Identifying novel disease genes in patients with skeletal muscle channelopathies.
Lead Research Organisation:
University College London
Department Name: Institute of Neurology
Abstract
Muscle channel diseases are a rare group of conditions that cause significant stiffness, pain, weakness and disability to its sufferers. Mutations in the muscle channel genes cause two main groups of disease: periodic paralysis and non-dystrophic myotonia. At present many of our patients remain undiagnosed and for those with a diagnosis there are relatively few treatments available.
With my research I hope to identify new genes and mutations responsible for causing these diseases. I will be using a new type of technology called targeted resequencing. This technique will enable specific analysis of all the skeletal muscle channels in our patients. When the genetic coding is compared with the reference human genome we can identify where it is different and therefore identify the mutations. Once the mutations are determined I will then look at the channels themselves using a technique called patch clamping. This will enable me to see how the mutations affect the channel‘s ability to function
This project will not only help diagnose many of our current patients and their families but also future patients. By finding new genes responsible for these diseases, new drugs may be developed to treat this group and those with similar diseases.
With my research I hope to identify new genes and mutations responsible for causing these diseases. I will be using a new type of technology called targeted resequencing. This technique will enable specific analysis of all the skeletal muscle channels in our patients. When the genetic coding is compared with the reference human genome we can identify where it is different and therefore identify the mutations. Once the mutations are determined I will then look at the channels themselves using a technique called patch clamping. This will enable me to see how the mutations affect the channel‘s ability to function
This project will not only help diagnose many of our current patients and their families but also future patients. By finding new genes responsible for these diseases, new drugs may be developed to treat this group and those with similar diseases.
Technical Summary
Genetic defects in voltage-gated ion channel genes are known to be responsible for a wide variety of diseases including skeletal muscle channelopathies where the two main clinical groups are periodic paralysis and non-dystrophic myotonia. In these conditions mutations in recognised sodium, chloride, calcium and potassium channel genes often cause significant stiffness, weakness and neurological disability but these genes only account for around 80% of cases.
The aim of this project is to improve our understanding of skeletal muscle channelopathies by:
1. Identifying novel disease-causing gene(s) in patients and families with undiagnosed muscle channelopathies.
2. Screening our cohort of undiagnosed channelopathy patients to establish range, frequency and ethnic variation of defects in these genes.
3. Investigating the pathogenicity of new mutations identified using an expression system.
We have identified the sample population utilising probably the world‘s largest database of channelopathy patients at the MRC Centre for Neuromuscular Diseases (linked to the UK National Channelopathy clinical service funded by the UK National Commissioning Group). From this we have selected the group of genetically undetermined patients with clear clinical and electrophysiological signs of muscle channelopathies. This currently consists of 296 patients (23% of 1300).
To identify novel disease-causing genes we have selected the 44 best probands from families that are negative for known genes. We will use targeted next-generation sequencing to analyse the coding and flanking intronic sequence of the 350 muscle channel genes. Four patients with known mutations in channel genes will be included as positive controls. Sequences will be aligned with the reference sequence and genetic variants cross-checked with public databases to identify true mutations and exclude normal polymorphisms. The 1000 genomes project will also be important in allowing us to align control sequences from individuals with differing ethnicity. Probable mutations will be rechecked with Sanger sequencing and the entire cohort screened with the identified disease genes.
Finally, to establish pathogenicity, we will use site-directed mutagenesis to develop mutated cDNA constructs to transfect HEK293 cells for expression studies. The expressed channels will be interrogated by patch clamping to identify the mutation‘s effect on its voltage-gating properties and help prove pathogenicity.
This project will expand the pool of scientific knowledge in muscle channelopathies by identifying novel genes, targeting an MRC research priority area, Genes and Disease. It will have an impact on both the patients we diagnose and future diagnostic testing. It will also provide new targets for treatment in a group of diseases which lack substantial treatment options.
The aim of this project is to improve our understanding of skeletal muscle channelopathies by:
1. Identifying novel disease-causing gene(s) in patients and families with undiagnosed muscle channelopathies.
2. Screening our cohort of undiagnosed channelopathy patients to establish range, frequency and ethnic variation of defects in these genes.
3. Investigating the pathogenicity of new mutations identified using an expression system.
We have identified the sample population utilising probably the world‘s largest database of channelopathy patients at the MRC Centre for Neuromuscular Diseases (linked to the UK National Channelopathy clinical service funded by the UK National Commissioning Group). From this we have selected the group of genetically undetermined patients with clear clinical and electrophysiological signs of muscle channelopathies. This currently consists of 296 patients (23% of 1300).
To identify novel disease-causing genes we have selected the 44 best probands from families that are negative for known genes. We will use targeted next-generation sequencing to analyse the coding and flanking intronic sequence of the 350 muscle channel genes. Four patients with known mutations in channel genes will be included as positive controls. Sequences will be aligned with the reference sequence and genetic variants cross-checked with public databases to identify true mutations and exclude normal polymorphisms. The 1000 genomes project will also be important in allowing us to align control sequences from individuals with differing ethnicity. Probable mutations will be rechecked with Sanger sequencing and the entire cohort screened with the identified disease genes.
Finally, to establish pathogenicity, we will use site-directed mutagenesis to develop mutated cDNA constructs to transfect HEK293 cells for expression studies. The expressed channels will be interrogated by patch clamping to identify the mutation‘s effect on its voltage-gating properties and help prove pathogenicity.
This project will expand the pool of scientific knowledge in muscle channelopathies by identifying novel genes, targeting an MRC research priority area, Genes and Disease. It will have an impact on both the patients we diagnose and future diagnostic testing. It will also provide new targets for treatment in a group of diseases which lack substantial treatment options.