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Clinical and Molecular Genetic Aspects of Hereditary Spastic Paraplegia

Kirsten Svenstrup  


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Accepted by: Faculty of Health Sciences University of Copenhagen
Defended on: August 28, 2009
Official opponents: Gunhild Waldemar , Chantal Tallaksen , Zeynep Tümer
Tutors: Lis Hasholt , Jørgen E. Nielsen

Published in the PhD Database: September 2, 2009


English abstract
Hereditary spastic paraplegia (HSP) is a group of rare, inherited, neurodegenerative diseases with the most prominent features being insidiously progressive spasticity and weakness in the lower limbs. These symptoms present alone in the pure form, whereas additional neurological symptoms such as mental retardation, ataxia and peripheral neuropathy may be present in the complex form. HSP is both clinically and genetically very heterogeneous. The inheritance pattern may be autosomal dominant, autosomal recessive, or X-linked. Thirty-eight loci associated with HSP have been found, and 19 of the underlying genes have been identified. It is possible that different proteins, encoded by these genes, can interact in a molecular network, and that changes in these different proteins can result in overlapping phenotypes.

This study from Section of Neurogenetics, Department of Cellular and Molecular Medicine and Memory Disorders Unit, Rigshospitalet, is based on clinical and molecular-genetic investigations of 104 index patients with HSP. The aim of the project was to characterize the mutation spectrum, describe phenotype-genotype relations, and to search for new genes in which changes could influence occurrence of HSP.

DNA samples from HSP patients were screened for disease-causing mutations in selected HSP genes (SPAST, SPG3A, HSPD1 and PLP1) using direct sequencing and multiplex ligation-dependent probe amplification (MLPA). Identification of sequence changes caused further clinical neurological characterization of the families in addition to paraclinical investigations in terms of MRI, MRS, PET scan, and neurophysiological investigation. Families with disease-causing sequence changes in PLP1 (one family), HSPD1 (one family), SPG3A (3 families) and in SPAST (16 families) were identified and characterized. Six disease-causing changes were not previously published, and further molecular genetic investigation was applied in these cases in order to interpret the causative effect. In SPAST, potentially pathogenic synonymous changes were identified in a region encoding a cryptic promoter.

In addition, selected candidate genes for HSP were screened. A missense variant was identified in VPS4B in a patient belonging to a family with autosomal dominant inheritance, and segregation between the variant and a variable phenotype with neurodegenerative features was shown. The variant was also found in one of 1320 control chromosomes. VPS4B was cloned into a plasmid and tagged with a reporter gene. The change was introduced into the gene by mutagenesis and the mutant VPS4B protein was expressed in cell-culture. The variant resulted in changed localisation of the protein in the cell, indicating a possible change of the function of the protein.

The study describes new genotype-phenotype relations for several specific changes in HSP genes, it contributes to a better understanding of the molecular background for HSP and it provides an extended knowledge of clinical and paraclinical consequences of specific changes. The study contributes to a more differentiated diagnostic foundation resulting in improved genetic counselling for HSP patients in the future.



Danish abstract
Hereditær spastisk paraparese (HSP) er en gruppe af sjældne, arvelige, neurodegenerative sygdomme, hvor de dominerende symptomer er langsomt fremadskridende spasticitet og parese i benene. De kliniske manifestationer forekommer alene i den såkaldte rene form, mens de i den komplekse form kan ledsages af andre neurologiske symptomer og fund, såsom mental retardering, ataxi og perifer neuropati. HSP er en både klinisk og genetisk meget heterogen sygdomsgruppe. Arvegangen kan være både autosomal dominant, autosomal recessiv og X-bunden. Der er fundet 38 loci, som er associeret med HSP, og man kender nu 19 af de tilgrundliggende gener. Det er muligt, at vidt forskellige proteiner, som disse gener koder for, interagerer i et molekylært netværk, og at ændringer i de forskellige proteiner kan give anledning til fænotyper, der ligner hinanden.

Denne afhandling, som udgår fra Sektion for Neurogenetik, Institut for Cellulær og Molekylær Medicin og Hukommelsesklinikken, Rigshospitalet, er baseret på kliniske og molekylær-genetiske undersøgelser af 104 probander med HSP. Formålet med projektet har været at karakterisere mutations-spektret, beskrive fænotype-genotype relationer og søge efter nye gener, hvori ændringer kan have betydning for forekomst af HSP.

DNA fra HSP patienter blev undersøgt for sygdomsfremkaldende mutationer i udvalgte kendte HSP gener (SPAST, SPG3A, HSPD1 og PLP1) ved hjælp af direkte sekventering og multiplex ligation-dependent probe amplification (MLPA). I de tilfælde hvor en mutation blev identificeret, karakteriseredes familien nærmere klinisk neurologisk samt i udvalgte tilfælde paraklinisk med MRI, MRS, PET scanning og neurofysiologisk undersøgelse. Familier med sygdomsfremkaldende ændringer i PLP1 genet (én familie), i HSPD1 genet (én familie), i SPG3A genet (3 familier) og i SPAST genet (16 familier) blev på denne måde identificeret og karakteriseret. Seks af de sygdomsfremkaldende mutationer var ikke tidligere rapporteret, og disse var genstand for yderligere molekylær-genetisk undersøgelse med henblik på vurdering af en potentielt sygdomsfremkaldende effekt. I SPAST genet blev potentielt patogene synonyme ændringer i en region, der koder for en kryptisk promoter, identificeret.

Desuden blev et udvalg af kandidat-gener for HSP screenet. I VPS4B genet fandt vi en missense ændring i en HSP patient fra en familie med autosomal dominant arvegang, og ændringen segregerede med et heterogent neurodegenerativt symptombillede i familien. Den blev desuden fundet i ét af 1320 kontrol kromosomer. VPS4B blev ved kloning indsat i et plasmid i fusion med et reporter gen. Ændringen blev introduceret i genet ved mutagenese og udtrykt i cellekultur. Ændringen gav anledning til en ændret lokalisation af VPS4B proteinet i cellen, hvilket indikerer, at den kan have betydning for proteinets funktion.

Studiet bidrager med beskrivelse af en udvidet fænotype-genotype relation for flere specifikke ændringer i kendte HSP gener og bidrager yderligere til bedre forståelse af den molekylære baggrund for HSP samt bedre kendskab til kliniske og parakliniske konsekvenser af specifikke ændringer. Hermed muliggøres en mere nuanceret tilgang til diagnostik og genetisk rådgivning af patienter med HSP i fremtiden.