A novel single nucleotide polymorphism of INSR gene for polycystic ovary syndrome

A novel single nucleotide polymorphism of INSR gene for polycystic ovary syndrome Eung-Ji Lee, M.Sc.,a Bermseok Oh, Ph.D.,b Jong-Young Lee, Ph.D.,b Kuchan Kimm, M.D., Ph.D.,b Sook-Hwan Lee, M.D., Ph.D.,a and Kwang-Hyun Baek, Ph.D.a a

Graduate School of Life Science and Biotechnology, Cell and Gene Therapy Research Institute, Pochon CHA University, CHA General Hospital, and b Center for Genome Science, National Institute of Health, Seoul, South Korea

Objective: To investigate several single nucleotide polymorphisms (SNPs) in the insulin receptor (INSR) gene that have significant associations with pathogenesis of polycystic ovary syndrome (PCOS) in a Korean population. Design: Case-control study. Setting: University-based hospital. Patient(s): 134 patients with PCOS and 100 healthy women as controls. Intervention(s): All exons of INSR in DNA samples from 100 healthy women and 134 women with PCOS were sequenced and compared. Main Outcome Measure(s): Frequencies of genotypes for several SNPs in INSR gene that were found as specifically expressed SNPs in a Korean population. Result(s): Among nine SNPs analyzed in a large population, the genotypic frequencies of eight SNPs were similar, and they had no statistically significant association with PCOS. However, the frequency of a minor allele for one novel SNP, þ176477 C>T, was higher in the control group than the patient group. Conclusion(s): Among the analyzed SNPs, þ176477 C>T, a novel SNP in the INSR gene, was associated with the pathogenesis of PCOS in a Korean population. (Fertil Steril 2008;89:1213–20. 2008 by American Society for Reproductive Medicine.) Key Words: Single nucleotide polymorphism, polycystic ovary syndrome, insulin receptor, type 2 diabetes

Polycystic ovary syndrome (PCOS) is a heterogeneous hormonal disorder that affects 4% to 12% of women of reproductive age (1). According to a recent report, 4.9% of women college students presented with PCOS in Korea (2). Based on the 2003 revised diagnostic criteria of the American Society for Reproductive Medicine/European Society of Human Reproduction and Embryology (ASRM/ ESHRE) Rotterdam consensus, PCOS is diagnosed when two out of three criteria are met by a patient, including oligomenorrhea or amenorrhea, clinical or biochemical hyperandrogenism, and ultrasonographic polycystic ovarian morphology. However, the PCOS diagnosis is excluded when the patient has other conditions such as nonclassic congenital adrenal hyperplasia (3, 4). Previous reports have shown that premenopausal women with PCOS have reproductive disorders due to anovulation, hyperandrogenism, polycystic ovaries, and obesity (5–7). In addition, postmenopausal women with PCOS can have metabolic syndromes such as diabetes, dyslipidemia, and hypertension (8). Insulin stimulates ovarian production and the secretion of androgens, affects ovarian steroidogenic responses to luteiReceived December 18, 2006; revised April 4, 2007; accepted May 11, 2007. Supported by a grant from Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (01-PJ10-PG6-01GN13-0002). Reprint requests: Kwang-Hyun Baek, Ph.D., Cell and Gene Therapy Research Institute, Pochon CHA University, CHA General Hospital, 606-16 Yeoksam 1-Dong, Kangnam-Gu, Seoul 135-907, Korea (FAX: þ82-2-3468-3264; E-mail: [email protected]).

0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.05.026

nizing hormone (LH) and follicle-stimulating hormone (FSH), and inhibits apoptosis in ovarian follicles, which can cause cyst formation (9, 10). The insulin receptor, a member of tyrosine kinase receptor family, consists of two a, b dimers that are linked by disulfide bonds. It is known that the a subunit contains a ligand-binding domain and that the b subunit has an associated tyrosine kinase activity (11). Binding of an insulin molecule activates the kinase activity of the receptor, and autophosphorylation of specific tyrosine residues occurs (12). Consequently, insulin receptor substrates (IRS) that have tyrosine-phosphorylated sites are phosphorylated (13, 14). To date, nine IRSs including IRS-1, IRS-2, IRS-3, IRS-4, Gab-1, p62dok and three isoforms of Shc have been identified (15). Several SH2 domain proteins, including phosphatidylinositol 3-kinase (PI3-kinase), bind to the tyrosine-phosphorylated site on the IRSs, inducing the activation of downstream signal pathways (12–16). Activation of PI3-kinase results in translocation of the insulin-responsive glucose transporter (GLUT4), which acts in glucose uptake, and activation of Akt (protein kinase B) and p70S6 kinase, which regulate glycogen synthesis (17–19). In addition to these responses, direct phosphorylation of Shc activates the Ras complex; the consequent activation of molecules involved in this signal transduction pathway occurs and then regulates the mitogenic actions of insulin (12, 15). As the insulin receptor (INSR) has an important role in insulin signaling, it is possible that single nucleotide

Fertility and Sterility Vol. 89, No. 5, May 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.

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polymorphisms in the INSR gene may be responsible for its abnormal function and may be associated with PCOS. Because INSR may be involved in PCOS, we investigated the effects of nine polymorphisms, þ109482 A>G, þ109665 C>T, þ125498 A>G, þ127527 G>A, þ143485 G>C, þ161822 G>A, þ168606 C>T, þ168828 T>A, and þ176477 C>T in the INSR gene in Korean patients with PCOS.

MATERIALS AND METHODS Patients For our patient group, we recruited 134 women with a PCOS diagnosis, based on the 2003 ASRM/ESHRE Rotterdam consensus revised diagnostic criteria (3, 4). We also recruited 100 healthy women who had a similar body mass index (BMI) as the control group (Table 1). All participants underwent a medical history, physical and pelvic examination, and complete blood chemistry. For the diagnostic criteria of PCOS, oligomenorrhea was defined as a reduction in the frequency of menses with intervals between 40 days and 6 months, and hyperandrogenism was defined as serum testosterone levels >0.6 ng/mL and/or serum dehydroepiandrosterone sulfate (DHEAS) levels R300 mg/dL (20, 21). All PCOS patients and controls in this study were Korean women, and all were recruited from the Fertility Center of CHA General Hospital in Seoul, South Korea. Written informed consent was obtained from all of the participants, and this study

with human blood samples was approved by an institutional review board. Blood samples were obtained from the women for biochemical assays and DNA sequencing analysis at 2 to 3 days after the start of menstruation. Blood samples for molecular genetic studies were collected in tubes containing ethylenediaminetetraacetic acid as an anticoagulant and were stored at 4 C. Genomic DNA was then extracted from the blood of patients with PCOS and the normal controls. Sequencing Analysis of the Human INSR We sequenced all exons of INSR to identify single nucleotide polymorphisms (SNPs) in 24 DNA samples from normal Korean women using the ABI PRISM 3730 DNA analyzer (Applied Biosystems, Foster City, CA). Twenty five primer sets for the amplification and sequencing analysis were designed based on GenBank sequences (Accession numbers: NM_000208 and NT_011255). Sequence variants were verified by chromatograms. Genotyping with TaqMan Analysis For genotyping of the polymorphic sites, amplifying primers and probes were designed for TaqMan analysis. Primer Express (Applied Biosystems) was used to design both the polymerase chain reaction (PCR) primers and the MGB TaqMan probes. One allelic probe was labeled with FAM dye and the

TABLE 1 Clinical and biochemical profiles of 100 normal controls and 134 patients with polycystic ovary syndrome (PCOS). Characteristics

Normal control (n [ 100)

PCOS patient (n [ 134)

BMI (kg/m2) Waist/hip ratio (WHR) Obesitya Polycystic ovaries and oligo- or amenorrhea Oligo- or amenorrhea and hyperandrogenism Polycystic ovaries, oligo- or amenorrhea, and hyperandrogenism FSH levels (mIU/mL) LH levels (mIU/mL) E2 levels (pg/mL) Prolactin levels (ng/mL) TSH levels (mIU/mL) DHEA-S levels (mg/dL) Testosterone (ng/mL) Fasting glucose (mg/dL) Insulin (mIU/mL)

20.73  2.36 (16.61–27.70) 0.80  0.07 (0.71–0.91) n ¼ 5 (5.00%) n ¼ 0 (0.00%)

23.22  3.88 (16.18–37.32) 0.81  0.09 (0.67–1.49) n ¼ 33 (24.63%) n ¼ 104 (77.61%)

n ¼ 0 (0.00%)

n ¼ 9 (6.72%)

n ¼ 0 (0.00%)

n ¼ 21 (15.67%)

6.54  3.04 (3.20–17.70) 3.24  1.48 (1.12–7.10) 32.10  14.37 (4.24–63.40) 12.11  5.84 (4.10–24.40) 2.28  1.17 (0.30–5.38) 157.86  64.22 (56.70–300.00) 0.28  0.13 (0.10–0.80) 91.00  11.24 (74.00–114.00) 12.88  6.33 (5.10–23.50)

5.81  4.53 (1.00–40.00) 8.37  6.45 (1.00–39.00) 40.2  21.45 (2.20–118.00) 12.07  4.47 (5.30–23.70) 2.28  1.15 (0.62–5.47) 199.10  77.50 (57.50–377.20) 0.68  0.56 (0.10–3.71) 93.88  13.63 (70.00–127.00) 13.93  4.20 (5.80–24.41)

a

BMI (body mass index) R25 kg/m2.

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other with fluorescent VIC dye. The PCR analyses were performed in the TaqMan Universal Master mix without uracilDNA glycosylase (UNG) (Applied Biosystems), and with PCR primer concentrations of 900 nM and TaqMan MGBprobe concentrations of 200 nM. Reactions were performed in a 384-well format in a total reaction volume of 5 mL using 20 ng of genomic DNA. The plates then were placed in a thermal cycler (PE 9700; Applied Biosystems) and heated at 50 C for 2 minutes and 95 C for 10 minutes followed by 40 cycles of 95 C for 15 seconds and 60 C for 1 minute. The TaqMan assay plates were transferred to a Prism 7900HT instrument (Applied Biosystems) where the fluorescence intensity in each well of the plate was read. Fluorescence data files for each plate were analyzed using automated software (SDS 2.1; Applied Biosystems). Association Test The genotype distributions of the INSR polymorphisms in PCOS patients and normal controls, and the association studies were analyzed with HapAnalyzer (NGRI, Seoul, South Korea; www.hap.ngri.re.kr) (22). P<.05 was considered statistically significant. Haplotype Analysis To test whether haplotypes in the INSR gene have an association with PCOS, we analyzed haplotypes composed of

SNPs, which were evaluated in this study by the use of HapAnalyzer (NGRI). Using the PL-EM program (Algorithm for Haplotype Construction of Single Nucleotide Polymorphism; Harvard University, Boston, MA; www.people.fas.harvard.edu/junliu/plem/click.html), we identified several haplotypes and their frequencies. Among the haplotypes analyzed, we selected five haplotypes that had frequencies over 0.05 and then performed an association test. RESULTS We have sequenced all exons of INSR to identify SNPs in samples from normal Korean patients using the ABI PRISM 3730 DNA analyzer. We identified 34 SNPs, and nine SNPs, including þ109482 A>G, þ109665 C>T, þ125498 A>G, þ127527 G>A, þ143485 G>C, þ161822 G>A, þ168606 C>T, þ168828 T>A, and þ176477 C>T. These were selected for a larger scale genotyping on the basis of linkage disequilibrium (LD) with other sites, allele frequencies, location, and haplotype-tagging status (Fig. 1). To analyze frequencies of genotypes for each SNP using TaqMan analysis, we used 134 samples from patients with PCOS and 100 control samples for this study. Because of sequencing errors, a few samples were not accounted for in the analysis. Table 1 shows the clinical and biochemical profiles of the PCOS patients and the normal control group. We identified

FIGURE 1 Map of INSR on chromosome 19p13.3/19p13.2 (177 kb). The black blocks indicate coding the exons and the white blocks indicate 50 and 30 UTR. 9 sites of SNPs marked with asterisks (*) were genotyped in a larger Korean population. Novel SNPs identified in this study are indicated using bold face. Numbers in parenthesis are the frequencies of minor alleles for each SNP.

Lee. Association between SNPs in INSR gene and PCOS. Fertil Steril 2008.

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specific characteristics of PCOS including obesity, polycystic ovaries, oligomenorrhea, amenorrhea, hyperandrogenism, and high levels of LH, DHEA-S, and testosterone. In the normal control group, 93% had no symptoms of PCOS. Although 5% of the women had irregular menstrual periods, and 2% had polycystic ovaries, they presented with only a single symptom; on the basis of the 2003 ASRM/ESHRE Rotterdam consensus criteria, we enrolled them into the normal control group. Table 2 shows results of genotyping using TaqMan analysis. All of the women in the PCOS patient and control groups had a major genotype for two SNPs, þ109482 A>G and þ168828 T>A. The genotypic distributions for the other SNPs also were similar between the PCOS patient and normal control groups, including þ109665 C>T, þ125498 A>G, þ127527 G>A, þ143485 G>C, þ161822 G>A, and þ168606 C>T. It is interesting that for the analysis of the novel SNP þ176477 C>T, we found the frequency of a minor allele T was lower in the PCOS patient group than in the control group (18.04% in the PCOS group and 28.28% in a control group, P¼.0401). After the analysis of genotypic frequencies of SNPs and their association with PCOS, we analyzed haplotypes that consisted of the SNPs that were identified in this study (Table 3). Among several haplotypes, we selected five haplotypes that have frequencies over 0.05; other haplotypes were included in ‘‘others.’’ After the selection of haplotypes with high frequencies, we analyzed the association between the selected haplotypes and PCOS. From these analyses, we found that these haplotypes are not associated with PCOS in this Korean population. P<.05 was considered statistically significant, and P>.05 for all codominant, dominant, and recessive of selected haplotypes. DISCUSSION It has been suggested that increased serine phosphorylation of the insulin receptor and downstream signaling molecules can cause insulin resistance in patients with PCOS (18). The IRS-1 can be serine-phosphorylated by protein kinase C (PKC), which can be activated by free fatty acids (FFAs) (15, 23, 24). Tumor necrosis factor a (TNF-a) induces serine-phosphorylation of IRS-1 resulting in insulin resistance, and plasma cell differentiation factor-1 (PC-1) inhibits the tyrosine kinase activity of the insulin receptor (25, 26). Although INSR has an important role in insulin signaling, which has an involvement in PCOS, several reports have suggested that the association between the INSR gene and PCOS is not clear. Studies with several markers for the insulin receptor have indicated that marker D19S884 on chromosome 19p13.2 may regulate transcription of INSR and has an association with PCOS (27–29). In contrast, a study with women from Spain and Italy identified that the region for marker D19S884 has no statistically significant association with PCOS (30). From studies with SNPs for exon 17 of the INSR gene, investigators from China suggested that the T to C variation in exon 17 (1008 bp) leads to an increased risk of insulin resistance in women with 1216

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PCOS, and investigators from both China and the United States have showed that a C to T variation in exon 17 (1058 bp) has an association with PCOS in a lean patient group based on restriction fragment length polymorphism (RFLP) analysis (31–33). However, investigators in both groups considered the C allele, which results from an undigested PCR product of 317 bp, which should be the T allele. This indicates that the results from both groups should be reconsidered with regard to the association of this SNP and PCOS. Recently, we identified that a C to T variation in exon 17 (1085 bp) is not associated with PCOS in a Korean population (34). Our study analyzed several candidate SNPs found in the INSR gene to determine if they have an association with PCOS in this Korean population. Among nine SNPs, þ109482 A>G, þ109665 C>T, þ125498 A>G, þ127527 G>A, þ143485 G>C, þ161822 G>A, þ168606 C>T, þ168828 T>A, and þ176477 C>T, genotypic distributions for all SNPs except a novel SNP þ176477 C>T, were similar; they were found to have no statistically significant association with PCOS. From the direct sequencing analysis for þ168606 C>T (SNP ID Number: rs1799817), our previous report was confirmed, indicating that the association test for the 1085His C>T polymorphism by RFLP analysis was reliable (34). In a previous study, we performed RFLP analysis for genotyping and found no association between the 1085His C>T polymorphism in exon 17 of the INSR gene and PCOS in a Korean population (34). Although we could not detect any statistically significant association of þ168606 C>T with PCOS in the Korean population, we did identify a novel SNP in the INSR gene (þ176477 C>T) that shows an association with PCOS. It is interesting that the frequency of this minor allele was higher in a control group than in the PCOS patient group at a statistically significant level (P¼.0401). From this result, we suggest that the minor allele T in þ176477 C>T of the INSR gene may have a protective effect for the pathogenesis of PCOS in this Korean population. However, the association between a novel SNP (þ176477 C>T) in the INSR gene and PCOS in other ethnic backgrounds remains to be investigated. Generally, it has been known that insulin signaling involving the insulin receptor pathway is related to the development of obesity; thus, many patients with PCOS are obese (27, 35–38). This suggests that polymorphisms in the INSR gene may be associated with PCOS patients who are obese. This led us to investigate the relationship between SNPs and PCOS with obesity in this Korean population. However, most of the women in both the normal control and PCOS patient groups had a body mass index (BMI) within the normal range, and the number of obese participant (Table 1; normal, n ¼ 5; PCOS, n ¼ 33) was too small to determine the association. In addition to association tests for SNPs in the INSR gene, we analyzed haplotypes that consisted of the SNPs identified in this study. However, the statistical analysis showed that these haplotypes were not associated with PCOS.

Association between SNPs in INSR gene and PCOS

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TABLE 2 The frequencies of genotypes of the analyzed single nucleotide polymorphisms. Co-dominant Loci þ109482

þ109665

þ125498

þ127527

þ143485

þ161822

þ168606

þ168828

þ176477

Dominant

Recessive

Genotype

Case (PCOS)

Control

OR (95%CI)

P

OR (95%CI)

P

OR (95%CI)

P

AA AG GG total CC CT TT total AA AG GG total GG GA AA total GG GC CC total GG GA AA total CC CT TT total TT TA AA total CC CT TT total

131 (100%) 0 (0%) 0 (0%) 131 100 (74.63%) 32 (23.88%) 2 (1.49%) 134 120 (90.23%) 11 (8.27%) 2 (1.5%) 133 77 (57.9%) 45 (33.83%) 11 (8.27%) 133 66 (50.38%) 50 (38.17%) 15 (11.45%) 131 101 (75.37%) 30 (22.39%) 3 (2.24%) 134 63 (47.73%) 59 (44.7%) 10 (7.57%) 132 133 (100%) 0 (0%) 0 (0%) 133 109 (81.96%) 23 (17.29%) 1 (0.75%) 133

99 (100%) 0 (0%) 0 (0%) 99 80 (80.81%) 18 (18.18%) 1 (1.01%) 99 87 (87.88%) 10 (10.1%) 2 (2.02%) 99 66 (66%) 28 (28%) 6 (6%) 100 46 (46.94%) 39 (39.8%) 13 (13.26%) 98 82 (82%) 17 (17%) 1 (1%) 100 46 (46%) 40 (40%) 14 (14%) 100 100 (100%) 0 (0%) 0 (0%) 100 71 (71.72%) 24 (24.24%) 4 (4.04%) 99

–

–

–

–

–

–

1.39 (0.77–2.50)

.2726

– 1.43 (0.76–2.70)

.2672

1.48 (0.13–16.61)

.7483

0.82 (0.42–1.62)

OR (95% CI) ¼ 1.38 (0.77–2.47), P¼ .3425 .5707 0.79 (0.34–1.80) .5692 0.74 (0.10–5.35)

.7658

1.31 (0.86–2.00)

OR (95% CI) ¼ 0.79 (0.37–1.67), P¼ .6627 .2134 1.41 (0.82–2.42) .2092 1.41 (0.50–3.96)

.5112

0.90 (0.61–1.31)

OR (95% CI) ¼ 1.35 (0.86–2.10), P¼ .2277 .5688 0.87 (0.52–1.47) .6061 0.85 (0.38–1.87)

.6785

1.46 (0.82–2.62)

OR (95% CI) ¼ 0.89 (0.60–1.32), P¼ .6193 .2010 1.49 (0.78–2.83) .2261 2.27 (0.23–22.13)

.4813

0.83 (0.56–1.23)

OR (95% CI) ¼ 1.48 (0.82–2.66), P¼ .2456 .3541 0.93 (0.55–1.57) .7941 0.50 (0.21–1.19)

.1167

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–

0.55 (0.32–0.96)

OR (95% CI) ¼ 0.83 (0.56–1.23), P¼ .4046 – – –

.0360

– 0.56 (0.30–1.04)

.0662

–

–

0.18 (0.02–1.64)

.1277

OR (95% CI) ¼ 0.54 (0.31–0.94), P¼ .0401

1218 Lee et al.

TABLE 3

ht

D109665 C>T

D125498 A>G

D127527 G>A

D143485 G>C

D161822 G>A

D168606 C>T

D168828 T>A

D176477 C>T

Co-dominant D109482 A>G

Association between SNPs in INSR gene and PCOS

Haplotypes of the INSR gene.

Frequency

ht1

A

C

A

G

G

G

C

T

C

0.33

0.79 (0.52–1.19)

ht2

A

C

A

G

G

G

T

T

C

0.13

1.42 (0.80–2.51)

ht3

A

C

A

G

C

G

C

T

C

0.11

0.81 (0.44–1.50)

ht4

A

C

A

G

G

G

T

T

T

0.09

0.64 (0.33–1.21)

ht5

A

C

A

G

C

A

C

T

C

0.05

1.05 (0.46–2.39)

Others

–

–

–

–

–

–

–

–

–

0.29

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Lee. Association between SNPs in INSR gene and PCOS. Fertil Steril 2008.

OR (95% CI)

P .256

Dominant

OR (95% CI)

Recessive

P

OR (95% CI)

0.67 .1407 1.00 (0.39–1.14) (0.40–2.48) OR (95% CI) ¼ 0.80 (0.54–1.19), P¼ .3155 .2296 1.43 .2638 2.28 (0.76–2.68) (0.23–22.27) OR (95% CI) ¼ 1.42 (0.80–2.51), P¼ .2853 .5014 0.76 .4051 N/A (0.41–1.44) OR (95% CI) ¼ 0.83 (0.46–1.48), P¼ .6215 .1657 0.62 .1917 0.37 (0.30–1.27) (0.03–4.14) OR (95% CI) ¼ 0.62 (0.32–1.19), P¼ .2 .9051 1.19 .7033 0.00 (0.49–2.87) (0.00–¢) OR (95% CI) ¼ 1.05 (0.46–2.42), P¼1 –

P 1

.4784

.9983

.4199

.9983

In our study, nine candidate SNPs were found in the INSR gene: þ109482 A>G, þ109665 C>T, þ125498 A>G, þ127527 G>A, þ143485 G>C, þ161822 G>A, þ168606 C>T, þ168828 T>A, and þ176477 C>T. Five haplotypes are not associated with PCOS, but the minor allele of the novel SNP þ176477 C>T has an association with the pathogenesis of PCOS in this Korean population. Because a number of PCOS patients show not only obesity but also type 2 diabetes, which results from abnormal insulin signaling, studies of SNPs in several genes involved in insulin signaling pathway and adipogenesis are required for a better understanding of PCOS. Therefore, further association studies of candidate SNPs of the genes involved in the insulin signaling pathway in various ethnic backgrounds are required to find SNPs related to the etiology of PCOS.

15.

16.

17. 18.

19.

20. Acknowledgments: The authors thank Byung Lae Park for his advice in our statistical analysis and members of the Fertility Center and Cell and Gene Therapy Research Institute at Pochon CHA University and CHA General Hospital.

21.

22.

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Association between SNPs in INSR gene and PCOS

Vol. 89, No. 5, May 2008