Although resistance to insulin action is a well-studied phenomenon in non-insulin-dependent diabetes and certain genetic syndromes, the role of inherited defects of the insulin receptor in these disorders is unknown. To facilitate the evaluation of that role, restriction fragment length polymorphisms (RFLPs) were identified using various portions of the insulin receptor cDNA to examine digested DNA from American Blacks, Pina Indians, and Caucasians. Five RFLPs were identified in Caucasians. Two of these were detected with a single 1.3-kilobase probe in Rsa I digests with minor allele frequencies of 0.48 and 0.23. An additional RFLP was noted with Bgl H and two more RFLPs with Sac I using a different 1.6-kilobase probe, with minor allele frequencies of 0.17 for Bgl II and 0.12 for both Sac I RFLPs. All RFLPs except for the second Sac I RFLP were present in American Blacks, while only the Rsa I RFLPs were present in Pima Indians. Pairwise analysis showed random association between all sites except for the Bgl II and second Rsa I sites, where the disequilibrium statistic, A, was -0.70 (different from 0 at P < 0.001). No association of any RFLP was noted with non-insulin-dependent diabetes in a small population. These studies show that this is a highly informative locus that should be important for mapping of chromosome 19p and for linkage studies.
Polymorphic sites adjacent to known genes can be used to examine the segregation of a disease relative to that gene in families, or to map the gene of interest relative to other loci. The polymorphic region 5' to the human insulin gene (5' FP) permits such analysis, but the three size classes previously identified are insufficient for many studies. More alleles are identified with restriction enzymes that generate small fragments (Pvu II). Nonetheless, sufficient polymorphism for informative family analyses is often not present. To facilitate such analyses, we searched for other polymorphisms in over 20 KB of DNA at the insulin locus in Pima Indians, American Blacks, and Caucasians. The previously described allelic variant at a Pst I site in the 3'-untranslated portion of the gene was not polymorphic in any race. An upstream Hinc II site (-62 BP) was present in only 48% of Black alleles, but was not polymorphic in Pima Indians or Caucasians. New polymorphisms were found at a Taq I site (-11,000 BP) and a Rsa I site (-13,000 BP). The Taq I site was present in 89% of Black alleles, 87% of Pima Indian alleles, and 84% of Caucasian alleles. In contrast, the Rsa I site was present in 60% of Black and Caucasian alleles, but in only 47% of Pima Indian alleles. The Hinc II, Rsa I, and Taq I sites show no obvious linkage with each other or the 5' FP. A fourth polymorphism, previously identified with Sac I, was found to be the creation of a new Sac I site at +2500 BP in 10% of Black alleles.(ABSTRACT TRUNCATED AT 250 WORDS)
Although non-insulin-dependent diabetes mellitus (NIDDM) is well recognized to be an inherited disease, the genetic lesion responsible remains to be determined. Several pedigrees have been reported in which defects of the insulin gene result in glucose intolerance or diabetes in affected members, but the role of insulin gene mutations in NIDDM is unknown. To evaluate this role, we ascertained 23 Caucasian pedigrees for a diabetic individual with at least one diabetic family member, sampled the unaffected individuals by a 75-g glucose tolerance test, and prepared leukocyte DNA on all family members. Included in the pedigrees ascertained were those with both predominantly lean and predominantly obese diabetic members and four pedigrees included as insulin-dependent diabetic individual. Insulin gene involvement was evaluated via previously described restriction-fragment-length polymorphisms (RFLPs) for the insulin gene and the nearby c-Ha-Ras oncogene (HRAS). Combination of these RFLPs resulted in the ability to trace the insulin alleles in all pedigrees studied. Analysis of individual pedigrees for sharing of insulin alleles was possible in 12 pedigrees, and lack of linkage was demonstrated in 6 of them. Neither linkage nor lack of linkage could be proved in the remaining pedigrees. Analysis of the pooled pedigree data failed to demonstrate linkage under several models, including autosomal-dominant and -recessive inheritance with different sporadic frequencies of diabetes and different prevalence figures. These results show that mutations of the insulin gene and the immediately surrounding area, including regulatory regions of the insulin gene, are unlikely to account for a significant subset of NIDDM in Caucasian individuals.
The cloning of the insulin receptor cDNA has permitted the definition of restriction fragment length polymorphisms at that locus. These polymorphisms were used to study the role of the insulin receptor in four pedigrees with maturity onset diabetes of the young through linkage analyses. When each pedigree was individually analysed, no linkage was demonstrated in the two larger pedigrees, implying that an insulin receptor defect was not responsible for the predisposition to diabetes in these pedigrees. One of these pedigrees was known to be hypoinsulinaemic, while insulin levels were unavailable in the second pedigree. In the two smaller pedigrees, however, a single haplotype cosegregated with diabetes. One of these pedigrees is known to be hyperinsulinaemic. The small size of the pedigrees which demon-strated cosegregation precluded statistical proof of linkage. Nonetheless, the presence of an uncommon insertional polymorphism which cosegregated with diabetes in both pedigrees was improbable and suggested that this insertion could be responsible for diabetes in these families. This study thus may be additional evidence for heterogeneity in maturity onset diabetes of the young. For the two larger pedigrees, the insulin gene and HLA region have already been eliminated as genetic markers. This study provides data which eliminate a third candidate gene in these two pedigrees.
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