Unraveling the genetic causes of hereditary ataxia and paraplegia using disease specific gene panels and next-generation sequencing

10 August 2015


Neurodegenerative (NDD) and neuromuscular (NMD) diseases are amongst the most frequent of rare diseases, affecting the life and mobility of more than 500,000 patients and families in Europe. In order to support research, the possibility to develop treatments and improve diagnosis for this large group of patients, the European Commission started the NeurOmics project. NeurOmics aim is to investigate 10 different kinds of rare NDD and NMD with a focus on so-called omics technologies. Hereditary ataxia and spastic paraplegia are among these rare diseases. They are highly heterogeneous and show autosomal dominant, recessive and X-linked forms of inheritance. A cure is still not available and treatment options are thus limited to mitigating symptoms. Many of the patients become wheelchair bound when the disease progresses.

The phenotypic spectrum of ataxia and spastic paraplegia is quite broad. Decreased motor abilities are the most prominent but by far not the only clinical features frequently observed. Depending on the genetic background and severity of the disease, patients may also suffer from impaired vision and eye disorders, swallowing difficulties (dysphagia), limited speech abilities (dysarthria), seizures and many other neurological symptoms. Different mutations in different genes are known to cause almost identical phenotypic manifestations of the disease. This makes it challenging for physicians to predict a patient’s disease progression and moreover limits the possibilities of effective genetic family counselling. With the development of next generation sequencing technologies, the possibility of diagnosing a genetic disease by deciphering parts of the patient’s DNA reaches a new level of efficiency, throughput and affordability.

The Institute of Medical Genetics and Applied Genomics in Tübingen has almost 8 years of experience in the field of next generation sequencing with a strong focus on diagnosing NDD. For the identification of disease-causing mutations within the NeurOmics project, our aim is to examine 100 patients per disease group using a targeted gene panel approach based on Illumina NGS technology. To enhance the diagnostic yield, we first developed inclusion criteria and selected patients accordingly. As patients may have developed acquired forms of ataxia or paraplegia due to physical trauma, tumors or severe infections of the nervous system, genetic testing wouldn’t lead to any result in these cases and we thus excluded such cases from the study. Moreover, including patients from families with two or more affected raises the probability of a genetic cause of the disease. Finally, we requested the recruiting physicians to exclude more common causes of hereditary ataxia (SCA1,2,3 etc.) and paraplegia (SPG4) prior to panel sequencing. Testing for mutations in distinct hot spots with a relatively high prevalence for the disease at hand is more cost-effective than running an NGS based targeted gene panel. Furthermore, NGS sequencing is not capable of detecting triplet repeat expansion based diseases. This makes a pre-NGS testing for the occurrence of more common triplet repeat expansions a reasonable decision.

To support a future genetic diagnosis, an MRI scan should be made available along with a detailed clinical record of each patient. We use PhenoTips, an open source database system, to store standardized and comprehensive clinical data. We established two disease specific gene panels (HaloPlex, Agilent) and kept the content updated on a regular basis as best practice procedure.

Our latest disease-specific gene panel covers 181 genes associated with ataxia (hpSCAv8) and 139 genes associated with spastic paraplegia (hpHSPv6), respectively. The underlying target region of 611kb (ataxia) / 391kb (paraplegia) is specifically enriched and sequenced on an Illumina MiSeq device (2 X 150 bp V2 chemistry). The read sequences were analyzed by an in-house bioinformatics pipeline based on BWA and FreeBayes. Currently, index patients from 97 families (ataxia: 76 / paraplegia: 21) are taking part in the study. We achieved a high efficiency with the Agilent HaloPlex platform [target region cov20X: >96% to >97,5% ; mean base coverage: 208±35 (ataxia)/ 366±130 (paraplegia)]. Read-mapping to the human genome followed by annotation using different databases (ClinVar, HGMD, OMIM) resulted in large variant lists: 317-433 (ataxia)/ 145–177 (paraplegia) which were further filtered for rare variants (in-house database, 1000g, esp6500, ExAcc) and for functional relevance. This led to a reduced number of 2-29 variants per patient. Panel sequencing elucidated the molecular basis in 14 out of 76 panel sequenced ataxia families (19%) and 8 out of 21 sequenced paraplegia families (38%) respectively. For both disease groups, potential disease causing variants of yet uncertain significance could be identified in another 35-50% of cases.

Figure 1 HSP and SCA sequencing panel
Figure 1 – Results of panel sequencing 1. The figure shows the outcome of diagnosing hereditary ataxia (SCA, n = 76 patients) and spastic paraplegia (HSP, n = 21 patients) using targeted next generation sequencing gene panels. For patients with an ambiguous result, variants of uncertain significance were found which needs further investigation (clinical feedback of attending physician or even functional analysis).

These quite convincing initial results prove the project’s inclusion criteria to be appropriate for both, research and diagnostic approaches. The relatively high proportion of ambiguous cases clearly shows the need of further investigations, like segregation analysis of candidate genes. Nevertheless, we could show that panel sequencing is a sensitive, fast and cost-efficient method to identify putative disease-causing mutations in a large number of cases. Within the NeurOmics project, for patients in whom no disease-causing mutation could be detected by panel sequencing, whole exome or genome sequencing will be added on a research basis. This should lead to the discovery of new disease genes which may be included into future panel versions and moreover expand the genotype/phenotype correlation for these rare neurodegenerative diseases.

The SCA and HSP panel gene list can be downloaded here.

~ Martin Schulze, the Institute of Medical Genetics and Applied Genomics, Tübingen