Absence of BiP Co-chaperone DNAJC3 Causes Diabetes Mellitus and Multisystemic Neurodegeneration
2014; Elsevier BV; Volume: 95; Issue: 6 Linguagem: Inglês
10.1016/j.ajhg.2014.10.013
ISSN1537-6605
AutoresMatthis Synofzik, Tobias B. Haack, Robert Kopajtich, Matteo Gorza, Doron Rapaport, Markus Greiner, Caroline Schönfeld, Clemens Freiberg, Stefan Schorr, Reinhard W. Holl, Michael Gonzalez, Andreas Fritsche, Petra Fallier‐Becker, Richard Zimmermann, Tim M. Strom, Thomas Meitinger, Stephan Züchner, Rebecca Schüle, Lüdger Schöls, Holger Prokisch,
Tópico(s)Heat shock proteins research
ResumoDiabetes mellitus and neurodegeneration are common diseases for which shared genetic factors are still only partly known. Here, we show that loss of the BiP (immunoglobulin heavy-chain binding protein) co-chaperone DNAJC3 leads to diabetes mellitus and widespread neurodegeneration. We investigated three siblings with juvenile-onset diabetes and central and peripheral neurodegeneration, including ataxia, upper-motor-neuron damage, peripheral neuropathy, hearing loss, and cerebral atrophy. Exome sequencing identified a homozygous stop mutation in DNAJC3. Screening of a diabetes database with 226,194 individuals yielded eight phenotypically similar individuals and one family carrying a homozygous DNAJC3 deletion. DNAJC3 was absent in fibroblasts from all affected subjects in both families. To delineate the phenotypic and mutational spectrum and the genetic variability of DNAJC3, we analyzed 8,603 exomes, including 506 from families affected by diabetes, ataxia, upper-motor-neuron damage, peripheral neuropathy, or hearing loss. This analysis revealed only one further loss-of-function allele in DNAJC3 and no further associations in subjects with only a subset of the features of the main phenotype. Our findings demonstrate that loss-of-function DNAJC3 mutations lead to a monogenic, recessive form of diabetes mellitus in humans. Moreover, they present a common denominator for diabetes and widespread neurodegeneration. This complements findings from mice in which knockout of Dnajc3 leads to diabetes and modifies disease in a neurodegenerative model of Marinesco-Sjögren syndrome. Diabetes mellitus and neurodegeneration are common diseases for which shared genetic factors are still only partly known. Here, we show that loss of the BiP (immunoglobulin heavy-chain binding protein) co-chaperone DNAJC3 leads to diabetes mellitus and widespread neurodegeneration. We investigated three siblings with juvenile-onset diabetes and central and peripheral neurodegeneration, including ataxia, upper-motor-neuron damage, peripheral neuropathy, hearing loss, and cerebral atrophy. Exome sequencing identified a homozygous stop mutation in DNAJC3. Screening of a diabetes database with 226,194 individuals yielded eight phenotypically similar individuals and one family carrying a homozygous DNAJC3 deletion. DNAJC3 was absent in fibroblasts from all affected subjects in both families. To delineate the phenotypic and mutational spectrum and the genetic variability of DNAJC3, we analyzed 8,603 exomes, including 506 from families affected by diabetes, ataxia, upper-motor-neuron damage, peripheral neuropathy, or hearing loss. This analysis revealed only one further loss-of-function allele in DNAJC3 and no further associations in subjects with only a subset of the features of the main phenotype. Our findings demonstrate that loss-of-function DNAJC3 mutations lead to a monogenic, recessive form of diabetes mellitus in humans. Moreover, they present a common denominator for diabetes and widespread neurodegeneration. This complements findings from mice in which knockout of Dnajc3 leads to diabetes and modifies disease in a neurodegenerative model of Marinesco-Sjögren syndrome. Nonautoimmune diabetes mellitus and neurodegeneration are common disorders for which shared genetic factors are still only partly known. Monogenic forms of diabetes include neonatal diabetes (MIM 606176) and maturity-onset diabetes of the young (MIM 606391), both of which arise from mutations that primarily reduce pancreatic β cell function.1Steck A.K. Winter W.E. Review on monogenic diabetes.Curr. Opin. Endocrinol. Diabetes Obes. 2011; 18: 252-258Crossref PubMed Scopus (44) Google Scholar Although monogenic forms account for only about 1%–2% of diabetes cases, they provide unique insights into the underlying basic disease mechanisms, such as endoplasmic reticulum (ER) stress.2Petrova K. Oyadomari S. Hendershot L.M. Ron D. Regulated association of misfolded endoplasmic reticulum lumenal proteins with P58/DNAJc3.EMBO J. 2008; 27: 2862-2872Crossref PubMed Scopus (109) Google Scholar, 3Fonseca S.G. Ishigaki S. Oslowski C.M. Lu S. Lipson K.L. Ghosh R. Hayashi E. Ishihara H. Oka Y. Permutt M.A. Urano F. Wolfram syndrome 1 gene negatively regulates ER stress signaling in rodent and human cells.J. Clin. Invest. 2010; 120: 744-755Crossref PubMed Scopus (286) Google Scholar Given that reduced mitigation of ER stress due to genetic mutations has been shown to cause both diabetes mellitus and multisystemic neurodegeneration (e.g., in Wolfram syndrome 1 [MIM 222300]3Fonseca S.G. Ishigaki S. Oslowski C.M. Lu S. Lipson K.L. Ghosh R. Hayashi E. Ishihara H. Oka Y. Permutt M.A. Urano F. Wolfram syndrome 1 gene negatively regulates ER stress signaling in rodent and human cells.J. Clin. Invest. 2010; 120: 744-755Crossref PubMed Scopus (286) Google Scholar, 4Fonseca S.G. Urano F. Weir G.C. Gromada J. Burcin M. Wolfram syndrome 1 and adenylyl cyclase 8 interact at the plasma membrane to regulate insulin production and secretion.Nat. Cell Biol. 2012; 14: 1105-1112Crossref PubMed Scopus (44) Google Scholar), it might present a shared mechanism linking diabetes with neurodegeneration. Mutations leading to loss of the ER protein DNAJC3—which serves to attenuate late phases of ER stress5Yan W. Frank C.L. Korth M.J. Sopher B.L. Novoa I. Ron D. Katze M.G. Control of PERK eIF2alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone P58IPK.Proc. Natl. Acad. Sci. USA. 2002; 99: 15920-15925Crossref PubMed Scopus (303) Google Scholar—have been shown to lead to pancreatic β cell failure and diabetes in mice.6Ladiges W.C. Knoblaugh S.E. Morton J.F. Korth M.J. Sopher B.L. Baskin C.R. MacAuley A. Goodman A.G. LeBoeuf R.C. Katze M.G. Pancreatic beta-cell failure and diabetes in mice with a deletion mutation of the endoplasmic reticulum molecular chaperone gene P58IPK.Diabetes. 2005; 54: 1074-1081Crossref PubMed Scopus (174) Google Scholar However, it is still unknown whether mutations in DNAJC3 (MIM 601184; RefSeq accession number NM_006260.4) also cause disease in humans. Here, we show in two index families that loss-of-function mutations in DNAJC3 and the resulting absence of ER protein DNAJC3 lead to diabetes mellitus and multisystemic neurodegeneration. Whole-exome sequencing (WES) was performed in two affected individuals (61691 and 61695, who are subjects II.2 and II.4, respectively, in family 1 in Figure 1A) of a consanguineous Turkish family affected by juvenile-onset diabetes and central and peripheral neurodegeneration, including early-onset ataxia, upper-motor-neuron damage, demyelinating peripheral neuropathy, neuronal hearing loss, and cerebral atrophy. This procedure and all following procedures reported in this manuscript were approved by the institutional review board of the University of Tübingen in Germany (reference number 598/20118O1), and proper informed consent was obtained from all subjects. After in-solution capture of exonic sequences (SureSelect Human All Exon 50 Mb Kit, Agilent Technologies), both samples were sequenced to an average of 126× and 173× coverage on the Illumina platform (Genome Analyzer II× System). For sequencing statistics, see Table S1 (available online). We used the Burrows-Wheeler Aligner (version 0.5.8) for read alignment to the human reference assembly (UCSC Genome Browser hg19) and SAMtools (version 0.1.7) for detection of single-nucleotide variants and small insertions and deletions. Given the rare combination of juvenile-onset diabetes and early-onset neurodegeneration, we assumed the causative mutations to be rare and to alter the protein sequence. We therefore excluded variants present in 4,517 exomes of control individuals with unrelated phenotypes and searched for nonsynonymous variants and splice-site mutations (Table 1). Given the autosomal-recessive pattern of inheritance, we filtered for genes harboring potential compound-heterozygous or homozygous rare variants that were predicted to be damaging by in silico prediction software tools (SIFT and PolyPhen-2). This approach left only one gene with mutations shared by both affected individuals: DNAJC3 (Table 1). The homozygous DNAJC3 mutation c.580C>T is predicted to cause premature truncation (p.Arg194∗) of more than 60% of the protein sequence (Figure 1B). Sanger sequencing of additional family members showed that all three affected siblings harbor the c.580C>T mutation in a homozygous state, whereas the healthy sister (subject II.1 in Figure 1A) has two wild-type alleles. Testing of parents demonstrated that both mutations are located in trans (for electropherograms of the Sanger sequences of all pedigree members, see Figure S1).Table 1Variants Identified by Exome Sequencing in Affected Individuals of Index Family 1II.2 (61691)II.4 (61695)Present in BothNSVs not present in 4,517 control individuals216238119At least two NSVs per gene11145 (PDCD11, DNAJC3, KIF23, CPT1C, MAGEC1)Two NSVs predicted to be damaging and present in both211 (DNAJC3)At least two loss-of-function alleles1 (DNAJC3)1 (DNAJC3)1 (DNAJC3)Nonsynonymous variants (NSVs) include missense, nonsense, stop-loss, and splice-site mutations and insertions and deletions. Variants were predicted to be damaging by at least one out of three software predictions (SIFT, PolyPhen-2, and MutationTaster). Open table in a new tab Nonsynonymous variants (NSVs) include missense, nonsense, stop-loss, and splice-site mutations and insertions and deletions. Variants were predicted to be damaging by at least one out of three software predictions (SIFT, PolyPhen-2, and MutationTaster). To confirm the significance of DNAJC3 mutations in the pathogenesis of diabetes-neurodegeneration syndromes, we screened the German multicenter DPV (Diabetes Patienten Verlaufsdokumentation) registry, which contains 226,194 pediatric and adult individuals with diabetes from 354 treatment centers. According to the key phenotypic characteristics of the affected members of index family 1 (for a detailed description, see Table 2 and the paragraph below), we used the following clinical screening criteria: (1) diabetes onset at age 10–20 years, (2) absence of β cell antibodies, (3) body mass index (BMI) below the median for age and gender, and (4) ataxia and/or hearing impairment. This filter left 35 individuals (31 with hearing impairment, two with ataxia, and two with hearing impairment and ataxia). Because the registry does not collect biomaterials, these 35 individuals were approached through their treatment centers (29 centers in total) by their local clinicians and were asked to provide a DNA sample for DNAJC3 analysis. DNA could be obtained from eight index subjects. Conventional Sanger sequencing (oligonucleotide sequences are available upon request) revealed no mutation in seven of eight subjects. In one of the two index subjects with hearing impairment and ataxia, we failed to amplify exons 6−12 by PCR, whereas PCR amplification delivered the expected DNA fragments for exons 1–5 in the same sample and for exons 1–12 in control samples, suggesting a potential deletion affecting exons 6–12. Breakpoints of the expected deletion were characterized by PCR and a primer-walking approach and were confirmed by Sanger sequencing (Figure 1B). This 72 kb homozygous deletion was also identified in the affected sibling (subject II.2 of family 2, Figure 1A) and was heterozygous in the parents (subjects I.1 and I.2 of family 2, Figure 1A).Table 2Characteristics of Subjects with Homozygous Loss-of-Function DNAJC3 Mutations and Healthy Sister 54829Index Family 1Index Family 2II.2 (61691)II.3 (61693)II.4 (61695)II.1 (54829)II.1 (66050)II.2 (66051)DNAJC3 variantc.580C>T (p.Arg194∗)c.580C>T (p.Arg194∗)c.580C>T (p.Arg194∗)NAdeletion of exons 6–12 (p.?)deletion of exons 6–12 (p.?)Age at examination39 years34 years20 years41 years20 years14 yearsSexmalefemalemalefemalefemalefemaleBelow-average body height (percentileaPercentiles of body weight and body height were taken from reference values for German-born Turkish children and adults (18 years).)+, 152 cm (<3%)+, 145 cm (<3%)+, 156 cm (25%)+, 155 cm (25%)+, 136 cm (<3%)+, 143 cm (3%)Below-average body weight (percentileaPercentiles of body weight and body height were taken from reference values for German-born Turkish children and adults (18 years).)+, 45 kg (<3%)+, 38 kg (<3%)+, 49 kg (10%)−, 65 kg (75%–90%)+, 39 kg (<3%)+, 39 kg (<3%)Reduced BMI (percentile)+, 19.5 (5%–10%)+, 18.1 (<3%)+, 20.1 (25%–50%)−, 27.1 (75%–90%)+, 21.1 (25%–50%)+, 19.4 (25%–50%)Age at onset of diabetes18 years18 years15 yearsno diabetes14 years11 yearsDiabetes insulin treatment+++−++HbA1c (ref: 4.3%–6.1%)7.4%6.9%12.1%NA7.5%7.9%IA-2 antibodies (ref: <0.9 U/ml)0.1 U/ml0.2 U/ml0.2 U/mlNA0.2 U/ml0.4 U/mlGAD2 antibodies (ref: 3.8 μV, >39 m/s)4.3 μV, 29 m/s (↓↓)no SNAP (↓↓)0.5 μV (↓↓), 25 m/s (↓↓)NDno SNAP (↓↓)no SNAP (↓↓)Radial (ref: >16 μV, >50 m/s)10.9 μV (↓), 37 m/s (↓↓)8.5 μV (↓), 32 m/s (↓↓)no SNAP (↓↓)NDno SNAP (↓↓)NDMotor NCScCompound muscle action potentials are given in mV, and motor nerve conduction velocities are given in m/s.Tibial (ref: >5 mV, >40 m/s)12.8 mV, 27 m/s (↓↓)13.6 mV, 23 m/s (↓↓)6.6 mV, 23 m/s (↓↓)ND0.2 mV (↓↓), 16 m/s (↓↓)9.5 mV, 20 m/s (↓↓)Ulnar (ref: >5 mV, >50 m/s)10.5 mV, 33 m/s (↓↓)14.9 mV, 32 m/s (↓↓)13.4 mV, 29 m/s (↓↓)ND11.6 mV, 24 m/s (↓↓)NDAbbreviations are as follows: BMI, body mass index; MEP, motor evoked potential; MMSE, Mini-Mental State Examination16Folstein M.F. Folstein S.E. McHugh P.R. "Mini-mental state". A practical method for grading the cognitive state of patients for the clinician.J. Psychiatr. Res. 1975; 12: 189-198Abstract Full Text PDF PubMed Scopus (71580) Google Scholar; NA, not applicable; NCS, nerve conduction study; ND, not done; ref, reference value; SARA, Scale for the Assessment and Rating of Ataxia; SEP, sensory evoked potential; SNAP, sensory nerve action potential; ↓, reduced value; and ↓↓, severely reduced value.a Percentiles of body weight and body height were taken from reference values for German-born Turkish children and adults (18 years).b SNAPs are given in μV, and sensory nerve conduction velocities are given in m/s.c Compound muscle action potentials are given in mV, and motor nerve conduction velocities are given in m/s. Open table in a new tab Abbreviations are as follows: BMI, body mass index; MEP, motor evoked potential; MMSE, Mini-Mental State Examination16Folstein M.F. Folstein S.E. McHugh P.R. "Mini-mental state". A practical method for grading the cognitive state of patients for the clinician.J. Psychiatr. Res. 1975; 12: 189-198Abstract Full Text PDF PubMed Scopus (71580) Google Scholar; NA, not applicable; NCS, nerve conduction study; ND, not done; ref, reference value; SARA, Scale for the Assessment and Rating of Ataxia; SEP, sensory evoked potential; SNAP, sensory nerve action potential; ↓, reduced value; and ↓↓, severely reduced value. To delineate the phenotypic and mutational spectrum and the genetic variability of DNAJC3, we screened a total of 8,603 exomes (4,303 from the Hussman Institute for Human Genomics [Miami] via the Genomes Management Application7Gonzalez M.A. Lebrigio R.F. Van Booven D. Ulloa R.H. Powell E. Speziani F. Tekin M. Schüle R. Züchner S. GEnomes Management Application (GEM.app): a new software tool for large-scale collaborative genome analysis.Hum. Mutat. 2013; 34: 842-846Crossref PubMed Scopus (64) Google Scholar and 4,300 from the Institute of Human Genetics [Munich]) for rare DNAJC3 variants. Among these 8,603 exomes, 506 unrelated index subjects with a family history consistent with recessive disease presented with at least one of the main phenotypic features of the DNAJC3 phenotypic cluster: diabetes (n = 30), early-onset ataxia (age of onset < 30 years; n = 69), hereditary spastic paraplegia (n = 161), Charcot-Marie-Tooth disease type 2 (n = 153), or deafness (n = 93). In a first step, we screened all 8,603 exomes for potential homozygous or compound-heterozygous variants in DNAJC3 by using the following filter criteria: low frequency in public databases (minor allele frequency < 1% in the NHLBI Exome Sequencing Project Exome Variant Server [ESP6500]) and genotype quality > 35. We identified two families affected by homozygous DNAJC3 missense mutations (family 1: c.207T>A [p.Asp69Glu]; family 2: c.1060G>C [p.Glu354Gln]) and one family harboring compound-heterozygous DNAJC3 missense mutations (c.641C>T [p.Ala214Val] and c.1037G>A [p.Arg346Gln]), but none of these mutations was considered to be disease causing (for details, see Table S2 and Figure S2). Apart from our index family 1, we did not identify any other family carrying two predicted loss-of-function variants in DNAJC3. Taken together, these findings suggest the following notion: although biallelic loss-of-function mutations in DNAJC3 cause diabetes and a multisystemic combination of ataxia, peripheral neuropathy, upper-motor-neuron disease, and hypacusis, they do not seem to be associated with only one of these features or a small subset of them. Next, we analyzed the overall occurrence of DNAJC3 variants to determine the genetic variability of DNAJC3 (same filters as above, but no inheritance filter). In 8,603 exomes, we identified 56 alleles with rare DNAJC3 variants, consisting of 41 unique variants (one stop [c.580C>T from index family 1], one frameshift, and 39 missense; see Table S3). This observation demonstrates that truncating variants in DNAJC3 are a very rare event (2 out of 17,206 alleles) and that genetic variability of DNAJC3 is low overall (56 events out of 17,206 alleles). This notion receives further independent support from the Residual Variation Intolerance Score (RVIS), which is based on the 6,500 exomes from NHLBI ESP6500.8Petrovski S. Wang Q. Heinzen E.L. Allen A.S. Goldstein D.B. Genic intolerance to functional variation and the interpretation of personal genomes.PLoS Genet. 2013; 9: e1003709Crossref PubMed Scopus (643) Google Scholar This score provides a measure of the departure from the (genome-wide) average number of common functional mutations found in genes with a similar amount of mutational burden (RVIS = 0 when the gene has an average number of common functional variants given its total mutational burden, RVIS < 0 when the gene has less common functional variation than predicted, and RVIS > 0 when the gene has more).8Petrovski S. Wang Q. Heinzen E.L. Allen A.S. Goldstein D.B. Genic intolerance to functional variation and the interpretation of personal genomes.PLoS Genet. 2013; 9: e1003709Crossref PubMed Scopus (643) Google Scholar DNAJC3 yields a RVIS of −0.47, ranking it among the 23% most intolerant of human genes.8Petrovski S. Wang Q. Heinzen E.L. Allen A.S. Goldstein D.B. Genic intolerance to functional variation and the interpretation of personal genomes.PLoS Genet. 2013; 9: e1003709Crossref PubMed Scopus (643) Google Scholar Genes such as DNAJC3, which are more intolerant of functional genetic variation, have been shown to be significantly more likely than other genes to harbor mutations that cause Mendelian diseases.8Petrovski S. Wang Q. Heinzen E.L. Allen A.S. Goldstein D.B. Genic intolerance to functional variation and the interpretation of personal genomes.PLoS Genet. 2013; 9: e1003709Crossref PubMed Scopus (643) Google Scholar DNAJC3 acts as co-chaperone of BiP (immunoglobulin heavy-chain binding protein), a major ER-localized member of the HSP70 family of molecular chaperones, which reversibly bind to contiguous segments of hydrophobic amino acids exposed in unfolded lumenal proteins to impede aggregation and promote adequate folding.9Gething M.J. Role and regulation of the ER chaperone BiP.Semin. Cell Dev. Biol. 1999; 10: 465-472Crossref PubMed Scopus (439) Google Scholar DNAJC3 is located in virtually all tissues in mice and humans and has especially high levels in the pancreas and liver.10Korth M.J. Lyons C.N. Wambach M. Katze M.G. Cloning, expression, and cellular localization of the oncogenic 58-kDa inhibitor of the RNA-activated human and mouse protein kinase.Gene. 1996; 170: 181-188Crossref PubMed Scopus (33) Google Scholar In mice, loss of DNAJC3 leads to hyperglycemia and glucosuria associated with increasing apoptosis of pancreatic β cells and reduced insulin levels.6Ladiges W.C. Knoblaugh S.E. Morton J.F. Korth M.J. Sopher B.L. Baskin C.R. MacAuley A. Goodman A.G. LeBoeuf R.C. Katze M.G. Pancreatic beta-cell failure and diabetes in mice with a deletion mutation of the endoplasmic reticulum molecular chaperone gene P58IPK.Diabetes. 2005; 54: 1074-1081Crossref PubMed Scopus (174) Google Scholar To validate the predicted loss-of-function character of the identified mutations in both families, we analyzed DNAJC3 in fibroblasts by immunoblotting. Neither full-length nor truncated DNAJC3 was detected in the affected individuals, whereas the two heterozygous parents showed normal protein levels (Figure 2). Given the well-established function of DNAJC3 in the downregulation of ER-associated proteins involved in the initial ER stress response, as well as the histopathological changes observed in some DNAJC3-deficient cell types,6Ladiges W.C. Knoblaugh S.E. Morton J.F. Korth M.J. Sopher B.L. Baskin C.R. MacAuley A. Goodman A.G. LeBoeuf R.C. Katze M.G. Pancreatic beta-cell failure and diabetes in mice with a deletion mutation of the endoplasmic reticulum molecular chaperone gene P58IPK.Diabetes. 2005; 54: 1074-1081Crossref PubMed Scopus (174) Google Scholar we investigated the ER morphology in fibroblasts from our individuals with DNAJC3 loss of function. No changes in ER morphology were observed by electron microscopy (Figure S3). This is in line with the previous finding that histological changes associated with the absence of DNAJC3 can be found only in some tissues (e.g., pancreatic islets), but not in others (e.g., pancreatic acini or other pancreatic parenchyma).6Ladiges W.C. Knoblaugh S.E. Morton J.F. Korth M.J. Sopher B.L. Baskin C.R. MacAuley A. Goodman A.G. LeBoeuf R.C. Katze M.G. Pancreatic beta-cell failure and diabetes in mice with a deletion mutation of the endoplasmic reticulum molecular chaperone gene P58IPK.Diabetes. 2005; 54: 1074-1081Crossref PubMed Scopus (174) Google Scholar We analyzed protein trafficking through the secretory pathway in cell lines from subjects with homozygous DNJAC3 mutations and healthy control individuals by monitoring the secretion of Gaussia princeps luciferase into the culture medium.12Badr C.E. Hewett J.W. Breakefield X.O. Tannous B.A. A highly sensitive assay for monitoring the secretory pathway and ER stress.PLoS ONE. 2007; 2: e571Crossref PubMed Scopus (107) Google Scholar Induction of ER stress by thapsigargin (an inhibitor of the ER Ca2+ ATPase) or tunicamycin or glucose deprivation (both of which interfere with N-linked protein glycosylation) resulted in impaired secretion in mutant and control cells. No significant differences were noted through induction or recovery from ER stress (Figure S4), indicating intact protein secretion in fibroblasts. In addition, we analyzed subjects' fibroblasts for a difference in ER calcium leakage. It has been shown for HeLa cells that a loss of BiP function leads to increased ER calcium leakage, as does replacement of BiP by an altered BiP variant, which cannot productively interact with its HSP40 co-chaperones.13Schäuble N. Lang S. Jung M. Cappel S. Schorr S. Ulucan O. Linxweiler J. Dudek J. Blum R. Helms V. et al.BiP-mediated closing of the Sec61 channel limits Ca2+ leakage from the ER.EMBO J. 2012; 31: 3282-3296Crossref PubMed Scopus (117) Google Scholar However, no increased ER calcium leakage was observed (Figure S5). Taken together, these experiments again point to the fact that fibroblasts might not be the most appropriate tissue for testing for DNAJC3-related ER stress and calcium leakage, most likely because of their low secretory activity and/or the compensatory mechanisms that are active in the affected subjects. However, no other tissue (e.g., pancreatic β cells or neurons) was available for testing. We aggregated clinical, electrophysiological, and imaging data from all five affected subjects belonging to the two index families affected by DNAJC3 loss-of-function mutations. Before identification of the DNAJC3 mutations, the phenotype in family 1 was clinically classified as a mitochondriopathy, and the phenotype in family 2 was classified as an atypical Shwachman-Bodian-Diamond syndrome (MIM 260400). All five affected subjects presented with young-onset diabetes melllitus diagnosed between 10 and 20 years of age, showed increased HbA1c levels (6.9%–12.1%), and received insulin treatment (Table 2). IA-2 antibodies were absent in all five subjects, and GAD2 antibodies were present in four of five subjects (Table 2). C-peptides—which reflect the degree of residual pancreatic β cell function—were assessed in fasting serum and upon response to standardized intravenous application with 1 mg of glucagon13Schäuble N. Lang S. Jung M. Cappel S. Schorr S. Ulucan O. Linxweiler J. Dudek J. Blum R. Helms V. et al.BiP-mediated closing of the Sec61 channel limits Ca2+ leakage from the ER.EMBO J. 2012; 31: 3282-3296Crossref PubMed Scopus (117) Google Scholar (Table S4). Residual endogenous insulin secretion was present in all subjects, and secretion could be stimulated by glucagon (both are characteristic of most monogenic forms of diabetes14Murphy R. Ellard S. Hattersley A.T. Clinical implications of a molecular genetic classification of monogenic beta-cell diabetes.Nat. Clin. Pract. Endocrinol. Metab. 2008; 4: 200-213Crossref PubMed Scopus (385) Google Scholar). However, all subjects showed an at least relative deficit in insulin secretion, given that fasting C-peptide levels were low in absolute levels and/or in relation to the actual glucose levels and HbA1c levels (Table S4). Fasting C-peptide levels were in the same low range as reported for other monogenic forms of diabetes (between 100 and 700 pmol/l14Murphy R. Ellard S. Hattersley A.T. Clinical implications of a molecular genetic classification of monogenic beta-cell diabetes.Nat. Clin. Pract. Endocrinol. Metab. 2008; 4: 200-213Crossref PubMed Scopus (385) Google Scholar). The presence of residual endogenous insulin stimulation by glucagon in subjects with >3 years of diabetes history and the absence of autoimmune antibodies clearly argues against a diabetes mellitus type 1.15Craig M.E. Jefferies C. Dabelea D. Balde N. Seth A. Donaghue K.C. Definition, epidemiology, and classification of diabetes in children and adolescents.Pediatr. Diabetes. 2014; 15: 4-17Crossref PubMed Scopus (219) Google Scholar All subjects exhibited multisystemic central and peripheral neurodegeneration. This included early-onset ataxia of combined cerebellar and afferent origin (age of onset = 2–34 years) and sensorimotor peripheral neuropathy predominantly of the demyelinating type (Table 2), which was observed even in the subject with the most recent disease onset of 6 months earlier (subject II.2 in family 2). Four of five (80%) individuals developed early-onset sensorineural hearing loss (range of onset = 2–27 years), and two of five (40%) showed pyramidal tract signs (Table 2). Cognitive screening by the Mini-Mental State Examination16Folstein M.F. Folstein S.E. McHugh P.R. "Mini-mental state". A practical method for grading the cognitive state of patients for the clinician.J. Psychiatr. Res. 1975; 12: 189-198Abstract Full Text PDF PubMed Scopus (71580) Google Scholar in family 1 revealed an isolated cognitive deficit in backward calculation of serial sevens, suggesting partial cerebrocortical dysfunction. All five affected subjects from the two index families had a below-average body weight (four were below the third percentile, and one was at the tenth percentile) and a below-normal BMI (one was below the third percentile, one was below the tenth percentile, and three were below the 50th percentile), whereas both body weight and BMI were above average in the healthy sister (II.1) from family 1 (each were between the 75th and 90th percentiles; Table 2; Figure 3H). All five affected subjects also showed a remarkably short body stature (four were below the third percentile, and one was at the 25th percentile; for an example, see Figure 3H), suggesting that it is highly likely that this is also part of the phenotype associated with DNAJC3 mutations. However, because body stature was also below average in the healthy sister (II.1) from family 1, future studies are warranted to confirm this feature. MRI available for index subjects II.2 (61691) of family 1 and II.1 (66050) of family 2 revealed generalized supra- and infratentorial atrophy pronounced in the cervical cord, cerebellar vermis, cerebellar hemispheres, and midbrain in both subjects and in the pre- and postcentral gyrus, crus cerebri, pons, and medulla in subject II.2 (Figure 3). Subject II.1 (66050) of family 2 also showed several small T2 hyperintense lesions without contrast enhancement bilaterally in frontoparietal and periventricular regions (Figure 3G). Dnajc3 knockout causes gradual onset of hyperglycemia and glucosuria associated with increasing apoptosis of pancreatic β cells in mice, thus mimicking disease processes in type 1 and late-stage type 2 diabetes.6Ladiges W.C. Knoblaugh S.E. Morton J.F. Korth M.J. Sopher B.L. Baskin C.R. MacAuley A. Goodman A.G. LeBoeuf R.C. Katze M.G. Pancreatic beta-cell failure and diabetes in mice with a deletion mutation of the endoplasmic reticulum molecular chaperone gene P58IPK.Diabetes. 2005; 54: 1074-1081Crossref PubMed Scopus (174) Google Scholar Our findings demonstrate that—analogous to the mouse model—homozygous DNAJC3 loss-of-function mutations cause young-onset diabetes in humans. Moreover, like Dnajc3-knockout mice,6Ladiges W.C. Knoblaugh S.E. Morton J.F. Korth M.J. Sopher B.L. Baskin C.R. MacAuley A. Goodman A.G. LeBoeuf R.C. Katze M.G. Pancreatic beta-cell failure and diabetes in mice with a deletion mutation of the endoplasmic reticulum molecular chaperone gene P58IPK.Diabetes. 2005; 54: 1074-1081Crossref PubMed Scopus (174) Google Scholar humans with DNAJC3 loss-of-function mutations also show a lower body weight, probably because of less body fat.6Ladiges W.C. Knoblaugh S.E. Morton J.F. Korth M.J. Sopher B.L. Baskin C.R. MacAuley A. Goodman A.G. LeBoeuf R.C. Katze M.G. Pancreatic beta-cell failure and diabetes in mice with a deletion mutation of the endoplasmic reticulum molecular chaperone gene P58IPK.Diabetes. 2005; 54: 1074-1081Crossref PubMed Scopus (174) Google Scholar Our findings also reveal that the absence of DNAJC3 is associated not only with diabetes mellitus but also with widespread neurodegeneration (which was not studied in detail in the Dnajc3-knockout mice6Ladiges W.C. Knoblaugh S.E. Morton J.F. Korth M.J. Sopher B.L. Baskin C.R. MacAuley A. Goodman A.G. LeBoeuf R.C. Katze M.G. Pancreatic beta-cell failure and diabetes in mice with a deletion mutation of the endoplasmic reticulum molecular chaperone gene P58IPK.Diabetes. 2005; 54: 1074-1081Crossref PubMed Scopus (174) Google Scholar). It has already been shown for mutations in other genes encoding ER-localized BiP co-chaperones that they can lead to multisystemic neurodegeneration. For example, mutations in SIL1 lead to Marinesco-Sjögren syndrome (MSS [MIM 248800]), a multisystemic syndrome including early-onset ataxia, cognitive deficits, short stature, and pyramidal tract signs,17Krieger M. Roos A. Stendel C. Claeys K.G. Sonmez F.M. Baudis M. Bauer P. Bornemann A. de Goede C. Dufke A. et al.SIL1 mutations and clinical spectrum in patients with Marinesco-Sjogren syndrome.Brain. 2013; 136: 3634-3644Crossref PubMed Scopus (56) Google Scholar, 18Senderek J. Krieger M. Stendel C. Bergmann C. Moser M. Breitbach-Faller N. Rudnik-Schöneborn S. Blaschek A. Wolf N.I. Harting I. et al.Mutations in SIL1 cause Marinesco-Sjögren syndrome, a cerebellar ataxia with cataract and myopathy.Nat. Genet. 2005; 37: 1312-1314Crossref PubMed Scopus (194) Google Scholar, 19Anttonen A.K. Mahjneh I. Hämäläinen R.H. Lagier-Tourenne C. Kopra O. Waris L. Anttonen M. Joensuu T. Kalimo H. Paetau A. et al.The gene disrupted in Marinesco-Sjögren syndrome encodes SIL1, an HSPA5 cochaperone.Nat. Genet. 2005; 37: 1309-1311Crossref PubMed Scopus (181) Google Scholar thus mirroring main features of the phenotype associated with DNAJC3 mutations. Both genes not only are linked phenotypically but also interact on a molecular level. Within the regulation of the BiP ATP-ADP cycle, DNAJC3 and SIL1 have opposing functions—loss of DNAJC3 has been shown to ameliorate cerebellar Purkinje cell death and ataxia in SIL1−/− mice.20Zhao L. Rosales C. Seburn K. Ron D. Ackerman S.L. Alteration of the unfolded protein response modifies neurodegeneration in a mouse model of Marinesco-Sjögren syndrome.Hum. Mol. Genet. 2010; 19: 25-35Crossref PubMed Scopus (77) Google Scholar Dysfunctions in the regulation of ER stress might thus serve as a common pathway linking diabetes mellitus and neurodegeneration. It has already been shown for mutations in other genes involved in the regulation of ER stress that they can lead to monogenic diabetes mellitus and multisystemic neurodegeneration, e.g., for mutations in the gene encoding ER-localized transmembrane protein WFS1.3Fonseca S.G. Ishigaki S. Oslowski C.M. Lu S. Lipson K.L. Ghosh R. Hayashi E. Ishihara H. Oka Y. Permutt M.A. Urano F. Wolfram syndrome 1 gene negatively regulates ER stress signaling in rodent and human cells.J. Clin. Invest. 2010; 120: 744-755Crossref PubMed Scopus (286) Google Scholar, 4Fonseca S.G. Urano F. Weir G.C. Gromada J. Burcin M. Wolfram syndrome 1 and adenylyl cyclase 8 interact at the plasma membrane to regulate insulin production and secretion.Nat. Cell Biol. 2012; 14: 1105-1112Crossref PubMed Scopus (44) Google Scholar WFS1 neurodegeneration includes early-onset ataxia, sensorineural hearing loss, and cognitive deficits (Wolfram syndrome 121Synofzik M. Weiss D. Erharhaghen J. Krüger R. Schöls L. Severe orthostatic dysregulation associated with Wolfram syndrome.J. Neurol. 2010; 257: 1751-1753Crossref PubMed Scopus (1) Google Scholar, 22Barrett T.G. Bundey S.E. Macleod A.F. Neurodegeneration and diabetes: UK nationwide study of Wolfram (DIDMOAD) syndrome.Lancet. 1995; 346: 1458-1463Abstract PubMed Scopus (258) Google Scholar), thus mirroring several of the systems also affected by DNAJC3-associated neurodegeneration. Likewise, mutations are well established for other genes encoding proteins directly involved in signaling of the endoplasmic unfolded protein response (UPR), which acts up- and downstream of DNAJC3 (Figure 4). For example, mutations in EIFKA3 (MIM 604032), coding for the UPR signaling protein PERK (PRKR-like endoplasmic reticulum kinase; Figure 4), cause Wolcott-Rallison syndrome (WRS [MIM 226980]), characterized by young-onset nonautoimmune insulin-requiring diabetes, skeletal dysplasia, and short body stature and thereby resembling several hallmarks of the DNAJC3-deficiency phenotype. Likewise, defects in SIL1 (MIM 608005), which encodes a protein upstream of DNAJC3 in the UPR (Figure 4), cause MSS.17Krieger M. Roos A. Stendel C. Claeys K.G. Sonmez F.M. Baudis M. Bauer P. Bornemann A. de Goede C. Dufke A. et al.SIL1 mutations and clinical spectrum in patients with Marinesco-Sjogren syndrome.Brain. 2013; 136: 3634-3644Crossref PubMed Scopus (56) Google Scholar, 18Senderek J. Krieger M. Stendel C. Bergmann C. Moser M. Breitbach-Faller N. Rudnik-Schöneborn S. Blaschek A. Wolf N.I. Harting I. et al.Mutations in SIL1 cause Marinesco-Sjögren syndrome, a cerebellar ataxia with cataract and myopathy.Nat. Genet. 2005; 37: 1312-1314Crossref PubMed Scopus (194) Google Scholar, 19Anttonen A.K. Mahjneh I. Hämäläinen R.H. Lagier-Tourenne C. Kopra O. Waris L. Anttonen M. Joensuu T. Kalimo H. Paetau A. et al.The gene disrupted in Marinesco-Sjögren syndrome encodes SIL1, an HSPA5 cochaperone.Nat. Genet. 2005; 37: 1309-1311Crossref PubMed Scopus (181) Google Scholar Clinical manifestations of MSS include early-onset ataxia and short stature, thus again mirroring several features of the DNAJC3-deficiency phenotype. In summary, we have identified DNAJC3 mutations as a cause of monogenic juvenile-onset diabetes combined with early-onset multisystemic neurodegeneration. DNAJC3 plays a crucial role in ER protein folding and the UPR, thus adding evidence that dysfunctions in regulation of ER stress might serve as a common pathway linking diabetes mellitus and neurodegeneration. DNAJC3 might be linked to neurodegeneration in other ER-stress-induced and UPR diseases (e.g., Wolfram syndrome 1, WRS, and MSS) not only on a phenotypic level but also on a molecular level. This study was supported by the German Federal Ministry of Education and Research (BMBF) (mitoNET grant 01GM0867 to H.P. and grant 01GM1113E to L.S. and D.R.), the European Union (grant F5-2012-305121 [NEUROMICS] to L.S. and grant PIOF-GA-2012-326681 [HSP/CMT Genetics] to R.S.), E-Rare grants to GENOMIT (01GM1207 to H.P.) and EUROSCAR (01GM1206 to L.S.), the Interdisciplinary Center for Clinical Research (IZKF) Tübingen (grants 2191-0-0 to M.S. and 1970-0-0 to R.S.), the BMBF Competence Network for diabetes (FKZ 01GI1106 to R.H.), and the NIH (grants 5R01NS072248, 1R01NS075764, and 5R01NS054132 to S.Z.). This study was also supported by a grant from the BMBF to the German Center for Diabetes Research (DZD e.V.) in Munich. We are grateful to Professor Hartwig Wolburg (Tübingen) for discussion of the electron-microscopy findings. Download .pdf (1.14 MB) Help with pdf files Document S1. Figures S1–S5 and Tables S1–S4 The URLs for data presented herein are as follows:Diabetes Patienten Verlaufsdokumentation (DPV), http://www.d-p-v.euOnline Mendelian Inheritance in Man (OMIM), http://www.omim.orgRefSeq, http://www.ncbi.nlm.nih.gov/RefSeq Absence of BiP Co-chaperone DNAJC3 Causes Diabetes Mellitus and Multisystemic NeurodegenerationSynofzik et al.The American Journal of Human GeneticsMarch 05, 2015In Brief(The American Journal of Human Genetics 96, 689–697; December 4, 2014) Full-Text PDF Open Archive
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