Molecular analysis of the Xlinked CMT (CMTX3) disease locus using NGS technologies
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Type
ThesisThesis type
Doctor of PhilosophyAuthor/s
Chaudhry, Rabia KhalidAbstract
Charcot-Marie-Tooth disease (CMT) is a genetically and clinically heterogeneous disorder of the peripheral nervous system affecting the motor and sensory neurons. X-linked CMT (CMTX) accounts for 15-20% of all cases making it the second most commonly inherited CMT. There are 6 ...
See moreCharcot-Marie-Tooth disease (CMT) is a genetically and clinically heterogeneous disorder of the peripheral nervous system affecting the motor and sensory neurons. X-linked CMT (CMTX) accounts for 15-20% of all cases making it the second most commonly inherited CMT. There are 6 reported loci (CMTX1, CMTX2, CMTX3, CMTX4, CMTX5 and CMTX6). Four genes, GJB1, AIFM1, PRPS1 and PDK3 are known to cause CMTX1, CMTX4, CMTX5 and CMTX6 respectively. The pathogenic cause for CMTX2 and CMTX3 remains unknown. In this study we re-analysed the three families (CMT623, CMT193-ext and US-PED2) that reported linkage to the CMTX3 interval on chromosome Xq26.3–27.1. To identify the pathogenic cause for CMTX3 disease several applications of next generation sequencing technologies including targeted capture, whole exome sequencing and RNA-sequencing were implemented to examine both the genome and transcriptome of CMTX3 patients. Targeted capture and sequence analyses of the CMTX3 interval identified a novel unreported non-coding variant (SNV7) located in the intergenic region that segregated with the disease phenotype in the two CMTX3 families (CMT623 and CMT193-ext) that were significantly linked to the CMTX3 interval. Bioinformatics analysis suggested the variant may reside in a regulatory element and possibly disrupt the binding of transcription factors required for regulation of genes important for axonal maintenance. The genomic region surrounding SNV7 was assessed for transcription properties by a reporter assay, however results suggested the variant may not have a transcriptional role in neuronal cells. In addition to the reporter assay studies, conservation analysis of the variant showed the region is not conserved which further suggests the region harbouring the SNV7 variant may not have a functional consequence. Although further studies are required to ensure the variant does not have a functional role in causing disease, it is possible the variant may represent a non-pathogenic rare polymorphism that segregates with the disease phenotype due to linkage disequilibrium within the CMTX3 linkage interval. As an alternative method to find the pathogenic cause for CMTX3 disease the transcriptome of an affected patient was analysed by RNA-seq technologies. Preliminary validation studies of the transcripts expressed and able to be detected in fibroblasts identified low expression of the FGF13 gene in the CMTX3 patients when compared to a normal control. Recent studies have shown that FGF13 is important for the architectural development of neurons and disruptions in the protein levels can affect the polarisation of neurons. Whilst the expression of FGF13 gene requires further validation it is possible that this gene is disrupted in CMTX3 disease. One of the key and unexpected findings of this study was the identification of a reported BSCL2 mutation (p.Asn88Ser) in the original CMTX3 (US-PED2) family using whole exome sequencing. Despite not identifying a mutation in the CMTX3 interval this family will be revisited once the pathogenic cause is discovered for the two large CMTX3 families showing significant linkage to the interval. Moreover, this discovery demonstrates the power of next generation sequencing technologies as a tool to identify gene mutations for small families in the absence of statistically significant linkage data. The search for the CMTX3 gene continues, however in this current study the use of next generation sequencing technologies has been instrumental in identifying a novel candidate SNV and a gene with altered expression which may have a role in CMTX3 disease. In addition to these findings this study has highlighted that the pathogenic cause is not likely to reside in the coding region of the CMTX3 interval. Therefore, in the event SNV7 and FGF13 gene are excluded for pathogenicity, future experiments and research strategies should focus on examining the non-coding region of the interval for potential structural variations as a cause for CMTX3 disease.
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See moreCharcot-Marie-Tooth disease (CMT) is a genetically and clinically heterogeneous disorder of the peripheral nervous system affecting the motor and sensory neurons. X-linked CMT (CMTX) accounts for 15-20% of all cases making it the second most commonly inherited CMT. There are 6 reported loci (CMTX1, CMTX2, CMTX3, CMTX4, CMTX5 and CMTX6). Four genes, GJB1, AIFM1, PRPS1 and PDK3 are known to cause CMTX1, CMTX4, CMTX5 and CMTX6 respectively. The pathogenic cause for CMTX2 and CMTX3 remains unknown. In this study we re-analysed the three families (CMT623, CMT193-ext and US-PED2) that reported linkage to the CMTX3 interval on chromosome Xq26.3–27.1. To identify the pathogenic cause for CMTX3 disease several applications of next generation sequencing technologies including targeted capture, whole exome sequencing and RNA-sequencing were implemented to examine both the genome and transcriptome of CMTX3 patients. Targeted capture and sequence analyses of the CMTX3 interval identified a novel unreported non-coding variant (SNV7) located in the intergenic region that segregated with the disease phenotype in the two CMTX3 families (CMT623 and CMT193-ext) that were significantly linked to the CMTX3 interval. Bioinformatics analysis suggested the variant may reside in a regulatory element and possibly disrupt the binding of transcription factors required for regulation of genes important for axonal maintenance. The genomic region surrounding SNV7 was assessed for transcription properties by a reporter assay, however results suggested the variant may not have a transcriptional role in neuronal cells. In addition to the reporter assay studies, conservation analysis of the variant showed the region is not conserved which further suggests the region harbouring the SNV7 variant may not have a functional consequence. Although further studies are required to ensure the variant does not have a functional role in causing disease, it is possible the variant may represent a non-pathogenic rare polymorphism that segregates with the disease phenotype due to linkage disequilibrium within the CMTX3 linkage interval. As an alternative method to find the pathogenic cause for CMTX3 disease the transcriptome of an affected patient was analysed by RNA-seq technologies. Preliminary validation studies of the transcripts expressed and able to be detected in fibroblasts identified low expression of the FGF13 gene in the CMTX3 patients when compared to a normal control. Recent studies have shown that FGF13 is important for the architectural development of neurons and disruptions in the protein levels can affect the polarisation of neurons. Whilst the expression of FGF13 gene requires further validation it is possible that this gene is disrupted in CMTX3 disease. One of the key and unexpected findings of this study was the identification of a reported BSCL2 mutation (p.Asn88Ser) in the original CMTX3 (US-PED2) family using whole exome sequencing. Despite not identifying a mutation in the CMTX3 interval this family will be revisited once the pathogenic cause is discovered for the two large CMTX3 families showing significant linkage to the interval. Moreover, this discovery demonstrates the power of next generation sequencing technologies as a tool to identify gene mutations for small families in the absence of statistically significant linkage data. The search for the CMTX3 gene continues, however in this current study the use of next generation sequencing technologies has been instrumental in identifying a novel candidate SNV and a gene with altered expression which may have a role in CMTX3 disease. In addition to these findings this study has highlighted that the pathogenic cause is not likely to reside in the coding region of the CMTX3 interval. Therefore, in the event SNV7 and FGF13 gene are excluded for pathogenicity, future experiments and research strategies should focus on examining the non-coding region of the interval for potential structural variations as a cause for CMTX3 disease.
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Date
2014-08-25Licence
The author retains copyright of this thesis. It may only be used for the purposes of research and study. It must not be used for any other purposes and may not be transmitted or shared with others without prior permission.Faculty/School
Sydney Medical School, Concord Clinical SchoolAwarding institution
The University of SydneyShare