The putative role of the hormone-sensitive lipase gene in the pathogenesis of Type II diabetes mellitus and abdominal obesity.

Impaired lipolysis has been proposed as a pathogenic factor contributing to clustering of abdominal obesity and dyslipidaemia in Type II (non-insulin-dependent) diabetes mellitus--that is, the metabolic syndrome (MSDR). As this syndrome clusters in families, alterations in the hormone-sensitive lipase (HSL) gene could contribute to the genetic predisposition to MSDR. To test this hypothesis we carried out population and intrafamily association studies in individuals with MSDR, using a polymorphic marker (LIPE) in the HSL gene. There was a significant difference in allele frequency distribution between 235 Type II diabetic patients and 146 control subjects (p = 0.002), particularly between 78 abdominally obese Type II diabetic patients with MSDR and the control group (p = 0.010). An extended transmission disequilibrium test (TDT) showed transmission disequilibrium of 66 alleles to 42 nondiabetic, abdominally obese offspring in families with Type II diabetes (p < 0.05). A slight difference in allele frequency distribution was seen between 71 individuals from the lowest and 71 from the highest tertile of isoprenaline-induced lipolysis in fat tissue (p = 0.07). No missense mutations were found with single-strand conformational polymorphism (SSCP) in 20 abdominally obese subjects with MSDR. In conclusion, our population and intrafamily association studies suggest that the LIPE marker in the HSL gene is in linkage disequilibrium with an allele and/or gene which increases susceptibility to abdominal obesity and thereby possibly to Type II diabetes.

No relationship between identified variants in the uncoupling protein 2 gene and energy expenditure.

OBJECTIVE: The uncoupling protein 2 (UCP2) uncouples respiration from the oxidative phosphorylation in most cell types, predominantly in white fat and skeletal muscle. Since a decreased basal metabolic rate (BMR) would increase the susceptibility to weight gain, genetic alterations in the UCP2 gene could contribute to the pathogenesis of obesity and the metabolic syndrome (MSDR). DESIGN AND METHODS: To test this hypothesis, we PCR amplified the introns of the UCP2 gene and sequenced the exon/intron boundaries. This information was used to construct intronic primers and to screen obese patients with low BMR for mutations in the coding regions of the UCP2 gene, using the single-strand conformational polymorphism technique. Furthermore, we examined whether there is an association between a biallelic marker in the UCP2 gene and BMR or MSDR. RESULTS: The UCP2 gene is composed of six coding exons, covering 5 kb of chromosome 11q13. One polymorphism, but no mutations, were identified in the coding regions of the UCP2 gene. There were no significant differences in the allele or genotype frequencies of the Ala55Val polymorphism between 55 patients with MSDR and 46 healthy controls. No association was found between the UCP2 gene and BMR in patients with MSDR or in healthy controls. CONCLUSIONS: Mutation screening and association studies suggest that mutations in the coding regions of the UCP2 gene do not affect BMR and do not contribute to increased susceptibility to obesity or MSDR. The results cannot, however, exclude the possibility that variants in regulatory elements of the gene could contribute to the development of obesity or MSDR.

Mutations in the liver glycogen synthase gene in children with hypoglycemia due to glycogen storage disease type 0.

Glycogen storage disease type 0 (GSD-0) is a rare form of fasting hypoglycemia presenting in infancy or early childhood and accompanied by high blood ketones and low alanine and lactate concentrations. Although feeding relieves symptoms, it often results in postprandial hyperglycemia and hyperlactatemia. The glycogen synthase (GS) activity has been low or immeasurable in liver biopsies, whereas the liver glycogen content has been only moderately decreased. To investigate whether mutations in the liver GS gene (GYS2) on chromosome 12p12.2 were involved in GSD-0, we determined the exon-intron structure of the GYS2 gene and examined nine affected children from five families for linkage of GSD-0 to the GYS2 gene. Mutation screening of the 16 GYS2 exons was done by single-strand conformational polymorphism (SSCP) and direct sequencing. Liver GS deficiency was diagnosed from liver biopsies (GS activity and glycogen content). GS activity in the liver of the affected children was extremely low or nil, resulting in subnormal glycogen content. After suggestive linkage to the GYS2 gene had been established (LOD score = 2.9; P < 0.01), mutation screening revealed several different mutations in these families, including a premature stop codon in exon 5 (Arg246X), a 5'-donor splice site mutation in intron 6 (G+1T--> CT), and missense mutations Asn39Ser, Ala339Pro, His446Asp, Pro479Gln, Ser483Pro, and Met491Arg. Seven of the affected children carried mutations on both alleles. The mutations could not be found in 200 healthy persons. Expression of the mutated enzymes in COS7 cells indicated severely impaired GS activity. In conclusion, the results demonstrate that GSD-0 is caused by different mutations in the GYS2 gene.

Mutations and variants of the epithelial sodium channel gene in Liddle's syndrome and primary hypertension.

Liddle's syndrome is a rare monogenic form of hypertension caused by truncating or missense mutations in the C termini of the epithelial sodium channel beta- or gamma-subunits. These mutations delete or alter a conserved proline-rich amino acid sequence referred to as the PY-motif. We report here a Liddle's syndrome family with a betaArg564X mutation with a premature stop codon deleting the PY-motif of the beta-subunit. This family shows marked phenotypic variation in blood pressure, serum potassium levels, and age of onset of hypertension. Given the similarity with primary hypertension, changes in the C termini of the beta- or gamma-subunits may contribute to the development of primary hypertension or to hypertension associated with diabetic nephropathy. Accordingly, the coding sequences for the cytoplasmic C termini of the beta- and gamma-subunits were screened for mutations with the use of polymerase chain reaction, single-strand conformation polymorphism, and direct DNA sequencing in 105 subjects with primary hypertension and 70 subjects with diabetic nephropathy. One frequent polymorphism was identified, but its frequency did not differ among subjects with primary hypertension, subjects with diabetic nephropathy, or control subjects. Two of the 175 subjects with primary hypertension or diabetic nephropathy showed variants that were not present in 186 control subjects. None of the variants changed the PY-motif sequence. In conclusion, a betaArg564X mutation is the likely cause of Liddle's syndrome in this Swedish family, but it is unlikely that mutations in the beta- and gamma-subunit genes of the epithelial sodium channel play a significant role in the pathogenesis of primary hypertension or diabetic nephropathy.

Novel mutations and a mutational hotspot in the MODY3 gene.

Maturity-onset diabetes of the young 3 (MODY3) is a type of NIDDM caused by mutations in the transcription factor hepatocyte nuclear factor-1alpha (HNF-1alpha) located on chromosome 12q. We have identified four novel HNF-1alpha missense mutations in MODY3 families. In four additional and unrelated families, we observed an identical insertion mutation that had occurred in a polycytidine tract in exon 4. Among those families, one exhibited a de novo mutation at this location. We propose that instability of this sequence represents a general mutational mechanism in MODY3. We observed no HNF-1alpha mutations among 86 unrelated late-onset diabetic patients with relative insulin deficiency. Hence mutations in this gene appear to be most strongly associated with early-onset diabetes.

Characterization of the MODY3 phenotype. Early-onset diabetes caused by an insulin secretion defect.

Maturity-onset diabetes of the young (MODY) type 3 is a dominantly inherited form of diabetes, which is often misdiagnosed as non-insulin-dependent diabetes mellitus (NIDDM) or insulin-dependent diabetes mellitus (IDDM). Phenotypic analysis of members from four large Finnish MODY3 kindreds (linked to chromosome 12q with a maximum lod score of 15) revealed a severe impairment in insulin secretion, which was present also in those normoglycemic family members who had inherited the MODY3 gene. In contrast to patients with NIDDM, MODY3 patients did not show any features of the insulin resistance syndrome. They could be discriminated from patients with IDDM by lack of glutamic acid decarboxylase antibodies (GAD-Ab). Taken together with our recent findings of linkage between this region on chromosome 12 and an insulin-deficient form of NIDDM (NIDDM2), the data suggest that mutations at the MODY3/NIDDM2 gene(s) result in a reduced insulin secretory response, that subsequently progresses to diabetes and underlines the importance of subphenotypic classification in studies of diabetes.

UKPDS 19: heterogeneity in NIDDM: separate contributions of IRS-1 and beta 3-adrenergic-receptor mutations to insulin resistance and obesity respectively with no evidence for glycogen synthase gene mutations. UK Prospective Diabetes Study.

Insulin receptor substrate-1 (IRS-1), beta 3-adrenergic-receptor (beta 3-AR) and glycogen synthase (GS) genes are candidate genes for non-insulin-dependent diabetes mellitus (NIDDM), insulin resistance, dyslipidaemia and obesity. We studied white Caucasian subjects with NIDDM, 227 being randomly selected, 49 NIDDM within the top two percentiles of insulin resistance; 54 with dyslipidaemia in the top quintile of triglyceride/insulin and the bottom quintile of HDL, and 166 non-diabetic control subjects. We examined the association of the simple tandem repeat DNA polymorphisms (STRPs) near the IRS-1 and GS genes, and the prevalence of mutations at codons of IRS-1 513 and 972, beta 3-AR 64 and GS 464 using restriction fragment length polymorphism (RFLP). The STRP alleles in IRS-1 were significantly different between NIDDM and control subjects (p = 0.015). The IRS-1 972 mutation was significantly different between the four groups with increased prevalence in the insulin resistant and dyslipidaemia subjects (18 and 26% compared with 11% in control subjects; p < 0.0005). Those with or without IRS-1 mutations had similar clinical characteristics and impaired insulin sensitivity. beta 3-AR 64 mutation was not significantly different between the four groups but those with the mutation were more obese, with a test for linear association between number of alleles and degree of obesity in an analysis of variance showing a significant association (p = 0.029). The GS 464 mutation was not detected in any of the diabetic or control subjects and the population association study using GS STRP showed no difference in allelic frequencies between NIDDM patients and control subjects. A mutation in lipoprotein lipase at codon 291, associated in the general population with low HDL cholesterol, was not at increased prevalence in the NIDDM patients with dyslipidaemia. In conclusion, IRS-1 972 had an increased prevalence in subjects with insulin resistance, with or without dyslipidaemia. beta 3-AR 64 was associated with increased obesity but not with insulin resistance or dyslipidaemia. These separate contributions to different features of NIDDM are an example of the polygenic inheritance of this heterogeneous disorder.

Polymorphism at the rad gene is not associated with NIDDM in Finns.

Recently, a trinucleotide repeat polymorphism at the rad (ras associated with diabetes) locus (RAD1) on chromosome 16q was described in association with NIDDM in white Americans. In an attempt to replicate this finding, we screened RAD1 and another microsatellite marker at the D16S265 loci, which is located near the rad locus, with a radioactive polymerase chain reaction method in 290 unrelated Finnish NIDDM patients and 270 control subjects and related the findings to measures of insulin sensitivity. Both groups were randomly selected from the western (189 NIDDM patients and 184 control subjects) and southern (101 NIDDM and 86 control subjects) parts of Finland. The allele frequency distributions of RAD1 and D16S265 did not differ between NIDDM patients and control subjects in the studied population groups. The genotype distribution was also analyzed by structural classes of the RAD1 polymorphism, and no difference was detected between the NIDDM and control groups. In addition, carriers of allele classes I, II, and IV (reported to be preferentially associated with NIDDM in white Americans) did not differ from the class III homozygotes with respect to age at onset of NIDDM, BMI, or rates of insulin-stimulated glucose disposal. In conclusion, we found no association between the rad locus and NIDDM or insulin resistance in Finnish NIDDM patients.

Lack of association between the Gly40Ser polymorphism in the glucagon receptor gene and NIDDM in Finland.

A heterozygous polymorphism changing GGT40 (Gly) to AGT40 (Ser) (Gly40Ser) in the glucagon receptor gene was reported to be associated with non-insulin-dependent diabetes mellitus (NIDDM). A possible involvement of this polymorphism in impaired glucose tolerance was also suggested in a French population. To replicate this finding we screened 311 unrelated NIDDM patients, 101 unrelated individuals with impaired glucose tolerance and 306 control subjects for the presence of the Gly40Ser polymorphism by use of polymerase chain reaction-restriction fragment length polymorphism in a Finnish population. None of the NIDDM or impaired glucose tolerant patients had this polymorphism. Instead, four of the control subjects (1.3%) were heterozygous carriers of the polymorphism (NS). The age, body mass index, 2-h blood glucose level, 2-h insulin level, and incremental insulin are of the four subjects with this polymorphism were similar to those of the control subjects homozygous for the wild type. Taken together, the data do not support the suggested involvement of the Gly40Ser polymorphism in impaired glucose tolerance and the hypothesis of an association between NIDDM and the glucagon receptor gene in this population.

Isolation and characterization of the human muscle glycogen synthase gene.

Impaired glycogen synthase (GS) activity in skeletal muscle has been considered to be an inherited trait in patients with non-insulin-dependent diabetes mellitus (NIDDM). We therefore isolated the human muscle GS gene from genomic libraries and determined the genomic structure. The entire coding region, the 5'-flanking region, and the exon-intron boundaries were sequenced. The gene consists of 16 exons spanning approximately 27 kb of DNA and exists as a single copy in the human genome. The negatively charged parts with all known phosphorylation sites were coded by the first and the last exon. A single transcription initiation site was located 167 nucleotides upstream of the initiation codon. All of the exons and the putative promoter region were analyzed by single-strand conformation polymorphism in 30 insulin-resistant Finnish NIDDM patients, and three polymorphic sites were found. A missense mutation Gly464/Ser in exon 11 was found in 2 of 228 NIDDM patients screened but in 0 of 154 control subjects. These two patients were characterized further by severe insulin resistance and premature arteriosclerosis. The characterization of the genomic structure of the human muscle GS gene will facilitate studies of its role in the development of insulin resistance and NIDDM.