No association of the insulin gene VNTR polymorphism with polycystic ovary syndrome in a Han Chinese population
© Xu et al; licensee BioMed Central Ltd. 2009
Received: 10 June 2009
Accepted: 01 December 2009
Published: 01 December 2009
Polycystic ovary syndrome (PCOS) is a common endocrine disorder associated with an increased risk of type II diabetes mellitus. The results of previous research about the association of the VNTR polymorphism in 5-prime flanking region of the insulin (INS) gene with PCOS have been inconsistent. The present study was to investigate the association of the INS-VNTR polymorphism with PCOS in a Han Chinese population.
The -23/HphI polymorphism as a surrogate marker of the INS-VNTR length polymorphism was genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) in 216 PCOS patients and 192 non-PCOS women as a control group. Allelic and genotypic frequencies were compared between patients and controls, and these results were analyzed in respect to clinical test data.
No significant differences were observed between the cases and controls groups either in allele (P = 0.996) or genotype (P = 0.802) frequencies of INS-VNTR polymorphism; Regarding anthropometric data and hormone levels, there were no significant differences between INS-VNTR genotypes in the PCOS group, as well as in the non-PCOS group.
The present study demonstrated for the first time that the INS-VNTR polymorphism is not a key risk factor for sporadic PCOS in the Han Chinese women. Further studies are needed to give a global view of this polymorphism in pathogenesis of PCOS in a large-scale sample, family-based association design or well-defined subgroups of PCOS.
Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting up to 4% of women of reproductive age. The disorder is characterized by hyperandrogenism, menstrual irregularities and often central obesity. Moreover, it is associated with an increased risk of type II diabetes mellitus . Colilla et al. demonstrated that there is a heritable component in beta-cell dysfunction in families of women with PCOS, and that heritability of beta-cell dysfunction is likely to be a significant factor in the predisposition to diabetes in PCOS .
The insulin (INS) gene locates at chromosome 11p15.5 and is one of established susceptibility locus to type II diabetes in Caucasians . In the 5-prime flanking region of the INS gene, a variable number of tandem repeat (VNTR) regulates transcription of the gene; the shortest (class I) alleles were found to increase, whereas the longest (class III) alleles were observed to decrease in the patients in comparison to the controls [4, 5]. It was reported that most class III alleles are associated with higher levels of INS transcription than class I alleles in the thymus [6, 7]. The higher INS expression may more efficiently induce tolerance to insulin. An A/T single nucleotide polymorphism (SNP) located at -23 bp is highly linked with the INS-VNTR length polymorphism. The SNP can be digested by the restriction endonuclease HphI and exhibits restriction fragment length polymorphism which is called "-23/Hph I polymorphism" as a surrogate marker of the INS-VNTR length polymorphism .
The association between the polymorphism and PCOS has been observed in Caucasians but the results were inconsistent. Waterworth et al were the first to provide evidence on a linkage of PCOS with the INS gene VNTR locus and an association between VNTR class III alleles and the subset of anovulatory PCOS subjects . A study of 74 UK women with PCOS reported an association between the class III allele and increasing severity of clinical phenotype . On the contrary, three independent research groups failed to replicate the association [11–13]. On account of these controversial reports, the association has to be confirmed by independent studies in different ethnic groups. The purpose of the present study is to examine the genetic association of the -23/Hph I polymorphism in the INS gene with PCOS in a Han Chinese population.
The subjects were recruited from patients who visited the Department of Obstetrics and Gynecology of the First Affiliated Hospital of Anhui Medical University during December 2005 and December 2006, including 216 PCOS patients and 192 non-PCOS women as a control group. The inclusion criteria of the PCOS group was based on the Rotterdam diagnostic criteria , while the eligibility criteria of non-PCOS women included: menstrual period < 35 d (most women had been pregnant or given birth to at least one child), precluding obesity, hirsutism, acne, excess sebum production and insulin resistance. All subjects were ethnic Han Chinese living in Anhui province and had not received hormonal therapy for at least 3 months before hormonal assays. After informed consent was obtained, blood was drawn from the subjects for hormonal assays and DNA analysis under the supervision of the Ethical Committee of the First Affiliated Hospital, Anhui Medical University. The peripheral blood was obtained between 8 AM and 9 AM after a 12-hour overnight fast during the 3rd to the 5th day of the menstrual cycle.
The level of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), total testosterone (T), estrogen (E2) and prolactin (PRL) were determined using Commercially available human chemiluminescence enzyme immunoassay kits (ROCHE DIAGNOSTICS GMBH, Mannheim, Germany). The intra- and inter-assay coefficients of variation of all the assays were less than 10%.
The standard X2 tests were used to compare allele or genotype frequencies in PCOS and non-PCOS groups and among different ethnic groups, and verify the Hardy-Weinberg equilibrium of genotype frequencies. The results of serum hormone levels were reported as MEANS ± SD. Differences in anthropometric data and serum hormone levels between PCOS and non-PCOS groups were assessed by using Student's t-test. P < 0.05 was considered significant for all tests. Statistical analyses were performed using the SPSS statistical software (SPSS Inc., Chicago, IL).
Anthropometric data and hormone levels of PCOS and non-PCOS groups
26.81 ± 4.25*
14.49 ± 1.62
23.08 ± 3.65*
6.38 ± 4.37
14.06 ± 8.38*
2.40 ± 1.27*
2.30 ± 1.09*
228.32 ± 166.28*
15.35 ± 8.53
31.53 ± 4.31
14.66 ± 2.41
21.27 ± 2.24
6.97 ± 2.08
4.46 ± 2.10
0.66 ± 0.32
1.65 ± 3.92
179.75 ± 143.00
18.42 ± 20.71
Allelic and genotypic frequencies of INS-23/Hph I polymorphism
Allele n (%)
Genotype n (%)
Anthropometric data and hormone levels of different INS genotypes in the PCOS and the non-PCOS groups
26.69 ± 4.28
27.94 ± 3.93
31.66 ± 4.27
30.74 ± 4.60
Menarch Age (years)
14.46 ± 1.58
14.88 ± 2.00
14.67 ± 2.52
14.57 ± 1.65
22.96 ± 3.55
24.30 ± 4.52
21.23 ± 2.16
21.49 ± 2.68
6.43 ± 4.55
5.88 ± 1.84
6.96 ± 2.17
7.05 ± 1.44
14.30 ± 8.64
11.68 ± 4.64
4.43 ± 2.09
4.64 ± 2.23
2.47 ± 1.29
2.12 ± 0.95
0.66 ± 0.31
0.68 ± 0.36
2.33 ± 1.29
1.99 ± 0.87
1.73 ± 4.22
1.24 ± 0.89
225.11 ± 163.90
260.93 ± 191.33
179.79 ± 139.70
179.53 ± 164.35
16.23 ± 12.15
16. 97 ± 8.31
16.97 ± 9.56
26.87 ± 49.01
The association of the VNTR polymorphism in 5-prime flanking region of the INS gene susceptible to type II diabetes with PCOS have been studied over the years in different populations, and the results observed have been inconsistent and controversial. No such data is available in the Han Chinese population. In the present study we have analyzed for the first time the association between the polymorphism of PCOS and anthropometric data and hormone levels.
Some of PCOS phenotypes have been linked to the INS-VNTR. Waterworth et al. were the first to provide the evidence on an association between VNTR class III alleles and the subset of anovulatory and hyperinsulinaemic PCOS subjects in a recessive genetic model . Ferk et al. also found that Class III INS-VNTR alleles were significantly more frequent in the Slovene PCOS patients and the interaction of obesity and the III/III INS VNTR genotype might be a risk factor in the development of PCOS .
In this study, we demonstrate that there is no association between INS-23/Hph I polymorphism and PCOS in the Han Chinese population. Moreover, further analysis showed that there was no significant difference regarding anthropometric data and hormone levels between INS-VNTR genotypes in the PCOS group, as well as in the non-PCOS group. The results were consistent to those previously reported [11–13]. After investigating 96 hyperandrogenic patients and 38 healthy control women, Calvo et al. concluded that there is no association between INS-VNTR and PCOS or hyperandrogenism, at least in Spanish women . Subsequently, in a large-scale research on 255 nuclear families and about 3000 subjects from two populations (Britisher/Irisher and Finlander), Powell et al. revealed that INS-VNTR was not a key factor in the pathogenesis and progress of PCOS . It should be noted that studies to date failed to confirm a significant association with the PCOS in general.
There are several possible explanations for these inconsistent results of association between the INS-VNTR and PCOS. Previous studies including this one showed the allele and genotype of the VNTR polymorphism in INS gene were different among ethical populations. Osawa et al. found the A allele frequency of -23/HphI polymorphism was 97.4% in Japanese subjects, whereas in Europeans it was about 30% . The results in this study showed the A allele frequency was about 93.5% (Table 2). Because of the predominance of class I over class III alleles in Asian population, further study with large-scale sample design is needed for us to ascertain the association between the INS-VNTR and PCOS. Case-control study is a common and powerful design to detect genetic contributions to complex diseases like PCOS. Unfortunately, a small portion of differences in allele frequencies between cases and controls could be attributable to diversity in background population, which is called population stratification. Family-based association designs like transmission disequilibrium test measure the over-transmission of an allele from heterozygous parents to affected offsprings. The designs do not use control groups and can overcome this disadvantage effectively , and would be an alternative way to assess the risk of PCOS.
In this study, we did not assess Insulin levels and Insulin resistance of the subjects. We will address the issue of whether subgroups of PCOS are associated with INS-VNTR polymorphism by investigating clinical and biochemical features related to glucose metabolism among PCOS women.
The present study demonstrated for the first time that the INS-VNTR polymorphism is not a key risk factor in sporadic PCOS in the Han Chinese women. Further studies are needed to give a global view of this polymorphism in pathogenesis of PCOS in a large-scale sample, family-based association design or well-defined subgroups of PCOS.
We thank Professor Faming Pan from Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University for statistical help. This work was supported by grants from the National Natural Science Foundation of China (30571954) and the Nature Basic Research Program of China (973 Program 2010CB945103).
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