Genotype/ phenotype correlation of two neuropsychiatric diseases. A genetic study of CDG1a and Rett syndrome

Abstract: The aim of this thesis was to investigate the effects of mutations in the genes that cause the two neuropsychiatric diseases congenital disorder of glycosylation (CDG) type Ia and Rett syndrome. These two diseases affect children's central and peripheral nervous systems, causing mental retardation and neuronal impairment. CDG type Ia patients also have cerebellar atrophy and, in most cases, liver dysfunction and skeletal abnormalities. CDG type Ia is a recessive autosomal disease. The gene involved in the pathogenesis of CDG type Ia is phosphomannomutase 2 (PMM2) located in chromosome region 16p13. Rett syndrome debutes after 6-18 months of normal development, and one of the most intriguing features of the disease is that it affects females almost exclusively. As the disease proceeds, they develop autistic behaviour, learning disabilities and characteristic hand-wringing movements, resembling hand washing. The majority of Rett syndrome cases are sporadic, and inheritance is dominant X-linked in the few familial cases. The methyl CpG-binding protein 2 gene (MECP2), located in chromosome region Xq28, contains mutations in approximately 80% of Rett patients.In summary, we detected 20 different mutations in a comprehensive mutation screening of PMM2 in a large group of CDG type Ia patients. We found PMM2 mutations with DNA sequencing and single-stranded conformation polymorphism (SSCP) in more than 95% of the patients. The dominating genotype was 357C>A/ 422G>A. The mutations detected in PMM2 were then used in an evaluation of the recently developed mutation detection technique denaturing high-performance liquid chromatography (DHPLC). We confirmed 19 of 20 mutations with DHPLC, and concluded that this technique is suitable for a first screening of PMM2 in CDG type Ia patients. Subsequenty, we performed a genotype/ phenotype correlation study with essentially the same group of patients. We were able to report some phenotypic trends in patient groups with the same mutation, i.e. mutation 548T>C correlated with mild disease, and mutation 691G>A correlated with severe disease. In contrast, the most common genotype (357C>A/ 422G>A) was related to the whole spectrum of clinical presentations. The enzyme activity of PMM, measured in fibroblasts from patients, did not correlate to either genotype or phenotype. Our haplotype study, combined with data on the geographical origin of the patients, resulted in the location of two mutations to regions in southern and mid-eastern Sweden, most likely as a result of founder mutations (common ancestry).Furthermore, we performed a mutation screening of MECP2 with DNA sequencing in 16 classical Rett women, and found that 13 had de novo MECP2 mutations. Three of these were novel, and three were hot-spot mutations. In order to improve the mutation detection rate, we tested another mutation detection method, multiplex ligation-dependent probe amplification (MLPA). Using this method, we found large deletions in three of eleven classical Rett patients, who had previously been considered to lack mutations, when analysed with DNA sequencing. We concluded that MLPA is useful as a complement to DNA sequencing for finding mutations in Rett patients.