Next Generation Gene Hunting in Amyotrophic Lateral Sclerosis
Lead Research Organisation:
King's College London
Department Name: Clinical Neuroscience
Abstract
There are no effective treatments for amyotrophic lateral aclerosis (ALS) because so little is known about what causes it. In most instances ALS appears out of the blue (called sporadic ALS) but in 10% the disease runs in families (familial ALS) due to a single defective gene, passed down from generation to generation. Four genes are known to cause ALS (SOD1, TARDBP, FUS and ANG) but they account for only 25% of all familial and 7% of sporadic ALS patients. The cause of the disease in 90% of patients is unknown but a genetic basis is strongly implicated.
The ?Next Generation? of DNA technology has transformed what can be achieved in genetic research. The human genome mapping project published in 2000 one person?s entire genetic code. It took 15 years and cost #~300 million. This can now be done in 1 month for #~10,000. This grant application aims to harness the extraordinary power of these new research tools in a global gene hunting effort.
Only ~2% of the human genetic code provides the blueprint for making proteins (the building blocks of all cells). Mutations in these genes accounts for most human diseases. Revolutionary DNA capture methods mean that we can pull out the protein encoding 2% of genes for further analysis. Single molecule DNA sequencing allows us to read the spelling of millions of different DNA fragments and rapidly identify the disease-causing spelling mistakes.
We are focusing our most intensive sequencing on DNA from 100 families and comparisons made to healthy controls. Potential mutations will be then screened in DNA samples held by a global network of ALS researchers, ( 2,000 familial and 10,000 sporadic samples). For the 30% of genes that are not covered by DNA capture methods we will identify chromosomal regions that contain ancient mutations and target genes implicated in known pathological ALS pathways.
Screening of new ALS genes can be offered to at-risk families and sporadic patients who are worried about the risk to other family members. The gene mutations we identify can be introduced into cells and mice allowing us to study the harmful effects of ALS gene mutations. This will dramatically improve our understanding of the underlying disease mechanisms and develop drugs capable of arresting the disease process and even preventing it occurring in genetically susceptible individuals.
The ?Next Generation? of DNA technology has transformed what can be achieved in genetic research. The human genome mapping project published in 2000 one person?s entire genetic code. It took 15 years and cost #~300 million. This can now be done in 1 month for #~10,000. This grant application aims to harness the extraordinary power of these new research tools in a global gene hunting effort.
Only ~2% of the human genetic code provides the blueprint for making proteins (the building blocks of all cells). Mutations in these genes accounts for most human diseases. Revolutionary DNA capture methods mean that we can pull out the protein encoding 2% of genes for further analysis. Single molecule DNA sequencing allows us to read the spelling of millions of different DNA fragments and rapidly identify the disease-causing spelling mistakes.
We are focusing our most intensive sequencing on DNA from 100 families and comparisons made to healthy controls. Potential mutations will be then screened in DNA samples held by a global network of ALS researchers, ( 2,000 familial and 10,000 sporadic samples). For the 30% of genes that are not covered by DNA capture methods we will identify chromosomal regions that contain ancient mutations and target genes implicated in known pathological ALS pathways.
Screening of new ALS genes can be offered to at-risk families and sporadic patients who are worried about the risk to other family members. The gene mutations we identify can be introduced into cells and mice allowing us to study the harmful effects of ALS gene mutations. This will dramatically improve our understanding of the underlying disease mechanisms and develop drugs capable of arresting the disease process and even preventing it occurring in genetically susceptible individuals.
Technical Summary
Amyotrophic lateral sclerosis (ALS, also known as motor neurone disease) causes progressive paralysis and takes the lives of 1,200 people in the UK every year. There are no effective treatments and little is known about its causes. Most ALS is sporadic but in 10% it is clearly familial (FALS), due to a single gene mutation inherited in an autosomal dominant fashion. Clinically and pathologically, familial and sporadic ALS are indistinguishable and many cases of sporadic ALS may be caused by gene defects with low penetrance. Most genetic neurodegenerative disorders are due to mutations that cause a change in the amino acid sequence. Four genes known to cause ALS account for only 25% of familial and 7% of sporadic ALS. We aim to identify the remaining FALS genes in order to offer comprehensive counselling and gene testing to at-risk families and concerned individuals with apparently sporadic disease. This will dramatically improve our understanding of disease pathogenesis and significantly advance drug discovery.
Here we propose an unprecedented gene hunting effort using revolutionary techniques including DNA capture, deep resequencing and high-density SNP genotyping. Using an initial cohort of cultured lymphoblast lines from 100 FALS cases we will extract high quality DNA, RNA and protein. Using the Nimblegen 2.1m array we will capture ~180,000 exons (comprising ~70% of the human genome) for sequencing on the Solexa GAII. We will also perform high density SNP genotyping on the Illumina Quad650 arrays looking for ancient common founder mutations. Lastly we will target genes implicated in pathways implicated in ALS pathogenesis not represented in the Nimblegen arrays.
Our five criteria for prioritising variants are those that (i) change the amino acid sequence, (ii) segregate with disease within kindreds, (iii) affect multiple families, (iv) have multiple mutations in multiple families and (v) have a low mutation frequency in multiple control populations. Variants that meet these criteria will be confirmed by Sanger sequencing and compared to the controls in SNP databases. Genes with confirmed mutations will be screened in a second cohort of local familial (200) and sporadic cases (2,000). We will notify our FALS Network partners of the most promising genes (frequency 1%) and request screening in their FALS and SALS cohorts to determine their global frequency and ethnic variation. Having identified mutations with a high likelihood of pathogenicity we will test for toxicity in cell lines, primary neurons and simple animal models (embryonic chicken and zebrafish).
Here we propose an unprecedented gene hunting effort using revolutionary techniques including DNA capture, deep resequencing and high-density SNP genotyping. Using an initial cohort of cultured lymphoblast lines from 100 FALS cases we will extract high quality DNA, RNA and protein. Using the Nimblegen 2.1m array we will capture ~180,000 exons (comprising ~70% of the human genome) for sequencing on the Solexa GAII. We will also perform high density SNP genotyping on the Illumina Quad650 arrays looking for ancient common founder mutations. Lastly we will target genes implicated in pathways implicated in ALS pathogenesis not represented in the Nimblegen arrays.
Our five criteria for prioritising variants are those that (i) change the amino acid sequence, (ii) segregate with disease within kindreds, (iii) affect multiple families, (iv) have multiple mutations in multiple families and (v) have a low mutation frequency in multiple control populations. Variants that meet these criteria will be confirmed by Sanger sequencing and compared to the controls in SNP databases. Genes with confirmed mutations will be screened in a second cohort of local familial (200) and sporadic cases (2,000). We will notify our FALS Network partners of the most promising genes (frequency 1%) and request screening in their FALS and SALS cohorts to determine their global frequency and ethnic variation. Having identified mutations with a high likelihood of pathogenicity we will test for toxicity in cell lines, primary neurons and simple animal models (embryonic chicken and zebrafish).