This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Powell, B. L. Articles by McCarthy, M. I. Search for Related Content PubMed PubMed Citation Articles by Powell, B. L. Articles by McCarthy, M. I. Related Collections Diabetes and Insulin Female Endocrinology

Analysis of Multiple Data Sets Reveals No Association between the Insulin Gene Variable Number Tandem Repeat Element and Polycystic Ovary Syndrome or Related Traits

Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN, and Oxford Centre for Diabetes, Endocrinology, and Metabolism (B.L.P., A.B., C.J.G., E.Z., M.I.M.), Churchill Hospital, Oxford OX3 7LJ, United Kingdom; Genomic Medicine Faculty of Medicine, (L.H., N.G., S.H., M.I.M.), and Institute of Reproductive and Developmental Biology (S.F.), Imperial College (Hammersmith Campus), London W12 0NN, United Kingdom; Reproductive Endocrinology Group, Department of Obstetrics and Gynaecology (K.R., M.J.G., S.F.) and Departments of Epidemiology and Public Health (U.S., M-R.J.), Imperial College (St. Mary’s Campus), London W2 1PG, United Kingdom; Department of Endocrinology (G.S.C.), University College, London W1T 3AA, United Kingdom; Departments of Clinical Chemistry (A.R., S.T.), Obstetrics and Gynecology (S.T., H.M., A.P., A.-L.H.), and Public Health Science and General Practice (M.-R.J., S.T., A.P.), Oulu University Hospital and University of Oulu, Oulu FIN-90014, Finland

Address all correspondence and requests for reprints to: Professor Mark McCarthy, Robert Turner Professor of Diabetes, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Churchill Hospital Site, Old Road, Headington, Oxford OX3 7LJ, United Kingdom. E-mail: mark.mccarthy{at}drl.ox.ac.uk.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Variation at the insulin gene VNTR (variable number tandem repeat) minisatellite has been reported to be associated with polycystic ovary syndrome (PCOS), but findings have been inconsistent and all studies have featured small sample sizes.

Objective: To gain a robust understanding of the role of the INS-VNTR in PCOS susceptibility.

Design: Case-control, family-based association and quantitative trait analyses.

Setting and Participants: A UK population comprising 255 parent-offspring trios, 185 additional cases, and 1062 control subjects (cases and controls all British/Irish) as well as 1599 women from a northern Finland population-based birth cohort characterized for PCO symptomatology and testosterone levels. VNTR class was inferred from genotyping of the –23HphI variant.

Intervention(s): None.

Main Outcome Measure(s): INS-VNTR genotype frequencies between subject groups, body mass index, and testosterone levels by genotype.

Results: Case-control analyses in both UK and Finnish samples failed to confirm previously reported class III allele associations with PCOS (UK, P = 0.43, Finnish, P = 0.31; Kruskal-Wallis ²). Transmission analysis in trios showed no excess transmission of either allele (P = 0.62), regardless of parent of origin (maternal: P = 0.73; paternal: P = 0.66). No association between genotype and testosterone levels was seen in any sample (UK PCOS subjects, P = 0.95; Finnish symptomatic cases, P = 0.38; Finnish control women, P = 0.58).

Conclusions: Despite the strong biological candidacy and supportive data from previous studies, we conclude that variation at the INS-VNTR has no major role in the development of PCOS.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
POLYCYSTIC OVARY SYNDROME (PCOS) is a common endocrine disorder affecting between 5 and 10% of women of reproductive age. The etiology of this condition remains controversial, although it is clear that disturbances of insulin secretion and action play an important part in sustaining the ovarian androgen hypersecretion, which is a key feature of this condition (1). Individuals with PCOS share many metabolic abnormalities that are characteristic of type 2 diabetes, including alterations in both insulin action and ß-cell function (2). In addition, women with PCOS are at substantially increased risk of developing type 2 diabetes and other components of the metabolic syndrome (3).

Despite familial clustering and other evidence that PCOS has a significant heritable component (1), progress with the identification of specific susceptibility genes has been slow. Of the many candidate genes and variants studied, some of the strongest evidence for association has been obtained for the variable number tandem repeat (VNTR) minisatellite element, which lies 5' to the insulin gene (4). Sequence and length diversity within this highly polymorphic element modifies insulin transcription in vitro (5, 6), raising the possibility that variation at this site might contribute to the disordered insulin secretion that is a feature of PCOS.

Waterworth et al. (7) were the first to provide evidence for a role of the insulin gene in PCOS susceptibility. They reported linkage of the insulin gene region on chromosome 11p15.5 and an association between VNTR class III alleles and the subset of anovulatory PCOS subjects. Analysis of a small number (n = 56) of parent-offspring trios suggested that transmission of the class III allele to affected offspring substantially favored the paternally derived allele (8), a finding consistent with known imprinting in the region (9). Given the physiological and epidemiological overlap between PCOS and type 2 diabetes (10), the credibility of these associations was increased by independent evidence suggesting that the paternally derived class III allele had a significant impact on susceptibility to type 2 diabetes (11).

Studies seeking to replicate this finding have generated conflicting results. A study of 74 UK women with polycystic ovaries (PCOs) reported an association between the class III allele and increasing severity of clinical phenotype (12). In contrast, analysis of 104 North American trios (13) and a small case-control sample from the Czech Republic (38 anovulatory women with PCOS; 22 healthy controls) (14) found no association.

However, the striking feature of all of these studies is that sample sizes were, at best, modest, especially in the context of the current understanding of the limited effect sizes to be expected for complex trait susceptibility loci. Small sample size is known to be a significant risk factor for the generation of association results that cannot be replicated on subsequent examination (15).

To obtain more robust estimates of the influence of INS-VNTR variation on susceptibility to PCOS and on PCOS-related traits [including androgen levels and body mass index (BMI)], we analyzed these relationships in more than 3500 subjects.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
UK subjects

Women with PCOS were ascertained from infertility and endocrine clinics, predominantly at St. Mary’s and the Middlesex Hospitals in London. Women were considered affected if they had polycystic ovaries on ultrasound (16), after presentation with menstrual disturbances (oligo- or amenorrhea) and/or hyperandrogenism. A diagnosis of hyperandrogenism required clinical (presence of hirsutism or acne) and/or biochemical evidence [serum total testosterone > 0.78 ng/ml (2.7 nmol/liter)]. Other potential endocrine and neoplastic causes of hyperandrogenemia were excluded (17). This diagnosis is consistent with the recent consensus on the diagnostic criteria for PCOS (17), i.e. presence of two of the following three features: clinical symptoms, ultrasonographic examination, and biochemical data. A total of 255 nuclear families (79% of British/Irish origin), comprising 229 complete families [both parents and at least one affected daughter (proband) with or without extra affected/unaffected siblings] and 26 part-families (only one parent available) were genotyped, together with 185 additional singleton cases (PCOS subjects with no relatives available; all of UK British/Irish origin). Family relationships in nuclear families were confirmed by genotyping with a panel of highly polymorphic microsatellite markers. Quantitative trait analyses (for BMI and testosterone) were performed in all British/Irish PCOS subjects (trios probands plus cases combined, after prior confirmation that there were no significant genotype or trait differences between the two component groups) with relevant data (n = 371 for BMI; n = 223 for testosterone, after exclusion of women taking hormonal therapy or metformin).

Two separate population-based samples were used as controls for the case-control design. The first consisted of DNA (n = 550) from a random UK sample (British UK origin) of blood donors available from the Centre for Applied Microbiological Research (CAMR, Salisbury, UK). The second sample consisted of DNA (n = 512) obtained from a random selection of women from the British 1958 birth cohort (B58C; http://www.cls.ioe.ac.uk/Cohort/Ncds/mainncds.htm). Metabolic and hor-monal status was not known for these individuals. It is well recognized that the misclassification that results from the use of population-based controls (rather than subjects in whom a diagnosis of PCOS has been excluded through relevant biochemical and endocrine tests) causes only a modest reduction in power and that this can readily be overcome by increasing the number of control subjects typed (18).

Finnish subjects

The Northern Finland Birth Cohort of 1966 comprises 12,058 subjects live born in the northernmost two provinces of Finland after pregnancies with expected dates of delivery during 1966. From this cohort, DNA and clinical information at age 31 was available for 5753 members (2975 women). For the current study, analyses were restricted to 1599 females who had been characterized for polycystic ovary symptomatology and successfully genotyped for the –23HphI variant (19). These comprised 530 women who had reported symptoms of hirsutism and/or oligo/amenorrhea at the 31-yr postal questionnaire (designated as symptomatic women) and 1069 women (cohort controls) randomly selected from among the cohort females reporting no such symptoms (generating an approximate 1:2 ratio of symptomatic women to controls, all matched for age given the birth cohort design). Women on oral contraceptives or using hormonal intrauterine devices were not included in either sample group. The symptomatic women have been shown as a group to have biochemical features consistent with PCOS (hyperandrogenemia, insulin resistance) (20), but, to date, in only a proportion (72 of 186 studied) has PCOS been confirmed by ultrasound scan (USS) (21). These 72 individuals are referred to as USS-confirmed cohort cases. Data were available for analyses of BMI in 977 cohort controls and 488 symptomatic women (after excluding women pregnant at the time of examination). For analysis of testosterone levels, numbers were further reduced to 972 for the cohort controls and 479 for the symptomatic women.

Clinical features of these sample sets are provided in Table 1. All clinical investigations were conducted in accordance with the guidelines in The Declaration of Helsinki, and the study was approved by the relevant ethics committees in the United Kingdom and Finland. All subjects provided fully informed consent.

In UK PCOS women, testosterone was measured with an in-house RIA using ether extraction and dextran-coated charcoal separation. In the Finnish subjects, serum testosterone was determined by automated chemiluminescence system (Ciba-Corning ACS-180; Diamond Diagnostics, Holliston, MA). SHBG was measured using the Immulite method (Diagnostic Products, Llanberis, UK). Free androgen index (FAI) was calculated as 100 x total testosterone/SHBG.

Genotyping

UK subjects. Genomic DNA was extracted from whole blood using the PUREGENE kit (Gentra Systems, Minneapolis, MN). The INS-VNTR locus was typed indirectly using a tetra-primer Amplification Refractory Mutation System (ARMS) assay (22) for the –23HphI polymorphic site upstream of the insulin gene. In Europeans, as in all other non-African populations, the INS-VNTR is in tight linkage disequilibrium with –23HphI (23). PCR was performed in a 10-µl volume containing 30 ng of genomic DNA, 200 µmol deoxynucleotide triphosphates, 2.5 mM MgCl₂, 1 U Amplitaq Gold Taq polymerase (Applied Biosystems, Foster City, CA), 5 µmol of the inner primers, and 0.5 µmol of the outer primers. Details of PCR conditions are available from the authors. The PCR product was loaded on a 2% agarose gel and visualized using ethidium bromide staining under UV light. There were no discrepancies in assignment of genotypes in 31 duplicate pairs. Approximately 45% of the samples were also genotyped using Pyrosequencing (Biotage, Uppsala, Sweden AB). Discrepant genotyping calls between the two methods were resolved by sequencing. We estimate the residual error rate to be less than 1%.

Finnish subjects. Genomic DNA was obtained from whole blood using phenol-chloroform extraction. The INS-VNTR locus was duplicate typed using PCR-restriction fragment-length polymorphism and a mass spectrometry assay (Sequenom, San Diego, CA) as previously described (19). Overall genotyping error rate for the Finnish study is estimated at less than 0.1%.

Statistical analyses

To test for genotypic associations in case-control analyses, the Kruskal-Wallis ² statistic was used. As necessary, exact significance values were obtained using StatXact 6 (Cytel Corp., Cambridge, MA). The pedigree disequilibrium test (PDT) was used to investigate the presence of association with the class III allele in families, making use of the full family structure (24). To detect parent-of-origin effects, trios (consisting of parents and one affected daughter) were analyzed using the extended transmission disequilibrium test (ETDT), examining paternal and maternal meioses separately (25). Quantitative trait analyses within unrelated British/Irish cases and in the Finnish samples (stratified by PCO symptomatic status) were conducted in SPSS (version 12.0 for Windows; SPSS Inc., Chicago, IL) using general linear models. BMI, testosterone, and FAI values were log transformed. Testosterone was analyzed with and without adjustment for BMI. Significance was assumed at the P = 0.05 level.

Power calculations

The UK case-control and transmission analyses had 92 and 85% power (respectively) at alpha = 0.05 (2-sided) to detect effect sizes [odds ratios (ORs)] of approximately 1.5, using a log-additive model. The power of the case-control comparison in the Finnish cohort was 98% under the same parameters (and remained above 70% assuming an OR of 1.3). Power to detect differences in testosterone levels differ with the sample size of each group, but in the Finnish control samples, between-genotype differences greater than 0.28 SD would have been detected (alpha = 0.05, power 95%).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
UK samples

Case-control analysis. In the case-control analysis, all samples were in Hardy-Weinberg equilibrium. There was no significant difference in genotype distribution between the cases and control groups (vs. CAMR controls, P = 0.65, vs. B58C controls, P = 0.31, vs. combined controls, P = 0.43; Kruskal-Wallis ², Table 2). Comparison of allele frequencies showed no difference in distribution of the class III allele [vs. CAMR controls, OR 1.02 (95% confidence interval [CI], 0.78, 1.33), P = 0.91, vs. B58C controls, OR 1.12 (0.85,1.46), P = 0.45, vs. combined controls, OR 1.07 (0.83,1.37), P = 0.64]. No association was seen when analysis was restricted to anovulatory cases (n = 126) (Table 2).

The PDT was used to perform family-based association analysis of the INS-VNTR locus. The INS-VNTR was in Hardy-Weinberg equilibrium in the probands and parents. There was no evidence for association between the INS-VNTR and PCOS (P = 0.66, PDT average test). Specifically, there was no evidence for excess class III allele transmission, within either trios (179 transmitted vs. 186 nontransmitted) or discordant sibling pairs (17 transmissions to affected and 10 transmissions to unaffected siblings). Similarly, there was no evidence for association in the anovulatory subset (P = 0.18, PDT average test).

In the complementary ETDT analysis, there were 201 informative transmissions. Again, no evidence of excess transmission of either allele was observed (P = 0.62). In addition, there was no suggestion of parent of origin effects in either the full sample (paternal 47.6% class III transmissions, P = 0.66; maternal 48.1%, P = 0.73) or the anovulatory subset (paternal 44.1%, P = 0.33; maternal 42.9%, P = 0.26) (Table 3).

BMI was analyzed with respect to genotype in the combined PCOS subjects (probands and cases, n = 371). There was no difference in mean BMI with respect to genotype distribution (P = 0.74). To test for association with testosterone, only women who were not undergoing any form of hormonal therapy or taking metformin were analyzed (n = 223). There was no association between genotype and mean testosterone levels with (P = 0.95) or without (P = 0.99) adjustment for BMI (Table 4). Similarly, no association was seen when FAI was used as an index of free testosterone (P = 0.38).

Case-control analysis. As previously observed (19, 26), the frequency of the class III allele is lower in Finns than other European populations. In the Finnish cohort, there was no significant association between INS-VNTR genotype and PCO symptomatic status (P = 0.31, Kruskal-Wallis ², Table 2). The class III allele was similarly distributed between symptomatic cases and controls, with an OR of 0.91 (95% CI 0.73, 1.13; P = 0.41). No association was seen when analysis was restricted to the USS-confirmed cohort cases (P = 0.09, Table 2).

Genotype/phenotype associations

Cohort controls and symptomatic women were analyzed separately with respect to BMI and testosterone levels. In symptomatic women, a nominally significant association with BMI was observed (P = 0.01), solely attributable to higher adiposity in heterozygotes. There was no replication of this association in Finnish control women (P = 0.76). When testosterone levels were examined, no association with genotype was seen, with or without adjustment for BMI (control women, P = 0.67, P = 0.58 adjusted; symptomatic women, P = 0.71, P = 0.38 adjusted) (Table 4).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
This is the largest study to date to analyze the relationship between INS-VNTR minisatellite variation and PCOS. We found no association with this condition or relevant continuous traits, most notably circulating levels of testosterone. These findings contrast with a number of previous studies that have reported significant positive associations between the class III VNTR allele and PCOS (7, 12) as well as paternal overtransmission of the class III allele from heterozygous fathers to affected daughters (8). These positive studies were consistent with other findings associating the class III allele with increased susceptibility to type 2 diabetes (11, 27) and altered insulin gene transcription in vitro (5, 6, 28). Studies that failed to detect PCOS associations (13, 14) involved modest sample sizes, consistent with the possibility of type 2 error. However, in recent years, inadequate sample size has become increasingly identified as a key contributor to the unreliability of much of the complex trait association literature (15): in this context, it is clear that all previous studies have been too small to offer any robust measurement of the true magnitude of any association between the INS-VNTR and risk of developing PCOS.

The current study employed substantially larger sample sizes and used several complementary analytical approaches, including case-control, family-based, and quantitative trait association methods. Each of the case-control studies and the transmission analysis had sufficient power (>85%) to detect an association, provided the effect size exceeded an OR of 1.5. Stratification of the case sample into those with and without regular menses further reduces sample size, but in some previous studies (7), INS-VNTR associations were evident only in the anovulatory subset. The family-based association analysis in the UK trios allowed us to avoid any adverse consequences arising from latent population stratification as well as providing the opportunity to detect parent-of-origin effects. Significant parent-of-origin effects have previously been detected at the INS-VNTR in relation to PCOS (8), type 1 diabetes (27, 28), type 2 diabetes (1), and childhood obesity (29) and are consistent with known imprinting in this region (9). To complicate matters, there is also strong evidence that the INS-VNTR region is prone to appreciable transmission ratio distortion, i.e. unequal transmission of parental alleles to unselected offspring (30). As a result, rates of class III transmission from heterozygous parents to unselected offspring are around 46% rather than the expected 50%. The modest (and decidedly nonsignificant) tendency for reduced class III transmission to PCOS offspring in the present study is entirely in line with these previous findings.

In both the UK PCOS cases and the Finnish birth cohort, we also sought evidence that INS-VNTR variation influences androgen levels. Direct stimulatory effects of insulin on ovarian steroidogenesis (31) appear to underlie the relationship between peripheral insulin resistance, ß-cell dysfunction, and ovarian hyperandrogenemia, which is characteristic of PCOS (1, 2). Despite the known effects of the INS-VNTR on insulin gene transcription in vitro (5, 6, 27), we found no genotype-related differences in testosterone levels. In the UK samples, this was true of both total and free testosterone (the latter as assessed by the FAI). In the Finns, only total testosterone levels were available, but adjustment for BMI (and therefore partial adjustment for undetected variation in SHBG levels) failed to generate any suggestion that INS-VNTR variation influences androgen levels. The nominal association with BMI in the Finnish symptomatic women is unlikely to have any biological significance because the deviation from expectation was solely attributable to increased BMI in heterozygotes. In addition, no evidence of this association was seen in UK PCOS subjects or the Finnish controls.

The overall impression from the current findings is that earlier reports have appreciably overestimated the effect of the INS-VNTR class III allele on PCOS susceptibility. It is important to realize that, whereas the current sample size is substantially larger than previous reports, the lack of association found in the current study cannot exclude a minor role of the INS-VNTR in PCOS. The present study was powered to detect only OR greater than 1.5 and differences in testosterone levels exceeding 0.3 SD. It is also important to recognize that differences between the Finnish and UK samples, together with the likely etiological heterogeneity of PCOS, may reduce power to detect association effects. Nevertheless, the consistency of the findings across the data sets studied here suggests this is not a major issue. Notwithstanding the above, the present study should clearly have detected effects on the scale suggested by the positive results in the previous literature.

An important related question is whether variants at the IGF2-INS-TH locus other than the INS-VNTR are involved in the modulation of PCOS risk. This seems unlikely on several counts. First, extensive studies of the linkage disequilibrium structure of the region (23) have shown that VNTR class (as defined by the –23HphI variant) tags the major haplotypic division in all non-African populations. Second, the evidence from type 1 diabetes points ever more strongly to the VNTR as the underlying susceptibility variant (32), although this does not, we recognize, necessarily imply that the same holds in PCOS and type 2 diabetes. Third, whereas some groups have suggested that variants within the neighboring IGF2 gene may contribute to variation in adiposity and other insulin-resistant traits (33), more recent studies, featuring an extensive haplotypic analysis of the IGF2-INS-TH region (34) indicate that much of the evidence for IGF2 association resides on class III-bearing haplotypes, once again focusing attention on the VNTR. Ultimately, large-scale studies of the full IGF2-INS-TH region will be necessary to exclude a role for additional variants.

In parallel with these studies in PCOS, large-scale association studies performed for other phenotypes previously reported to be associated with this minisatellite are inviting similar revision of the likely importance of INS-VNTR variation. Early positive associations between the class III allele and type 2 diabetes (11, 27, 35) and adiposity (29, 34) have not been substantiated in larger studies (Ref. 36 and our unpublished observations). Similarly, an effect of the INS-VNTR on early fetal growth (37) has not been replicated in studies of larger birth cohorts (19). Taken together, these data certainly cast serious doubt on the hypothesis that variation at the insulin gene locus contributes significantly to phenotypic variation in metabolic, endocrine, and ovarian function.

	Acknowledgments

 
We acknowledge the many patients, relatives, nurses, and physicians who contributed to the ascertainment of the various clinical samples used in this study.

	Footnotes

 
This work was supported by the UK Medical Research Council (program Grant G9700120), the Academy of Finland, the European Commission (Framework 5 award QLG1-CT-2000-01643), and the Wellcome Trust (project Grant GR069224MA). We acknowledge use of DNA from the British 1958 Birth Cohort collection, funded by the Medical Research Council Grant G0000934 and the Wellcome Trust Grant 068545/Z/02.

First Published Online February 10, 2005

Abbreviations: B58C, British 1958 birth cohort; BMI, body mass index; CI, confidence interval; ETDT, extended transmission disequilibrium test; FAI, free androgen index; OR, odds ratio; PCO, polycystic ovary; PCOS, polycystic ovary syndrome; PDT, pedigree disequilibrium test; USS, ultrasound scan; VNTR, variable number tandem repeat.

Received December 17, 2004.

Accepted February 2, 2005.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Franks S 1995 Polycystic ovary syndrome. N Engl J Med 333:853–861[Free Full Text]
2. Goodarzi MO, Erickson S, Port SC, Jennrich RI, Korenman SG 2004 ß-Cell function: a key pathological determinant in polycystic ovary syndrome. J Clin Endocrinol Metab 90:310–315
3. Legro RS, Kunselman AR, Dodson WC, Dunaif A 1999 Prevalence and predictors of risk for type 2 diabetes mellitus and impaired glucose tolerance in polycystic ovary syndrome: a prospective, controlled study in 254 affected women. J Clin Endocrinol Metab 84:165–169[Abstract/Free Full Text]
4. Franks S, Gharani N, McCarthy M 2001 Candidate genes in polycystic ovary syndrome. Hum Reprod Update 7:405–410[Abstract/Free Full Text]
5. Vafiadis P, Bennett ST, Colle E, Grabs R, Goodyer CG, Polychronakos C 1996 Imprinted and genotype-specific expression of genes at the IDDM2 locus in pancreas and leucocytes. J Autoimmun 9:397–403[CrossRef][Medline]
6. Bennett ST, Lucassen AM, Gough SC, Powell EE, Undlien DE, Pritchard LE, Merriman ME, Kawaguchi Y, Dronsfield MJ, Pociot F, Nerup J, Bouzekri N, Cambon-Thomsen A, Rønningen KS, Barnett AH, Bain SC, Todd JA 1995 Susceptibility to human type 1 diabetes at IDDM2 is determined by tandem repeat variation at the insulin gene minisatellite locus. Nat Genet 9:284–292[CrossRef][Medline]
7. Waterworth DM, Bennett ST, Gharani N, McCarthy MI, Hague S, Batty S, Conway GS, White D, Todd JA, Franks S, Williamson R 1997 Linkage and association of insulin gene VNTR regulatory polymorphism with polycystic ovary syndrome. Lancet 349:986–990[CrossRef][Medline]
8. Bennett ST, Todd JA, Waterworth DM, Franks S, McCarthy MI 1997 Association of insulin gene VNTR polymorphism with polycystic ovary syndrome. Lancet 349:1771–1772[Medline]
9. Moore GE, Abu-Amero SN, Bell G, Wakeling EL, Kingsnorth A, Stanier P, Jauniaux E, Bennett ST 2001 Evidence that insulin is imprinted in the human yolk sac. Diabetes 50:199–203[Abstract/Free Full Text]
10. Ovalle F, Azziz R 2002 Insulin resistance, polycystic ovary syndrome, and type 2 diabetes mellitus. Fertil Steril 77:1095–1105[CrossRef][Medline]
11. Huxtable SJ, Saker PJ, Haddad L, Walker M, Frayling TM, Levy JC, Hitman GA, O’Rahilly S, Hattersley AT, McCarthy MI 2000 Analysis of parent-offspring trios provides evidence for linkage and association between the insulin gene and type 2 diabetes mediated exclusively through paternally transmitted class III variable number tandem repeat alleles. Diabetes 49:126–130[Abstract]
12. Michelmore K, Ong K, Mason S, Bennett S, Perry L, Vessey M, Balen A, Dunger D 2001 Clinical features in women with polycystic ovaries: relationships to insulin sensitivity, insulin gene VNTR and birth weight. Clin Endocrinol (Oxf) 55:439–446[CrossRef][Medline]
13. Urbanek M, Legro RS, Driscoll DA, Azziz R, Ehrmann DA, Norman RJ, Strauss 3rd JF, Spielman RS, Dunaif A 1999 Thirty-seven candidate genes for polycystic ovary syndrome: strongest evidence for linkage is with follistatin. Proc Natl Acad Sci USA 96:8573–8578[Abstract/Free Full Text]
14. Vankova M, Vrbikova J, Hill M, Cinek O, Bendlova B 2002 Association of insulin gene VNTR polymorphism with polycystic ovary syndrome. Ann NY Acad Sci 967:558–565[Medline]
15. Ioannidis JP, Trikalinos TA, Ntzani EE, Contopoulos-Ioannidis DG 2003 Genetic associations in large versus small studies: an empirical assessment. Lancet 361:567–571[CrossRef][Medline]
16. Adams J, Polson DW, Franks S 1986 Prevalence of polycystic ovaries in women with anovulation and idiopathic hirsutism. Br Med J (Clin Res Ed) 293:355–359
17. The Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group 2004 Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 19:41–47[Abstract/Free Full Text]
18. Colhoun HM, McKeigue PM, Davey Smith G 2003 Problems of reporting genetic associations with complex outcomes. Lancet 361:865–872[CrossRef][Medline]
19. Bennett AJ, Sovio U, Ruokonen A, Martikainen H, Pouta A, Taponen S, Hartikainen AL, King VJ, Elliott P, Jarvelin MR, McCarthy MI 2004 Variation at the insulin gene VNTR (variable number tandem repeat) polymorphism and early growth: studies in a large Finnish birth cohort. Diabetes 53:2126–2131[Abstract/Free Full Text]
20. Taponen S, Martikainen H, Jarvelin MR, Laitinen J, Pouta A, Hartikainen AL, Sovio U, McCarthy MI, Franks S, Ruokonen A 2003 Hormonal profile of women with self-reported symptoms of oligomenorrhea and/or hirsutism: Northern Finland birth cohort 1966 study. J Clin Endocrinol Metab 88:141–147[Abstract/Free Full Text]
21. Taponen S, Ahonkallio S, Martikainen H, Koivunen R, Ruokonen A, Sovio U, Hartikainen AL, Pouta A, Laitinen J, King V, Franks S, McCarthy MI, Jarvelin MR 2004 Prevalence of polycystic ovaries in women with self-reported symptoms of oligomenorrhoea and/or hirsutism: Northern Finland Birth Cohort 1966 Study. Hum Reprod 19:1083–1088[Abstract/Free Full Text]
22. Ye S, Dhillon S, Ke X, Collins AR, Day IN 2001 An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res 29:e88
23. Stead JD, Jeffreys AJ 2002 Structural analysis of insulin minisatellite alleles reveals unusually large differences in diversity between Africans and non-Africans. Am J Hum Genet 71:1273–1284[CrossRef][Medline]
24. Martin ER, Monks SA, Warren LL, Kaplan NL 2000 A test for linkage and association in general pedigrees: the pedigree disequilibrium test. Am J Hum Genet 67:146–154[CrossRef][Medline]
25. Sham PC, Curtis D 1995 An extended transmission/disequilibrium test (TDT) for multi-allele marker loci. Ann Hum Genet 59(Pt 3):323–336
26. Metcalfe KA, Hitman GA, Fennessy MJ, McCarthy MI, Tuomilehto J, Tuomilehto-Wolf E 1995 In Finland insulin gene region encoded susceptibility to IDDM exerts maximum effect when there is low HLA-DR associated risk. DiMe (Childhood Diabetes in Finland) Study Group. Diabetologia 38:1223–1229[Medline]
27. Bennett ST, Todd JA 1996 Human type 1 diabetes and the insulin gene: principles of mapping polygenes. Annu Rev Genet 30:343–370[CrossRef][Medline]
28. Julier C, Hyer RN, Davies J, Merlin F, Soularue P, Briant L, Cathelineau G, Deschamps I, Rotter JI, Froguel P, Boitard C, Bell JI, Lathrop GM 1991 Insulin-IGF2 region on chromosome 11p encodes a gene implicated in HLA-DR4-dependent diabetes susceptibility. Nature 354:155–159[CrossRef][Medline]
29. Le Stunff C, Fallin D, Bougneres P 2001 Paternal transmission of the very common class I INS VNTR alleles predisposes to childhood obesity. Nat Genet 29:96–99[CrossRef][Medline]
30. Eaves IA, Bennett ST, Forster P, Ferber KM, Ehrmann D, Wilson AJ, Bhattacharyya S, Ziegler AG, Brinkmann B, Todd JA 1999 Transmission ratio distortion at the INS-IGF2 VNTR. Nat Genet 22:324–325[CrossRef][Medline]
31. Cara JF, Rosenfield RL 1988 Insulin-like growth factor I and insulin potentiate luteinizing hormone-induced androgen synthesis by rat ovarian thecal-interstitial cells. Endocrinology 123:733–739[Abstract/Free Full Text]
32. Barratt BJ, Payne F, Lowe CE, Hermann R, Healy BC, Harold D, Concannon P, Gharani N, McCarthy MI, Olavesen MG, McCormack R, Guja C, Ionescu-Tirgoviste C, Undlien DE, Ronningen KS, Gillespie KM, Tuomilehto-Wolf E, Tuomilehto J, Bennett ST, Clayton DG, Cordell HJ, Todd JA 2004 Remapping the insulin gene/IDDM2 locus in type 1 diabetes. Diabetes 53:1884–1889[Abstract/Free Full Text]
33. Gaunt TR, Cooper JA, Miller GJ, Day IN, O’Dell SD 2001 Positive associations between single nucleotide polymorphisms in the IGF2 gene region and body mass index in adult males. Hum Mol Genet 10:1491–1501[Abstract/Free Full Text]
34. Rodriguez S, Gaunt TR, O’Dell SD, Chen XH, Gu D, Hawe E, Miller GJ, Humphries SE, Day IN 2004 Haplotypic analyses of the IGF2-INS-TH gene cluster in relation to cardiovascular risk traits. Hum Mol Genet 13:715–725[Abstract/Free Full Text]
35. Ong KK, Phillips DI, Fall C, Poulton J, Bennett ST, Golding J, Todd JA, Dunger DB 1999 The insulin gene VNTR, type 2 diabetes and birth weight. Nat Genet 21:262–263[CrossRef][Medline]
36. Hansen SK, Gjesing AP, Rasmussen SK, Glumer C, Urhammer SA, Andersen G, Rose CS, Drivsholm T, Torekov SK, Jensen DP, Ekstrom CT, Borch-Johnsen K, Jorgensen T, McCarthy MI, Hansen T, Pedersen O 2004 Large-scale studies of the HphI insulin gene variable-number-of-tandem-repeats polymorphism in relation to type 2 diabetes mellitus and insulin release. Diabetologia 47:1079–1087[Medline]
37. Dunger DB, Ong KK, Huxtable SJ, Sherriff A, Woods KA, Ahmed ML, Golding J, Pembrey ME, Ring S, Bennett ST, Todd JA 1998 Association of the INS VNTR with size at birth. ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood. Nat Genet 19:98–100[Medline]

This article has been cited by other articles:

				M. Simoni, C.B. Tempfer, B. Destenaves, and B.C.J.M. Fauser Functional genetic polymorphisms and female reproductive disorders: Part I: polycystic ovary syndrome and ovarian response Hum. Reprod. Update, September 1, 2008; 14(5): 459 - 484. [Abstract] [Full Text] [PDF]

				S. Le Fur, C. Auffray, F. Letourneur, C. Cruaud, C. Le Stunff, and P. Bougneres Heterogeneity of class I INS VNTR allele association with insulin secretion in obese children Physiol Genomics, May 16, 2006; 25(3): 480 - 484. [Abstract] [Full Text] [PDF]

				K. Qin, D. A. Ehrmann, N. Cox, S. Refetoff, and R. L. Rosenfield Identification of a Functional Polymorphism of the Human Type 5 17{beta}-Hydroxysteroid Dehydrogenase Gene Associated with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., January 1, 2006; 91(1): 270 - 276. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Powell, B. L. Articles by McCarthy, M. I. Search for Related Content PubMed PubMed Citation Articles by Powell, B. L. Articles by McCarthy, M. I. Related Collections Diabetes and Insulin Female Endocrinology