wherein the subject being heterozygous or homozygous for the NOTCH3 missense variant nucleic acid molecule is an indication that the subject has an increased risk of developing the cerebrovascular disease.

2. 2. The method of claim 1, wherein the NOTCH3 missense variant nucleic acid molecule is a nucleic acid molecule encoding Arg1178Cys, Arg1231Cys, or Arg1182Cys.

3. 2. The method of claim 1, wherein the NOTCH3 missense variant nucleic acid molecule is a nucleic acid molecule encoding Arg1231Cys.

4. The NOTCH3 missense variant nucleic acid molecule comprises: A genomic nucleic acid molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 21,944 according to SEQ ID NO:2; an mRNA molecule having a nucleotide sequence comprising uracil at a position corresponding to position 3,781 according to SEQ ID NO:8, at a position corresponding to position 3,767 according to SEQ ID NO:9, at a position corresponding to position 3,532 according to SEQ ID NO:10, at a position corresponding to position 3,769 according to SEQ ID NO:11, or at a position corresponding to position 3,544 according to SEQ ID NO:12; or A cDNA molecule produced from an mRNA molecule, said cDNA molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 3,781 according to SEQ ID NO:18, at a position corresponding to position 3,767 according to SEQ ID NO:19, at a position corresponding to position 3,532 according to SEQ ID NO:20, at a position corresponding to position 3,769 according to SEQ ID NO:21, or at a position corresponding to position 3,544 according to SEQ ID NO:

22. The method according to claim 2, wherein

5. The method according to any one of claims 1 to 4, wherein the cerebrovascular disease comprises subcortical stroke, ischemic stroke, hemorrhagic stroke, parenchymal stroke, or cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL).

6. Use of a therapeutic agent that treats or inhibits cerebrovascular disease in the preparation of a medicament for treating cerebrovascular disease in a subject identified as having a NOTCH3 missense variant nucleic acid molecule encoding neurogenic locus Notch homolog protein 3 (NOTCH3) Arg1178Cys, Arg1231Cys, or Arg1182Cys.

7. The NOTCH3 missense variant nucleic acid molecule, A genomic nucleic acid molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 21,944 according to SEQ ID NO:2, or a complement thereof; an mRNA molecule having a nucleotide sequence comprising uracil or its complement at a position corresponding to position 3781 according to SEQ ID NO:8; uracil or its complement at a position corresponding to position 3767 according to SEQ ID NO:9; uracil or its complement at a position corresponding to position 3532 according to SEQ ID NO:10; uracil or its complement at a position corresponding to position 3769 according to SEQ ID NO:11; or uracil or its complement at a position corresponding to position 3544 according to SEQ ID NO:12; or A cDNA molecule produced from an mRNA molecule, said cDNA molecule having a nucleotide sequence comprising: a thymine or its complement at a position corresponding to position 3781 according to SEQ ID NO:18; a thymine or its complement at a position corresponding to position 3767 according to SEQ ID NO:19; a thymine or its complement at a position corresponding to position 3532 according to SEQ ID NO:20; a thymine or its complement at a position corresponding to position 3769 according to SEQ ID NO:21; or a thymine or its complement at a position corresponding to position 3544 according to SEQ ID NO:

22. The use according to claim 6, wherein 8. The use of claim 6, wherein the medication comprises tissue plasminogen activator (tPA), an anticoagulant, a diuretic, an ACE inhibitor, a beta blocker, a calcium channel blocker, or a statin, or any combination thereof.

9. The anticoagulant comprises apixaban, dabigatran, edoxaban, rivaroxaban, or warfarin, the diuretic comprises bumetanide, ethacrynic acid, furosemide, or torsemide; the ACE inhibitor comprises benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, or trandolapril; the beta blocker comprises acebutolol, atenolol, bisoprolol, metoprolol, nadolol, nebivolol, or propranolol; the calcium channel blocker comprises amlodipine, felodipine, isradipine, nicardipine, nifedipine, nimodipine, or nitrendipine; The statin comprises atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, or simvastatin; 9. The use according to claim 8.

10. The use described in any one of claims 6 to 9, wherein the cerebrovascular disease comprises subcortical stroke, ischemic stroke, hemorrhagic stroke, parenchymal stroke, or cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL).

11. Use of a neurogenic locus Notch homolog protein 3 (NOTCH3) agent in the preparation of a medicament for treating cerebrovascular disease in a subject.

12. The use described in claim 11, wherein the NOTCH3 agent is an inhibitory nucleic acid molecule.

13. The use according to claim 12, wherein the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes with a NOTCH3 nucleic acid molecule.

14. The use described in claim 11, wherein the NOTCH3 agent comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within a NOTCH3 genomic nucleic acid molecule.

15. The use according to claim 14, wherein the Cas protein is Cas9 or Cpf1.

16. The use of claim 14, wherein the gRNA recognition sequence comprises or is adjacent to position 21,944 according to SEQ ID NO:

2.

17. The use of claim 14, wherein the gRNA recognition sequence is located about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides away from a position corresponding to position 21,944 according to SEQ ID NO:

2.

18. The use of claim 14, wherein a protospacer adjacent motif (PAM) sequence is located about 2 to about 6 nucleotides downstream of the gRNA recognition sequence.

19. The use of claim 14, wherein the gRNA comprises from about 17 to about 23 nucleotides.

20. The use according to claim 14, wherein the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs: 29 to 43.

21. The use of claim 11, wherein the subject is heterozygous or homozygous for a NOTCH3 reference type or a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide.

22. The use of claim 21, wherein the NOTCH3 missense variant nucleic acid molecule encodes NOTCH3 Arg1178Cys, Arg1231Cys, or Arg1182Cys.

23. The object, A genomic nucleic acid molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 21,944 according to SEQ ID NO:2, or a complement thereof; an mRNA molecule having a nucleotide sequence comprising uracil or its complement at a position corresponding to position 3781 according to SEQ ID NO:8; uracil or its complement at a position corresponding to position 3767 according to SEQ ID NO:9; uracil or its complement at a position corresponding to position 3532 according to SEQ ID NO:10; uracil or its complement at a position corresponding to position 3769 according to SEQ ID NO:11; or uracil or its complement at a position corresponding to position 3544 according to SEQ ID NO:12; or A cDNA molecule produced from an mRNA molecule, said cDNA molecule having a nucleotide sequence comprising: a thymine or its complement at a position corresponding to position 3781 according to SEQ ID NO:18; a thymine or its complement at a position corresponding to position 3767 according to SEQ ID NO:19; a thymine or its complement at a position corresponding to position 3532 according to SEQ ID NO:20; a thymine or its complement at a position corresponding to position 3769 according to SEQ ID NO:21; or a thymine or its complement at a position corresponding to position 3544 according to SEQ ID NO:

22. The use according to claim 11, comprising:

24. The use described in claim 11, wherein the cerebrovascular disease includes subcortical stroke, ischemic stroke, hemorrhagic stroke, parenchymal stroke, or cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL).

25. The use of any one of claims 11 to 24, wherein the NOTCH3 agent comprises an anti-aggregating antibody or an agonist antibody that increases processing.

26. A pharmaceutical composition for treating cerebrovascular disease in a subject, comprising a neurogenic locus Notch homolog protein 3 (NOTCH3) agent as an active ingredient.

27. ​​The pharmaceutical composition of claim 26, wherein the NOTCH3 agent comprises an inhibitory nucleic acid molecule that hybridizes with a NOTCH3 nucleic acid molecule.

28. The pharmaceutical composition of claim 27, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

29. The pharmaceutical composition of claim 26, wherein the cerebrovascular disease comprises subcortical stroke, ischemic stroke, hemorrhagic stroke, parenchymal stroke, or cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL).

30. The pharmaceutical composition of claim 26, wherein the subject is heterozygous or homozygous for a NOTCH3 reference type or a NOTCH3 missense variant nucleic acid molecule encoding a NOTCH3 predicted gain-of-function polypeptide.

31. The pharmaceutical composition of claim 30, wherein the NOTCH3 missense variant nucleic acid molecule encodes NOTCH3 Arg1178Cys, Arg1231Cys, or Arg1182Cys.

32. The object, A genomic nucleic acid molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 21,944 according to SEQ ID NO:2, or a complement thereof; an mRNA molecule having a nucleotide sequence comprising uracil or its complement at a position corresponding to position 3781 according to SEQ ID NO:8; uracil or its complement at a position corresponding to position 3767 according to SEQ ID NO:9; uracil or its complement at a position corresponding to position 3532 according to SEQ ID NO:10; uracil or its complement at a position corresponding to position 3769 according to SEQ ID NO:11; or uracil or its complement at a position corresponding to position 3544 according to SEQ ID NO:12; or A cDNA molecule produced from an mRNA molecule, said cDNA molecule having a nucleotide sequence comprising: a thymine or its complement at a position corresponding to position 3781 according to SEQ ID NO:18; a thymine or its complement at a position corresponding to position 3767 according to SEQ ID NO:19; a thymine or its complement at a position corresponding to position 3532 according to SEQ ID NO:20; a thymine or its complement at a position corresponding to position 3769 according to SEQ ID NO:21; or a thymine or its complement at a position corresponding to position 3544 according to SEQ ID NO:

22. The pharmaceutical composition according to any one of claims 26 to 31, comprising:

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