Treatment of cognitive impairment with α-N-acetylgalactosaminide α-2,6-sialyltransferase 5 (ST6GALNAC5) inhibitors

Abstract

The present disclosure provides methods of treating a subject having or at risk of developing a cognitive disorder, and methods of identifying a subject having an increased risk of developing a cognitive disorder.

Description

[Technical field]

[0001] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically as a text file of 634 kilobytes in size under the title 18923808002SEQ, created on June 25, 2022. This Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates broadly to the treatment of subjects with cognitive impairment with α-N-acetylgalactosaminide α-2,6-sialyltransferase 5 (ST6GALNAC5) inhibitors, and to methods of identifying subjects at high risk for developing cognitive impairment. [Background technology]

[0003] Cognitive impairment may be characterized by an individual's difficulty in remembering, learning new things, concentrating, or making decisions that affect daily life. Cognitive impairment can range from mild to severe. With mild impairment, an individual may begin to notice changes in cognitive function, but still be able to perform daily activities. Severe levels of impairment may lead to loss of ability to understand the meaning or significance of things and the ability to speak or write, resulting in an inability to live independently. Cognitive impairment includes, but is not limited to, myelin integrity loss, neurodegeneration, gray matter / white matter contrast (GWC) loss, and conversion from mild cognitive impairment to dementia.

[0004] GWC can be used across a wide range of brain regions to assess cognitive impairment. GWC is a measure of the blurring between the boundaries of gray matter / white matter brain compartments, and is considered to be an indicator of regional variations in tissue integrity and myelin degradation, increased water content in white matter, or iron deposition (Non-Patent Document 1). Lower GWC is associated with aging and lower cognitive index (Non-Patent Document 2), as well as increased conversion rate from mild cognitive impairment to dementia (Non-Patent Document 3).

[0005] α-N-acetylgalactosaminide α-2,6-sialyltransferase 5 (ST6GALNAC5) is a gene that catalyzes the biosynthesis of ganglioside GD1α from GM1b in the brain (Non-Patent Document 4). The ST6GALNAC5 gene was previously identified as one of the genes that mediate brain metastasis of breast cancer. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Uribe et al., Front.Aging Neurosci., 2018, 10, 89 [Non-Patent Document 2] Lewis et al., Neuroimage., 2018, 173, 341-350 [Non-Patent Document 3] Jefferson et al., Brain Imaging Behav.,2015,9,141-8 [Non-Patent Document 4] Okajima et al., J. Biol. Chem., 1999, 274, 30557-30562 Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides a method of treating a subject having or at risk of developing a cognitive impairment, the method comprising administering to the subject an ST6GALNAC5 inhibitor. The present disclosure also provides a method of treating a subject having or at risk of developing reduced myelin integrity, the method comprising administering to the subject an ST6GALNAC5 inhibitor.

[0008] The present disclosure also provides a method of treating a subject having or at risk of developing neurodegeneration, the method comprising administering to the subject an ST6GALNAC5 inhibitor. The present disclosure also provides a method of treating a subject having or at risk of developing gray / white matter contrast (GWC) reduction, the method comprising administering to the subject an ST6GALNAC5 inhibitor.

[0009] The present disclosure also provides a method of treating a subject experiencing or at risk of conversion from mild cognitive impairment to dementia, the method comprising administering to the subject an ST6GALNAC5 inhibitor.

[0010] The disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits a cognitive disorder, the subject having or at risk of developing a cognitive disorder, the method comprising determining whether the subject has a ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 by obtaining or obtaining a biological sample from the subject and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype that includes a ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5; and i) administering to the subject a standard dose of a therapeutic agent that treats or inhibits a cognitive disorder. or ii) administering or continuing to administer a therapeutic agent that treats or inhibits cognitive impairment, at the same or a lower dose than a standard dose, to a subject who is heterozygous for an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, and / or administering an ST6GALNAC5 inhibitor to the subject; wherein the presence of a genotype having an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 indicates that the subject has a low risk of developing a cognitive disorder.

[0011] The present disclosure also provides a method for identifying a subject having an elevated risk of developing a cognitive disorder, the method comprising determining or having determined the presence or absence of an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 in a biological sample obtained from the subject; wherein if the subject is ST6GALNAC5 criterion, the subject has an elevated risk of developing a cognitive disorder, and if the subject is heterozygous or homozygous for an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the subject has a lower risk of developing a cognitive disorder.

[0012] The disclosure also provides a method for detecting an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 or its complement in a subject, the method comprising assaying a biological sample obtained from the subject to determine whether the nucleic acid molecule in the biological sample is: i) a genomic nucleic acid molecule having a nucleotide sequence that includes a cytosine or its complement at a position corresponding to position 176,867 set forth in SEQ ID NO:2; ii) a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:17; or its complement; or an mRNA molecule having a nucleotide sequence which comprises a cytosine or its complement at a position corresponding to position 600 as set forth in SEQ ID NO: 18; or iii) a cDNA molecule generated from the mRNA molecule in the biological sample, the cDNA molecule having a nucleotide sequence which comprises a cytosine or its complement at a position corresponding to position 600 as set forth in SEQ ID NO: 27; a cytosine or its complement at a position corresponding to position 639 as set forth in SEQ ID NO: 28; a cytosine or its complement at a position corresponding to position 562 as set forth in SEQ ID NO: 29; a cytosine or its complement at a position corresponding to position 515 as set forth in SEQ ID NO: 30; a cytosine or its complement at a position corresponding to position 579 as set forth in SEQ ID NO: 31; a cytosine or its complement at a position corresponding to position 416 as set forth in SEQ ID NO: 32; a cytosine or its complement at a position corresponding to position 639 as set forth in SEQ ID NO: 33; or a cytosine or its complement at a position corresponding to position 600 as set forth in SEQ ID NO: 34.

[0013] The present disclosure also provides a therapeutic agent for treating, inhibiting, or preventing cognitive impairment, comprising: i) a genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, having a nucleotide sequence including a cytosine or its complement at a position corresponding to position 176,867 set forth in SEQ ID NO:2; or a complement thereof; ii) an mRNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence including a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:15; or a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:16. or its complement; a cytosine or its complement at a position corresponding to position 639 as set forth in SEQ ID NO: 17; or a cytosine or its complement at a position corresponding to position 600 as set forth in SEQ ID NO: 18; or iii) a cDNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 as set forth in SEQ ID NO: 27; a cytosine or its complement at a position corresponding to position 639 as set forth in SEQ ID NO: 28; a cytosine or its complement at a position corresponding to position 562 as set forth in SEQ ID NO: 29; a cytosine or its complement at a position corresponding to position 515 as set forth in SEQ ID NO: 30; a cytosine or its complement at a position corresponding to position 579 as set forth in SEQ ID NO: 31; a cytosine or its complement at a position corresponding to position 416 as set forth in SEQ ID NO: 32; a cytosine or its complement at a position corresponding to position 639 as set forth in SEQ ID NO: 33;or a cytosine at a position corresponding to position 600 of SEQ ID NO: 34, or its complement, for use in treating or preventing a cognitive disorder (or for use in the preparation of a medicament for treating a cognitive disorder) in a subject identified as having said cDNA molecule.

[0014] The present disclosure also provides an ST6GALNAC5 inhibitor, comprising: a) a genomic nucleic acid molecule that is referenced to an ST6GALNAC5 genomic nucleic acid molecule, an ST6GALNAC5 mRNA molecule, or an ST6GALNAC5 cDNA molecule, or b) a genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 having a nucleotide sequence that includes i) a cytosine or its complement at a position corresponding to position 176,867 set forth in SEQ ID NO:2; ii) a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:17. or a complement thereof; or an mRNA molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 having a nucleotide sequence comprising a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO: 18; or a complement thereof; or iii) a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO: 27; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO: 28; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO: 29; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO: 30; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO: 31; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO: 32; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO: 33;or a cDNA molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 having a nucleotide sequence including a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:34, or a complement thereof, for use in treating or preventing a cognitive disorder in a subject (or for use in the preparation of a medicament for treating or preventing a cognitive disorder). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Various terms relating to the aspects of the present disclosure are used throughout the specification and claims. Unless otherwise indicated, such terms are to be given their ordinary meaning in the art. Other terms that are specifically defined are to be interpreted in a manner consistent with the definitions set forth herein.

[0016] Unless expressly stated otherwise, any method or embodiment set forth herein is in no way intended to be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically specifies in the claim or description that the steps are to be limited to a particular order, it is in no way intended to dictate order in any respect. This also applies to any possible implicit criteria of interpretation, including logical matters regarding the arrangement of steps or work flow, general meanings derived from grammatical construction or punctuation, or the number or type of embodiments described herein.

[0017] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. As used herein, the term "about" means that a cited numerical value is approximate, and small variations do not significantly affect the practice of the disclosed embodiments. When a numerical value is used, unless otherwise indicated by context, the term "about" means that the numerical value can vary by ±10% and remain within the range of the disclosed embodiments.

[0018] As used herein, the term "comprising" may in certain embodiments be replaced with "consisting" or "consisting essentially of," as desired.

[0019] 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., more than 95% pure or more 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.

[0020] 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.

[0021] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs), pet animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, rabbits), and non-human primates (e.g., apes and monkeys). In some embodiments, the subject is a human. In some embodiments, the subject is a patient under the care of a physician.

[0022] The present disclosure has identified rare loss-of-function variants in the ST6GALNAC5 gene that are associated with a low risk of developing cognitive impairment in humans. For example, it has been observed that a genetic variant that changes thymine to cytosine at position 176,867 of the ST6GALNAC5 reference genomic nucleic acid molecule (see SEQ ID NO: 1) indicates that subjects with such variants may have a low risk of developing cognitive impairment. Variants of the ST6GALNAC5 gene or protein are not believed to have a known association with cognitive impairment. In summary, the genetic analysis described herein surprisingly shows that the ST6GALNAC5 gene, and in particular, variants of the ST6GALNAC5 gene, are associated with a low risk of developing cognitive impairment. Thus, subjects with ST6GALNAC5 references that have a high risk of developing cognitive impairment, such as myelin integrity loss, neurodegeneration, GWC loss, and conversion of mild cognitive impairment to dementia, can be treated to prevent cognitive impairment, reduce symptoms, and / or inhibit the onset of symptoms. Thus, the present disclosure provides methods that utilize the identification of such variants in a subject to identify or stratify the risk in such a subject of developing a cognitive disorder such as myelin integrity loss, neurodegeneration, GWC loss, and conversion of mild cognitive impairment to dementia, or to diagnose a subject as having an increased risk of developing a cognitive disorder such as myelin integrity loss, neurodegeneration, GWC loss, and conversion of mild cognitive impairment to dementia, such that at-risk subjects or subjects with active disease can be treated accordingly.

[0023] For the purposes of this disclosure, any particular subject can be classified as having one of three ST6GALNAC5 genotypes: i) ST6GALNAC5 standard; ii) heterozygous for ST6GALNAC5 missense variant nucleic acid molecule encoding predicted loss-of-function polypeptide of ST6GALNAC5; or iii) homozygous for ST6GALNAC5 missense variant nucleic acid molecule encoding predicted loss-of-function polypeptide of ST6GALNAC5. If the subject does not have a copy of ST6GALNAC5 missense variant nucleic acid molecule encoding predicted loss-of-function polypeptide of ST6GALNAC5, the subject is ST6GALNAC5 standard. If the subject has a single copy of ST6GALNAC5 missense variant nucleic acid molecule encoding predicted loss-of-function polypeptide of ST6GALNAC5, the subject is heterozygous for ST6GALNAC5 missense variant nucleic acid molecule encoding predicted loss-of-function polypeptide of ST6GALNAC5. As used herein, an ST6GALNAC5 missense variant nucleic acid molecule is any ST6GALNAC5 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes an ST6GALNAC5 polypeptide with partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. A subject having an ST6GALNAC5 missense variant nucleic acid molecule that encodes a predicted ST6GALNAC5 loss-of-function polypeptide with partial loss of function (or predicted partial loss of function) is hypomorphic for ST6GALNAC5. ST6GALNAC5 missense variant nucleic acid molecules that encode predicted ST6GALNAC5 loss-of-function polypeptides include ST6GALNAC5 Val135Ala, Val4 ... * , or Val135Ala *It may be any nucleic acid molecule encoding a polypeptide. If a subject has two copies of a ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, then the subject is homozygous for the ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5.

[0024] For subjects who are genotyped or determined to be ST6GALNAC5-based, such subjects have an increased risk of developing cognitive impairment, such as myelin integrity loss, neurodegeneration, GWC loss, and conversion of mild cognitive impairment to dementia. For subjects who are genotyped or determined to be ST6GALNAC5-based or heterozygous for a ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, such subjects can be treated with an ST6GALNAC5 inhibitor.

[0025] In any of the embodiments described throughout this disclosure, the ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 can be any ST6GALNAC5 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes an ST6GALNAC5 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function. For example, the ST6GALNAC5 missense variant nucleic acid molecule can be any ST6GALNAC5 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes an ST6GALNAC5 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function. * , or Val135Ala *In some embodiments, the ST6GALNAC5 missense variant nucleic acid molecule encodes ST6GALNAC5 Val135Ala. In some embodiments, the ST6GALNAC5 missense variant nucleic acid molecule encodes ST6GALNAC5 Val45Ala. In some embodiments, the ST6GALNAC5 missense variant nucleic acid molecule encodes ST6GALNAC5 Val45Ala. * In some embodiments, the ST6GALNAC5 missense variant nucleic acid molecule encodes ST6GALNAC5 Val135Ala * Code the following:

[0026] In any of the embodiments described throughout this disclosure, the predicted loss-of-function polypeptide of ST6GALNAC5 can be any ST6GALNAC5 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function. In any of the embodiments described throughout this disclosure, the predicted loss-of-function polypeptide of ST6GALNAC5 can be, for example, ST6GALNAC5 Val135Ala, Val45Ala, Val45Ala. * , or Val135Ala * In some embodiments, the predicted loss-of-function polypeptide of ST6GALNAC5 is ST6GALNAC5 Val135Ala. In some embodiments, the predicted loss-of-function polypeptide of ST6GALNAC5 is ST6GALNAC5 Val45Ala. In some embodiments, the predicted loss-of-function polypeptide of ST6GALNAC5 is ST6GALNAC5 Val45Ala. * In some embodiments, the predicted loss-of-function polypeptide of ST6GALNAC5 is ST6GALNAC5 Val135Ala * It is.

[0027] In any of the embodiments described throughout the disclosure, the cognitive impairment is myelin integrity loss, neurodegeneration, GWC loss, or conversion of mild cognitive impairment to dementia. In any of the embodiments described throughout the disclosure, the cognitive impairment is myelin integrity loss. In any of the embodiments described throughout the disclosure, the cognitive impairment is neurodegeneration. In any of the embodiments described throughout the disclosure, the cognitive impairment is GWC loss. In any of the embodiments described throughout the disclosure, the cognitive impairment is conversion of mild cognitive impairment to dementia.

[0028] Symptoms of cognitive impairment include, but are not limited to, memory loss, frequently asking the same questions or repeating the same story several times, not recognizing familiar people and places, difficulty making decisions (such as knowing what to do in an emergency), changes in mood or behavior, vision problems, and difficulty planning and carrying out tasks.

[0029] The present disclosure provides a method of treating a subject having or at risk of developing a cognitive impairment, the method comprising administering to the subject an ST6GALNAC5 inhibitor. The present disclosure also provides a method of treating a subject having or at risk of developing reduced myelin integrity, the method comprising administering to the subject an ST6GALNAC5 inhibitor.

[0030] The present disclosure also provides a method of treating a subject having or at risk of developing neurodegeneration, the method comprising administering to the subject an ST6GALNAC5 inhibitor. The present disclosure also provides a method of treating a subject having or at risk of developing reduced GWC, the method comprising administering to the subject an ST6GALNAC5 inhibitor.

[0031] The present disclosure also provides a method of treating a subject having or at risk of converting mild cognitive impairment to dementia, the method comprising administering to the subject an ST6GALNAC5 inhibitor.

[0032] In some embodiments, the ST6GALNAC5 inhibitor 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 an ST6GALNAC5 nucleic acid molecule, such as an mRNA molecule. In some embodiments, the inhibitory nucleic acid molecule hybridizes with a sequence within an ST6GALNAC5 genomic nucleic acid molecule or an mRNA molecule and reduces the expression of the ST6GALNAC5 polypeptide in a cell of a subject. In some embodiments, the ST6GALNAC5 inhibitor comprises an antisense molecule that hybridizes with an ST6GALNAC5 genomic nucleic acid molecule or an mRNA molecule and reduces the expression of the ST6GALNAC5 polypeptide in a cell of a subject. In some embodiments, the ST6GALNAC5 inhibitor comprises an siRNA that hybridizes with an ST6GALNAC5 genomic nucleic acid molecule or an mRNA molecule and reduces the expression of the ST6GALNAC5 polypeptide in cells of a subject.In some embodiments, the ST6GALNAC5 inhibitor comprises an shRNA that hybridizes with an ST6GALNAC5 genomic nucleic acid molecule or an mRNA molecule and reduces the expression of the ST6GALNAC5 polypeptide in cells of a subject.

[0033] The inhibitory nucleic acid molecule can comprise RNA, DNA, or both RNA and DNA. The inhibitory nucleic acid molecule can also be linked or fused to a heterologous nucleic acid sequence, for example in a vector, or a heterologous label. For example, the inhibitory nucleic acid molecule can be present as an exogenous donor sequence in or containing a vector that contains the inhibitory nucleic acid molecule and the heterologous nucleic acid sequence. The inhibitory nucleic acid molecule can also be linked or fused to a heterologous label. The label can be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, an enzyme, or a fluorophore quencher). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, whereby an enzyme-dependent secondary generation of a signal occurs. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a binding molecule such that the binding molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and probed using a colorimetric (e.g., tetramethylbenzidine (TMB)) or fluorogenic substrate to detect the presence of HRP. 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, fluorescent and chemiluminescent substrates, as well as other labels.

[0034] Inhibitory 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.

[0035] The inhibitory nucleic acid molecule 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, sugar, 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 various purine or pyrimidine bases, such as, for example, pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-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 cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (cytosine), and thymine. Examples of uracils and cytosines include, but are not limited to, 4-isopropyl uracil, 4-isopropyl uracil, 8 ...

[0036] Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose, as well as synthetic modifications. 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-alkyl groups. 1~10 Alkyl or C 2~10 Alkenyl, and C 2~10 Exemplary 2' sugar modifications 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 1~10Examples of suitable substituents 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 may be made at other positions on the sugar, particularly the 3' position of the sugar in the 3' terminal nucleotide or 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.

[0037] 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. Such phosphate or modified phosphate linkages between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages 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 substitutes also include peptide nucleic acids (PNAs).

[0038] In some embodiments, the antisense nucleic acid molecule is a gapmer, whereby the first 1-7 nucleotides of the 5'-end and the 3'-end, respectively, have a 2'-methoxyethyl (2'-MOE) modification. In some embodiments, the first 5 nucleotides of the 5'-end and the 3'-end, respectively, have a 2'-MOE modification. In some embodiments, the first 1-7 nucleotides of the 5'-end and the 3'-end are RNA nucleotides. In some embodiments, the first 5 nucleotides of the 5'-end and the 3'-end are RNA nucleotides. In some embodiments, each of the internucleotide backbone linkages is a phosphorothioate linkage.

[0039] In some embodiments, the siRNA molecule has terminal modification.In some embodiments, the 5'-end of the antisense strand is phosphorylated.In some embodiments, a 5'-phosphate analog that cannot be hydrolyzed, such as 5'-(E)-vinyl-phosphonate, is used.

[0040] In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linking consecutive ribose nucleosides have been shown to increase the stability and bioavailability of siRNA in vivo. Non-ester groups (-OH, =O) of the phosphodiester bond can be replaced with sulfur, boron, or acetate to obtain phosphorothioate, boranophosphate, and phosphonoacetate linkages. In addition, the phosphodiester group can be replaced with a phosphotriester to facilitate cellular uptake of the siRNA and retention in serum components by removing its negative charge. In some embodiments, the siRNA molecule has a sugar modification. In some embodiments, the sugar is deprotonated (a reaction catalyzed by exonucleases and endonucleases), allowing the 2'-hydroxyl to act as a nucleophile and attack the adjacent phosphorus of the phosphodiester bond. Such alternatives include 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro modifications.

[0041] In some embodiments, the siRNA molecule has base modifications, in some embodiments, the bases may be replaced with modified bases such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0042] In some embodiments, siRNA molecules are bound to lipid.Lipid can be bound to 5'-end or 3'-end of siRNA, and can improve their bioavailability in vivo by associating with serum lipoprotein.Representative lipids include, but are not limited to, cholesterol and vitamin E, and fatty acids such as palmitic acid and tocopherol.

[0043] In some embodiments, an exemplary siRNA has the following formula: Sense: mN * mN * / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / * mN * / 32FN / Antisense: / 52FN / * / i2FN / * mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN * N * N where "N" is a base; "2F" is a 2'-F modification; "m" is a 2'-O-methyl modification; "I" is an internal base; * " are phosphorothioate backbone linkages.

[0044] The present disclosure also provides a vector comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the vector comprises any one or more of the inhibitory nucleic acid molecules 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.

[0045] The present disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules. 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-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution such as PBS, HBSS, and the like.

[0046] In some embodiments, the ST6GALNAC5 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks in the recognition sequence(s) in the ST6GALNAC5 genomic nucleic acid molecule or in a DNA binding protein that binds to the recognition sequence. The recognition sequence can be located in the coding region of the ST6GALNAC5 gene or in a regulatory 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 non-protein coding region. The recognition sequence can include or be adjacent to the start codon of the ST6GALNAC5 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 containing or adjacent to a start codon, and the other targeting a nuclease recognition sequence containing or adjacent to a stop codon, and cleavage by these 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-strand break at 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.

[0047] Suitable nuclease agents and DNA binding proteins 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 interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary, but includes, for example, recognition sequences that are about 30-36 bp for zinc finger proteins or ZFN pairs, about 15-18 bp for each ZFN, about 36 bp for TALE proteins or TALEN, and about 20 bp for CRISPR / Cas guide RNA.

[0048] In some embodiments, the CRISPR / Cas system can be used to modify the ST6GALNAC5 genomic nucleic acid molecule in a cell. The methods and compositions disclosed herein can employ the CRISPR-Cas system by utilizing a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of the ST6GALNAC5 nucleic acid molecule.

[0049] Cas proteins generally contain at least one RNA recognition domain or RNA binding domain that can interact with gRNA. Cas proteins can also contain nuclease domains (e.g., 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 (e.g., FnCpf1). Cas proteins can have full cleavage activity to create double-stranded breaks in ST6GALNAC5 genomic nucleic acid molecules, or can be nickases that create single-stranded breaks in ST6GALNAC5 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), Cas proteins include, but are not limited to, Cse4 (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 variants 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 transcriptional activation domain, or a transcriptional repressor domain. Cas proteins can be provided in any form. For example, the Cas protein can be provided in the form of a protein, e.g., a Cas protein complexed with a gRNA.Alternatively, the Cas protein can be provided in the form of a nucleic acid molecule, e.g., RNA or DNA, encoding the Cas protein.

[0050] In some embodiments, targeted genetic modification of ST6GALNAC5 genomic nucleic acid molecules can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize with one or more gRNA recognition sequences in a target genomic locus in the ST6GALNAC5 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 176,867 as set forth in 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 176,867 as set forth in SEQ ID NO:1. The gRNA recognition sequence can include or be adjacent to the start codon of the ST6GALNAC5 genomic nucleic acid molecule or the stop codon of the ST6GALNAC5 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 away from the start codon or the stop codon.

[0051] The gRNA recognition sequence in the target genomic locus in the ST6GALNAC5 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 transport Cas9 anywhere in the genome for gene editing, but cannot edit at sites 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 (e.g., when using Cas9 from S. pyogenes or a closely related Cas9), 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 is 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.

[0052] gRNA is an RNA molecule that binds to Cas protein and targets Cas protein to a specific position in ST6GALNAC5 genomic nucleic acid molecule. An exemplary gRNA is a gRNA that is effective for inducing Cas enzyme to bind to or cleave ST6GALNAC5 genomic nucleic acid molecule, wherein the gRNA comprises a DNA targeting segment that hybridizes with a gRNA recognition sequence in ST6GALNAC5 genomic nucleic acid molecule that includes or is adjacent to the position corresponding to position 176,867 in SEQ ID NO:1. For example, the gRNA can be selected to hybridize with a gRNA recognition sequence located 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 away from the position corresponding to position 176,867 in SEQ ID NO:1. Other exemplary gRNAs comprise a DNA targeting segment that hybridizes with the gRNA recognition sequence present in the ST6GALNAC5 genomic nucleic acid molecule that includes or is adjacent to the start codon or stop codon.For example, gRNAs can be selected to hybridize with the gRNA recognition sequence located 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 away 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 away from the stop codon. A suitable gRNA can comprise about 17 to about 25 nucleotides, about 17 to about 23 nucleotides, about 18 to about 22 nucleotides, or about 19 to about 21 nucleotides. In some embodiments, the gRNA can comprise 20 nucleotides.

[0053] Examples of suitable gRNA recognition sequences located within the ST6GALNAC5 reference gene are set forth in Table 1 as SEQ ID NOs: 43-62.

[0054] [Table 1]

[0055] The Cas protein and gRNA form a complex, and the Cas protein cuts the target ST6GALNAC5 genomic nucleic acid molecule. The Cas protein can cut the nucleic acid molecule at a site inside or outside the nucleic acid sequence present in the target ST6GALNAC5 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds. For example, the formation of a CRISPR complex (comprising a gRNA that hybridizes with a gRNA recognition sequence and complexes with a Cas protein) can result in the cleavage of one or both strands within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 50 or more base pairs) the nucleic acid sequence present in the ST6GALNAC5 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds.

[0056] Such a method can result in ST6GALNAC5 genomic nucleic acid molecule in which, for example, a region of SEQ ID NO:1 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 ST6GALNAC5 genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (e.g., a second gRNA that hybridizes with a second gRNA recognition sequence), the cleavage by Cas protein can create two or more double-strand breaks or two or more single-strand breaks.

[0057] In some embodiments, the ST6GALNAC5 inhibitor comprises a small molecule. In some embodiments, the ST6GALNAC5 inhibitor is myricetin. In some embodiments, the treatment method further comprises detecting the presence or absence of a ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 in a biological sample obtained from a subject. As used throughout this disclosure, an "ST6GALNAC5 missense variant nucleic acid molecule" is any ST6GALNAC5 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes an ST6GALNAC5 polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function.

[0058] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits cognitive impairment. In some embodiments, the subject has a cognitive impairment. In some embodiments, the method includes determining whether the subject has a ST6GALNAC5 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of ST6GALNAC5 by obtaining or obtaining a biological sample from the subject, and performing or performing a sequence analysis on the biological sample to determine whether the subject has a genotype that includes the ST6GALNAC5 missense variant nucleic acid molecule. If the subject is ST6GALNAC5 criterion, the therapeutic agent that treats or inhibits cognitive impairment is administered or continues to be administered to the subject at a standard dosage, and / or an ST6GALNAC5 inhibitor is administered to the subject. If the subject is heterozygous for the ST6GALNAC5 missense variant nucleic acid molecule, the therapeutic agent that treats or inhibits cognitive impairment is administered or continues to be administered to the subject at a standard dosage or less, and / or an ST6GALNAC5 inhibitor is administered to the subject. The presence of a genotype having a ST6GALNAC5 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of ST6GALNAC5 indicates that the subject has a low risk of developing cognitive impairment.In some embodiments, the subject is ST6GALNAC5 standard.In some embodiments, the subject is heterozygous for the ST6GALNAC5 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of ST6GALNAC5.

[0059] For subjects who have been genotyped or determined to be heterozygous for an ST6GALNAC5 missense variant nucleic acid molecule that is an ST6GALNAC5 standard or encodes a predicted loss-of-function polypeptide of ST6GALNAC5, such subjects can be treated with an ST6GALNAC5 inhibitor as described herein.

[0060] Detecting the presence or absence of an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 in a biological sample from a subject and / or determining whether a subject has an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 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 ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 can be present in a cell obtained from the subject.

[0061] In some embodiments, if the subject is ST6GALNAC5 criterion, the subject is also administered a standard dose of a therapeutic agent that treats or inhibits cognitive impairment. In some embodiments, if the subject is heterozygous for a ST6GALNAC5 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of ST6GALNAC5, the subject is also administered a standard dose of a therapeutic agent that treats or inhibits cognitive impairment.

[0062] In some embodiments, the method of treatment further comprises detecting the presence or absence of predicted loss-of-function polypeptide of ST6GALNAC5 in a biological sample from the subject.In some embodiments, if the subject does not have a predicted loss-of-function polypeptide of ST6GALNAC5, the subject is also administered a therapeutic agent for treating or inhibiting cognitive impairment at a standard dosage.In some embodiments, if the subject has a predicted loss-of-function polypeptide of ST6GALNAC5, the subject is also administered a therapeutic agent for treating or inhibiting cognitive impairment at a dosage equal to or less than the standard dosage.

[0063] The present disclosure also provides a method of treating a subject with a therapeutic agent for treating or preventing cognitive impairment. In some embodiments, the subject has a cognitive impairment. In some embodiments, the subject is at risk of developing a cognitive impairment. In some embodiments, the method includes determining whether the subject has a predicted loss-of-function polypeptide of ST6GALNAC5 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 predicted loss-of-function polypeptide of ST6GALNAC5. If the subject does not have a predicted loss-of-function polypeptide of ST6GALNAC5, the therapeutic agent for treating or inhibiting cognitive impairment is administered or continues to be administered to the subject at a standard dosage, and / or an ST6GALNAC5 inhibitor is administered to the subject. If the subject has a predicted loss-of-function polypeptide of ST6GALNAC5, the therapeutic agent for treating or inhibiting cognitive impairment is administered or continues to be administered to the subject at a standard dosage or less, and / or an ST6GALNAC5 inhibitor is administered to the subject. The presence of a predicted loss-of-function polypeptide of ST6GALNAC5 indicates that the subject has a low risk of developing cognitive impairment. In some embodiments, the subject has a predicted loss-of-function polypeptide of ST6GALNAC5. In some embodiments, the subject does not have a predicted loss-of-function polypeptide of ST6GALNAC5.

[0064] Detecting the presence or absence of a predicted loss-of-function ST6GALNAC5 polypeptide in a biological sample from a subject and / or determining whether the subject has a predicted loss-of-function ST6GALNAC5 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 predicted loss-of-function ST6GALNAC5 polypeptide can be present in a cell obtained from the subject.

[0065] Examples of therapeutic agents for treating or preventing cognitive impairment include, but are not limited to, cholinesterase inhibitors, vitamin E, and ginkgo. In some embodiments, the therapeutic agent for treating or inhibiting cognitive impairment is a cholinesterase inhibitor. In some embodiments, the therapeutic agent for treating or inhibiting cognitive impairment is vitamin E. In some embodiments, the therapeutic agent for treating or inhibiting cognitive impairment is ginkgo. In some embodiments, the cholinesterase inhibitor is ARICEPT® or ARICEPT® ODT (donepezil), COGNEX® (tacrine), EXELON® or EXELON® patch (rivastigmine), RAZADYNE® (galantamine), NAMZARIC® (memantine / donepezil), MYTELASE® (ambenonium), or BLOXIVERZ® (neostigmine). In some embodiments, the cholinesterase inhibitor is donepezil, tacrine, rivastigmine, galantamine, memantine / donepezil, ambenonium, or neostigmine. In some embodiments, the cholinesterase inhibitor is donepezil. In some embodiments, the cholinesterase inhibitor is tacrine. In some embodiments, the cholinesterase inhibitor is rivastigmine. In some embodiments, the cholinesterase inhibitor is galantamine. In some embodiments, the cholinesterase inhibitor is memantine / donepezil. In some embodiments, the cholinesterase inhibitor is ambenonium. In some embodiments, the cholinesterase inhibitor is neostigmine.

[0066] In some embodiments, the dose of a therapeutic agent for treating or preventing cognitive impairment can be reduced (i.e., less than the standard dose) for a subject who is heterozygous for a ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, compared to a subject who is ST6GALNAC5 norm (which may receive a standard dose). In some embodiments, the dose of a therapeutic agent for treating or preventing cognitive impairment can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, the dose of a therapeutic agent for treating or preventing cognitive impairment in a subject who is heterozygous for a ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 can be administered less frequently than a subject who is ST6GALNAC5 norm.

[0067] The administration of the therapeutic agent and / or ST6GALNAC5 inhibitor for treating or preventing cognitive impairment 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. The repeated administration can be the same dose or different doses. The administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. For example, according to a particular dosing regimen, a subject can be treated for an extended period of time, such as, for example, 6 months, 1 year, or more. Furthermore, the therapeutic agent and / or ST6GALNAC5 inhibitor for treating or preventing cognitive impairment can be administered sequentially or simultaneously. Furthermore, the therapeutic agent and / or ST6GALNAC5 inhibitor for treating or preventing cognitive impairment can be administered in separate compositions or together in the same composition.

[0068] Administration of the therapeutic agent and / or ST6GALNAC5 inhibitor for treating or preventing cognitive impairment can occur 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., a single dose for administration). Pharmaceutical compositions can be formulated using one or more physiologically and pharmacologic acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. 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.

[0069] As used herein, the terms "treat", "treating", and "treatment" and "prevent", "prevention", and "prevention" refer to eliciting a desired biological response, such as a therapeutic effect and a prophylactic effect, respectively. In some embodiments, the therapeutic effect includes one or more of the following after administration of the agent or a composition comprising the agent: reduction / alleviation of cognitive impairment, reduction / alleviation of the severity of cognitive impairment (e.g., reduction or suppression of the onset of cognitive impairment), reduction / alleviation of symptoms and cognitive impairment-related effects, delay in the onset of symptoms and cognitive impairment-related effects, reduction in the severity of symptoms of cognitive impairment-related effects, reduction in the severity of acute episodes, reduction in the number of symptoms and cognitive impairment-related effects, reduction in the latency period of symptoms and cognitive impairment-related effects, improvement in symptoms and cognitive impairment-related effects, reduction in secondary symptoms, reduction in secondary infections, prevention of recurrence of cognitive impairment, 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, enhancement of remission, acceleration of recovery, or increase in the effectiveness or reduction in resistance to alternative therapeutic agents, 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) of the onset / progression of cognitive impairment following administration of a treatment protocol, and increased survival time of an affected host animal. Treatment of cognitive impairment includes treatment of a subject already diagnosed with some form of cognitive impairment, either at a clinical stage or clinical symptoms, delaying the onset or progression or aggravation or worsening of symptoms or signs of cognitive impairment, and / or preventing and / or reducing the severity of cognitive impairment.

[0070] The present disclosure also provides a method for identifying a subject with high risk of developing cognitive impairment. In some embodiments, the method comprises determining or determining the presence or absence of ST6GALNAC5 missense variant nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule and / or cDNA molecule) encoding a predicted loss-of-function polypeptide of ST6GALNAC5 in a biological sample obtained from a subject. If a subject lacks ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 (i.e., the subject is classified as ST6GALNAC5 standard by genotyping), the subject has high risk of developing cognitive impairment. If a subject has ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 (i.e., the subject is heterozygous or homozygous for ST6GALNAC5 missense variant nucleic acid molecule), the subject has low risk of developing cognitive impairment compared to a subject who is ST6GALNAC5 standard.

[0071] Having a single copy of an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 further protects a subject from developing a cognitive disorder than not having a copy of an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5. Without intending to be limited to a particular theory or mechanism of action, it is believed that a single copy of an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 (i.e., heterozygous for the ST6GALNAC5 missense variant nucleic acid molecule) is believed to protect a subject from developing a cognitive disorder, and it is also believed that having two copies of an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 (i.e., homozygous for the ST6GALNAC5 missense variant nucleic acid molecule) may further protect a subject from developing a cognitive disorder compared to a subject having a single copy. Thus, in some embodiments, a single copy of a ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 may not completely protect a subject from developing cognitive impairment, but may instead provide partial or incomplete protection. Without wishing to be bound by any particular theory, there may be additional factors or molecules involved in the development of cognitive impairment that are still present in a subject having a single copy of a ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, which may result in less than complete protection from developing cognitive impairment.

[0072] Detecting the presence or absence of an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 in a biological sample from a subject and / or determining whether a subject has an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 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 ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 can be present in a cell obtained from the subject.

[0073] In some embodiments, once a subject is identified as having a high risk of developing a cognitive disorder, the subject is further treated with a therapeutic agent that treats or inhibits the cognitive disorder and / or an ST6GALNAC5 inhibitor, as described herein. For example, if a subject is ST6GALNAC5-based and therefore has a high risk of developing a cognitive disorder, the subject is administered an ST6GALNAC5 inhibitor. In some embodiments, such a subject is administered a therapeutic agent that treats or inhibits the cognitive disorder. In some embodiments, if the subject is heterozygous for a ST6GALNAC5 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of ST6GALNAC5, the subject is administered a therapeutic agent that treats or inhibits the cognitive disorder and / or an ST6GALNAC5 inhibitor at a dose equal to or less than the standard dose. In some embodiments, the subject is ST6GALNAC5-based. In some embodiments, the subject is heterozygous for a ST6GALNAC5 missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of ST6GALNAC5.

[0074] The present disclosure also provides a method for detecting the presence or absence of ST6GALNAC5 missense variant genomic nucleic acid molecules encoding predicted loss-of-function polypeptides of ST6GALNAC5 in a biological sample obtained from a subject, and / or ST6GALNAC5 missense variant mRNA molecules encoding predicted loss-of-function polypeptides of ST6GALNAC5 in a biological sample obtained from a subject, and / or ST6GALNAC5 missense variant cDNA molecules encoding predicted loss-of-function polypeptides of ST6GALNAC5 generated from mRNA molecules in a biological sample obtained from a subject. It is understood that gene sequences within a population, and the mRNA molecules encoded by such genes, may differ due to polymorphisms, such as single nucleotide polymorphisms. The sequences provided herein for ST6GALNAC5 variant genomic nucleic acid molecules, ST6GALNAC5 variant mRNA molecules, and ST6GALNAC5 variant cDNA molecules are merely exemplary sequences. Other sequences for ST6GALNAC5 variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules are also possible.

[0075] The biological sample can be derived from any cell, tissue, or biological fluid of a subject. The biological sample may include any clinically relevant tissue, such as, for example, a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of a bodily fluid, such as, for example, blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine. In some embodiments, the biological sample includes a buccal swab. The biological sample used in the methods disclosed herein may vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. The biological sample may be subjected to different treatments depending on the assay employed. For example, when detecting ST6GALNAC5 mutant nucleic acid molecules, a pretreatment designed to isolate or enrich the biological sample for ST6GALNAC5 mutant nucleic acid molecules may be employed. For this purpose, various techniques may be used. When detecting the level of ST6GALNAC5 mutant mRNA molecules, various techniques may be used to enrich the biological sample containing mRNA molecules. Various methods may be used to detect the presence or level of mRNA molecules, or the presence of a particular mutant genomic DNA locus.

[0076] The present disclosure also provides a method for detecting an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 or its complement in a subject, the method comprising assaying a biological sample obtained from the subject to determine whether a nucleic acid molecule in the biological sample is an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5.

[0077] In some embodiments, an ST6GALNAC5 missense mutant nucleic acid molecule or its complement encoding a predicted loss-of-function polypeptide of ST6GALNAC5 is a genomic nucleic acid molecule having a nucleotide sequence including a cytosine or its complement at a position corresponding to position 176,867 set forth in SEQ ID NO:2.

[0078] In some embodiments, an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, or its complement, is an mRNA molecule having a nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:17; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:18.

[0079] In some embodiments, an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, or its complement, is a cDNA molecule generated from an mRNA molecule in a biological sample having a nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:27; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:28; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:29; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:30; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:31; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:32; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:33; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:34.

[0080] In some embodiments, the ST6GALNAC5 missense variant nucleic acid molecule is selected from the group consisting of i) a cytosine at a position corresponding to position 176,867 set forth in SEQ ID NO:2 (for a genomic nucleic acid molecule); ii) a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:11, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:12, a cytosine at a position corresponding to position 562 set forth in SEQ ID NO:13, a cytosine at a position corresponding to position 515 set forth in SEQ ID NO:14, a cytosine at a position corresponding to position 579 set forth in SEQ ID NO:15, a cytosine at a position corresponding to position 416 set forth in SEQ ID NO:16, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:17, or a cytosine at position 600 set forth in SEQ ID NO:18. or iii) a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:27, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:28, a cytosine at a position corresponding to position 562 set forth in SEQ ID NO:29, a cytosine at a position corresponding to position 515 set forth in SEQ ID NO:30, a cytosine at a position corresponding to position 579 set forth in SEQ ID NO:31, a cytosine at a position corresponding to position 416 set forth in SEQ ID NO:32, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:33, or a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:34 (in the case of a cDNA molecule obtained from an mRNA molecule).

[0081] 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 ST6GALNAC5 genomic nucleic acid molecules or mRNA molecules, and, if mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays can comprise, for example, determining the identity of these positions of a particular ST6GALNAC5 nucleic acid molecule. In some embodiments, the methods are in vitro methods.

[0082] In some embodiments, the determining, detecting, or sequence analysis step comprises sequencing at least a portion of the nucleotide sequence of an ST6GALNAC5 genomic nucleic acid molecule, an ST6GALNAC5 mRNA molecule, or an ST6GALNAC5 cDNA molecule generated from the mRNA molecule in a biological sample, wherein the sequenced portion contains one or more mutations that cause or are predicted to cause loss of function (partial or complete).

[0083] In some embodiments, the determining, detecting or sequence analysis step comprises: i) sequencing at least a portion of the nucleotide sequence of an ST6GALNAC5 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 176,867 set forth in SEQ ID NO:2 or its complement; ii) sequencing at least a portion of the nucleotide sequence of an ST6GALNAC5 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 600 set forth in SEQ ID NO:11 or its complement; position 639 set forth in SEQ ID NO:12 or its complement; position 562 set forth in SEQ ID NO:13 or its complement; position 515 set forth in SEQ ID NO:14 or its complement; position 579 set forth in SEQ ID NO:15 or its complement; position 416 set forth in SEQ ID NO:16 or its complement; position 639 set forth in SEQ ID NO:17 or its complement; or position 600 set forth in SEQ ID NO:18 or its complement; and / or iii) sequencing at least a portion of the nucleotide sequence of an ST6GALNAC5 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 600 set forth in SEQ ID NO:11 or its complement; position 639 set forth in SEQ ID NO:12 or its complement; position 562 set forth in SEQ ID NO:13 or its complement; position 515 set forth in SEQ ID NO:14 or its complement; position 579 set forth in SEQ ID NO:15 or its complement; position 416 set forth in SEQ ID NO:16 or its complement; position 639 set forth in SEQ ID NO:17 or its complement; or position 600 set forth in SEQ ID NO:18 or its complement; It comprises sequencing at least a portion of the nucleotide sequence of the cDNA molecule, wherein the sequenced portion comprises a position corresponding to position 600 set forth in SEQ ID NO:27 or its complement; position 639 set forth in SEQ ID NO:28 or its complement; position 562 set forth in SEQ ID NO:29 or its complement; position 515 set forth in SEQ ID NO:30 or its complement; position 579 set forth in SEQ ID NO:31 or its complement; position 416 set forth in SEQ ID NO:32 or its complement; position 639 set forth in SEQ ID NO:33 or its complement; or position 600 set forth in SEQ ID NO:34 or its complement).The sequenced portion of the ST6GALNAC5 nucleic acid molecule in the biological sample was found to contain a cytosine at a position corresponding to position 176,867 set forth in SEQ ID NO:2, a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:11, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:12, a cytosine at a position corresponding to position 562 set forth in SEQ ID NO:13, a cytosine at a position corresponding to position 515 set forth in SEQ ID NO:14, a cytosine at a position corresponding to position 579 set forth in SEQ ID NO:15, a cytosine at a position corresponding to position 416 set forth in SEQ ID NO:16, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:17, a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:18, a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:27, a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:29, a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:30, a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:31, a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:32, a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:33, a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:34, a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:35, a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:3 If the ST6GALNAC5 nucleic acid molecule in the biological sample contains a cytosine at a position corresponding to position 0, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:28, a cytosine at a position corresponding to position 562 set forth in SEQ ID NO:29, a cytosine at a position corresponding to position 515 set forth in SEQ ID NO:30, a cytosine at a position corresponding to position 579 set forth in SEQ ID NO:31, a cytosine at a position corresponding to position 416 set forth in SEQ ID NO:32, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:33, or a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:34, the ST6GALNAC5 nucleic acid molecule in the biological sample is an ST6GALNAC5 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5.

[0084] In some embodiments, the determining, detecting, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the ST6GALNAC5 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 176,867 set forth in SEQ ID NO: 2, or a complement thereof. If the sequenced portion of the ST6GALNAC5 nucleic acid molecule in the biological sample comprises a cytosine at a position corresponding to position 176,867 set forth in SEQ ID NO: 2, then the ST6GALNAC5 nucleic acid molecule in the biological sample is an ST6GALNAC5 missense variant genomic nucleic acid molecule that encodes a predicted loss-of-function polypeptide of ST6GALNAC5.

[0085] In some embodiments, the determining, detecting or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the ST6GALNAC5 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 600 or its complement set forth in SEQ ID NO:11; position 639 or its complement set forth in SEQ ID NO:12; position 562 or its complement set forth in SEQ ID NO:13; position 515 or its complement set forth in SEQ ID NO:14; position 579 or its complement set forth in SEQ ID NO:15; position 416 or its complement set forth in SEQ ID NO:16; position 639 or its complement set forth in SEQ ID NO:17; or position 600 or its complement set forth in SEQ ID NO:18. If the sequenced portion of the ST6GALNAC5 mRNA molecule in the biological sample comprises a cytosine at a position corresponding to 600 set forth in SEQ ID NO:11, a cytosine at a position corresponding to 639 set forth in SEQ ID NO:12, a cytosine at a position corresponding to 562 set forth in SEQ ID NO:13, a cytosine at a position corresponding to 515 set forth in SEQ ID NO:14, a cytosine at a position corresponding to 579 set forth in SEQ ID NO:15, a cytosine at a position corresponding to 416 set forth in SEQ ID NO:16, a cytosine at a position corresponding to 639 set forth in SEQ ID NO:17, or a cytosine at a position corresponding to 600 set forth in SEQ ID NO:18, then the ST6GALNAC5 nucleic acid molecule in the biological sample is an ST6GALNAC5 missense mutant mRNA molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5.

[0086] In some embodiments, the determining, detecting, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of an ST6GALNAC5 cDNA molecule generated from an mRNA molecule in a biological sample, wherein the sequenced portion comprises a position corresponding to position 600 or its complement set forth in SEQ ID NO:27; position 639 or its complement set forth in SEQ ID NO:28; position 562 or its complement set forth in SEQ ID NO:29; position 515 or its complement set forth in SEQ ID NO:30; position 579 or its complement set forth in SEQ ID NO:31; position 416 or its complement set forth in SEQ ID NO:32; position 639 or its complement set forth in SEQ ID NO:33; or position 600 or its complement set forth in SEQ ID NO:34. If the sequenced portion of the ST6GALNAC5 cDNA molecule in the biological sample comprises a cytosine at a position corresponding to 600 set forth in SEQ ID NO:27, a cytosine at a position corresponding to 639 set forth in SEQ ID NO:28, a cytosine at a position corresponding to 562 set forth in SEQ ID NO:29, a cytosine at a position corresponding to 515 set forth in SEQ ID NO:30, a cytosine at a position corresponding to 579 set forth in SEQ ID NO:31, a cytosine at a position corresponding to 416 set forth in SEQ ID NO:32, a cytosine at a position corresponding to 639 set forth in SEQ ID NO:33, or a cytosine at a position corresponding to 600 set forth in SEQ ID NO:34, then the ST6GALNAC5 nucleic acid molecule in the biological sample is an ST6GALNAC5 missense variant cDNA molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5.

[0087] In some embodiments, the determining, detecting or sequence analyzing step comprises: a) detecting a genomic nucleic acid molecule adjacent to a position corresponding to: i) position 176,867 set forth in SEQ ID NO:2 or its complement; ii) position 600 set forth in SEQ ID NO:11 or its complement; position 639 set forth in SEQ ID NO:12 or its complement; position 562 set forth in SEQ ID NO:13 or its complement; position 515 set forth in SEQ ID NO:14 or its complement; position 579 set forth in SEQ ID NO:15 or its complement; position 416 set forth in SEQ ID NO:16 or its complement. position 639 as set forth in SEQ ID NO: 17 or its complement; or an mRNA molecule or its complement adjacent to a position corresponding to position 600 as set forth in SEQ ID NO: 18 or its complement; and / or iii) position 600 as set forth in SEQ ID NO: 27 or its complement; position 639 as set forth in SEQ ID NO: 28 or its complement; position 562 as set forth in SEQ ID NO: 29 or its complement; position 515 as set forth in SEQ ID NO: 30 or its complement; position 579 as set forth in SEQ ID NO: 31 or its complement; position 416 as set forth in SEQ ID NO: 32 or its complement; position 639 as set forth in SEQ ID NO: 33 or its complement; or a primer that hybridizes to a portion of the nucleotide sequence of the cDNA molecule or its complement adjacent to a position corresponding to position 600 set forth in SEQ ID NO:34 or its complement; and b) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the cDNA molecule or its complement adjacent to a position corresponding to position 600 set forth in SEQ ID NO:34 or its complement; or a primer that hybridizes to a portion of the nucleotide sequence of the cDNA molecule or its complement adjacent to a position corresponding to position 600 set forth in SEQ ID NO:34 or its complement; an mRNA molecule or its complement corresponding to position 515 as set forth in SEQ ID NO: 14 or its complement; position 579 as set forth in SEQ ID NO: 15 or its complement; position 416 as set forth in SEQ ID NO: 16 or its complement; position 639 as set forth in SEQ ID NO: 17 or its complement; or position 600 as set forth in SEQ ID NO: 18 or its complement; and / or iii) position 600 as set forth in SEQ ID NO: 27 or its complement; position 639 as set forth in SEQ ID NO: 28 or its complement; position 562 as set forth in SEQ ID NO: 29 or its complement; position 515 as set forth in SEQ ID NO: 30 or its complement;or position 600 or its complement as set forth in SEQ ID NO:34; and c) extending the primer at least through a position in the nucleotide sequence of the cDNA molecule or its complement corresponding to position 579 or its complement as set forth in SEQ ID NO:31; position 416 or its complement as set forth in SEQ ID NO:32; position 639 or its complement as set forth in SEQ ID NO:33; or position 600 or its complement as set forth in SEQ ID NO:34; and or a complement thereof at a position corresponding to position 639 as set forth in SEQ ID NO: 17; a cytosine or its complement at a position corresponding to position 600 as set forth in SEQ ID NO: 18; a cytosine or its complement at a position corresponding to position 600 as set forth in SEQ ID NO: 27; a cytosine or its complement at a position corresponding to position 639 as set forth in SEQ ID NO: 28; a cytosine or its complement at a position corresponding to position 562 as set forth in SEQ ID NO: 29; a cytosine or its complement at a position corresponding to position 515 as set forth in SEQ ID NO: 30; a cytosine or its complement at a position corresponding to position 579 as set forth in SEQ ID NO: 31; a cytosine or its complement at a position corresponding to position 416 as set forth in SEQ ID NO: 32; a cytosine or its complement at a position corresponding to position 639 as set forth in SEQ ID NO: 33; or a cytosine or its complement at a position corresponding to position 600 as set forth in SEQ ID NO: 34.

[0088] In some embodiments, the determining, detecting, or sequence analyzing step includes: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an ST6GALNAC5 genomic nucleic acid molecule or its complement adjacent to a position corresponding to position 176,867 or its complement set forth in SEQ ID NO:2; b) extending the primer at least through the position of the nucleotide sequence of an ST6GALNAC5 genomic nucleic acid molecule or its complement corresponding to position 176,867 or its complement set forth in SEQ ID NO:2; and c) determining whether the extension product of the primer includes a cytosine or its complement at the position corresponding to position 176,867 set forth in SEQ ID NO:2.

[0089] In some embodiments, the determining, detecting or sequence analyzing step is performed by determining: a) a region of ST6GALNAC5 adjacent to a position corresponding to: position 600 set forth in SEQ ID NO:11 or its complement; position 639 set forth in SEQ ID NO:12 or its complement; position 562 set forth in SEQ ID NO:13 or its complement; position 515 set forth in SEQ ID NO:14 or its complement; position 579 set forth in SEQ ID NO:15 or its complement; position 416 set forth in SEQ ID NO:16 or its complement; position 639 set forth in SEQ ID NO:17 or its complement; or position 600 set forth in SEQ ID NO:18 or its complement. contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the mRNA molecule or its complement; and b) detecting a primer corresponding to position 600 set forth in SEQ ID NO: 11 or its complement; position 639 set forth in SEQ ID NO: 12 or its complement; position 562 set forth in SEQ ID NO: 13 or its complement; position 515 set forth in SEQ ID NO: 14 or its complement; position 579 set forth in SEQ ID NO: 15 or its complement; position 416 set forth in SEQ ID NO: 16 or its complement; position 639 set forth in SEQ ID NO: 17 or its complement; or position 600 set forth in SEQ ID NO: 18 or its complement. extending the primer at least through a position in the nucleotide sequence of the mRNA molecule; and c) determining whether the extension product of the primer comprises a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:16; a 639 or its complement set forth in SEQ ID NO:17; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:18.

[0090] In some embodiments, the determining, detecting or sequence analyzing step is performed by determining: a) a sequence of ST6GALNAC5 adjacent to a position corresponding to: position 600 set forth in SEQ ID NO:27 or its complement; position 639 set forth in SEQ ID NO:28 or its complement; position 562 set forth in SEQ ID NO:29 or its complement; position 515 set forth in SEQ ID NO:30 or its complement; position 579 set forth in SEQ ID NO:31 or its complement; position 416 set forth in SEQ ID NO:32 or its complement; position 639 set forth in SEQ ID NO:33 or its complement; or position 600 set forth in SEQ ID NO:34 or its complement. contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the cDNA molecule or its complement; and b) detecting a primer corresponding to position 600 set forth in SEQ ID NO:27 or its complement; position 639 set forth in SEQ ID NO:28 or its complement; position 562 set forth in SEQ ID NO:29 or its complement; position 515 set forth in SEQ ID NO:30 or its complement; position 579 set forth in SEQ ID NO:31 or its complement; position 416 set forth in SEQ ID NO:32 or its complement; position 639 set forth in SEQ ID NO:33 or its complement; or position 600 set forth in SEQ ID NO:34 or its complement. extending the primer at least through a position in the nucleotide sequence of the cDNA molecule; and c) determining whether the extension product of the primer comprises a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:27; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:28; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:29; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:30; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:31; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:32; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:33; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:34.

[0091] In some embodiments, the entire nucleic acid molecule is sequenced. In some embodiments, only the ST6GALNAC5 genomic nucleic acid molecule is analyzed. In some embodiments, only the ST6GALNAC5 mRNA is analyzed. In some embodiments, only the ST6GALNAC5 cDNA obtained from the ST6GALNAC5 mRNA is analyzed.

[0092] In some embodiments, the determining, detecting, or sequence analyzing step comprises: a) amplifying at least a portion of the ST6GALNAC5 nucleic acid molecule or its complement in the biological sample, wherein the amplified portion is selected from the group consisting of: a cytosine or its complement at a position corresponding to position 176,867 in SEQ ID NO:2; a cytosine or its complement at a position corresponding to position 600 in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 in SEQ ID NO:17; a cytosine or its complement at a position corresponding to position 600 in SEQ ID NO:18; a cytosine or its complement at a position corresponding to position 600 in SEQ ID NO:27. a cytosine or its complement at a position corresponding to position 639 in SEQ ID NO:28; a cytosine or its complement at a position corresponding to position 562 in SEQ ID NO:29; a cytosine or its complement at a position corresponding to position 515 in SEQ ID NO:30; a cytosine or its complement at a position corresponding to position 579 in SEQ ID NO:31; a cytosine or its complement at a position corresponding to position 416 in SEQ ID NO:32; a cytosine or its complement at a position corresponding to position 639 in SEQ ID NO:33; or a cytosine or its complement at a position corresponding to position 639 in SEQ ID NO:34; a) amplifying a nucleic acid molecule comprising a cytosine or its complement at a position corresponding to position 600 of SEQ ID NO:4; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe detecting a cytosine or its complement at a position corresponding to position 176,867 of SEQ ID NO:2; a cytosine or its complement at a position corresponding to position 600 of SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 of SEQ ID NO:12;A cytosine or its complement at a position corresponding to position 562 in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 in SEQ ID NO:17; a cytosine or its complement at a position corresponding to position 600 in SEQ ID NO:18; a cytosine or its complement at a position corresponding to position 600 in SEQ ID NO:27; a cytosine or its complement at a position corresponding to position 639 in SEQ ID NO:28; a sequence number 29, or its complement; SEQ ID NO: 30, or its complement; SEQ ID NO: 31, or its complement; SEQ ID NO: 32, or its complement; SEQ ID NO: 33, or its complement; SEQ ID NO: 34, or its complement;

[0093] In some embodiments, the determining, detecting, or sequence analyzing step comprises: a) amplifying at least a portion of an ST6GALNAC5 genomic nucleic acid molecule or its complement in a biological sample, the portion comprising a cytosine or its complement at a position corresponding to position 176,867 of 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 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 cytosine or its complement at a position corresponding to position 176,867 of SEQ ID NO:2; and d) detecting the detectable label.

[0094] In some embodiments, the determining, detecting, or sequence analyzing step comprises: a) amplifying at least a portion of ST6GALNAC5 mRNA molecules or their complements in the biological sample, the portion comprising a cytosine or its complement at a position corresponding to 600 as set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to 639 as set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to 562 as set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to 515 as set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to 579 as set forth in SEQ ID NO:15; a cytosine or its complement at a position corresponding to 416 as set forth in SEQ ID NO:16; a cytosine or its complement at a position corresponding to 639 as set forth in SEQ ID NO:17; or a cytosine or its complement at a position corresponding to 600 as set forth in SEQ ID NO:18; b) labeling the amplified nucleic acid molecules with a detectable label; and c) coupling the labeled nucleic acid molecules to a support comprising a mutation-specific probe. a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:17; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:18; and d) detecting the detectable label.

[0095] In some embodiments, the determining, detecting, or sequence analyzing step comprises: a) amplifying at least a portion of an ST6GALNAC5 cDNA molecule or its complement in the biological sample, the portion comprising: a cytosine or its complement at a position corresponding to 600 as set forth in SEQ ID NO:27; a cytosine or its complement at a position corresponding to 639 as set forth in SEQ ID NO:28; a cytosine or its complement at a position corresponding to 562 as set forth in SEQ ID NO:29; a cytosine or its complement at a position corresponding to 515 as set forth in SEQ ID NO:30; a cytosine or its complement at a position corresponding to 579 as set forth in SEQ ID NO:31; a cytosine or its complement at a position corresponding to 416 as set forth in SEQ ID NO:32; a cytosine or its complement at a position corresponding to 639 as set forth in SEQ ID NO:33; or a cytosine or its complement at a position corresponding to 600 as set forth in SEQ ID NO:34; b) labeling the amplified nucleic acid molecule with a detectable label; and c) coupling the labeled nucleic acid molecule to a support comprising a mutation-specific probe. a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:27; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:28; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:29; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:30; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:31; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:32; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:33; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:34; and d) detecting the detectable label.

[0096] 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. In some embodiments, the determining, detecting, or sequence analyzing step comprises: contacting an ST6GALNAC5 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe detects a cytosine or its complement at a position corresponding to position 176,867 in SEQ ID NO:2; a cytosine or its complement at a position corresponding to position 600 in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 in SEQ ID NO:17; a cytosine or its complement at a position corresponding to position 600 in SEQ ID NO:18; a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:27; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:28; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:29; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:30; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:31; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:32; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:33; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:34; and detecting the detectable label.

[0097] In some embodiments, the determining step, detecting step, or sequence analysis comprises: contacting an ST6GALNAC5 genomic nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an ST6GALNAC5 genomic nucleic acid molecule or its complement that comprises a cytosine at a position corresponding to position 176,867 set forth in SEQ ID NO:2, or its complement; and detecting the detectable label.

[0098] In some embodiments, the determining, detecting, or sequence analyzing step comprises: contacting ST6GALNAC5 mRNA molecules or their complements in the biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a cytosine or its complement at a position corresponding to position 600 in SEQ ID NO: 11; a cytosine or its complement at a position corresponding to position 639 in SEQ ID NO: 12; a cytosine or its complement at a position corresponding to position 562 in SEQ ID NO: 13; a cytosine or its complement at a position corresponding to position 515 in SEQ ID NO: 14; a cytosine or its complement at a position corresponding to position 579 in SEQ ID NO: 15; a cytosine or its complement at a position corresponding to position 416 in SEQ ID NO: 16; a cytosine or its complement at a position corresponding to position 639 in SEQ ID NO: 17; or a cytosine or its complement at a position corresponding to position 600 in SEQ ID NO: 18. contacting, comprising a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the mRNA molecule or its complement; and detecting the detectable label.

[0099] In some embodiments, the determining, detecting, or sequence analyzing step comprises: contacting an ST6GALNAC5 cDNA molecule, or its complement, generated from an mRNA molecule in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:27; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:28; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:29; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:30; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:31; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:32; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:33; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:34. contacting, comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the cDNA molecule or its complement; and detecting the detectable label.

[0100] In some embodiments, the ST6GALNAC5 nucleic acid molecule is present in a cell obtained from the subject. Mutation-specific polymerase chain reaction techniques can be used to detect mutations such as SNPs in nucleic acid sequences. Mutation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0101] In some embodiments, the determining, detecting, or sequence analysis step comprises contacting the biological sample with a primer or probe, such as a mutation-specific primer or a mutation-specific probe, that specifically hybridizes to the ST6GALNAC5 variant genomic sequence, variant mRNA sequence, or variant cDNA sequence under stringent conditions and does not hybridize to the corresponding ST6GALNAC5 reference sequence, and determining whether hybridization occurs.

[0102] In some embodiments, the assay involves RNA sequencing (RNA-Seq). In some embodiments, the assay also involves reverse transcribing mRNA into cDNA, for example, by reverse transcription polymerase chain reaction (RT-PCR).

[0103] In some embodiments, the method utilizes probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and / or identify polynucleotides comprising ST6GALNAC5 mutant genomic nucleic acid molecules, mutant mRNA molecules, or mutant cDNA molecules. Hybridization or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length that is sufficient for use in an optimal detection method, including any assay described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under highly stringent hybridization conditions. The probes and primers can have a perfect nucleotide sequence identity of consecutive nucleotides in the target nucleotide sequence, although probes that are different 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.

[0104] In some embodiments, the ST6GALNAC5 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in the biological sample contains a cytosine at a position corresponding to position 176,867 set forth in SEQ ID NO:2, a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:11, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:12, a cytosine at a position corresponding to position 562 set forth in SEQ ID NO:13, a cytosine at a position corresponding to position 515 set forth in SEQ ID NO:14, a cytosine at a position corresponding to position 579 set forth in SEQ ID NO:15, a cytosine at a position corresponding to position 416 set forth in SEQ ID NO:16, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:17, To determine whether the nucleotide sequence contains a cytosine at a position corresponding to 600 set forth in SEQ ID NO:18, a cytosine at a position corresponding to 600 set forth in SEQ ID NO:27, a cytosine at a position corresponding to 639 set forth in SEQ ID NO:28, a cytosine at a position corresponding to 562 set forth in SEQ ID NO:29, a cytosine at a position corresponding to 515 set forth in SEQ ID NO:30, a cytosine at a position corresponding to 579 set forth in SEQ ID NO:31, a cytosine at a position corresponding to 416 set forth in SEQ ID NO:32, a cytosine at a position corresponding to 639 set forth in SEQ ID NO:33, or a cytosine at a position corresponding to 600 set forth in SEQ ID NO:34,A cytosine at a position corresponding to position 867, a cytosine at a position corresponding to position 600 in SEQ ID NO: 11, a cytosine at a position corresponding to position 639 in SEQ ID NO: 12, a cytosine at a position corresponding to position 562 in SEQ ID NO: 13, a cytosine at a position corresponding to position 515 in SEQ ID NO: 14, a cytosine at a position corresponding to position 579 in SEQ ID NO: 15, a cytosine at a position corresponding to position 416 in SEQ ID NO: 16, a cytosine at a position corresponding to position 639 in SEQ ID NO: 17, a cytosine at a position corresponding to position 600 in SEQ ID NO: 18, a cytosine at a position corresponding to position 200 in SEQ ID NO: 27, a first primer derived from a 5' flanking sequence adjacent to a cytosine at a position corresponding to position 600, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:28, a cytosine at a position corresponding to position 562 set forth in SEQ ID NO:29, a cytosine at a position corresponding to position 515 set forth in SEQ ID NO:30, a cytosine at a position corresponding to position 579 set forth in SEQ ID NO:31, a cytosine at a position corresponding to position 416 set forth in SEQ ID NO:32, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:33, or a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:34, and a second primer derived from a 5' flanking sequence adjacent to a cytosine at a position corresponding to position 176,cytosine at a position corresponding to position 867, cytosine at a position corresponding to position 600 in SEQ ID NO:11, cytosine at a position corresponding to position 639 in SEQ ID NO:12, cytosine at a position corresponding to position 562 in SEQ ID NO:13, cytosine at a position corresponding to position 515 in SEQ ID NO:14, cytosine at a position corresponding to position 579 in SEQ ID NO:15, cytosine at a position corresponding to position 416 in SEQ ID NO:16, cytosine at a position corresponding to position 639 in SEQ ID NO:17, cytosine at a position corresponding to position 600 in SEQ ID NO:18, cytosine at a position corresponding to position 600 in SEQ ID NO:27 a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:28, a cytosine at a position corresponding to position 562 set forth in SEQ ID NO:29, a cytosine at a position corresponding to position 515 set forth in SEQ ID NO:30, a cytosine at a position corresponding to position 579 set forth in SEQ ID NO:31, a cytosine at a position corresponding to position 416 set forth in SEQ ID NO:32, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:33, or a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:34,A cytosine at a position corresponding to position 867, a cytosine at a position corresponding to position 600 in SEQ ID NO: 11, a cytosine at a position corresponding to position 639 in SEQ ID NO: 12, a cytosine at a position corresponding to position 562 in SEQ ID NO: 13, a cytosine at a position corresponding to position 515 in SEQ ID NO: 14, a cytosine at a position corresponding to position 579 in SEQ ID NO: 15, a cytosine at a position corresponding to position 416 in SEQ ID NO: 16, a cytosine at a position corresponding to position 639 in SEQ ID NO: 17, a cytosine at a position corresponding to position 600 in SEQ ID NO: 18, a cytosine at a position corresponding to position 200 in SEQ ID NO: 27, amplicons can be generated that indicate the presence of a SNP at a position that encodes a cytosine at position corresponding to 600 of SEQ ID NO: 28, a cytosine at position corresponding to 639 of SEQ ID NO: 29, a cytosine at position corresponding to 515 of SEQ ID NO: 30, a cytosine at position corresponding to 579 of SEQ ID NO: 31, a cytosine at position corresponding to 416 of SEQ ID NO: 32, a cytosine at position corresponding to 639 of SEQ ID NO: 33, or a cytosine at position corresponding to 600 of SEQ ID NO: 34. In some embodiments, the length of the amplicon can range from a combination of the length of the primer pair plus one nucleotide base pair to any length of an amplicon that can be generated 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 is a cytosine at position 176,cytosine at a position corresponding to position 867, cytosine at a position corresponding to position 600 in SEQ ID NO:11, cytosine at a position corresponding to position 639 in SEQ ID NO:12, cytosine at a position corresponding to position 562 in SEQ ID NO:13, cytosine at a position corresponding to position 515 in SEQ ID NO:14, cytosine at a position corresponding to position 579 in SEQ ID NO:15, cytosine at a position corresponding to position 416 in SEQ ID NO:16, cytosine at a position corresponding to position 639 in SEQ ID NO:17, cytosine at a position corresponding to position 600 in SEQ ID NO:18, A position including a cytosine at a position corresponding to 600 in sequence number 27, a cytosine at a position corresponding to 639 in sequence number 28, a cytosine at a position corresponding to 562 in sequence number 29, a cytosine at a position corresponding to 515 in sequence number 30, a cytosine at a position corresponding to 579 in sequence number 31, a cytosine at a position corresponding to 416 in sequence number 32, a cytosine at a position corresponding to 639 in sequence number 33, or a cytosine at a position corresponding to 600 in sequence number 34, and a cytosine at a position corresponding to 176,A cytosine at a position corresponding to position 867, a cytosine at a position corresponding to position 600 in SEQ ID NO: 11, a cytosine at a position corresponding to position 639 in SEQ ID NO: 12, a cytosine at a position corresponding to position 562 in SEQ ID NO: 13, a cytosine at a position corresponding to position 515 in SEQ ID NO: 14, a cytosine at a position corresponding to position 579 in SEQ ID NO: 15, a cytosine at a position corresponding to position 416 in SEQ ID NO: 16, a cytosine at a position corresponding to position 639 in SEQ ID NO: 17, a cytosine at a position corresponding to position 600 in SEQ ID NO: 18, a cytosine at a position corresponding to position 600 in SEQ ID NO: 27 at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of a position that includes a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:28, a cytosine at a position corresponding to position 562 set forth in SEQ ID NO:29, a cytosine at a position corresponding to position 515 set forth in SEQ ID NO:30, a cytosine at a position corresponding to position 579 set forth in SEQ ID NO:31, a cytosine at a position corresponding to position 416 set forth in SEQ ID NO:32, a cytosine at a position corresponding to position 639 set forth in SEQ ID NO:33, or a cytosine at a position corresponding to position 600 set forth in SEQ ID NO:34.

[0105] 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 inspected and primers manually specified using known guidelines.

[0106] 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 (fluorescence in situ hybridization (FISH)), including the use of labeled primers or labeled probes on purified DNA, amplified DNA, and fixed cell preparations. In some methods, the target nucleic acid molecule may 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).

[0107] Hybridization techniques can employ stringent conditions so that the probe or primer specifically hybridizes with 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 above background, including more than 10-fold above background. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 4-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than other nucleotide sequences, more than 10-fold above background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0108] Suitable stringent 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 will be 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.

[0109] The present disclosure also provides a method for detecting the presence of a predicted loss-of-function polypeptide of ST6GALNAC5, comprising performing an assay on a biological sample obtained from a subject to determine whether the ST6GALNAC5 polypeptide in the biological sample contains one or more mutations that cause the polypeptide to have a loss of function (partial or complete) or a predicted loss of function (partial or complete). The predicted loss-of-function polypeptide of ST6GALNAC5 can be any of the predicted loss-of-function polypeptides of ST6GALNAC5 described herein. In some embodiments, the method includes detecting ST6GALNAC5 Val135Ala, Val4 ... * , or Val135Ala * In some embodiments, the method detects the presence of ST6GALNAC5 Val135Ala. In some embodiments, the method detects the presence of ST6GALNAC5 Val45Ala. In some embodiments, the method detects the presence of ST6GALNAC5 Val45Ala. * In some embodiments, the method detects the presence of ST6GALNAC5 Val135Ala. * Detect the presence of

[0110] In some embodiments, the method includes performing an assay on a biological sample obtained from the subject to determine whether an ST6GALNAC5 polypeptide in the biological sample comprises an alanine at a position corresponding to 135 set forth in SEQ ID NO:39, an alanine at a position corresponding to 45 set forth in SEQ ID NO:40, an alanine at a position corresponding to 45 set forth in SEQ ID NO:41, or an alanine at a position corresponding to 135 set forth in SEQ ID NO:42.

[0111] In some embodiments, the detection step comprises sequencing at least a portion of the ST6GALNAC5 polypeptide comprising a position corresponding to position 135 set forth in SEQ ID NO:39, position 45 set forth in SEQ ID NO:40, position 45 set forth in SEQ ID NO:41, or position 135 set forth in SEQ ID NO:42, or SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.

[0112] In some embodiments, the detection step comprises an immunoassay to detect the presence of an ST6GALNAC5 polypeptide comprising a position corresponding to position 135 set forth in SEQ ID NO:39, position 45 set forth in SEQ ID NO:40, position 45 set forth in SEQ ID NO:41, or position 135 set forth in SEQ ID NO:42, or SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.

[0113] In some embodiments, if a subject does not have a predicted loss-of-function polypeptide of ST6GALNAC5, the subject has a high risk of developing cognitive impairment or any of myelin integrity loss, neurodegeneration, GWC loss, and conversion of mild cognitive impairment to dementia. In some embodiments, if a subject has a predicted loss-of-function polypeptide of ST6GALNAC5, the subject has a low risk of developing cognitive impairment or any of myelin integrity loss, neurodegeneration, GWC loss, and conversion of mild cognitive impairment to dementia.

[0114] The present disclosure also provides isolated nucleic acid molecules that hybridize with ST6GALNAC5 missense variant genomic nucleic acid molecules, ST6GALNAC5 missense variant mRNA molecules, and / or ST6GALNAC5 missense variant cDNA molecules (e.g., any of the genomic variant nucleic acid molecules, mRNA variant molecules, and cDNA variant molecules disclosed herein). In some embodiments, such isolated nucleic acid molecules hybridize with ST6GALNAC5 missense variant nucleic acid molecules under stringent conditions. Such nucleic acid molecules can be used, for example, as probes, primers, mutation-specific probes, or mutation-specific primers as described or exemplified herein.

[0115] In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an ST6GALNAC5 missense nucleic acid molecule comprising a position corresponding to: position 176,867 set forth in SEQ ID NO:2, position 600 set forth in SEQ ID NO:11, position 639 set forth in SEQ ID NO:12, position 562 set forth in SEQ ID NO:13, position 515 set forth in SEQ ID NO:14, position 579 set forth in SEQ ID NO:15, position 416 set forth in SEQ ID NO:16, position 639 set forth in SEQ ID NO:17, position 600 set forth in SEQ ID NO:18, position 600 set forth in SEQ ID NO:27, position 639 set forth in SEQ ID NO:28, position 562 set forth in SEQ ID NO:29, position 515 set forth in SEQ ID NO:30, position 579 set forth in SEQ ID NO:31, position 416 set forth in SEQ ID NO:32, position 639 set forth in SEQ ID NO:33, or position 600 set forth in SEQ ID NO:34.

[0116] 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.

[0117] 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 ST6GALNAC5 missense variant genomic nucleic acid molecule, a ST6GALNAC5 missense variant mRNA molecule, and / or a ST6GALNAC5 missense 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.

[0118] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to the nucleotide sequence of a portion of an ST6GALNAC5 missense nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 or its complement. In some embodiments, the portion comprises position 176,867 or its complement as set forth in SEQ ID NO:2; position 600 or its complement as set forth in SEQ ID NO:11; position 639 or its complement as set forth in SEQ ID NO:12; position 562 or its complement as set forth in SEQ ID NO:13; position 515 or its complement as set forth in SEQ ID NO:14; position 579 or its complement as set forth in SEQ ID NO:15; position 416 or its complement as set forth in SEQ ID NO:16; position 639 or its complement as set forth in SEQ ID NO:17 ...62 or its complement as set forth in SEQ ID NO:13; position 515 or its complement as set forth in SEQ ID NO:14; position 579 or its complement as set forth in SEQ ID NO:15; position 416 or its complement as set forth in SEQ ID NO:16; position 639 or its complement as set forth in SEQ ID NO:17; position 562 or its complement as set forth in SEQ ID NO:18. position 600 or its complement set forth in SEQ ID NO:27; position 639 or its complement set forth in SEQ ID NO:28; position 562 or its complement set forth in SEQ ID NO:29; position 515 or its complement set forth in SEQ ID NO:30; position 579 or its complement set forth in SEQ ID NO:31; position 416 or its complement set forth in SEQ ID NO:32; position 639 or its complement set forth in SEQ ID NO:33; or a position corresponding to position 600 or its complement set forth in SEQ ID NO:34.In some embodiments, the portion is selected from the group consisting of positions 176,866 to 176,868 set forth in SEQ ID NO:2, or a complement thereof; positions 599 to 601 set forth in SEQ ID NO:11, or a complement thereof; positions 638 to 640 set forth in SEQ ID NO:12, or a complement thereof; positions 561 to 563 set forth in SEQ ID NO:13, or a complement thereof; positions 514 to 516 set forth in SEQ ID NO:14, or a complement thereof; positions 578 to 580 set forth in SEQ ID NO:15, or a complement thereof; positions 415 to 417 set forth in SEQ ID NO:16, or a complement thereof; positions 638 to 640 set forth in SEQ ID NO:17, or a complement thereof; positions 578 to 580 set forth in SEQ ID NO:18, or a complement thereof. positions 599 to 601 or their complements set forth in SEQ ID NO:27; positions 638 to 640 or their complements set forth in SEQ ID NO:28; positions 561 to 563 or their complements set forth in SEQ ID NO:29; positions 514 to 516 or their complements set forth in SEQ ID NO:30; positions 578 to 580 or their complements set forth in SEQ ID NO:31; positions 415 to 417 or their complements set forth in SEQ ID NO:32; positions 638 to 640 or their complements set forth in SEQ ID NO:33; or positions 599 to 601 or their complements set forth in SEQ ID NO:34.

[0119] In some embodiments, the mutation-specific probe and the mutation-specific primer comprise DNA. In some embodiments, the mutation-specific probe and the mutation-specific primer comprise RNA.

[0120] 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 probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0121] In some embodiments, the primers can be used in second generation or high throughput sequencing, including mutation specific primers. Sometimes the primers can be modified, including mutation 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 steps. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. The adapters can contain 5'-biotin tags for immobilizing the DNA library on streptavidin-coated beads.

[0122] The probes and primers described herein can be used to detect nucleotide variations within any of the ST6GALNAC5 missense variant genomic nucleic acid molecules, ST6GALNAC5 missense variant mRNA molecules, and / or ST6GALNAC5 missense variant cDNA molecules disclosed herein. The primers described herein can be used to amplify ST6GALNAC5 missense variant genomic nucleic acid molecules, ST6GALNAC5 missense variant mRNA molecules, or ST6GALNAC5 missense variant cDNA molecules, or fragments thereof.

[0123] 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 thymine (rather than cytosine) at a position corresponding to position 176,867 of SEQ ID NO:1 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 reference genomic nucleic acid molecule. Conversely, if one of the 3' ends of the primer hybridizes with cytosine (rather than thymine) at a position corresponding to position 176,867 of SEQ ID NO:2 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 missense variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at a position corresponding to position 176,867 of SEQ ID NO:2 can be at the 3' end of the primer.

[0124] If one of the 3' ends of the primer hybridizes to uracil (rather than cytosine) at a position corresponding to position 600 set forth in SEQ ID NO: 3 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to cytosine (rather than uracil) at a position corresponding to position 600 set forth in SEQ ID NO: 11 in a particular ST6GALNAC5 mRNA molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at a position corresponding to position 600 set forth in SEQ ID NO: 11 can be at the 3' end of the primer.

[0125] If one of the 3' ends of the primer hybridizes to uracil (rather than cytosine) at a position corresponding to position 639 of SEQ ID NO: 4 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to cytosine (rather than uracil) at a position corresponding to position 639 of SEQ ID NO: 12 in a particular ST6GALNAC5 mRNA molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at a position corresponding to position 639 of SEQ ID NO: 12 can be at the 3' end of the primer.

[0126] If one of the 3' ends of the primer hybridizes to uracil (rather than cytosine) at a position corresponding to position 562 of SEQ ID NO:5 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to cytosine (rather than uracil) at a position corresponding to position 562 of SEQ ID NO:13 in a particular ST6GALNAC5 mRNA molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 562 of SEQ ID NO:13 can be at the 3' end of the primer.

[0127] If one of the 3' ends of the primer hybridizes to uracil (rather than cytosine) at a position corresponding to position 515 of SEQ ID NO: 6 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to cytosine (rather than uracil) at a position corresponding to position 515 of SEQ ID NO: 14 in a particular ST6GALNAC5 mRNA molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at a position corresponding to position 515 of SEQ ID NO: 14 can be at the 3' end of the primer.

[0128] If one of the 3' ends of the primer hybridizes to uracil (rather than cytosine) at a position corresponding to position 579 of SEQ ID NO: 7 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to cytosine (rather than uracil) at a position corresponding to position 579 of SEQ ID NO: 15 in a particular ST6GALNAC5 mRNA molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 579 of SEQ ID NO: 15 can be at the 3' end of the primer.

[0129] If one of the 3' ends of the primer hybridizes to uracil (rather than cytosine) at a position corresponding to position 416 set forth in SEQ ID NO:8 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to cytosine (rather than uracil) at a position corresponding to position 416 set forth in SEQ ID NO:16 in a particular ST6GALNAC5 mRNA molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at a position corresponding to position 416 set forth in SEQ ID NO:16 can be at the 3' end of the primer.

[0130] If one of the 3' ends of the primer hybridizes to uracil (rather than cytosine) at a position corresponding to position 639 of SEQ ID NO: 9 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to cytosine (rather than uracil) at a position corresponding to position 639 of SEQ ID NO: 17 in a particular ST6GALNAC5 mRNA molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 639 of SEQ ID NO: 17 can be at the 3' end of the primer.

[0131] If one of the 3' ends of the primer hybridizes to uracil (rather than cytosine) at a position corresponding to position 600 set forth in SEQ ID NO: 10 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to cytosine (rather than uracil) at a position corresponding to position 600 set forth in SEQ ID NO: 18 in a particular ST6GALNAC5 mRNA molecule, the presence of an amplified fragment indicates the presence of an ST6GALNAC5 missense variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at a position corresponding to position 600 set forth in SEQ ID NO: 18 can be at the 3' end of the primer.

[0132] If one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 600 set forth in SEQ ID NO: 19 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 600 set forth in SEQ ID NO: 27 in a particular ST6GALNAC5 cDNA molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 600 set forth in SEQ ID NO: 27 can be at the 3' end of the primer.

[0133] If one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 639 of SEQ ID NO:20 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 639 of SEQ ID NO:28 in a particular ST6GALNAC5 cDNA molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 639 of SEQ ID NO:28 can be at the 3' end of the primer.

[0134] If one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 562 of SEQ ID NO:21 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 562 of SEQ ID NO:29 in a particular ST6GALNAC5 cDNA molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 562 of SEQ ID NO:29 can be at the 3' end of the primer.

[0135] If one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 515 set forth in SEQ ID NO:22 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 515 set forth in SEQ ID NO:30 in a particular ST6GALNAC5 cDNA molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at a position corresponding to position 515 set forth in SEQ ID NO:30 can be at the 3' end of the primer.

[0136] If one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 579 of SEQ ID NO: 23 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 579 of SEQ ID NO: 31 in a particular ST6GALNAC5 cDNA molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 579 of SEQ ID NO: 31 can be at the 3' end of the primer.

[0137] If one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 416 set forth in SEQ ID NO:24 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 416 set forth in SEQ ID NO:32 in a particular ST6GALNAC5 cDNA molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at a position corresponding to position 416 set forth in SEQ ID NO:32 can be at the 3' end of the primer.

[0138] If one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 639 of SEQ ID NO: 25 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 639 of SEQ ID NO: 33 in a particular ST6GALNAC5 cDNA molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 639 of SEQ ID NO: 33 can be at the 3' end of the primer.

[0139] If one of the 3' ends of the primer hybridizes to a thymine (rather than a cytosine) at a position corresponding to position 600 set forth in SEQ ID NO: 26 in a particular ST6GALNAC5 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to a cytosine (rather than a thymine) at a position corresponding to position 600 set forth in SEQ ID NO: 34 in a particular ST6GALNAC5 cDNA molecule, the presence of an amplified fragment indicates the presence of a ST6GALNAC5 missense variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 600 set forth in SEQ ID NO: 34 can be at the 3' end of the primer.

[0140] 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 ST6GALNAC5-based genomic nucleic acid molecule, a ST6GALNAC5-based mRNA molecule, and / or a ST6GALNAC5-based cDNA molecule.

[0141] In any of the embodiments described throughout this disclosure, the probe (e.g., the mutation-specific probe) can include a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0142] 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, usually containing one array per well. In some embodiments, the support is a microarray.

[0143] The present disclosure also provides a molecular complex comprising or consisting of any of the ST6GALNAC5 missense nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the mutation-specific primers or mutation-specific probes described herein. In some embodiments, the ST6GALNAC5 missense nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) or their complements in the molecular complex are single-stranded. In some embodiments, the ST6GALNAC5 missense nucleic acid molecule is any of the genomic nucleic acid molecules described herein. In some embodiments, the ST6GALNAC5 missense nucleic acid molecule is any of the mRNA molecules described herein. In some embodiments, the ST6GALNAC5 missense nucleic acid molecule is any of the cDNA molecules described herein. In some embodiments, the molecular complex comprises or consists of any of the ST6GALNAC5 missense nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the mutation-specific primers described herein. In some embodiments, the molecular complex comprises or consists of any of the ST6GALNAC5 missense nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the mutation-specific probes described herein.

[0144] In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe hybridized to an ST6GALNAC5 genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, wherein the mutation-specific primer or the mutation-specific probe hybridizes to the ST6GALNAC5 genomic nucleic acid molecule at a position corresponding to position 176,867 set forth in SEQ ID NO:2 or its complement.

[0145] In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe hybridized to the GCG codon at a position corresponding to positions 176,866 to 176,868 of SEQ ID NO:2.

[0146] In some embodiments, the molecular complex comprises or consists of a genomic nucleic acid molecule comprising SEQ ID NO:2. In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe hybridized to an ST6GALNAC5 mRNA molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, wherein the mutation-specific primer or the mutation-specific probe hybridizes to the ST6GALNAC5 mRNA molecule at a position corresponding to position 600 or its complement set forth in SEQ ID NO: 11; position 639 or its complement set forth in SEQ ID NO: 12; position 562 or its complement set forth in SEQ ID NO: 13; position 515 or its complement set forth in SEQ ID NO: 14; position 579 or its complement set forth in SEQ ID NO: 15; position 416 or its complement set forth in SEQ ID NO: 16; position 639 or its complement set forth in SEQ ID NO: 17; or position 600 or its complement set forth in SEQ ID NO: 18.

[0147] In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe hybridized to a GCG codon at a position corresponding to positions 599-601 of SEQ ID NO:11, a GCG codon at a position corresponding to positions 638-640 of SEQ ID NO:12, a GCG codon at a position corresponding to positions 561-563 of SEQ ID NO:13, a GCG codon at a position corresponding to positions 514-516 of SEQ ID NO:14, a GCG codon at a position corresponding to positions 578-580 of SEQ ID NO:15, a GCG codon at a position corresponding to positions 415-417 of SEQ ID NO:16, a GCG codon at a position corresponding to positions 638-640 of SEQ ID NO:17, or a GCG codon at a position corresponding to positions 599-601 of SEQ ID NO:18.

[0148] In some embodiments, the molecular complex comprises or consists of an mRNA molecule comprising SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

[0149] In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe hybridized to an ST6GALNAC5 cDNA molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, wherein the mutation-specific primer or the mutation-specific probe hybridizes to the ST6GALNAC5 cDNA molecule at a position corresponding to position 600 or its complement set forth in SEQ ID NO:27; position 639 or its complement set forth in SEQ ID NO:28; position 562 or its complement set forth in SEQ ID NO:29; position 515 or its complement set forth in SEQ ID NO:30; position 579 or its complement set forth in SEQ ID NO:31; position 416 or its complement set forth in SEQ ID NO:32; position 639 or its complement set forth in SEQ ID NO:33; or position 600 or its complement set forth in SEQ ID NO:34.

[0150] In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe hybridized to a GCG codon at a position corresponding to positions 599-601 of SEQ ID NO:27, a GCG codon at a position corresponding to positions 638-640 of SEQ ID NO:28, a GCG codon at a position corresponding to positions 561-563 of SEQ ID NO:29, a GCG codon at a position corresponding to positions 514-516 of SEQ ID NO:30, a GCG codon at a position corresponding to positions 578-580 of SEQ ID NO:31, a GCG codon at a position corresponding to positions 415-417 of SEQ ID NO:32, a GCG codon at a position corresponding to positions 638-640 of SEQ ID NO:33, or a GCG codon at a position corresponding to positions 599-601 of SEQ ID NO:34.

[0151] In some embodiments, the molecular complex comprises or consists of a cDNA molecule comprising SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34.

[0152] In some embodiments, the molecular complex comprises a mutation-specific probe or a mutation-specific primer that comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin. In some embodiments, the molecular complex further comprises a non-human polymerase.

[0153] The nucleotide sequence of the ST6GALNAC5 reference genomic nucleic acid molecule is set forth in SEQ ID NO: 1. With reference to SEQ ID NO: 1, position 176,867 is a thymine. There is an ST6GALNAC5 missense variant genomic nucleic acid molecule in which the thymine at position 176,867 is replaced by a cytosine (rs756654226; chr1:77044346 in the GRCh38 / hg38 human genome assembly). The nucleotide sequence of this ST6GALNAC5 missense variant genomic nucleic acid molecule is set forth in SEQ ID NO:2.

[0154] The nucleotide sequence of an ST6GALNAC5 referenced mRNA molecule is set forth in SEQ ID NO:3. With reference to SEQ ID NO:3, position 600 is uracil. The nucleotide sequence of another ST6GALNAC5 referenced mRNA molecule is set forth in SEQ ID NO:4. With reference to SEQ ID NO:4, position 639 is uracil. The nucleotide sequence of another ST6GALNAC5 referenced mRNA molecule is set forth in SEQ ID NO:5. With reference to SEQ ID NO:5, position 562 is uracil. The nucleotide sequence of another ST6GALNAC5 referenced mRNA molecule is set forth in SEQ ID NO:6. With reference to SEQ ID NO:6, position 515 is uracil. The nucleotide sequence of another ST6GALNAC5 referenced mRNA molecule is set forth in SEQ ID NO:7. With reference to SEQ ID NO:7, position 579 is uracil. The nucleotide sequence of another ST6GALNAC5 referenced mRNA molecule is set forth in SEQ ID NO:8. With reference to SEQ ID NO:8, position 416 is uracil. The nucleotide sequence of another ST6GALNAC5-based mRNA molecule is set forth in SEQ ID NO: 9. With reference to SEQ ID NO: 9, position 639 is uracil. The nucleotide sequence of another ST6GALNAC5-based mRNA molecule is set forth in SEQ ID NO: 10. With reference to SEQ ID NO: 10, position 600 is uracil.

[0155] There exists a ST6GALNAC5 missense variant mRNA molecule in which uracil at position 600 is replaced by cytosine. The nucleotide sequence of this ST6GALNAC5 missense variant mRNA molecule is set forth in SEQ ID NO:11.

[0156] Another ST6GALNAC5 missense variant mRNA molecule exists in which uracil at position 639 is replaced by cytosine. The nucleotide sequence of this ST6GALNAC5 missense variant mRNA molecule is set forth in SEQ ID NO:12.

[0157] Another ST6GALNAC5 missense variant mRNA molecule exists in which uracil at position 562 is replaced by cytosine. The nucleotide sequence of this ST6GALNAC5 missense variant mRNA molecule is set forth in SEQ ID NO:13.

[0158] Another ST6GALNAC5 missense variant mRNA molecule exists in which uracil at position 515 is replaced by cytosine, the nucleotide sequence of which is set forth in SEQ ID NO:14.

[0159] Another ST6GALNAC5 missense variant mRNA molecule exists in which uracil at position 579 is replaced by cytosine. The nucleotide sequence of this ST6GALNAC5 missense variant mRNA molecule is set forth in SEQ ID NO:15.

[0160] Another ST6GALNAC5 missense variant mRNA molecule exists in which uracil at position 416 is replaced by cytosine. The nucleotide sequence of this ST6GALNAC5 missense variant mRNA molecule is set forth in SEQ ID NO:16.

[0161] Another ST6GALNAC5 missense variant mRNA molecule exists in which uracil at position 639 is replaced by cytosine. The nucleotide sequence of this ST6GALNAC5 missense variant mRNA molecule is set forth in SEQ ID NO:17.

[0162] Another ST6GALNAC5 missense variant mRNA molecule exists in which uracil at position 600 is replaced by cytosine. The nucleotide sequence of this ST6GALNAC5 missense variant mRNA molecule is set forth in SEQ ID NO:18.

[0163] The nucleotide sequence of a ST6GALNAC5-based cDNA molecule is set forth in SEQ ID NO: 19. With reference to SEQ ID NO: 19, position 600 is a thymine. The nucleotide sequence of another ST6GALNAC5-based cDNA molecule is set forth in SEQ ID NO: 20. With reference to SEQ ID NO: 20, position 639 is a thymine. The nucleotide sequence of another ST6GALNAC5-based cDNA molecule is set forth in SEQ ID NO: 21. With reference to SEQ ID NO: 21, position 562 is a thymine. The nucleotide sequence of another ST6GALNAC5-based cDNA molecule is set forth in SEQ ID NO: 22. With reference to SEQ ID NO: 22, position 515 is a thymine. The nucleotide sequence of another ST6GALNAC5-based cDNA molecule is set forth in SEQ ID NO: 23. With reference to SEQ ID NO: 23, position 579 is a thymine. The nucleotide sequence of another ST6GALNAC5-based cDNA molecule is set forth in SEQ ID NO: 24. With reference to SEQ ID NO: 24, position 416 is a thymine. The nucleotide sequence of another ST6GALNAC5-based cDNA molecule is set forth in SEQ ID NO: 25. With reference to SEQ ID NO: 25, position 639 is a thymine. The nucleotide sequence of another ST6GALNAC5-based cDNA molecule is set forth in SEQ ID NO: 26. With reference to SEQ ID NO: 26, position 600 is a thymine.

[0164] There exists an ST6GALNAC5 missense variant cDNA molecule in which the thymine at position 600 is replaced by a cytosine, the nucleotide sequence of which is set forth in SEQ ID NO:27.

[0165] Another ST6GALNAC5 missense variant cDNA molecule exists in which the thymine at position 639 is replaced by a cytosine. The nucleotide sequence of this ST6GALNAC5 missense variant cDNA molecule is set forth in SEQ ID NO:28.

[0166] Another ST6GALNAC5 missense variant cDNA molecule exists in which the thymine at position 562 is replaced by a cytosine. The nucleotide sequence of this ST6GALNAC5 missense variant cDNA molecule is set forth in SEQ ID NO:29.

[0167] Another ST6GALNAC5 missense variant cDNA molecule exists in which the thymine at position 515 is replaced by a cytosine, the nucleotide sequence of which is set forth in SEQ ID NO:30.

[0168] Another ST6GALNAC5 missense variant cDNA molecule exists in which the thymine at position 579 is replaced by a cytosine. The nucleotide sequence of this ST6GALNAC5 missense variant cDNA molecule is set forth in SEQ ID NO:31.

[0169] Another ST6GALNAC5 missense variant cDNA molecule exists in which the thymine at position 416 is replaced by a cytosine, the nucleotide sequence of which is set forth in SEQ ID NO:32.

[0170] Another ST6GALNAC5 missense variant cDNA molecule exists in which the thymine at position 639 is replaced by a cytosine. The nucleotide sequence of this ST6GALNAC5 missense variant cDNA molecule is set forth in SEQ ID NO:33.

[0171] Another ST6GALNAC5 missense variant cDNA molecule exists in which a thymine is replaced by a cytosine at position 600. The nucleotide sequence of this ST6GALNAC5 missense variant cDNA molecule is set forth in SEQ ID NO:34.

[0172] 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 human or another organism (e.g., 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 only exemplary sequences. Other sequences are also possible.

[0173] 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.

[0174] 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 as exogenous donor sequences in or containing a vector that includes the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to heterologous labels. The labels can be directly detectable (e.g., fluorophores) or indirectly detectable (e.g., haptens, enzymes, or fluorophore quenchers). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The labels can also be, for example, chemiluminescent materials; metal-containing materials; or enzymes, where enzyme-dependent secondary generation of a signal occurs. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a binding molecule such that the binding molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and probed using a colorimetric (e.g., tetramethylbenzidine (TMB)) or fluorogenic substrate to detect the presence of HRP. 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, fluorescent and chemiluminescent substrates, as well as other labels.

[0175] The isolated nucleic acid molecule or its complement can be present in a host cell. In some embodiments, the host cell can comprise a vector comprising any of the nucleic acid molecules described herein or their complements. In some embodiments, the nucleic acid molecule is operably linked to a promoter active in the host cell. In some embodiments, the promoter is an exogenous promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the host cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the host cell is an insect cell. In some embodiments, the host cell is a mammalian cell.

[0176] 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.

[0177] The nucleic acid molecules disclosed herein may also include one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide that contains modifications to either the base, sugar, 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 various purine or pyrimidine bases, such as, for example, pseudouridine, uracil-5-yl, hypoxanthine-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 cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (cytosine), and thymine. Examples of uracils and cytosines include, but are not limited to, 4-isopropyl uracil, 4-isopropyl uracil, 8 ...

[0178] Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose, as well as synthetic modifications. 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-alkyl groups. 1~10 Alkyl or C 2~10 Alkenyl, and C 2~10Exemplary 2' sugar modifications 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 Examples of suitable substituents 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 may be made at other positions on the sugar, particularly the 3' position of the sugar in the 3' terminal nucleotide or 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.

[0179] 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. Such phosphate or modified phosphate linkages between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages 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 substitutes also include peptide nucleic acids (PNAs).

[0180] 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 into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector in 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.

[0181] 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, such as native immunoglobulin promoters and actin promoters. Methods for expressing polypeptides in bacterial or fungal cells, such as 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).

[0182] Percent identity (%) (or percent complementarity) between specific stretches of nucleotide sequences in nucleic acid molecules or amino acid sequences in polypeptides can be routinely determined using the BLAST program (base local sequence comparison 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 the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings that use the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). When referring to percent sequence identity in this specification, a higher percent sequence identity is preferred over a lower one.

[0183] The present disclosure also provides compositions comprising 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-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution such as PBS, HBSS, and the like.

[0184] As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in the context of numbering a particular nucleotide or sequence of nucleotides or position, refers to the numbering of the specified reference sequence when the particular nucleotide or sequence of nucleotides is compared to a reference sequence (e.g., SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:19). In other words, the residue (e.g., nucleotide or amino acid) number or residue (e.g., nucleotide or amino acid) position of a particular polymer is specified with reference to the reference sequence, not by the actual position number of the 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 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.

[0185] For example, an ST6GALNAC5 missense nucleic acid molecule comprising a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence of which comprises a cytosine at position corresponding to position 176,867 of SEQ ID NO:2, means that when the nucleotide sequence of the ST6GALNAC5 genomic nucleic acid molecule is aligned against the sequence of SEQ ID NO:2, the sequence of ST6GALNAC5 has a cytosine residue at position corresponding to position 176,867 of SEQ ID NO:2. The same applies to an ST6GALNAC5 missense mRNA molecule comprising a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence of which comprises a cytosine at position corresponding to position 600 of SEQ ID NO:11, and to an ST6GALNAC5 missense cDNA molecule comprising a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence of which comprises a cytosine at position corresponding to position 600 of SEQ ID NO:27. In other words, these terms refer to a nucleic acid molecule encoding an ST6GALNAC5 polypeptide, wherein a genomic nucleic acid molecule has a nucleotide sequence that includes a cytosine residue that is homologous to the cytosine residue at position 176,867 of SEQ ID NO:2 (or an mRNA molecule has a nucleotide sequence that includes a cytosine residue that is homologous to the cytosine residue at position 600 of SEQ ID NO:11, or a cDNA molecule has a nucleotide sequence that includes a cytosine residue that is homologous to the cytosine residue at position 600 of SEQ ID NO:27).

[0186] As described herein, the position in the ST6GALNAC5 missense genomic nucleic acid molecule corresponding to position 176,867 of SEQ ID NO:2 can be identified, for example, by performing sequence comparison between the nucleotide sequence of a particular ST6GALNAC5 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 comparison to identify the position of the nucleotide corresponding to position 176,867 of SEQ ID NO:2. For example, sequence comparison 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.

[0187] The amino acid sequences of the ST6GALNAC5 reference polypeptide are set forth in SEQ ID NO:35 (isoform 1), SEQ ID NO:36 (isoform 2), SEQ ID NO:37 (isoform 3), and SEQ ID NO:38 (isoform 4). With reference to SEQ ID NO:35 (isoform 1), the ST6GALNAC5 reference polypeptide is 336 amino acids long. With reference to SEQ ID NO:35, position 135 is a valine. With reference to SEQ ID NO:36 (isoform 2), the ST6GALNAC5 reference polypeptide is 146 amino acids long. With reference to SEQ ID NO:36, position 45 is a valine. With reference to SEQ ID NO:37 (isoform 3), the ST6GALNAC5 reference polypeptide is 246 amino acids long. With reference to SEQ ID NO:37, position 45 is a valine. With reference to SEQ ID NO:38 (isoform 4), the ST6GALNAC5 reference polypeptide is 236 amino acids long. With reference to SEQ ID NO:38, position 135 is a valine.

[0188] The amino acid sequences of the predicted loss-of-function polypeptides of ST6GALNAC5 are SEQ ID NO: 39 (isoform 1; Val135Ala), SEQ ID NO: 40 (isoform 2; Val45Ala), SEQ ID NO: 41 (isoform 3; Val45Ala), * ), and SEQ ID NO: 42 (isoform 4; Val135Ala * ) With reference to SEQ ID NO:39, position 135 is alanine. With reference to SEQ ID NO:40, position 45 is alanine. With reference to SEQ ID NO:41, position 45 is alanine. With reference to SEQ ID NO:42, position 135 is alanine.

[0189] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5'-end of the sequence and proceeding toward the 3'-end (i.e., from left to right in each sequence). Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by reference to the shown strand. The amino acid sequences follow the standard convention of beginning at the amino-terminus of the sequence and proceeding toward the carboxy-terminus (i.e., from left to right in each sequence).

[0190] The present disclosure also provides a therapeutic agent for treating or preventing a cognitive disorder, the therapeutic agent being for use in treating a cognitive disorder (or for use in the preparation of a medicament for treating a cognitive disorder) in a subject having any of the ST6GALNAC5 missense variant genomic nucleic acid molecules, missense variant mRNA molecules, and / or missense variant cDNA molecules encoding the predicted loss-of-function polypeptides of ST6GALNAC5 described herein. The therapeutic agent for treating or preventing a cognitive disorder may be any of the therapeutic agents for treating or preventing a cognitive disorder described herein.

[0191] In some embodiments, the subject is identified as having a genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the genomic nucleic acid molecule having a nucleotide sequence that includes a cytosine or its complement at a position corresponding to position 176,867 of SEQ ID NO:2.

[0192] In some embodiments, the subject is identified as having an mRNA molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the mRNA molecule having a nucleotide sequence including: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:17; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:18.

[0193] In some embodiments, the subject is identified as having a cDNA molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the cDNA molecule having a nucleotide sequence including: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:27; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:28; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:29; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:30; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:31; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:32; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:33; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:34.

[0194] In some embodiments, the subject is provided with: i) a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence comprising a cytosine or its complement at a position corresponding to position 176,867 set forth in SEQ ID NO:2; ii) an mRNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:17. or its complement; or an mRNA molecule comprising a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO: 18; or iii) a cDNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO: 27; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO: 28; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO: 29; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO: 30; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO: 31; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO: 32; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO: 33; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO: 34.

[0195] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence including a cytosine or its complement at a position corresponding to position 176,867 of SEQ ID NO:2.

[0196] In some embodiments, the subject is identified as having an mRNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:17; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:18.

[0197] In some embodiments, the subject is identified as having a cDNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:27; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:28; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:29; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:30; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:31; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:32; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:33; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:34.

[0198] In some embodiments, the subject is identified as having a predicted loss-of-function polypeptide of ST6GALNAC5 that includes an alanine at a position corresponding to 135 set forth in SEQ ID NO:39, an alanine at a position corresponding to 45 set forth in SEQ ID NO:40, an alanine at a position corresponding to 45 set forth in SEQ ID NO:41, or an alanine at a position corresponding to 135 set forth in SEQ ID NO:42.

[0199] The present disclosure also provides an ST6GALNAC5 inhibitor for use in treating a cognitive disorder (or for use in the preparation of a medicament for treating a cognitive disorder) in a subject who is heterozygous for any of the ST6GALNAC5 missense variant genomic nucleic acid molecules, missense variant mRNA molecules, and / or missense variant cDNA molecules encoding predicted loss-of-function polypeptides of ST6GALNAC5 described herein, or who is a reference for an ST6GALNAC5 genomic nucleic acid molecule, mRNA molecule, or cDNA molecule. The ST6GALNAC5 inhibitor may be any of the ST6GALNAC5 inhibitors described herein.

[0200] In some embodiments, the subject is a reference for a ST6GALNAC5 genomic nucleic acid molecule, a ST6GALNAC5 mRNA molecule, or a ST6GALNAC5 cDNA molecule. In some embodiments, the subject is a reference for a ST6GALNAC5 genomic nucleic acid molecule. In some embodiments, the subject is a reference for a ST6GALNAC5 mRNA molecule. In some embodiments, the subject is a reference for a ST6GALNAC5 cDNA molecule.

[0201] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 having a nucleotide sequence that includes a cytosine or its complement at a position corresponding to position 176,867 of SEQ ID NO:2.

[0202] In some embodiments, the subject is identified as heterozygous for an mRNA molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the mRNA molecule having a nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:17; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:18.

[0203] In some embodiments, the subject is identified as heterozygous for a cDNA molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the cDNA molecule having a nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:27; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:28; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:29; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:30; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:31; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:32; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:33; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:34.

[0204] In some embodiments, the subject is provided with: i) a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence comprising a cytosine or its complement at a position corresponding to position 176,867 set forth in SEQ ID NO:2; ii) an mRNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:17. or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO: 18; or iii) a cDNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO: 27; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO: 28; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO: 29; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO: 30; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO: 31; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO: 32; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO: 33; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO: 34.

[0205] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence comprising a cytosine or its complement at a position corresponding to position 176,867 of SEQ ID NO:2.

[0206] In some embodiments, a subject is identified as heterozygous for an mRNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:11; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:12; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:13; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:14; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:15; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:16; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:17; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:18.

[0207] In some embodiments, the subject is identified as heterozygous for a cDNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of ST6GALNAC5, the nucleotide sequence comprising: a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:27; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:28; a cytosine or its complement at a position corresponding to position 562 set forth in SEQ ID NO:29; a cytosine or its complement at a position corresponding to position 515 set forth in SEQ ID NO:30; a cytosine or its complement at a position corresponding to position 579 set forth in SEQ ID NO:31; a cytosine or its complement at a position corresponding to position 416 set forth in SEQ ID NO:32; a cytosine or its complement at a position corresponding to position 639 set forth in SEQ ID NO:33; or a cytosine or its complement at a position corresponding to position 600 set forth in SEQ ID NO:34.

[0208] 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 document was specifically and individually indicated to be so incorporated by reference. Where various versions of a sequence are associated with accession numbers at different 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 to which the accession number refers, if applicable. Similarly, where different versions of publications, websites, etc. have been 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 practiced within the scope of the appended claims.

[0209] The following examples are provided to further illustrate the embodiments. They are intended to illustrate, not limit, the claimed embodiments. The following examples provide those skilled in the art with a disclosure and explanation of how the compounds, compositions, articles, devices and / or methods described herein are made and evaluated, and are intended to be merely illustrative and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some error and deviation can be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric pressure. EXAMPLES

[0210] Example 1: Association of rare variants with brain imaging traits The exomes of 454,787 UKB study participants were sequenced, covering 95.8% of the target bases at a depth of more than 20x, as previously described (Szustakowski, Advancing Human Genetics Research and Drug Discovery through Exome Sequencing of the UK Biobank. bioRxiv, 2021; and Van Hout et al., Nature, 2020). 12 million variants were identified in 39 million base pairs across the coding regions of 18,659 genes (data not shown). Among the variants identified, there were 3,375,252 (median 10,260 per subject) synonymous variants, 7,689,495 (9,284 per subject) missense variants, and 889,957 (212 per subject) putative loss-of-function (pLOF) variants (data not shown), of which approximately half were observed only once in this dataset (singleton variants; data not shown).

[0211] We analyzed the imaging component of the UK Biobank, which includes 36,968 subjects. Conditional on GWAS signal, we performed exome-wide association analyses for 2,077 brain imaging phenotypes derived from magnetic resonance imaging (MRI) to separate rare variant associations from common variant signal. Overall, 343 associations with 52 genes were found with P ≤ 10 -7 was found to be more conservative with a significance threshold of P ≤ 10 -10 found 98 associations with seven genes (data not shown). The strongest association with increasing GWC was with a deleterious missense variant in ST6GALNAC5 (rs756654226, 9 carriers; effect = 1.7 SD units, 95% CI 1.1 to 2.4, P = 8.2 × 10 -8 ). This is consistent with recent evidence that the relative abundance of specific gangliosides in the brain changes with age and in common neurological pathologies including Huntington's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, multiple sclerosis, and epilepsy.

[0212] In addition to those described herein, various modifications of the described subject matter will become 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 and for all purposes.

Claims

**Claim 1** An in vitro method for identifying a subject's susceptibility to the onset of cognitive impairment, reduced myelin integrity, neurodegeneration, reduced gray matter / white matter contrast (GWC), or conversion from mild cognitive impairment to dementia, said method comprising determining or having determined the presence or absence of an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of α-N-acetylgalactosaminide α-2,6-sialyltransferase 5 (ST6GALNAC5) in a biological sample obtained from said subject; wherein a subject who is ST6GALNAC5-positive is shown to be at high risk of developing cognitive impairment, reduced myelin integrity, neurodegeneration, reduced gray matter / white matter contrast (GWC), or conversion from mild cognitive impairment to dementia; and a subject who is heterozygous or homozygous for an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of said ST6GALNAC5 is shown to be at low risk of developing cognitive impairment, reduced myelin integrity, neurodegeneration, reduced gray matter / white matter contrast (GWC), or conversion from mild cognitive impairment to dementia. **Claim 2** The ST6GALNAC5 missense variant nucleic acid molecule is ＊ , or Val135Ala ＊ The method of claim 1 , wherein the **Claim 3** The ST6GALNAC5 missense variant nucleic acid molecule is: a genomic nucleic acid molecule having a nucleotide sequence comprising cytosine at a position corresponding to position 176,867 as set forth in SEQ ID NO: 2; an mRNA molecule having a nucleotide sequence comprising cytosine at a position corresponding to position 600 as set forth in SEQ ID NO: 11, cytosine at a position corresponding to position 639 as set forth in SEQ ID NO: 12, cytosine at a position corresponding to position 562 as set forth in SEQ ID NO: 13, cytosine at a position corresponding to position 515 as set forth in SEQ ID NO: 14, cytosine at a position corresponding to position 579 as set forth in SEQ ID NO: 15, cytosine at a position corresponding to position 416 as set forth in SEQ ID NO: 16, cytosine at a position corresponding to position 639 as set forth in SEQ ID NO: 17, or cytosine at a position corresponding to position 600 as set forth in SEQ ID NO: 18; or The method according to claim 2, which is a cDNA molecule generated from an mRNA molecule and has a nucleotide sequence containing cytosine at a position corresponding to position 600 described in SEQ ID NO: 27, cytosine at a position corresponding to position 639 described in SEQ ID NO: 28, cytosine at a position corresponding to position 562 described in SEQ ID NO: 29, cytosine at a position corresponding to position 515 described in SEQ ID NO: 30, cytosine at a position corresponding to position 579 described in SEQ ID NO: 31, cytosine at a position corresponding to position 416 described in SEQ ID NO: 32, cytosine at a position corresponding to position 639 described in SEQ ID NO: 33, or cytosine at a position corresponding to position 600 described in SEQ ID NO:

34.

4. Use of a therapeutic agent for treating or inhibiting cognitive impairment, myelin integrity reduction, neurodegeneration, gray matter / white matter contrast (GWC) reduction, or conversion from mild cognitive impairment to dementia in the preparation of a medicament for treating or preventing cognitive impairment, myelin integrity reduction, neurodegeneration, gray matter / white matter contrast (GWC) reduction, or conversion from mild cognitive impairment to dementia in a subject, wherein the subject is heterozygous for a ST6GALNAC5 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of α-N-acetylgalactosaminide α-2,6-sialyltransferase 5 (ST6GALNAC5).

5. Use of an α-N-acetylgalactosaminide α-2,6-sialyltransferase 5 (ST6GALNAC5) inhibitor in the preparation of a medicament for treating or preventing cognitive impairment, myelin integrity reduction, neurodegeneration, gray matter / white matter contrast (GWC) reduction, or conversion from mild cognitive impairment to dementia in a subject, wherein the subject is a) a reference for a ST6GALNAC5 genomic nucleic acid molecule or a ST6GALNAC5 mRNA molecule, or b) heterozygous for a ST6GALNAC5 missense mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5.

6. The use according to claim 5, which is an inhibitory nucleic acid molecule.

7. The use according to claim 6, wherein the inhibitory nucleic acid molecule is an antisense nucleic acid molecule that hybridizes with an ST6GALNAC5 nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

8. The use according to claim 5, comprising a Cas protein and a guide RNA (gRNA) that hybridizes with a guide RNA (gRNA) recognition sequence within an ST6GALNAC5 genomic nucleic acid molecule.

9. The use according to claim 8, wherein the Cas protein is Cas9 or Cpf1.

10. The use according to claim 8 or claim 9, wherein the gRNA recognition sequence comprises or is proximate to position 176,867 as set forth in SEQ ID NO:

1.

11. The use according to claim 8 or claim 9, 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 the position corresponding to position 176,867 as set forth in SEQ ID NO:

1.

12. The use according to claim 8 or claim 9, wherein the protospacer adjacent motif (PAM) sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence.

13. The use according to claim 8 or claim 9, wherein the gRNA comprises about 17 to about 23 nucleotides.

14. The use according to claim 8 or claim 9, wherein the gRNA recognition sequence comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 43 to 62.

15. The use according to claim 4, wherein the ST6GALNAC5 missense mutant nucleic acid molecule encodes ST6GALNAC5 Val135Ala, Val45Ala, Val45Ala*, or Val135Ala*.

16. The ST6GALNAC5 missense mutant nucleic acid molecule is: A genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of α-N-acetylgalactosaminide α-2,6-sialyltransferase 5 (ST6GALNAC5) or its complement, wherein the genomic nucleic acid molecule has a nucleotide sequence comprising cytosine at a position corresponding to position 176,867 as set forth in SEQ ID NO: 2; or An mRNA molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 or its complement, wherein the mRNA molecule has a nucleotide sequence comprising cytosine at a position corresponding to position 600 of SEQ ID NO: 11, cytosine at a position corresponding to position 639 of SEQ ID NO: 12, cytosine at a position corresponding to position 562 of SEQ ID NO: 13, cytosine at a position corresponding to position 515 of SEQ ID NO: 14, cytosine at a position corresponding to position 579 of SEQ ID NO: 15, cytosine at a position corresponding to position 416 of SEQ ID NO: 16, cytosine at a position corresponding to position 639 of SEQ ID NO: 17, or cytosine at a position corresponding to position 600 of SEQ ID NO: 18, or its complement, The use according to claim 4, comprising **Claim 17** The use according to any one of claims 4, 15 and 16, wherein the therapeutic agent comprises a cholinesterase inhibitor, vitamin E, or Ginkgo biloba. **Claim 18** The use according to claim 17, wherein the cholinesterase inhibitor comprises donepezil, tacrine, rivastigmine, galantamine, memantine / donepezil, ambenonium, or neostigmine. **Claim 19** The use according to any one of claims 5 to 9, wherein the ST6GALNAC5 missense mutant nucleic acid molecule encodes ST6GALNAC5 Val135Ala, Val45Ala, Val45Ala*, or Val135Ala*. **Claim 20** The ST6GALNAC5 missense mutant nucleic acid molecule is: A genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 or its complement, wherein the genomic nucleic acid molecule has a nucleotide sequence comprising cytosine at a position corresponding to position 176,867 of SEQ ID NO: 2, or its complement; or An mRNA molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5 or its complement, wherein the mRNA molecule has a nucleotide sequence comprising cytosine at a position corresponding to position 600 as set forth in SEQ ID NO: 11, cytosine at a position corresponding to position 639 as set forth in SEQ ID NO: 12, cytosine at a position corresponding to position 562 as set forth in SEQ ID NO: 13, cytosine at a position corresponding to position 515 as set forth in SEQ ID NO: 14, cytosine at a position corresponding to position 579 as set forth in SEQ ID NO: 15, cytosine at a position corresponding to position 416 as set forth in SEQ ID NO: 16, cytosine at a position corresponding to position 639 as set forth in SEQ ID NO: 17, or cytosine at a position corresponding to position 600 as set forth in SEQ ID NO: 18, or its complement. The use according to any one of claims 5 to 9, comprising the same. **Claim 21** The use according to any one of claims 5 to 9, wherein the ST6GALNAC5 inhibitor comprises myricetin. **Claim 22** A pharmaceutical composition for treating cognitive impairment, reduced myelin integrity, neurodegeneration, reduced gray matter / white matter contrast (GWC), or conversion from mild cognitive impairment to dementia in a patient, wherein the patient is heterozygous for an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 (ST6GALNAC5), and the pharmaceutical composition comprises, as an active ingredient, a therapeutic agent for treating or preventing cognitive impairment, reduced myelin integrity, neurodegeneration, reduced gray matter / white matter contrast (GWC), or conversion from mild cognitive impairment to dementia.

23. A pharmaceutical composition for treating cognitive impairment, reduced myelin integrity, neurodegeneration, reduced gray / white matter contrast (GWC), or conversion from mild cognitive impairment to dementia in a patient, wherein the patient is a) a reference for an α-N-acetylgalactosaminide α-2,6-sialyltransferase 5 (ST6GALNAC5) genomic nucleic acid molecule, or an ST6GALNAC5 mRNA molecule, or b) heterozygous for an ST6GALNAC5 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of ST6GALNAC5, and the pharmaceutical composition comprises an ST6GALNAC5 inhibitor as an active ingredient.