The distal hereditary motor neuropathies (dHMN) are a clinically and genetically heterogeneous group of disorders that primarily affect motor neurons, without significant sensory involvement. Using genome wide linkage analysis in a large Australian family (CMT54), a form of dHMN was previously mapped by this laboratory, to a 12.98 Mb interval on chromosome 7q34-q36. The axonal neuropathy seen in this family was classified as dHMN1; with autosomal dominant inheritance, early but variable age of onset, and muscle weakness and wasting affecting the lower limbs.
In this project, genetic linkage analysis of the chromosome 7q34-q36 disease interval was carried out in the original family (CMT54) and 20 smaller families from an Australian dHMN cohort. Fine mapping in family CMT54, including unaffected individuals suggested a minimum probable candidate interval of 6.92 Mb, flanked by markers D7S615 and D7S2546 within the 12.98 Mb critical disease interval. Of the additional dHMN families, one (family CMT44) achieved suggestive linkage to the chromosome 7q34-q36 disease locus with a LOD score of 2.02.
Mutation screening was carried out in family CMT54 at the chromosome 7q34-q36 locus. The 12.9 Mb disease interval contains 89 annotated protein-coding genes, of which 60 lay within the prioritised 6.92 Mb interval. A combination of methods was used to screen these genes for a putative pathogenic mutation. Functional candidate genes were identified via a literature and database search. The coding exons of 35 prioritised candidate genes were sequenced and no pathogenic mutation was identified. Cytogenetic analysis excluded large scale chromosomal abnormalities. Array based comparative genomic hybridisation of the 7q34-q36 interval in patients did not identify any pathogenic duplications or deletions.
Next generation sequencing (NGS) techniques were used to identify sequence variants within the remaining genes within the 7q34-q36 interval and elsewhere in the genome. Two NGS based approaches were applied to mutation screening in family CMT54. Initially, the chromosome 7q34-q36 disease interval was analysed in one affected individual using a custom designed DNA capture microarray and 454 GS FLX (Roche) sequencing. Approximately 80% of patient coding exons were captured, sequenced and no pathogenic mutations were identified. The chromosome 7q34-q36 target captured DNA sample was also re-sequenced along with an additional two affected individuals and one unaffected parent using exome capture and Solexa (Illumina) sequencing. Combined, 99.5% of coding exons were sequenced in the chromosome 7q34-q36 interval and all sequence variants that were identified were excluded from a pathogenic role. Sequence variants identified elsewhere in the exome were also excluded from a pathogenic role.
Exome sequencing of dHMN family CMT44 did not identify any putative pathogenic mutation at the chromosome 7q34-q36 locus. The exomes of four affected and one unaffected individuals were sequenced. Exome wide analysis identified a potential digenic inheritance in CMT44 of a previously published MFN2 mutation causing a mild CMT2 phenotype and a second mutation causing a dHMN phenotype. Potential candidate mutations for dHMN were identified in two genes, PCDHGA4 and DNAH11. PCDHGA4, was previously shown to function in the brain and spinal cord, and deletion of PCDHG genes in a mouse model causes a severe neurodegenerative phenotype.
The gene mutation causing dHMN that maps to chromosome 7q34-q36 remains to be identified. The disease mutation may lie in a coding region not captured by current exome platforms, a non-coding region, or the mutation may cause disease through an alternate mechanism not detected by the methods employed in this thesis. Future studies should concentrate on transcriptome analysis by next-gen RNA sequencing, which may identify unknown transcripts and exons that map to chromosome 7q34-q36 or highlight sequence variants located in regulatory elements.
Identification of new gene mutations is critical to further understanding the biochemical and cellular processes underlying dHMN. Although the causative mutation for dHMN on 7q34-q36 was not identified, a significant proportion of the disease interval has been excluded using a combination of traditional and new technologies.
The purpose of this thesis is to identify new gene mutations causing dHMN. The genetic and functional data presented here suggest this will be a difficult task; the genetic heterogeneity complicates genetic analysis and the multiple molecular mechanisms implicated to date make it difficult to pinpoint specific candidate genes. The identification of additional genes and genetic modifiers is necessary to increase our understanding of the disease mechanisms causing dHMN and related neuropathies. This will directly aid in the diagnosis and classification of these neurodegenerative diseases and may lead to new therapeutics and treatment strategies.