Genetic defects in patients with mitochondrial encephalomyopathies

Summary This thesis is a contribution to the fast growing field devoted to the improvement of the diagnostics in patients with mitochondrial encephalomyopathies at the DNA level and is inspired amongst others by the hypothesis of intergenomic crosstalk between the nuclear genome (e.g. the 24 kDa subunit of complex I) and the mitochondrial genome. It presents the results of clinical, biochemical and molecular genetic studies that have been performed at the Department of Human Genetics, University Hospital Nijmegen, Nijmegen; at the Division of Genetics, University of Maastricht, Maastricht; at the Department of Neurology, Erasmus MC -University Medical Center Rotterdam, The Netherlands and at the Institute of Neurology, The National Hospital, Queen Square, London, UK (Introduction, Chapter 1). In this thesis, different strategies are described to discriminate the cause of a mitochondrial encephalomyopathy to be located either in the mitochondrial or in the nuclear genome (Chapter 2-7). First a PCR-based test is described to detect the whole spectrum of large deletions as can be found in Progressive External Ophthalmoplegia and in the Kearns-Sayre syndrome. The advantage of this PCR-based test compared to Southern blot analysis is the sensitivity of the method for detecting deletions. In many cases patient’s leukocyte DNA is sufficient for making a diagnosis saving the patient a muscle biopsy procedure (Chapter 2). A single stranded conformation analysis (SSCP) screening method for mitochondrial tRNA mutations revealed in a patient with a mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS-pheno-type), a mutation in the tRNAVal gene (G1642A) being the second report of this mutation. Our observation was done independently and our patient’s phenotype is the same as in the former report confirming its likely pathogenicity. The phenotype of this patient differed from other MELAS patients because of the involvement of small cerebral arteries in the disease process. This involvement has not been reported before (Chapter 3). Next the analysis of five patients, with a biochemical phenotype of a complex III deficiency, for mutations in the only mitochondrially encoded subunit of complex III, the cytochrome b gene is described. In one patient a four base pair deletion-mutation at position 14787 and a homoplasmic SUMMARY 187 polymorphism are found. The mutation is heteroplasmic and present in 95% in muscle. In the clinically unaffected mother no mutation is detected. This frame shift mutation is predicted to cause a severe disruption of the synthesis of the cytochrome b protein. The phenotype of the patient, a Parkinsonism-MELAS overlap syndrome, has not been described before, neither in association with a complex III deficiency nor with a mutation in the cytochrome b gene. In two out of the four other patients the mutation analysis revealed two different homoplasmic polymorphisms (Chapter 4). In a patient with symptoms as seen in patients with a Leigh syndrome is a de novo arisen T8993C mitochondrial mutation reported. Hypotheses are formulated to explain this de novo event and the rise in mutant rate from 0% in the mother’s muscle mtDNA to 79% in the patient. Nuclear encoded, modifier genes are very likely necessary to understand the high percentage of heteroplasmy for this mutation (Chapter 5). Hereafter is the use of denaturing high performance liquid chromatography (DHPLC) technology demonstrated as an answer to the increasing number of different tests to exclude all the possible mtDNA mutations. The DHPLC method is a fast, reliable and sensitive method to detect heteroduplexes that result from heteroplasmic strands. Therefore this method is particularly suited for mtDNA screening, because most mutations in the mtDNA are heteroplasmic. A mtDNA-DHPLC protocol was developed that enables a complete mtDNA mutation analysis within one day. Levels of heteroplasmy as low as 0.5% for the A8344G mutation can be detected. The first six mitochondrial encephalomyopathy patients screened with this method showed a mutation in three out of six patients tested. Exclusion of mtDNA involvement supports a subsequent investigation of nuclear genes and has important implications for counselling (Chapter 6). There are only a few therapeutic possibilities for patients with mitochondrial encephalomyopathies. In case of a mtDNA mutation as the causative factor for disease also the possibilities for prenatal diagnosis are limited because of the complicated way the mutation is transmitted. For the first time a prenatal diagnosis and transmission findings are reported in a family with Leigh syndrome associated with the T9176C (ATPase6 gene) mutation (Chapter 7). In the second part of the thesis the characterization and mutation screening of the three flavoprotein fraction genes, the NDUFV1, NDUFV2, NDUFV3 genes, of the complex I are described. Using chaotropic agents 188 SUMMARY complex I can be divided in three fractions. One of the fractions, the flavoprotein fraction, consists of three subunits of 51, 24 and 10 kDa and is functionally important for complex I and the oxidative phosphorylation (Chapter 8-11). First the cloning and mapping of the gene coding for the smallest of the three subunits the NDUFV3 or 10 kDa-gene is described. The human cDNA sequence was elucidated by screening a human renal cDNA library with the known bovine 10-kDa cDNA. The 5' end of the cDNA was obtained with the rapid amplification of cDNA ends (RACE) procedure. Northern blot procedures showed the gene to be ubiquitously expressed. The gene contains three exons and spans about 20 kb. A Southern blot panel with human/hamster somatic cell hybrids showed that the gene is localized on chromosome 21. A 10 kDa gene containing cosmid was derived from a chromosome 21-specific cosmid library and used for a fluorescence in situ hybridization (Fish) procedure to refine the chromosome 21 location to 21q22.3 (Chapter 8). Next the cloning and characterization of the Fe-S cluster containing 24 kDa subunit gene is reported. The homologous bovine 24 kDa cDNA was used to screen a human cosmid library. The search was complicated by the presence of a pseudogene. The 24kDa cDNA cosmids were mapped by screening a Southern blot panel of human/hamster somatic cell hybrids to two different genes. One large fragment mapped to chromosome 19 and three smaller fragments to chromosome 18. Further refinement of the mapping was done with somatic cell hybrids containing either chromosome 18 or 19 fragments. With this procedure the locus could be assigned to 18p11.2-pter and to chromosome 19q13.3-qter. In a Fish procedure the loci were further refined to 18p11.2-11.31 and 19qter. The two genes were sequenced and revealed that the chromosome 19 locus represented a pseudogene and that the chromosome 18 locus represented the active 24 kDa gene. Northern blot analysis showed an ubiquitous gene expression (Chapter 9). Then the structure of the NDUFV1 gene, encoding the 51 kDa flavoprotein subunit of complex I, is described. The structure of the gene was clarified by using the known bovine 51 kDa cDNA sequence. With primers derived from the cDNA, PCR fragments from genomic DNA were generated. The gene appeared to contain 10 exons coding for 464 amino acids and spanned about 5 kb of the human genome. Northern blot analysis showed ubiquitous gene expression with the highest expression in pan- SUMMARY 189 creas. For testis mRNA a unique mRNA length fragment was present (Chapter 10). Following the characterisation of the three flavoprotein subunit genes NDUFV1, NDUFV2 and NDUFV3 the mutation analysis is described. For this comprehensive mutation analysis twenty patients with a mitochondrial encephalomyopathy and an isolated complex I deficiency were selected. No mutations in this group of patients were detected. Three polymorphisms were found in the NDUFV2 gene. This study supports the idea that the flavoprotein fraction of complex I is not a hotspot for mutations (Chapter 11). Finally the new findings presented in this thesis are put in perspective and directions for future research are discussed (Chapter 12).