P. J. Saker

Studies of Association between the Gene for Calpain-10 and Type 2 Diabetes Mellitus in the United Kingdom

Variation in CAPN10, the gene encoding the ubiquitously expressed cysteine protease calpain-10, has been associated with type 2 diabetes in Mexican Americans and in two northern-European populations, from Finland and Germany. We have studied CAPN10 in white subjects of British/Irish ancestry, using both family-based and case-control studies. In 743 sib pairs, there was no evidence of linkage at the CAPN10 locus, which thereby excluded it as a diabetes-susceptibility gene, with an overall sib recurrence risk, lambda(S), of 1.25. We examined four single-nucleotide polymorphisms (SNP-44, -43, -19, and -63) previously either associated with type 2 diabetes or implicated in transcriptional regulation of calpain-10 expression. We did not find any association between SNP-43, -19, and -63, either individually or as part of the previously described risk haplotypes. We did, however, observe significantly increased (P=.033) transmission of the less common C allele at SNP-44, to affected offspring in parents-offspring trios (odds ratio 1.6). An independent U.K. case-control study and a small discordant-sib study did not show significant association individually. In a combined analysis of all U.K. studies (P=.015) and in combination with a Mexican American study (P=.004), the C allele at SNP-44 is associated with type 2 diabetes. Sequencing of the coding region of CAPN10 in a group of U.K. subjects revealed four coding polymorphisms-L34V, T504A, R555C, and V666I. The T504A polymorphism was in perfect linkage disequilibrium with the diabetes-associated C allele at SNP-44, suggesting that the synthesis of a mutant protein and/or altered transcriptional regulation could contribute to diabetes risk. In conclusion, we were not able to replicate the association of the specific calpain-10 alleles identified by Horikawa et al. but suggest that other alleles at this locus may increase type 2 diabetes risk in the U.K. population.

An uncoupling protein 2 gene variant is associated with a raised body mass index but not Type II diabetes

Aims/hypothesis. Linkage between markers close to the uncoupling protein 2 and 3 genes (11q13) and resting metabolic rate and a pre-diabetic phenotype have been found. The syntenic region in mouse has been found to be linked to quantitative traits associated with obesity and diabetes. UCP2 and UCP3 could therefore have an important role in body weight regulation and susceptibility to diabetes. We investigated a recently identified variant of the UCP2 gene in exon 8 as a marker for glucose and weight homeostasis. Methods. Length variation of the UCP2 exon 8 variant was studied by the polymerase chain reaction and agarose gel electrophoresis. Sequence variants of the UCP3 gene were sought by semi-automated DNA sequencing. Results. In 453 South Indian subjects, we found an association in women between the UCP2 exon variant and body mass index (p = 0.018). These findings were replicated in a separate group of South Indian subjects (n = 143, p < 0.001) irrespective of sex. Although no association was found between the UCP2 exon 8 variant and overt obesity in British subjects, the UCP2 genotype of obese women (n = 83) correlated with fasting serum leptin concentration (p = 0.006) in the presence of extreme obesity. These observations could not be explained by tight linkage disequilibrium with a coding region variant in the region of the UCP3 gene of biological significance. Lastly, no association was found between UCP2 and Type II (non-insulin-dependent) diabetes using either a family based design (85 families) or case control study (normal glucose tolerance n = 335, impaired glucose tolerance n = 42, Type II diabetes n = 76). Conclusion/interpretation. We have described a UCP2 gene exon 8 variant that may affect susceptibility to weight gain by influencing regulation of leptin. [Diabetologia (1999) 42: 688–693]

Evidence that single nucleotide polymorphism in the uncoupling protein 3 (UCP3) gene influences fat distribution in women of European and Asian origin

Aims/hypothesis. Uncoupling proteins are mitochondrial transmembrane carriers implicated in the regulation of energy balance. Dysfunction of UCP3 (the predominant uncoupling protein in skeletal muscle) might therefore be expected to reduce thermogenic capacity, alter energy homeostasis and influence predisposition to obesity and Type II (non-insulin-dependent) diabetes mellitus. A variant in the putative promoter region of UCP3 (–55 c→t) has recently been identified, and an association with obesity reported in French subjects. Our aim was to study the pathophysiological role of this variant in diabetes-related and obesity-related traits using two distinct ethnic populations. Methods. The –55 c→t variant was genotyped in 85 South Indian and 150 European parent-offspring trios ascertained through Type II diabetic probands and in 455 South Indian subjects initially recruited to an urban survey into the prevalence of diabetes. Results. In South Indian and European parent-offspring trios there was no preferential transmission of either allele at the –55 c→t polymorphism to diabetic offspring (South Indians, p = 0.60; Europeans, p = 0.15). When family members were analysed for intermediate traits, the t-allele was associated with increased waist-to-hip ratio but only in females (South Indian mothers p = 0.036, daughters p = 0.032: European mothers p = 0.037, daughters p = 0.14). These findings were replicated in South Indian females from the population-based survey (p = 0.039). Conclusion/interpretation. The consistent association between the t-allele at this locus and increased waist-to-hip ratio in women from three separate data sets indicates that variation at this polymorphism (or another locus with which it is in linkage disequilibrium) influences fat distribution but that this effect is restricted to females. [Diabetologia (2000) 43: 1558–1564]

Uncoupling protein 3 genetic variants in human obesity: the c-55t promoter polymorphism is negatively correlated with body mass index in a UK Caucasian population

OBJECTIVE: To investigate whether genetic variation at the UCP3 locus contributes to human obesity.SUBJECTS: Ninety-one obese children (BMI>4 standard deviations from age related mean) and 419 Caucasian adults from the Isle of Ely Study.DESIGN: Single strand conformation polymorphism (SSCP) analysis was used to scan the coding region of the UCP3 gene in 91 severely obese children. A common polymorphism identified in this gene (c-55t) has been shown to associate with lower UCP3 mRNA expression. Polymerase chain reaction-based forced restriction digestion was used to detect this allele in Caucasian adults. Multiple regression analysis was used to determine associations between the c-55t genotype and anthropometric, energetic and biochemical indices relevant to obesity.MEASUREMENTS: For the obese children, SSCP analysis and sequencing of variants were carried out. For the Isle of Ely Study, c-55t genotype and anthropometric (body mass index, waist–hip ratio, percentage body fat), energetic (dietary fat intake, physical activity index, adjusted metabolic rate, maximum oxygen consumption) and biochemical indices (pre- and post-glucose challenge plasma triglycerides, non-esterified fatty acids, insulin and glucose) were determined.RESULTS: A previously reported missense mutation (V102I) was detected in a single obese Afro-Carribean child. Twenty-one percent of the genes examined in the Isle of Ely study carried the c-55t promoter variant. Age-adjusted body mass index (BMI) was significantly (P=0.0037) lower in carriers of this variant.CONCLUSION: Mutations in the coding sequence of UCP3 are unlikely to be a common monogenic cause of severe human obesity. In a Caucasian population the UCP3 c-55t polymorphism is negatively associated with BMI.International Journal of Obesity (2001) 25, 472–477

Glucokinase mutations in a phenotypically selected multiethnic group of women with a history of gestational diabetes

Gestational diabetes mellitus (GDM) frequently provides the ®rst presentation of glucose intolerance in women with mutations in the glucokinase gene (GCK). Identifying such women during, or immediately after, their period of antenatal care is desirable since a molecular diagnosis of GCK de®ciency provides valuable prognostic information on the expected future patterns of glucose homeostasis of both mother and fetus [1,2]. However, molecular screening for GCK mutations is labour-intensive and currently impractical for universal use in GDM, and, as a result, preselection remains a necessary component of any molecular screening strategy. Ellard et al. recently described a set of clinical criteria, which when applied to a large cohort of GDM women, successfully preselected 15 European GDM women yielding a high prevalence (80%) of GCK mutations [2]. We have applied similar (but, in our case, purely biochemical) criteria to our local multiethnic antenatal population, namely: (i) mild fasting hyperglycaemia (5.5±8.0 mmol/l) in pregnancy; (ii) persisting mild fasting hyperglycaemia outside pregnancy (5.5±8.0 mmol/l); and (iii) an increment between the fasting and 2-h plasma glucose concentrations < 3.5 mmol/l during the 75-g OGTT postpartum. These criteria were met by 17 women of multiethnic origin (Table 1) with a median (interquartile range) age of 37.5 (34.0±40.3) years and a median parity of 2. Singlestranded conformational polymorphism analysis followed by direct sequencing was used to screen these women for GCK variants. Functional variants, previously reported, were detected in two of the 17 (12%) subjects: R403fsdelC (exon 9) and T228M (exon 7). In fact, neither subject would have been selected for screening according to the non-biochemical criteria included in previous studies [2] as neither had received insulin during pregnancy and only one had a family history of diabetes. R403fsdelC (exon 9) is a frameshift mutation and hence of probable functional signi®cance [2] and T228M (exon 7) is a missense mutation previously shown to cosegregate with Type 2 diabetes [3]. In addition, three novel and one previously reported intronic variants (IVS4nt±48insC, IVS9nt±18C®A, IVS9nt+49G®A, IVS9nt+8T®C), all considered neutral polymorphisms, were identi®ed in three South Asian subjects. The prevalence of functional GCK mutations detected in our study (12%) is higher than reported in some studies [4± 6], but considerably lower (P = 0.0001) than that reported by Ellard et al. [2]. There are three principal differences between the Ellard study [2] and ours. First, Ellard et al. applied additional non-biochemical preselection criteria, including family history of diabetes and insulin treatment during pregnancy. Given that the two subjects found in our study to have GCK-coding mutations would not have been preselected for molecular screening had the Ellard criteria been applied, we suggest that these non-biochemical criteria may be unnecessarily strict. Second, Ellard et al. screened only European women. It seems probable that the high prevalence and young onset of (non-GCK) Type 2 diabetes in Asian and Afro-Caribbean populations may reduce the relative proportion of GDM attributable to GCK mutations in these groups. Third, we note that our study subjects were fairly overweight which would be less typical of women with GCK mutations [1]. In summary, whilst a strong case can be made for molecular screening of the glucokinase gene in women with GDM, the most appropriate strategy remains to be determined. On current evidence, we recommend that GDM women with modestly elevated postpartum fasting glucose and a small glucose increment during an OGTT represent the most suitable group for genetic screening.

Non-linkage of the glucagon-like peptide 1 receptor gene with maturity onset diabetes of the young

SummaryGlucagon-like peptide-1 (GLP-1) is a hormone derived from the preproglucagon molecule that is secreted by intestinal L cells and stimulates insulin secretion from betacells. The GLP-1 receptor is a candidate gene for diabetes mellitus, as mutations may induce the impaired insulin response that is a characteristic feature of NIDDM. To study the relationship between the GLP-1 receptor gene and NIDDM, linkage of a microsatellite polymorphism flanking the GLP-1 receptor gene with diabetes was investigated in three Caucasian families with MODY and in the nuclear families of 12 NIDDM probands. A cumulative LOD score −8.50 excludes linkage in these MODY pedigrees. A LOD score of −1.24 in the NIDDM nuclear pedigrees makes linkage improbable. Mutations in or near the GLP-1 receptor gene are unlikely to be the major cause of the inherited predisposition to NIDDM in Caucasian pedigrees, but we cannot exclude a role for this locus in a polygenic model or a major role in some pedigrees.

Prohormone Convertase 1 in Obesity, Gestational Diabetes Mellitus, and NIDDM: No Evidence for a Major Susceptibility Role

Improved understanding of the primary molecular events underlying NIDDM and obesity is essential if more effective therapies are to be devised. Individual susceptibility to these interrelated conditions is under genetic influence (1), and clues to the identity of the major aetiological genes may come from physiological studies that pinpoint candidate pathways. A characteristic feature of NIDDM and certain prediabetic states, such as gestational diabetes mellitus (GDM), is a marked increase in the proportion of circulating insulin precursor molecules (proinsulin and split proinsulin intermediates) (2,3). Release of disproportionate amounts of these biologically inactive precursors may contribute to the relative insulin deficiency apparent in GDM and NIDDM (2). The processing of proinsulin to mature insulin is catalyzed by prohormone convertase (PC) enzymes active in (3-cell granules. PCI (also named PC3) cleaves intact proinsulin to produce 32,33 split proinsulin. PC2 and carboxypeptidase E (CPE) catalyze subsequent reactions (4). Functional defects in any of these candidate genes could contribute to the NIDDM phenotype. Furthermore, recent studies indicate a role for these loci in the determination of obesity. Mutations in the Cpe gene resulting in absent enzyme activity in islets and pituitary underlie the phenotype of the fat/fat mouse (5). More recently, Jackson et al. (6) reported on a family segregating two distinctPCi mutations. The mother was a compound heterozygote who had presented with childhood obesity, GDM, and a variety of

Candidate gene studies in pedigrees with maturity-onset diabetes of the young not linked with glucokinase

SummaryMaturity-onset diabetes of the young (MODY) is a form of non-insulin-dependent diabetes mellitus characterised by an early age of onset and an autosomal dominant mode of inheritance. Only a proportion of cases are due to mutations in the glucokinase gene. We have studied five Caucasian MODY families, including the first MODY family to be described, with five candidate genes implicated in regulation of insulin secretion. The affected subjects showed more marked hyperglycaemia than that found in subjects with glucokinase mutations. We assessed polymorphic markers close to the genes for glucokinase, hexokinase II, adenosine deaminase, pituitary adenylate cyclase-activating polypeptide receptor, and glucagon-like peptide-1 receptor. Linkage analysis with diabetes gave cumulative log of the odds (LOD) scores of less than -3, implying that mutations in these genes are unlikely to provide a major genetic contribution to this form of MODY.

Mitochondrial FAD-Glycerophosphate Dehydrogenase and G-Protein—Coupled Inwardly Rectifying K+ Channel: No Evidence for Linkage in Maturity-Onset Diabetes of the Young or NIDDM

Two genes that have potentially important regulatory roles in insulin secretion are both located on chromosome 2q24.1. G-protein–coupled muscarinic potassium channel (GIRK1) is an inwardly rectifying K+ channel that helps to maintain the resting potential and excitability of cells. Mitochondrial FAD-linked glycerophosphate dehydrogenase (m-GDH) catalyzes a rate-limiting step of the glycerol phosphate shuttle in pancreatic islets. Reduced m-GDH activity has been demonstrated in islets isolated from diabetic subjects compared with islets from nondiabetic control subjects and from the diabetic GK rat. To study the relationship between these candidate genes and NIDDM, we have examined a simple tandem-repeat polymorphism (STRP) close to both the KCN J3 (GIRK1) locus and the m-GDH locus. In a linkage study of three maturity-onset diabetes of the young (MODY) pedigrees, not linked to MODY1, MODY2, or MODY3, a cumulative score of −9.6 at a recombination fraction of θ = 0 excluded linkage. In a population-association study, no linkage disequilibrium for the STRP was found between 190 unselected NIDDM patients and 60 geographically and age-matched white nondiabetic subjects (χ2 = 1.51 on 3 df, P = 0.68). Thus, mutations involving the genes for GIRK1 or FAD-glycerophosphate dehydrogenase are unlikely to cause MODY, and a common mutation in either gene is unlikely to contribute to NIDDM in whites. These data do not exclude mutations in some families or other ethnic groups.

Mutation Screening Using PCR-SSCP

Screening for mutations prior to sequencing can reduce the time and costs of identifying mutations. When the DNA sequence is known, the technique of detecting mutations as single-stranded conformational polymorphisms (SSCP) is a convenient method of screening for possible mutations. SSCP was originally developed by Orita et al. (1). It has the ability of detecting a single base change, and has been applied to a number of genes, including the insulin receptor (2), GLUT 4 (3), glucokinase (4), and the mitochondrial genome (5).