Dicsover putative disease-causing mutations in patients with ataxia or paraplegia by using Next -Generation -Sequencing panels

Within the last decade, knowledge could greatly be expanded about genetics of neurodegenerative diseases such as highly heterogeneous hereditary ataxia and paraplegias. To date, more than 100 genes have been identified to be involved in Mendelian causes of ataxia and paraplegia. Despite this knowledge, only a subset of these genes has been tested systematically for putative disease-causing mutations in patient cohorts. Here we investigated two NeurOmics cohorts for the occurrence of disease-causing variants.

To accomplish our aim, we investigated samples which are from pre-screened patients from families with at least two affected members. Therefore we established two separate ataxia and paraplegia specific selector probe based enrichment assays (HaloPlex, Agilent). The ataxia probe set contains 140 genes (classical, mitochondrial and metabolic ataxia) including genes from rare diseases showing a partial ataxia phenotype. The paraplegia panel contains 98 genes. A total target region of 582kbp (ataxia) / 267kbp (paraplegia) was specifically enriched and sequenced by use of an Illumina MiSeq next generation sequencing platform with 2x 150bp read paired-end runs. Sequenced reads were analysed by an in-house bioinformatics pipeline.

Currently, 31 NeurOmics patients (22 with ataxia, 9 with paraplegia) have been analysed. We achieved a high efficiency with the HaloPlex platform. On average, >95% (ataxia group) / >97% (paraplegia group) of the target region were covered by >20 reads with a mean base coverage of 176 ± 16 (ataxia) and 285 ± 131 (paraplegia).

Mapping the reads to the human genome (hg19) followed by annotations to different databases (ANNOVAR) resulted in a large number of variants (350 – 416 for ataxia / 158 – 177 for paraplegia). These lists of variants were further filtered for rare variants (in-house NGS database, 1000g, esp6500) and for functional relevance (ns, ss, indel) which led to a reduced number of 14 – 16 (ataxia) and 8 – 13 (paraplegia) variants per patient. Several rare, potentially disease causing mutations were found in different genes for diseases with ataxia (CACNA1A, FGF14, SPG7) and paraplegia (SPG11, KIF1C).

The HaloPlex target enrichment method followed by Illumina MiSeq NGS sequencing has shown relevant technical advantages in contrast to other approaches (solid hybridization or exome sequencing). This workflow is more sensitive, relatively fast and cost-effective especially for diagnostic questions. It is useful for screening large patient cohorts with an unknown genetic cause. Within the NeurOmics project, for patients in whom no disease causing mutation could be detected by panel sequencing, whole exome sequencing might be an opportunity to find putative causal mutation.