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The INS VNTR Locus Does Not Associate with Smallness for Gestational Age (SGA) but Interacts with SGA to Increase Insulin Resistance in Young Adults

INSERM Unité 690 (T.-A.V.-H., S.D., P.C., C.L.-M.) and Department of Biochemistry (D.C.), Robert Debré Hôpital, 75019 Paris, France; CNRS-8090 (E.D., P.B., D.M., P.F.), Pasteur Institute, 59021 Lille, France; and Department of Genomic Medicine (P.F.), Hammersmith Hospital, Imperial College, London W12 ONN, United Kingdom

Address all correspondence and requests for reprints to: Claire Levy-Marchal, M.D., Institut National de la Santé et de la Recherche Médicale Unit 690, Robert Debré Hospital, 48, Boulevard Serurier, 75019 Paris, France. E-mail: clairelm{at}rdebre.inserm.fr.

	Abstract

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Context: Both adverse intrauterine events and genetic background have been suggested to promote insulin resistance in subjects born small for gestational age (SGA). Among candidate genes that potentially influence both fetal growth and glucose metabolism is insulin. The potential effect of the insulin gene VNTR (INS) on birth weight has been controversial so far.

Objective: The present association study aimed at testing for the contribution of the INS VNTR locus on birth weight and on the metabolic profile of young adults born SGA (mean age, 22 yr). Two groups of subjects were selected on birth data: SGA (birth weight < 10th percentile; n = 735), and appropriate for gestational age (AGA; birth weight between 25th and 75th percentiles; n = 886). All subjects were genotyped for rs689 A/T single nucleotide polymorphism, in complete linkage disequilibrium with the INS VNTR classes I and III, respectively.

Results: Class I INS frequencies were similar in the two groups (70% in AGA; 72% in SGA; P = 0.42). There was significant effect on mean birth weight in neither SGA (P = 0.99) nor AGA (P = 0.18). Although the INS VNTR locus did not associate with anomalies of insulin resistance indices in the AGA group, in the SGA group, INS VNTR class III allele was associated with higher insulin resistance (quantitative insulin sensitivity check index = 0.38 vs. 0.39; P = 0.05). Furthermore, there was evidence of an interaction between the SGA/AGA status and INS VNTR locus on insulin resistance indices (P = 0.01) in a multivariate analysis.

Conclusion: The INS VNTR locus does not associate in a major way with SGA in the French population. However, our data support an interaction between severe fetal growth restriction and INS VNTR locus, which were associated with insulin resistance in young adults born SGA.

	Introduction

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SMALLNESS FOR GESTATIONAL age (SGA) is epidemiologically defined as a birth weight less than the 10th percentile according to sex and gestational age. This status is mostly due to well-known environmental causes such as maternal smoking, chronic maternal diseases, maternal undernutrition, or placental anomalies. However, a large proportion of SGA remains unexplained. Evidence for a genetic contribution has been reported. Familial recurrence of SGA (1, 2) or parental contribution to SGA (3) have been described. The increased risk of recurrence conferred by the father (3) emphasizes the role of a genetic contribution.

SGA predisposes to insulin resistance and metabolic syndrome in adults (4). Indeed, in a case-control cohort, comprising 1621 subjects, of which half were born SGA and half were born with an appropriate for gestational age (AGA) birth weight, Jaquet et al. (5) reported that subjects born SGA are more likely to develop insulin resistance in adulthood, especially from 20 yr of age. Both adverse intrauterine events and genetic background have been suggested to promote insulin resistance in subjects born SGA (6, 7). Among candidate genes that potentially influence both fetal growth and glucose metabolism is insulin. Fetal circulating insulin levels are positively correlated to birth weight (8, 9). Besides, the variations of the insulin gene (INS) VNTR (variable number of tandem repeats) have been reported to modify insulin gene transcription and pancreatic insulin expression in vitro (10). In vivo, INS VNTR class III allele has been shown to be associated with alterations of insulin secretion (11, 12). The potential effect of the INS VNTR locus on birth weight has been controversial so far. The class III allele has been reported to be associated with a reduction of 140 g in birth weight in Pima Indians (13). It has also been shown to be associated with larger head circumference at birth as well as higher birth weight and birth length in the British ALSPAC cohort (14). These findings have not been confirmed in other large cohorts from different origins but has been replicated in the ALSPAC cohort (15, 16, 20).

The relationship between this polymorphism and the metabolic syndrome remains unclear. The INS VNTR class III allele was reported to be associated with glucose intolerance or diabetes in subjects whose weight was stable during the first 2 yr of life (17). In combined cohorts defined by birth weight, Mitchell et al. (15) reported an association of the same variant with an alteration of insulin resistance indices. However, other studies did not replicate these observations (18, 19).

The aim of our study was to assess the contribution of the INS VNTR locus on SGA and the metabolic profile of young adults born SGA (mean age 22 yr), in a case-control cohort comprising 1621 subjects, of which half were born SGA and half had an AGA birth weight.

	Subjects and Methods

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The Haguenau case-control cohort

Subjects born between 1971 and 1985 were identified from a population-based registry encompassing more than 20,000 births in the metropolitan area of the city of Haguenau, France. Only singletons were included. Gestational age was determined from the date of the mother’s last menstrual period and by physical examination during pregnancy, confirmed by ultrasound measurements when available (>80%). Two groups were selected on birth data derived from the local reference curves drawn for gender and gestational age: SGA (birth weight < 10th percentile; n = 735 subjects) and AGA (birth weight between 25th and 75th percentile; n = 886 subjects).

Birth parameters, consisting of birth weight, length, and ponderal index, were collected from the registry. At a mean age of 22 yr, all subjects underwent a medical examination to assess anthropometric parameters (weight, height, waist to hip ratio). Glucose tolerance and insulin resistance were evaluated during an oral glucose tolerance test. After a glucose load of 75 g, blood samples were taken at 0, 30, and 120 min to measure plasma glucose and serum insulin concentrations. Insulin resistance was assessed by quantitative insulin sensitivity check index (QUICKI) (QUICKI = 1/[log(serum insulin) + log(glycemia)]). Insulin secretion was assessed by the insulinogenic index [(serum insulin at 30 min) – (fasting serum insulin)]/[(plasma glucose at 30 min) – (fasting plasma glucose)]. Total cholesterol, high-density lipoprotein (HDL)-cholesterol, and triglyceride concentrations were measured fasting.

The study protocol was reviewed and approved by the faculty ethics committee, and all subjects and parents gave signed written consent.

Genotyping

We studied rs689 (also called –23HphI), a single nucleotide polymorphism (SNP) in complete linkage disequilibrium with the INS VNTR locus, identifying two alleles: A and T. The rs689 A allele is representative for class I VNTR allele, whereas rs689 T allele is representative for class III.

DNA, extracted from blood samples of the 1621 subjects, was genotyped for rs689.

Genotyping was performed by LightTyper assay (Roche Diagnostics, Meylan, France), based on fluorescence resonance energy transfer method, after DNA amplification by PCR. The SNP rs689 was amplified using oligonucleotides 5'-AGCAGGTCTGTTCCAAGG-3' and 5'-CTTGGGTGTGTAGAAGAAGC-3' according to the LightTyper 384 PCR kit (Roche) in 4 mmol/liter MgCl₂ and Tanneal 62 C conditions. Genotyping was performed by LightTyper assay (Roche), based on the fluorescence resonance energy transfer method, using 5'-tcAcccagatcactgt-3'and 5'-gcctgcctcagccctgc-3' probes.

Statistical analyses

The SNP rs689 complied with Hardy-Weinberg proportions.

The ² test was applied to compare allelic frequencies between case and controls. Given a 30% allelic frequency for the dominant allele and a frequency of 10% of SGA, the power of the study was 75% to observe odds ratio = 1.3.

Quantitative trait analyses for birth parameters and metabolic syndrome criteria were performed using the Student’s t test (dominant model). The relationship between rs689 genotype and phenotypes of interest was examined by multiple general linear regression modeling using the SAS software (version 8; SAS Institute Inc., Cary, NC). We considered the two genotype models: dominant, in which the T allele (class III) is dominant, and additive (results not shown). Each quantitative phenotypic trait was an explained variable; rs689 genotype and AGA/SGA status were the explanatory variables. This model took known covariables into account: gender and gestational age when testing for birth weight, birth length and ponderal index; age at examination and gender when testing for adult weight, adult height, body mass index (BMI), and waist to hip ratio; age at examination, gender, BMI, familial history of dyslipidemia, fasting serum insulin when testing for total plasma cholesterol, HDL, and triglyceride; age at examination, gender, BMI, familial history of diabetes when testing for fasting plasma glucose, fasting insulin, QUICKI, and insulinogenic index.

All tests were two tailed, and P 0.05 was considered to be statistically significant.

	Results

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Study population characteristics

Regarding birth parameters, mean birth weight was 2609 ± 302 g in SGA vs. 3370 ± 267 g in AGA. At a mean age of 22 yr, adult height and adult weight were significantly lower in SGA (167.5 ± 8.9 vs. 172 ± 8.9 cm and 63.1 ± 14 vs. 67.2 ± 13.6 kg, respectively; P < 0.0001), whereas mean BMI (22.4 ± 4.3 vs. 22.6 ± 3.9 kg/m²) was similar in the two groups. The prevalence of impaired glucose tolerance was significantly higher in the SGA group (4.2%) than the AGA group (1.8%; P = 0.01); no case of type 2 diabetes was diagnosed. Fasting serum insulin was significantly higher in SGA (5.6 ± 4.2 mIU/liter) than AGA (4.8 ± 2.5 mIU/liter; P < 0.0001); mean QUICKI was significantly lower in SGA (0.38 ± 0.04) than AGA (0.39 ± 0.03; P = 0.02); mean insulinogenic index was not significantly different between the two groups (102.2 ± 224.8 vs. 100.1 ± 242.3; P = 0.87); total cholesterol was similar in the two groups (4.77 ± 0.99 vs. 4.72 ± 0.95 mmol/liter); plasma triglyceride concentration was significantly higher in SGA than AGA (1.10 ± 0.59 vs. 1.03 ± 0.51 mmol/liter; P = 0.006); plasma HDL concentration was significantly lower in SGA than AGA (1.40 ± 0.36 vs. 1.44 ± 0.35 mmol/liter; P = 0.005).

rs689 genotype and birth parameters

Class I allele frequencies were similar in the two groups (70% in AGA; 72% in SGA; P = 0.42).

Birth parameters were not significantly influenced by INS VNTR genotype in either group (Table 1).

No significant effect of the INS VNTR genotype was detected on any of the metabolic parameters in the AGA group (Table 1).

In the SGA group, however, the class III allele was associated with reduced QUICKI (adjusted means of QUICKI = 0.38 vs. 0.39; P = 0.05), suggesting higher insulin resistance (Fig. 1). Furthermore, there was a significant interaction between the SGA/AGA status and INS VNTR alleles on QUICKI (P = 0.01). The same pattern was found for fasting serum insulin (Table 1). Comparisons yield similar results with respect to birth order (first born and others).

View larger version (43K): [in this window] [in a new window]   FIG. 1. Insulin resistance measured by QUICKI in SGA and AGA subjects, according to INS VNTR alleles. I/I, Homozygous for common class I INS VNTR; III+, [I/III] + [III, III], presence of class III INS VNTR dominant model.

	Discussion

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The INS variants have already been tested for their association with birth weight and the long-term metabolic consequences. Indeed, the INS VNTR class III allele has been associated with an increased risk of type 2 diabetes (17). Moreover, insulin is a major fetal growth factor. However, the influence of INS VNTR locus on birth weight has been subject to controversial data so far. This variant has been reported to have a minor effect, if any, on birth weight (13, 14, 15, 16, 17, 20). As for the interaction between SGA and the insulin gene variants on the long-term metabolic consequences, no literature data are available so far. We took advantage of a large homogeneous Caucasian study population selected on birth data, made up of two groups, highly contrasted in terms of birth status. Despite these specifications, no association could be evidenced between the INS VNTR and SGA status. INS VNTR class III was found to be associated with increased birth weight in nonchanger children only, who showed no catch-up growth during the first 2 postnatal years (14), but this result has never been replicated (16, 17, 20). This variant has been associated with a reduction of birth weight in Pima Indians, a population in whom the distribution of birth weight is strongly influenced by maternal factors such as obesity and diabetes (13).

In our population, at the age of 22 yr, insulin resistance and the components of the metabolic syndrome are moderately, although statistically, different between SGA and AGA adults. Despite this subtle difference in phenotype, insulin resistance was influenced by the INS VNTR locus in SGA of our study population, whereas it has no significant effect in the AGA group. This distinct effect has not been identified previously and has probably been evidenced here because of the contrasted selection of the two study groups. This interaction may promote insulin resistance described in SGA subjects in several studies and visible early in life (5). Although modest in magnitude, the effect of the genetic variant was observed in the SGA group, reminiscent of what was observed by our group and others with the Pro12Ala peroxisomal proliferator-activated receptor- polymorphism, and indicative of a physiological interaction between this genetic variant, intrauterine environment, and long-term consequences, the nature of which remains to be understood.

Although the INS VNTR class III allele has been shown to reduce insulin secretion, such an effect was not observed in our study population but is consistent with the fact that we never could identify a defect in ß-cell function in our SGA population (21, 22).

In contrast with some other studies, we did not find a strong effect of the INS VNTR class III allele on either birth weight or insulin resistance associated with SGA. One flaw could be a point of methodology.

As the –23HphI A and T alleles present a concordance of 99.6% with the adjacent insulin VNTR class I and III alleles (23), –23HphI was commonly used by us and others to infer insulin VNTR genetic variation (18, 24). Therefore, we assume that it may represent an oversimplification of a more complex phenomenon, taking into account the high diversity of tandem repeat number at the insulin VNTR and possible functional effect of the –23HphI itself (25).

Conclusion

In conclusion, the INS VNTR locus was not associated with SGA in a carefully selected French population. However, our data support an interaction between severe fetal growth restriction and the insulin gene in the development of insulin resistance in young adults born SGA.

	Footnotes

 
This work was supported by grants from Institut National de la Santé et de la Recherche Médicale (PROGRES 2000), ALFEDIAM-NOVO (2003), Pfizer-France, and Fondation de France (2003). T.-A.V.-H. was supported by a fellowship from SFE-IPSEN.

First Published Online April 4, 2006

Abbreviations: AGA, Appropriate for gestational age; BMI, body mass index; HDL, high-density lipoprotein; INS, insulin gene; QUICKI, quantitative insulin sensitivity check index; SGA, small or smallness for gestational age; SNP, single nucleotide polymorphism; VNTR, variable number of tandem repeats.

Received October 11, 2005.

Accepted March 23, 2006.

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This Article Abstract Full Text (PDF) All Versions of this Article: 91/6/2437    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart 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 Vu-Hong, T.-A. Articles by Levy-Marchal, C. Search for Related Content PubMed PubMed Citation Articles by Vu-Hong, T.-A. Articles by Levy-Marchal, C. Related Collections Pediatric Endocrinology Metabolism