PHOX2B polymorphisms as hirschsprung's disease diagnostic markers and methods based thereon

An isolated nucleic acid molecule comprising at least 6 contiguous nucleotides of SEQ ID NO:

• 1 including a nucleotide substitution at position 2250 by a nucleotide base that is not adenine and that hybridizes to a nucleic acid molecule consisting of:
• (a) the nucleic acid sequence of SEQ ID NO:
• 1;
• or (b) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:
• 2, under moderately stringent conditions, wherein the moderately stringent conditions comprise hybridization in 0.5 M NaHPO4, 7% sodium dodecyl sulfate, 1 mM EDTA at 65° C., and washing in 0.5 M NaHPO4, 7% sodium dodecyl sulfate, 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% sodium dodecyl sulfate at 42° C., or the complement thereof.

An isolated nucleic acid molecule comprising at least 6 contiguous nucleotides of SEQ ID NO:

• 1 including a nucleotide substitution at position 3493 by a nucleotide base that is not adenine and that hybridizes to a nucleic acid molecule consisting of:
• (a) the nucleic acid sequence of SEQ ID NO:
• 1;
• or (b) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:
• 2, under moderately stringent conditions, wherein the moderately stringent conditions comprise hybridization in 0.5 M NaHPO4, 7% sodium dodecyl sulfate, 1 mM EDTA at 65° C., and washing in 0.5 M NaHPO4, 7% sodium dodecyl sulfate, 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% sodium dodecyl sulfate at 42° C., or the complement thereof.

An isolated nucleic acid molecule comprising at least 6 contiguous nucleotides of SEQ ID NO:

• 1 including one or more nucleotides at positions 3495 to 3499 and that hybridizes to a nucleic acid molecule consisting of:
• (a) the nucleic acid sequence of SEQ ID NO:
• 1;
• or (b) a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:
• 2, under moderately stringent conditions, wherein the moderately stringent conditions comprise hybridization in 0.5 M NaHPO4, 7% sodium dodecyl sulfate, 1 mM EDTA at 65° C., and washing in 0.5 M NaHPO4, 7% sodium dodecyl sulfate, 1 mMEDTA at 65° C., and washing in 0.2×SSC/0.1% sodium dodecyl sulfate at 42° C., or the complement thereof.

A method for screening a subject predisposed or susceptible to HSCR associated with a genetic polymorphism in the PHOX2B gene, said method comprising:

• (a) providing a biological sample from the subject;
• and
• (b) testing the sample for the presence of a nucleotide mutation at nucleotide position 2250 of SEQ ID NO:
• 1, wherein, the presence of the mutation indicates that the subject is genetically predisposed to HSCR.

A method for screening a subject predisposed or susceptible to HSCR associated with a genetic polymorphism in the PHOX2B gene, said method comprising:

• (a) providing a biological sample from the subject;
• and
• (b) testing the sample for the presence of one or more nucleotide mutations from nucleotide positions 1-2250 of SEQ ID NO:
• 1, wherein, the presence of the mutation indicates that the subject is genetically predisposed to HSCR.

A method for diagnosing a subject with increased risk of HSCR associated with a genetic polymorphism in the PHOX2B gene, said method comprising:

• (a) providing a biological sample from the subject;
• and
• (b) testing the sample for the presence of a nucleotide mutation at nucleotide position 2250 of SEQ ID NO:
• 1, wherein, the presence of the mutation indicates that the subject is genetically predisposed to HSCR.

A method for diagnosing a subject with increased risk of HSCR associated with a genetic polymorphism in the PHOX2B gene, said method comprising:

• (a) providing a biological sample from the subject;
• and
• (b) testing the sample for the presence of one or more nucleotide mutations from nucleotide positions 1-2250 of SEQ ID NO:
• 1, wherein, the presence of the mutation indicates that the subject is genetically predisposed to HSCR.

An oligonucleotide of at least 8 nucleotides in length selected from nucleotides 1-46, 48-123;

• 120-369;
• 365-394;
• 390-540;
• 538-646;
• 643-1004;
• 1001-1080;
• 1083-1109;
• 1106-1304;
• 1301-1366;
• 1363-1386;
• 1389-1514;
• 1516-1778;
• 1773-1917;
• 1921-2010;
• 2051-2146;
• 2154-2209;
• 2234-2368;
• 2367-2422;
• 2420-2464;
• 2465-2491;
• 2488-2568;
• 2872-2901;
• 2902-2934;
• 2936-2954;
• 2449-3001;
• 3000-3042;
• 3420-3435;
• 3451-3708;
• 3703-3754;
• 3750-3770;
• 3774-3840;
• 3840-3962;
• 3964-3978;
• 3974-3992;
• 3990-4157;
• 4153-4251;
• 4257-4282;
• 4284-4321;
• 4316-4333;
• 4337-4391;
• 4386-4400;
• 4398-4436;
• 4444-4547;
• 4572-4714;
• 4709-4777;
• 5165-5397;
• 5394-5508 of SEQ ID NO:
• 1, 3 or 4.

A method of determining the source of a first biological sample from a subject compared to a second biological sample, said method comprising:

• (a) providing a first biological sample from the subject;
• (b) testing the first biological sample for the presence of a mutation of the PHOX2B gene at nucleotide positions 2250, 3493, or 3495-3499 of SEQ ID NO:
• 1;
• (c) testing the second biological sample for the presence of a mutation of the PHOX2B gene at nucleotide positions 2250, 3493, or 3495-3499 of SEQ ID NO:
• 1;
• and
• (d) comparing the mutations present in step (b) and (c);
• wherein the presence of identical mutation in the first biological sample compared to the second biological sample indicates that the first and second biological samples are obtained from an identical source.