EIF2AK3, encoding translation initiation factor 2-alpha kinase 3, is mutated in patients with Wolcott-Rallison syndrome.

Wolcott-Rallison syndrome (WRS) is a rare, autosomal recessive disorder characterized by permanent neonatal or early infancy insulin-dependent diabetes. Epiphyseal dysplasia, osteoporosis and growth retardation occur at a later age. Other frequent multisystemic manifestations include hepatic and renal dysfunction, mental retardation and cardiovascular abnormalities. On the basis of two consanguineous families, we mapped WRS to a region of less than 3 cM on chromosome 2p12, with maximal evidence of linkage and homozygosity at 4 microsatellite markers within an interval of approximately 1 cM. The gene encoding the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3) resides in this interval; thus we explored it as a candidate. We identified distinct mutations of EIF2AK3 that segregated with the disorder in each of the families. The first mutation produces a truncated protein in which the entire catalytic domain is missing. The other changes an amino acid, located in the catalytic domain of the protein, that is highly conserved among kinases from the same subfamily. Our results provide evidence for the role of EIF2AK3 in WRS. The identification of this gene may provide insight into the understanding of the more common forms of diabetes and other pathologic manifestations of WRS.

Susceptibility to insulin dependent diabetes mellitus maps to a 4.1 kb segment of DNA spanning the insulin gene and associated VNTR.

Recent studies have demonstrated that a locus at 11p15.5 confers susceptibility to insulin dependent diabetes mellitus (IDDM). This locus has been shown to lie within a 19 kb region. We present a detailed sequence comparison of the predominant haplotypes found in this region in a population of French Caucasian IDDM patients and controls. Identification of polymorphisms both associated and unassociated with IDDM has allowed us to define further the region of association to 4.1 kb. Ten polymorphisms within this region are in strong linkage disequilibrium with each other and extend across the insulin gene locus and the variable number tandem repeat (VNTR) situated immediately 5' to the insulin gene. These represent a set of candidate disease polymorphisms one or more of which may account for the susceptibility to IDDM.

A missense mutation in the glucagon receptor gene is associated with non-insulin-dependent diabetes mellitus.

Non-insulin-dependent diabetes mellitus (NIDDM) affects about 5% of the world population. The disease presents a polygenic mode of inheritance, but mechanisms and genes involved in late-onset NIDDM are largely unknown. We report the association of a single heterozygous Gly to Ser missense mutation in the glucagon receptor gene with late-onset NIDDM. This mutation was highly associated with NIDDM in a pooled set of French and Sardinian patients (chi 2 = 14.4, P = 0.0001) and showed some evidence for linkage to diabetes in 18 sibships from 9 French pedigrees (chi 2 = 6.63, P < 0.01). Receptor binding studies using cultured cells expressing the Gly40Ser mutation demonstrate that this mutation results in a receptor which binds glucagon with a three-fold lower affinity compared to the wild type receptor.

Confirmation of novel type 1 diabetes risk loci in families.

AIMS/HYPOTHESIS: Over 50 regions of the genome have been associated with type 1 diabetes risk, mainly using large case/control collections. In a recent genome-wide association (GWA) study, 18 novel susceptibility loci were identified and replicated, including replication evidence from 2,319 families. Here, we, the Type 1 Diabetes Genetics Consortium (T1DGC), aimed to exclude the possibility that any of the 18 loci were false-positives due to population stratification by significantly increasing the statistical power of our family study. METHODS: We genotyped the most disease-predicting single-nucleotide polymorphisms at the 18 susceptibility loci in 3,108 families and used existing genotype data for 2,319 families from the original study, providing 7,013 parent-child trios for analysis. We tested for association using the transmission disequilibrium test. RESULTS: Seventeen of the 18 susceptibility loci reached nominal levels of significance (p < 0.05) in the expanded family collection, with 14q24.1 just falling short (p = 0.055). When we allowed for multiple testing, ten of the 17 nominally significant loci reached the required level of significance (p < 2.8 × 10(-3)). All susceptibility loci had consistent direction of effects with the original study. CONCLUSIONS/INTERPRETATION: The results for the novel GWA study-identified loci are genuine and not due to population stratification. The next step, namely correlation of the most disease-associated genotypes with phenotypes, such as RNA and protein expression analyses for the candidate genes within or near each of the susceptibility regions, can now proceed.

Evidence for association of the TCF7 locus with type I diabetes.

The Type I Diabetes Genetics Consortium (T1DGC) has collected thousands of multiplex and simplex families with type I diabetes (T1D) with the goal of identifying genes involved in T1D susceptibility. These families have been genotyped for the HLA class I and class II loci and, recently, for a genome-wide panel of single-nucleotide polymorphisms (SNPs). In addition, multiple SNPs in specific candidate genes have been genotyped in these families in an attempt to evaluate previously reported T1D associations, including the C883A (Pro-Thr) polymorphism in exon 2 of TCF7, a T-cell transcription factor. The TCF7 883A allele was associated with T1D in subjects with T1D not carrying the high-risk HLA genotype DR3/DR4. A panel of 11 SNPs in TCF7 was genotyped in 2092 families from 9 cohorts of the T1DGC. SNPs at two positions in TCF7 were associated with T1D. One associated SNP, C883A (rs5742913), was reported earlier to have a T1D association. A second SNP, rs17653687, represents a novel T1D susceptibility allele in TCF7. After stratification on the high T1D risk DR3/DR4 genotype, the variant (A) allele of C883A was significantly associated with T1D among non-DR3/DR4 cases (transmission=55.8%, P=0.004; OR=1.26) but was not significantly associated in the DR3/DR4 patient subgroup, replicating the earlier report. The reference A allele of intronic SNP rs17653687 was modestly associated with T1D in both DR3/DR4 strata (transmission=54.4% in DR3/DR4; P=0.03; transmission=52.9% in non-DR3/DR4; P=0.03). These results support the previously reported association of the non-synonymous Pro-Thr SNP in TCF7 with T1D, and suggest that other alleles at this locus may also confer risk.

Evaluation of IL12B as a candidate type I diabetes susceptibility gene using data from the Type I Diabetes Genetics Consortium.

As part of its efforts to identify genes affecting the risk of type I diabetes (T1D), the Type I Diabetes Genetics Consortium commissioned an extensive survey of variants associated with genes reported earlier to have an association with disease susceptibility. In this report, we present the analysis of a set of single-nucleotide polymorphisms (SNPs) within and flanking the IL12B gene, which encodes the p40 subunit of the cytokines interleukin (IL)-12 and IL-23. No SNP showed individually significant association in the population as a whole. Nevertheless, subjects stratified according to genotype at the earlier reported SNP in the IL12B 3'UTR, rs3212227, confirmed small, but significant, differences in age of disease onset with a relative hazard=0.88 (P=0.005). The protective effect of rs3212227 allele 2 was gender specific (P=0.004 overall and P=0.0003 when unaffected siblings were considered). Among females, the 2.2 genotype was more protective, with relative hazard=0.75. We conclude that while there was no major effect of IL12B polymorphisms on T1D susceptibility in the entire study group, they have an impact on a subset of at-risk individuals.

The Type I Diabetes Genetics Consortium 'Rapid Response' family-based candidate gene study: strategy, genes selection, and main outcome.

Candidate gene studies have long been the principal method for identification of susceptibility genes for type I diabetes (T1D), resulting in the discovery of HLA, INS, PTPN22, CTLA4, and IL2RA. However, many of the initial studies that relied on this strategy were largely underpowered, because of the limitations in genomic information and genotyping technology, as well as the limited size of available cohorts. The Type I Diabetes Genetic Consortium (T1DGC) has established resources to re-evaluate earlier reported genes associated with T1D, using its collection of 2298 Caucasian affected sib-pair families (with 11 159 individuals). A total of 382 single-nucleotide polymorphisms (SNPs) located in 21 T1D candidate genes were selected for this study and genotyped in duplicate on two platforms, Illumina and Sequenom. The genes were chosen based on published literature as having been either 'confirmed' (replicated) or not (candidates). This study showed several important features of genetic association studies. First, it showed the major impact of small rates of genotyping errors on association statistics. Second, it confirmed associations at INS, PTPN22, IL2RA, IFIH1 (earlier confirmed genes), and CTLA4 (earlier confirmed, with distinct SNPs) loci. Third, it did not find evidence for an association with T1D at SUMO4, despite confirmed association in Asian populations, suggesting the potential for population-specific gene effects. Fourth, at PTPN22, there was evidence for a novel contribution to T1D risk, independent of the replicated effect of the R620W variant. Fifth, among the candidate genes selected for replication, the association of TCF7-P19T with T1D was newly replicated in this study. In summary, this study was able to replicate some genetic effects, reject others, and provide suggestions of association with several of the other candidate genes in stratified analyses (age at onset, HLA status, population of origin). These results have generated additional interesting functional hypotheses that will require further replication in independent cohorts.

Overview of the Type I Diabetes Genetics Consortium.

The Type I Diabetes Genetics Consortium (T1DGC) is an international, multicenter research program with two primary goals. The first goal is to identify genomic regions and candidate genes whose variants modify an individual's risk of type I diabetes (T1D) and help explain the clustering of the disease in families. The second goal is to make research data available to the research community and to establish resources that can be used by, and that are fully accessible to, the research community. To facilitate the access to these resources, the T1DGC has developed a Consortium Agreement (http://www.t1dgc.org) that specifies the rights and responsibilities of investigators who participate in Consortium activities. The T1DGC has assembled a resource of affected sib-pair families, parent-child trios, and case-control collections with banks of DNA, serum, plasma, and EBV-transformed cell lines. In addition, both candidate gene and genome-wide (linkage and association) studies have been performed and displayed in T1DBase (http://www.t1dbase.org) for all researchers to use in their own investigations. In this supplement, a subset of the T1DGC collection has been used to investigate earlier published candidate genes for T1D, to confirm the results from a genome-wide association scan for T1D, and to determine associations with candidate genes for other autoimmune diseases or with type II diabetes that may be involved with beta-cell function.

Current status and the future for the genetics of type I diabetes.

The Type I Diabetes Genetics Consortium (T1DGC) is an international collaboration whose primary goal is to identify genes whose variants modify an individual's risk of type I diabetes (T1D). An integral part of the T1DGC's mission is the establishment of clinical and data resources that can be used by, and that are fully accessible to, the T1D research community (http://www.t1dgc.org). The T1DGC has organized the collection and analyses of study samples and conducted several major research projects focused on T1D gene discovery: a genome-wide linkage scan, an intensive evaluation of the human major histocompatibility complex, a detailed examination of published candidate genes, and a genome-wide association scan. These studies have provided important information to the scientific community regarding the function of specific genes or chromosomal regions on T1D risk. The results are continually being updated and displayed (http://www.t1dbase.org). The T1DGC welcomes all investigators interested in using these data for scientific endeavors on T1D. The T1DGC resources provide a framework for future research projects, including examination of structural variation, re-sequencing of candidate regions in a search for T1D-associated genes and causal variants, correlation of T1D risk genotypes with biomarkers obtained from T1DGC serum and plasma samples, and in-depth bioinformatics analyses.

rs2476601 T allele (R620W) defines high-risk PTPN22 type I diabetes-associated haplotypes with preliminary evidence for an additional protective haplotype.

Protein tyrosine phosphatase non-receptor type 22 (PTPN22) is the third major locus affecting risk of type I diabetes (T1D), after HLA-DR/DQ and INS. The most associated single-nucleotide polymorphism (SNP), rs2476601, has a C->T variant and results in an arginine (R) to tryptophan (W) amino acid change at position 620. To assess whether this, or other specific variants, are responsible for T1D risk, the Type I Diabetes Genetics Consortium analyzed 28 PTPN22 SNPs in 2295 affected sib-pair (ASP) families. Transmission Disequilibrium Test analyses of haplotypes revealed that all three haplotypes with a T allele at rs2476601 were overtransmitted to affected children, and two of these three haplotypes showed statistically significant overtransmission (P=0.003 to P=5.9E-12). Another haplotype had decreased transmission to affected children (P=3.5E-05). All haplotypes containing the rs2476601 T allele were identical for all SNPs across PTPN22 and only varied at centromeric SNPs. When considering rs2476601 'C' founder chromosomes, a second haplotype (AGGGGC) centromeric of PTPN22 in the C1orf178 region was associated with protection from T1D (odds ratio=0.81, P=0.0005). This novel finding requires replication in independent populations. We conclude the major association of PTPN22 with T1D is likely due to the recognized non-synonymous SNP rs2476601 (R620W).

Association analysis of SNPs in the IL4R locus with type I diabetes.

The Type I Diabetes Genetics Consortium (T1DGC) has collected thousands of multiplex and simplex families with type I diabetes (T1D) with the goal of identifying genes involved in T1D susceptibility. These families have all been genotyped for the HLA class I and class II loci and a subset of samples has been typed for an major histocompatibility complex (MHC) single-nucleotide polymorphism (SNP) panel. In addition, the T1DGC has genotyped SNPs in candidate genes to evaluate earlier reported T1D associations. Individual SNPs and SNP haplotypes in IL4R, which encodes the alpha-chain of the IL4 and IL13 receptors, have been associated with T1D in some reports, but not in others. In this study, 38 SNPs in IL4R were genotyped using the Sequenom iPLEX Gold MassARRAY technology in 2042 multiplex families from nine cohorts. Association analyses (transmission-disequilibrium test and parental-disequilibrium test) were performed on individual SNPs and on three-SNP haplotypes. Analyses were also stratified on the high-risk HLA DR3/DR4-DQB1*0302 genotype. A modest T1D association in HBDI families (n=282) was confirmed in this larger collection of HBDI families (n=424). The variant alleles at the non-synonymous SNPs (rs1805011 (E400A), rs1805012 (C431R), and rs1801275 (Q576R)), which are in strong linkage disequilibrium, were negatively associated with T1D risk. These SNPs were more associated with T1D among non-DR3/DR4-DQB1*0302 genotypes than DR3/DR4-DQB1*0302 genotypes. This association was stronger, both in terms of odds ratio and P-values, than the initial report of the smaller collection of HBDI families. However, the IL4R SNPs and the three-SNP haplotype containing the variant alleles were not associated with T1D in the total data. Thus, in the overall families, these results do not show evidence for an association of SNPs in IL4R with T1D.

Genetics of insulin-dependent diabetes mellitus.

Recent studies that have focused on the detection of non-MHC susceptibility loci in insulin-dependent diabetes mellitus are reviewed. It has been confirmed that the region on human chromosome 11p contains such a susceptibility locus and recent research has attempted to identify the causative DNA variants and their functional role in disease.

Reverse cascade screening of newborns for hereditary haemochromatosis: a model for other late onset diseases?

BACKGROUND: Genetic testing can determine those at risk for hereditary haemochromatosis (HH) caused by HFE mutations before the onset of symptoms. However, there is no optimum screening strategy, mainly owing to the variable penetrance in those who are homozygous for the HFE Cys282Tyr (C282Y) mutation. The objective of this study was to identify the majority of individuals at serious risk of developing HFE haemochromatosis before they developed life threatening complications. METHODS: We first estimated the therapeutic penetrance of the C282Y mutation in people living in la Somme, France, using genetic, demographic, biochemical, and follow up data. We examined the benefits of neonatal screening on the basis of increased risk to relatives of newborns carrying one or two copies of the C282Y mutation. Between 1999 and 2002, we screened 7038 newborns from two maternity hospitals in the north of France for the C282Y and His63Asp (H63D) mutations in the HFE gene, using bloodspots collected on Guthrie cards. Family studies and genetic counselling were undertaken, based on the results of the baby's genotype. FINDINGS: In la Somme, we found that 24% of the adults homozygous for the C282Y mutation required at least 5 g iron to be removed to restore normal iron parameters (that is, the therapeutic penetrance). In the reverse cascade screening study, we identified 19 C282Y homozygotes (1/370), 491 heterozygotes (1/14) and 166 compound heterozygotes (1/42) in 7038 newborns tested. The reverse cascade screening strategy resulted in 80 adults being screened for both mutations. We identified 10 previously unknown C282Y homozygotes of whom six (four men and two women) required venesection. Acceptance of neonatal screening was high; parents understood the risks of having HH and the benefits of early detection, but a number of parents were reluctant to take the test themselves. Neonatal screening for HH is straightforward. Reverse cascade screening increased the efficiency of detecting affected adults with undiagnosed haemochromatosis. This strategy allows almost complete coverage for HH and could be a model for efficient screening for other late onset genetic diseases.

Evaluation of the SA locus in human hypertension.

The SA gene is expressed at 10-fold greater levels in the kidney of the spontaneously hypertensive rat compared with the normotensive Wistar-Kyoto rat. The gene is linked to blood pressure levels in a number of crosses involving the spontaneously hypertensive rat and other strains of genetically hypertensive rats. To assess its role in human hypertension, a human SA cDNA was cloned from a liver library. The cDNA was 1513 bp in length and exhibited a high identity with the published rat SA cDNA sequence in the coding region. A microsatellite marker was developed from a yeast artificial chromosome clone containing SA and mapped by linkage to human chromosome 16p13.11-12.3. Polymerase chain reaction amplification of human genomic DNA revealed two introns located in the SA gene, one of which contains a frequent polymorphism due to a single nucleotide substitution (cytosine to thymidine at residue 79 of the intron). Association and linkage studies in a large sample of hypertensive patients, normotensive control subjects, and multiplex sibships with these markers and other microsatellites in close proximity to SA revealed no evidence favoring involvement of the gene in the disease in humans. The methodology used in this study can be applied to the evaluation of other novel candidate genes obtained from investigations of experimental models of hereditary hypertension.

Human variable number of tandem repeat probes as a source of polymorphic markers in experimental animals.

Human VNTR (Variable Number of Tandem Repeat) markers are examined as a source of polymorphism for linkage studies in inbred strains of mice and rats. High frequencies of cross-hybridization are found under fingerprinting conditions that detect many distinct minisatellite loci in these species. Linkage studies suggest that minisatellite markers are widely distributed in the mouse genome, in contrast to humans where they are clustered, particularly in telomeric regions. Human VNTR probes can be used to screen in mouse genomic libraries to isolate mouse specific VNTR sequences. Some of these sequences reveal fingerprint patterns under stringent hybridization conditions.

Linkage analysis in juvenile neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinosis (NCL, Batten disease) is an autosomal recessive disease characterized by progressive mental retardation, cortical atrophy, seizures, and retinal degeneration. Several subtypes have been delineated on the basis of age-at-onset and histological characteristics; the most common is the juvenile (JNCL) form. Recently, the gene for JNCL was shown to reside on chromosome 16 through linkage studies to the haptoglobin locus and anonymous DNA markers using numerous European families. We have now examined 8 families from North America with JNCL for linkage to markers in 16q21-23. Results in 3 families tend to support linkage to chromosome 16;3 families remained uninformative, and 2 families produced negative lod scores in this region. A test of homogeneity was suggestive, but could not significantly reject the null hypothesis of homogeneity. We are continuing to collect families, particularly those with multiple living affecteds, and are identifying other probes in this region. Given close localization on chromosome 16 for JNCL, molecular strategies, including candidate gene strategies, are being explored.

[Genomics and type I diabetes]. / Génomique et diabète de type I.

Type I diabetes is the multifactorial disease for which genome-wide studies were conducted for the first time, both in human and in animal models. Today, two susceptibility loci have been identified: one, at the HLA locus, which represents a major genetic determinant to the disease, and the other at the insulin gene. Studies are in progress to try to map and identify other genetic factors involved in disease susceptibility. The task is made difficult due to the large number of susceptibility genes involved, interacting between themselves and with environmental factors, and most of which carrying only minor effects, which may be heterogeneous between populations. The combination of genetic and biological approaches will be essential in order to elucidate the nature and function of these genes. Recent progress on the knowledge of the human genome, as well as the availability of adequate technologies will accelerate these researches.

[HLA-DR:DQ genotypes and insulin-dependent diabetes in Senegal]. / Génotypes HLA-DR:DQ et diabète insulino-dépendant en milieu sénégalais.

At the opposite of HLA-DR, HLA-DQ was not well documented in homogeneous negroïd populations. So, 93 IDDM and 115 control patients, all black senegalese people, were studied. The results showed three HLA-DQ IDDM-related susceptibility genotypes and also a high risk conferred by HLA-DR4/DR9 usually described in Mongoloïd people. Furthermore, DR:DQ associations allowed the identification of three IDDM predisposition genotypes, each of them with a characteristic mean age for disease diagnosis.