Polymorphisms in six genes and association study of SSCs and controls using allelic frequencies

SNP ID Location Minor allele frequency c2 P dbSNP rs# Contig Reference (function) position SSC Control FOXO1A (NT_024524) FO1 Intron 0.357 (87/244) 0.385 (94/244) 0.430 0.512 2297626 22214002 FO2 Intron 0.299 (73/244) 0.328 (80/244) 0.467 0.495 2297627 22213931 FO3 Intron 0.131 (32/244) 0.160 (39/244) 0.808 0.369 – 22123190 Bonafe et al. (2003) FO4 Intron – 0.332 (61/184) – – 3751436 22115038 INSR (NT_011255) IN1 Intron 0.230 (56/244) 0.201 (49/244) 0.595 0.441 3745544 7207939 IN2 Intron 0.102 (25/244) 0.143 (35/244) 1.900 0.168 3745546 7151816 IN3 Intron 0.430 (105/244) 0.516 (126/244) 3.625 0.057 3745548 7092703 IN4 Intron 0.234 (57/244) 0.303 (74/244) 3.016 0.083 2252673 7090418 IN5 Exon (syn) 0.340 (83/244) 0.287 (70/244) 1.609 0.205 1799817 7065297 IN6 Intron 0.459 (112/244) 0.508 (124/244) 1.182 0.277 2288404 7064986 IRS1 (NT_005403) IR1 Exon (syn) 0.344 (84/244) 0.332 (81/244) 0.082 0.774 1801123 77870455 IR2 Exon 0.045 (11/244) 0.029 (7/244) 0.923 0.337 1801278 77869956 Bonafe et al. (R971G) (2003) PIK3CB (NT_005612) 3B1 Promoter 0.037 (9/242) 0.041 (10/244) 0.047 0.829 361072 44973698 Bonafe et al. (2003) 3B2 Promoter – – – – – 44973642 Bonafe et al. (2003) 3B3 Intron 0.475 (116/244) 0.434 (106/244) 0.826 0.363 2305268 44879227 PIK3CG (NT_079596) 3G1 Intron 0.270 (66/244) 0.332 (81/244) 2.190 0.139 3779501 5908409 PPARGC1A (NT_006316) PP1 Exon 0.492 (119/242) 0.525 (128/244) 0.525 0.469 8192678 14491020 Ek et al. (S482G) (2001) PP2 Exon 0.169 (41/242) 0.148 (36/244) 0.436 0.509 3736265 14490065 Ek et al. (M612T) (2001)

Syn, synonymous change.

FOXOIAFOLFO2   IN| IN2 IN3. IN4 INS ING    

ac.jp/) using the criteria that minor allele frequencies were more than 10% in the Japanese population. Five SNPs in the FOXO1A, IRS1 and PIK3CB gene loci (1, 1, 1, and 2 SNPs, respectively) were from Bonafe et al. (2003). Additionally 2 non-synonymous SNPs in the PPARGC1A gene locus were selected from Ek et al. (2001) (Table 2). The genomic DNA sequences of FOXO1A, INSR, IRS1, PIK3CB, PIK3CG, and PPARGC1A were obtained from the National Center for Biotechnology Information (NCBI, USA) (accession num- bers NT_024524, NT_011255, NT_005403, NT_005612, NT_079596, and NT_006316, respectively). For each polymorphism not obtained from JSNP, we ensured that there was a sufficiently high frequency in our subjects by testing 24 control subjects. Polymorphisms were typed by DNA sequencing using the BigDye Terminator cycle sequencing kit and an ABI Prism 3700 DNA analyzer (Applied Biosystems, Foster City, CA, USA) or by real-time pyrophosphate DNA sequencing (Ronaghi et al., 1996, 1998) using a PSQ 96 system (Pyrosequencing AB, Uppsala, Sweden) according to the manufacturer’s instructions.

2.3. Statistical analysis

Fig. 1. Pairwise LD in FOXO1A, INSR, IRS1, PIK3CB, and PPARGC1A evaluated by jD0j and r2 estimations. The LD between all pairs of SNPs was evaluated by measuring jD0j and r2 values. Designated SNP IDs are shown in Table 2. Pairwise LD was determined in 92 younger controls. SNP pairs in high LD (jD0jO0.5, r2O0.5) are shown as gray boxes. Upper right triangles show values of jD0j and lower left triangles show values of r2.

The chi-square test was performed between SSCs and control subjects for each allelic and haplotypic frequency. Statistical significance was inferred when P!0.05. Pairwise linkage disequilibrium (LD) was estimated as DZ x Kp q , 11 1 1 wherex 11isthefrequencyofhaplotypeA 1B 1,andp 1andq 1are the frequencies of alleles A and B at locus A and B, 1 1 respectively. A standardized LD coefficient, r, is given by D/(p p q q )1/2wherep andq arethefrequenciesoftheother 1 2 1 2 2 2 alleles at locus A and B, respectively (Hill and Robertson, 1968). Lewontin’s coefficient D0 is given by D/D , max where D Z min[q p ,p q ] when DO0 (Lewontin, 1964). max 1 2 1 2 Haplotype frequencies for multiple loci were estimated by the expectation-maximization method.

An additional 122 SSCs and 30 healthy younger controls were genotyped for an association analysis using 16 SNPs in six genes (Table 2). Two SNPs (IN3 and IN4) in INSR showed a weak difference between SSCs and controls. These SNPs are in LD with each other (jD0jZ 0.899) and are within 2.4 kb of each other (Fig. 1).

Computations were performed using SNPAlyze software (Dynacom, Mobara, Japan).

Haplotypes were constructed on the basis of the genotype data from these SNPs in INSR. The expectation-maximiza- tion algorithm, with phase-unknown samples, was used to estimate haplotype frequencies. The MM haplotype (M: major allele) was more frequent in SSCs (57.0%) than Z in controls (47.3%) (P 0.030) (Table 3).

3.1. Pairwise LD in 5 genes

Among the SNPs not from the JSNP database, 3B2 in PIK3CB was not polymorphic in our 24 control samples (Table 2). Consequently this SNP was excluded from further experiments. The 92 healthy controls were genotyped for each of the 17 selected SNPs. The strength of LD for each SNP pair within each gene was measured using the jD0j and the r2 values (Fig. 1). This figure shows that FO1 and FO4 in FOXO1A locus are in very tight LD with each other (r2Z 0.789). FO1 was selected as the representative SNP for this SNP pair and was examined in further analysis. FO4 was excluded from further analysis.

Case control study of SSCs and controls using estimated haplotype frequencies in INSR

Haplo- SNP ID Frequency c2 P type ID IN3 IN4 SSC Control 1 M M 0.570 0.473 4.729 0.030 2 m M 0.197 0.224 0.603 0.437 3 M m 0.000 0.011 3.019 0.082 4 m m 0.234 0.292 2.076 0.150

M, major allele; m, minor allele.

4. Discussion

study. This work was supported in part by a grant to RIKEN GSC from the Ministry of Education, Culture, Sports, Science and Technology of Japan.