Ali-Reza Moslemi

The incidence of mitochondrial encephalomyopathies in childhood: Clinical features and morphological, biochemical, and DNA abnormalities

In this study we present incidence, point prevalence, and mortality figures of mitochondrial encephalomyopathies in a population‐based study of children from western Sweden. Through the screening of registers and review of medical records, we identified 32 patients under 16 years of age from the study population who were diagnosed between January 1, 1984, and December 31, 1998. The incidence of mitochondrial encephalomyopathies in preschool children (<6 years of age) was 1 out of 11,000. The preschool incidence of Leighs syndrome was 1 out of 32,000, and the preschool incidences of both Alpers syndrome and infantile mitochondrial myopathy with cytochrome C oxidase deficiency were 1 out of 51,000. The point prevalence (January 1, 1999) of mitochondrial encephalomyopathies in children under 16 years of age was 1 out of 21,000. The median survival for patients with infantile onset was until 12 years of age. We identified 4 cases with mitochondrial DNA point mutations, 2 cases with mitochondrial DNA deletions, and 2 cases with nuclear mutations in the SURF1 gene. We conclude that mitochondrial encephalomyopathies are relatively common neurometabolic disorders in childhood. Ann Neurol 2001;49:377–383

Ageing muscle: clonal expansions of mitochondrial DNA point mutations and deletions cause focal impairment of mitochondrial function

Although mitochondrial DNA deletions have been shown to accumulate in cytochrome c oxidase deficient muscle fibres of ageing muscle, this has not been demonstrated for point mutations. In this study, we investigated the occurrence of mitochondrial DNA alterations (point mutations and deletions) in cytochrome c oxidase deficient muscle fibres from 14 individuals, without muscle disease, aged 69-82 years. Immunohistochemical investigation showed that the majority of the cytochrome c oxidase deficient muscle fibres expressed reduced levels of subunit II of cytochrome c oxidase, which is encoded by mitochondrial DNA, whereas there was normal or increased expression of subunit IV of cytochrome c oxidase, which is encoded by nuclear DNA. This pattern is typical for mitochondrial DNA mutations causing impaired mitochondrial translation. Single muscle fibres (109 cytochrome c oxidase deficient and 109 normal fibres) were dissected and their DNA extracted. Mitochondrial DNA point mutations were searched for in five tRNA genes by denaturing gradient gel electrophoresis while deletions were looked for by polymerase chain reaction amplification. High levels of clonally expanded point mutations were identified in eight cytochrome c oxidase deficient fibres but in none of the normal ones. They included the previously described pathogenic tRNALeu(UUR)A3243G and tRNALysA8344G mutations and three original mutations: tRNAMetT4460C, tRNAMetG4421A, and a 3-bp deletion in the tRNALeu(UUR) gene. Four different large-scale mitochondrial DNA deletions were identified in seven cytochrome c oxidase deficient fibres and in one of the normal ones. There was no evidence of depletion of mitochondrial DNA by in situ hybridisation experiments. Our data show that mitochondrial DNA point mutations, as well as large-scale deletions, are associated with cytochrome c oxidase deficient muscle fibre segments in ageing. Their focal accumulation causes significant impairment of mitochondrial function in individual cells in spite of low overall levels of mitochondrial DNA mutations in muscle.

Mitochondrial DNA Deletions in Muscle Fibers in Inclusion Body Myositis

Abstract . Inclusion body myositis (IBM) is an autoimmune, inflammatory myopathy where morphological changes of muscle, including ragged red fibers, have indicated mitochondrial dysfunction in some muscle fibers. In this study enzyme histochemical analysis showed that cytochrome c oxidase (COX)-deficient muscle fibers were present at a frequency ranging from 0.5 to 5% of the muscle fibers in a series of 20 IBM patients. In age-matched controls, only occasional COX-deficient muscle fibers were present. Polymerase chain reaction (PCR) analysis of DNA extracted from muscle tissue of the IBM patients showed multiple mtDNA deletions. PCR analysis of isolated, single muscle fibers showed presence of mtDNA with only one type of deletion and deficiency of wild-type mtDNA in each COχ-deficient muscle fiber. This finding was supported by results from in situ hybridization using different mtDNA probes on consecutive sections. A 5 kb deletion was identified in all 20 IBM patients. DNA sequencing of the breakpoint region showed that this deletion was the so-called “common deletion.” Most but not all of the investigated deletion breakpoints were flanked by direct repeats. COχ-deficient fibers were more frequent among fibers with positive immunostaining with antibodies directed toward a regeneration marker, the Leu-19 antigen, than in the entire fiber population. These results show that COχ deficiency in muscle fiber segments in IBM is associated with deletions of mtDNA. Clonal expansion of mtDNA with deletions may take place in regenerating muscle fibers following segmental necrosis.

Mitochondrial abnormalities in inclusion-body myositis.

Mitochondrial changes are frequently encountered in sporadic inclusion-body myositis (s-IBM). Cytochrome c oxidase (COX)-deficient muscle fibers and large-scale mitochondrial DNA (mtDNA) deletions are more frequent in s-IBM than in age-matched controls. COX deficient muscle fibers are due to clonal expansion of mtDNA deletions and point mutations in segments of muscle fibers. Such segments range from 75 μm to more than 1,000 μm in length. Clonal expansion of the 4977 bp “common deletion” is a frequent cause of COX deficient muscle fiber segments, but many other deletions also occur. The deletion breakpoints cluster in a few regions that are similar to what is found in human mtDNA deletions in general. Analysis in s-IBM patients of three nuclear genes associated with multiple mtDNA deletions, POLG1, ANT1 and C10orf2, failed to demonstrate any mutations. In s-IBM patients with high number of COX-deficient fibers, the impaired mitochondrial function probably contribute to muscle weakness and wasting. Treatment that has positive effects in mitochondrial myopathies may be tried also in s-IBM.

POLG1 Mutations Associated With Progressive Encephalopathy in Childhood

Abstract We have identified compound heterozygous missense mutations in POLG1, encoding the mitochondrial DNA polymerase gamma (Pol &ggr;), in 7 children with progressive encephalopathy from 5 unrelated families. The clinical features in 6 of the children included psychomotor regression, refractory seizures, stroke-like episodes, hepatopathy, and ataxia compatible with Alpers-Huttenlocher syndrome. Three families harbored a previously reported A467T substitution, which was found in compound with the earlier described G848S or the W748S substitution or a novel R574W substitution. Two families harbored the W748S change in compound with either of 2 novel mutations predicted to give an R232H or M1163R substitution. Muscle morphology showed mitochondrial myopathy with cytochrome c oxidase (COX)-deficient fibers in 4 patients. mtDNA analyses in muscle tissue revealed mtDNA depletion in 3 of the children and mtDNA deletions in the 2 sibling pairs. Neuropathologic investigation in 3 children revealed widespread cortical degeneration with gliosis and subcortical neuronal loss, especially in the thalamus, whereas there were only subcortical neurodegenerative findings in another child. The results support the concept that deletions as well as depletion of mtDNA are involved in the pathogenesis of Alpers-Huttenlocher syndrome and add 3 new POLG1 mutations associated with an early-onset neurodegenerative disease.

Molecular basis of infantile reversible cytochrome c oxidase deficiency myopathy

Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as ‘benign cytochrome c oxidase deficiency myopathy’ is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T>C mt-tRNAGlu mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNAGlu may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis.

Glycogenin-1 Deficiency and Inactivated Priming of Glycogen Synthesis

Glycogen, which serves as a major energy reserve in cells, is a large, branched polymer of glucose molecules. We describe a patient who had muscle weakness, associated with the depletion of glycogen in skeletal muscle, and cardiac arrhythmia, associated with the accumulation of abnormal storage material in the heart. The skeletal muscle showed a marked predominance of slow-twitch, oxidative muscle fibers and mitochondrial proliferation. Western blotting showed the presence of unglucosylated glycogenin-1 in the muscle and heart. Sequencing of the glycogenin-1 gene, GYG1, revealed a nonsense mutation in one allele and a missense mutation, Thr83Met, in the other. The missense mutation resulted in inactivation of the autoglucosylation of glycogenin-1 that is necessary for the priming of glycogen synthesis in muscle.

Analysis of multiple mitochondrial DNA deletions in inclusion body myositis

Inclusion body myositis (IBM) is a sporadic progressive myopathy, which is morphologically characterized by inflammatory cell infiltrates and rimmed vacuoles in muscle fibers. Mitochondrial changes are regularly present with ragged‐red fibers showing deficiency of cytochrome c oxidase. In these muscle fiber segments, there is accumulation of mitochondria with mitochondrial DNA (mtDNA) deletions. There are different deletions in different muscle fibers. In this study, we have sequenced for the first time the multiple mtDNA deletions in muscle from four patients with IBM. The deletion breakpoints were sequenced from cloned polymerase chain reaction (PCR)‐amplified mtDNA fragments. The sequencing was performed directly from the bacterial colonies used for cloning. Of 122 analyzed clones, 33 different deletions were identified. The majority of these have not previously been described. There was a marked predominance of deletion breakpoints in certain regions of mtDNA. These predominant breakpoint regions are similar to those described in other conditions with multiple deletions, such as autosomal dominant progressive external ophthalmoplegia (adPEO) and normal aging, but different from those described in diseases due to single deletions such as Kearns‐Sayre syndrome and sporadic PEO. These findings indicate that common factors are involved in the development of multiple mtDNA deletions in IBM, adPEO, and aging. Hum Mutat 10:381–386, 1997.

A novel homozygous RRM2B missense mutation in association with severe mtDNA depletion

This report describes two brothers, both deceased in infancy, with severe depletion of mitochondrial DNA (mtDNA) in muscle tissue. Both had feeding difficulties, failure to thrive, severe muscular hypotonia and lactic acidosis. One of the boys developed a renal proximal tubulopathy. A novel homozygous c.686 G-->T missense mutation in the RRM2B gene, encoding the p53-inducible ribonucleotide reductase subunit (p53R2), was identified. This is the third report on mutations in RRM2B associated with severe mtDNA depletion, which further highlights the importance of de novo synthesis of deoxyribonucleotides (dNTPs) for mtDNA maintenance.

Autosomal dominant progressive external ophthalmoplegia Distribution of multiple mitochondrial DNA deletions

Objective: To relate signs and symptoms to morphologic changes and presence of multiple mitochondrial DNA (mtDNA) deletions in a patient with autosomal dominant progressive external ophthalmoplegia (adPEO) and mitochondrial myopathy. Background: An etiologic association between the somatic multiple mtDNA deletions in adPEO and clinical manifestations other than the myopathy has so far not been demonstrated. Methods: The authors investigated a patient with adPEO and multiorgan system manifestations including levodopa-responsive parkinsonism. She died at age 61 years of pancreatic carcinoma. Autopsy tissue specimens were investigated for morphologic alterations and occurrence of mtDNA deletions by Southern blot and long-extension PCR analyses. Results: The patient had carcinoma of the pancreas with metastases to liver, lymph nodes, and bone marrow. The brain revealed slight gliosis of the gray and white matter and degeneration of the substantia nigra. The myocardium showed focal areas with loss and atrophy of myocytes and fibrosis. Analysis of mtDNA revealed multiple deletions in different regions of the brain, skeletal muscle, and myocardium. Twenty-five different mtDNA deletions were identified. Most of these were flanked by large direct-sequence repeats. Six identical deletions were found in muscle and brain. Conclusions: These findings indicate that somatic multiple mtDNA deletions are associated with degenerative tissue changes and clinical manifestations in adPEO.