Annabelle Chaussenot

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.

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.

Prevalence of rare mitochondrial DNA mutations in mitochondrial disorders

Abstract Background Mitochondrial DNA (mtDNA) diseases are rare disorders whose prevalence is estimated around 1 in 5000. Patients are usually tested only for deletions and for common mutations of mtDNA which account for 5–40% of cases, depending on the study. However, the prevalence of rare mtDNA mutations is not known. Methods We analysed the whole mtDNA in a cohort of 743 patients suspected of manifesting a mitochondrial disease, after excluding deletions and common mutations. Both heteroplasmic and homoplasmic variants were identified using two complementary strategies (Surveyor and MitoChip). Multiple correspondence analyses followed by hierarchical ascendant cluster process were used to explore relationships between clinical spectrum, age at onset and localisation of mutations. Results 7.4% of deleterious mutations and 22.4% of novel putative mutations were identified. Pathogenic heteroplasmic mutations were more frequent than homoplasmic mutations (4.6% vs 2.8%). Patients carrying deleterious mutations showed symptoms before 16 years of age in 67% of cases. Early onset disease (<1 year) was significantly associated with mutations in protein coding genes (mainly in complex I) while late onset disorders (>16 years) were associated with mutations in tRNA genes. MTND5 and MTND6 genes were identified as ‘hotspots’ of mutations, with Leigh syndrome accounting for the large majority of associated phenotypes. Conclusions Rare mitochondrial DNA mutations probably account for more than 7.4% of patients with respiratory chain deficiency. This study shows that a comprehensive analysis of mtDNA is essential, and should include young children, for an accurate diagnosis that is now accessible with the development of next generation sequencing technology.

Fatal heart failure associated with CoQ10 and multiple OXPHOS deficiency in a child with propionic acidemia.

The role of a secondary respiratory chain deficiency as an additional mechanism to intoxication, leading to development of long-term energy-dependent complications, has been recently suggested in patients with propionic acidemia (PA). We show for the first time a coenzyme Q(10) (CoQ(10)) functional defect accompanied by a multiple organ oxidative phosphorylation (OXPHOS) deficiency in a child who succumbed to acute heart failure in the absence of metabolic stress. Quinone-dependent activities in the liver (complex I+III, complex II+III) were reduced, suggesting a decrease in electron transfer related to the quinone pool. The restoration of complex II+III activity after addition of exogenous ubiquinone to the assay system suggests CoQ(10) deficiency. Nevertheless, we disposed of insufficient material to perform direct measurement of CoQ(10) content in the patients liver. Death occurred before biochemical diagnosis of OXPHOS deficiency could be made. However, this case highlights the usefulness of rapidly identifying CoQ(10) defects secondary to PA since this OXPHOS disorder has a good treatment response which could improve heart complications or prevent their appearance. Nevertheless, further studies will be necessary to determine whether CoQ(10) treatment can be useful in PA complications linked to CoQ(10) deficiency.

A novel CISD2 mutation associated with a classical Wolfram syndrome phenotype alters Ca2+ homeostasis and ER-mitochondria interactions

&NA; Wolfram syndrome (WS) is a progressive neurodegenerative disease characterized by early‐onset optic atrophy and diabetes mellitus, which can be associated with more extensive central nervous system and endocrine complications. The majority of patients harbour pathogenic WFS1 mutations, but recessive mutations in a second gene, CISD2, have been described in a small number of families with Wolfram syndrome type 2 (WFS2). The defining diagnostic criteria for WFS2 also consist of optic atrophy and diabetes mellitus, but unlike WFS1, this phenotypic subgroup has been associated with peptic ulcer disease and an increased bleeding tendency. Here, we report on a novel homozygous CISD2 mutation (c.215A > G; p.Asn72Ser) in a Moroccan patient with an overlapping phenotype suggesting that Wolfram syndrome type 1 and type 2 form a continuous clinical spectrum with genetic heterogeneity. The present study provides strong evidence that this particular CISD2 mutation disturbs cellular Ca2+ homeostasis with enhanced Ca2+ flux from the ER to mitochondria and cytosolic Ca2+ abnormalities in patient‐derived fibroblasts. This Ca2+ dysregulation was associated with increased ER‐mitochondria contact, a swollen ER lumen and a hyperfused mitochondrial network in the absence of overt ER stress. Although there was no marked alteration in mitochondrial bioenergetics under basal conditions, culture of patient‐derived fibroblasts in glucose‐free galactose medium revealed a respiratory chain defect in complexes I and II, and a trend towards decreased ATP levels. Our results provide important novel insight into the potential disease mechanisms underlying the neurodegenerative consequences of CISD2 mutations and the subsequent development of multisystemic disease.

Quantitative multiplex PCR of short fluorescent fragments for the detection of large intragenic POLG rearrangements in a large French cohort.

Polymerase gamma (POLG) is the gene most commonly involved in mitochondrial disorders with mitochondrial DNA instability and causes a wide range of diseases with recessive or dominant transmission. More than 170 mutations have been reported. Most of them are missense mutations, although nonsense mutations, splice-site mutations, small deletions and insertions have also been identified. However, to date, only one large-scale rearrangement has been described in a child with Alpers syndrome. Below, we report a large cohort of 160 patients with clinical, molecular and/or biochemical presentation suggestive of POLG deficiency. Using sequencing, we identified POLG variants in 22 patients (18 kindreds) including five novel pathogenic mutations. Two patients with novel mutations had unusual clinical presentation: the first exhibited an isolated ataxic neuropathy and the second was a child who presented with endocrine signs. We completed the sequencing step by quantitative multiplex PCR of short fluorescent fragments (QMPSF) analysis in 37 patients with either only one POLG heterozygous variant or a family history suggesting a dominant transmission. We identified a large intragenic deletion encompassing part of intron 21 and exon 22 of POLG in a child with refractory epilepsia partialis continua. In conclusion, we describe the first large French cohort of patients with POLG mutations, expanding the wide clinical and molecular spectrum observed in POLG disease. We confirm that large deletions in the POLG gene are rare events and we highlight the importance of QMPSF in patients with a single heterozygous POLG mutation, particularly in severe infantile phenotypes.

Assembly defects of multiple respiratory chain complexes in a child with cardiac hypertrophy associated with a novel ACAD9 mutation

Patients carrying Acyl-CoA dehydrogenase 9 (ACAD9) mutations reported to date mainly present with severe hypertrophic cardiomyopathy and isolated complex I (CI) dysfunction. Here we report a novel ACAD9 mutation in a young girl presenting with severe hypertrophic cardiomyopathy, isolated CI deficiency and interestingly multiple respiratory chain complexes assembly defects. We show that ACAD9 analysis has to be performed in first intention in patients presenting with cardiac hypertrophy even in the presence of multiple assembly defects.

Targeted next generation sequencing with an extended gene panel does not impact variant detection in mitochondrial diseases

BackgroundSince the advent of next generation sequencing (NGS), several studies have tried to evaluate the relevance of targeted gene panel sequencing and whole exome sequencing for molecular diagnosis of mitochondrial diseases. The comparison between these different strategies is extremely difficult. A recent study analysed a cohort of patients affected by a mitochondrial disease using a NGS approach based on a targeted gene panel including 132 genes. This strategy led to identify the causative mutations in 15.2% of cases. The number of novel genes responsible for respiratory chain deficiency increases very rapidly.MethodsIn order to determine the impact of larger panels used as a first screening strategy on molecular diagnosis success, we analysed a cohort of 80 patients affected by a mitochondrial disease with a first mitochondrial DNA (mtDNA) NGS screening and secondarily a targeted mitochondrial panel of 281 nuclear genes.ResultsPathogenic mtDNA abnormalities were identified in 4.1% (1/24) of children and 25% (14/56) of adult patients. The remaining 65 patients were analysed with our targeted mitochondrial panel and this approach enabled us to achieve an identification rate of 21.7% (5/23) in children versus 7.1% (3/42) in adults.ConclusionsOur results confirm that larger gene panels do not improve diagnostic yield of mitochondrial diseases due to (i) their very high genetic heterogeneity, (ii) the ongoing discovery of novel genes and (iii) mutations in genes apparently not related to mitochondrial function that lead to secondary respiratory chain deficiency.