A novel mtDNA point mutation in tRNAVal is associated with hypertrophic cardiomyopathy and MELAS

Background. Pathological mutations of mitochondrial (mt) DNA may cause specific diseases such as cardiomyopathies or hearing loss, or syndromes such as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode (MELAS) syndrome. We describe a novel mtDNA mutation in a patient with severe hypertrophic cardiomyopathy associated with MELAS. The familial phenotype included 1) hypertrophic cardiomyopathy and MELAS, 2) clinically mild cardiac hypertrophy, and 3) deafness. Methods. The proband and her first degree relatives underwent echo and electrocardiograms, and biochemical tests. Magnetic resonance imaging of the brain was performed in the proband. mtDNA was fully analyzed by sequencing. DNA purification, polymerase chain reaction and direct automated sequencing were performed following standard procedures. Heteroplasmy of the novel mutation was quantified by densitometric analysis. Results. A novel G1644A transition affecting the tRNAVal was identified in the proband and maternal relatives. The mutation has been interpreted as pathological because the G at the 1644 position is a highly conserved base, is heteroplasmic with higher levels of mutant DNA in the proband than in the relatives, is located in the unique tRNAVal, is very close to a mutation described as causative of MELAS, and finally has not been found in 100 healthy controls. Conclusions. Although it is rare for patients with MELAS to be referred to cardiological evaluation because of coexisting cardiomyopathy, cardiologists should be aware of this association as well as of the non cardiac signs that may address the diagnosis to mtDNA defect-related disease in families with a variable phenotype. © 2004 CEPI Srl

Mitochondrial DNA Variant Discovery and Evaluation in Human Cardiomyopathies through Next-Generation Sequencing

Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We “shotgun” sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300× average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20× while heteroplasmic variants required >200× coverage. Several Sanger “misses” were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease

Mitochondrial cardiomyopathies: how to identify candidate pathogenic mutations by mitochondrial DNA sequencing, MITOMASTER and phylogeny

Pathogenic mitochondrial DNA (mtDNA) mutations leading to mitochondrial dysfunction can cause cardiomyopathy and heart failure. Owing to a high mutation rate, mtDNA defects may occur at any nucleotide in its 16 569 bp sequence. Complete mtDNA sequencing may detect pathogenic mutations, which can be difficult to interpret because of normal ethnic/geographic-associated haplogroup variation. Our goal is to show how to identify candidate mtDNA mutations by sorting out polymorphisms using readily available online tools. The purpose of this approach is to help investigators in prioritizing mtDNA variants for functional analysis to establish pathogenicity. We analyzed complete mtDNA sequences from 29 Italian patients with mitochondrial cardiomyopathy or suspected disease. Using MITOMASTER and PhyloTree, we characterized 593 substitution variants by haplogroup and allele frequencies to identify all novel, non-haplogroup-associated variants. MITOMASTER permitted determination of each variant's location, amino acid change and evolutionary conservation. We found that 98% of variants were common or rare, haplogroup-associated variants, and thus unlikely to be primary cause in 80% of cases. Six variants were novel, non-haplogroup variants and thus possible contributors to disease etiology. Two with the greatest pathogenic potential were heteroplasmic, nonsynonymous variants: m.15132T>C in MT-CYB for a patient with hypertrophic dilated cardiomyopathy and m.6570G>T in MT-CO1 for a patient with myopathy. In summary, we have used our automated information system, MITOMASTER, to make a preliminary distinction between normal mtDNA variation and pathogenic mutations in patient samples; this fast and easy approach allowed us to select the variants for traditional analysis to establish pathogenicity

Prevalenza dei difetti del gene della distrofina in pazienti maschi adulti affetti da cardiomiopatia dilatativa

Dottorato di ricerca in patologia umana. 11. ciclo. A.a. 1998-99. Coordinatore Marco FraccaroConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Mitochondrial DNA Variant Discovery and Evaluation in Human Cardiomyopathies through Next-Generation Sequencing

Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We “shotgun” sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300× average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20× while heteroplasmic variants required >200× coverage. Several Sanger “misses” were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease

Mitochondrial cardiomyopathies: how to identify candidate pathogenic mutations by mitochondrial DNA sequencing, MITOMASTER and phylogeny.

Pathogenic mitochondrial DNA (mtDNA) mutations leading to mitochondrial dysfunction can cause cardiomyopathy and heart failure. Owing to a high mutation rate, mtDNA defects may occur at any nucleotide in its 16 569 bp sequence. Complete mtDNA sequencing may detect pathogenic mutations, which can be difficult to interpret because of normal ethnic/geographic-associated haplogroup variation. Our goal is to show how to identify candidate mtDNA mutations by sorting out polymorphisms using readily available online tools. The purpose of this approach is to help investigators in prioritizing mtDNA variants for functional analysis to establish pathogenicity. We analyzed complete mtDNA sequences from 29 Italian patients with mitochondrial cardiomyopathy or suspected disease. Using MITOMASTER and PhyloTree, we characterized 593 substitution variants by haplogroup and allele frequencies to identify all novel, non-haplogroup-associated variants. MITOMASTER permitted determination of each variant's location, amino acid change and evolutionary conservation. We found that 98% of variants were common or rare, haplogroup-associated variants, and thus unlikely to be primary cause in 80% of cases. Six variants were novel, non-haplogroup variants and thus possible contributors to disease etiology. Two with the greatest pathogenic potential were heteroplasmic, nonsynonymous variants: m.15132T>C in MT-CYB for a patient with hypertrophic dilated cardiomyopathy and m.6570G>T in MT-CO1 for a patient with myopathy. In summary, we have used our automated information system, MITOMASTER, to make a preliminary distinction between normal mtDNA variation and pathogenic mutations in patient samples; this fast and easy approach allowed us to select the variants for traditional analysis to establish pathogenicity

Autosomal recessive paediatric sick sinus syndrome associated with novel compound mutations in SCN5A

We report the case of a boy who was first addressed to medical attention when he was 2-years-old because of an episode of Salmonellosis. His cardiologic evaluation was normal. There were no clinical signs of myocarditis. Three years later he was admitted to the hospital due to a prolonged episode of flu with persistent fever. At that time, his electrocardiogram (ECG) showed junctional rhythm (JR) at 35 bpm. Further ECGs revealed sinus rhythm (SR) with first degree atrio-ventricular block, episodes of sinoatrial (SA) exit blocks, sinus arrests and phases of JR, supporting the diagnosis of sick sinus syndrome (SSS) [1]. Although the boy did not complain of symptoms, Holter monitoring showed frequent pauses of sinus arrest (max interval = 5.4 s) and chronotropic incompetence. At the age of 8 years, he underwent permanent pacemaker (PM) implantation. Although ventricular pacing is less indicated in SSS [2], the patient was treated with VVIR modality because an attempt at right atrial catheter positioning failed, due to inexcitability of the atrium. Since then, the boy has been asymptomatic. Further Holter monitoring showed episodes of paroxysmal atrial fibrillation (Fig. 1) and showed rate responsive pacing with an acceptable mean heart rate of 66 bp

Analysis of Vlambda-Jlambda expression in plasma cells from primary (AL) amyloidosis and normal bone marrow identifies 3r (lambda III) as a new amyloid-associated germline gene segment

Primary (AL) amyloidosis is a plasma cell dyscrasia characterized by extracellular deposition of monoclonal light-chain variable region (V) fragments in the form of amyloid fibrils. Light-chain amyloid is rare, and it is not fully understood why it occurs in only a fraction of patients with a circulating monoclonal component and why it typically associates with lambda isotype and lambdaVI family light-chain proteins. To provide insights into these issues, we obtained complete nucleotide sequences of monoclonal V(lambda) regions from 55 consecutive unselected cases of primary amyloidosis and the results were compared with the light-chain expression profile of polyclonal marrow plasma cells from 3 healthy donors (a total of 264 sequences). We demonstrated that: (1) the lambdaIII family is the most frequently used both in amyloidosis (47%) and in polyclonality (43%); (2) both conditions are characterized by gene restriction; (3) a very skewed repertoire is a feature of amyloidosis, because just 2 germline genes belonging to the lambdaIII and lambdaVI families, namely 3r (22% of cases, lambdaIII) and 6a (20%, lambdaVI), contributed equally to encode 42% of amyloid V(lambda) regions; (4) these same 2 gene segments have a strong association with amyloidosis if their prevalences are compared with those in polyclonal conditions (3r, 8.3%, P =.024; 6a, 2.3%, P =.0008, chi2 test); (5) the J(lambda)2/3 segment, encoding the fourth framework region, appears to be slightly overrepresented in AL (83% versus 67%, P =.03), and this might be related to preferential J(lambda)2/3 rearrangement in amyloid (11 of 12 cases) versus polyclonal 3r light chains (13 of 22 cases). These findings demonstrate that V(lambda)-J(lambda) expression is more restricted in plasma cells from amyloidosis than from polyclonal bone marrow and identify 3r as a new disease-associated gene segment. Overusage of just 2 gene segments, 3r and 6a, can thus account for the lambda light-chain overrepresentation typical of this disorder