1 Génétique des Maladies Infectieuses et Autoimmunes, INSERM E102, Institut Pasteur, Paris, France
2 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana
3 Centre National de Génotypage, Evry, France
4 Medical and Molecular Genetics, The Medical School, University of Birmingham, Birmingham, U.K
5 INSERM U383, G. Hospitalier Necker-Enfants Malades, Paris, France
6 Endocrinologie et Diabétologie Infantiles, Hôpital Debrousse, Lyon, France
7 Department of Endocrinology, Birmingham Childrens Hospital, Birmingham, U.K
8 G Hospitalier Necker-Enfants Malades, Paris, France
9 Department of Pediatrics, Antwerp University Hospital, Edegem, Belgium
10 Service de Génétique Médicale, Hôpital Sainte-Justine, Montréal, Canada
11 Department of Pediatrics, King Faisal Specialist Hospital, Riyadh, Kingdom of Saudi Arabia
12 Division of Pediatric Endocrinology, King Faisal Specialist Hospital and Research Center, Jeddah, Kingdom of Saudi Arabia
13 Childrens Hospital, University of Mainz, Mainz, Germany
14 Dipartimento di Scienze Pediatriche e dellAdolescenza, Servizio di Genetica Clinica, Torino, Italy
15 Pediatric Endocrinology, Cukurova University, Adana, Turkey
16 Institute of Child Health, University of Birmingham, Birmingham, U.K
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ABSTRACT |
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The identification of susceptibility genes for multifactorial disorders, such as type 1 diabetes, presents many challenges. To date, only two susceptibility loci for type 1 diabetes have been unambiguously identified, IDDM1 (HLA locus) and IDDM2 (insulin gene locus). Genetic studies of monogenic forms of diabetes-related disorders can significantly increase our knowledge of ß-cell function and may point to potential candidate genes for the common forms of diabetes. Much information has been gained on studying syndromes such as maturity-onset diabetes of the young (1,2), Wolfram syndrome (3), and, more recently, Wolcott-Rallison syndrome (WRS) (4). WRS is a very rare syndrome, with less than 20 cases described in the world literature (417). It associates permanent neonatal or early-childhood insulin-dependent diabetes and epiphyseal dysplasia. Other clinical features that show variability between WRS cases include mental retardation, hepatic and kidney dysfunction, cardiac abnormalities, exocrine pancreatic dysfunction, and neutropenia. Based on genetic studies of two consanguineous families, we previously identified the gene responsible for this disorder as EIF2AK3 (or PEK), the pancreatic eukaryotic initiation factor 2 (eIF2
) kinase (4). In addition, two independent knockout mice have been produced and studied that show a phenotype remarkably similar to human WRS, with neonatal diabetes, skeletal defects, and small size with delayed growth (18,19). Recent reports have begun to identify mutations in EIF2AK3 genes linked with WRS (4,16,17). Together, these studies definitively establish that EIF2AK3 mutations are responsible for WRS. However, the variability of the clinical manifestations observed in WRS remains unexplained. In the present study, we have assembled and studied a large collection of cases and families with WRS in order to extend the clinical and genetic investigation of this syndrome and to explore the determinism of its clinical variability.
A total of 12 families (18 patients with WRS) were studied, and detailed clinical information was obtained on these patients. This is the largest collection of WRS patients studied to date. The summary description of these patients is shown in Table 1. Patients were from various population origins, and, in most cases, their parents were known to be consanguineous (families WRS1, -2, -3, -4, -5, -6, -7, -8, -10, and -12) or likely to be since they lived in the same village (WRS9). There was no evidence of consanguinity in one case (WRS11). The age at onset was generally very young: 15 had an onset between the ages of 5 weeks and 6 months, but 2 had a significantly older age at onset, at 18 months (WRS12-1) and 30 months (WRS10-1). Excluding these two outliers, the mean age at onset was 3 months. Most of these cases were still alive (aged between 3 months and 13 years), and some had died (aged between 3 months and 35 years). Diabetes required insulin therapy from the onset of the disease. With one possible exception, there was no evidence of autoantibodies (islet cell antibody, insulin autoantibody, IA-2 antigen, and GADA) in the patients at the onset of disease when this was tested. Slightly elevated values were found for patient WRS12-1 at disease onset (GADA: 2.3 IU) but within normal range (0.94 IU) 3 months later. In addition to the multiple epiphyseal dysplasia common to all patients, there were various degrees of osteopenia, ranging from undetected or mild (possibly detected by bone densitometry) to severe, with multiple fractures. The mental and psychomotor development was assessed based on the sitting/walking age, speech and communication, and school performance. There was slight to severe mental retardation or developmental delay in the majority of the patients, but some were within the normal range (WRS9-1, 10-1, 11-1, and 12-1). Epilepsy was not reported in any of these patients, in contrast with other reports (5). Recurrent episodes of hepatitis occurred in the majority of the patients. These were major events, requiring hospitalization, often accompanied by acute renal failure and sometimes resulting in death. Hepatic and renal functions returned to normal for those surviving these episodes. In all cases where it was explored, no causative viral infection could be identified. In addition, progressive chronic renal insufficiency was noted in three patients, WRS8-1, WRS9-1, and WRS9-2, who were also the ones who lived to older ages (between 11 and 35 years). Four patients showed signs of exocrine pancreas dysfunction, WRS2-2, WRS4-1, WRS5-1, and WRS10-1, with pancreatic hypotrophy in WRS10-1 (12) and fibrosis infiltrations in pancreas biopsy in WRS2-2 (11). Central hypothyroidism was noted in four patients (WRS1-1, WRS4-1, WRS7-1, and WRS7-2) (14,15), but thyroid function was normal in the others. Neutropenia was reported in nine patients (WRS2-1, WRS2-2, WRS2-3, WRS3-1, WRS4-1, WRS5-1, WRS6-1, WRS10-1, and WRS11-1), who also tended to suffer from frequent infections (bacterial, viral, and fungic). The phenotype of the parents and heterozygous siblings was not remarkable; in particular, none had diabetes.
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No homozygous or heterozygous mutations were detected in patient WRS12-1 or in the EIF2AK3 exons and 3' untranslated and promoter regions. Genotyping of five adjacent microsatellite polymorphisms encompassing the EIF2AK3 gene excluded linkage to WRS in this family (Fig. 3). In addition, the sequence of EIF2AK3 exons in this family showed that the patient was heterozygous at four genetic variants within this gene (data not shown). Although it is formally possible that this patient has inherited independent EIF2AK3 mutations from each parent, which would both be located in distant regions of the gene or in introns, this is highly unlikely given the fact that the 15 EIF2AK3 mutations identified so far in WRS affect the amino acid sequence. Together, these observations support the idea that the WRS in this patient is not caused by EIF2AK3 mutations. Since his parents are consanguineous, his syndrome is likely to result from a recessive mutation in another single gene, although we cannot exclude that the observed diabetes and bone dysplasia occurred by coincidence in this patient. In the first hypothesis, the responsible gene may be one of the alternative eIF2 kinase, another gene involved in this pathway, or interacting with EIF2AK3. Using microsatellite markers encompassing GCN2, HRI, and PKR genes, we could exclude linkage of WRS in WRS12 family to the regions of these three alternative eIF2
kinases (data not shown).
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Apart from age at onset, there was poor concordance overall between the nature of the EIF2AK3 mutation and the variable features of the disease, suggesting that other genetic factors (modifier genes) or environmental factors may be involved. In particular, some affected siblings from the same family were discordant for mental retardation (family WRS9), the presence of acute or chronic hepatic or kidney dysfunction (families WRS2 and WRS8), and exocrine pancreas dysfunction (family WRS2). The patients who had chronic kidney dysfunction were the older living patients (age >11 years), suggesting that this feature may be a long-term complication of this syndrome. However, the absence of acute episodes or chronic conditions may not be a significant phenotype in young children, who may develop these at a later age. Possible familial aggregation was found for neutropenia/frequent infections, osteopenia, and hypothyroidism; however, it is difficult to conclude whether these complications are related to specific mutations because of the limited number of observations. Overall, our data suggest that factors unrelated to EIF2AK3 gene contribute to a large extent to the clinical variability of WRS. Variation in environmental conditions that lead to endoplasmic reticulum stress or genetic variation in other genes involved in the response to endoplasmic reticulum stress are likely to modulate the severity and clinical characteristics of the disease. Such an effect of environmental stress factors on disease severity and progression has been shown in a rare recessive neurological disorder, leukoencephalothay with vanishing white matter, which is caused by mutations in subunits of the eIF2B translation initiation factor (22,23).
Parents and heterozygous siblings of WRS patients from this study had no remarkable features; in particular none had type 1 or type 2 diabetes or bone disorder. This suggests that EIF2AK3 may not play a major role in the susceptibility to frequent forms of diabetes, at least as a single gene, despite our observation of linkage of the EIF2AK3 region to type 1 diabetes in the Scandinavian population (24).
Our studies suggest the existence of a variant form of WRS, which does not present any of the complications frequently observed in this syndrome and may be associated with an older age at onset. In addition, the only patient who carried a homozygous EIF2AK3 mutation with a slight residual activity in our functional studies was found to have a significantly delayed age at onset. Therefore, we propose to extend the age-at-onset definition in WRS to "early-infancy onset" instead of the generally used "neonatal" diabetes. Based on our observations, a diagnosis of WRS should be considered in patients presenting with insulin-dependent diabetes starting at older ages, in case of an association with epiphyseal dysplasia, and the EIF2AK3 gene screened for mutations in these patients. In addition to these genetic heterogeneity factors, part of the variability of WRS is likely to be due to additional factors, such as modifier genes or factors related to the environment or patients management, or to the natural progression of the disease.
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RESEARCH DESIGN AND METHODS |
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Mutation screening.
DNA was extracted from peripheral blood collected on EDTA, and EIF2AK3 mutation screening was performed on genomic DNA on all the coding regions of the gene in the cases and their parents (when available), as previously described (4). Alternatively, RNA was extracted from peripheral blood and sequencing performed on the cDNA (4). Additional sequencing was performed in one patient and his parents (family WRS12) to cover the 3' untranslated region of the gene, as previously described (4), and exon 1 and >1,500 bp of sequence 5' of the starting ATG of the gene, using three sets of overlapping primers: 5p3f/73-377R, 5p2f/5p2r, and 5p1f/5p1r (template sizes: 962, 554, and 574 bp, respectively), with 5p3f: 5'-GTCAGAATCCGCCACGTAGT-3', 73377R: 5'-CGCGCGTAAACAAGTTG-3'; 5p2f: 5'-AGTTCAAATGCCTTGGCTGA-3', 5p2r: 5'-GTCTGCGCTAACTGCCTCTT-3'; and 5p1f: 5'-ACCCATATTGCCAACACCTT-3', 5p1r: 5'-GGGCAGAGTGGAGAAGACTG-3'.
Sequencing reactions were performed using the amplification primers and an additional primer in the case of 5p3f/73-377R: 5'-CGAGATAGGCTGTCACTCAGG-3'.
Microsatellite genotyping.
Microsatellite genotyping was performed using fluorescence-labeled primers on an ABI3700 sequencer, using standard methods. For assessing linkage to the EIF2AK3 region, the following microsatellite markers were used: D2S1331, D2S2216, D2S2181, D2S113, and D2S2175.
Analysis of EIF2AK3 activity.
Functional assays for EIF2AK3 were performed using the yeast strain H1894 (MATa ura352 leu23, 112 gcn2 trp1
-63), which is deleted for its sole endogenous eIF2
kinase. The EIF2AK3 gene was inserted downstream of a galactose-inducible promoter in the URA3-marked low-copy plasmid p416. Each plasmid construct consisted of an NH2-terminal GST tag fused in-frame with the EIF2AK3 catalytic domain, including residues 5511,115. Five mutant constructs were generated, corresponding to WRS mutations R587Q, L645P, N655K, W898C, and L1057P. Positive and negative controls were wild-type EIF2AK3 and the kinase-deficient K621M mutant, respectively (21). Plasmids were transformed into H1894 cells using uracil prototrophy, and equal volumes of the yeast cultures at A600 = 0.25 were spotted onto agar plates containing synthetic minimal mediumcontaining glucose (SD) or in synthetic medium containing galactose (SGAL), which induces EIF2AK3 expression. Strains were grown for 34 days at 30°C on the agar plates and electronically imaged.
H1894 cells expressing the indicated EIF2AK3 proteins were grown in SGAL medium for 4 h at 30°C, and the levels of eIF2 phosphorylation were measured by immunoblot analysis. Phosphorylated eIF2
was visualized using an affinity-purified antibody that specifically recognizes eIF2
phosphorylated at serine-51 (Biosource International), and total eIF2
protein levels were measured by using polyclonal antibody prepared against total yeast eIF2
. The eIF2
-antibody complex was visualized by using horseradish peroxidaselabeled anti-rabbit secondary antibody and chemiluminescent substrate. To measure the steady-state levels of wild-type and mutant versions of EIF2AK3 in yeast, cells were inoculated into SGAL medium at A600 = 0.1 and incubated with constant shaking at 30°C for 20 h. Cells were collected, broken with glass beads, and lysates were clarified by centrifugation as described (20). The GST-tagged EIF2AK3 proteins were purified from 50 µg yeast lysate by using glutathione sepharose. Proteins associated with the sepharose were removed by boiling in SDS sample buffer and separated by electrophoresis in a 10% SDS polyacrylamide gel. Proteins were transferred to nitrocellulose filters, and immunoblot analysis was performed with either an antibody that recognizes total levels of the GST-tagged EIF2AK3 or an antibody specific for EIF2AK3 phosphorylated at T980. Kinase reactions were carried out by using EIF2AK3 expressed in yeast strain J82, which contains a mutant version of eIF2
-S51A that blocks EIF2AK3 inhibition of translation in the galactose-inducing medium (20). The GST-tagged EIF2AK3 proteins were purified by using glutathione sepharose and assayed for phophorylation of recombinant eIF2
in a reaction containing 10 µCi [
-32P]ATP in a final concentration of 50 µmol/l, as described (20). Reaction mixtures were incubated at 30°C for 4 min, a time point that was found to be in the linear range of the assay.
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ACKNOWLEDGMENTS |
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We thank Drs. M. Ovicova, M. Korada, and S. Iyer for providing clinical information and blood samples on some WRS patients. We thank the Hospices Civils de Lyon for their support.
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FOOTNOTES |
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C.H. is currently affiliated with the Institut de Radioprotection et de Sûreté Nucléaire, Fontenay-aux-Roses, France.
Address correspondence and reprint requests to Cécile Julier, Génétique des Maladies Infectieuses et Autoimmunes, 28, rue du Docteur Roux, 75724 Paris cedex 15, France. E-mail: cjulier{at}pasteur.fr
Received for publication January 30, 2004 and accepted in revised form March 22, 2004
eIF2, eukaryotic initiation factor 2
; GST, glutathione S-transferase; WRS, Wolcott-Rallison syndrome
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REFERENCES |
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