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Insulin Gene Variable Number of Tandem Repeat Genotype and the Low Birth Weight, Precocious Pubarche, and Hyperinsulinism Sequence

Endocrine Unit, Hospital Sant Joan de Déu, University of Barcelona (L.I.), 08950 Barcelona, Spain; Department of Pediatrics, University of Cambridge (K.O., D.D.), Cambridge CB2 2QQ, United Kingdom; Hormonal Laboratory, Hospital Materno-Infantil Vall d’Hebron, Autonomous University of Barcelona (N.P.), Barcelona, Spain; Endocrine Unit, Consorci Hospitalari de Terrassa (M.V.M.), 08227 Barcelona, Spain; and Department of Pediatrics, University of Leuven (F.d.Z.), 3000 Leuven, Belgium

Address all correspondence and requests for reprints to: Prof. David B. Dunger, Department of Pediatrics, University of Cambridge, Level 8, Addenbrookes Hospital, Box 116, Cambridge, United Kingdom CB2 2QQ. E-mail: dbd25{at}cam.ac.uk

Abstract

Low birth weight associations with hyperinsulinemia and other adulthood disease risk factors have been described in several cohorts, including girls who present with precocious pubarche (pubic hair <8 yr). We hypothesized that these associations might be influenced by the insulin gene (INS) variable number of tandem repeat (VNTR), a common polymorphism related to INS transcription levels.

In 141 Caucasian girls, who presented with precocious pubarche, hyperinsulinemia was assessed from mean insulin levels during an oral glucose load (MSI), and insulin sensitivity was determined from fasting glucose and insulin levels. Fasting blood lipid profiles were also measured. DNA was genotyped for INS VNTR allele class (I or III) in precocious pubarche girls and in 140 age- and body mass index-matched control girls.

INS VNTR genotype distribution was similar in precocious pubarche and control girls. However among precocious pubarche girls, INS VNTR genotype was related to the severity of phenotype; I/I and I/III genotypes had lower birth weights (P < 0.01), higher MSI (P < 0.005), and lower insulin sensitivity (P < 0.005) than III/III girls. In precocious pubarche girls, birth weight was also inversely related to MSI (r = -0.29; P < 0.0005), total cholesterol (r = -0.38; P < 0.0005), and low density lipoprotein cholesterol (r = -0.44; P < 0.0005). Using logistic regression, additive adverse effects of I/* genotype and low birth weight were seen on MSI (P = 0.03 and P = 0.004, respectively) and total cholesterol levels (P = 0.01 and P < 0.0001).

In summary, in girls who presented with precocious pubarche, hyperinsulinemia and dyslipidemia were related to both low birth weight and INS VNTR class I alleles. A similar interaction between genotype and intrauterine growth restraint may underlie other reported links between low birth weight and adulthood disease risks.

THERE HAS BEEN considerable interest in the long-term outcomes of low birth weight, which, particularly when followed by catch-up growth in childhood, has been associated with metabolic markers for cardiovascular disease and type 2 diabetes in adulthood, such as reduced insulin sensitivity and hyperlipidemia (1). In girls who presented with precocious pubarche, defined as the appearance of pubic hair before the age of 8 yr, around 50% of subjects develop features of ovarian hyperandrogenism, hyperinsulinemia, and hyperlipidemia at adolescence (2, 3, 4), and this risk is inversely related to birth weight (5) In these subjects, insulin sensitization therapy results in normalization of hirsutism, hyperandrogenism, menstrual disturbances, dyslipidemia, and insulin levels (6), indicating that insulin resistance with compensatory hyperinsulinemia is a key pathogenic factor in this condition (7), as has been suggested in other links between low birth weight, catch-up growth, and adulthood disease risks (8).

We hypothesized that this low birth weight association could also be influenced by a common polymorphism at the insulin gene (INS) VNTR on chromosome 11 (class I or class III alleles) that is associated with levels of INS mRNA transcription (9). Class I alleles have been associated with smaller size at birth (10), increased postnatal weight gain (11), and increased insulin secretion in obese children (12). Thus, class I alleles could contribute to the association among low birth weight, catch-up growth, and risk of hyperinsulinemia described in girls with precocious pubarche. We therefore aimed to determine whether INS VNTR genotype might be related to precocious pubarche in girls and in particular to explore the effect of genotype on the relationship between low birth weight and their subsequent development of hyperinsulinemia and other associated metabolic abnormalities.

Subjects and Methods

Subjects

We enrolled 141 Caucasian girls who presented with precocious pubarche and were followed in the Endocrine Unit (Barcelona, Spain). Recruitment and biochemical investigations in these subjects have been well documented (3, 5). Girls with precocious pubarche were only eligible for the study if the entity was attributable to pronounced adrenarche, as suggested by elevated dehydroepiandrosterone sulfate (DHEAS) levels at precocious pubarche diagnosis and corroborated by an ACTH test to exclude nonclassic adrenal hyperplasia (2). None of the girls had acanthosis nigricans, thyroid dysfunction, Cushing’s syndrome, hyperprolactinemia, or a family or personal history of diabetes mellitus or was receiving oral contraceptive medication or a drug known to affect carbohydrate or lipid metabolism. Longitudinal clinical and/or endocrine data were collected from all girls at birth, at presentation (age range, 4.0–7.6 yr), and at inclusion (age range, 4–19 yr). Their baseline characteristics are described in Table 1.

Clinical assessment

Birth weight and gestational age data from controls were obtained from hospital records or were self-reported at clinical interview; in precocious pubarche girls the data were obtained from hospital records. Birth weights were transformed into gestational age-adjusted SD scores, as previously described (5). Body mass index, at both presentation and inclusion, was transformed into SD scores according to published normative data (13).

Endocrine-metabolic assessment

After 3 d of a high carbohydrate diet (300 g/d) and an overnight fast, a standard 1.75 g/kg (maximum, 75 g) 2-h oral glucose tolerance test was performed in precocious pubarche girls, starting at 0800 h. Blood was sampled 0, 30, 60, and 120 min after oral glucose administration for glucose and immunoreactive insulin measurements (3). Areas under the curve for insulin [mean serum insulin (MSI)] were calculated according to the trapezoidal rule. Individual MSI levels were transformed into SD scores according to published normative data (3). Estimates of insulin sensitivity were derived from fasting serum insulin and glucose levels by the homeostasis model of assessment (HOMA) method (14). These HOMA estimates have been shown to correlate closely with independent measurements of insulin sensitivity (14, 15) All subjects had normal glucose tolerance, according to the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus criteria (16).

Serum lipids, lipoproteins, and SHBG levels were measured from the baseline fasting blood samples during the oral glucose tolerance test. The 17-hydroxyprogesterone (17-OHP) response to leuprolide acetate, a GnRH agonist (Procrin, Abbott, Madrid, Spain; 500 µg, sc) was measured as an indicator of ovarian hyperandrogenism (2). Postmenarcheal girls were studied in the follicular phase (d 3–8) of the menstrual cycle.

Hormonal assays

Serum glucose was measured by the glucose oxidase method. Immunoreactive insulin was assayed by IMX (Abbott Diagnostics, Santa Clara, CA). The mean intra- and interassay coefficients of variation were 4.7% and 7.2%, respectively. Serum total cholesterol, high density lipoprotein cholesterol, and triglycerides were measured by the CHOD-PAP- and GPO-PAP-based methods, as previously described (4); low density lipoprotein cholesterol was calculated by the Friedewald formula. Serum 17-OHP was determined using a commercially available RIA kit (5), and serum DHEAS and SHBG were measured by enzymo-immunochemiluminiscence (17). Serum samples were stored at -20 C until assay.

Genotype

To identify INS VNTR genotypes, DNA from precocious pubarche girls and control subjects was collected from whole blood EDTA samples; parental DNA was collected by mouth swabs. DNA was isolated using a rapid purification method; samples were genotyped for the -23 bp A/T diallelic polymorphism, which is recognized by the restriction enzyme HphI, using PCR and restriction enzyme digestion as previously described (9). This polymorphism is in very tight linkage disequilibrium with INS VNTR class; in people of European descent there is a 99.6% concordance between -23/HphI alleles and INS VNTR allele class (18).

Calculations and statistics

Anthropometric data and hormonal results are expressed as the mean ± SEM, unless stated otherwise. Comparison of means was performed by ANOVA in a generalized linear model, adjusting for pubertal stage. Birth weight distribution according to INS VNTR genotype was analyzed by Fisher’s exact test. Multiple logistic analysis and conditional regression analysis were performed to analyze the independent roles of birth weight and VNTR genotype on the different endocrine-metabolic variables.

Results

The INS VNTR genotype distribution was similar in precocious pubarche girls (I/I, 53%; I/III, 39%; III/III, 8%) and normal birth weight controls (I/I, 49%; I/III, 44%; III/III, 7%), and these distributions are comparable to those reported in other Caucasian populations (18). In 32 of 55 heterozygous precocious pubarche girls, the parental origin of INS VNTR alleles was determined; the frequency of transmission of the class I allele was similar from fathers (n = 15; 32%) and from mothers (n = 17; 35%). In the remaining 23 heterozygous girls transmission could not be determined either because both parents were heterozygous (n = 8) or parental DNA was unavailable (n = 15).

Within precocious pubarche girls, class I homozygotes and heterozygotes had lower birth weights than class III homozygotes (Table 2), and they also had more evidence of endocrinopathy, with significantly reduced insulin sensitivity, higher stimulated insulin levels, reduced SHBG, higher 17-OHP levels post-GnRH agonist, and adverse lipid profiles compared with class III homozygotes despite no difference in body mass index at time of assessment (Table 2). Birth weights and all biochemical variables were similar in I/I and I/III genotypes and these were pooled (I/¹ group) in subsequent analyses.

To assess the independent effects of INS VNTR genotype and low birth weight on biochemical markers, we divided the precocious pubarche group into normal and low birth weight subgroups according to a cut-off level of -2.0 SD. Class III homozygotes and class III alleles were significantly underrepresented in the low birth weight group (0%) compared with the normal birth weight group (10%; by Fisher’s exact test for allele frequency, P = 0.01; Table 3). Comparison of endocrine-metabolic data between the normal and low birth weight I/¹ groups (Table 4), therefore, allows assessment of the impact of low birth weight in the absence of genotype differences, whereas any differences between the normal birth weight I/¹ and III/III groups are independent of low birth weight effects. We documented adverse effects of both I/¹ genotype and low birth weight on MSI SD scores and blood lipid profiles (Table 4); Fig. 1 shows that these effects were additive. I/¹ genotype-only adverse effects were seen on HOMA insulin sensitivity and SHBG, whereas low birth weight-only effects were seen on the 17-OHP responses to GnRH agonist (Table 4).

View larger version (9K): [in this window] [in a new window]   Figure 1. Additive effects of INS VNTR class I/* genotype and low birth weight (birth weight less than -2 SD score) on insulin levels during oral glucose tolerance testing (MSI SD score; top panel) and lipid parameters (lower panels) in precocious pubarche girls: without I/* genotype and without low birth weight (-/-), with I/* genotype and without low birth weight (±), and with I/* genotype and with low birth weight (+/+).

Low birth weight associations with reduced insulin sensitivity and hyperinsulinism have been attributed to programming of metabolism in response to fetal undernutrition, as demonstrated in animal models (8, 19). Girls who presented with precocious pubarche often have hyperinsulinemia and a history of low birth weight (5), and this link has been confirmed in other low birth weight groups (20, 21). Postnatally, girls with precocious pubarche have more rapid childhood growth (22), and despite the fact that they do not become obese, hyperinsulinemia is often already present at diagnosis of precocious pubarche and may underpin the subsequent development of a constellation of endocrine and metabolic abnormalities in many of these girls (3, 6). In this well characterized population of girls with precocious pubarche, we now report evidence for a genetic/environmental interaction that affects the severity of this endocrine-metabolic phenotype. Insulin resistance, hyperinsulinemia, dyslipidemia, and 17-OHP levels post-GnRH (indicative of ovarian hyperandrogenism) were greater in girls with INS VNTR class I/I or I/III genotypes compared with III/III genotype. I/¹ genotype and low birth weight had additive adverse effects on insulin levels postglucose. Thus, hyperinsulinemia, dyslipidemia, and ovarian hyperandrogenism were most severe in low birth weight I/¹ girls.

The influence of the INS VNTR class I alleles may reflect their association with smaller size at birth (10), resulting in the observed over-representation of the I/¹ genotype among low birth weight precocious pubarche girls. INS VNTR class I alleles are also associated with increased levels of pancreatic INS mRNA (9), increased fasting insulin levels in obese children (12), and increased pulsatility of insulin secretion in response to glucose in adults (23), and these factors could contribute to the hyperinsulinemia in precocious pubarche girls. After fetal growth restraint, compensatory rapid, or catch-up, postnatal growth may be a crucial factor in the development of insulin resistance and long-term disease risk (24, 25). During infancy, these rapid growth rates are probably nutritionally mediated (26) and could be further enhanced by increased insulin secretion. Class I alleles confer susceptibility to type 1 diabetes (27), and rapid infancy weight gain is often observed in such children (28), supporting the possibility that the contribution of class I alleles to the disease risks related to low birth weight may in part be mediated through more rapid rates of postnatal catch-up growth.

In our study, there were no differences between class I/I and I/III genotype girls, indicating that class I alleles may have had a dominant effect. However, in other studies the I/III groups have shown varying similarities with either class I or class III homozygotes (9, 10, 12, 18). There is evidence that INS VNTR genotype effects may be parent of origin specific (29, 30, 31), possibly due to genetic imprinting of the embryonic INS and IGFS genes (32, 33), and thus differences in parent of origin of alleles could account for this variation in the heterozygote group.

Precocious pubarche in these girls was attributable to pronounced adrenarche, as characterized by elevated DHEAS levels and the exclusion of other conditions, such as nonclassic adrenal hyperplasia. The INS VNTR genotype did not appear to determine the development of precocious pubarche, and thus other genetic factors may underlie the wide population and racial differences in the prevalence of this entity in children. About 50% of girls with precocious pubarche develop clinical signs and symptoms of androgen excess in adolescence, including hirsutism, menstrual disturbances, anovulation, and hyperandrogenemia (5), and these abnormalities have been likened to features of the polycystic ovary syndrome (PCOS) (7, 34). Ovarian hyperandrogenism and hyperinsulinemia are principal characteristics of PCOS; however, the United Kingdom/European criteria for PCOS diagnosis differ from those used in the U.S. (35) by the further requirement for the presence of polycystic ovarian appearances on ultrasound (36). The absence of polycystic ovarian morphology in these girls with precocious pubarche (37) may suggest a heterogeneity in the pathogeneses of these PCOS-like conditions. Indeed, in contrast to the ovarian hyperandrogenism and hyperinsulinemia associated with low birth weight and INS VNTR class I alleles in precocious pubarche girls, paradoxically PCOS based on ultrasound in older women in the United Kingdom has been associated with larger birth weight (38) and linked to paternal transmission of INS VNTR class III alleles (29, 30, 39) and may thus represent a distinct entity from the low birth weight-associated phenotype.

In conclusion, in these girls who presented with precocious pubarche, both INS VNTR class I/¹ genotype and low birth weight contributed to the risk of hyperinsulinemia and dyslipidemia. These results indicate that the INS VNTR class I, low birth weight, catch-up growth, hyperinsulinemia, and ovarian hyperandrogenism sequence in precocious pubarche girls may be genetically distinct from those in women who present with similar endocrine abnormalities and ultrasound evidence of polycystic ovaries, for which links are described with larger birth weight. These data require further confirmation, but highlight potentially important early genetic/environmental interactions in the pathogenesis of subsequent links between birth weight, early growth rates, hyperinsulinemia, and other risk factors for adulthood disease.

Acknowledgments

We are extremely grateful to all the girls who took part in this study and to their families. We also thank Montserrat Gallart for collecting the samples, Miguel Carballo for performing patients’ DNA extraction, Sam Mason and Louisa Hull for assistance with parental DNA extraction and genotyping, and Karin Vanweser and Inge Laleeuwe for editorial assistance.

Footnotes

This work was supported by a scholarship from the European Society for Pediatric Endocrinology, and laboratory costs in Barcelona were also supported in part by Eli Lilly & Co.

¹ Medical Research Council, United Kingdom, Clinical Training Fellow.

² Clinical Research Investigator of the Fund for Scientific Research, Flanders, Belgium.

Abbreviations: DHEAS, Dehydroepiandrosterone sulfate; HOMA, homeostasis model of assessment; MSI, mean serum insulin; 17-OHP, 17-hydroxyprogesterone; PCOS, polycystic ovary syndrome; VNTR, variable number of tandem repeat.

Received April 30, 2001.

Accepted August 29, 2001.

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