Analysis of dystrophin point mutations

Foskett, Pierre (2005) Analysis of dystrophin point mutations. MSc(R) thesis, University of Glasgow.

Abstract

Point mutation in the dystrophin gene accounts for around 20-35% of all cases of Duchenne and Becker muscular dystrophies. Detection of the causative mutation in each case allows accurate genetic advice to be given to families, opens up the possibility of using emerging therapies and gives insight into the pathology of the disorder in each case. Novel reports of mutation/polymorphism frequency and type will add to the knowledge of the dystrophin gene and protein function. The overall aims of this study were to search for and characterise point mutations in the dystrophin gene. This involved the development of a rapid protocol to analyse all 79 dystrophin coding exons that could be used in a clinical setting. Robotic PCR followed by automated dHPLC of a single patient sample at a time identified amplicons containing variant sequences. Sequencing was then used to characterise the changes in the nucleotide sequence in each of these fragments by comparison to a normal control. All the data was analysed and stored in a patient specific database. Restriction digests were designed to confirm each of the mutations and the inheritance of one mutation was studied within several pedigrees. Genotype-phenotype associations in dysfrophin were assessed based on reports of increased frequency of mutation in the isoform Dp71 (exons 63-79) in individuals with mental retardation. No increase in the frequency of mutation within the brain specific franscript was observed in a cohort of individuals with learning difficulties, suggesting that the distribution of cognitive impairment in affected individuals is too broad to screen this region alone in these cases. In this study the sensitivity of dHPLC for the coding region was shown to be equal to sequencing. A study into the incidence of mutation within a single exon in a cohort of 46 individuals with DMD/BMD showed that a single mutation within exon 70 occurred three times at the same hypermutable CpG dinucleotide. Haplotype studies using microsatellite markers and SNP analysis showed that the 3 mutations most likely originated on independent backgrounds. This was confirmed by analysis of mitochondrial HV1 haplotypes showing that a greater degree of similarity existed with a random control population than between the patients with the same mutation. This evidence suggests that certain gene regions may have an increased incidence of mutations. To assess variance across the dystrophin gene: all 79 coding exons of the muscle transcript Dp427 were examined in a total of 22 patients with DMD and 1 with BMD. The overall sensitivity of this technique was 96% for recognised and potentially pathogenic mutations. Several novel mutations were found, nonsense mutations: 9100C>T, 5159T>A, 754G>T, 5255T>G, 1357C>T, 5089C>T the splice site mutation: 1150-1G>T and deletion insertion mutations: 10205delA, 8438delC, 8405delC, 3036-3037delinsC1021-1022insC and 3201delT. Previously reported mutations were: 10171C>T (3 times), 313A>T, 9337 C>T (all nonsense changes). Two single codon deletions were identified - a type of change reported only once before in the literature. 10099-101 OldelGAA has been seen once before and is associated with a less severe form of DMD. This variant is believed to affect an uncharacterised region of the C-terminal section of the protein. The second single codon deletion is novel: 482-484delCCA is expected to delete an amino acid within an internal a- helix of the actin-binding domain in the N-terminal of dystrophin. This variant is believed to affect the binding affinity of dystrophin to actin and is associated with a BMD phenotype in this study. Two uncharacterised intronic variants were also identified: 1332- 9A>G is a suggested cryptic splice site mutation within intron 11 and has been reported once before; 7098+105_7098+106_insTATTTAATACTTTG is a novel intronic change shown to be absent in over 500 chromosomes from the same population. Splice site and ESE/ESS analysis of both these changes was performed and has not been reported before. Polymorphism frequency within the study population was described with the identification of the novel variants: 358-80T>C, 5448+168A>T and 8669-750G. One previously uncharacterised complex variant is reported as a polymorphism in this study (8729_8734delinsTGGTCG). It was also shown that mutation is associated with stretches of repeated sequences and CpG dinucleotides distributed throughout the coding sequence. Analysis for point mutations in the dystrophin gene of patients with DMD/BMD identified several novel variants. Characterisation of these variants has contributed to the understanding of dysfunction in the dysfrophin gene and identified areas for further study. PCR and dHPLC was shown to be a rapid and sensitive means of analysing genetic sequence, suitable to large genes with infrequent (but often urgent) referrals.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Keywords: Genetics.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Boxer, Dr. Maureen and Connor, Prof. Michael
• Date of Award: 2005
• Depositing User: Enlighten Team
• Unique ID: glathesis:2005-71488
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 May 2019 09:31
• Last Modified: 12 Aug 2021 15:24
• URI: https://theses.gla.ac.uk/id/eprint/71488