Molecular Pathology Detection Strategies for Three Autosomal Dominant Neurodegenerative Diseases

Elshafey, Alaa E (1996) Molecular Pathology Detection Strategies for Three Autosomal Dominant Neurodegenerative Diseases. PhD thesis, University of Glasgow.

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The present study aimed to optimise mutation detection strategies for three autosomal dominant neurological diseases, myotonic dystrophy (DM), amyotrophic lateral sclerosis (ALS) and tuberous sclerosis complex (TSC). (A) Myotonic dystrophy: After exploring different methods that have been used for the detection of CTG repeat expansions in DM patients, a strategy was chosen, optimised and applied to screen 49 DM families (86 DM affected and 96 apparently normal individuals). Using published primer sets, both radiolabelled and non-radiolabelled PCR amplification of the area containing the CTG repeat were optimised using the published conditions as a starting point. After PCR optimisation, DM alleles carrying up to 90 CTG repeats were properly amplified however, amplification of > 90 CTG repeats was not possible due to PCR limitations. To detect such expansions, Southern blot analysis using the BglI enzyme and the p5B1.4 DNA probe was optimised. The running time and the voltage used for gel electrophoresis were modified to get clear separation of the second band that represents the expanded CTG repeats. Meanwhile, normal individuals showed only a single band so that they were not confused with the affected persons. Using this strategy, all CTG repeat expansions between 50 and several thousands were detected. Analysis of the results obtained revealed the following: 1) A correlation between the intergenerational repeat expansion and the patient phenotype giving legitimacy to the phenomenon of anticipation. 2) Two cases of reduction of the repeat size upon paternal transmission. Since the age of both asymptomatic daughters were younger than the age at onset of the disease in their fathers, it was not possible to anticipate their clinical outcome. 3) Lastly, a severely affected child with mental retardation and onset in infancy was found to be paternally transmitted. (B) Amyotrophic lateral sclerosis: Two mutations in the superoxide dismutase (SOD-1) gene, Ala4Val and Ilell3Thr, were previously shown to be prevalent among familial amyotrophic lateral sclerosis (FALS) patients. These two mutations changed restriction enzyme recognition sites, so that restriction digestion of the appropriate PCR products was optimised and used to screen for their presence in the studied amyotrophic lateral sclerosis patients. SSCP analysis was also optimised and applied as a screening method for detection of unknown mutations in the SOD-1 gene using DNA samples from 2 familial and 67 sporadic amyotrophic lateral sclerosis (ALS) patients. Any experiment with a positive SSCP screening result was repeated and a few false positive results due to PCR errors and/or errors in the gel preparations, loading or electrophoresis were detected. A reproducible SSCP band shift was detected in exon 4 from one of the two familial cases. Sequencing of that exon revealed a G277?C point mutation which caused a Gly93Arg missense change. This mutation was confirmed to be present in all affected members of that family. Gly93 is a neutral and polar amino acid and is highly conserved among 18 different species and it was substituted by the basic arginine. Mapping of Gly93 to the crystallographic structure of the SOD-1 gene revealed that it is one of the critical glycine residues that allow main chain conformation and packing interactions so that a mutation affecting this residue should have a deleterious effect on the conformation and stability of the enzyme dimer. In this FALS family, the affected members showed an early age of onset of the disease (26-40 years). Analysis of the results obtained revealed that: 1) In the two FALS families screened one SOD-1 mutation was detected in the two screened FALS patients. 2) No SOD-1 mutations could be detected in any of the sporadic cases. These results suggested that other gene(s) may be involved in the familial form of the disease and make it unlikely that SOD-1 mutations are major determinants of sporadic ALS. (C) Tuberous sclerosis: In order to screen the tuberous sclerosis complex (TSC) patients for point mutations within the TSC2 gene, chemical cleavage of the mismatch (CCM) analysis was optimised and applied to four RT-PCR amplified (from 22 patients) and two DNA PCR amplified (from 32 patients for one segment and from 10 patients for the other segment) TSC2 segments. These segments were chosen as they include proposed important functional parts of the gene. Using this approach, nine cleavage products were detected from the screened patients. Sequence analysis of the corresponding cDNA and/or DNA segments revealed three missense mutations in three sporadic TSC patients and three polymorphic changes. No mutations could be detected in the screened promoter area of the gene. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J M Connor
Keywords: Genetics, Molecular biology, Neurosciences
Date of Award: 1996
Depositing User: Enlighten Team
Unique ID: glathesis:1996-74917
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 15:12
Last Modified: 27 Sep 2019 15:12

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