Studying the Genetics of Charcot-Marie-Tooth Neuropathy

James Radke

Charcot-Marie-Tooth (CMT) neuropathy is an inherited disorder affecting the peripheral motor and sensory neurons. Although it is an inherited condition, the main signs and symptoms such as decreased muscle size and weakness may not present until adolescence of early adulthood.

Signs and symptoms of Charcot-Marie-Tooth disease vary greatly but may include:
At present, there is no cure for CMT and much of the research is focused on the 80+ genes associated with the disease. In this exclusive interview with Rare Disease Report, Marina Kennerson PhD of the ANZAC Research Institute and Principal Research Fellow at Sydney Medical School talks about some of the research her lab is conducting to better understand the genetics of CMT and its possible implications to other neuropathies.

RDR:  What is Charcot-Marie-Tooth (CMT) neuropathy?

Dr Kennerson: CMT is an inherited disorder of the peripheral nervous system. The motor and sensory neurons are affected. Patients present with muscle wasting of legs and arms, foot deformities and sensory symptoms. It is the most common disorder presenting in neuromuscular clinics affecting 1 in 2500 individuals. The neurons of the peripheral nervous system represent a unique cell type as they can be up to a meter in length. Due to the unique physical property our neurons they are very vulnerable to injuries that disrupt communication between the cell body and the end of the nerve.

We know that a length dependent ‘dying back’ of the nerves (also known as axonal degeneration) underlies the phenotype seen in patients with CMT and is a common process in many neurodegenerative disorders.  The genes identified (80 to date) for CMT are providing clues to pathways and molecules involved with this disease causing process at the end of the nerves.

Whilst CMT is a rare disorder the broader implications and long term aim of our research will be to develop therapies that prevent or ameliorate axonal degeneration which may be relevant to other neurodegenerative diseases involving long nerves such as amyotrophic lateral sclerosis (ALS), Parkinson’s Disease and Alzheimers’ Disease.

What are the genetic components associated with CMT?

CMT can be inherited as an autosomal dominant, autosomal recessive or X-linked trait. 80 genes are known to cause CMT and associated disorders.

Your lab identifies CMT genes via multiple research avenues. Can you briefly discuss the difference/importance of studying a rare disease such as CMT via family studies vs gene sequencing studies?

Where possible, our lab uses a combination gene mapping strategies to help to validate potential pathogenic DNA variants, which have been identified by next generation sequencing. Family studies are particularly powerful when you have large families in which CMT has been inherited.

Using linkage analysis to identify candidate region where the faulty gene will be located. This information can then be combined with NGS information which expedites the identification of potential pathogenic DNA variants. Many of the families we work on are small nuclear  This approach is not unique to CMT and can be used for other rare diseases which are inherited in families.

Can you describe the family studies your lab is conducting to better understand the genetics and/or natural history of CMT?

Currently we are working on a form of X-linked CMT (CMTX3) to identify the culprit gene. This study has used linkage analysis to identify the region on chromosome X (5.7 million base pairs) where we should focus our efforts to identify the gene. We have employed whole genome sequencing (WGS) to identify all novel variants (both single base changes as well as structural variants) within our candidate interval.

We suspect the mutation causing CMTX3 disease will be in a non-coding region as we have been able to rule out mutations in the known annotated genes in the CMTX3 interval. There are two large families which are significantly linked to the CMTX3 region and it is likely the mutation has arisen from a common founder based on the genetic information we know.

What is High Resolution Melt analysis and how is it being used to discover mutations in CMT patients?

High Resolution Melt (HRM) analysis is a DNA scanning method used in our laboratory to validate putative variants identified by NGS in normal controls. The method relies on generating a DNA melt profile of a particular region of DNA using PCR and acquisition of fluorescence melt data at different temperature time points. A DNA fragment will give a signature melt profile based on the sequence information. If a base change occurs in the fragment an altered melt profile will then be observed.

When a novel variant is identified we design an assay that will be able to generate a normal profile and a variant profile. Using this method we can genotype 100s of samples by PCR and generate a melt profile to immediately determine which samples contain a variant of interest. This approach reduces the sanger sequencing burden for the variant validation aspects of our gene discovery projects.

Can you describe whole-exome sequencing and methods such as Clinical Sequence Analyzer and Sequence Miner to help your lab identify rare variants in CMT patients?

The use of NextCode Clinical Sequence Analyser (CSA) and Sequence Miner has been a helpful web based tool to manage the WGS data for our CMTX3 project. Using the CSA program we used a customized gene list to eliminate mutations in all the genes known to cause CMT and associated syndromes in the two large families linked to the CMTX3 locus. The NextCode Sequence Miner has allowed us to view both novel single nucleotide variants and structural variants and determine if they segregate in the family. We are able to extract the genomic regions of interest and use this to design validation experiments for the novel variants.

Of particular importance to this study is the analysis of structural variation in noncoding regions of the genome. The bioinformatics pipelines developed by NextCode have allowed us to visualize this important class of DNA variants. To prove pathogenicity of a variant will require rigorous genomic validation and the use of cell/animal models to demonstrate the DNA variant results in a functional cellular pathogenic change.    

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