Primers used to amplify and sequence the human Indy gene

Primer name 5?(cid:1)3? sequence Annealing / Size bp Temperature (8C) DMSO Indy Ex 1 Top ACACTCTGATTGCCAGACTGG 343 60 NO Indy Ex 1 Bot GAAGTTATGCTGGATCCTAAGG Indy Ex 2 Top TCTGACAGACAGGAGGTGGCAT 286 62 NO Indy Ex 2 Bot CCTTTGTCAACAAGGGCTGAG Indy Ex 3 Top AATGTCAGTGACTGCATCCCAT 631 62 NO Indy Ex 4 Bot TCCAAGATGACCAGCCCTCG Indy Ex 5 Top TGGGCTGTTCACAGACAAGGT 601 62 NO Indy Ex 6 Bot TTTTCCACATCTGCACCCAACT Indy Ex 7 Top CCTCAGCCTTTAGATGGTAGG 404 60 NO Indy Ex 7 Bot GTTACCTCCTGTTCTGCAATG Indy Ex 8 Top GGCTTGTCTATGGGAGAGGCT 276 62 5% Indy Ex 8 Bot CTGCCAAGTGTGGTGGAAGGT Indy Ex 9 Top AAGGCCTGTTTGTTCTTTGGTG 257 60 5% Indy Ex 9 Bot CCTCTGTGGGACAGAAGCAG Indy Ex 10 Top GCAGGAGCCTCATAGAGCCT 328 62 NO Indy Ex 10 Bot AGGATACTCTGCCCAGCAGG Indy Ex 11 Top GTTCCCCAGAGAAGCAGGAG 314 62 NO Indy Ex 11 Bot TGGCTTTAAGCTGCTTTGTGGT Indy Ex 12 Top TTGCTGGTAGGAGGGCTCCT 451 60 NO Indy Ex 12 Bot TTCCAGCACATAGGAAACTGAG

enegydnIehtfoseicneuqerfepytolpaH selella68(cid:4)nycneuqerFI155I3561tn21xEV055I8461tn21xE83(cid:2)01SVI04(cid:2)9SVIA99A792tn3xEL08V832tn3xEC5roC401(cid:2)2SVIL26F681tn2xEepytolpaH //// 5645.0CACTGG4C1 6232.0CGCTAG5C2 3611.0CGCTGG5C3 9430.0CACTGG5C4 6110.0TGCTGC4Ca5 1850.0CACTAG5Ca6 9100.0CGroACTGG5ro4Ga7 .epytolpahderrefnIa



Fig. 1. Polymorphisms in the Indy gene. All numbering begins with the initiator methionine, ATG, as 1.

between species; it is a threonine and valine in D. Melanogaster Indy and Indy-2, respectively, and alanine and leucine in rat SDCT1 and 2, respectively (Rogina et al., 2000).

Two coding region polymorphisms which do not encode a change in amino acid were identified, a G0 / A at nt 297 (A99A), and a C0T polymorphism at nt / 1653 (I551I) (Fig. 1). The A99A polymorphism occurred at a frequency of 0.2097 (n(cid:4)62 alleles). The I551I / polymorphism was found on one allele in the same African American subject carrying the V80L mutation. These synonymous mutations were not examined further.

enegydnIehtfoseicneuqerfepytolpaH

3.1.5. Intronic SNPs

Three intron polymorphisms were also observed, a C deletion at IVS2(cid:2)10, a T0A at IVS9(cid:2)40 and a C0T / / / / at IVS10(cid:2)38 (Fig. 1). The IVS2(cid:2)10 C deletion / / occurred at polymorphic frequencies (0.6129 in 62 alleles) which were not significantly different between the young and old group. The IVS9(cid:2)40 and IVS10(cid:2)38 / / polymorphisms may be in linkage disequilibrium since they occurred in a single individual, a 45-year old Hispanic female. These intronic mutations were not examined further.

Table 4

Number of subjects and gene frequencies of the Indy nt 1648 A0G mutation in different ethnic groups /

Genotype Ethnic group Alla Non-Hisp White Hispanic Asian African(cid:1)American / A/A 974 866 32 23 10 A/G 1050 891 77 25 17 G/G 342 265 35 14 13 Gene frequency (G) 0.3664 0.3514 0.5104 0.4274 0.5375

a Includes subjects of mixed ancestry, and is therefore, greater than the sum of the other columns.

3.2. Haplotype

frequency of the two alleles in white women over 79 years of age did not differ significantly from those under 50 years of age (x2 (cid:4)0.7585; P(cid:4)0.38). Fig. 2 shows the / / cumulative age distributions of subjects with different genotypes as a quantile(cid:1)quantile plot (Wilk and Gna- / nadesikan, 1968).

Seven haplotypes could be identified from the single nucleotide polymorphisms found by direct sequencing of the 43 subjects (Table 3). The first four haplotypes were observed in the homozygous state and the latter three haplotypes were inferred from heterozygous sub- jects. The relative ratio of the seven haplotypes was not statistically significant between subjects of ages 90(cid:1)99 / and subjects of ages 549 using a x2-test for indepen- / dence (P(cid:4)0.1979). /

The V80L polymorphism occurs in the African American population at an allele frequency of 0.0083 (600 alleles) and was absent in the white (1522 alleles) and Asian (66 alleles) populations examined. Since the age range of the five African American subjects hetero- zygous for the V80L polymorphism ranged from 40 to 57 years, an association between the V80L polymorph- ism and aging could not be made. The valine at amino acid 80 is also not conserved between species in that it is an arginine and lysine in Drosophila Indy and Indy-2, respectively, and isoleucine and proline in rat SDCT1 and 2, respectively (Rogina et al., 2000).

3.3.1. I550V

The I550V polymorphism was common in the four ethnic groups examined (Table 4). The number of white subjects within different age groups and I550V geno- types are shown in Table 5. These data demonstrate that women of age (cid:3)80 had a slightly higher frequency of / the G allele (0.3764) than women B80 (0.3427), a / difference that was not quite statistically significant on a x2-test (P(cid:4)0.0762). The frequency of the 1648 G allele / in women of age (cid:3)90 was also not quite statistically / significant from women of age B90 (P(cid:4)0.0551). The / /

The F62L polymorphism is a rare polymorphism or a sporadic mutation since it was not observed in 1390 additional alleles that were examined. The phenylala-

Table 5

Number of subjects with each Indy nt 1648 genotype at different ages among white, non-hispanic subjects

Age Female Male A/A A/G G/G ALL A/A A/G G/G ALL B40 40 (55) 25 (35) 7 (10) 72 26 (39) 32 (49) 8 (12) 66 / 40(cid:1)49 39 (34) 58 (51) 17 (15) 114 45 (41) 52 (48) 12 (11) 109 / 50(cid:1)59 70 (44) 67 (42) 23 (14) 160 71 (42) 76 (46) 20 (12) 167 / 60(cid:1)69 80 (45) 72 (40) 26 (15) 178 72 (46) 66 (42) 18 (12) 156 / 70(cid:1)79 96 (46) 89 (43) 22 (11) 207 63 (48) 49 (38) 18 (14) 130 / 80(cid:1)89 231 (39) 279 (47) 80 (14) 590 21 (50) 16 (38) 5 (12) 42 / 90(cid:1)99 6 (35) 4 (24) 7 (41) 17 6 (43) 6 (43) 2 (14) 14 / G allele nt 1648 frequency 579 0.3427 (n(cid:4)1462) 0.3400 (n(cid:4)1256) / / / Age B80 0.3764a (n(cid:4)1214) 0.3214 (n(cid:4)112) / / /