Julie C. Evans

Missense mutations in the insulin promoter factor-1 gene predispose to type 2 diabetes

The transcription factor insulin promoter factor-1 (IPF-1) plays a central role in both the development of the pancreas and the regulation of insulin gene expression in the mature pancreatic β cell. A dominant-negative frameshift mutation in the IPF-l gene was identified in a single family and shown to cause pancreatic agenesis when homozygous and maturity-onset diabetes of the young (MODY) when heterozygous. We studied the role of IPF-1 in Caucasian diabetic and nondiabetic subjects from the United Kingdom. Three novel IPF-1 missense mutations (C18R, D76N, and R197H) were identified in patients with type 2 diabetes. Functional analyses of these mutations demonstrated decreased binding activity to the human insulin gene promoter and reduced activation of the insulin gene in response to hyperglycemia in the human β-cell line Nes2y. These mutations are present in 1% of the population and predisposed the subject to type 2 diabetes with a relative risk of 3.0. They were not highly penetrant MODY mutations, as there were nondiabetic mutation carriers 25‐53 years of age. We conclude that mutations in the IPF-1 gene may predispose to type 2 diabetes and are a rare cause of MODY and pancreatic agenesis, with the phenotype depending upon the severity of the mutation. J. Clin. Invest. 104:R33-R39 (1999).

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.

Meta-Analysis and a Large Association Study Confirm a Role for Calpain-10 Variation in Type 2 Diabetes Susceptibility

To the Editor: Variation in the calpain-10 gene (CAPN10 [MIM 605286]) was recently linked and associated with type 2 diabetes mellitus (T2DM) susceptibility (Horikawa et al. 2000). The initial linkage of T2DM to chromosome 2 was found in a population of Mexican Americans from Starr County, Texas (Hanis et al. 1996). Specific combinations of three intronic variants, designated “SNP-43,” “SNP-19,” and “SNP-63,” that capture most of the haplotype diversity at CAPN10 were associated with a three-fold increased risk of T2DM in this population and could account for the observed linkage (Horikawa et al. 2000). Subsequent association and linkage studies of these three polymorphisms in other populations have produced conflicting results, with association being observed in some populations (Baier et al. 2000 [Pima Indian]; Cassell et al. 2002 [South Indian]; Garant et al. 2002 [African American]; Malecki et al. 2002 [Polish]; Orho-Melander et al. 2002 [Finnish/Botnia]), but not others (Evans et al. 2001 [British]; Hegele et al. 2001 [Oji-Cree Indians]; Tsai et al. 2001 [Samoan]; Xiang et al. 2001 [Chinese]; Daimon et al. 2002 [Japanese]; Elbein et al. 2002 [whites from Utah]; Fingerlin et al. 2002 [Finnish]; Rasmussen et al. 2002 [Danish and Swedish]; Horikawa et al. 2003 [Japanese]). We previously reported that another variant, SNP-44 (designated “CAPN10-g4841T→C”; minor allele frequency 16%), located in intron 3 and 11 bp from SNP-43, was independently associated with T2DM in whites from the United Kingdom (Evans et al. 2001). Further studies have provided tentative support for a role of SNP-44 in T2DM and related traits: associations with polycystic ovary syndrome (Gonzalez et al. 2002) and with measures of oral glucose tolerance (Wang et al. 2002; Tschritter et al. 2003) have been reported. Functional studies suggest that SNP-44 is located in an enhancer element and might affect CAPN10 expression (Horikawa et al. 2000). Also, in the U.K., German, Japanese, and South Indian populations, SNP-44 is in perfect linkage disequilibrium (r2=1) with a missense mutation Thr504Ala (SNP-110) and two polymorphisms in the 5′-UTR (SNP-134 and SNP-135) (Evans et al. 2001; Cassell et al. 2002; Y. Horikawa and P. E. Schwarz, unpublished data). To assess the association of SNP-44 with T2DM more comprehensively, we performed a meta-analysis of all published SNP-44/T2DM association study data. To identify all relevant published studies, we searched PubMed using the keywords “calpain 10,” “diabetes,” “44,” “SNP 44,” “CAPN10,” and “type 2,” in different combinations. When necessary, authors were contacted to obtain exact genotype numbers, so that precise odds ratios (ORs) from each study could be calculated. Our search identified 10 published case/control studies, consisting of 3,303 subjects. The studies were spread across a number of ethnic groups: British (three studies, Evans et al. 2001); Chinese (Wang et al. 2002); Japanese (Daimon et al. 2002; Horikawa et al. 2003); Finnish/Botnia (two studies, Orho-Melander et al. 2002); South Indian (Cassell et al. 2002); and Mexican American (Horikawa et al. 2000). The frequency of the T2DM-associated SNP-44 C allele (allele 2) ranged from 6% in Mexican Americans to 25% in the Botnia I control population. There was no evidence for OR heterogeneity (Q test P=.27), and, although these studies are only a small sample from the many existing T2DM genetic resources, a funnel-plot analysis (Egger et al. 1997) suggested an absence of publication bias (P=.44). A Mantel-Haenszel meta-analysis of these studies showed that the C allele was associated with increased risk of T2DM (OR 1.17 [1.02–1.34], P=.02). Three transmission/disequilibrium tests (TDT) had been performed (Evans et al. 2001; Cassell et al. 2002; Orho-Melander et al. 2002). The combined TDT results demonstrated that the C allele was significantly overtransmitted (117 transmitted vs. 77 not transmitted, P=.004) from heterozygous parents to diabetic offspring. Although this result cannot be considered independent replication, as proband data was included in the case/control meta-analysis from two of the TDT studies (Evans et al. 2001; Cassell et al. 2002), it provides evidence that the association is not due to population stratification. Of the 10 studies in the meta-analysis, only 1 reported a significant (P<.05) association (Evans et al. 2001). However, these studies were small and the mean power to detect an OR of 1.17 at P<.05 was ∼11% (range 5%–14%). In the context of genetic association studies, which test many polymorphisms in numerous candidate genes, a P value of .02 can only be considered evidence suggestive of a real association. We therefore genotyped SNP-44 in an additional 4,213 subjects: 3,274 white European subjects from four case/control studies (one British, two German, and one Czech); 691 Japanese subjects from two case/control studies; and 248 Mexican (mestizo) subjects from Mexico City and Orizaba City from one case/control study. Overall, this provided 2,056 subjects with T2DM and 2,157 controls, and a power of ∼80% to detect an OR of 1.17. Clinical details of the study subjects are presented in table 1; further details are available as supplementary information from the authors. All studies were approved by the relevant ethics committee, and all subjects gave their informed consent. Table 1 Clinical Characteristics of Subjects in Study[Note] When all the studies were combined, there was no evidence for between-studies OR heterogeneity (Q test P=.23); a Mantel-Haenszel fixed-effects model was therefore used for subsequent analysis. Meta-analysis of the new studies gave an OR for the SNP-44 C allele of 1.18 (1.04–1.34), P=.01 (fig. 1). A combined meta-analysis of all previously published data and our new data gave an OR of 1.17 (1.07–1.29), P=.0007. All study populations were in Hardy-Weinberg equilibrium except the T2DM cohort of Horikawa et al. 2003 (P=.005) and the control population of the third Japanese study (P=.02). Although these deviations may be due to random fluctuation and multiple-hypothesis testing, they contributed a large amount to heterogeneity (27% of the Q statistic); excluding these studies, the SNP-44 C allele OR for the new studies was 1.23 (1.07–1.40), P=.003; the overall OR was 1.19 (1.08–1.31), P=.0005. This OR is of similar magnitude to that of E23K (Gloyn et al. 2003; Love-Gregory et al. 2003; Nielsen et al. 2003) and Pro12Ala (Altshuler et al. 2000), the other common variants confirmed as T2DM-susceptibility polymorphisms. An OR of 1.17 is low and may help explain why there is little evidence for linkage of the CAPN10 region to T2DM in most populations. The haplotypes responsible for the CAPN10 linkage seen in the Mexican American population were associated with a higher T2DM OR (∼3.0) and were more likely to be detected by linkage analysis (Horikawa et al. 2000). These haplotypes are less common in other populations. Figure 1 Mantel-Haenszel OR meta-analysis plot (fixed effects) for SNP-44 association with T2DM. Point estimates and 95% CLs for each previously published, new, and combined case/control study. SNP-44 is in perfect linkage disequilibrium (r2=1) with the missense mutation, Thr504Ala, and two SNPs (SNP-134 and SNP-135) in the 5′-UTR and therefore may not be the causal variant. Further haplotype and functional analyses are required to confirm which of these polymorphisms contribute to T2DM susceptibility. In conclusion, our results have confirmed that a CAPN10 haplotype defined by the SNP-44 polymorphism predisposes to T2DM. Meta-analyses of published genetic associations, combined with large replication studies, are a powerful approach to detecting susceptibility variants in common disease.

Analysis of haematopoietic chimaerism by quantitative real-time polymerase chain reaction.

Summary:Allogeneic bone marrow transplantation (BMT) with marrow ablative conditioning is the treatment of choice for haematopoietic malignancies. The use of nonmyeloablative stem cell transplants has allowed the treatment of patients previously ineligible for BMT because of age or other disease. These reduced conditioning regimes allow the persistence initially of some recipient cells in the blood and bone marrow (haematopoietic chimaerism). Monitoring of the relative proportion of donor and recipient cells is required to assess the success of the procedure, to predict subsequent rejection or impending relapse and to guide the use of donor lymphocyte infusions. We present a quantitative real-time PCR approach for the measurement of haematopoietic chimaerism using the TaqMan™. This approach exploits the presence of single-nucleotide polymorphisms (SNPs) to distinguish cells of patient or donor origin. We have designed and validated a panel of seven allele-specific probes to quantify the contribution of patient and donor cells in the haematopoietic population from 12 patient and donor pairs. We have compared the performance of this approach with an existing method and proved it to be superior in both accuracy and sensitivity. The use of more sensitive and accurate techniques permits earlier intervention for improved clinical outcome.

Role of the D76N polymorphism of insulin promoter factor-1 in predisposing to Type 2 diabetes

To the Editor: Insulin promoter factor-1 (IPF-1, PDX-1) is a transcription factor and homeodomain protein required for development of the pancreas and initiation and maintenance of the insulin-secreting phenotype of the beta cell [1]. Reduced levels of IPF-1/PDX-1 are associated with pancreatic agenesis and hyperglycaemia in rodent models. A dominant negative mutation in man causes pancreatic agenesis [2] and MODY [3]. Therefore IPF-1/PDX-1 is an excellent candidate gene for Type 2 diabetes. Coding variants in IPF-1/PDX-1 were found in 3 to 5% of young-onset familial Type 2 diabetes compared to 0.5 to 1% of control subjects with normoglycaemia in the UK [4], France [5] and Sweden [6]. However, these variants were rare in lateonset Type 2 diabetes [7, 8, 9]. In vitro studies of the naturally occurring IPF-1/PDX-1 mutations have shown diminished activity in most [4, 5, 6] but not all studies [7]. Physiological assessment of non-diabetic carriers of IPF-1/PDX-1 mutations showed higher glucose levels during an OGTT than those found in control subjects [4, 6] and a lower insulin response to a glucose load [5]. Most studies in Type 2 diabetes have been small (~200 cases) and the variants identified individually are rare and not found in all populations. In most European populations studied, only one polymorphism, D76N (CAG→AAG), causing an amino acid change of aspartic acid to asparagine, has been reported in both diabetic subjects and control subjects. It was also the most frequent variant found in the initial UK and French studies [4, 5]. The aim of our study was to assess whether D76N predisposed to young-onset familial Type 2 diabetes in a large UK case-control study. All subjects were white UK citizens. The Type 2 diabetic subjects had been diagnosed with diabetes by World Health Organization criteria or were on treatment for diabetes. Genetic and autoimmune subtypes were excluded. The Type 2 diabetic subjects (n=860) were recruited from three sources: (i) youngonset (≥18 and ≤45 years at diagnosis) subjects (the youngonset Type 2 diabetes cohort described in [10]); (ii) probands of parent–offspring trios; and (iii) probands of affected sib-pairs. All subjects were therefore selected either for early-onset or for familial diabetes consistent with an increased genetic susceptibility. Population control subjects (n=1275) were recruited from two sources: (i) non-diabetic parents from a consecutive birth cohort (the Exeter Family Study) and (ii) a population control group of blood donors without known diabetes from the European Collection of Cell Cultures. Genotyping was either performed on the ABI 7000 Taqman (Applied Biosystems, Warrington, UK) or by a previously described RFLP method [4]. Taqman reactions were carried out in a total volume of 25 μl containing 12.5 μl Taqman universal PCR master mix, 0.5 μl of probes 1 and 2, and 1.125 μl of forward and reverse primers (F primer CAGCCCCCCGGACATCT, R primer CGGGAGGTGTGGTGAAGGT, probe 1 CTCGCCGACGACC, probe 2 CTCGCCGACAACC). The default thermal cycling cycle was used for all experiments followed by an allelic discrimination step. Data were analysed and scored with the Prism 7000 Sequence Detection System (Applied Biosystems). Samples that could not be scored after three repeats were excluded from the final analysis. All samples scored as containing the rare allele were confirmed by direct sequencing. Using a control frequency of 1% for D76N (as in [4]), we had 80% power at p=0.05 to detect an Odds Ratio (OR) of 2.15. Statistical analysis was performed using Stats direct v2.2. Chi square analysis was used to calculate the OR and 95% CI. Recent studies have shown that the evidence for or against genetic associations in common phenotypes needs to be assessed over many studies. We therefore performed a meta-analysis of the current plus previously published studies [4, 5, 6, 7, 8, 9] examining the D76N variant in Type 2 diabetes, and calculated a Mantel-Haenszel pooled odds ratio. Using STATXACT with calculation of EXACT estimations did not make any difference to the result, suggesting there were no significant departures from asymptotic expectation. One study [8], where the rare allele was not detected in diabetic or control subjects, was not included in the meta-analysis, as it is impossible to calculate an odds ratio in this situation. All genotypes were in Hardy-Weinberg equilibrium. The rate of failed and/or unscoreable samples was 3%. Table 1 shows the genotype frequency in the case and control subjects, as well as the case subjects diagnosed at 45 years of age or earlier. There were no rare homozygotes (AA). Of case and control subjects 1.7% and 1.6% respectively were heterozygous (GA) for the rare allele, giving an OR of 1.1 (CI: 0.52 to 2.1, p=0.86). In the subjects diagnosed at 45 years or earlier, 2.5% were heterozygous, giving an OR of 1.5 (CI: 0.7 to 3.2, p=0.26). Meta-analysis showed that a Mantel-Haenszel pooled estimate of the OR was 1.7 (CI: 1.0 to 2.8, p=0.07). When only study populations with a mean age of onset of 45 years or earlier were included [4, 5, 6], the Mantel-Haenszel pooled OR was 2.5 (CI: 1.4 to 4.6, p=0.004). We compared the phenotype of the subjects with the D76N variant to wild-type, but no difference was observed in the age of onset, parental history of diabetes, BMI or current insulin treatment. Diabetologia (2004) 47:957–958

Clinical Consequences of Defects in β-Cell Genes

Genes critical for β-cell function have been identified by the genetic analysis of families with maturity onset diabetes of the young (MODY). The clinical description of MODY families by Fajans and Tattersall (1,2) and the development of appropriate molecular genetic techniques were both critical in the definition of these genes. In this chapter, we describe the clinical and physiological features of patients with known defects in β-cell genes. This has led to the demonstration of previously un-recognized heterogeneity in clinically defined MODY. In addition, it has given fascinating insights into the role of the genes in the normal β-cell both in post-natal and foetal life.