Thesis (M.Sc.). / Written for the Division of Experimental Medicine. Title from title page of PDF (viewed 2009/06/18). Includes bibliographical references.

There is a large variety of diseases caused by single-gene mutations. Although most of these conditions are rare, together they impose a significant burden to the population. This thesis describes clinical and genetic studies of two single-gene diseases: 1) Adult-onset autosomal dominant leukodystrophy with autonomic symptoms (ADLD) caused by LMNB1 gene duplications, and characterized by autonomic, pyramidal and cerebellar symptoms. Spinal cords of patients with ADLD were studied by MRI and found to be thin, with high signal intensity in white matter. Histopathology showed loss of myelinated fibres with some reactive gliosis. DNA samples from four different families with ADLD were obtained, and the LMNB1 gene was screened for duplications. Single nucleotide polymorphism array revealed LMNB1 duplications in all ADLD families. LMNB1 mRNA and protein levels were assessed in white blood cells using quantitative polymerase chain reaction and Western blot, and increased levels of LMNB1 mRNA and lamin B1 protein could be demonstrated. We concluded that spinal cord atrophy in patients with ADLD is a valuable differential diagnostic sign, and that increased levels of LMNB1 can be detected in peripheral blood. 2) Rippling muscle disease (RMD) is caused by CAV3 gene mutations. Clinical features are percussion-induced muscle mounding, –rapid contractions and undulating muscle contractions (rippling). The CAV3 gene was sequenced in 38 members of a family with RMD. Twenty-two individuals had clinical features of RMD. No muscle weakness was seen. All patients with signs of RMD carried the p.A46T CAV3 mutation, showing that the p.A46T mutation was benign and that the diagnosis can be made clinically. In vitro contracture test results from 10 of the subjects were collected, but no association between pathological test results and RMD was found.

Thesis (Ph. D.)--University of Washington, 2007. / Includes bibliographical references (leaves 79-99).

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13568号 / 論医博第2294号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 森田 智視, 教授 石見 拓, 教授 佐藤 俊哉 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Introdução: As Distrofias Musculares Congênitas (DMCs) são um grupo clínica e geneticamente heterogêneo de doenças musculares que se manifestam ao nascimento ou no primeiro ano de vida, sendo caracterizadas por hipotonia, fraqueza muscular, retardo do desenvolvimento motor e retrações fibrotendíneas. O músculo esquelético apresenta-se distrófico, mas sem alterações estruturais específicas. Em quase metade dos casos a doença é causada pela deficiência da laminina alfa;-2 (merosina). Outras deficiências proteicas descritas incluem: colágeno VI, selenoproteína N1, várias glicosiltransferases responsáveis pela glicosilação da alfa- distroglicana e lamina A/C. Vários genes já foram identificados. Objetivo: o objetivo deste estudo foi a caracterização clínica, histológica e molecular das crianças com DMC por deficiência de lamina A/C. Método: Foram incluídos 13 pacientes com diagnóstico clínico e histológico de DMC, com expressão muscular normal para distrofina, sarcoglicanas, merosina, colágeno 6 e disferlina. Os pacientes foram reavaliados segundo protocolo clínico e neurológico. As biópsias musculares realizadas previamente foram revisadas e o estudo das mutações no gene da lamina A/C foi realizado através de sequenciamento de toda região codificadora do gene. Resultados: Identificamos mutações em 30,7% dos pacientes (quatro casos) com fenótipo clínico de DMC por deficiência de lamina A/C. Todas as mutações encontradas (p.E358K, p.R249W, e p.N39S) ocorreram em heterozigose e de novo e já haviam sido descritas na literatura em pacientes com distrofias musculares. Em geral, estes pacientes apresentavam um grave comprometimento motor com o característico aspecto de cabeça caída, com início dos sintomas nos primeiros dois anos de vida. A CPK estava elevada entre 2 a 6 vezes o padrão superior da normalidade. O padrão histológico variou desde um músculo levemente até gravemente distrófico. Curiosamente, no estudo histológico do músculo, um dos pacientes apresentou agregados intracitoplasmáticos. Um outro paciente apresentava associadamente alterações neurogênicas ao estudo eletroneuromiográfico. Em todos os casos observamos complicações respiratórias, cardíacas e distúrbios de deglutição. Houve um caso de morte súbita, provavelmente em decorrência de arritmia cardíaca. Conclusões: A correlação genótipo-fenótipo permanece difícil, mas todos os casos apresentaram sinal da cabeça caída, comprometimento respiratório, cardíaco e biópsia muscular distrófica. A ampliação do conhecimento clínico e histológico pode orientar o diagnóstico e direcionar para o estudo molecular adequado, além de permitir o diagnóstico precoce das complicações, tão frequentes na DMC por deficiência de lamina A/C. Os exons 1, 4 e 6 são os mais frequentemente mutados e devem ser pesquisados inicialmente. Esta série de casos contribui também por demonstrar a distribuição universal da doença / Background: The Congenital Muscular Dystrophies (CMD) are a clinically and genetically heterogeneous group of myopathies characterized by muscle hypotonia, delayed motor development and early onset of progressive muscle weakness with dystrophic pattern on muscle biopsy. The clinical course is broadly variable and can comprise the involvement of the brain and eyes. Almost half of the cases is caused by deficiency of laminin-alfa 2 (merosin). Other protein deficiencies described include: collagen VI, selenoprotein N1, several glycosyltransferases responsible for glycosylation of alfa-dystroglycan and lamin A/C. Several genes have been identified and the increased knowledge of new clinical and histological forms of CMD can guide diagnosis and direct appropriate molecular studies. LMNA-related CMD is often characterized by muscle weakness and a dropped head developed in the early years of life. Regarding lamin A/C deficiency, the immunohistochemical findings can be normal, probably because the protein change is functional only; this makes diagnosis using muscle samples more difficult. Objectives: The aim of this study was to characterize the clinical, histological and molecular aspects in patients with CMD related to deficiency of lamin A/C. Methods: thirteen children with clinical and histological diagnosis of CMD with normal muscle expression for dystrophin, merosin, collagen 6, sarcoglycans and dysferlin were included in this study. The LMNA gene was sequenced after amplification of all coding exons. In addition, the muscle biopsies were revised. Results: In 30.7% (four cases) of our patients with typical clinical phenotype of lamin A/C deficiency were detected mutations on LMNA gene and all of them presented dropped-head syndrome, restrictive ventilator insufficiency, cardiac changes, increased serum CPK level and myopathic/dystrophic aspect on muscle biopsy. Two of the patients had normal motor development milestones in the first months of life and subsequently developed cervical and limb weakness. The other two patients presented a more severe motor involvement and failure to walk. One patient showed associated peripheral neuropathy. Curiously one case had myofibrillar aggregates on muscle biopsy. All mutations (p.E358K, p.R249W and p.N39S) were heterozygous and de novo and had been previously described in patients with muscular dystrophy. Conclusion: Genotype/phenotype correlation in CMD remains difficult. However patients with LMNA mutation and CMD seems to have a more homogeneous phenotype characterized by dropped head, severe motor disability, and cardiac and pulmonary involvement. Mutations on exons 1, 4 and 6 should be tested first. This case series also contributes for showing the universal distribution of the disease

Introdução: As Distrofias Musculares Congênitas (DMCs) são um grupo clínica e geneticamente heterogêneo de doenças musculares que se manifestam ao nascimento ou no primeiro ano de vida, sendo caracterizadas por hipotonia, fraqueza muscular, retardo do desenvolvimento motor e retrações fibrotendíneas. O músculo esquelético apresenta-se distrófico, mas sem alterações estruturais específicas. Em quase metade dos casos a doença é causada pela deficiência da laminina alfa;-2 (merosina). Outras deficiências proteicas descritas incluem: colágeno VI, selenoproteína N1, várias glicosiltransferases responsáveis pela glicosilação da alfa- distroglicana e lamina A/C. Vários genes já foram identificados. Objetivo: o objetivo deste estudo foi a caracterização clínica, histológica e molecular das crianças com DMC por deficiência de lamina A/C. Método: Foram incluídos 13 pacientes com diagnóstico clínico e histológico de DMC, com expressão muscular normal para distrofina, sarcoglicanas, merosina, colágeno 6 e disferlina. Os pacientes foram reavaliados segundo protocolo clínico e neurológico. As biópsias musculares realizadas previamente foram revisadas e o estudo das mutações no gene da lamina A/C foi realizado através de sequenciamento de toda região codificadora do gene. Resultados: Identificamos mutações em 30,7% dos pacientes (quatro casos) com fenótipo clínico de DMC por deficiência de lamina A/C. Todas as mutações encontradas (p.E358K, p.R249W, e p.N39S) ocorreram em heterozigose e de novo e já haviam sido descritas na literatura em pacientes com distrofias musculares. Em geral, estes pacientes apresentavam um grave comprometimento motor com o característico aspecto de cabeça caída, com início dos sintomas nos primeiros dois anos de vida. A CPK estava elevada entre 2 a 6 vezes o padrão superior da normalidade. O padrão histológico variou desde um músculo levemente até gravemente distrófico. Curiosamente, no estudo histológico do músculo, um dos pacientes apresentou agregados intracitoplasmáticos. Um outro paciente apresentava associadamente alterações neurogênicas ao estudo eletroneuromiográfico. Em todos os casos observamos complicações respiratórias, cardíacas e distúrbios de deglutição. Houve um caso de morte súbita, provavelmente em decorrência de arritmia cardíaca. Conclusões: A correlação genótipo-fenótipo permanece difícil, mas todos os casos apresentaram sinal da cabeça caída, comprometimento respiratório, cardíaco e biópsia muscular distrófica. A ampliação do conhecimento clínico e histológico pode orientar o diagnóstico e direcionar para o estudo molecular adequado, além de permitir o diagnóstico precoce das complicações, tão frequentes na DMC por deficiência de lamina A/C. Os exons 1, 4 e 6 são os mais frequentemente mutados e devem ser pesquisados inicialmente. Esta série de casos contribui também por demonstrar a distribuição universal da doença / Background: The Congenital Muscular Dystrophies (CMD) are a clinically and genetically heterogeneous group of myopathies characterized by muscle hypotonia, delayed motor development and early onset of progressive muscle weakness with dystrophic pattern on muscle biopsy. The clinical course is broadly variable and can comprise the involvement of the brain and eyes. Almost half of the cases is caused by deficiency of laminin-alfa 2 (merosin). Other protein deficiencies described include: collagen VI, selenoprotein N1, several glycosyltransferases responsible for glycosylation of alfa-dystroglycan and lamin A/C. Several genes have been identified and the increased knowledge of new clinical and histological forms of CMD can guide diagnosis and direct appropriate molecular studies. LMNA-related CMD is often characterized by muscle weakness and a dropped head developed in the early years of life. Regarding lamin A/C deficiency, the immunohistochemical findings can be normal, probably because the protein change is functional only; this makes diagnosis using muscle samples more difficult. Objectives: The aim of this study was to characterize the clinical, histological and molecular aspects in patients with CMD related to deficiency of lamin A/C. Methods: thirteen children with clinical and histological diagnosis of CMD with normal muscle expression for dystrophin, merosin, collagen 6, sarcoglycans and dysferlin were included in this study. The LMNA gene was sequenced after amplification of all coding exons. In addition, the muscle biopsies were revised. Results: In 30.7% (four cases) of our patients with typical clinical phenotype of lamin A/C deficiency were detected mutations on LMNA gene and all of them presented dropped-head syndrome, restrictive ventilator insufficiency, cardiac changes, increased serum CPK level and myopathic/dystrophic aspect on muscle biopsy. Two of the patients had normal motor development milestones in the first months of life and subsequently developed cervical and limb weakness. The other two patients presented a more severe motor involvement and failure to walk. One patient showed associated peripheral neuropathy. Curiously one case had myofibrillar aggregates on muscle biopsy. All mutations (p.E358K, p.R249W and p.N39S) were heterozygous and de novo and had been previously described in patients with muscular dystrophy. Conclusion: Genotype/phenotype correlation in CMD remains difficult. However patients with LMNA mutation and CMD seems to have a more homogeneous phenotype characterized by dropped head, severe motor disability, and cardiac and pulmonary involvement. Mutations on exons 1, 4 and 6 should be tested first. This case series also contributes for showing the universal distribution of the disease

The nuclear lamina protein, lamin A is produced by proteolytic cleavage of a 74 kDa precursor protein, prelamin A. The conversion of this precursor to mature lamin A is mediated by a specific endoprotease, prelamin A endoprotease. Subnuclear fractionation indicates that the prelamin A endoprotease is localized at the nuclear membrane. The enzyme appears to be an integral membrane protein, as it can only be removed from the nuclear envelope with detergent. It is effectively solubilized by the detergent n-octyl-beta-D-glucopyranoside and can be partially-purified (approximately 1200-fold) by size exclusion and cation exchange (Mono S) chromatography. Prelamin A endoprotease from HeLa cells was eluted from Mono S with 0.3 M sodium chloride as a single peak of activity. SDS-PAGE analysis of this prelamin A endoprotease preparation shows that it contains one major polypeptide at 65 kDa and smaller amounts of a second 68 kDa polypeptide. Inhibition of the enzyme activity in this preparation by specific serine protease inhibitors is consistent with the enzyme being a serine protease.