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Erratum sheet pasted on front end-paper. / Bibliography: p. 220-239. / xxi, 239 p. : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The objectives of this project are to investigate the development of metachromatic leukodystrophy (MLD) in the patient population referred to the Women's and Children's Hospital to gain further insight into the molecular and biochemical defects which cause disease. This study hypothesises that alleles which cause pseudodeficiencies of arylsulphatase A gene (ASA) may play a role in modifying the course of MLD or may be responsible for the development of the disease. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1997?

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22737号 / 医博第4655号 / 新制||医||1046(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 伊佐 正, 教授 林 康紀, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

As a novel therapy for neurodegenerative diseases, transplantation of multipotent mesenchymal stromal cells (MSCs) requires extensive optimization in animal models before being implemented in clinical trials. It is a goal of our laboratory to understand the mechanism of action of these cells and to improve their therapeutic efficacy. To address these goals, this study aims to optimize the cell dosage, cell type, administration route and timing, and/or donor age for stem cell therapy in two mouse models of demyelinating diseases: globoid cell leukodystrophy (GLD; Krabbe’s disease) and experimental autoimmune encephalomyelitis (EAE). GLD is a neurodegenerative lysosomal storage disease caused by the deficiency of galactocerebrosidase (GALC). Accumulation of toxic byproducts in myelin producing oligodendrocytes leads to the demyelination of neurons and increase in brain inflammation. The twitcher mouse model of GLD was used to test the therapeutic effects of MSCs after injection through intracerebroventricular (ICV) or intraperitoneal (IP) routes. Weekly MSC IP injections and single IP GALC-transduced MSC injections were performed. Other twitcher mouse cohorts received temporal vein (TV) or intracerebral (IC) injections of GALC-containing adeno-associated virus serotype 9 (AAV9-GALC) with or without IP MSC injections. All GLD affected mice treated with peripheral MSC and/or vector therapy had extended lifespans with improved motor function. The ameliorating effects of MSCs were related to their potent anti-apoptotic and anti-inflammatory effects on the peripheral and central nervous systems. These results indicate a promising future for peripheral administration of MSCs and vectors as non-invasive, adjunct therapies for patients affected with GLD. A similar study was performed using the EAE mouse model of multiple sclerosis (MS), which is a demyelinating disease due to an autoimmune reaction to myelin. The results demonstrated that biological age of the donor reduces the ability of MSCs to alleviate symptoms and improve pathology in the EAE mouse model. Upon transplantation, the young, but not old, MSCs provided neuroprotective effects through immunomodulation and remyelination in the central nervous system (CNS). The age-related therapeutic differences corroborate recent findings that biologic aging occurs in stem cells and highlight the potential need for allogeneic transplantation of MSCs in older MS patients. / acase@tulane.edu

<p>The aim of this thesis has been to identify genes and gene regions underlying four different disorders. In papers I-IV, positional cloning methods, such as linkage, association and haplotype analysis have been used for the identification of genomic regions associated with the ichthyosis prematurity syndrome (IPS), adult-onset autosomal dominant leukodystrophy (ADLD) and Kostmann disease. </p><p>IPS is a rare autosomal recessive skin disorder, which includes a premature birth of the affected child. We mapped the IPS locus to a region on chromosome 9q34, and within this region a haplotype is shared by IPS patients, which suggests a strong founder effect. The haplotype spans 76 kb, which includes four known genes. No sequence or mRNA expression alterations could be detected for the four genes in IPS patients. </p><p>A candidate region for an adult-onset leukodystrophy (ADLD) on chromosome 5 was investigated in a large Swedish family with ADLD. A significant multipoint LOD score of 9.45 was obtained for markers in the chromosome 5 region and fine-mapping of recombination events restricts a candidate gene region to 1.5 Mb. </p><p>Kostmann disease is an autosomal recessive form of severe congenital neutropenia. We have identified a 1.2 Mb region on chromosome 1q22 associated with the disease in the original Kostmann family. The region contains 37 genes.</p><p>In paper V, cDNA microarrays were used to asses the mRNA levels of 7,700 genes in lymphoblastoid cell lines derived from autistic and control samples. The <i>SEMA5A</i> gene, which is involved in axonal guidance, was found downregulated in the cells derived from autistic individuals, and this was confirmed by quantitative PCR. </p><p>In summary, candidate genes or gene regions have been identified for all four disorders and further studies are needed to confirm their roles in the pathogenesis of the disorders. </p>

The aim of this thesis has been to identify genes and gene regions underlying four different disorders. In papers I-IV, positional cloning methods, such as linkage, association and haplotype analysis have been used for the identification of genomic regions associated with the ichthyosis prematurity syndrome (IPS), adult-onset autosomal dominant leukodystrophy (ADLD) and Kostmann disease. IPS is a rare autosomal recessive skin disorder, which includes a premature birth of the affected child. We mapped the IPS locus to a region on chromosome 9q34, and within this region a haplotype is shared by IPS patients, which suggests a strong founder effect. The haplotype spans 76 kb, which includes four known genes. No sequence or mRNA expression alterations could be detected for the four genes in IPS patients. A candidate region for an adult-onset leukodystrophy (ADLD) on chromosome 5 was investigated in a large Swedish family with ADLD. A significant multipoint LOD score of 9.45 was obtained for markers in the chromosome 5 region and fine-mapping of recombination events restricts a candidate gene region to 1.5 Mb. Kostmann disease is an autosomal recessive form of severe congenital neutropenia. We have identified a 1.2 Mb region on chromosome 1q22 associated with the disease in the original Kostmann family. The region contains 37 genes. In paper V, cDNA microarrays were used to asses the mRNA levels of 7,700 genes in lymphoblastoid cell lines derived from autistic and control samples. The SEMA5A gene, which is involved in axonal guidance, was found downregulated in the cells derived from autistic individuals, and this was confirmed by quantitative PCR. In summary, candidate genes or gene regions have been identified for all four disorders and further studies are needed to confirm their roles in the pathogenesis of the disorders.

No description available.

La Leucoencefalopatía Megalencefálica con Quistes Subcorticales (MLC) es un tipo raro de leucodistrofia vacuolizante, que presenta como principales características clínicas macrocefalia, deterioro de las funciones motoras, epilepsia y retraso mental medio. Sin embargo, el diagnóstico de MLC se confirma mediante imágenes de resonancia magnética, donde el encéfalo se presenta atrofiado e hinchado, muestra una sustancia blanca anormalmente difusa y hay presencia de quistes subcorticales. Desde el punto de vista fisiopatológico, una biopsia obtenida de un paciente de MLC muestra la presencia de numerosas vacuolas situadas en las láminas más externas de la mielina. Se ha encontrado un primer gen responsable de la enfermedad en el 75% de los pacientes afectados, denominado MLC1. Se han descrito alrededor de 60 mutaciones, aunque existen pacientes que manifiestan las características clínicas de la enfermedad pero no presentan mutaciones en MLC1 ni presentan ligamiento con su locus, sugiriendo que existe al menos otro gen involucrado en la enfermedad. En el 25% de pacientes restantes, la enfermedad se manifiesta de dos maneras diferentes: en un caso, los enfermos presentan las mismas características clínicas que los pacientes con mutaciones en MLC1; y en el otro, presentan síntomas transitorios y los pacientes mejoran, llegando incluso a que la enfermedad remitiera. El gen MLC1 codifica para una proteína transmembrana que lleva el mismo nombre. Su función es todavía desconocida. Aunque muestra un bajo grado de homología con el canal de potasio Kv1.1 no se ha podido detectar actividad de canal iónico en diferentes sistemas heterólogos. No obstante, dicha homología, su confinamiento en la membrana plasmática y el fenotipo característico vacuolizante de los pacientes sugieren que la proteína podría estar mediando la translocación de iones a través de la superficie celular. El total desconocimiento del rol preciso de la proteína MLC1 ha imposibilitado el entendimiento del mecanismo patofisiológico de la enfermedad, y por ello, no se ha podido desarrollar ningún tratamiento efectivo para los pacientes afectados. Es por ello que nuestro grupo quiso apostar por estrategias innovadoras (combinación de bioquímica y genética) para poder encontrar otros genes responsables de la enfermedad. Usando técnicas de purificación por afinidad combinada con métodos de proteómica cuantitativa encontramos a GlialCAM como una proteína que estaba asociada con MLC1. Es por eso que decidimos estudiar (en colaboración) si los pacientes que no tenían mutaciones en MLC1 podían presentar mutaciones en GLIALCAM. Tras el análisis de 40 de estos pacientes encontramos que cuando los enfermos tenían las características clínicas típicas de MLC presentaban dos mutaciones en GLIALCAM (herencia recesiva); mientras que en el caso de aquellos que mejoraban a lo largo del tiempo, éstos solo presentaban una mutación (herencia dominante), demostrando que GLIALCAM es el segundo gen de MLC. En este estudio también se ha podido determinar que mutaciones dominantes en GLIALCAM podían también causar otras enfermedades como la macrocefalia familiar benigna y la macrocefalia con retraso mental, con o sin autismo. Estudios bioquímicos posteriores han permitido avanzar en el entendimiento de la relación que existe entre MLC1 y GlialCAM. Así se ha demostrado que GlialCAM actúa como una molécula escolta, necesaria para localizar específicamente a MLC1 en uniones celulares. De esta forma pudimos descubrir que las mutaciones en GLIALCAM provocaban un defecto en el tráfico de la proteína debido a una deficiente oligomerización. Como consecuencia, estas mutaciones provocaban la deslocalización de los complejos de MLC1-GlialCAM en las uniones astrocitarias. De forma interesante, GlialCAM permite estabilizar la proteína MLC1, sugiriendo nuevas aproximaciones terapéuticas para los pacientes afectos con MLC. Tras el descubrimiento de GlialCAM como segundo gen de MLC gracias a la aproximación proteómica, y tras comprobar que no todo GlialCAM estaba asociado a MLC1, nos planteamos volver a realizar estudios de proteómica para intentar encontrar posibles proteínas que pudiesen estar interaccionando con GlialCAM. De esta manera encontramos que el canal de cloruro ClC-2, estaba asociado con GlialCAM, y pudimos comprobar que GlialCAM también actuaba como molécula escolta para localizar específicamente a ClC-2 en las uniones entre células. Además, también era capaz de modificar sus propiedades de canal, así como aumentar su función, demostrándose interacción directa entre ambas proteínas. Igualmente que para el caso de MLC1, las mutaciones encontradas en GLIALCAM fallaban en la capacidad de concentrar a ClC-2 en las uniones astrocitarias. Por tanto, la función de GlialCAM podría ser necesaria para agrupar tanto a MLC1 como a ClC-2 en tales uniones, particularmente en los pies terminales astrocitarios, donde podrían estar llevando a cabo su función. ClC-2 podría ser necesario para desarrollar un flujo de Cl- transcelular o para compensar gradientes electroquímicos iónicos que pueden estar ocurriendo en dichas uniones durante cambios en la osmolaridad. El descubrimiento de GlialCAM como una subunidad auxiliar de ClC-2 incrementa la compleja regulación de este canal y proporciona nuevas ideas acerca del papel que ClC-2 puede estar desempeñando en las células gliales así como se sugiere que pueda estar involucrado en la fisiopatología de MLC. / Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by early-onset macrocephaly and delayed-onset neurological deterioration. Recessive MLC1 mutations are observed in 75% of patients with MLC. Genetic-linkage studies failed to identify another gene. We have showed that some patients without MLC1 mutations display the classical phenotype; others improve or become normal but retain macrocephaly. To find another MLC-related gene, we used quantitative proteomic analysis of affinity-purified MLC1 as an alternative approach and found that GlialCAM, an IgG-like cell adhesion molecule, is a direct MLC1-binding partner. Analysis of 40 MLC patients without MLC1 mutations revealed multiple different GLIALCAM mutations. Patients with the classical phenotype had two mutations, and patients with the improving phenotype had one mutation. In addition, patients with dominant GLIALCAM mutations, could also had macrocephaly and mental retardation with or without autism. Therefore, we found that GLIALCAM is the second gene found to be mutated in MLC. Furthermore, we demonstrated that GlialCAM functions as an MLC1 beta-subunit, needed for proper localization of MLC1 in cell-cell junctions. We also demonstrated that MLC1 and GlialCAM form homo- and hetero-complexes and that MLC-causing mutations in GLIALCAM mainly reduce the formation of GlialCAM homo-complexes, leading to a defect in the trafficking of GlialCAM alone to cell junctions. GLIALCAM mutations also affect the trafficking of its associated molecule MLC1, explaining why GLIALCAM and MLC1 mutations lead to the same disease: MLC. In this thesis, we also identify GlialCAM as a chloride channel ClC-2 binding partner. GlialCAM and ClC-2 colocalize in Bergmann glia, in astrocyteastrocyte junctions at astrocytic end-feet around blood vessels, and in myelinated fiber tracts. GlialCAM targets ClC-2 to cell junctions, increases ClC- 2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. Hence, we describes the first auxiliary subunit of ClC-2 and suggests that ClC-2 may play a role in the pathology of MLC disease.

Depuis déjà plusieurs décennies, nous sommes en mesure d'identifier les mutations responsable de diverses maladies mendéliennes. La découverte des gènes responsables de ces maladies permet non seulement un meilleur diagnostic clinique pour ces familles, mais aussi de mieux comprendre les mécanismes physiopathologiques de ces maladies ainsi que mieux définir la fonction normale des gènes causales. Ultimement, ces découvertes mènent à l'identification de cibles thérapeutiques pour le traitement de ces maladies. Les progrès technologiques sont depuis toujours un facteur très important dans la découverte de ces gènes mutés. De l'approche traditionnelle de clonage positionnel en passant par la première séquence du génome humain et maintenant les technologies de séquençage à grande échelle, de plus en plus de maladies ont maintenant une entité génétique. Dans le cadre de ce projet de doctorat, nous avons utilisé tant les approches traditionnelles (leucodystrophies) que les nouvelles technologies de séquençage (polyneuropathie douloureuse) qui ont mené à l'identification du gène causal pour plusieurs de nos familles. L'efficacité de ces deux approches n'est plus à démontrer, chacune d'entre elles possèdent des avantages et des inconvénients. Dans le cadre de ces projets, nous avons utilisé la population canadienne-française connue pour ces effets fondateurs et la présence, encore aujourd'hui, de grandes familles. Les différents projets ont permis d'établir certains avantages et inconvénients quant à l'utilisation de ces techniques et de la population canadienne-française. Dans le cadre d'un phénotype assez homogène et bien défini comme celui du projet leucodystrophie, l'approche traditionnel par gène candidat nous a permis d'identifier le gène causal, POLR3B, sans trop de difficulté. Par contre, pour les autres projets où nous sommes en présence d'une hétérogénéité clinique et génétique une approche non-biaisée utilisant le séquençage exomique a obtenu un plus grand succès. La présence de grandes familles est un grand avantage dans les deux approches. Dans le projet polyneuropathie douloureuse, une grande famille originaire du Saguenay-Lac-St-Jean nous a permis d'identifier le gène NAGLU comme responsable suite à l'exclusion des autres variants candidats par analyse de ségrégation. Comme NAGLU était déjà associé à un phénotype qui diffère sur plusieurs points à celui de notre famille, une approche traditionnelle n'aurait pas été en mesure d'identifier NAGLU comme le gène causal. Dans l'analyse de nos données de séquençage exomique, nous avons observé que plusieurs variants rares, absents des bases de données, étaient partagés entre les différents individus Canadiens français. Ceci est probablement dû à la démographie génétique particulière observée chez les Canadiens français. En conclusion, les technologies de séquençage à grande échelle sont avantageuses dans l'étude de maladies hétérogènes au niveau clinique et génétique. Ces technologies sont en voie de modifier l'approche d'identification de gènes en permettant une analyse de génétique inversée, c'est-à-dire de la génétique vers la clinique. / Since many decades we are able to identify mutations responsible for Mendelian diseases. The identification of the causative gene not only allows a better diagnostic to these families, but also allows a better understanding of the pathophysiological mechanisms of these disorders and of the normal function of a gene. Ultimately, those discoveries lead to the identification of therapeutic targets that will enable clinicians to treat these diseases. Technological progress has forever driven gene identification. Starting with traditional approaches like positional cloning passing through the first sequence of the human genome and finally the recent high-throughput sequencing technologies, more and more diseases are now linked to a genetic cause. During my PhD, I had the opportunity to use traditional approaches (leukodystrophy) as well as new sequencing technologies (painful sensory polyneuropathy) which both led to gene identification for some of our families. The efficiency of these approaches is well known, each of them has advantages and disadvantages. In these projects, we used the french-canadian population well known for its founder effect and the presence, still today, of large families. Working on these projects allowed us to establish advantages and disadvantages concerning the use of those technologies and the french-canadian population. In the presence of a homogenous and well defined phenotype, like the leukodystrophy project, the traditional approach enables us to rapidly identify the causative gene (POLR3B). On the other hand, the unbiased exome sequencing approach has had more success for diseases characterized with clinical and genetic heterogeneity. Large families are a great advantage for both methods. In the painful sensory polyneuropathy project, a large family originated from the Saguenay-Lac-St-Jean region allowed us to identify the NAGLU gene as responsible for the disease after exclusion of the candidate variants by segregation analysis. NAGLU has already been associated with a phenotype that differs in many points with the clinical features observed in our family. In this case a traditional approach would have failed to identify NAGLU as the causative gene. In the analysis of our exome sequencing results, we observed many rare variants absent from databases but shared between french-canadian individuals. This enrichment in rare variants is probably due to the particular genetic demography of Quebec. In conclusion, high-throughput sequencing technologies are advantageous in the study of clinically and genetically heterogeneous diseases. These technologies are changing the gene identification approach towards reverse genetics, meaning genetics towards clinic.

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