Development of a next-generation sequencing gene panel for neurogenetic disorders J E Martindale, R Crookes, L Crooks, N J Beauchamp, A Dalton Sheffield Diagnostic Genetics Service (SDGS) Introduction Results Clinical Diagnoses: Patient B A total of 73 variants (mutations and SNPs) previously detected by Sanger sequencing were also identified by the NGS analysis, with 100% concordance. Previously unidentified sequence variants were also detected and those determined to be clinically significant were confirmed by Sanger sequencing. Sanger sequencing services for some genes implicated in hereditary spastic paraplegia (HSP), episodic ataxia, dopa-responsive dystonia and amyotrophic lateral sclerosis (ALS) are currently available at SDGS. Sequential testing is time-consuming and only covers a small proportion of the genes implicated in these disorders. We have developed a next-generation sequencing panel targeting 96 genes involved in neurogenetic disorders, some of which show a degree of phenotypic overlap. Analysis of the relevant genes is performed according to the clinical presentation of individual patients. Patient B was a male first referred in 2003 for HSP testing. The information provided with the referral stated that there was an anecdotal family history, with an aunt who had a diagnosis of multiple sclerosis and a cousin reported to have spastic paraplegia. No mutation had been found in the SPAST, ATL1 or REEP1 genes. The patient was homozygous for the c.1183C>T mutation in exon 6 of the CYP27A1 gene, predicted to result in p.Arg395Cys. This variant is listed on dbSNP as a pathogenic mutation (rs121908096) and in silico analysis supported a pathogenic role. A more extensive review of the family history showed no convincing evidence of other affected family members. CYP27A1 is associated with the AR disorder cerebrotendinous xanthomatosis (CTX). Deficiency in sterol 27-hydroxylase leads to a deficiency of chenodeoxycholic acid (CDCA) and thus the storage of cholestanol and cholesterol in many tissues and the development of tendon xanthomas. However, a milder form described as spinal xanthomatosis has been reported1, and this can present as spastic paraplegia. CDCA replacement therapy is effective in treatment of CTX. Clinical diagnoses: Patient A Patient A was a female referred in 2013 for HSP testing. There was no reported family history, but her father had died at an early age. No mutation had been found in the SPAST, ATL1 or REEP1 genes. The NGS analysis showed that the patient was heterozygous for both the c.223A>T mutation in exon 2 and the c.1354C>T mutation in exon 6 of the CYP7B1 gene, predicted to result in the p.Lys75* and p.Arg452* protein changes. Neither of these mutations have been previously reported; however, if present in trans would confirm a diagnosis of autosomal recessive HSP type 5A (parents are yet to be tested to confirm phase). This diagnosis will enable the patient to avoid a scheduled muscle biopsy and will mean that any offspring are at very low risk of becoming affected. Aims Transfer existing Sanger sequencing diagnostic services for HSP, ataxia, dystonia and ALS onto a next-generation sequencing platform. Significantly extend the scope of testing by introducing a large number of additional genes for these disorders into the panel. Patients & Methods To validate the panel, 21 patients previously tested for neurogenetic disorders by Sanger sequencing were screened. These were grouped according to phenotype as follows: 9 HSP, 4 ataxia, 4 dystonia and 4 ALS patients. Shearing of DNA was followed by end repair, A tailing and ligation of adaptors using the SureSelectXT library system (Agilent Technologies). SureSelect target enrichment was performed using custom in house designed probes, followed by sequencing on the Illumina MiSeq using the MiSeq Reagent Kit v2, performing 2 x 150 bp end paired reads. Data analysis was based on the open source ‘Best Practices’ workflow by the Broad Institute. Gene coverage was assessed and gaps filled by Sanger sequencing. The results were compared with the results of Sanger sequencing for the relevant genes to establish the specificity of the panel. Figure 2: Major bile acid and neurosteroid biosynthetic pathways2. CYP27A1 and CYP7B1 are involved in the same pathway and hence both patients might be treatable with CDCA. Conclusions SDGS is now able to offer services for an extended NGS panel of genes associated with neurogenetic disorders. This includes 40 genes for HSP, 37 genes for hereditary ataxia / hemiplegic migraine, 27 genes for dystonia and 22 genes for ALS. Figure 1: Sanger sequencing traces confirming the CYP7B1 mutations in Patient A. References 1 Verrips et al (1999) Brain 122: 1589-95 2 Goizet et al (2009) Brain 132: 1589-1600 Contact details: joanne.martindale@sch.nhs.uk