Mike Gerards

Clinical expression of Leber hereditary optic neuropathy is affected by the mitochondrial DNA-haplogroup background.

Leber hereditary optic neuropathy (LHON) is due primarily to one of three common point mutations of mitochondrial DNA (mtDNA), but the incomplete penetrance implicates additional genetic or environmental factors in the pathophysiology of the disorder. Both the 11778G-->A and 14484T-->C LHON mutations are preferentially found on a specific mtDNA genetic background, but 3460G-->A is not. However, there is no clear evidence that any background influences clinical penetrance in any of these mutations. By studying 3,613 subjects from 159 LHON-affected pedigrees, we show that the risk of visual failure is greater when the 11778G-->A or 14484T-->C mutations are present in specific subgroups of haplogroup J (J2 for 11778G-->A and J1 for 14484T-->C) and when the 3460G-->A mutation is present in haplogroup K. By contrast, the risk of visual failure is significantly less when 11778G-->A occurs in haplogroup H. Substitutions on MTCYB provide an explanation for these findings, which demonstrate that common genetic variants have a marked effect on the expression of an ostensibly monogenic mtDNA disorder.

Riboflavin-responsive oxidative phosphorylation complex I deficiency caused by defective ACAD9: new function for an old gene

Mitochondrial complex I deficiency is the most common oxidative phosphorylation defect. Mutations have been detected in mitochondrial and nuclear genes, but the genetics of many patients remain unresolved and new genes are probably involved. In a consanguineous family, patients presented easy fatigability, exercise intolerance and lactic acidosis in blood from early childhood. In muscle, subsarcolemmal mitochondrial proliferation and a severe complex I deficiency were observed. Exercise intolerance and complex I activity was improved by a supplement of riboflavin at high dosage. Homozygosity mapping revealed a candidate region on chromosome three containing six mitochondria-related genes. Four genes were screened for mutations and a homozygous substitution was identified in ACAD9 (c.1594 C>T), changing the highly conserved arginine-532 into tryptophan. This mutation was absent in 188 ethnically matched controls. Protein modelling suggested a functional effect due to the loss of a stabilizing hydrogen bond in an α-helix and a local flexibility change. To test whether the ACAD9 mutation caused the complex I deficiency, we transduced fibroblasts of patients with wild-type and mutant ACAD9. Wild-type, but not mutant, ACAD9 restored complex I activity. An unrelated patient with the same phenotype was compound heterozygous for c.380 G>A and c.1405 C>T, changing arginine-127 into glutamine and arginine-469 into tryptophan, respectively. These amino acids were highly conserved and the substitutions were not present in controls, making them very probably pathogenic. Our data support a new function for ACAD9 in complex I function, making this gene an important new candidate for patients with complex I deficiency, which could be improved by riboflavin treatment.

Exome sequencing reveals a novel Moroccan founder mutation in SLC19A3 as a new cause of early-childhood fatal Leigh syndrome

Leigh syndrome is an early onset, often fatal progressive neurodegenerative disorder caused by mutations in the mitochondrial or nuclear DNA. Until now, mutations in more than 35 genes have been reported to cause Leigh syndrome, indicating an extreme genetic heterogeneity for this disorder, but still only explaining part of the cases. The possibility of whole exome sequencing enables not only mutation detection in known candidate genes, but also the identification of new genes associated with Leigh syndrome in small families and isolated cases. Exome sequencing was combined with homozygosity mapping to identify the genetic defect in a Moroccan family with fatal Leigh syndrome in early childhood and specific magnetic resonance imaging abnormalities in the brain. We detected a homozygous nonsense mutation (c.20C>A; p.Ser7Ter) in the thiamine transporter SLC19A3. In vivo overexpression of wild-type SLC19A3 showed an increased thiamine uptake, whereas overexpression of mutant SLC19A3 did not, confirming that the mutation results in an absent or non-functional protein. Seventeen additional patients with Leigh syndrome were screened for mutations in SLC19A3 using conventional Sanger sequencing. Two unrelated patients, both from Moroccan origin and one from consanguineous parents, were homozygous for the same p.Ser7Ter mutation. One of these patients showed the same MRI abnormalities as the patients from the first family. Strikingly, patients receiving thiamine had an improved life-expectancy. One patient in the third family deteriorated upon interruption of the thiamine treatment and recovered after reinitiating. Although unrelated, all patients came from the province Al Hoceima in Northern Morocco. Based on the recombination events the mutation was estimated to have occurred 1250-1750 years ago. Our data shows that SLC19A3 is a new candidate for mutation screening in patients with Leigh syndrome, who might benefit from high doses of thiamine and/or biotin. Especially, Moroccan patients with Leigh syndrome should be tested for the c.20C>A founder mutation in SLC19A3.

Nonsense mutations in CABC1/ADCK3 cause progressive cerebellar ataxia and atrophy

Hereditary ataxias are genetic disorders characterized by uncoordinated gait and often poor coordination of hands, speech, and eye movements. Frequently, atrophy of the cerebellum occurs. Many ataxias are autosomal dominant, but autosomal recessive (AR) disease occurs as well. Homozygosity mapping in a consanguineous family with three affected children with progressive cerebellar ataxia and atrophy revealed a candidate locus on chromosome 1, containing the CABC1/ADCK3 (the chaperone, ABC1 activity of bc1 complex homologue) gene. CABC1/ADCK3 is the homologue of the yeast Coq8 gene, which is involved in the ubiquinone biosynthesis pathway. Mutation analysis of this gene showed a homozygous nonsense mutation (c.1042C>T, p.R348X). Eight additional patients with AR cerebellar ataxia and atrophy were screened for mutations in the CABC1/ADCK3 gene. One patient was compound heterozygous for the same c.1042C>T mutation and a second nonsense mutation (c.1136T>A, p.L379X). Both mutations created a premature stop codon, triggering nonsense mediated mRNA decay as the pathogenic mechanism. We found no evidence of a Dutch founder for the c.1042C>T mutation in AR ataxia. We report here the first nonsense mutations in CABC1 that most likely lead to complete absence of a functional CABC1 protein. Our results indicate that CABC1 is an important candidate for mutation analysis in progressive cerebellar ataxia and atrophy on MRI to identify those patients, who may benefit from CoQ10 treatment.

Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome

Background Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. Methods and results A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30–40% of mature complex I present in patients and 70–90% in carriers. Conclusions A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.

Chip-based mtDNA mutation screening enables fast and reliable genetic diagnosis of OXPHOS patients

Purpose: Oxidative phosphorylation is under dual genetic control of the nuclear and the mitochondrial DNA (mtDNA). Oxidative phosphorylation disorders are clinically and genetically heterogeneous, which makes it difficult to determine the genetic defect, and symptom-based protocols which link clinical symptoms directly to a specific gene or mtDNA mutation are falling short. Moreover, approximately 25% of the pediatric patients with oxidative phosphorylation disorders is estimated to have mutations in the mtDNA and a standard screening approach for common mutations and deletions will only explain part of these cases. Therefore, we tested a new CHIP-based screening method for the mtDNA.Methods: MitoChip (Affymetrix) resequencing was performed on three test samples and on 28 patient samples.Results: Call rates were 94% on average and heteroplasmy detection levels varied from 5–50%. A genetic diagnosis can be made in almost one-quarter of the patients at a potential output of 8 complete mtDNA sequences every 4 days. Moreover, a number of potentially pathogenic unclassified variants (UV) were detected.Conclusions: The availability of long-range PCR protocols and the predominance of single nucleotide substitutions in the mtDNA make the resequencing CHIP a very fast and reliable method to screen the complete mtDNA for mutations.

Leigh syndrome: Resolving the clinical and genetic heterogeneity paves the way for treatment options.

Leigh syndrome is a progressive neurodegenerative disorder, affecting 1 in 40,000 live births. Most patients present with symptoms between the ages of three and twelve months, but adult onset Leigh syndrome has also been described. The disease course is characterized by a rapid deterioration of cognitive and motor functions, in most cases resulting in death due to respiratory failure. Despite the high genetic heterogeneity of Leigh syndrome, patients present with identical, symmetrical lesions in the basal ganglia or brainstem on MRI, while additional clinical manifestations and age of onset varies from case to case. To date, mutations in over 60 genes, both nuclear and mitochondrial DNA encoded, have been shown to cause Leigh syndrome, still explaining only half of all cases. In most patients, these mutations directly or indirectly affect the activity of the mitochondrial respiratory chain or pyruvate dehydrogenase complex. Exome sequencing has accelerated the discovery of new genes and pathways involved in Leigh syndrome, providing novel insights into the pathophysiological mechanisms. This is particularly important as no general curative treatment is available for this devastating disorder, although several recent studies imply that early treatment might be beneficial for some patients depending on the gene or process affected. Timely, gene-based personalized treatment may become an important strategy in rare, genetically heterogeneous disorders like Leigh syndrome, stressing the importance of early genetic diagnosis and identification of new genes/pathways. In this review, we provide a comprehensive overview of the most important clinical manifestations and genes/pathways involved in Leigh syndrome, and discuss the current state of therapeutic interventions in patients.

Novel pathogenic SLC25A46 splice-site mutation causes an optic atrophy spectrum disorder

The inherited optic neuropathies comprise a group of genetically heterogeneous disorders causing optic nerve dysfunction. In some cases, optic neuropathies are associated with cerebellar atrophy which mainly affects the vermis. Here, we describe a Moroccan girl of consanguineous parents with optic atrophy and cerebellar atrophy. Exome sequencing revealed a novel homozygous mutation (c.283+3G>T) in the donor splice site for exon 1 of SLC25A46. RNA analysis revealed that an alternative splice site within exon 1 was used leading to a premature termination codon within exon 2. SLC25A46 mRNA expression showed there is no wild‐type transcript present in the patient and the mutant transcript does not undergo nonsense‐mediated mRNA decay. Futhermore, we observed c.283+3G>T SLC25A46 mutation induces mitochondrial fragmentation. An additional 10 patients with optic atrophy and cerebellar atrophy, which were negative for mtDNA and OPA1 variants, were tested for pathogenic mutations in the SLC25A46 gene. However, no additional variants were identified. Our findings confirm the recent report of pathogenic SLC25A46 mutations as a novel cause for optic atrophy spectrum disorder.

Inherited mitochondrial variants are not a major cause of age-related hearing impairment in the European population.

Mitochondrial DNA (mtDNA) mutations have been implicated in various age-related diseases. To further clarify the role of mtDNA variants in age-related hearing impairment (ARHI), we determined the DNA sequence of the entire mitochondrial genome of 400 individuals using the Affymetrix Human Mitochondrial Resequencing Array. These were the 200 worst hearing and the 200 best hearing from a collection of 947 Belgian samples. We performed association tests with individual mitochondrial variants, comparison of the mutation load, and association with European haplogroups and their interaction with environmental risk factors. We also tested the influence of rare variants on ARHI. None of these tests showed any association with ARHI.

Pathogenic CWF19L1 variants as a novel cause of autosomal recessive cerebellar ataxia and atrophy.

Autosomal recessive cerebellar ataxia (ARCA) is a group of neurological disorders characterized by degeneration or abnormal development of the cerebellum and spinal cord. ARCA is clinically and genetically highly heterogeneous, with over 20 genes involved. Exome sequencing of a girl with ARCA from non-consanguineous Dutch parents revealed two pathogenic variants c.37G>C; p.D13H and c.946A>T; p.K316* in CWF19L1, a gene with an unknown function, recently reported to cause ARCA in a Turkish family. Sanger sequencing showed that the c.37G>C variant was inherited from the father and the c.946A>T variant from the mother. Pathogenicity was based on the damaging effect on protein function as the c.37G>C variant changed the highly conserved, negatively charged aspartic acid to the positively charged histidine and the c.946A>T variant introduced a premature stop codon. In addition, 27 patients with ARCA were tested for pathogenic variants in CWF19L1, however, no pathogenic variants were identified. Our data confirm CWF19L1 as a novel but rare gene causing ARCA.