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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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1. Jacob R, Barret E, Plewe G, Fagin KD, Sherwin RJ. 1989 Acute effects of insulin-like growth factor-I on glucose and amino acid metabolism in awake fasted rat. J Clin Invest. 83:1717–1723.
2. Moses AC, Young SCJ, Morrow LA, O’Brien M, Clemmons DR. 1996 Recombinant human insulin-like growth factor I increases insulin sensitivity and improves glycemic control in type II diabetes. Diabetes. 45:91–100.[Abstract]
3. Liu J.-P, Baker J, Perkins AS, Robertson EJ, Efstratiadis A. 1993 Mice carrying null mutations of the genes encoding insulin-like factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell. 75:59–72.[Medline]
4. Han VKM, Lund PK, Lee DC, D’Ercole AJ. 1988 Expression of somatomedin/insulin-like growth factor messenger ribonucleic acids in the human fetus: identification, characterization, and tissue distribution. J Clin Endocrinol Metab. 66:422–429.[Abstract/Free Full Text]
5. Giudice LC, de Zegher F, Gargosky SE, et al. 1995 Insulin-like growth factors and their binding proteins in the term and preterm human fetus and neonate with normal and extremes of intrauterine growth. J Clin Endocrinol Metab. 80:1548–1555.[Abstract/Free Full Text]
6. Woods KA, Camacho-Hübner C, Savage MO, Clark AJL. 1996 Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. N Engl J Med. 335:1363–1367.[Free Full Text]
7. Roback EW, Barakat AJ, Dev VG, Mbikay M, Chrétien M, Butler MG. 1991 An infant with deletion of the distal long arm of chromosome 15 (q26.1qter) and loss of insulin-like growth factor 1 receptor gene. Am J Med Gen. 38:74–79.[CrossRef][Medline]
8. Phillips DIW, Barker DJP, Hales CN, Hirst S, Osmond C. 1994 Thinness at birth and insulin resistance in adult life. Diabetologia. 37:150–154.[CrossRef][Medline]
9. Lithell HO, McKeigue PM, Berglund L, Mohsen R, Lithell UB, Leon DA. 1996 Relation of size at birth to non-insulin dependent diabetes and insulin concentrations in men aged 50–60 years. Br Med J. 312:406–410.[Abstract/Free Full Text]
10. McCance DR, Pettitt DJ, Hanson RL, Jacobsson LTH, Knowler WC, Bennett PH. 1994 Birth weight and non-insulin dependent diabetes: thrifty genotype, thrifty phenotype, or surviving small baby genotype? Br Med J. 308:942–945.[Abstract/Free Full Text]
11. Poulsen P, Vaag AA, Kyvik KO, Jensen DM, Beck-Nielsen H. 1997 Low birth weight is associated with NIDDM in discordant monozygotic and dizygotic twin pairs. Diabetologia. 40:439–446.[CrossRef][Medline]
12. Hattersley AT, Beards F, Ballantyne E, Appleton M, Harvey R, Ellard S. 1998 Mutations in the glucokinase gene of the fetus result in reduced birth weight. Nat Genet. 19:268–270.[CrossRef][Medline]
13. Yang-Feng TL, Brissenden JE, Ullrich A, Francke U. 1985 Sub-regional localization of human genes for insulin-like growth factors I (IGF1) and II (IGF2) by in situ hybridization. Cytogenet Cell Genet. 40:782 only.
14. Mahtani MM, Widen E, Lehto M, et al. 1996 Mapping of a gene for type 2 diabetes associated with an insulin secretion defect by a genome scan in Finnish families. Nat Genet. 14:90–94.[CrossRef][Medline]
15. Francke U, Yang-Feng TE, Brissenden JE, Ullrich A. 1986 Chromosomal mapping of genes involved in growth control. Spring Harbor symp. Quant Biol. 51:855–866.
16. Cox NJ, Frigge M, Nicolae DL, et al. 1999 Loci on chromosome 2 (NIDDM1) and 15 interact to increase susceptibility to diabetes in Mexican Americans. Nat Genet. 21:213–215.[CrossRef][Medline]
17. Clausen JO, Borch-Johnsen K, Ibsen H, et al. 1996 Insulin sensitivity index, acute insulin response, and glucose effectiveness in a population based sample of 380 young healthy caucasians. J Clin Invest. 98:1195–1209.[Medline]
18. Hansen L, Hansen T, Vestergaard H, et al. 1995 A widespread amino acid polymorphism at codon 905 of the glycogen-associated regulatory subunit of protein phosphatase-1 is associated with insulin resistance and hypersecretion of insulin. Hum Mol Genet. 4:1313–1320.[Abstract/Free Full Text]
19. Hansen L, Echwald SM, Hansen T, Urhammer SA, Clausen JO, Pedersen O. 1997 Amino acid polymorphism in the ATP-regulatable inward rectifier Kir6.2 and their relationships to glucose- and tolbutamide-induced insulin secretion, the insulin sensitivity index, and NIDDM. Diabetes. 508–512:1997.
20. Abu-Amero S, Price S, Wakeling E., et al. 1997 Lack of hemizygosity for the insulin-like growth factor I receptor gene in a quantitative study of 33 Silver Russel syndrome probands and their families. Eur J Hum Genet. 5:235–241.[Medline]
21. Rotwein P, Pollock KM, Didier DK, Krivi GG. 1986 Organization and sequence of the human insulin-like growth factor I gene. J Biol Chem. 261:4828–4832.[Abstract/Free Full Text]
22. Abbott AM, Bueno R, Pedrini MT, Murray JM, Smith RJ. 1992 Insulin-like growth factor I receptor gene structure. J Biol Chem. 267:10759–10763.[Abstract/Free Full Text]

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