De Novo Mutations in EIF2B1 Affecting eIF2 Signaling Cause Neonatal/Early-Onset Diabetes and Transient Hepatic Dysfunction.
ABSTRACT: Permanent neonatal diabetes mellitus (PNDM) is caused by reduced ?-cell number or impaired ?-cell function. Understanding of the genetic basis of this disorder highlights fundamental ?-cell mechanisms. We performed trio genome sequencing for 44 patients with PNDM and their unaffected parents to identify causative de novo variants. Replication studies were performed in 188 patients diagnosed with diabetes before 2 years of age without a genetic diagnosis. EIF2B1 (encoding the eIF2B complex ? subunit) was the only gene with novel de novo variants (all missense) in at least three patients. Replication studies identified two further patients with de novo EIF2B1 variants. In addition to having diabetes, four of five patients had hepatitis-like episodes in childhood. The EIF2B1 de novo mutations were found to map to the same protein surface. We propose that these variants render the eIF2B complex insensitive to eIF2 phosphorylation, which occurs under stress conditions and triggers expression of stress response genes. Failure of eIF2B to sense eIF2 phosphorylation likely leads to unregulated unfolded protein response and cell death. Our results establish de novo EIF2B1 mutations as a novel cause of permanent diabetes and liver dysfunction. These findings confirm the importance of cell stress regulation for ?-cells and highlight EIF2B1's fundamental role within this pathway.
Project description:Heterozygous activating mutations in the KCNJ11 gene encoding the pore-forming Kir6.2 subunit of the pancreatic beta cell K(ATP) channel are the most common cause of permanent neonatal diabetes (PNDM). Patients with PNDM due to a heterozygous activating mutation in the ABCC8 gene encoding the SUR1 regulatory subunit of the K(ATP) channel have recently been reported. We studied a cohort of 59 patients with permanent diabetes who received a diagnosis before 6 mo of age and who did not have a KCNJ11 mutation. ABCC8 gene mutations were identified in 16 of 59 patients and included 8 patients with heterozygous de novo mutations. A recessive mode of inheritance was observed in eight patients with homozygous, mosaic, or compound heterozygous mutations. Functional studies of selected mutations showed a reduced response to ATP consistent with an activating mutation that results in reduced insulin secretion. A novel mutational mechanism was observed in which a heterozygous activating mutation resulted in PNDM only when a second, loss-of-function mutation was also present.
Project description:Leukoencephalopathy with Vanishing White Matter (VWM) is an autosomal recessive disorder caused by germline mutations in the genes EIF2B1-5, which encode the 5 subunits of the eukaryotic translation initiation factor eIF2B. To date, analysis of the biochemical effects of mutations in the EIF2B2-5 genes has been carried out, but no study has been performed on mutations in the EIF2B1 gene. This gene encodes eIF2B?, the smallest subunit in eIF2B which has an important role in both the structure and regulation of the eIF2B complex.eIF2B subunits were overexpressed in HEK293 cells and isolated from the resulting cell lysates by affinity chromatography. Formation of the eIF2B complex and binding of its substrate, eIF2, was assessed by western blot. Assays of the guanine nucleotide exchange (GEF) activity were also carried out.Of the 5 eIF2B? mutations studied, we found 3 that showed loss or reduction of binding of eIF2B? to the rest of the complex, one with increased GEF activity, and one where no effects on activity or complex formation were observed.This is the first study on eIF2B? VWM mutations. We show that some mutations cause expected decreases in GEF activity or complex formation, similar to a majority of observed VWM mutations. However, we also observe some unexpected changes which hint at other effects of these mutations on as yet undescribed functions of eIF2B.
Project description:This is a study estimating diagnostic accuracy of CSF asialotransferrin to transferrin ratio measurement in eIF2B related disorders by using clinical evaluation and EIF2B mutation analysis as the reference standard. eIF2B-related disorder is a relatively common leukodystrophy with broad phenotypic variation that is caused by mutations in any of the five EIF2B genes. There is a need for a simple and clinically valid screening tool for physicians evaluating patients with an unclassified leukodystrophy.CSF two-dimensional gel (2DG) electrophoresis analyses to measure asialotransferrin to transferrin ratios were performed in 60 subjects including 6 patients with documented EIF2B gene mutations, patients with other types of leukodystrophy, and patients with no leukodystrophy.All six patients with mutation proven eIF2B-related disease showed low to nearly undetectable amounts of asialotransferrin in their CSF when compared to 54 unaffected controls by CSF 2DG analyses in this study. eIF2B-like patients, with clinically similar presentations but no mutations in EIF2B1-5, were distinguished from patients with mutations in EIF2B1-5 by this biomarker. Patients with mutations in EIF2B1-5 had asialotransferrin/transferrin ratio levels significantly different from the group as a whole (p < 0.001). Using 8% asialotransferrin/transferrin ratio as a cutoff, this biomarker has a 100% sensitivity (95% CI = 52-100%) and 94% specificity (95% CI = 84-99%).Decreased asialotransferrin/transferrin ratio in the CSF of patients with eIF2B-related disorder is highly sensitive and specific. This rapid (<48 hours) and inexpensive diagnostic tool for eIF2B-related disorders has the potential to identify patients with likely eIF2B-related disorder for mutation analysis.
Project description:BACKGROUND:Neonatal diabetes mellitus (NDM) is a rare condition that occurs within the first six months of life. Permanent NDM (PNDM) is caused by mutations in specific genes that are known for their expression at early and/or late stages of pancreatic beta- cell development, and are either involved in beta-cell survival, insulin processing, regulation, and release. The native population in Qatar continues to practice consanguineous marriages that lead to a high level of homozygosity. To our knowledge, there is no previous report on the genomics of NDM among the Qatari population. The aims of the current study are to identify patients with NDM diagnosed between 2001 and 2016, and examine their clinical and genetic characteristics. METHODS:To calculate the incidence of PNDM, all patients with PNDM diagnosed between 2001 and 2016 were compared to the total number of live births over the 16-year-period. Whole Genome Sequencing (WGS) was used to investigate the genetic etiology in the PNDM cohort. RESULTS:PNDM was diagnosed in nine (n = 9) patients with an estimated incidence rate of 1:22,938 live births among the indigenous Qatari. Seven different mutations in six genes (PTF1A, GCK, SLC2A2, EIF2AK3, INS, and HNF1B) were identified. In the majority of cases, the genetic etiology was part of a previously identified autosomal recessive disorder. Two novel de novo mutations were identified in INS and HNF1B. CONCLUSION:Qatar has the second highest reported incidence of PNDM worldwide. A majority of PNDM cases present as rare familial autosomal recessive disorders. Pancreas associated transcription factor 1a (PTF1A) enhancer deletions are the most common cause of PNDM in Qatar, with only a few previous cases reported in the literature.
Project description:Identifying new causes of permanent neonatal diabetes (PNDM) (diagnosis <6 months) provides important insights into ?-cell biology. Patients with Down syndrome (DS) resulting from trisomy 21 are four times more likely to have childhood diabetes with an intermediate HLA association. It is not known whether DS can cause PNDM. We found that trisomy 21 was seven times more likely in our PNDM cohort than in the population (13 of 1,522 = 85 of 10,000 observed vs. 12.6 of 10,000 expected) and none of the 13 DS-PNDM patients had a mutation in the known PNDM genes that explained 82.9% of non-DS PNDM. Islet autoantibodies were present in 4 of 9 DS-PNDM patients, but DS-PNDM was not associated with polygenic susceptibility to type 1 diabetes (T1D). We conclude that trisomy 21 is a cause of autoimmune PNDM that is not HLA associated. We propose that autoimmune diabetes in DS is heterogeneous and includes coincidental T1D that is HLA associated and diabetes caused by trisomy 21 that is not HLA associated.
Project description:The aim of this study was to investigate the genetic aetiology of permanent diabetes mellitus with onset in the first 12 months of age.We studied 46 probands with permanent, insulin-requiring diabetes with onset within the first 6 months of life (permanent neonatal diabetes mellitus [PNDM]/monogenic diabetes of infancy [MDI]) (group 1) and eight participants with diabetes diagnosed between 7 and 12 months of age (group 2). KCNJ11, INS and ABCC8 genes were sequentially sequenced in all patients. For those who were negative in the initial screening, we examined ERN1, CHGA, CHGB and NKX6-1 genes and, in selected probands, CACNA1C, GCK, FOXP3, NEUROG3 and CDK4. The incidence rate for PNDM/MDI was calculated using a database of Italian patients collected from 1995 to 2009.In group 1 we found mutations in KCNJ11, INS and ABCC8 genes in 23 (50%), 9 (19.5%) and 4 (8.6%) patients respectively, and a single homozygous mutation in GCK (2.1%). In group 2, we identified one incidence of a KCNJ11 mutation. No genetic defects were detected in other loci. The incidence rate of PNDM/MDI in Italy is estimated to be 1:210,287.Genetic mutations were identified in ~75% of non-consanguineous probands with PNDM/MDI, using sequential screening of KCNJ11, INS and ABCC8 genes in infants diagnosed within the first 6 months of age. This percentage decreased to 12% in those with diabetes diagnosed between 7 and 12 months. Patients belonging to the latter group may either carry mutations in genes different from those commonly found in PNDM/MDI or have developed an early-onset form of autoimmune diabetes.
Project description:EIF2AK1 and EIF2AK2 encode members of the eukaryotic translation initiation factor 2 alpha kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of nine unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/9) or EIF2AK2 (8/9). Features seen in these nine individuals include white matter alterations (9/9), developmental delay (9/9), impaired language (9/9), cognitive impairment (8/9), ataxia (6/9), dysarthria in probands with verbal ability (6/9), hypotonia (7/9), hypertonia (6/9), and involuntary movements (3/9). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and proband-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate eukaryotic translation initiation factor 2 subunit 1 (EIF2S1, also known as EIF2?), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter (CACH/VWM), a leukodystrophy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.
Project description:One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting the interaction of eIF2 with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of yeast eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.2?Å. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2?-D1, which contains the phosphorylated Ser51. eIF2?-D1 is mainly inserted between the N-terminal helix bundle domains of ? and ? subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2B? and indirectly by inducing contacts of eIF2? helix 58-63 with eIF2B? leading to a competition with Met-tRNAi.
Project description:Phosphorylation of eIF2? controls translation initiation by restricting the levels of active eIF2-GTP/Met-tRNAi ternary complexes (TC). This modulates the expression of all eukaryotic mRNAs and contributes to the cellular integrated stress response. Key to controlling the activity of eIF2 are translation factors eIF2B and eIF5, thought to primarily function with eIF2-GDP and TC respectively. Using a steady-state kinetics approach with purified proteins we demonstrate that eIF2B binds to eIF2 with equal affinity irrespective of the presence or absence of competing guanine nucleotides. We show that eIF2B can compete with Met-tRNAi for eIF2-GTP and can destabilize TC. When TC is formed with unphosphorylated eIF2, eIF5 can out-compete eIF2B to stabilize TC/eIF5 complexes. However when TC/eIF5 is formed with phosphorylated eIF2, eIF2B outcompetes eIF5 and destabilizes TC. These data uncover competition between eIF2B and eIF5 for TC and identify that phosphorylated eIF2-GTP translation initiation intermediate complexes can be inhibited by eIF2B.
Project description:Eukaryotic translation initiation factor 2 (eIF2) is a heterotrimeric GTPase, which plays a critical role in protein synthesis regulation. eIF2-GTP binds Met-tRNAi to form the eIF2-GTP•Met-tRNAi ternary complex (TC), which is recruited to the 40S ribosomal subunit. Following GTP hydrolysis, eIF2-GDP is recycled back to TC by its guanine nucleotide exchange factor (GEF), eIF2B. Phosphorylation of the eIF2? subunit in response to various cellular stresses converts eIF2 into a competitive inhibitor of eIF2B, which triggers the integrated stress response (ISR). Dysregulation of eIF2B activity is associated with a number of pathologies, including neurodegenerative diseases, metabolic disorders, and cancer. However, despite decades of research, the underlying molecular mechanisms of eIF2B action and regulation remain unknown. Here we employ a combination of NMR, fluorescence spectroscopy, site-directed mutagenesis, and thermodynamics to elucidate the mechanisms of eIF2B action and its regulation by phosphorylation of the substrate eIF2. We present: (i) a novel mechanism for the inhibition of eIF2B activity, whereby eIF2? phosphorylation destabilizes an autoregulatory intramolecular interaction within eIF2?; and (ii) the first structural model for the complex of eIF2B with its substrate, eIF2-GDP, reaction intermediates, apo-eIF2 and eIF2-GTP, and product, TC, with direct implications for the eIF2B catalytic mechanism.