Assessment of copy number variations in the nebulin gene and other nemaline myopathy-causing genes
Year: 2015
Type: dissertation
Study design: Observational Study
Abstract: Nemaline myopathy (NM) and related disorders constitute a heterogeneous group of congenital myopathies. Mutations in the nebulin gene (NEB) are the main cause of the recessively inherited form. NEB is one of the largest genes in the human genome consisting of 249 kb of genomic sequence. NEB contains 183 exons and a 32 kb homologous triplicate region (TRI) where eight exons are repeated three times. The aims of this Doctoral Thesis study were to develop and implement into diagnostics new efficient variant analysis methods for NEB and other NM-causing genes. The first aim was to design and validate a custom copy number microarray targeting the NM-causing genes for the detection of copy number variations. MLPA (multiplex ligation-dependent probe amplification) and Sanger sequencing were also used. The second aim was to utilise whole-exome sequencing to search for novel disease-causing variants in the known NM genes and try to identify novel NM genes. Lastly, the aim was to collect more data in order to try to find genotype-phenotype correlations of NEB-caused NM. The design and validation of the NM-CGH microarray was successful. Of the total sample cohort of 356 NM families, 196 NM families were studied using the custom-made NM-CGH array. Nine different novel large causative variants were identified in ten NM families. The size of these variants varies greatly, covering only a part of one NEB exon on up to dozens of NEB exons (72bp - 133 kb). In addition, a novel recurrent variation of the NEB TRI region was identified in 13% of the NM families and in 10% of the studied 60 control samples. Deviations of one copy are suggested to be benign but gains of two or more copies might be pathogenic. One novel homozygous deletion was also identified in another NM gene, TPM3, in a patient with severe NM. Furthermore, ten samples were studied using exome sequencing, and for six of those samples, novel disease-causing variant(s) were identified. Two variants were identified in one family in a novel, putative NM gene that is currently under further investigation. 165 NM families from the total cohort of 356 NM families have been identified thus far with two pathogenic NEB variants. Altogether 220 different pathogenic variants were identified in these 165 families, accentuating that the patients in the majority (84%) of the families are compound heterozygous for two different NEB variants. Most of the variants are small single nucleotide changes whereas large variants are more rare. However, copy number variations are much more frequent than previously thought: pathogenic copy number variants were identified in 16% of these 356 NM-families. Genotype-phenotype correlations between the type of NEB mutation and the NM subtypes remained, however, unobtainable. The NM-CGH microarray has been implemented into molecular diagnostics of NM. Using the NM-CGH microarray followed by exome-sequencing has accelerated mutation detection. This combination has increased the coverage of the NM genes and thus improved the diagnostics of NM and NM-related disorders.
Source: Työväentutkimus Vuosikirja
Author: K. Kiiski
Language: English
Topic: Genomics and Rare Diseases
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1915016762