L.P.W.J. van den Heuvel

A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy

Purpose: To identify the biochemical and molecular genetic defect in a 16-year-old patient presenting with apical hypertrophic cardiomyopathy and neuropathy suspected for a mitochondrial disorder. Methods: Measurement of the mitochondrial energy-generating system (MEGS) capacity in muscle and enzyme analysis in muscle and fibroblasts were performed. Relevant parts of the mitochondrial DNA were analysed by sequencing. Transmitochondrial cybrids were obtained by fusion of 143B206 TK− rho zero cells with patient-derived enucleated fibroblasts. Immunoblotting techniques were applied to study the complex V assembly. Results: A homoplasmic nonsense mutation m.8529G→A (p.Trp55X) was found in the mitochondrial ATP8 gene in the patient’s fibroblasts and muscle tissue. Reduced complex V activity was measured in the patient’s fibroblasts and muscle tissue, and was confirmed in cybrid clones containing patient-derived mitochondrial DNA. Immunoblotting after blue native polyacrylamide gel electrophoresis showed a lack of holocomplex V and increased amounts of mitochondrial ATP synthase subcomplexes. An in-gel activity assay of ATP hydrolysis showed activity of free F1-ATPase in the patient’s muscle tissue and in the cybrid clones. Conclusion: We describe the first pathogenic mutation in the mitochondrial ATP8 gene, resulting in an improper assembly and reduced activity of the complex V holoenzyme.

Natural disease course and genotype-phenotype correlations in Complex I deficiency caused by nuclear gene defects: what we learned from 130 cases

Mitochondrial complex I is the largest multi-protein enzyme complex of the oxidative phosphorylation system. Seven subunits of this complex are encoded by the mitochondrial and the remainder by the nuclear genome. We review the natural disease course and signs and symptoms of 130 patients (four new cases and 126 from literature) with mutations in nuclear genes encoding structural complex I proteins or those involved in its assembly. Complex I deficiency caused by a nuclear gene defect is usually a non-dysmorphic syndrome, characterized by severe multi-system organ involvement and a poor prognosis. Age at presentation may vary, but is generally within the first year of life. The most prevalent symptoms include hypotonia, nystagmus, respiratory abnormalities, pyramidal signs, dystonia, psychomotor retardation or regression, failure to thrive, and feeding problems. Characteristic symptoms include brainstem involvement, optic atrophy and Leigh syndrome on MRI, either or not in combination with internal organ involvement and lactic acidemia. Virtually all children ultimately develop Leigh syndrome or leukoencephalopathy. Twenty-five percent of the patients died before the age of six months, more than half before the age of two and 75xa0% before the age of ten years. Some patients showed recovery of certain skills or are still alive in their thirties . No clinical, biochemical, or genetic parameters indicating longer survival were found. No clear genotype-phenotype correlations were observed, however defects in some genes seem to be associated with a better or poorer prognosis, cardiomyopathy, Leigh syndrome or brainstem lesions.

Major depression in adolescent children consecutively diagnosed with mitochondrial disorder

A higher incidence of major depression has been described in adults with a primary oxidative phosphorylation disease. Intriguingly however, not all patients carrying the same mutation develop symptoms of major depression, pointing out the significance of the interplay of genetic and non-genetic factors in the etiology. In a series of paediatric patients evaluated for mitochondrial dysfunction, out of 35 children with a biochemically and genetically confirmed mitochondrial disorder, we identified five cases presenting with major depression prior to the diagnosis. The patients were diagnosed respectively with mutations in MTTK, MTND1, POLG1, PDHA1 and the common 4977 bp mtDNA deletion. Besides cerebral lactic acidemia protein and glucose concentrations, immunoglobins, anti-gangliosides and neurotransmitters were normal. No significant difference could be confirmed in the disease progression or the quality of life, compared to the other, genetically confirmed mitochondrial patients. In three out of our five patients a significant stress life event was confirmed. We propose the abnormal central nervous system energy metabolism as the underlying cause of the mood disorder in our paediatric patients. Exploring the genetic etiology in children with mitochondrial dysfunction and depression is essential both for safe medication and adequate counselling.

Clinical heterogeneity in patients with mutations in the NDUFS4 gene of mitochondrial complex I

Summary: A comparison of the clinical presentation, disease course and results of laboratory and imaging studies of all patients so far published with a NDUFS4 mutation are presented. This reveals marked clinical heterogeneity, even in patients with the same genotype.

Carnitine-acylcarnitine carrier deficiency: identification of the molecular defect in a patient

M. HUIZING1, U. WENDEL2, W. RUITENBEEK1*, V. IACOBAZZI3, L. IJLST4, P. VEENHUIZEN1, P. SAVELKOUL1, L. P. VAN DEN HEUVEL1, J. A. M. SMEITINK1, R. J. A. WANDERS4, J. M. F. TRIJBELS1 and F. PALMIERI3 of 1University Hospital, Department Pediatrics, Nijmegen, T he Netherlands ; of of 2University Hospital, Department Pediatrics, Dux8e sseldorf, Germany ; 3University of of Bari, Department Pharmaco-Biology, Bari, Italy ; 4University Amsterdam, of Clinical Chemistry and Departments Pediatrics, Amsterdam, T he Netherlands

Brody syndrome: A clinically heterogeneous entity distinct from Brody disease: A review of literature and a cross-sectional clinical study in 17 patients

Brody disease is a rare inherited myopathy due to reduced sarcoplasmic reticulum Ca(2+) ATPase (SERCA)1 activity caused by mutations in ATP2A1, which causes delayed muscle relaxation and silent cramps. So far the disease has mostly been diagnosed by measurement of SERCA1 activity. Since mutation analysis became more widely available, it has appeared that not all patients with reduced SERCA1 activity indeed have ATP2A1 mutations, and a distinction between Brody disease (with ATP2A1 mutations) and Brody syndrome (without ATP2A1 mutations) was proposed. We aim to compare the clinical features of patients with Brody disease and those with Brody syndrome and detect clinical features which help to distinguish between the two. In addition, we describe the Brody syndrome phenotype in more detail. We therefore performed a literature review on clinical features of both Brody disease and Brody syndrome and a cross-sectional clinical study consisting of questionnaires, physical examination, and a review of medical files in 17 Brody syndrome patients in our centre. The results showed that Brody disease presents with an onset in the 1st decade, a generalized pattern of muscle stiffness, delayed muscle relaxation after repetitive contraction on physical examination, and autosomal recessive inheritance. Patients with Brody syndrome more often report myalgia and experience a considerable impact on daily life. Future research should focus on the possible mechanisms of reduction of SERCA activity in Brody syndrome and other genetic causes, and on evaluation of treatment options.

The X-chromosomal NDUFA1 gene of complex I in mitochondrial encephalomyopathies: Tissue expression and mutation detection

electron transport chain consists of four protein enzyme complexes, of which The the NADH:ubiquinone oxidoreductase (complex I) is the largest. Complex I contains at least 41 subunits, 7 of which are encoded by the mitochondrial DNA (ND16 and ND4L) ; nuclear genes encode the remainder (HateÐ 1985 ; Walker 1992 ; Complex I catalyses the transfer of electrons from NADH to ubiRobinson 1993). quinone, which is coupled to the translocation of protons across the inner mitochondrial membrane. Patients described with a (partial) complex I deÐciency can generally be categorized into two major clinical phenotypes : an isolated myopathy and a multisystem disorder with predominantly encephalopathy. Respiratory chain defects may be inherited as autosomal, or X-linked Mendelian traits et al et al or in the case of certain muta(Orstavik 1993 ; Zhuchenko 1996), tions in mitochondrial DNA as maternal traits. To date, no mutations in a nuclearencoded subunit of complex I have been described. In our biochemically proven complex I-deÐcient patients as well as among the a†ected siblings, (the latter currently not all biochemically evaluated), we observed a strong male preponderance, suggestive of an X-linked inheritance. Recently, the NDUFA1 gene, one of the nuclear-encoded complex I genes, was isolated and mapped to chromosome Xq24 et al The NDUFA1 (Zhuchenko 1996). gene is composed of three exons, and spans about 5.0 kb of genomic DNA. It shows 80% homology with the bovine MWFE subunit of complex I. The knowledge of function of the human NDUFA1, and the bovine MWFE subunit is very limited. The bovine MWFE subunit is thought to be situated in the extrinsic membrane domain of complex I (Walker 1992).

Normal biochemical analysis of the oxidative phosphorylation (OXPHOS) system in a child with POLG mutations: A cautionary note

SummaryWe report a 5-year-old child carrying polymerase gamma (POLG1) mutations, but strikingly normal oxidative phosphorylation analysis in muscle, fibroblasts and liver. Mutations in POLG1 have so far been described in children with severe combined oxidative phosphorylation (OXPHOS) deficiencies and with the classical Alpers–Huttenlocher syndrome. The patient presented with a delayed psychomotor development and ataxia during the first two years of life. From the third year of life he developed epilepsy and regression in development, together with symptoms of visual impairment and sensorineuronal deafness. Cerebrospinal fluid showed elevated lactic acid and protein concentrations. An elder brother had died due to combined OXPHOS deficiencies. Despite the clinical similarity with the elder brother, except for liver involvement, the OXPHOS system analysis in a frozen muscle biopsy was normal. For this reason a fresh muscle biopsy was performed, which has the advantage of the possibility of measuring the substrate oxidation rates and ATP production, part of the mitochondrial energy-generating system (MEGS). During the same session, biopsies of liver and fibroblasts were taken. These three tissues showed normal measurements of the MEGS capacity. Based on the phenotype of Alpers–Huttenlocher syndrome in the elder brother, we decided to screen the POLG1 gene. Mutation analysis showed compound heterozygosity with two known mutations, A467T and G848S. The normal MEGS capacity in this patient expands the already existing complexity and heterogeneity of the childhood POLG1 patients and, on the basis of the high frequency of POLG1 mutations in childhood, warrants a liberal strategy with respect to mutation analysis.

Sequence variants in four candidate genes (NIPSNAP1, GBAS, CHCHD1 and METT11D1) in patients with combined oxidative phosphorylation system deficiencies

SummaryThe oxidative phosphorylation (OXPHOS) system, comprising five enzyme complexes, is located in the inner membrane of mitochondria and is the final biochemical pathway in oxidative ATP production. Defects in this energy-generating system can cause a wide range of clinical symptoms; these diseases are often progressive and multisystemic. Numerous genes have been implicated in OXPHOS deficiencies and many mutations have been described. However, in a substantial number of patients with decreased enzyme activities of two or more OXPHOS complexes, no mutations in the mitochondrial DNA or in nuclear genes known to be involved in these disorders have been found. In this study, four nuclear candidate genes—NIPSNAP1, GBAS, CHCHD1 and METT11D1—were screened for mutations in 22 patients with a combined enzymatic deficiency of primarily the OXPHOS complexes I, III and IV to determine whether a mutation in one of these genes could explain the mitochondrial disorder. For each variant not yet reported as a polymorphism, 100 control samples were screened for the presence of the variant. This way we identified 14 new polymorphisms and 2 presumably non-pathogenic mutations. No mutations were found that could explain the mitochondrial disorder in the patients investigated in this study. Therefore, the genetic defect in these patients must be located in other nuclear genes involved in mtDNA maintenance, transcription or translation, in import, processing or degradation of nuclear encoded mitochondrial proteins, or in assembly of the OXPHOS system.

Mutation detection in four candidate genes (OXA1L, MRS2L, YME1L and MIPEP) for combined deficiencies in the oxidative phosphorylation system.

SummaryMitochondria are the main energy-producing organelles of the cell. Five complexes embedded in the mitochondrial inner membrane, together constituting the oxidative phosphorylation (OXPHOS) system, comprise the final steps in cellular energy production. Many patients with a mitochondrial defect suffer from a so-called combined deficiency, meaning that the enzymatic activities of two or more complexes of the OXPHOS system are decreased. Numerous mutations have been described in nuclear genes that are involved in the functioning of a single complex of the OXPHOS system. However, little attention has been paid to patients with a deficiency of more than one complex of this particular system. In this study we have investigated four nuclear genes (OXA1L, MRS2L, YME1L and MIPEP) that might be involved in the pathology of combined enzymatic deficiencies of the OXPHOS system. Based on the results of yeast knockouts of these four proteins, we have sequenced the open reading frame of OXA1L in eight patients with an enzymatic deficiency of complexes I and IV. MRS2L, YME1L and MIPEP have been sequenced in three patients with a combined defect of complexes III and IV. No mutations were detected in these patients, showing that at least in these patients the OXPHOS system deficiency cannot be explained by a mutation in these four genes.