Whole exome sequencing and DNA methylation analysis in a clinical amyotrophic lateral sclerosis cohort

Abstract

BackgroundGene discovery has provided remarkable biological insights into amyotrophic lateral sclerosis (ALS). One challenge for clinical application of genetic testing is critical evaluation of the significance of reported variants.MethodsWe use whole exome sequencing (WES) to develop a clinically relevant approach to identify a subset of ALS patients harboring likely pathogenic mutations. In parallel, we assess if DNA methylation can be used to screen for pathogenicity of novel variants since a methylation signature has been shown to associate with the pathogenic C9orf72 expansion, but has not been explored for other ALS mutations. Australian patients identified with ALS‐relevant variants were cross‐checked with population databases and case reports to critically assess whether they were “likely causal,” “uncertain significance,” or “unlikely causal.”ResultsPublished ALS variants were identified in >10% of patients; however, in only 3% of patients (4/120) could these be confidently considered pathogenic (in SOD1 and TARDBP). We found no evidence for a differential DNA methylation signature in these mutation carriers.ConclusionsThe use of WES in a typical ALS clinic demonstrates a critical approach to variant assessment with the capability to combine cohorts to enhance the largely unknown genetic basis of ALS.

Whole-exome sequencing in amyotrophic lateral sclerosis suggests NEK1 is a risk gene in Chinese

“…For familial probands, 38% (5 out of 13) were carriers of a likely causal variant. This is on the lower end of the range (30–70%) compared to what has previously been reported in European ancestry populations [ 35 , 40 ]. The lower proportion of identified likely causal variants in both sALS and fALS cases is likely to be explained by a lower prevalence of the C9orf72 repeat expansion which accounts for up to 7% of sALS and 40% of fALS in European populations [ 2 ] compared to just 0.3% in sALS cases in this study (as found in other Chinese samples [ 41 , 42 ]).…”

“…2 ). This was slightly lower than the proportion of ALS cases with a known causal variant in an Australian clinical ALS cohort (~ 90% European ancestry) which was 10% using an identical filtering technique [ 35 ]. For familial probands, 38% (5 out of 13) were carriers of a likely causal variant.…”

“…they occur at a population frequency that is inconsistent with the assumed disease prevalence and penetrance), we ignored any known variants identified in our cohort for which the frequency in ExAC populations of any ethnicity (the ‘popmax’ approach [ 33 ]) was > 0.01. To identify novel variants in relevant genes we used a previously curated hierarchical gene-set [ 35 ] (Additional file 1 : Table S4) and restricted the analysis to non-synonymous (missense), stop-gain/loss (nonsense) and splicing (first and last two bases of each intron) variants. To enhance pathogenicity call rates [ 36 ], any missense variants classified as ‘tolerated’ by both MetaLR [ 37 ] and MetaSVM_pred [ 37 ] (integration of 18 current deleteriousness-scoring methods) were excluded.…”

“…Final variant lists were cross-checked with clinical databases (OMIM, Clinvar [ 38 ]) and the literature for case reports to assess pathogenicity. In examining the curated set of genes [ 35 ] (Additional file 1 : Table S4), variants passing all filters present in ≥ 1 individual (case and/or control) were identified.…”

“…Putatively pathogenic indels were screened for in a subset of 21 genes, with prior evidence for causative indels and/or LOF variants [ 35 ] (Additional file 1 : Table S4). These were separated into non-truncating (in-frame) and truncating (frame-shift) insertions and deletions, which were subsequently cross-checked for pathogenicity as above.…”