1 Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford, U.K
2 Department of Epidemiology and Public Health, Imperial College (St. Marys Campus), London, U.K
3 Department of Clinical Chemistry, University of Oulu, Oulu, Finland
4 Department of Obstetrics and Gynaecology, University of Oulu, Oulu, Finland
5 Department of Public Health Science and General Practice, University of Oulu, Oulu, Finland
6 Wellcome Trust Centre for Human Genetics, Churchill Hospital, Oxford, U.K
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ABSTRACT |
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There are two main explanations for the widely observed relationship between restricted early growth and increased susceptibility to type 2 diabetes. One mechanism, encapsulated in the thrifty-phenotype hypothesis, links poor intrauterine nutrition to permanent metabolic changes ("programming") that predispose to subsequent diabetes (1). Amply supported by studies in animal models (2), evidence that this mechanism is important in humans is less convincing (3,4). A complementary explanation attributes these associations to variation in genes with effects on both early growth and metabolic phenotypes (5). Evidence that paternal diabetes is associated with lower offspring birth weight (6,7) supports such a genetic explanation, and analyses within families segregating rare variants (e.g., in glucokinase) provide proof of principle that genes influencing insulin secretion (and/or action) can have pleiotropic effects on early growth (5). Such rare variants cannot, however, explain the observed population associations.
In this context, several groups have sought to establish the role of insulin gene polymorphisms with respect to early growth. Variation at the insulin gene VNTR (variable number of tandem repeats) minisatellite has been implicated in susceptibility to type 2 diabetes (8,9), polycystic ovarian syndrome (10), and obesity (11). The importance of insulin as a major growth factor in early life, and evidence that the VNTR has a direct effect on insulin (and IGF2) transcription (12,13), provides strong grounds for suspecting that INS-VNTR variation also influences early growth.
In the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort (n = 1,049), Dunger et al. (14) found that VNTR class III homozygote infants had larger head circumference at birth than children of other genotypes. In infants displaying limited postnatal growth realignment ("nonchangers"), in whom birth size may more closely reflect fetal genotype, these class III associations extended to greater birth weight and length. However, there are some difficulties with these data (15). First, the direction of the association is contrary to expectation, given the consensus view that VNTR class III alleles reduce pancreatic insulin gene transcription (12,13) and increase risk of adult metabolic phenotypes (810). Second, a study of 418 offspring of Pima origin (16) reported an association between class III alleles and birth weight in the opposite direction, whereas in a recent study (17) of 1,184 infants from the U.K., no significant associations with birth weight were observed. Most recently, a study (18) of 452 additional subjects from the ALSPAC cohort confirmed the class III association with greater head size but failed to corroborate the association with birth weight.
Since much of the inconsistency that has troubled complex trait association studies has resulted from the interpretation of findings from inadequately sized samples (19), we studied the relationship between the VNTR genotype and early growth phenotypes (birth weight, birth length, ponderal index, placental weight, and head circumference at 1 year) in 5,753 subjects from the Northern Finland Birth Cohort of 1966 (NFBC66). In this sample, as in other longitudinal cohorts (20), birth weight variation is significantly associated with adult metabolic traits (U.S., M.-R.J., unpublished observations). Of these subjects, 5,646 were successfully genotyped for the 23HphI variant, a close to perfect proxy for VNTR class in non-African populations (21). Overall, 68.3% (3,859 subjects) were homozygous for the A allele, 29.2% (1,646) were heterozygotes, and 2.5% (141) T allele homozygotes. The frequency of the 23HphI T allele (equivalent to VNTR class III) is, as previously noted (22,23), lower in Finns than in other European populations (17% in NFBC66). Genotype frequencies did not deviate significantly from Hardy-Weinberg equilibrium. Characteristics of the typed subjects are provided in Table 1.
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What are the possible explanations for this apparent discrepancy? Genotyping error in the current study seems unlikely given the duplicate genotyping and documented low error rate. Neither is the answer likely to lie in ethnic differences in VNTR subclass composition, presence (or absence) of nearby modifying variants, or variable local linkage disequilibrium relationships because these are known to be broadly similar in all non-African populations (21). Although the comparatively low class III allele frequency in this northern Finnish population reduces the power to detect effects restricted to class III homozygotes, the increased overall sample size should more than compensate (the number of class III homozygotes is twice that in the ALSPAC study). The fact that our population-based sample cannot detect (and allow for) parent-of-origin effects implicated in VNTR effects on both early growth (16) and subsequent phenotypes (9,11) represents an intrinsic limitation of the current study, but, again, cannot explain the failure to detect the VNTR association observed in the similarly constrained ALSPAC sample (14).
According to the fetal insulin hypothesis, diabetes-susceptibility alleles are expected to reduce fetal size through compromised insulin secretion or action; however, where the mother also carries susceptibility alleles, and is therefore predisposed to gestational diabetes mellitus (GDM), such associations might be obscured by consequent fetal macrosomia (5). No systematic information on blood glucose levels during pregnancy or GDM status is available for the NFBC66 mothers because at the time of cohort recruitment, GDM was not widely recognized and diagnostic criteria not established. However, undiagnosed GDM is unlikely to explain the failure to detect class III associations with increased birth size. The ALSPAC data indicate that, in the case of the INS-VNTR, the diabetes-associated (class III) allele leads to increased, not decreased, birth size (14,18). In this situation, GDM (presumably associated with maternal class III) would exacerbate, not obscure, the class III association with birth size. In addition, no VNTR associations were revealed when we excluded offspring with negative postnatal growth realignment on the basis that most offspring born following pregnancies complicated by GDM-associated macrosomia would be "change-downers." Four of the NFBC66 mothers had a preexisting diagnosis of diabetes; their exclusion from the analyses had no impact on the findings.
Two possible explanations remain. The first attributes the discrepant findings to biological differences between the various study samples (e.g., environmental exposures, antenatal management, secular trends) and/or to study designrelated issues (ascertainment schemes, accuracy, and choice of measures of early growth) that have an effect on the power to detect VNTR association effects. In particular, it is important to note that we did not have data on head circumference at birth, the phenotype most strongly associated with the VNTR genotype in the ALSPAC cohort (14,18). Nonetheless, the persistence of the VNTR association with head circumference from birth to 7 years of age in the ALSPAC study (18) suggests that this is not a complete answer. The second explanation is that certain of the analyses in the smaller sets have been subject to type 1 error and effect-size inflation ("the winners curse") (19), which have led to an overestimation of the evidence that VNTR class and early growth are truly associated. The available data do not allow us to distinguish between these alternatives, which are, in any event, not mutually exclusive.
Preliminary analyses of the 31-year data from the NFBC66 cohort have failed to find any clear evidence of a relationship between VNTR class variation and adult metabolic phenotypes (A.J.B., M.-R.J., M.I.M., unpublished observations). Therefore, while we can conclude that studies of this large population-based cohort have failed to generate convincing evidence that insulin gene VNTR class variation influences early growth, these studies of the insulin gene do not allow us to discriminate between genetic and environmental explanations for the observed associations between early growth and adult metabolic phenotypes.
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RESEARCH DESIGN AND METHODS |
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Genotyping.
The 23HphI variant (rs689) was typed as a surrogate for VNTR class because in Finns, as in other non-African populations, these are in tight linkage disequilibrium (21). Genotyping was performed in duplicate, using both a PCRrestriction fragmentlength polymorphism and a mass-spectrometry assay. The amplicon for the former was generated using oligonucleotides 5'-AGCAGGTCTGTTCCAAGG and 5'-CTTGGGTGTGTAGAAGAAGC and included an obligate restriction site; following restriction, products were separated on a 7.5% acrylamide gel. The mass-spectrometry assay used an amplicon generated with primers 5'-ACGTTGGATGTCCACAGGGCCATGGCAGAAG and 5'-ACGTTGGATGTGGCCTTCAGCCTGCCTCAG. Following dNTP removal with shrimp alkaline phosphatase, the sequencing oligo (5'-CAGAAGGACAGTGATCTGGG) was extended using thermosequenase, and the products were resin captured, arrayed, and analyzed in a Bruker Biflex III mass spectrometer according to the manufacturers protocol (Sequenom, San Diego, CA). Discrepant calls were resolved using a four-primer amplification refractory mutation system (ARMS)-PCR assay designed to use the flanking oligos, 5'CTCAGCCCTCCAGGACAGGCTGCATCAGA and 5'-AGAGCTTCCACCAGGTGTGAGCCGCACA, and allele-specific oligos, 5'-TCAGCCTGCCTCAGCCCTGCCTGACA (class I) and 5'-GGCCATGGCAGAAGGACAGTGATCTGCGA (class III). Amplification products were separated on a 5% acrylamide gel. In addition, class III homozygote genotypes were reconfirmed (with no discrepancies detected) by ARMS-PCR, direct sequencing, and/or Pyrosequencing. A final round of ARMS-PCR retyping of 384 samples identified only one genotype discrepancy compared with the assigned genotype. Thus, we estimate our overall error rate as <0.1%. Additional details on assay design and quality control data are available from the authors.
Statistical analysis.
The relationship between the 23HphI genotype and phenotypes of interest was examined by linear multivariate regression modeling using the SAS (version 8.2) and SPSS (version 11.5 for Windows) programs. We considered two different genotype models. Given previous findings (14), the first ("recessive") model compared TT (class III) homozygotes against all other genotypes. The second ("additive") model assumed a linear relationship between the number of T alleles and the trait of interest. Analyses were optionally adjusted for potential confounding and explanatory variables, with four such adjustments considered (none; sex alone; sex and gestational age; and sex, gestational age, and maternal background factors, including maternal BMI and height, depression, employment, smoking during pregnancy, and parental socioeconomic status). The full list of variables included in these adjustments is shown in Table 1. Unless otherwise stated, all P values reported are those for the fully adjusted analyses. Analyses were repeated after stratification by postnatal growth realignment (14) and birth order. The former divided subjects into "nonchangers," "change-downers," and "change-uppers" based on comparison of sex- and gestational ageadjusted SD scores for weight at birth and 1 year (the latter available for 4,574 individuals). The boundaries for each stratum were set at a change in SD score of ±0.67 (14). Stratification by parity considered first borns and those from second or subsequent pregnancies ("later borns"). Additional adjustment for actual age at the 12-month visit had no impact on the findings (data not shown).
Power.
Our power to detect an effect size of 25% of an SD (130 g for birth weight) with a significant P value of 0.05 was >80% (given that the at-risk genotype was present in only 2.5% of the sample). The additive analysis had equivalent power to detect a difference in the parameter of interest of <10% of an SD.
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ACKNOWLEDGMENTS |
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We acknowledge the many patients, relatives, nurses, and physicians who have contributed to the ascertainment of the various clinical samples used in this study.
Address correspondence and reprint requests to Prof. Mark McCarthy, Robert Turner Professor of Diabetes, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital Site, Old Road, Headington, Oxford, OX3 7LJ, U.K. E-mail: mark.mccarthy{at}drl.ox.ac.uk
Received for publication February 2, 2004 and accepted in revised form April 26, 2004
ALSPAC, Avon Longitudinal Study of Parents and Children; ARMS, amplification refractory mutation system; GDM, gestational diabetes mellitus; NFBC66, Northern Finland Birth Cohort of 1966; VNTR, variable number of tandem repeats
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REFERENCES |
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