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Studies of the Variability of the Genes Encoding the Insulin-Like Growth Factor I Receptor and Its Ligand in Relation to Type 2 Diabetes Mellitus¹

Steno Diabetes Center and Hagedorn Research Institute (S.K.R., L.H., S.M.E., T.H., C.T.E., S.A.U., K.B.-J., O.P.), DK-2820, Gentofte, Denmark; Institut Universitaire de Recherche Clinique, Molecular Endocrinology Laboratory (C.L., F.G.), 34093 Montpellier, France; Joslin Diabetes Center, Harvard Medical School (R.J.S.), Boston, Massachusetts 02215; and Center of Preventive Medicine, Glostrup University Hospital (K.B.-J.), DK-2820 Glostrup, Denmark

Address all correspondence and requests for reprints to: Søren K. Rasmussen, M.Sc., Steno Diabetes Center and Hagedorn Research Institute, Niels Steensens Vej 2-6, DK-2820, Gentofte, Denmark.

	Abstract

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Insulin-like growth factor I (IGF-I) is an important regulator of many aspects of growth, differentiation, and development, and as low birth weight has been associated with impaired glucose tolerance and overt type 2 diabetes in adult life, we considered the genes encoding the IGF-I and the IGF-I receptor (IGF-IR) as candidates for low birth weight, insulin resistance, and type 2 diabetes. Here we report the mutational analysis of the coding regions of the IGF-I and IGF-IR performed on genomic DNA from probands of 82 Danish type 2 diabetic families. No mutations predicting changes in the amino acid sequences of the IGF-I or IGF-IR genes were detected, but several silent and intronic polymorphisms were found. The impact of the most prevalent polymorphism, GAG1013GAA of the IGF-IR, was evaluated in a population-based sample of 349 young healthy subjects, where the variant had an allele frequency of 0.44 (95% confidence interval, 0.40–0.48). No significant relationships between this variant and birth weight, birth length, or insulin sensitivity index were detected. In addition, we did not observe any significant differences in allelic frequencies of the codon 1013 variant between 395 type 2 diabetic patients (allele frequency, 0.52; 95% confidence interval, 0.49–0.55) and 238 matched glucose-tolerant control subjects (allelic frequency, 0.47; 95% confidence interval, 0.43–0.50). In conclusion, variability in the coding regions of IGF-I and the IGF-IR does not associate with reduced birth weight, insulin sensitivity index, or type 2 diabetes in the Danish population.

	Introduction

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INSULIN-LIKE growth factor I (IGF-I) plays a major role in mediating the growth-promoting effects of GH in postnatal life, but IGF-I is also an important intrauterine growth factor. In addition, IGF-I has the potential to elicit more insulin-like metabolic effects (1, 2). These effects may be mediated through cross-reactions, due to the structural homology of insulin and IGF-I as well as that of their receptors, or through hybrids of the receptors. However, the IGF-I receptor (IGF-IR) is present in skeletal muscle, and the metabolic effects of IGF-I in this tissue are probably mediated through its own receptor (2). Administration of IGF-I to type 2 diabetic patients increases insulin sensitivity and improves glycemic control (2), and defects in the genes encoding IGF-I/IGF-IR could have an impact on glucose tolerance in adult life.

Mouse knockouts have clearly demonstrated the critical importance of IGF-I and the IGF-IR for embryonic and postnatal growth, with mice homozygous null for IGF-I or the IGF-IR having birth weights 60% and 45% those of wild-type animals, respectively (3). Although there is no direct evidence that IGF-I has a prominent role in human fetal growth, fetal tissues express IGF-I from an early stage, and circulating fetal IGF-I concentrations are correlated with fetal size (4, 5). Furthermore, a patient with severe prenatal and postnatal growth failure probably caused by a homozygous partial deletion of the IGF-I gene has been reported (6), and a patient has been described with a deletion of the region of chromosome 15 containing the IGF-IR gene and severe prenatal and postnatal growth failure (7).

There is accumulating evidence that impaired intrauterine growth is one of the factors that contributes to the pathogenesis of type 2 diabetes, and associations between low birth weight, insulin resistance, and type 2 diabetes in adult life have repeatedly been reported (8, 9, 10). The intrauterine growth retardation may be caused by genetic and/or environmental factors, such as intrauterine malnutrition. Although a twin study has shown that the association between birth weight and type 2 diabetes is partly independent of genotype (11), a recent study has shown that mutations in the glucokinase gene result in reduced birth weight when sibling pairs discordant for the presence of the genetic variability were compared (12), thereby indicating that some of the variability in birth weight is genetically determined.

Thus, IGF-I and the IGF-IR represent logical biochemical/functional candidate genes for type 2 diabetes, and variants in these genes could explain part of the association between birth weight and impaired glucose tolerance in adult life. Both IGF-I and the IGF-IR are positional candidates genes, as well. The IGF-I gene is assigned to chromosome 12q22-q24.1 (13), which is proximal to the NIDDM2 locus at 12q24.2 (14). The IGF-IR gene is assigned to 15q25-q26 (15) and is therefore localized relatively close to a locus on chromosome 15 that has recently been shown to interact with NIDDM1 locus and increase susceptibility to diabetes in Mexican Americans (16). The objectives of this study were to search for mutations in the IGF-I and IGF-IR genes in probands of type 2 diabetic families and to examine potential genetic variability in these two proteins as contributors to type 2 diabetes, alterations in insulin sensitivity, and birth weight and length.

	Subjects and Methods

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Subjects and clinical and biochemical variables

The primary mutation analyses were performed on genomic DNA isolated from 82 probands of Danish Caucasian type 2 diabetic families who were recruited from the Danish family resource bank at the Department of Human Genetics, University of Copenhagen, or from the out-patient clinic at the Steno Diabetes Center. The families were ascertained through one type 2 diabetic proband with 4 or more nondiabetic offspring. The patients comprised 45 males and 37 females, with a mean age of 67 yr (range, 43–87), a mean body mass index (BMI) of 30.6 kg/m² (range, 22.3–48.8), and a mean reported disease onset at 56 yr (range, 27–75). Eleven percent of the patients were treated with diet alone, 83% were treated with oral hypoglycemic agents, and 6% were treated with insulin. All were negative for anti-glutamic acid decarboxylase antibodies. An association study was performed in 395 type 2 diabetic patients recruited from the out-patient clinic at the Steno Diabetes Center and 238 age-matched and glucose-tolerant Danish Caucasians. The 238 control subjects had a mean age of 52 yr (range, 30–88) and a mean BMI of 25.4 kg/m² (range, 17.5–43.3). The diabetic patients had a mean age of 55 yr (30–84), a mean BMI of 29.0 kg/m² (range, 16.5–52.3), and a reported average duration of diabetes of 6 yr (range, 0–28). Twenty-seven percent of the patients were treated with diet alone, 60% were treated with oral hypoglycemic agents, and 13% were treated with insulin. All tested negative for anti-glutamic acid decarboxylase antibodies. For studies of birth length, birth weight, and insulin sensitivity index, 349 subjects were randomly recruited from a population-based sample of young individuals, aged 18–32 yr. The physiological characteristics of these subjects have been previously described (17), with data on birth length and weight obtained from midwife records. Fasting plasma glucose, fasting serum insulin, and fasting serum C-peptide were taken after a 12-h overnight fast and analyzed as previously reported (17). The insulin sensitivity index and acute insulin response were determined after a combined iv glucose and tolbutamide tolerance test (17).

Type 2 diabetes in all affected patient groups was diagnosed in accordance with the 1985 WHO criteria. All study participants were Danish Caucasians by self-identification. The study was approved by the ethical committee of Copenhagen and was in accordance with the principles of the Declaration of Helsinki II.

Mutation analysis of the IGF-I and IGF-IR genes

Genomic DNA was obtained from human leukocyte nuclei isolated from whole blood. DNA derived from the 82 probands of Danish Caucasian type 2 diabetes patients was examined for mutations in the IGF-I and IGF-IR genes by single strand conformational polymorphism (SSCP) and heteroduplex formation analysis under 2 different experimental conditions as previously reported (18). All of the coding regions of the 5 exons of IGF-I and the 21 exons of the IGF-IR (total of 25 SSCP segments for the IGF-IR due to the examination of 3 overlapping segments in exon 2 and 2 overlapping segments in exons 3 and 21) were analyzed. The sizes of PCR segments ranged from 169–338 bp. SSCP/heteroduplex variants were sequenced in both directions as previously described (19). The sequences of primers and the PCR conditions are detailed in Table 1.

Restriction fragment length polymorphism was used to identify individuals with the GAG-GAA variant. Segments of exon 16 were amplified by PCR, and the carriers of the GAA polymorphism were demonstrated by the loss of an MnlI restriction site.

Statistical analysis

Fisher’s exact test was applied to test for differences in carrier frequencies. Differences between groups were tested with a generalized linear model using Statistical Package of Social Science for Windows (version 9.0, SPSS, Inc., Chicago, IL). Analyses included gender and genotype as fixed factors, and BMI and age as covariate factors. BMI and age were not included for the analyses of birth length, birth weight, or ponderal index.

	Results

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No nonsense, frameshift, or missense mutations were found in the IGF-I or IGF-IR genes in the 82 probands of type 2 diabetes families (Fig. 1). Six silent variants and 4 intron variants were detected in the IGF-IR gene. The 6 variants in the coding region of the IGF-IR were: codon 211, GCG (Ala)GCA (Ala) (allelic frequency, 0.01); codon 261, AGC (Ser)AGT (Ser) (allelic frequency, 0.01); codon 271, GGC (Gly)GGA (Gly) (allelic frequency, 0.02); codon 736, ACC (Thr)ACT (Thr) (allelic frequency, 0.05); codon 1013 [previously reported (20)], GAG (Glu)GAA (Glu) (allelic frequency, 0.49); and codon 1316, TAC (Tyr)TAT (Tyr) (allelic frequency, 0.05). Only 2 variants were identified in the IGF-I gene; 1 intron variant (Fig. 1) and 1 variant in codon 69 (AGG (Arg)AGA (Arg) (allelic frequency, 0.02).

View larger version (18K): [in this window] [in a new window]   Figure 1. Diagrammatic representation of the variants identified in the IGF-IR (A) and the IGF-I (B) genes. Variants in exons are shown with codon number and amino acid. Intron sequence changes are shown with nucleotide change and nucleotide position relative to splice site [+ (upstream)/- (downstream) of splice site]. Numbering for IGF-I and IGF-IR corresponds to those in Refs. 21 and 22, respectively. Boxes depict exons, coding regions are black, and noncoding regions are white.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

After the identification of a prevalent silent polymorphism at exon 16 (GAG1013GAA), we also tested whether this polymorphism could serve as a DNA marker for individual variations in birth weight, birth length, and insulin sensitivity index. Using this approach, it is possible to identify/exclude effects on the mentioned variable due to a common polymorphism that is in tight linkage disequilibrium with the GAG1013GAA polymorphism. On the other hand, effects from variants in low linkage disequilibrium or rare variants can probably not be identified/excluded by this approach. We were not able to identify any significant differences between genotype status and the mentioned variables. However, among 349 young healthy individuals we found that heterozygous carriers of the GAG1013GAA variant had significantly lower fasting serum insulin levels than wild-type and homozygous carriers. We interpreted this finding as false positive, since a gene dosage was not observed, the finding could not be replicated among 238 glucose-tolerant middle-aged subjects, and a case-control study did not show any association between the variant and type 2 diabetes.

In conclusion, no mutations with predicted functional impact were detected in the coding regions of the IGF-I and IGF-IR genes. Variability in the coding regions of the IGF-I and the IGF-IR genes can be excluded as a common cause of type 2 diabetes mellitus among Danes.

	Acknowledgments

 
We thank Helle Fjorvang, Sandra Urioste, Annemette Forman, Lene Aabo, Bente Mottlau, Susanne Kjelberg, Jane Brønnum, and Quan Truong for technical assistance, and Grete Lademann for secretarial support.

	Footnotes

 
¹ This work was supported by grants from the Danish Medical Research Council (Cellular Growth and Regeneration), the Danish Research Academy, the Danish Diabetes Association, the Velux Foundation, and the European Economic Community (BMH4-CT98-3084).

² Recipient of Bayer-EASD Travel Fellowship Award-1997.

Received September 22, 1999.

Revised November 30, 1999.

Accepted December 15, 1999.

	References

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