WO2011059895A1

WO2011059895A1 - Hexa mutations

Info

Publication number: WO2011059895A1
Application number: PCT/US2010/055675
Authority: WO
Inventors: Noh Jin Park; Denise Z. Salazar; Weimin Sun; Julie A. Neidich; Charles M. Strom
Original assignee: Quest Diagnostics Investments Incorporated
Priority date: 2009-11-11
Filing date: 2010-11-05
Publication date: 2011-05-19

Abstract

Provided herein are novel mutations in the HEXA nucleic acid and HEXA protein. Also provided are compositions and methods useful for diagnosing Tay-Sachs disease and identifying an individual as a carrier of Tay-Sachs disease.

Description

HEXA MUTATIONS

FIELD OF THE INVENTION

[0001] This invention relates to the field of medical diagnostics, and in particular for the detection of mutations in the HEXA gene and diagnosis of Tay-Sachs disease.

BACKGROUND OF THE INVENTION

[0002] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the invention.

[0003] Tay-Sachs disease (TSD) is an autosomal recessive, neurodegenerative disorder caused by insufficient β-hexosaminidase A (Hex A) activity, leading to excess storage of G_M2 ganglioside. Tay-Sachs disease is an inherited disease that occurs when both parents carry one or more mutations in β-hexosaminidase a gene (HEXA) and each parent transmits the defective gene to their child. A person with defective HEXA gene in one of the two copies of the gene may be perfectly healthy, but is a TSD carrier. Biological parents of a fetus or child with any form of TSD are defined as obligate carriers. Patients with the classical infant form manifest after the first few months of life. Death typically occurs between the ages of 2 and 5 years. Delayed onset (late infant, juvenile, or adult) forms occur less frequently; the later the onset, the less severe the disease. High-risk populations include Ashkenazi Jews (carrier frequency -1/30), French Canadians, Cajuns, and Pennsylvania Dutch Carrier frequency in other populations is 1/300.

[0004] Five mutations in the HEXA gene account for most of the common mutations resulting in TSD. Three mutations account for 92% to 98 % of carriers among Ashkenazi Jewish individuals (Grebner et al; Am J Hum Genet. 1991;48: 604-607; Triggs-Raine et al; N Engl J Med. 1990;323: 6-12). The most common mutation is a TATC insertion at exon 11 (1278insTATC). Transversion of G to C at the 5' splice site in intron 12 (1421+1G→C, also designated +1 IVS12 G→C) is the second most common mutation, and a glycine to serine mutation in exon 7 (G805A, also known as G269S) is the third most common mutation. The 1278insTATC and the G269S mutations are also commonly found in non- Ashkenazi Jewish populations, along with an intron 9 splice site mutation (1073 +1G>A, also termed +1 IVS9 G>A) and a 7.6 kb deletion (7.6-KB DEL, EX1). Screening is currently based on biochemical analysis of HEXA activity supplemented with molecular analysis for detection of the 5 most common HEXA gene mutations.

[0005] Biochemical assays are useful initial screening assays but do not identify the specific HEXA mutation in the patient and do not always yield definite diagnostic results. First, there is an overlapping range in which results from carriers and non-carriers overlap. As many as 10% of samples can have results in this range (Bach et al.; Am J Med Genet. 2001; 99: 70-75). Second, false-positive results occur in the serum of pregnant women and those taking oral contraceptives owing to the presence of another form of hexosaminidase i.e., HEXP (Navon et al. Clin Genet. 1973; 4: 286-287; Nitowsky et al; Am J Obstet Gynecol. 1979; 134: 642-647). HEXP, which is not heat labile, reduces the calculated percentage of HEXA, and the woman may be incorrectly classified as a carrier. To circumvent this problem, the assay can be performed using white blood cells, which lack HEXP. Testing white blood cells, though accurate, is labor-intensive and not cost-effective. Third, false-positive results (2% in Ashkenazi Jewish, 35% in other populations) occur in individuals who carry a pseudo deficiency allele (Triggs-Raine et al; Am J Hum Genet. 1992;51 : 793-801; Cao et al; Am J Hum Genet. 1993; 53; 1198-1205). There are 2 such alleles: 7390T, causing the ARG247TRP (R247W) amino acid substitution, and 7450T, causing the ARG249TRP (R249W) amino acid substitution. These alleles code for HEXA enzymes with reduced activity against the artificial MUG substrate but not against the cellular GM2 ganglioside. Thus, biochemical assay results are likely to be consistent with a TSD carrier, though the phenotype is benign. Fourth, a Bl variant phenotype can result in a false-negative test (individual classified as normal rather than as a carrier) (Kytzia et al. A. J Biol Chem. 1985; 260: 7568-7572; Rozenberg et al; J Child Neurol. 2006; 21 : 540-544). Five HEXA mutations have been identified that cause this phenotype. Their protein product has reduced activity against cellular GM2 ganglioside, but normal activity against the artificial MUG substrate. Fifth, a rare thermolabile HEXB can result in an apparent increase in HEXA activity, potentially masking a TSD carrier. Finally, biochemical assays cannot differentiate carriers of the milder juvenile/adult mutations from carriers of the more severe, infantile mutations. SUMMARY OF THE INVENTION

[0006] The present inventions are based on the identification of previously unknown mutations in the HEXA nucleic acid and protein, including the mutations shown in Table 1. The compositions and methods useful in the diagnosis and prognosis of Tay-Sachs diseases are also provided.

[0007] Provided are isolated polynucleotides encoding at least a portion of a HEXA nucleic acid encoding one or more of the following mutations: G759A, A775G, A965T, 118delT, G1292A, 1214-1215delAAinsG, 759-774dup, G5461A, C9T, C1177T, and G1393A. In one example, the polynucleotide is about 10, 15, 20, 25, 30, 50, 100, 200, or more nucleotides in length that encodes one of the foregoing mutations, and is otherwise substantially identical to the sequence showing in SEQ ID NO: 1.

[0008] The G759A mutation in HEXA gene is a silent mutation in which the amino acid valine at position 253 in the HEXA protein (SEQ ID NO: 7) is unchanged. An exemplary nucleic acid sequence of the HEXA gene having the G759A mutation is SEQ ID NO: 3. In one example, an isolated polynucleotide including G759A mutation has a nucleic acid sequence that is at least 95% identical to 20 contiguous nucleotides of SEQ ID NO: 8. The C9T is also a silent mutation resulting in no change of amino acid sequence. The A775G mutation in HEXA gene encodes a HEXA protein having a T259A substitution. An exemplary nucleic acid sequence of the HEXA gene having the A775G mutation is SEQ ID NO: 4. In one example, an isolated polynucleotide including A775G mutation has a nucleic acid sequence that is at least 95% identical to 20 contiguous nucleotides of SEQ ID NO: 9. The A965T mutation in HEXA gene encodes a HEXA protein having a D332V substitution. An exemplary nucleic acid sequence of the HEXA gene having the A965T mutation is SEQ ID NO: 5. In one example, an isolated polynucleotide including A965T mutation has a nucleic acid sequence that is at least 95% identical to 20 contiguous nucleotides of SEQ ID NO: 12. The CI 177T mutations results in a substitution of Arg to Thr at amino acid position 393 corresponding to SEQ ID NO: 7. The G 1393 A mutation results in a substitution of Asp to Asn at amino acid position 465 corresponding to SEQ ID NO: 7. The 118 delT mutation results in a premature stop codon in which only the first 98 amino acids of HEXA protein are translated. An exemplary nucleic acid sequence of the HEXA gene having the 118delT mutation is SEQ ID NO: 6. In one example, an isolated polynucleotide including 118delT mutation has a nucleic acid sequence that is at least 95% identical to 20 contiguous nucleotides of SEQ ID NO: 15. The G 1292 A mutation results in a truncated HEXA protein where the HEXA protein truncates after first 430 amino acids. An exemplary nucleic acid sequence of the HEXA gene having the G1292A mutation is SEQ ID NO: 64. In one example, an isolated polynucleotide including G1292A mutation has a nucleic acid sequence that is at least 95% identical to 20 contiguous nucleotides of SEQ ID NO: 65. The 1214- 1215delAAinsG mutation results in a truncated HEXA protein with a different C-terminal amino acids. The mutant protein includes a stretch of 17 amino acids after amino acid position 404 which are not present in the wild-type sequence. An exemplary nucleic acid sequence of the HEXA gene having the 1214-1215delAAinsG mutation is SEQ ID NO: 67. In one example, an isolated polynucleotide including 1214-1215delAAinsG mutation has a nucleic acid sequence that is at least 95% identical to 20 contiguous nucleotides of SEQ ID NO: 68. The 759-774dup mutation results in a truncated HEXA protein with a different C- terminal amino acids. The mutant protein includes a stretch of 4 amino acids after amino acid position 258 which are not present in the wild-type sequence. An exemplary nucleic acid sequence of the HEXA gene having the 759-774dup mutation is SEQ ID NO: 71. In one example, an isolated polynucleotide including 759-774dup mutation has a nucleic acid sequence that is at least 95% identical to 20 contiguous nucleotides of SEQ ID NO: 72. The G5461A mutation results in a splice variant of HEXA mRNA by disrupting the "GT" splice donor site in intron 1. The amino acid sequence of the resulting protein will differ considerably from the wild-type HEXA protein. An exemplary nucleic acid sequence of the HEXA gene having the G5461A mutation is SEQ ID NO: 75. In one example, an isolated polynucleotide including G5461A mutation has a nucleic acid sequence that is at least 95% identical to 20 contiguous nucleotides of SEQ ID NO: 76.

[0009] Also provided are isolated polypeptides corresponding to at least a portion of the HEXA protein and encoding one or more substitution mutations. In one example, the substitution mutation in the HEXA protein includes a substitution of threonine to an alanine at position 259 (T259A) corresponding to SEQ ID NO: 7. An exemplary amino acid sequence of HEXA mutant protein having T259A mutation is shown as SEQ ID NO: 10. In one example an isolated polypeptide having T259A mutation is at least 95% identical to 20 contiguous amino acids of SEQ ID NO: 1 1. In another example the substitution mutation in the HEXA protein includes a substitution of aspartic acid to valine at position 332 (D332V) corresponding to SEQ ID NO: 7. An exemplary amino acid sequence of HEXA mutant protein having D332V mutation is shown as SEQ ID NO: 13. In one example the isolated polypeptide having D332V mutation is at least 95% identical to 20 contiguous amino acids of SEQ ID NO: 14. In another example, the isolated polypeptides corresponding to at least a portion of the HEXA protein may include both substitution mutations T259A and D332V.

[0010] Additionally, HEXA truncation mutants are also provided. In one example, the truncated protein encoded by HEXA gene having 1 18delT mutation has first 98 amino acids of SEQ ID NO: 7. In one example the isolated polypeptide having a portion of the truncated protein encoded by HEXA gene having 1 18delT mutation is at least 95% identical to 20 contiguous amino acids of SEQ ID NO: 16. In one example, the truncated protein encoded by HEXA nucleic acid having G1292A has first 430 amino acids of SEQ ID NO: 7. An exemplary amino acid sequence of the truncated HEXA protein is SEQ ID NO: 66. In one example the isolated polypeptide having a portion of the truncated protein encoded by HEXA gene having G 1292 A mutation is at least 95% identical to 20 contiguous amino acids of SEQ ID NO: 66. In another example, the truncated protein encoded by HEXA nucleic acid having 1214-1215delAAinsG has a different C-terminus. An exemplary amino acid sequence of the truncated HEXA protein encoded by HEXA gene having 1214-1215delAAinsG is SEQ ID NO: 69. In one example the isolated polypeptide having a portion of the truncated protein encoded by HEXA gene having 1214-1215delAAinsG mutation is at least 95% identical to 20 contiguous amino acids of SEQ ID NO: 70. In another example, the truncated protein encoded by HEXA nucleic acids having 759-774dup has a different C-terminus. An exemplary amino acid sequence of the truncated HEXA protein encoded by HEXA gene having 759-774dup is SEQ ID NO: 73. In one example the isolated polypeptide having a portion of the truncated protein encoded by HEXA gene having 759-774dup mutation is at least 95% identical to 20 contiguous amino acids of SEQ ID NO: 74.

[0011] Also provided are antibodies that specifically bind to the polypeptides having at least a portion of the HEXA protein in which the polypeptides include one or more mutations in HEXA protein such as T259A, D332V, truncated proteins encoded by 1 18 delT, G1292A, 1214-1215delAAinsG, 759-774dup mutations, and the protein encoded by G5461A mutation. In one example, the antibodies can specifically bind to polypeptides having an amino acid sequence for example, SEQ ID NOS: 11, 14, 16, 70 or 74.

[0012] Also provided are methods of determining whether a human is predisposed to Tay- Sachs disease. The method includes determining whether the human is homozygous for one or more mutations selected from the group consisting of A775G, A965T, 1 18delT, G1292A, 1214-1215delAAinsG, 759-774dup, G5461A, C1177T, and G 1393 A in the beta- hexosaminidase A (HEXA) gene and identifying the human as having a predisposition to Tay-Sachs disease when the human is homozygous.

[0013] Also provided are methods for determining the Tay-Sachs disease status of a human. The methods include (a) determining the presence or absence of one or more mutations such as A775G, A965T, 118delT, G1292A, 1214-1215delAAinsG, 759-774dup, G5461A, CI 177T, and G1393A in both alleles of the beta-hexosaminidase A (HEXA) gene of the human. The human is identified as having Tay-Sachs disease or being predisposed to Tay- Sachs disease when the human is homozygous for one or more of these mutations in the HEXA gene. The human is identified as being Tay-Sachs disease carrier when the human is heterozygous for one or more of these mutations in the HEXA gene. The human is identified as having no predisposition or carrier status caused by one or more of these mutations when one or more of these mutations are absent from both alleles of the HEXA gene.

[0014] Also provided are methods of identifying a human with an increased likelihood of having an offspring predisposed to Tay-Sachs disease. The methods include determining the presence of one or more mutations selected from the group consisting of A775G, A965T, 118delT, G1292A, 1214-1215delAAinsG, 759-774dup, G5461A, C1177T, and G1393A of the beta-hexosaminidase A (HEXA) gene and identifying the human as having an increased likelihood of having an offspring predisposed to Tay-Sachs disease when one or more of these mutations in HEXA gene are present in at least one allele.

[0015] In one example of the above methods, assessing HEXA nucleic acid is done by one or more methods such as sequencing, oligonucleotide probe hybridization, single nucleotide primer extension, or allele-specific primer extension. In another example, the HEXA gene is assessed for the presence or absence of mutations using genomic DNA. In another example, the HEXA gene is assessed for the presence or absence of mutations using cDNA.

[0016] In one example, the above methods further includes assessing HEXA nucleic acid from the sample for the presence or absence of one or more common HEXA mutations such as 1278insTATC, +1IVS 12 G→C, G805A, +1 IVS9G→A, or 7.6 KB DEL EX1 (Grebner et al; Am J Hum Genet. 1991;48: 604-607; Triggs-Raine et al; N Engl J Med. 1990;323: 6-12). The presence of these mutations together with one or more mutations such as A775G, A965T, 118delT, G1292A, 1214-1215delAAinsG, 759-774dup, or G5461A is indicative of the severity of the Tay-Sachs disease or the increased likelihood of having an offspring predisposed to Tay-Sachs disease.

[0017] In one example, the individual is homozygous for the mutations such as G759A, A775G, A965T, 118delT, G1292A, 1214-1215delAAinsG, 759-774dup, G5461A, C9T, CI 177T, or G1393A. In another example, the individual is heterozygous for the mutations such as G759A, A775G, A965T, 118delT, G1292A, 1214-1215delAAinsG, 759-774dup, G5461A, C9T, CI 177T, or G1393A.

[0018] Also provided are kits for assessing the HEXA nucleic acid from the sample for the presence or absence of one or more mutations such as A775G, A965T, 118delT, G1292A, 1214-1215delAAinsG, 759-774dup, G5461A, C9T, CI 177T, or G1393A. The kit may include one or more primers or probes that specifically hybridize to HEXA nucleic acid. In one example, the primers may be allele-specific primers. In one example, the primers may be suitable for diagnostic primer extension reactions including single base primer extension (e.g. SNaPshot™ primers). In one example the primers or probes may be detectably labeled. Optionally, kits containing SNaPshot™ primers further contain one or more labeled nucleotides (e.g., ddNTPs). Also provided are antibodies and kits containing antibodies that specifically bind to the a portion of the HEXA protein having the T259A, D332V, R393T, or D465N amino acid substitutions or the new C-terminus of truncated HEXA proteins encoded by HEXA 1214-1215delAAinsG, 759-774dup nucleic acids. In one example, the antibodies can specifically bind to polypeptides having an amino acid sequence for example, SEQ ID NOS: 11, 14, 16, 70 or 74. [0019] As used herein the terms "diagnose" or "diagnosis" or "diagnosing" refer to distinguishing or identifying a disease, syndrome or condition or distinguishing or identifying a person having a particular disease, syndrome or condition.

[0020] The term "carrier" refers to a person who may have one or more mutations in HEXA nucleic acid but is asymptomatic for Tay-Sachs or a Tay Sachs-related condition. Most commonly, carriers are heterozygous for a HEXA gene mutation that would cause disease in a homozygote.

[0021] The term "sample" or "patient sample" is meant to include biological samples such as cells, tissues and bodily fluids. "Bodily fluids" may include, but are not limited to, blood, serum, plasma, saliva, cerebral spinal fluid, pleural fluid, tears, lactal duct fluid, lymph, sputum, urine, amniotic fluid, and semen. A sample may include a bodily fluid that is "acellular." An "acellular bodily fluid" includes less than about 1% (w/w) whole cellular material. Plasma or serums are examples of acellular bodily fluids.

[0022] "Heterozygous" refers to having different alleles at one or more genetic loci in homologous chromosome segments. As used herein "heterozygous" may also refer to a sample, a cell, a cell population, or an organism in which different alleles at one or more genetic loci may be detected. Heterozygous samples may also be determined via methods known in the art such as, for example, nucleic acid sequencing. For example, if a sequencing electropherogram shows two peaks at a single locus and both peaks are roughly the same size, the sample may be characterized as heterozygous. Or, if one peak is smaller than another, but is at least about 25% the size of the larger peak, the sample may be characterized as heterozygous. In some embodiments, the smaller peak is at least about 15% of the larger peak. In other embodiments, the smaller peak is at least about 10% of the larger peak. In other embodiments, the smaller peak is at least about 5% of the larger peak. In other embodiments, a minimal amount of the smaller peak is detected.

[0023] As used herein, "homozygous" refers to having identical alleles at one or more genetic loci in homologous chromosome segments. "Homozygous" may also refer to a sample, a cell, a cell population, or an organism in which the same alleles at one or more genetic loci may be detected. Homozygous samples may be determined via methods known in the art, such as, for example, nucleic acid sequencing. For example, if a sequencing electropherogram shows a single peak at a particular locus, the sample may be termed "homozygous" with respect to that locus.

[0024] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, which may be single or double stranded, or partially double stranded and represent the sense or antisense strand. A nucleic acid may include DNA or RNA, and may be of natural or synthetic origin and may contain deoxyribonucleotides, ribonucleotides, or nucleotide analogs in any combination. Nucleic acid may comprise a detectable label. Although a sequence of the nucleic acids may be shown in the form of DNA, a person of ordinary skill in the art recognizes that the corresponding RNA sequence will have a similar sequence with the thymine being replaced by uracil i.e. "t" with "u".

[0025] Non-limiting examples of nucleic acid include a gene or gene fragment, genomic DNA, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant nucleic acid, branched nucleic acid, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, synthetic nucleic acid, nucleic acid probes and primers. Nucleic acid may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. A nucleic acid may be modified such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of chemical entities for attaching the polynucleotide to other molecules such as proteins, metal ions, labeling components, other nucleic acid, or a solid support. Nucleic acid may include nucleic acid that has been amplified (e.g., using polymerase chain reaction).

[0026] A "substantially pure" nucleic acid or a polypeptide, is one that represents more than 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, or 99% of the nucleic acid or polypeptide in a sample. The nucleic acid or polypeptide sample may exist in solution or as a dry preparation.

[0027] The term "substantially identical" in the context of polynucleotide and a reference polypeptide and means the polypeptide is at least 65%, at least 70%> or at least 75%, at least 80% or at least 85%, at least 90%, at least 95%, at least 99% identical to or identical to the reference polypeptide.

[0028] By "isolated", when referring to a nucleic acid (e.g., an oligonucleotide such as RNA, DNA, or a mixed polymer) or a polypeptide is meant a naturally-occurring nucleic acid or a polypeptide (or fragments thereof) that is substantially free from the naturally-occurring molecules and cellular components with which it is naturally associated. For example, any nucleic acid or a polypeptide that has been produced synthetically is considered to be isolated. Nucleic acids that are recombinantly expressed, cloned, produced by a primer extension reaction (e.g., PCR), or otherwise excised from a genome are also considered to be isolated. Similarly, polypeptides that are recombinantly produced are also considered to be isolated. However, cDNA preparations, and the like, are not considered isolated because they do not contain naturally-occurring molecules.

[0029] As used herein, "a portion of in the context of a polynucleotide means that the polynucleotide is at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 1000 nucleotides or more in length.

[0030] As used herein, "a portion of in the context of a polypeptide means at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500 amino acids or more.

[0031] "Specific hybridization" is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after any subsequent washing steps. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may occur, for example, at 65°C in the presence of about 6^XSSC. Stringency of hybridization may be expressed, in part, with reference to the temperature under which the wash steps are carried out. Such temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Equations for calculating Tm and conditions for nucleic acid hybridization are known in the art. [0032] "Stringent hybridization conditions" as used herein refers to hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5x SSC, 50 mM NaH₂P0₄, pH 6.8, 0.5%> SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5x Denhart's solution at 42° C. overnight; washing with 2x SSC, 0.1 % SDS at 45° C; and washing with 0.2x SSC, 0.1% SDS at 45° C. In another example, stringent hybridization conditions should not allow for hybridization of two nucleic acids which differ over a stretch of 20 contiguous nucleotides by more than two bases.

[0033] By "complement" is meant the complementary sequence to a nucleic acid according to standard Watson/Crick pairing rules. A complement sequence can also be a sequence of RNA complementary to the DNA sequence or its complement sequence, and can also be a cDNA.

[0034] By "substantially complementary" is meant that two sequences hybridize under stringent hybridization conditions. The skilled artisan will understand that substantially complementary sequences need not hybridize along their entire length.

[0035] Oligonucleotides used as primers or probes for specifically amplifying (i.e., amplifying a particular target nucleic acid sequence) or specifically detecting (i.e., detecting a particular target nucleic acid sequence) a target nucleic acid generally are capable of specifically hybridizing to the target nucleic acid.

[0036] The term "oligonucleotide" is understood to be a molecule that has a sequence of bases on a backbone comprised mainly of identical monomer units at defined intervals. The bases are arranged on the backbone in such a way that they can enter into a bond with a nucleic acid having a sequence of bases that are complementary to the bases of the oligonucleotide. The most common oligonucleotides have a backbone of sugar phosphate units. A distinction may be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group in this position. Oligonucleotides also may include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group. Oligonucleotides of the method which function as primers or probes are generally at least about 10-15 nucleotides long and more preferably at least about 15 to 25 nucleotides long, although shorter or longer oligonucleotides may be used in the method. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The

oligonucleotide may be generated in any manner, including, for example, chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof. The oligonucleotide may be modified. For example, the oligonucleotide may be labeled with an agent that produces a detectable signal (e.g., a fluorophore).

[0037] "Primer" refers to an oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated (e.g., primer extension associated with an application such as PCR). An oligonucleotide "primer" may occur naturally, as in a purified restriction digest or may be produced synthetically. "Primer" refers to a sequence of nucleic acid, preferably DNA that hybridizes to a substantially complementary target sequence and is recognized by DNA polymerase to begin DNA replication.

[0038] A "probe" refers to an oligonucleotide that interacts with a target nucleic acid via hybridization. A probe may be fully complementary to a target nucleic acid sequence or partially complementary. The level of complementarity will depend on many factors based, in general, on the function of the probe. A probe or probes can be used, for example to detect the presence or absence of a mutation in a nucleic acid sequence by virtue of the sequence characteristics of the target. Probes can be labeled or unlabeled, or modified in any of a number of ways well known in the art. A probe may specifically hybridize to a target nucleic acid.

[0039] As used herein, the term "about" means in quantitative terms, plus or minus 10%.

BRIEF DESCRIPTION OF THE FIGURES

[0040] FIG. 1 shows a picture of agarose gel electrophoresis. All 14 HEXA gene exons are separately amplified via PCR, and PCR amplicons were visualized by agarose gel electrophoresis. Lane M: Marker, Lane 1-14: amplicons of HEXA exons 1-14 respectively.

[0041] FIG. 2 is a representative sequencing chromatograms identifying G759A mutation. FIG. 2 discloses SEQ ID NOs: 26-36. [0042] FIG. 3 is a representative sequencing chromatograms identifying A775G mutation. FIG. 3 discloses SEQ ID NOS: 37-42.

[0043] FIG. 4 is a representative sequencing chromatograms identifying A965T mutation. FIG. 4 discloses SEQ ID NOS: 43-52.

[0044] FIG. 5 is a representative sequencing chromatograms identifying 118 delT mutation. FIG. 5 discloses SEQ ID NOS: 53-63, respectively.

[0045] FIG. 6 is a representative sequencing chromatograms identifying G 1292 A mutation. Fig. 6 discloses SEQ ID NOS 89-95 respectively in the order of appearance.

[0046] FIG. 7 is a representative sequencing chromatograms identifying 1214- 1215delAAinsG mutation. Fig. 7 discloses SEQ ID NOS: 96-106 respectively in the order of appearance.

[0047] FIG. 8 is a representative sequencing chromatograms identifying 759-774dup mutation. Fig. 8 discloses SEQ ID NOS: 107-125 respectively in the order of appearance.

[0048] FIG. 9 is a representative sequencing chromatograms identifying G5461A mutation. Fig. 9 discloses SEQ ID NOS 126-144 respectively in the order of appearance.

DETAILED DESCRIPTION OF THE INVENTION

[0049] Mutations in the HEXA gene which have been associated with Tay-Sachs disease are provided.

[0050] HEXA Gene and HEXA Protein

[0051] Beta-hexosaminidase A (HEXA) enzyme is a heterodimer of a and β subunits encoded by the human HEXA and HEXB genes respectively. Several mutations in HEXA gene are known to affect the Hex A enzymatic activity and often associated with Tay-Sachs disease. Human HEXA gene is located in chromosome 15 (15q23-q24). Exemplary sequences of human chromosome 15 include but are not limited to GenBank Accession numbers: NW_001838218, NW_925884, and NT_010194. Exemplary HEXA genomic nucleic acid includes but not limited to GenBank Accession number NG 009017. These sequences are incorporated herein by reference. Sequence of reference HEXA genomic nucleic acid is listed as SEQ ID NO: 1.

[0052] Full length and partial sequences of HEXA mRNA are known. Exemplary HEXA mRNA sequences include but not limited to GenBank Accession numbers: NM 000520, AK222502, BC084537, BC018927, BC021030, BC001138, AH007351, S76984, S76982, S77043, S61298, J04178, and M13520. These sequences are incorporated herein by reference. One reference sequence for HEXA cDNA is provided as SEQ ID NO: 2.

[0053] Several variations of HEXA protein sequence are known in the art including sequences of the full length protein and fragments. Exemplary HEXA protein sequences include but not limited to the NCBI protein database accession numbers: NP 000511, P06865, AAD13932, EAW77902, AAH21030, AAB33748, AAA51827, P06865, and AAB00965. An exemplary amino acid sequence of full length HEXA protein is listed in SEQ ID NO: 7.

[0054] HEXA Mutations

[0055] Mutations in the HEXA gene are may be associated with Tay-Sachs disease. Common mutations in the HEXA gene include: 1278insTATC, +1IVS12 G→C, G805A, +1 IVS9G→A, and 7.6 KB DEL, EX1 (Grebner et al. Am J Hum Genet. 1991; 48: 604-607; Triggs-Raine et al. N Engl J Med. 1990; 323:6-12).

[0056] Provided herein are several mutations identified in the HEXA gene and are shown below.

[0057] Table 1. HEXA Gene Mutations

ND: Not determined

[0058] G759A

[0059] A substitution of G to A in HEXA gene at nucleotide position 759 of SEQ ID NO: 2 results in a silent mutation, leaving the valine at position 253 of SEQ ID NO: 7 unchanged. An exemplary HEXA nucleic acid sequence encoding the G759A mutation is provided in SEQ ID NO: 3. In one example, the G759A HEXA mutation is identified by assessing a target sequence including the sequence of SEQ ID NO: 8.

5'-CAGCACAGGATGTGAAGGAGGTCATTGAATACGCACGGCTCCGGGGTA

TCCGTGTACTTGCAGAGTTTGACACTCCTGGCCACAC-3' (SEQ ID NO: 8)

[0060] The amino acid sequence of the HEXA protein encoded by the polynucleotide of SEQ ID NO: 3 is provided as SEQ ID NO: 7. [0061] A775G

[0062] A substitution of A to G in HEXA gene at nucleotide position 775 ("A775G") in the genomic HEXA sequence of SEQ ID NO: 2 results in the T259A substitution in the HEXA protein (SEQ ID NO: 10). An exemplary nucleic acid sequence of HEXA A775G is listed as SEQ ID NO: 4. In one example, the A775G mutation is identified by assessing a target sequence including the sequence of SEQ ID NO: 9.

5'-GAATACGCACGGCTCCGGGGTATCCGTGTGCTTGCAGAGTTTGAC

GCTCCTGGCCACACTTTGTCCTGGGGACCAG-3' (SEQ ID NO: 9)

[0063] The effect of T259A mutation on HEXA protein structure is assessed by the Polypen and SHIFT software essentially as described by Ng et al. (Ann Rev Genom Hum Genet 2006; 7: 61-80) and Ramensky et al. (Nucleic Acids Res 2002; 30: 3894-3900) and may be used to assess the effect of any of the protein mutations described herein. The T259A mutation had minimal effect on the HEXA protein structure. However, the HEXA enzyme activity was lower than the normal range in sample with only A775G mutation present (Table 1). Thus, this mutation contributes to lower HEXA activity and is involved in Tay-Sachs disease.

[0064] Also provided are methods for assessing the T259A HEXA protein, including antibodies that specifically bind to the mutated protein. One HEXA protein fragment that may be assessed or used to generate useful antibodies in provided as SEQ ID NO: 11 which shows amino acids 241-270 of SEQ ID NO: 7 and having T259A mutation.

EVIEYARLRGIRVLAEFDAPGHTLSWGPGI (SEQ ID NO: 11)

[0065] A965T

[0066] A substitution of A to T in HEXA gene at nucleotide position 965 ("A965G") in the HEXA nucleic acid sequence of SEQ ID NO: 2 results in the D322V substitution in the HEXA protein (SEQ ID NO: 13). An exemplary nucleic acid sequence of HEXA A965G is listed as SEQ ID NO: 5. In one example, the A965G mutation is identified by assessing a target sequence including the sequence of SEQ ID NO: 12.

5 ' -T AGAAGTC AGCTCTGTCTTCCC AGATTTTTATCTTC ATCTTGGAGGA

GTTGAGGTTGATTTCACCTGCTG-3 ' (SEQ ID NO: 12)

[0067] The D322V mutation altered the HEXA protein folding and affected the ligand binding site of HEXA protein. The HEXA enzyme activity was lower than the normal range in sample with only A965T mutation present (Table 1). Thus, this mutation contributes to lower HEXA activity and is involved in Tay-Sachs disease.

[0068] Also provided are methods for assessing the D322V HEXA protein, including antibodies that specifically bind to the mutated protein. One HEXA protein fragment that may be assessed or used to generate useful antibodies in provided as SEQ ID NO: 14 which shows amino acids 311-340 of SEQ ID NO: 7 and having D322V mutation.

VFPDFYLHLGGVEVDFTCWKSNPEIQDFMR (SEQ ID NO: 14)

[0069] 118 delT

[0070] A deletion of a single nucleotide "T" in HEXA gene at nucleotide position 118 of the genomic HEXA sequence of SEQ ID NO: 2 results in a frameshift mutation leading to a truncated HEXA protein. Exemplary nucleic acid sequence of HEXA 118 delT is listed as SEQ ID NO: 6. In one example, the 118delT mutation is identified by assessing a target sequence including the sequence of SEQ ID NO: 15.

5'-GCCTCAGAACTTCCAAACCTCCGACCAGCGCTACGTCCTTACCCG

AACAACTTTCAATTCCAGTACGATGTCAGCTCGGCCGCGC-3' (SEQ ID NO: 15)

[0071] The frameshift resulting from the 118delT mutation in HEXA nucleic acid produces a truncated HEXA protein such that the HEXA protein truncates after first 98 amino acids from the N-terminus. An exemplary sequence of which is provided as SEQ ID NO: 16. MTSSRLWFSLLLAAAFAGRATALWPWPQNFQTSDQRYVLTRTTFNSSTMSARPRSP AAQSSTRPSSAIVTCFSVPGLGPVLTSQGNGIHWRRMCWLSL (SEQ ID NO: 16)

[0072] The mutation significantly alters the HEXA protein folding. The HEXA enzyme activity was lower than the normal range when only 118 delT mutation present (Table 1). Thus, this mutation contributes to lower HEXA activity and is involved in Tay-Sachs disease.

[0073] G 1292 A

[0074] Nucleic acid samples obtained from obligate carriers of Tay-Sachs disease indicated a substitution of G to A in HEXA gene at nucleotide position 1292 ("G1292A") in the HEXA nucleic acid sequence of SEQ ID NO: 2. The G 1292 A mutation results in a truncated of HEXA protein. An exemplary nucleic acid sequence of HEXA G 1292 A is listed as SEQ ID NO: 64. In one example, the G1292A mutation is identified by assessing a target sequence including the sequence of SEQ ID NO: 65.

5'-TGCCCCCTGGTACCTGAACCGTATATCCTATGGCCCTGACTAGA

AGGATTTCTACGTAGT GGAACCCCTGGCATTTGAAG-3 ' (SEQ ID NO: 65)

[0075] The amino acid sequence of the HEXA protein encoded by the polynucleotide of SEQ ID NO: 64 truncates after amino acid 430 of SEQ ID NO: 7. An exemplary amino acid sequence of the truncated protein is provided as SEQ ID NO: 66.

[0076] The truncation of HEXA protein is likely to disrupt the HEXA activity and thus is likely to be involved in Tay-Sachs disease.

[0077] 1214-1215delAAinsG

[0078] Nucleic acid samples obtained from obligate carriers of Tay-Sachs disease indicated a deletion of dinucleotide "AA" and an insertion of "G" in place of the deleted dinucleotide at nucleotide position 1214-1215 ("1214-1215delAAinsG") in the HEXA sequence of SEQ ID NO: 2. The mutation results in a truncated of HEXA protein with a different C-terminal amino acid sequence than the wild-type protein. An exemplary nucleic acid sequence of HEXA 1214-1215delAAinsG is listed as SEQ ID NO: 67. In one example, the 1214- 1215delAAinsG mutation is identified by assessing a target sequence including the sequence of SEQ ID NO: 68.

5'-AGTGAACTATATGAAGGAGCTGGGCTGGTCACCAAGGCCGGCTT

CCGGGCCCTTCTCTCTGCCCCCTGG-3' (SEQ ID NO: 68)

[0079] Exemplary amino acid sequence of the truncated HEXA protein with a different c- terminus encoded by the polynucleotide of SEQ ID NO: 67 is provided as SEQ ID NO: 69. The mutated HEXA protein has 17 new amino acids at the C-terminus after amino acid 404 corresponding to SEQ ID NO: 7. An exemplary amino acid sequence of the new C-terminus of the protein is shown below.

GWSPRPASGPFSLPPGT (SEQ ID NO: 70)

[0080] Also provided are methods for assessing the mutated HEXA protein, including antibodies that specifically bind to the mutated protein. One HEXA protein fragment that may be assessed or used to generate useful antibodies is SEQ ID NO: 70.

[0081] The truncation of HEXA protein and the alteration of the C-terminus is likely to disrupt the HEXA activity and thus is likely to be involved in Tay-Sachs disease.

[0082] 759-774 dup

[0083] Nucleic acid samples obtained from obligate carriers of Tay-Sachs disease indicated a duplication of 16 nucleotides corresponding to nucleotides 759-774 ("759-774 dup") in the HEXA nucleic acid sequence of SEQ ID NO: 2. The mutation results in a truncated HEXA protein with a different C-terminal amino acid sequence than the wild-type protein. An exemplary nucleic acid sequence of HEXA 759-774dup is listed as SEQ ID NO: 71. In one example, the HEXA 759-774dup mutation is identified by assessing a target sequence including the sequence of SEQ ID NO: 72.

5'-GATGTGAAGGAGGTCATTGAATACGCACGGCTCCGGGGTATCCGTGT

GCTTGCAGAGTTTGACGCTTGCAGAGTTTGACACTCCTGGCCACACTTTGTCCTG GGG-3' (SEQ ID NO: 72)

[0084] Exemplary amino acid sequence of the truncated HEXA protein with a different C- terminus encoded by the polynucleotide of SEQ ID NO: 71 is provided as SEQ ID NO: 73. The mutated HEXA protein has 4 new amino acids "ACRV" (SEQ ID NO: 145) at the C- terminus after amino acid 258 corresponding to SEQ ID NO: 7. An exemplary amino acid sequence comprising the new C-terminus of the protein is shown below.

RGIRVLAEFDACRV (SEQ ID NO: 74)

[0085] The truncation of HEXA protein and the alteration of the C-terminus is likely to disrupt the HEXA activity and thus is likely to be involved in Tay-Sachs disease.

[0086] Also provided are methods for assessing the mutated HEXA protein, including antibodies that specifically bind to the mutated protein. One HEXA protein fragment that may be assessed or used to generate useful antibodies is SEQ ID NO: 74.

[0087] G5461A

[0088] Nucleic acid samples obtained from obligate carriers of Tay-Sachs disease indicated a substitution of G to A in HEXA genomic nucleic acid in intron 1 at nucleotide position 5461 ("G5461A") corresponding to SEQ ID NO: 1. The mutation results in a splice variant of HEXA mRNA. This mutation disrupts the "GT" splice donor site. As a result some other cryptic splice site within intron 1 or further downstream will be utilized. The resulting mRNA due to such altered splicing will comprise HEXA exon 1 and a downstream sequence that will vary considerably from the normal transcript. An exemplary nucleic acid sequence of G5461A is listed as SEQ ID NO: 75.

[0089] In one example, the G5461A mutation is identified by assessing a target sequence including the sequence of SEQ ID NO: 76.

5'-ccgggtcttg gccccgtcct tacctcacag atgagtcgga cttgtcccgc cctgttcctg -3' (SEQ ID NO: 76)

[0090] The protein encoded by the spliced variant will differ considerably in amino acid sequence with respect to wild-type HEXA protein. The mutation is likely to disrupt the HEXA activity and thus is likely to be involved in Tay-Sachs disease. [0091] C9T

[0092] The substitution of 'C to ' at position 9 corresponding to SEQ ID NO: 2 is a silent mutation resulting in no change of amino acid.

[0093] C1177T

[0094] The substitution of 'C to 'T' at position 1177 corresponding to SEQ ID NO: 2 results in a change of amino acid from Arg to Thr at position 393 corresponding to SEQ ID NO: 7.

[0095] G1393A

[0096] The substitution of 'G' to 'A' at position 1393 corresponding to SEQ ID NO: 2 results in a change of amino acid from Asp to Asn at position 465 corresponding to SEQ ID NO: 7.

[0097] Biological Sample Collection and Preparation

[0098] The methods and compositions of this invention may be used to detect mutations in the HEXA nucleic acid and/or HEXA protein using a biological sample obtained from an individual. The nucleic acid (DNA or RNA) may be isolated from the sample according to any methods well known to those of skill in the art. If necessary the sample may be collected or concentrated by centrifugation and the like. The cells of the sample may be subjected to lysis, such as by treatments with enzymes, heat, surfactants, ultrasonication, or a combination thereof. The lysis treatment is performed in order to obtain a sufficient amount of nucleic acid derived from the individual's cells to detect using polymerase chain reaction.

Alternatively, mutations in the HEXA nucleic acid may be detected using an acellular bodily fluid according to the methods described in U.S. Patent Application 11/408,241 (Publication No. US 2007-0248961), hereby incorporated by reference. Any liquid or solid material believed to contain HEXA nucleic acids can be an appropriate sample. In one example, the sample is whole blood. In another example, the sample is leukocytes. In one example, the sample is obtained from an individual who is suspected of having a disease, or a genetic abnormality. In another example the sample is obtained from a healthy individual who is assumed of having no disease, or a genetic abnormality. In preferred embodiments, the sample is obtained from Tay-Sachs disease patients. In another example, the sample is obtained from carriers of Tay-Sachs disease.

[0099] In one embodiment, the nucleic acid may be mRNA, cDNA generated from mRNA, or total RNA. RNA may be isolated from cells or tissue samples using standard techniques, see, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989), Cold Spring Harbor Press, Plainview, NY. Reagents and kits for isolating RNA from any biological sample such as whole blood, plasma, serum, buffy coat, bone marrow, other body fluids, lymphocytes, cultured cells, tissue, and forensic specimens are commercially available e.g., RNeasy Protect Mini kit, RNeasy Protect Cell Mini kit, QIAamp RNA Blood Mini kit, RNeasy Protect Saliva Mini kit, Paxgene Blood RNA kit from Qiagen; MELT™,

RNaqueous®, ToTALLY RNA™, RiboPure™-Blood, Poly(A)Purist™ from Applied Biosystems; TRIZOL® reagent, Dynabeads® mRNA direct kit from Invitrogen.

[0100] Nucleic Acid Extraction

[0101] The nucleic acid to be amplified may be from a biological sample such as an organism, cell culture, tissue sample, and the like. The biological sample can be from a subject which includes any animal, preferably a mammal. A preferred subject is a human, which may be a patient presenting to a medical provider for diagnosis or treatment of a disease. The biological sample may be obtained from a stage of life such as a fetus, young adult, adult, and the like. Particularly preferred subjects are humans being tested for the existence of a HEXA mutation.

[0102] Various methods of extraction are suitable for isolating the DNA or RNA. Suitable methods include phenol and chloroform extraction. See Maniatis et al., Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press, page 16.54 (1989). Numerous commercial kits also yield suitable DNA and RNA including, but not limited to, QIAamp™ mini blood kit, Agencourt Genfind™, Roche Cobas® Roche MagNA Pure® or

phenol: chloroform extraction using Eppendorf Phase Lock Gels®, and the NucliSens extraction kit (Biomerieux, Marcy l'Etoile, France). In one example, DNA may be extracted using Gentra Autopure LS (Minneapolis, MN) using manufacturer's protocol. In some examples, DNA can be extracted using Qiagen Biorobot 9604 platform using MegAttract® DNA blood M96 kit (Valencia, CA).

[0103] Nucleic Acid Amplification

[0104] Nucleic acids may be amplified by various methods known to the skilled artisan. Nucleic acid amplification may be linear or exponential. Amplification is generally carried out using polymerase chain reaction (PCR) technologies known in the art. See e.g., Mullis and Faloona, Methods Enzymol. (1987), 155:335, U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,800,159.

[0105] Alternative methods to PCR include for example, isothermal amplification methods, rolling circle methods, Hot-start PCR, real-time PCR, Allele-specific PCR, Assembly PCR or Polymerase Cycling Assembly (PCA), Asymmetric PCR, Colony PCR, Emulsion PCR, Fast PCR, Real-Time PCR, nucleic acid ligation, Gap Ligation Chain Reaction (Gap LCR), Ligation-mediated PCR, Multiplex Ligation-dependent Probe Amplification, (MLPA), Gap Extension Ligation PCR (GEXL-PCR), quantitative PCR (Q-PCR), Quantitative real-time PCR (QRT-PCR), multiplex PCR, Helicase-dependent amplification, Intersequence-specific (ISSR) PCR, Inverse PCR, Linear-After-The-Exponential-PCR (LATE-PCR), Methylation- specific PCR (MSP), Nested PCR, Overlap-extension PCR, PAN-AC assay, Reverse

Transcription PCR (RT-PCR), Rapid Amplification of cDNA Ends (RACE PCR), Single molecule amplification PCR (SMA PCR), Thermal asymmetric interlaced PCR (TAIL-PCR), Touchdown PCR, long PCR, nucleic acid sequencing (including DNA sequencing and RNA sequencing), transcription, reverse transcription, duplication, DNA or RNA ligation, and other nucleic acid extension reactions known in the art. The skilled artisan will understand that other methods may be used either in place of, or together with, PCR methods, including enzymatic replication reactions developed in the future. See, e.g., Saiki, "Amplification of Genomic DNA" in PCR Protocols, Innis et al, eds., Academic Press, San Diego, CA, 13-20 (1990); Wharam, et al, 29(11) Nucleic Acids Res, E54-E54 (2001); Hafner, et al, 30(4) Biotechniques, 852-6, 858, 860 passim (2001). [0106] Nucleic Acid Detection

[0107] Nucleic acids can be detected by any of a number of methods well-known in the art such as gel electrophoresis, column chromatography, hybridization with a probe, or sequencing. Detectable labels can be used to identify the probe hybridized to a genomic nucleic acid or reference nucleic acid. Detectable labels include but are not limited to

32 33 35 3 14 125 131

fluorophores, isotopes (e.g., P, P, S, H, C, I, I), electron-dense reagents (e.g. , gold, silver), nanoparticles, enzymes commonly used in an ELISA (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminiscent compound, colorimetric labels (e.g., colloidal gold), magnetic labels (e.g., Dynabeads™), biotin, digoxigenin, haptens, proteins for which antisera or monoclonal antibodies are available, ligands, hormones, oligonucleotides capable of forming a complex with the corresponding oligonucleotide complement.

[0108] Mutation in a nucleic acid such as in HEXA nucleic acid can be detected by various methods known in the art. Exemplary methods include but are not limited to sequencing, allele-specific PCR, hybridization of specific probes, single-base primer extension assay for example, SNaPshot® assay, detection by size such as by capillary electrophoresis or column chromato graphy .

[0109] Detection of nucleic acid by real-time PCR assays

[0110] In one example, detection of a HEXA nucleic acid with or without mutation, such as is performed using the TaqMan® assay, which is also known as the 5' nuclease assay (U.S. Pat. Nos. 5,210,015 and 5,538,848). The TaqMan® assay detects the accumulation of a specific amplified product during PCR. The TaqMan® assay utilizes an oligonucleotide probe labeled with a fluorescent reporter dye and a quencher dye. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye. The DNA polymerase cleaves the probe between the reporter dye and the quencher dye only if the probe hybridizes to the target SNP-containing template which is amplified during PCR, and the probe is designed to hybridize to the target SNP site only if a particular SNP allele is present. [0111] TaqMan® primer and probe sequences can readily be determined using the variant and associated nucleic acid sequence information provided herein. A number of computer programs, such as Primer Express (Applied Biosystems, Foster City, Calif), can be used to rapidly obtain optimal primer/probe sets. Modifications of the TaqMan® assay well known in the art such as the use of Molecular Beacon probes (U.S. Pat. Nos. 5,118,801 and

5,312,728) and other variant formats (U.S. Pat. Nos. 5,866,336 and 6,117,635).

[0112] Scorpion™ probes are highly sensitive, sequence-specific, bi-labeled fluorescent probe/primer hybrids, designed for real-time quantitative PCR (Thelwell et al. Nucleic Acids Res. 2000; 28(19): 3752-61). The Scorpion primer carries a Scorpion probe element at the 5' end. The probe is a self-complementary stem sequence with a fluorophore at one end and a quencher at the other. The Scorpion primer sequence is modified at the 5'end. It contains a PCR blocker at the start of the hairpin loop (e.g. HEG monomers can be added as blocking agent). After one cycle of PCR extension completes, the newly synthesized target region will be attached to the same strand as the probe. Following the second cycle of denaturation and annealing, the probe and the target hybridize. The denaturation of the hairpin loop requires less energy than the new DNA duplex produced. Consequently, the hairpin sequence hybridizes to a part of the newly produced PCR product. This results in the separation of the fluorophore from the quencher and causes emission.

[0113] Oligonucleotide probes can be designed which are between about 10 and about 100 nucleotides in length and hybridize to the amplified region. Oligonucleotides probes are preferably 12 to 70 nucleotides; more preferably 15-60 nucleotides in length; and most preferably 15-25 nucleotides in length. The probe may be labeled. Amplified fragments may be detected using standard gel electrophoresis methods. For example, in preferred embodiments, amplified fractions are separated on an agarose gel and stained with ethidium bromide by methods known in the art to detect amplified fragments.

[0114] Assay controls may be used in the assay for detecting carriers and individuals afflicted with Tay Sachs disease. Positive controls for normal or wild type HEXA gene may be used. [0115] Suitable fluorescent moieties include but are not limited to the following fluorophores working individually or in combination:

[0116] 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor^® 350, Alexa Fluor^® 488, Alexa Fluor^® 546, Alexa Fluor^® 555, Alexa Fluor^® 568, Alexa Fluor^® 594, Alexa Fluor^® 647 (Molecular Probes); 5 -(2'-aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino- N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino- l-naphthyl)maleimide; anthranilamide; Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes: BODIPY^® R-6G, BOPIPY^® 530/550, BODIPY^® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2^®, Cy3^®, Cy3.5^®, Cy5^®, Cy5.5^®; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"-dibromopyrogallol- sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5- [dimethylamino]naphthalene-l -sulfonyl chloride (DNS, dansyl chloride); 4-(4'- dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'- isothiocyanate (DABITC); Eclipse™ (Epoch Biosciences Inc.); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfiuorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)amino fluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6-carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescein (TET); fluorescamine; IR144; IR1446; lanthamide phosphors; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin, R-phycoerythrin;

allophycocyanin; o-phthaldialdehyde; Oregon Green^®; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 -pyrene butyrate; QSY^® 7; QSY^® 9; QSY^® 21; QSY^® 35 (Molecular Probes); Reactive Red 4 (Cibacron^® Brilliant Red 3B-A); rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, riboflavin, rosolic acid, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);

terbium chelate derivatives; N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA);

tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC).

[0117] Other fluorescent nucleotide analogs can be used, see, e.g., Jameson, Meth.

Enzymol. (1997), 278:363-390; Zhu et al, Nucleic Acids Res. (1994), 22:3418-3422. U.S. Patent Nos. 5,652,099 and 6,268,132 also describe nucleoside analogs for incorporation into nucleic acids, e.g., DNA and/or RNA, or oligonucleotides, via either enzymatic or chemical synthesis to produce fluorescent oligonucleotides. U.S. Patent No. 5,135,717 describes phthalocyanine and tetrabenztriazaporphyrin reagents for use as fluorescent labels.

[0118] The detectable label can be incorporated into, associated with or conjugated to a nucleic acid. Label can be attached by spacer arms of various lengths to reduce potential steric hindrance or impact on other useful or desired properties. See, e.g., Mansfield, Mol. Cell. Probes (1995), 9: 145-156.

[0119] Detectable labels can be incorporated into nucleic acid probes by covalent or non- covalent means, e.g. , by transcription, such as by random-primer labeling using Klenow polymerase, or nick translation, or, amplification, or equivalent as is known in the art. For example, a nucleotide base is conjugated to a detectable moiety, such as a fluorescent dye, e.g., Cy3^™ or Cy5™ and then incorporated into nucleic acid probes during nucleic acid synthesis or amplification. Nucleic acid probes can thereby be labeled when synthesized using Cy3™- or Cy5™-dCTP conjugates mixed with unlabeled dCTP.

[0120] Nucleic acid probes can be labeled by using PCR or nick translation in the presence of labeled precursor nucleotides, for example, modified nucleotides synthesized by coupling allylamine-dUTP to the succinimidyl-ester derivatives of the fluorescent dyes or haptens (such as biotin or digoxigenin) can be used; this method allows custom preparation of most common fluorescent nucleotides, see, e.g., Henegariu et al, Nat. Biotechnol. (2000), 18:345- 348,

[0121] Nucleic acid probes may be labeled by non-covalent means known in the art. For example, Kreatech Biotechnology's Universal Linkage System® (ULS®) provides a non- enzymatic labeling technology, wherein a platinum group forms a co-ordinative bond with DNA, RNA or nucleotides by binding to the N7 position of guanosine. This technology may also be used to label proteins by binding to nitrogen and sulfur containing side chains of amino acids. See, e.g., U.S. Patent Nos. 5,580,990; 5,714,327; and 5,985,566; and European Patent No. 0539466.

[0122] Labeling with a detectable label also can include a nucleic acid attached to another biological molecule, such as a nucleic acid, e.g. , an oligonucleotide, or a nucleic acid in the form of a stem-loop structure as a "molecular beacon" or an "aptamer beacon". Molecular beacons as detectable moieties are well known in the art; for example, Sokol (Proc. Natl. Acad. Sci. USA (1998), 95: 11538-11543) synthesized "molecular beacon" reporter oligodeoxynucleotides with matched fluorescent donor and acceptor chromophores on their 5' and 3' ends. In the absence of a complementary nucleic acid strand, the molecular beacon remains in a stem-loop conformation where fluorescence resonance energy transfer prevents signal emission. On hybridization with a complementary sequence, the stem-loop structure opens increasing the physical distance between the donor and acceptor moieties thereby reducing fluorescence resonance energy transfer and allowing a detectable signal to be emitted when the beacon is excited by light of the appropriate wavelength. See also, e.g., Antony (Biochemistry (2001), 40:9387-9395,), describing a molecular beacon consist of a G- rich 18-mer triplex forming oligodeoxyribonucleotide. See also U.S. Patent Nos. 6,277,581 and 6,235,504.

[0123] Aptamer beacons are similar to molecular beacons; see, e.g., Hamaguchi, Anal. Biochem. (2001), 294: 126-131; Poddar, Mol. Cell. Probes (2001), 15:161-167; Kaboev, Nucleic Acids Res. (2000), 28:E94. Aptamer beacons can adopt two or more conformations, one of which allows ligand binding. A fluorescence-quenching pair is used to report changes in conformation induced by ligand binding. See also, e.g., Yamamoto et al, Genes Cells (2000), 5:389-396; Smimov et al, Biochemistry (2000), 39: 1462-1468.

[0124] The nucleic acid probe may be indirectly detectably labeled via a peptide. A peptide can be made detectable by incorporating predetermined polypeptide epitopes recognized by a secondary reporter {e.g., leucine zipper pair sequences, binding sites for secondary

antibodies, transcriptional activator polypeptide, metal binding domains, epitope tags). A label may also be attached via a second peptide that interacts with the first peptide (e.g., S-S association).

[0125] As readily recognized by one of skill in the art, detection of the complex containing the nucleic acid from a sample hybridized to a labeled probe can be achieved through use of a labeled antibody against the label of the probe. In a preferred example, the probe is labeled with digoxigenin and is detected with a fluorescent labeled anti-digoxigenin antibody. In another example, the probe is labeled with FITC, and detected with fluorescent labeled anti- FITC antibody. These antibodies are readily available commercially. In another example, the probe is labeled with FITC, and detected with anti-FITC antibody primary antibody and a labeled anti-anti FITC secondary antibody.

[0126] Detection of nucleic acid by size:

[0127] Methods for detecting the presence or amount of nucleic acid are well known in the art and any of them can be used in the methods described herein so long as they are capable of separating individual nucleic acid by the difference in size of the amplicons. The separation technique used should permit resolution of nucleic acid as long as they differ from one another by at least one nucleotide. The separation can be performed under denaturing or under non-denaturing or native conditions~i.e., separation can be performed on single- or double-stranded nucleic acids. It is preferred that the separation and detection permits detection of length differences as small as one nucleotide. It is further preferred that the separation and detection can be done in a high-throughput format that permits real time or contemporaneous determination of amplicon abundance in a plurality of reaction aliquots taken during the cycling reaction. Useful methods for the separation and analysis of the amplified products include, but are not limited to, electrophoresis (e.g., agarose gel electrophoresis, capillary electrophoresis (CE)), chromatography (HPLC), and mass spectrometry.

[0128] DNA sequencing:

[0129] In some examples, detection of nucleic acid is by DNA sequencing. Sequencing may be carried out by the dideoxy chain termination method of Sanger et al. (Proceedings of the National Academy of Sciences USA (1977), 74, 5463-5467) with modifications by Zimmermann et al. (Nucleic Acids Res. (1990), 18:1067). Sequencing by dideoxy chain termination method can be performed using Thermo Sequenase (Amersham Pharmacia, Piscataway, NJ), Sequenase reagents from US Biochemicals or Sequatherm sequencing kit (Epicenter Technologies, Madison, Wis.). Sequencing may also be carried out by the "RR dRhodamine Terminator Cycle Sequencing Kit" from PE Applied Biosystems (product no. 403044, Weiterstadt, Germany), Taq DyeDeoxy™ Terminator Cycle Sequencing kit and method (Perkin-Elmer/ Applied Biosystems) in two directions using an Applied Biosystems Model 373 A DNA or in the presence of dye terminators CEQ™ Dye Terminator Cycle Sequencing Kit, (Beckman 608000). Alternatively, sequencing can be performed by a method known as Pyrosequencing (Pyrosequencing, Westborough, Mass.). Detailed protocols for Pyrosequencing can be found in: Alderborn et al, Genome Res. (2000), 10: 1249-1265.

[0130] Detection of mutation using allele-specific primers and probes

[0131] For analyzing mutation in HEXA nucleic acid, it may be appropriate to use oligonucleotides specific for alternative alleles. Such oligonucleotides which detect single nucleotide variations in target sequences may be referred to by such terms as "allele-specific probes", or "allele-specific primers". The design and use of allele-specific probes for analyzing polymorphisms is described in, e.g., Mutation Detection A Practical Approach, ed. Cotton et al. Oxford University Press, 1998; Saiki et al, Nature, 324: 163-166 (1986);

Dattagupta, EP235,726; and Saiki, WO 89/11548. In one example, a probe or primer may be designed to hybridize to a segment of target DNA such that the mutation site aligns with either the 5' most end or the 3' most end of the probe or primer.

[0132] In some examples, the amplification may include a labeled primer, thereby allowing detection of the amplification product of that primer. In one example, the amplification may include a multiplicity of labeled primers; typically, such primers are distinguishably labeled, allowing the simultaneous detection of multiple amplification products.

[0133] In one type of PCR-based assay, an allele-specific primer hybridizes to a region on a target nucleic acid molecule that overlaps a mutation site of HEXA nucleic acid {e.g., G759A, A775G, A965T, 118delT, G1292A, 1214-1215delAAinsG, 759-774dup, and G5461A of HEXA nucleic acid) and only primes amplification of an allelic form to which the primer exhibits perfect complementarity (Gibbs, 1989, Nucleic Acid Res., 17:2427-2448). Typically, the primer's 3'-most nucleotide is aligned with and complementary to the mutation site of the target nucleic acid molecule. This primer is used in conjunction with a second primer that hybridizes at a distal site. Amplification proceeds from the two primers, producing a detectable product that indicates which allelic form is present in the test sample. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the mutation site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification or substantially reduces amplification efficiency, so that either no detectable product is formed or it is formed in lower amounts or at a slower pace. The method generally works most effectively when the mismatch is at the 3'-most position of the oligonucleotide (i.e., the 3'-most position of the oligonucleotide aligns with the target mutation position) because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456). Exemplary allele- specific primer sequences for detecting the G759A, A775G, A965T, 118delT, G1292A, 1214-1215delAAinsG, 759-774dup, and G5461A mutations of the HEXA nucleic acid are shown in Table 2 below.

[0134] In one example, a primer contains a sequence substantially complementary to a segment of a mutation-containing target nucleic acid molecule except that the primer has a mismatched nucleotide in one of the three nucleotide positions at the 3'-most end of the primer, such that the mismatched nucleotide does not base pair with a particular allele at the mutation site. The mismatched nucleotide in the primer can be the first, second or the third nucleotide from the last nucleotide at the 3'-most position of the primer. In some examples, primers and/or probes are labeled with detectable labels.

Table 2. Exemplary Allele-Specific Primers

Forward WT (A775) GTGTGCTTGCAGAGTTTGACA SEQ ID NO: 19 Allele-Specific Primer

Forward Mutant (G775) GTGTGCTTGCAGAGTTTGACG SEQ ID NO: 20 Allele-Specific Primer

Forward WT (A965) TTATCTTCATCTTGGAGGAGA SEQ ID NO: 21 Allele-Specific Primer

Forward Mutant (T965) TTATCTTCATCTTGGAGGAGT SEQ ID NO: 22 Allele-Specific Primer

Forward WT (118 delT) CCAGCGCTACGTCCTTT SEQ ID NO: 23 Allele-Specific Primer

Forward Mutant (118 CCAGCGCTACGTCCTTA SEQ ID NO: 24 delT)

Allele-Specific Primer

Reverse Primer 1 CTGTATGATTGTGTCTGGCTG SEQ ID NO: 25

Forward WT (G1292) GTATATCCTATGGCCCTGACTG SEQ ID NO: 77 Allele-Specific Primer

Forward Mutant (A 1292) GTATATCCTATGGCCCTGACTA SEQ ID NO: 78 Allele-Specific Primer

Forward WT (1214- AGTGAACTATATGAAGGAGCTGGA SEQ ID NO: 79 1215delAAinsG)

Allele-Specific Primer

Forward Mutant (1214- AGTGAACTATATGAAGGAGCTGGG SEQ ID NO: 80 1215delAAinsG)

Allele-Specific Primer

Forward WT (G5461 A) GCC CC GTC CTT AC CTC AC AGG SEQ ID NO: 81 Allele-Specific Primer

Forward Mutant GCC CC GTC CTT AC CTC AC AG A SEQ ID NO: 82 (G5461A)

Allele-Specific Primer

Reverse Primer 2 CCCCTGCTCTGGGCCAGAGCCTG SEQ ID NO: 83

ATCACCTTAGATTTCTGCTGGCACA SEQ ID NO: 84

Reverse Primer 3

[0135] In one example, HEXA mutants G759A, A775G, A965T, 118delT can be detected with reverse primer 1 (SEQ ID NO: 25) with the combination of corresponding forward primers (mutant and wild-type). In another example, HEXA mutants G1292A and 1214- 1215delAAinsG can be detected with reverse primer 2 (SEQ ID NO: 83) with the combination of corresponding forward primers (mutant and wild-type). In another example, HEXA mutant G5461A can be detected with reverse primer 3 (SEQ ID NO: 84) with corresponding wild-type and mutant forward primers SEQ ID NO: 81 and 82 respectively.

[0136] Primer extension assay for detecting mutation

[0137] Single nucleotide primer extension assay can be used to identify mutation in HEXA nucleic acid. An oligonucleotide primer hybridizes to a complementary region along the nucleic acid, to form a duplex, with the primer's terminal 3' end directly adjacent to the nucleotide base to be identified. The oligonucleotide primer is enzymatically extended a single base by a nucleotide terminator complementary to the nucleotide being identified. The terminator prevents additional nucleotides from being incorporated. Many different approaches can be taken for determining the identity of a terminator, including fluorescence labeling, mass labeling for mass spectrometry, measuring enzyme activity using a protein moiety, and isotope labeling. Several commercially available methods are available to detect mutation using single nucleotide primer extension such as SNaPshot® (Applied Biosystems).

[0138] In the SNaPshot® assay, each primer binds to a complementary template in the presence of fluorescently labeled ddNTPs and DNA Polymerase. The polymerase extends the primer by one nucleotide, adding a single ddNTP to its 3 ^' end. The amplification products can be separated by size, for example, by capillary electrophoresis and can be analyzed using suitable software such as GeneScan® Analysis Software (Applied

Biosystems). A multiplexed SNaPshot® assay can be used to detect multiple mutations simultaneously.

[0139] In one example, the nucleic acid sequences for the primers used in the SNaPshot® assay to detect G759A, A775G, A965T, 118delT, G1292A, 1214-1215delAAinsG, 759- 774dup, and G5461 A mutations of the HEXA nucleic acid may be identical to the allele specific forward primers described in Table 2 but lack the 3 '-terminal nucleotide. For example, the primer for the SNaPshot® assay to detect the G759A mutation will have the same sequence as SEQ ID NO: 18 but lack the 3 '-terminal "A" nucleotide, the primer to detect A775G mutation will have the same sequence as SEQ ID NO: 20 but lack the 3'- terminal "G" nucleotide, the primer to detect A965T mutation will have the same sequence as SEQ ID NO: 22 but lack the 3 '-terminal "T" nucleotide, the primer to detect 118delT mutation will have the same sequence as SEQ ID NO: 24 but lack the 3 '-terminal "A" nucleotide, the primer to detect G 1292 A mutation will have the same sequence as SEQ ID NO: 78 but lack the 3'-terminal "A" nucleotide, the primer to detect 1214-1215delAAinsG mutation will have the same sequence as SEQ ID NO: 80 but lack the 3 '-terminal "G" nucleotide, the primer to detect G5461A mutation will have the same sequence as SEQ ID NO: 82 but lack the 3 '-terminal "A" nucleotide. In one example, more than one mutation can be detected by multiplexed SNaPshot® assay.

[0140] In an alternative approach, tagged allele specific primer pairs can be used to detect mutation in HEXA nucleic acid (Strom et al. Genet Med. 2005;7:633-63). In one example, two tagged allele-specific primers overlap the mutation site in the target DNA, only the correctly hybridized primer(s) will be extended to generate a labeled product(s). A non- complementary primer will not be extended or labeled due to the 3' mismatched base. The labeled extended product can be detected based on the detectable label. The tagged extended primers can be captured on solid support such as beads that are coupled to anti-tag sequences. The immobilized extended primer product can be detected by commercially available means such as Luminex 100 LabMAP™ (Luminex Corporation, Austin TX).

[0141] Detection of HEXA mutation by hybridization of a nucleic acid probe

[0142] HEXA mutation may be detected by hybridization of a nucleic acid probe to HEXA nucleic acid or to a portion of amplified nucleic acid comprising the mutation. Probes may encompass the mutation site (for G759A: nucleotide 759 corresponding to SEQ ID NO: 2; for A775G: nucleotide 775 corresponding to SEQ ID NO: 2; for A965T: nucleotide 965 corresponding to SEQ ID NO: 2; for 118 del T: nucleotide 118 corresponding to SEQ ID NO: 2, for G1292A: nucleotide 1292 of SEQ ID NO: 2, for 1214-1215delAAinsG: nucleotide 1214-1215 of SEQ ID NO: 2, for 759-774dup: nucleotides 759-774 of SEQ ID NO: 2, for G5461A: nucleotide 5461 of SEQ ID NO: 1) of HEXA nucleic acid.

[0143] Detection of Mutated HEXA Proteins

[0144] The presence or absence of HEXA mutations can also be determined by analyzing the HEXA protein encoded by the mutated HEXA nucleic acid. The mutations include those shown in Table 1. Detection of HEXA mutations at the protein level can be detected by any method well known in the field. In one example, detection of HEXA mutations is carried out by isolating HEXA protein and subjecting it to amino acid sequence determination. This may require fragmenting the protein by proteolytic or chemical means prior to sequencing.

[0145] Several methods for detection of proteins are well known in the art. Detection of the proteins could be by resolution of the proteins by SDS polyacrylamide gel electrophoresis (SDS PAGE), followed by staining the proteins with suitable stain for example, Coomassie Blue. The HEXA proteins with and without mutation can be differentiated from each other and also from other proteins based on their molecular weight and migration on SDS PAGE.

[0146] Detection of mutated HEXA proteins can be accomplished using, for example, antibodies, aptamers, ligands/substrates, other proteins or protein fragments, other protein- binding agents, or mass spectrometry analysis of fragments. Preferably, protein detection agents are specific for the mutated HEXA protein and can therefore discriminate between a mutated protein and the wild-type protein or another variant form. This can generally be accomplished by, for example, selecting or designing detection agents that bind to the region of a protein that differs between the variant and wild-type protein.

[0147] One preferred agent for detecting a mutated HEXA protein is an antibody capable of selectively binding to a variant form of the protein. Antibodies capable of distinguishing between wild-type and mutated HEXA protein may be created by any suitable method known in the art. The antibodies may be monoclonal or polyclonal antibodies, single chain or double chain, chimeric or humanized antibodies or portions of immunoglobulin molecules containing the portions known in the state of the art to correspond to the antigen binding fragments.

[0148] In vitro methods for detection of the mutated HEXA proteins also include, for example, enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), Western blots, immunoprecipitations, immunofluorescence, and protein arrays/chips (e.g., arrays of antibodies or aptamers). For further information regarding immunoassays and related protein detection methods, see Current Protocols in Immunology, John Wiley & Sons, N.Y., and Hage, "Immunoassays", Anal Chem. 1999 Jun. 15; 71(12):294R-304R. Additional analytic methods of detecting amino acid variants include, but are not limited to, altered electrophoretic mobility (e.g., 2-dimensional electrophoresis), altered tryptic peptide digest, altered HEXA activity in cell-based or cell-free assay, alteration in ligand or antibody- binding pattern, altered isoelectric point, and direct amino acid sequencing.

[0149] HEXA proteins with and without mutation can be differentiated from each other and from other proteins and also can be identified by Western blot analysis. Methods of Western blot are well known in the art and described for example in W. Burnette W.N. Anal.

Biochem. 1981; 112 (2): 195-203. Briefly, HEXA proteins can be subjected to SDS PAGE. Following the gel electrophoresis, the proteins can be transferred on nitrocellulose or polyvinylidene fluoride (PVDF) membrane. The membranes are blocked with suitable blocking agents to prevent non-specific binding of antibody to the membrane. Suitable blocking agents include bovine serum albumin, non-fat dry milk. After blocking and several washes with suitable buffer, antibodies that specifically bind to the HEXA protein without any mutation and antibodies that specifically bind to HEXA protein with mutation are allowed to bind to the protein of interest. Following the binding of primary antibody to the protein of interest, the excess antibodies are washed away with suitable buffer. A suitable secondary antibody that is able to bind to the primary antibody is applied. The secondary antibody is detectably labeled. Excess secondary antibody is washed away with suitable buffer and the detectable label of the secondary antibody is detected. Detection of the detectable label of the secondary antibody indicates the presence of the protein of interest. If primary antibodies specific for the mutant HEXA protein is used, then the mutant HEXA protein can be identified.

[0150] In preferred examples, Flow Cytometry may be applied to detect the wild-type or mutant HEXA protein. Antibodies specific for the wild-type or mutant HEXA protein can be coupled to beads and can be used in the Flow Cytometry analysis.

[0151] Determination of HEXA activity

[0152] In one example, the effect of the mutation of HEXA protein can be assessed by determining HEXA activity. The biochemical assay for determining HEXA activity uses an uncharged, artificial substrate, 4-methlyumbelliferyl-P-N-acetyl glucosaminide (MUG), to measure total hexosaminidase activity in serum. Total hexosaminidase activity comprises the total activities of HEXA and hexosaminidase B (HEXB) activities. HEXA is labile to heating at 50°C where as HEXB is stable under this condition. The HEXA activity is determined indirectly by calculating the difference of total hexaminidase activity and HEXB activity (O'Brien et al. N Engl J Med. 1970; 283: 15-20). The HEXB activity is measured after heat inactivation of the HEXA in a second serum aliquot. Serum sample is heated at 50°C for 2-4 hours at pH 4.4; 95% of HEXA activity and 5% or less of HEXB activity is inactivated after 4 hours. After the measurement of total hexaminidase activity and HEXB activity, HEXA activity is then calculated as the difference between the total hexosaminidase and HEXB activities. Results are expressed as the percentage of HEXA activity relative to the total hexosaminidase activity. Reference ranges for HEXA activity are: Normal: 55- 75%, Tay-Sachs disease carrier: 25-50%), and Tay-Sachs disease patient: below 25%.

[0153] Antibody Production and Screening

[0154] Various procedures known in the art may be used for the production of antibodies to epitopes of the HEXA protein and the mutants of HEXA protein. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Antibodies that specifically bind to an epitope of SEQ ID NOs: 7, 10, 13, 16, 66, 69, or 73 are useful for detection and diagnostic purposes.

[0155] In one example, the antibodies may bind specifically to an epitope comprising at least 10 contiguous amino acids of SEQ ID NO: 11, 14, 16, 70, 74. Such antibodies are useful for detection and diagnostic purposes.

[0156] Monoclonal antibodies to HEXA protein and the mutant of HEXA protein may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature (1975), 256:495-497), the human B-cell hybridoma technique (Kosbor et al, Immunology Today (1983), 4:72; Cote et al. Proc. Natl. Acad. Sci. (1983), 80:2026-2030) and the EBV-hybridoma technique (Cole et al,

Monoclonal Antibodies and Cancer Therapy (1985), Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al, Proc. Natl. Acad. Sci. USA (1984), 81 :6851-6855; Neuberger et al, Nature (1984), 312:604- 608; Takeda et al, Nature (1985), 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce HEXA protein and mutant of HEXA protein-specific single chain antibodies.

[0157] Antibody fragments which contain specific binding sites of HEXA protein and mutants of HEXA protein may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al, Science. 1989; 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity to HEXA protein and mutant of HEXA protein.

[0158] Kits

[0159] Kits for diagnostic are provided. A diagnostic system may include a kit which contains, in an amount sufficient for at least one assay, any of the hybridization assay probes, amplification primers, and/or antibodies against HEXA wild type and mutant in a packaging material. Typically, the kits will also include instructions recorded in a tangible form (e.g., contained on paper or an electronic medium) for using the packaged probes, primers, and/or antibodies in a detection assay for determining the presence or amount of HEXA variant nucleic acid or HEXA mutant protein in a test sample.

[0160] The various components of the diagnostic systems may be provided in a variety of forms. For example, the required enzymes, the nucleotide triphosphates, the probes, primers, and/or antibodies may be provided as a lyophilized reagent. These lyophilized reagents may be pre-mixed before lyophilization so that when reconstituted they form a complete mixture with the proper ratio of each of the components ready for use in the assay. In addition, the diagnostic systems may contain a reconstitution reagent for reconstituting the lyophilized reagents of the kit. In preferred kits for amplifying target nucleic acid derived from a Tay- Sachs disease patients, the enzymes, nucleotide triphosphates and required cofactors for the enzymes are provided as a single lyophilized reagent that, when reconstituted, forms a proper reagent for use in the present amplification methods.

[0161] In one example, the kit may comprise at least three lyophilized oligonucleotides: a primer pair to amplify a portion of HEXA nucleic acid, and a detectably labeled probe capable of hybridizing to the amplicon generated. In some preferred kits, at least three lyophilized oligonucleotides are the primers for amplification of at least a portion of HEXA nucleic acid by semi-nested PCR.

[0162] Some preferred kits may further comprise to a solid support for anchoring the nucleic acid of interest on the solid support. The target nucleic acid may be anchored to the solid support directly or indirectly through a capture probe anchored to the solid support and capable of hybridizing to the nucleic acid of interest. Examples of such solid support include but are not limited to beads, microp articles (for example, gold and other nano particles), microarray, microwells, multiwell plates. The solid surfaces may comprise a first member of a binding pair and the capture probe or the target nucleic acid may comprise a second member of the binding pair. Binding of the binding pair members will anchor the capture probe or the target nucleic acid to the solid surface. Examples of such binding pairs include but are not limited to biotin/streptavidin, hormone/receptor, ligand/receptor, antigen/antibody.

[0163] In other preferred kits, lyophilized antibodies against HEXA wild type and mutant protein are provided. In some preferred kits a primary/secondary antibody pair may be provided. Some preferred kits may further comprise to a solid support for anchoring the HEXA wild type and mutant proteins. Such anchoring of the HEXA wild type and mutant proteins may be through biotin/streptavidin, antigen/antibody interactions.

[0164] Typical packaging materials would include solid matrices such as glass, plastic, paper, foil, micro-particles and the like, capable of holding within fixed limits hybridization assay probes, and/or amplification primers. Thus, for example, the packaging materials can include glass vials used to contain sub-milligram (e.g., picogram or nanogram) quantities of a contemplated probe, primer, or antibodies or they can be microtiter plate wells to which probes, primers, or antibodies have been operatively affixed, i.e., linked so as to be capable of participating in an amplification and/or detection methods. [0165] The instructions will typically indicate the reagents and/or concentrations of reagents and at least one assay method parameter which might be, for example, the relative amounts of reagents to use per amount of sample. In addition, such specifics as maintenance, time periods, temperature, and buffer conditions may also be included.

[0166] The diagnostic systems contemplate kits having any of the hybridization assay probes, amplification primers, or antibodies described herein, whether provided individually or in one of the preferred combinations described above, for use in determining the presence or amount of HEXA mutant mRNA or HEXA mutant protein in a test sample.

EXAMPLE 1

Samples and Study Protocol

[0167] Individuals were solicited who were either affected with TSD (all adult onset patients), self-reported parents of children or adults affected with TSD, or relatives of these index cases who have been told they were carriers of TSD. Obligate carrier status was known in only 9 of the 33 (27%) self-reported, unaffected carriers. Informed consent was obtained and samples were collected for both serum and blood/white blood cell preparations.

[0168] HEXA gene sequencing, HEXA DNA mutation test, and the HEXA enzyme assay were performed on 33 self-reported TSD carriers, 7 late-onset TSD patients, and 1 pseudodeficiency allele carrier. None of the carriers claimed to be of Ashkenazi Jewish descent, and none were pregnant at time of sample collection.

[0169] DNA were extracted from most of the samples using Gentra Autopure LS

(Minneapolis, MN) exactly as described by the manufacturer, and some samples were extracted using Qiagen Biorobot 9604 platform using MegAttract® DNA blood M96 kit (Valencia, CA). Whole blood was used for DNA extraction in most cases, and in some cases, leukocytes were used. No differences in sequencing quality between different extraction platforms and different starting materials were observed.

EXAMPLE 2

HEXA Gene Sequencing

[0170] DNA sequencing and PCR were performed essentially as described by Huang et al. (Clin Colorectal Cancer. Nov 2004; 4:275-279) except that DNA purification post-PCR was done using exonuclease (USB Corporation, Cleveland, OH)/calf intestinal alkaline phosphatase (Promega, Madison, WI) digestion, and post dye terminator reaction DNA was purified by ethanol precipitation and resuspended in Hi-Di formamide (ABI, Foster City, CA). Fourteen amplicons, each amplifying one of 14 exons, were generated through individual PCR reactions. Amplification of the 14 exons were performed using the primers primers shown in Table 3 below. Amplification efficiency and correctness of sizes were verified by agarose gel electrophoresis (Fig. 1). Sequencing was done with M13F and M13R primers. Sequencing covered exons 1 through 14 of HEXA gene and at least 20bp flanking sequences. Samples were resolved on an ABI PRISM® 3730 Genetic Analyzer and data were analyzed using ABI Prism® SeqScape software (Applied Biosystems, Foster City, CA, USA).

Table 3 : Primers used for amplification and sequencing

Primer Seq ID Name Primer Sequence*

No:

Exon tgtaaaacgacggccagtACGTGATTCGCCGATAAGTC 146

Exon _1R caggaaacagctatgaccCAGGCAGGCACTCTCAGG 147

Exon 2F tgtaaaacgacggccagtCCATGTCCTCCCTTTCCTTT 148

Exon 2R caggaaacagctatgaccAGGCCATCCAGAGTTACAGC 149

Exon 3F tgtaaaacgacggccagtATATCTGGTCTATAATCTGAGAAT 150

AGAAAAC

Exon 3R caggaaacagctatgaccGGGAAGATTGCTTGAGCCTA 151

Exon 4F tgtaaaacgacggccagtTTTTCCCTGTGTACCCAAATG 152

Exon 4R caggaaacagctatgaccGATCCAACCCCAGAGATGAA 153

Exon _5F tgtaaaacgacggccagtATCTCCCTGTGCCCCCATAGTAA 154

Exon 5R caggaaacagctatgaccTGCTCCATCACCCTAGAACTCTTA 155

Exon 6F tgtaaaacgacggccagtAGGCTGAAACCGGAGAGACT 156

Exon 6R caggaaacagctatgaccTCCAAATCCAGTGTCCTTCC 157

Exon _7F tgtaaaacgacggccagtGCTATGGGCAGGTTTTCTGA 158

Exon 7R caggaaacagctatgaccCTGGGATATGCCACTTCCAT 159

Exon 8F tgtaaaacgacggccagtTGCGTGGGGGTTTATGTATT 160

Exon 8R caggaaacagctatgaccGGCAGAGGAAGGCCTAAGAC 161

Exon 9F tgtaaaacgacggccagtCAGGCATTAGGCTTTCAGGATGTT 162 Exon 9R caggaaacagctatgaccGGTATGGAAAGGGAGGACCC 163

Exon 10F tgtaaaacgacggccagtACTGGCACAAACAGTCTAGAACC 164

Exon 10R caggaaacagctatgaccAGTCTCTGTAGAGGCAGGGAGG 165

Exon 11F tgtaaaacgacggccagtAATAAGGCCCAGGAATCTCC 166

Exon 11R caggaaacagctatgaccCTTCCTGCCTCCCATCCTGT 167

Exon 12F tgtaaaacgacggccagtGAAGGTGGTTGAGAGGGACC 168

Exon 12R caggaaacagctatgaccGGGATTGGGTCTCTAAGGGAG 169

Exon 13F tgtaaaacgacggccagtCTGGCTGTGGCTCATAACAA 170

Exon 13R caggaaacagctatgaccCGTGGATGAGGGCTGACTAT 171

Exon 14F tgtaaaacgacggccagtCCCTTCTGAGGGGCTTATCT 172

Exon 14R caggaaacagctatgaccCCTTTCTCTCCAAGCACAGG 173

M13F y- TGTAAAACGACGGCCAGT -3' (for sequencing) 174

M13R y- CAGGAAACAGCTATGACC -3 (for sequencing) 175

[0171] Sequencing the HEXA gene in the carriers identified eight novel mutations. A single nucleotide deletion, 118 delT, in exon 1 was found in 1 carrier which leads to a one nucleotide frame shift resulting in a truncated protein ending at exon 2. This is expected to be a disease-causing mutation. A substitution mutation of 965 A— >T (D322V), causes an amino acid change from a negatively charged aspartic acid to a hydrophobic valine. This is likely to be disease-causing since 1 of the 2 individuals with this mutation is an obligate carrier and has a HEXA activity result in the carrier range and no other detected mutation. Additionally, analysis of the effect of these mutations using PolyPen and SHIFT web-based tools suggest damage at the ligand-binding site that interferes with protein folding. (Ng PC and Henikoff S., Ann Rev Genomics Hum Genet. 2006;7:61-80; Ramensky et al. Nucleic Acids Res. 2002; 30: 3894-3900).

[0172] Another missense mutation, 775 A— »G changes a hydrophilic threonine to a hydrophobic alanine at amino acid position 259 (T259A). It may be a disease-causing mutation since the enzyme activity was in the carrier level (51% HEXA) and no other TSD- associated mutation was detected. However, the effect on predicted protein folding appears to be minimal according to PolyPen and SHIFT web-based tools. [0173] A silent mutation, 759G— >A (V253V), in exon 7 was detected in a carrier who also has a 1278insTATC allele. Thus, 759G—A is mostly likely a benign polymorphism.

[0174] A substitution mutation G1292A was found in obligate carriers of Tay-Sachs disease. The mutation causes a premature truncation of HEXA protein after amino acid position 430. This is expected to be a disease-causing mutation since the enzyme activity of the HEXA protein is likely to be disrupted due to premature truncation.

[0175] A deletion and insertion mutation was found in obligate carriers of Tay-Sachs disease. A deletion of dinucleotide "AA" and an insertion of G at position 1214-1215 of HEXA cDNA (1214-1215delAAinsG) causes a premature truncation of HEXA protein after amino acid position 404 corresponding to the wild-type protein sequence (SEQ ID NO: 7). Additionally, the mutation also results in generating a new C-terminus of the truncated protein having 17 amino acids which are not present in the wild-type protein. This is expected to be a disease-causing mutation since the enzyme activity of the HEXA protein is likely to be disrupted due to premature truncation.

[0176] A duplication of 16 nucleotides at position 759-774 (759-774dup) was found in obligate carriers of Tay-Sachs disease. The 759-774dup mutation causes a premature truncation of HEXA protein after amino acid position 258 corresponding to the wild-type protein sequence (SEQ ID NO: 7). Additionally, the mutation also results in generating a new C-terminus of the truncated protein having 4 amino acids which are not present in the wild-type protein. This is expected to be a disease-causing mutation since the enzyme activity of the HEXA protein is likely to be disrupted due to premature truncation.

[0177] A substitution mutation G5461A was found in obligate carriers of Tay-Sachs disease. This mutation in intron 1 of HEXA nucleic acid disrupts the "GT" splice donor site resulting in spliced variant of HEXA mRNA. The protein encoded by the spliced variant will differ considerably in amino acid sequence with respect to wild-type HEXA protein. This is expected to be a disease-causing mutation since the enzyme activity of the HEXA protein is likely to be disrupted due to altered protein sequence encoded by the splice variant. [0178] The mutations: G759A, A965T, A775G, 118 delT, G1292A, 1214-1215delAAinsG, 759-774dup and G5461A are shown in Table 1.

[0179] Sequencing the HEXA gene of 33 self-reported, unaffected carriers identified common mutations prevalent among Ashkenazi Jewish (AJ) population and known mutations prevalent among non- Ashkenazi Jewish population and shown in Table 4 below.

Table 4. Common HEXA mutations detected by DNA sequencing

[0180] Sequencing the HEXA gene correctly identified both mutant alleles in 5 out of 7 adult onset TSD-affected individuals. However, sequencing the HEXA gene identified only one of the two mutant alleles in the other two TSD-affected individuals. Thus, gene sequencing correctly classified 71% of TSD-affected individuals (5 out of 7) and

misclassified the remaining two TSD-affected individuals as carriers. Sequencing the HEXA gene of 33 self-reported, unaffected carriers correctly classified 29 of 33 individuals.

Sequencing detected a novel silent mutation (A775G) in one of the remaining carriers and failed to detect any mutation in the remaining 3 carriers. Sequencing the HEXA gene of a TSD pseudodeficiency carrier identified a pseudodeficiency mutation (739C— >T [R247W]). The sensitivity of the HEXA gene sequencing method is shown in Table 5. Table 5. Results from HEXA Enzyme, DNA Mutation, and Gene Sequencing Assays

a DNA mutation assay includes detection of 7.6-KB DEL, EXl; 739C→T; 745C→T; 805G→A; 1073 +1G→A; 1278insTATC; and 1421+1G→C.

[0181] The samples that were misclassified by gene sequencing method were further analyzed by HEXA enzyme assay and known DNA mutation analysis. In one sample (TS54) from a self reported carrier, no mutation was detected either by gene sequencing or by the DNA mutation test, and the HEXA enzyme activity was found to be in the normal range. The results are shown in Table 6.

Table 6. Sequencing Results

A/C, in affected/carrier range; N, in normal range.

a DNA mutation assay includes detection of 7.6-KB DEL, EXl; 739C→T; 745C→T; 805G→A;

1073 +1G→A; 1278insTATC; and 1421+1G→C.

b Novel mutation; no change in phenotype expected. EXAMPLE 3

HEXA DNA Common Mutation Test

[0182] Allele specific primer extension assays were performed to detect 5 common mutations and 2 pseudoalleles: 1278insTATC, +1 IVS 12 G→C, G805A, +1 IVS9 G>A, and 7.6-KB DEL, EX1 , EX1 ; C739T (ARG247TRP [R247W]); and C745T (ARG249TRP

[R249W]). Allele-specific primer-extension to detect these mutations as described by Strom et al. (Genet Med. Nov-Dec 2005; 7(9):633-639). Briefly, tagged allele-specific elongation primers were used in a multiplex allele-specific primer extension assay. Tagged allele specific primers comprised of a 24-mer universal tag sequence on the 5'- end and a variable length allele-specific sequence on the 3'-end. The 3 '-end of the allele-specific primers were unmodified. Allele-specific primer extension (ASPE) was performed in a 20-μΙ_^ reaction containing a 5-μί aliquot of treated PCR product. Each reaction consisted of 20 mmol/L Tris-HCl, pH8.4, 50 mmol/L KC1, 1.25 mmol/L MgC12, 4.5 units of Platinum Tsp

polymerase (Invitrogen), 8 μιηοΙ/L each of biotin-dCTP, dATP, dGTP, and dTTP (Roche), and 24 nM ASPE primer pool containing the universally tagged primers. The ASPE reactions were incubated for 2 minutes at 96°C and then subjected to 40 cycles for 30 seconds at 95°C, 30 seconds at 52°C, and 60 seconds at 74°C. Reactions were maintained at 4°C until further use. Allele specific primer extension products were hybridized to a population of Luminex microspheres couple to complimentary anti-tag sequences. The hybridized microspheres were analyzed on Luminex xAMP instrument at ambient temperature.

EXAMPLE 4

HEXA Enzyme Assay

[0183] The serum HEXA enzyme assay has been described previously (O'Brien et al; N Engl J Med. 1970; 283(1): 15-20). Briefly, the biochemical assay uses an uncharged, artificial substrate, 4-methlyumbelliferyl-P-N-acetyl glucosaminide (MUG), to measure total hexosaminidase activity in serum. Total hexosaminidase activity consists of the HEXA and hexosaminidase B (HEXB) activities. HEXA is labile to heating at 50°C where as HEXB is stable under this condition. HEXB activity is measured after heat inactivation of the HEXA in a second serum aliquot. HEXA activity is then calculated as the difference between the total hexosaminidase and HEXB activities. Results are expressed as the percentage of HEXA activity relative to the total hexosaminidase activity. The normal HEXA activity level was set at 57-80 % and affected/carrier level at <51 %.

[0184] Based on the cutoff level, the HEXA enzyme assay identified all seven individuals with Tay-Sachs disease as affected; 29 out of 33 carriers of Tay-Sachs disease as carriers. The Enzyme assay results for the pseudo carrier of TSD was indeterminate. The results of the enzyme assay are shown in Table 2. Gene sequencing and HEXA mutation tests were performed for samples in which the enzyme assay results failed to correlate with the phenotype. The results are shown in Table 7.

Table 7. Enzyme Assay Results

I, in indeterminate range; N, in normal range; A, above normal range.

[0185] The instant application contains a Sequence Listing which is submitted along with the application in ASCII format via EFS-Web and is incorporated by reference in its entirety. Said ASCII copy, created on October 7, 2010, is named 3482731.txt and is 178,986 bytes in size.

[0186] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0187] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0188] Thus, it should be understood that although the invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0189] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0190] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0191] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

Claims

What is claimed is:

1. A method of determining whether a human is predisposed to Tay-Sachs disease, comprising determining whether said human is homozygous for one or more mutations selected from the group consisting of A775G, A965T, 118delT, G1292A, 1214- 1215delAAinsG, 759-774dup, G5461A, CI 177T, and G1393A in the beta-hexosaminidase A (HEXA) gene and identifying said human as having a predisposition to Tay-Sachs disease when said human is homozygous.

2. The method of claim 1, wherein said one or more mutations in HEXA gene is assessed by one or more methods selected from the group consisting of sequencing, oligonucleotide hybridization, single nucleotide primer extention, and allele-specific primer extension.

3. The method of claim 2, wherein said one or more mutations in HEXA gene is assessed by nucleic acid sequencing.

4. The method of claim 1, wherein said one or more mutations is assessed using genomic DNA.

5. The method of claim 1, wherein said one or more mutations is assessed using cDNA.

6. The method of claim 1 further comprising assessing HEXA gene from a sample in said human for the presence or absence of one or more mutations selected from the group consisting of 1278insTATC, +1IVS12 G→C, G805A, +1 IVS9G→A, and 7.6 KB DEL EX1.

7. A method of determining the Tay-Sachs disease status of a human, comprising:

(a) determining the presence or absence of one or more mutations selected from the group consisting of A775G, A965T, 118delT, G1292A, 1214- 1215delAAinsG, 759-774dup, G5461A, CI 177T, and G1393A in both alleles of the beta-hexosaminidase A (HEXA) gene of the human, and

(b) identifying said human:

(i) as having Tay-Sachs disease or being predisposed to Tay-Sachs disease when said human is homozygous for said one or more mutations in the HEXA gene, or

(ii) as being Tay-Sachs disease carrier when said human is heterozygous for said one or more mutations in the HEXA gene, or

(iii) as having no predisposition or carrier status caused by said one or more mutations when said one or more mutations are absent from both alleles of the HEXA gene.

8. The method of claim 7, wherein said one or more mutations in HEXA gene is assessed by one or more methods selected from the group consisting of nucleic acid sequencing, oligonucleotide hybridization, single nucleotide primer extention, and allele- specific primer extension.

9. The method of claim 8, wherein said one or more mutations in HEXA gene is assessed by nucleic acid sequencing.

10. The method of claim 7, wherein said one or more mutations is assessed using genomic DNA.

11. The method of claim 7, wherein said one or more mutations is assessed using cDNA.

12. The method of claim 7, further comprising assessing HEXA gene from a sample in said human for the presence or absence of one or more mutations selected from the group consisting of 1278insTATC, +1IVS12 G→C, G805A, +1 IVS9G→A, and 7.6 KB DEL EX1.

13. A method of identifying a human with an increased likelihood of having an offspring predisposed to Tay-Sachs disease, comprising determining the presence of one or more mutations selected from the group consisting of A775G, A965T, 118delT, G1292A, 1214-1215delAAinsG, 759-774dup, G5461A, C1177T, and G1393A of the beta- hexosaminidase A (HEXA) gene and identifying said human as having an increased likelihood of having an offspring predisposed to Tay-Sachs disease when said one or more mutations in HEXA gene are present in at least one allele.

14. The method of claim 13, wherein said one or more mutations in HEXA gene is assessed by one or more methods selected from the group consisting of sequencing, oligonucleotide hybridization, single nucleotide primer extention, and allele-specific primer extension.

15. The method of claim 14, wherein said one or more mutations in HEXA gene is assessed by nucleic acid sequencing.

16. The method of claim 13, wherein said one or more mutations is assessed using genomic DNA.

17. The method of claim 13, wherein said one or more mutations is assessed using cDNA.

18. The method of claim 13, further comprising assessing HEXA gene from a sample in said human for the presence or absence of one or more mutations selected from the group consisting of 1278insTATC, +1IVS12 G→C, G805A, +1 IVS9G→A, and 7.6 KB DEL EX1.

19. An isolated polynucleotide encoding at least a portion of the HEXA gene, wherein said polynucleotide encodes one or more mutations in HEXA gene selected from the group consisting of A775G, A965T, 118delT, G1292A, 1214-1215delAAinsG, 759-774dup, G5461A, CI 177T, and G 1393 A.

20. The polynucleotide of claim 19, wherein said polynucleotide is about 20-200 nucleotides in length.

21. The polynucleotide of claim 19, wherein said polynucleotide comprises a sequence at least 95% identical to 20 contiguous nucleotides of SEQ ID NO: 8 and encodes the G759A mutation.

22. The polynucleotide of claim 19, wherein said polynucleotide comprises a sequence at least 95% identical to 20 contiguous nucleotides of SEQ ID NO: 9 and encodes the A775G mutation.

23. The polynucleotide of claim 19, wherein said polynucleotide comprises a sequence, at least 95% identical to at least 20 contiguous nucleotides of SEQ ID NO: 12 and encodes the A965T mutation.

24. The polynucleotide of claim 19, wherein said polynucleotide comprises a sequence at least 95% identical to at least 20 contiguous nucleotides of SEQ ID NO: 15 and encodes the 118delT mutation.

25. The polynucleotide of claim 19, wherein said polynucleotide comprises a sequence at least 95% identical to at least 20 contiguous nucleotides of SEQ ID NO: 65 and encodes the G 1292 A mutation.

26. The polynucleotide of claim 19, wherein said polynucleotide comprises a sequence at least 95% identical to at least 20 contiguous nucleotides of SEQ ID NO: 68 and encodes the 1214-1215delAAinsG mutation.

27. The polynucleotide of claim 19, wherein said polynucleotide comprises a sequence at least 95% identical to at least 20 contiguous nucleotides of SEQ ID NO: 72 and encodes the 759-774dup mutation.

28. The polynucleotide of claim 19, wherein said polynucleotide comprises a sequence at least 95% identical to at least 20 contiguous nucleotides of SEQ ID NO: 76 and encodes the G5461A mutation.

29. A kit for detecting one or more mutations in HEXA nucleic acid comprising at least one oligonucleotide of claim 19.

30. An isolated polypeptide comprising at least a portion of the HEXA protein, wherein said polypeptide comprises one or more mutations in HEXA protein selected from the group consisting of T259A, D332V, R393T, D465N, truncated proteins encoded by 118 delT, G1292A, 1214-1215delAAinsG, 759-774dup mutations, and the protein encoded by G5461 A mutation.

31. The polypeptide of claim 30, wherein said polypeptide comprises T259A mutation and have a sequence at least 95% identical to at least 20 contiguous amino acids of SEQ ID NO: 11.

32. The polypeptide of claim 30, wherein said polypeptide comprises D322V mutation and have a sequence at least 95% identical to at least 20 contiguous amino acids of SEQ ID NO: 14.

33. The polypeptide of claim 30, wherein said polypeptide comprises a trunctated protein having a sequence at least 95% identical to SEQ ID NO: 16.

34. The polypeptide of claim 30, wherein said polypeptide comprises a trunctated protein having a sequence at least 95% identical to SEQ ID NO: 70.

35. The polypeptide of claim 30, wherein said polypeptide comprises a trunctated protein having a sequence at least 95% identical to SEQ ID NO: 74.

36. An antibody that specifically binds to a polypeptide of claim 30.

37. The antibody of claim 36, wherein said antibody binds specifically to the poypeptides having an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 14, 16, 70, 74.