Véronique Paquis-Flucklinger

Mutation of RRM2B , encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion

Mitochondrial DNA (mtDNA) depletion syndrome (MDS; MIM 251880) is a prevalent cause of oxidative phosphorylation disorders characterized by a reduction in mtDNA copy number. The hitherto recognized disease mechanisms alter either mtDNA replication (POLG (ref. 1)) or the salvage pathway of mitochondrial deoxyribonucleosides 5′-triphosphates (dNTPs) for mtDNA synthesis (DGUOK (ref. 2), TK2 (ref. 3) and SUCLA2 (ref. 4)). A last gene, MPV17 (ref. 5), has no known function. Yet the majority of cases remain unexplained. Studying seven cases of profound mtDNA depletion (1–2% residual mtDNA in muscle) in four unrelated families, we have found nonsense, missense and splice-site mutations and in-frame deletions of the RRM2B gene, encoding the cytosolic p53-inducible ribonucleotide reductase small subunit. Accordingly, severe mtDNA depletion was found in various tissues of the Rrm2b−/− mouse. The mtDNA depletion triggered by p53R2 alterations in both human and mouse implies that p53R2 has a crucial role in dNTP supply for mtDNA synthesis.

Maternally Inherited Diabetes and Deafness: A Multicenter Study

Mitochondrial (mt) gene abnormalities cause disease due to defects in oxidative production of energy (1). In 1990, Goto and colleagues (2) described the co-segregation of a syndrome called mitochondrial encephalopathy, lactic acidosis, and strokelike episodes (which is sometimes associated with diabetes) and an A to G transition at position 3243 of mtDNA, encoding transfer RNA leucine (tRNALeu [UUR]). In 1992, a subtype of diabetes called maternally inherited diabetes and deafness (MIDD) was reported to co-segregate with the same point mutation (3, 4). Maternally inherited diabetes and deafness was initially characterized by matrilineal transmission, associated hearing loss, or both, without major neurologic defects. In MIDD, diabetes seems to be due primarily to a defect in insulin secretion (4-9), while insulin sensitivity is unaltered (8, 10). An estimated 0.5% to 2.8% of diabetic patients have MIDD (9, 11-16). Because most reported series have been small, the clinical description of MIDD and its course, particularly the occurrence of diabetic complications, remains incomplete. The aims of our study were to delineate the clinical presentation of MIDD, including involvement of other organs, in a large series of patients and to assess the prevalence of diabetic microvascular and macrovascular complications. Methods Patients We conducted this prospective multicenter descriptive study between September 1995 and December 1999. A questionnaire was sent to all members of the French Association for the Study of Diabetes (ALFEDIAM) in order to assess the clinical presentation of MIDD in a large series of patients. A total of 52 patients were included as a result of the questionnaire, and 19 patients were recruited directly from Lariboisire Hospital. The 3243 mtDNA mutation was identified in 40 probands who were selected primarily because they had both diabetes and deafness. Family screening identified 31 additional carriers of the mutation. All patients were white and European, except one who was of Caribbean origin. Three patients had mitochondrial encephalopathy, lactic acidosis, and strokelike episodes associated with diabetes. Among the 68 remaining patients, 54 had overt diabetes, 2 had impaired glucose tolerance, and 12 were healthy carriers. The study conformed to the principles of the Declaration of Helsinki, and all patients gave informed consent. Measurements We used a structured interview and a standardized examination of the patients and of their relatives to ascertain a family history of diabetes and hearing loss and to determine glucose tolerance disorders, treatment, diabetic complications, and associated manifestations. Abnormalities in glucose tolerance were diagnosed on the basis of the 1997 criteria outlined by the American Diabetes Association (17). Accordingly, diabetes was defined as a fasting plasma glucose level of 7 mmol/L (126 mg/dL) or greater on two occasions, a plasma glucose level of 11 mmol/L (200 mg/dL) or greater 2 hours after a 75-g oral glucose load, or both. In our study, we report only on the 54 patients with overt diabetes. Obesity was defined as a body mass index (BMI) of 30 kg/m2 or greater, body weight excess was defined as a BMI of 25 kg/m2 or greater, and low body weight was defined as a BMI less than 18.5 kg/m2 (18). Hemoglobin A1c level was assayed by using high-performance liquid chromatography. Islet-cell antibodies, antibodies to glutamic acid decarboxylase 65, or IA2 antibodies were determined in 32 cases. Hypertension was defined as a blood pressure exceeding 140/85 mm Hg on two occasions after 10 minutes in the resting position or as the use of antihypertensive treatment (19). Diagnosis of macrovascular complications (coronary heart disease, lower-limb arteriopathy, cerebrovascular disease) and diagnosis of cardiomyopathy were based on medical records, clinical examination, and appropriate tests (echocardiography, ultrasonography). We determined 24-hour urinary albumin excretion, proteinuria, and plasma creatinine concentration. A standardized ophthalmologic examination was performed in 49 patients, including ophthalmoscopy after pupillary dilation, color photographs, and fluorescein angiography, all of which were read by the same investigator. The retinal epithelium alterations were graded according to published criteria (20, 21). Molecular Studies The A to G 3243 mtDNA mutation was identified in peripheral blood leukocytes in 70 patients and from a buccal smear in 1 patient. Total DNA was extracted from the peripheral blood by using the conventional salting-out procedures. The 3243 mutation was detected by using 2% agarose electrophoresis after polymerase chain reaction amplification of a 294base pair fragment and enzymatic digestion with Apa I, as described elsewhere (2, 22). Statistical Analysis Data were stored and analyzed by using SPSS for Windows (SPSS Inc., Chicago, Illinois). Data are expressed as the mean (SD), with ranges indicated in parentheses. Differences in BMI and hemoglobin A1c levels among patient groups were assessed by using KruskalWallis analysis of variance. Correlations were analyzed by using the Spearman nonparametric rank correlation coefficient. Results Diabetes All 54 patients with overt diabetes (21 men, 33 women) had a fasting plasma glucose level greater than 7 mmol/L (126 mg/dL) when diabetes was diagnosed. Diabetes was diagnosed by systematic screening in 32 patients (59%). In the 22 remaining patients (41%), diabetes was revealed by the occurrence of polyuria, which was associated with ketoacidosis in 4 patients (7%). Age at diagnosis of diabetes was 38.8 9.6 years (range, 12 to 67 years); in 25 patients, diabetes was diagnosed before age 35. At the time of the study, the mean patient age was 50.0 10.3 years (range, 31 to 71 years), and diabetes duration was 11.8 8.7 years (range, 0 to 37 years). Diabetes was noninsulin-dependent in 22 patients (41%); 9 of 22 were treated with diet alone, and 13 were treated with sulfonylureas, metformin, or both. Twenty-five patients (46%) required insulin after experiencing secondary failure with a combination of maximally dosed sulfonylureas and metformin 9.9 5.8 years (range, 1 to 28 years) after diabetes was diagnosed. In 7 patients (13%), diabetes was insulin-dependent from its onset. Islet-cell antibodies were present in only 1 patient. A first-degree family history of diabetes was present in 33 of 40 probands (83%), and a maternal family history of diabetes was present in 29 probands (73%). No patient with MIDD was obese (Table), and 19 of 50 patients (38%) had low body weight. No correlation was found between BMI and hemoglobin A1c level, age, or diabetes duration. However, BMI and hemoglobin A1c values differed among patient groups according to treatment (Table). Table. Hemoglobin A1c Level and Body Mass Index in 54 Patients with Maternally Inherited Diabetes and Deafness Deafness Bilateral neurosensory hearing loss was present in 53 of 54 patients (98%). Hearing loss was clinically significant in all patients and was documented by audiography in 28 patients. Fourteen patients (26%) required a prosthetic hearing aid. Age at diagnosis of deafness was 34.6 13.9 years (range, 2 to 61 years). Twenty-five of 40 probands (63%) had a maternal history of deafness. Diabetes was the first clinical manifestation of the disease in 24 patients, and hearing loss was the first manifestation in 23 patients. In 7 patients, both conditions were diagnosed simultaneously. Macular Pattern Dystrophy We previously reported the presence of a characteristic macular pattern dystrophy in patients with MIDD (20). In our current study, macular pattern dystrophy was present in 42 of 49 examined patients (86%). Age at discovery was 46.5 10.8 years (range, 27 to 71 years). In 6 patients, the pigmented lesions were very small and were localized to the macula (grade 1) (Figure 1, top). In 27 patients, the deposits were more extensive and were localized around the macula and the optic disc (grade 2). In the 9 patients with advanced macular pattern dystrophy, the macula had patches of retinal atrophy (grade 3) (Figure 1, bottom). No correlation was found between grade of macular pattern dystrophy and age or diabetes duration. Figure 1. Macular pattern dystrophy. Top. Bottom. Visual acuity was normal in 43 of 49 patients (88%). Among patients with retinal atrophy, 4 had a visual acuity of 20/50 to 20/32 and 2 had a visual acuity below 20/63. Of the latter 2 patients, 1 had severe astigmatism and 1 had diabetic macular edema. Refraction was between +3 and 3 diopters in all patients with MIDD, except in 1 patient with severe astigmatism. No ocular nerve atrophy was observed. Neuromuscular Disorders Muscular disorders were observed in 22 of 51 documented cases of MIDD (43.1%). Patients reported painful muscle weakness that affected lower limbs during prolonged walking or running. In the 6 patients in whom it was performed, muscle biopsy showed ragged-red fibers typical of mitochondrial myopathy (Figure 2). Eight patients had cardiomyopathy; echocardiography showed typical left ventricular hypertrophy in all. Symptoms of congestive heart failure were present in 2 patients. A preexcitation syndrome (WolffParkinsonWhite) was present in 2 patients, and atrial fibrillation was seen in 1 patient. Mitochondrial myopathy was present in 4 of 8 patients with cardiomyopathy. Coronary heart disease was present in 4 of 54 patients (7%), and clinically significant peripheral artery lesions were observed in 2 of 54 patients. Hypertension was present in 15 of 53 patients (28%). Ocular motor palsy was present in 2 patients from the same family, and cerebellar ataxia with cerebellar atrophy on nuclear magnetic resonance imaging was seen in another patient. Atrophic changes in the brain were observed in the 4 other patients who underwent nuclear magnetic resonance imaging. Neuropsychiatric disturbances were present in 9 of 51 patients (18%). The

A mitochondrial origin for frontotemporal dementia and amyotrophic lateral sclerosis through CHCHD10 involvement

Mitochondrial DNA instability disorders are responsible for a large clinical spectrum, among which amyotrophic lateral sclerosis-like symptoms and frontotemporal dementia are extremely rare. We report a large family with a late-onset phenotype including motor neuron disease, cognitive decline resembling frontotemporal dementia, cerebellar ataxia and myopathy. In all patients, muscle biopsy showed ragged-red and cytochrome c oxidase-negative fibres with combined respiratory chain deficiency and abnormal assembly of complex V. The multiple mitochondrial DNA deletions found in skeletal muscle revealed a mitochondrial DNA instability disorder. Patient fibroblasts present with respiratory chain deficiency, mitochondrial ultrastructural alterations and fragmentation of the mitochondrial network. Interestingly, expression of matrix-targeted photoactivatable GFP showed that mitochondrial fusion was not inhibited in patient fibroblasts. Using whole-exome sequencing we identified a missense mutation (c.176C>T; p.Ser59Leu) in the CHCHD10 gene that encodes a coiled-coil helix coiled-coil helix protein, whose function is unknown. We show that CHCHD10 is a mitochondrial protein located in the intermembrane space and enriched at cristae junctions. Overexpression of a CHCHD10 mutant allele in HeLa cells led to fragmentation of the mitochondrial network and ultrastructural major abnormalities including loss, disorganization and dilatation of cristae. The observation of a frontotemporal dementia-amyotrophic lateral sclerosis phenotype in a mitochondrial disease led us to analyse CHCHD10 in a cohort of 21 families with pathologically proven frontotemporal dementia-amyotrophic lateral sclerosis. We identified the same missense p.Ser59Leu mutation in one of these families. This work opens a novel field to explore the pathogenesis of the frontotemporal dementia-amyotrophic lateral sclerosis clinical spectrum by showing that mitochondrial disease may be at the origin of some of these phenotypes.

MSH4 acts in conjunction with MLH1 during mammalian meiosis

MSH4 is a meiosis‐specific MutS homolog. In yeast, it is required for reciprocal recombination and proper segregation of homologous chromosomes at meiosis I. MLH1 (MutL homolog 1) facilitates both mismatch repair and crossing over during meiosis in yeast. Germ‐line mutations in the MLH1 human gene are responsible for hereditary nonpolyposis cancer, but the analysis of MLH1‐deficient mice has revealed that MLH1 is also required for reciprocal recombination in mammals. Here we show that hMSH4 interacts with hMLH1. The two proteins are coimmunoprecipitated regardless of the presence of DNA or ATP, suggesting that the interaction does not require the binding of MSH4 to DNA. The domain of hMSH4 responsible for the interaction is in the amino‐terminal part of the protein whereas the region that contains the ATP binding site and helix‐turn‐helix motif does not bind to hMLH1. Immunolocalization analysis shows that MSH4 is present at sites along the synaptonemal complex as soon as homologous chromosomes synapse. The number of MSH4 foci decreases gradually as pachynema progresses. During this transition, MLH1 foci begin to appear and colocalize with MSH4. These results suggest that MSH4 is first required for chromosome synapsis and that this MutS homologue is involved later with MLH1 in meiotic reciprocal recombination.–Santucci‐Darmanin, S., Walpita, D., Lespinasse, F., Desnuelle, C., Ashley, T., Paquis‐Flucklinger, V. MSH4 acts in conjunction with MLH1 during mammalian meiosis. FASEB J. 14, 1539–1547 (2000)

Neurologic features and genotype-phenotype correlation in Wolfram syndrome.

Wolfram syndrome (WS) is a rare neurodegenerative disorder characterized by juvenile‐onset diabetes mellitus and optic atrophy. Our aim was to describe the nature and the frequency of the neurologic manifestations, which had been poorly studied until now.

Screening of CHCHD10 in a French cohort confirms the involvement of this gene in frontotemporal dementia with amyotrophic lateral sclerosis patients

Mutations in the CHCHD10 gene have been recently identified in a large family with a complex phenotype variably associating frontotemporal dementia (FTD) with amyotrophic lateral sclerosis (ALS), cerebellar ataxia, myopathy, and hearing impairment. CHCHD10 encodes a protein located in the mitochondrial intermembrane space and is likely involved in mitochondrial genome stability and maintenance of cristae junctions. However, the exact contribution of CHCHD10 in FTD and ALS diseases spectrum remains unknown. In this study, we evaluated the frequency of CHCHD10 mutations in 115 patients with FTD and FTD-ALS phenotypes. We identified 2 heterozygous variants in 3 unrelated probands presenting FTD and ALS, characterized by early and predominant bulbar symptoms. This study demonstrates the implication of CHCHD10 in FTD and ALS spectrum. Although the frequency of mutations is low in this series (2.6%), our work suggests that CHCHD10 mutations should be searched particularly when bulbar symptoms are present at onset.

A common pattern of brain MRI imaging in mitochondrial diseases with complex I deficiency

Objective To identify a consistent pattern of brain MRI imaging in primary complex I deficiency. Complex I deficiency, a major cause of respiratory chain dysfunction, accounts for various clinical presentations, including Leigh syndrome. Human complex I comprises seven core subunits encoded by mitochondrial DNA (mtDNA) and 38 core subunits encoded by nuclear DNA (nDNA). Moreover, its assembly requires six known and many unknown assembly factors. To date, no correlation between genotypes and brain MRI phenotypes has been found in complex I deficiencies. Design and subjects The brain MRIs of 30 patients carrying known mutation(s) in genes involved in complex I were retrospectively collected and compared with the brain MRIs of 11 patients carrying known mutations in genes involved in the pyruvate dehydrogenase (PDH) complex as well as 10 patients with MT-TL1 mutations. Results All complex I deficient patients showed bilateral brainstem lesions (30/30) and 77% (23/30) showed anomalies of the putamen. Supratentorial stroke-like lesions were only observed in complex I deficient patients carrying mtDNA mutations (8/19) and necrotising leucoencephalopathy in patients with nDNA mutations (4/5). Conversely, the isolated stroke-like images observed in patients with MT-TL1 mutations, or the corpus callosum malformations observed in PDH deficient patients, were never observed in complex I deficient patients. Conclusion A common pattern of brain MRI imaging was identified with abnormal signal intensities in brainstem and subtentorial nuclei with lactate peak as a clue of complex I deficiency. Combining clinico-biochemical data with brain imaging may therefore help orient genetic studies in complex I deficiency.

Refractory epilepsy and mitochondrial dysfunction due to GM3 synthase deficiency

We report two children, born from consanguineous parents, who presented with early-onset refractory epilepsy associated with psychomotor delay, failure to thrive, blindness and deafness. Polarographic and spectrophotometric analyses in fibroblasts and liver revealed a respiratory chain (RC) dysfunction. Surprisingly, we identified a homozygous nonsense mutation in the GM3 synthase gene by using exome sequencing. GM3 synthase catalyzes the formation of GM3 ganglioside from lactosylceramide, which is the first step in the synthesis of complex ganglioside species. Mass spectrometry analysis revealed that the complete absence of GM3 ganglioside and its biosynthetic derivatives was associated with an upregulation of the alternative globoside pathway in fibroblasts. The accumulation of Gb3 and Gb4 globosides likely has a role in RC dysfunction and in the decrease of mitochondrial membrane potential leading to apoptosis, which we observed in fibroblasts. We show for the first time that GM3 synthase deficiency, responsible for early-onset epilepsy syndrome, leads to a secondary RC dysfunction. Our study highlights the role of secondary mitochondrial disorders that can interfere with the diagnosis and the evolution of other metabolic diseases.

Identification of novel mutations in WFS1 and genotype-phenotype correlation in Wolfram syndrome.

Mutations in the WFS1 gene have been reported in Wolfram syndrome (WS), an autosomal recessive disorder defined by early onset of diabetes mellitus (DM) and progressive optic atrophy. Because of the low prevalence of this syndrome and the recent identification of the WFS1 gene, few data are available concerning the relationships between clinical and molecular aspects of the disease. Here, we describe 12 patients from 11 families with WS. We report on eight novel (A214fsX285, L293fsX303, P346L, I427S, V503fsX517, R558C, S605fsX711, P838L) and seven previously reported mutations. We also looked for genotype–phenotype correlation both in patients included in this study and 19 additional WS patients that were previously reported. Subsequently, we performed a systematic review and meta‐analysis of five published clinical and molecular studies of WFS1 for genotype–phenotype correlation, combined with our current French patient group for a total of 96 patients. The presence of two inactivating mutations was shown to predispose to an earlier age of onset of both DM and optic atrophy. Moreover, the clinical expression of WS was more complete and occurred earlier in patients harboring no missense mutation.

Reply: CHCHD10 mutations in Italian patients with sporadic amyotrophic lateral sclerosis

Sir, We read with interest the paper recently published in Brain (Bannwarth et al. , 2014) reporting a mutation in CHCHD10 (c.176C > T, p.Ser59Leu) in familial amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD). Interestingly, the mutated patients also showed signs of muscle mitochondrial pathology consisting of cytochrome c oxidase (COX)-negative fibres, ultrastructural mitochondrial abnormalities, impaired respiratory chain activity, and altered mitochondrial DNA (mtDNA) maintenance (multiple deletions). Additional CHCHD10 mutations were reported by Muller et al. (2014) who identified the c.44C > A variant (p.Arg15Leu) in two German familial ALS cases and the variant c.197C > A (p.Gly66Val) in a Finnish patient with familial motor neuron disease with predominant lower motor neuron involvement. Chaussenot et al. (2014) screened CHCHD10 in a cohort of 80 French patients with sporadic FTD-ALS, disclosing the p.Pro34Ser mutation in two independent subjects. Mutated patients also featured sensorineural hypoacusia typically associated with mitochondrial disease, but mitochondrial dysfunction was not formally documented. Finally Johnson et al. (2014) investigated 85 independent North American cases with familial ALS, reporting the p.Arg15Leu mutation in three of them. Here we report clinical, biochemical, and molecular findings of an Italian patient affected by sporadic early-onset ALS and muscle mitochondrial pathology associated with a novel CHCHD10 mutation. Moreover we investigated a cohort of Italian sporadic ALS patients, supporting the modest, but not negligible causative role of CHCHD10 variations. We previously found severe histochemical COX deficiency in 7 of 50 muscle biopsies from patients with sporadic ALS (Crugnola et al. , 2010). Sequence analysis of ALS-related genes was negative in all patients but two (SOD1: p.Gln22Arg and TDP43: p.Ala382Thr). We sequenced CHCHD10 coding regions in the remaining five patients, disclosing the novel heterozygous transition c.239C > T in exon 2, resulting in the amino acid change p.Pro80Leu, in one of them (Fig. 1 …