Sylvie Bannwarth

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.

Molecular analysis of ANT1, TWINKLE and POLG in patients with multiple deletions or depletion of mitochondrial DNA by a dHPLC-based assay

ANT1, TWINKLE and POLG genes affect mtDNA stability and are involved in autosomal dominant PEO, while mutations in POLG are responsible for numerous clinical presentations, including autosomal recessive PEO, sensory ataxic neuropathy, dysarthria and ophthalmoparesis (SANDO), spino-cerebellar ataxia and epilepsy (SCAE) or Alpers syndrome. In this study, we report on the mutational analysis of ANT1, TWINKLE and POLG genes in 15 unrelated patients, using a dHPLC-based protocol. This series of patients illustrates the large array of clinical presentations associated with mtDNA stability defects, ranging from isolated benign PEO to fatal Alpers syndrome. A total of seven different mutations were identified in six of 15 patients (40%). Six different recessive mutations were found in POLG, one in TWINKLE while no mutation was identified in ANT1. Among the POLG mutations, three are novel and include two missense and one frameshift changes. Seventeen neutral changes and polymorphisms were also identified, including four novel neutral polymorphisms. Overall, this study illustrates the variability of phenotypes associated with mtDNA stability defects, increases the mutational spectrum of POLG variants and provides an efficient and reliable detection protocol for ANT1, TWINKLE and POLG mutational screening.

Rapid identification of unknown heteroplasmic mutations across the entire human mitochondrial genome with mismatch-specific Surveyor Nuclease

Mitochondrial DNA (mtDNA) mutations are responsible for mitochondrial diseases in numerous patients. But, until now, no rapid method was available for the identification of unknown deleterious point mutations. Here, we describe a new strategy for the rapid identification of heteroplasmic mtDNA mutations using mismatch-specific Surveyor Nuclease. This protocol involves the following three steps: (i) PCR amplification of the entire human mitochondrial genome in 17 overlapping fragments; (ii) localization of mtDNA mismatch(es) after digestion of the 17 amplicons by Surveyor Nuclease; and (iii) identification of the mutation by sequencing the region containing the mismatch. This Surveyor Nuclease-based strategy allows a systematic screening of the entire mtDNA to identify a mutation within 2 days. It represents an important diagnostic approach for mitochondrial diseases that can be routinely used in molecular diagnostic laboratories.

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.

The human MSH5 (MutSHomolog 5) protein localizes to mitochondria and protects the mitochondrial genome from oxidative damage.

MutS homologs play a central role in maintaining genetic stability. We show that MSH5 (MutSHomolog 5) is localized into the mitochondria of germ and somatic cells. This protein binds to mtDNA and interacts with the Twinkle helicase and the DNA polymerase gamma. hMSH5 stimulates mtDNA repair in response to DNA damage induced by oxidative stress. Furthermore, we observed a subsarcolemmal accumulation of hMSH5 in COX negative muscle fibers of patients presenting a mitochondrial myopathy. We report a novel localization for hMSH5 suggesting that this protein may have functions other than those known in meiotic recombination.

A Novel Unstable Mutation in Mitochondrial DNA Responsible for Maternally Inherited Diabetes and Deafness

OBJECTIVE The m.3243A>G mutation in mitochondrial DNA (mtDNA) is responsible for maternally inherited diabetes and deafness (MIDD). Other mtDNA mutations are extremely rare. RESEARCH DESIGN AND METHODS We studied a patient presenting with diabetes and deafness who does not carry the m.3243A>G mutation. RESULTS We identified a deficiency of respiratory chain complex I in the patient’s fibroblasts. mtDNA sequencing revealed a novel mutation that corresponds to an insertion of one or two cytosine residues in the coding region of the MT-ND6 gene (m.14535_14536insC or CC), leading to premature stop codons. This heteroplasmic mutation is unstable in the patient’s somatic tissues. CONCLUSIONS We describe for the first time an unstable mutation in a mitochondrial gene coding for a complex I subunit, which is responsible for the MIDD phenotype. This mutation is likely favored by the m.14530T>C polymorphism, which is homoplasmic and leads to the formation of an 8-bp polyC tract responsible for genetic instability.

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.

Detection of Low Levels of the Mitochondrial tRNALeu(UUR) 3243A>G Mutation in Blood Derived from Patients with Diabetes

AbstractBackground: Mutations in the human mitochondrial genome have been suspected to play a significant role in the etiological development of mitochondrial diabetes. Detection of the 3243A>G mutation in the mitochondrial transfer RNALeu(UUR) gene (MTTL1), especially at low heteroplasmy levels, is highly desirable since it facilitates the diagnosis and subsequent management of the disease. The proportions of mutant mitochondrial DNA (mtDNA) can vary between tissues and are usually significantly higher in muscle than in blood, but muscle biopsies from patients with diabetes are rarely available. Methods: Here, we describe a technique that can not only determine the presence of MTTL1 3243A>G, but can also estimate the percentage of mutant DNA. The technique is based on the use of the WAVE® system for the high-performance liquid chromatography (HPLC)-mediated analysis of mutation-specific restriction fragments derived from mutant PCR amplicons. PCR amplicon restriction fragment analysis by HPLC (PARFAH) can also be used for the detection of other mutations. Results: This PARFAH analytical approach led to the discovery of the 3243A>G mutation in blood samples from a series of patients who had initially been reported to lack the mutation, even though matrilineal relatives had been shown to harbor the mutation associated with maternally inherited diabetes and deafness (MIDD) or mitochondrial myopathy encephalopathy lactic acidosis stroke-like episodes (MELAS) phenotypes. We have established that the PARFAH method can reliably detect as little as 1% mutant DNA in a sample, which would normally be missed by commonly used gel electrophoresis or sequencing methods. Conclusions: The PARFAH method not only provides a sensitive, high-throughput, and cost-effective strategy for the detection of low levels of mtDNA mutations in peripheral tissues, but also facilitates the estimation of the percentage of mutant DNA in the sample. The fact that samples can be readily obtained from peripheral tissues in many cases will avoid the need for invasive muscle biopsies. Our ability to detect low levels of mtDNA mutations in blood samples of carriers will allow us to reassess the prevalence of the MTTL1 3243A>G mutation in patients with diabetes.

Inactivation of Pif1 helicase causes a mitochondrial myopathy in mice

Mutations in genes coding for mitochondrial helicases such as TWINKLE and DNA2 are involved in mitochondrial myopathies with mtDNA instability in both human and mouse. We show that inactivation of Pif1, a third member of the mitochondrial helicase family, causes a similar phenotype in mouse. pif1-/- animals develop a mitochondrial myopathy with respiratory chain deficiency. Pif1 inactivation is responsible for a deficiency to repair oxidative stress-induced mtDNA damage in mouse embryonic fibroblasts that is improved by complementation with mitochondrial isoform mPif1(67). These results open new perspectives for the exploration of patients with mtDNA instability disorders.