1 Molecular Endocrinology Laboratory, Institut Universitaire de Recherche Cilnique, Montpellier, France
2 Department of Endocrinology, Lapeyronie Hospital, Montpellier, France
3 Joslin Diabetes Center, Boston, Massachusetts
ABSTRACT
To assess the role of insulin receptor, insulin receptor substrate (IRS)-1, and IRS-2 genes in insulin resistance, we explored the genomic DNA in women with polycystic ovary syndrome (PCOS) and a variable degree (mean ± SE) of insulin resistance (homeostasis model assessment index for insulin resistance [HOMAIR] 3.2 ± 0.6, n = 53; control subjects 1.56 ± 0.34, n = 102) using direct sequencing. Whereas no novel mutations were found in these genes, gene-dosage effects were found on fasting insulin for the Gly972Arg IRS-1 variant and on 2-h plasma glucose for the Gly1057Asp IRS-2 variant. The Gly972Arg IRS-1 variant was more prevalent in insulin-resistant patients compared with noninsulin-resistant individuals or control subjects (39.3 vs. 4.0 and 16.6%, P < 0.0031, respectively). A multivariate model that included BMI as a variable revealed significant effects of the Gly1057Asp IRS-2 variant on insulin resistance (P < 0.016, odds ratio [OR] 7.2, 95% CI 1.2943.3). HOMAIR was higher in carriers of both IRS variants than in those with IRS-2 mutations only or those with wild-type variants (6.2 ± 2.3, 2.8 ± 0.5, and 1.8 ± 0.2, respectively; P < 0.01), and it was significantly associated with this genotype (P < 0.0085, OR 1.7, 95% CI 1.092.99). We conclude that polymorphic alleles of both IRS-1 and IRS-2, alone or in combination, may have a functional impact on the insulin-resistant component of PCOS.
Severe insulin resistance is a prominent feature of rare genetic syndromes in humans and, albeit in a milder form, of common diseases such as obesity, type 2 diabetes, and polycystic ovary syndrome (PCOS) (1,2,3). Although clear evidence was provided for involvement of mutations in the insulin receptor (IR) in genetic Type A syndrome and leprechaunism, in complex diseases the role of IR remains elusive, and most of the defects in insulin action are expected at the postreceptor level (2). IR substrate (IRS) genes encoding for key proteins in insulin transduction are attractive candidates, particularly IRS-2, as suggested by recent transgenic animal models (4,5). In humans, the role of IRS genes was suggested by the identification of several more prevalent allelic variants in type 2 diabetes (6,7,8,9,10,11). PCOS is another good model to study influent genes, because in this complex disease, characterized by chronic anovulation and hyperandrogenism in women, insulin resistance is a major component (3,12). In common forms of PCOS, IR mutations have not been found, and linkage studies excluded IR and IRS-1 as major genes (13,14). To understand genetic determinants of insulin resistance, we investigated the effects of two variants of IRS-1 and IRS-2 in PCOS (n = 53) with values (mean ± SE) for homeostasis model assessment index for insulin resistance (HOMAIR) twofold higher than the control population (1.56 ± 0.034, n = 102) (Table 1). For the purpose of this study, the PCOS population was stratified into two groups with normal (group A) and elevated (group B) fasting insulin.
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When carriers of the Gly972 wild type variant of IRS-1 (G/G, n = 41) were compared with heterozygous Gly972Arg (G/C) carriers (n = 12), a gene-dosage effect was found on fasting insulin (12.16 ± 1.36 vs. 29.58 ± 9.71 µU/ml, P < 0.0031) or HOMAIR (2.37 ± 0.28 vs. 5.94 ± 2.16, P < 0.0065, Mann-Whitney U test). Similarly, when carriers of the wild- type Gly1057 variant of IRS-2 (G/G, n = 19) were compared with heterozygous (G/A) and homozygous (A/A) carriers of Gly1057Asp (n = 34), the gene-dosage effect was observed for 2-h glucose (5.16 ± 0.23 vs. 6.54 ± 0.47 mmol/l P <0.02) and 2-h insulin (42.4 ± 6.4 vs. 95.7 ± 14.5 µU/ml, P < 0.033, Mann-Whitney U test). None of the IRS-2 wild-type (G/G) carriers were glucose-intolerant, but 24% of the Gly1057Asp carriers displayed impaired glucose tolerance (IGT) (P < 0.032).
As shown in Table 3, when the population was stratified as a function of insulin resistance, the prevalence of the Gly972Arg IRS-1 variant in group B was 10-fold higher than in group A (P < 0.001) and 2.3-fold higher than in control women (n = 102, P < 0,0,031) or in the general population (15.2%, n = 224). The Gly1057Asp variant of IRS-2 also appeared more prevalent in group B than in group A, although the difference did not reach statistical significance. Some level of significance (not supported by Bonferroni correction) was reached when prevalence rates in group A and group B were compared with control women (Table 3). Multivariate analysis performed to explain differences between group A and group B indicated a role of BMI (P < 0.0001, odds ratio [OR] 1.2, 95% CI, 1.071.38) and an additional effect of IRS-2 variant (P < 0,016, OR 7.2, 95% CI 1.2943.3). Some effects may also be expected from the IRS-1 variant (P < 0.01, OR 12.5, 95% CI 1.2128.2), but CI values suggested inadequacy of the sample size. In addition to heterogeneity at the gene and phenotype levels, the understanding of IRS effects on insulin resistance seems complicated by the fact that 35.7% of the subjects in group B are carriers of both allelic variants (designated as double-mutated). Introduction in the multivariate model of a genetic variable corresponding to genotype I (carriers of the wild type IRS variants), genotype II (carriers of a mutation on IRS-2 only), and genotype III (carriers of mutations on both IRS-1 and IRS-2) indicated again the influence of the IRS-2 variant (P < 0.0003, OR 7.9, 95% CI 1.2650.6). The effect of the double mutation was difficult to estimate because of the inadequacy of the sample size (P < 0.0003, OR 79.2, 95% CI 4.91,260).
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RESEARCH DESIGN AND METHODS
Caucasian women of European extraction were recruited in accordance with the Helsinki Declaration (as revised in 1983) after informal consent. Diagnosis of PCOS was based on prolonged oligomenorrhea and/or amenorrhea and two of the following criteria: 1) hyperandrogenism (androstenedione and/or testosterone), 2) increased LH-to-FSH ratio (>2), and 3) criteria for PCOS in a transvaginal ultrasound scan (3,18). Patients with Cushings syndrome, nonclassical adrenal 21-hydroxylase deficiency, hyperprolactinemia, androgen-secreting neoplasms, Type A syndrome of severe insulin resistance (fasting insulin >100 µU/ml), and type 2 diabetes (fasting blood glucose >7 mmol/l) were excluded. Before DNA sampling, patients were maintained on a free diet containing 300 g carbohydrates daily and were submitted to a standard (75 g glucose) oral glucose tolerance test (OGTT). No upper limit was considered in the recruitment of obese (BMI >27 kg/m2) patients, whereas positive family history of diabetes was considered when at least first- or second-degree relatives had type 2 diabetes.
A population (n = 154) was registered over a period of 2 years, and 53 case subjects were consecutively included for the genetic study, based on the absence of medication before DNA sampling. They were recruited as group A (with normal fasting insulin) and group B (with high fasting insulin). The cutoff value was established at 10.8 µU/ml insulin, which represented the mean + 2 SD of insulin in 111 normal individuals (7.8 ± 1.7 µU/ml, mean ± SD). This value was close to the median (11 µU/ml insulin) of the PCOS population and corresponds to a stratification of hyperinsulinemic patients (group B) >50th percentile of fasting insulin (13).
Significant differences were detected between group A and group B concerning the prevalence of acanthosis nigricans (12.0 vs. 46.4%, P < 0.006), and values (mean ± SE) for BMI (25.9 ± 1.4 vs. 33.4 ± 1.4, P < 0.001), the area under the curve (AUC) for glucose (AUCgluc; 11.1 ± 0.5 vs. 14.1 ± 0.9, P < 0.003), 2-h glucose (5.2 ± 0.3 vs. 6.4 ± 0.5, P < 0.01), the AUC for insulin (AUCins; 76.0 ± 11.9 vs. 179.9 ± 24.8, P < 0.0008) and 2-h insulin (41.0 ± 0.1 vs. 103.7 ± 15.2, P < 0.002, Mann-Whitney U test), respectively. Patients in group B had a higher prevalence of obesity (78.5 vs. 40.0%) and IGT (25 vs. 4.8%) than group A, respectively. Their HOMAIR (4.8 ± 1.0) was 3.4-fold higher than in group A (1.4 ± 0.1) and twofold higher than the value of 2.17 ± 1.5 found in a consecutive series (n = 51) of obese women without PCOS, matched for BMI and age (J.F. Brun, unpublished results). Differences in the degree of insulin resistance between group A and group B were further confirmed by intravenous glucose tolerance test in a limited number of patients (n = 7 in group A and n = 6 in group B). The index for in vivo insulin sensitivity was almost twofold lower in group B than in group A (3.9 ± 1.5 vs. 7.4 ± 2.3 103 pmol/l · min-1 insulin). Because two Type A patients with lean phenotype, acanthosis nigricans, and confirmed mutations in the IR were excluded, the remaining patients with hyperadrogenism, insulin resistance, and acanthosis nigricans in group B were designated as HAIRAN syndrome (19).
Allelic frequencies or prevalence rates of IRS variants were determined in a control population (n = 224) randomly selected from 1,000 individuals representative of the general population and registered by the Nîmes Obstetricians and Hematologist Association (NOHA) in the Languedoc-Roussillon Region in southern France. The control population had a female-to-male ratio of 1.2, age (mean ± SE) 40.8 ± 0.6 years, and BMI 24.3 ± 1.7 and was recruited based on: 1) the absence of medication for at least 3 months before DNA sampling, 2) no personal history of diabetes (fasting blood glucose <7 mmol/l, mean 4.7 ± 0.5 mmol/l, n = 224), 3) no cardiovascular complications (diastolic blood pressure <90 mmHg at rest, mean cholesterol 4.2 ± 0.8, and triglycerides 1.1 ± 0.4 mmol/l), and 4) no family history (parents) of hypertension or type 1 or type 2 diabetes. Allelic frequencies in the PCOS were compared with this large population of 224 individuals and were also compared with the subpopulation of women (n = 102) who were fertile (at least one child) and who had no episodes of hypertension, intrauterine death, or growth retardation during pregnancy and no personal history of PCOS (i.e., an absence of menstrual dysfunction).
Genotyping.
Genomic DNA was obtained from whole blood using a Nucleon BACC 3 DNA isolation kit (Amersham, Buckingham, U.K.). Candidate genes (IR and IRS-1) were investigated by direct sequencing of the entire coding regions under conditions previously described (20). For the IRS-2 gene, fragments were amplified by polymerase chain reaction (PCR) with 22 oligonucleotide pairs. Two pairs of primers corresponded to 751 bp covering 638 bp at 5' untranslated region (UTR) and the first 112 bp of exon 1 (GenBank AF 073310). We used 19 pairs of primers to amplify fragments from 239 to 371 bp corresponding to the large exon 1 (Genome Data Base accession no. AB000732) and exon 1intron boundary (AF074850). Another pair of primers was used to amplify a fragment of 268 bp that covered the intronexon 2 boundary and 3' UTR (AF 074851). PCR products were purified by Centricon 100 (Amicon; Millipore, Bedford, MA) and then sequenced from both ends on an ABI 373A DNA sequencer using the AmpliTaq FS ABI Prism Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Roissy, France), as previously described (19). High quality chromatograms were obtained in each case and visually inspected, and litigious sequences were repeated by sequencing from both ends. Preliminary experiments indicated an efficiency <85% in the screening of the Gly1057Asp IRS-2 mutation with BanI (261-bp fragment); therefore, all subjects were genotyped by direct sequencing for the IRS-2 variant.
Assays.
Plasma glucose was measured with a Beckman analyzer, and hormones were measured by radioimmunoassay. Plasma insulin was measured with a P2796 kit (DiaSorin, Antony, France), which displayed 28% cross-reactivity with proinsulin. FSH and LH were measured with Kryptor kits (CisBio International, Gif-sur-Yvette, France), whereas androstenedione and total testosterone were assayed with kits from Immunotech (Tassigny, Marseille, France).
Data and statistical analysis.
AUCgluc and AUCins were obtained from OGTT values (30, 60, 90, and 120 min) by the trapezoidal method on Microsoft Excel. Numerical variables were expressed as the mean ± SE and analyzed with nonparametrical tests (Mann-Whitney U or Krukal-Wallis), whereas nominal variables were analyzed by 2 test. Significant values were considered at P < 0.05 (StatView 5.0; Abacus Concepts, Berkley, CA). For the logistic regression analysis, one model was tested to define genetic factors (IRS alleles) compared with other confounding variables in the phenotype that would explain insulin resistance in groups A and B. All variables with P < 0.2 were included in the model. A second model (plurimodal) was tested to compare three distinct genotypes (I, II, and III, as indicated in Table 4). One patient with a mutation only on IRS-1 was excluded. A unique variable was introduced for insulin resistance (fasting insulin or HOMAIR), and another was introduced for glucose intolerance (2-h glucose or IGT).
ACKNOWLEDGMENTS
This work was supported by the Fondation pour la Recherche Médicale (2000, to F.G.), an European Association for the Study of Diabetes, Bayer Travel fellowship (1997, to C.L. ), and a ALFEDIAM-LIPHA award (1998, to F.M.).
We thank M.C. Bozonnat of the Department of Epidemiology and Statistics at IURC and J.F. Brun and J. Mercier (Lapeyronie Hospital) for providing unpublished data. We also thank F. Boulet, S. Clouet, and F. Galtier for help with the collection of clinical data.
FOOTNOTES
Address correspondence and reprint requests to Florin Grigorescu, IURC, Molecular Endocrinology, 641 Ave. Du Doyen Gaston Giraud, 34093 Montpellier Cedex 5, France. E-mail: florin{at}iurc1.iurc.montp.inserm.fr.
Received for publication 18 August 2000 and accepted in revised form 21 May 2001.
AUC, area under the curve; AUCgluc, AUC for glucose; AUCins, AUC for insulin; FSH, follicle-stimulating hormone; LH, leutenizing hormone; HOMAIR, homeostasis model assessment index for insulin resistance; IGT, impaired glucose tolerance; IR, insulin receptor; IRS, IR substrate; OGTT, oral glucose tolerance test; OR, odds ratio; PCOS, polycystic ovary syndrome; PCR, polymerase chain reaction; UTR, untranslated region.
REFERENCES