Progressive myoclonus epilepsies-Residual unsolved cases have marked genetic heterogeneity including dolichol-dependent protein glycosylation pathway genes

Abstract

Progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous rare diseases. Over 70% of PME cases can now be molecularly solved. Known PME genes encode a variety of proteins, many involved in lysosomal and endosomal function. We performed whole-exome sequencing (WES) in 84 (78 unrelated) unsolved PME-affected individuals, with or without additional family members, to discover novel causes. We identified likely disease-causing variants in 24 out of 78 (31%) unrelated individuals, despite previous genetic analyses. The diagnostic yield was significantly higher for individuals studied as trios or families (14/28) versus singletons (10/50) (OR = 3.9, p value = 0.01, Fisher's exact test). The 24 likely solved cases of PME involved 18 genes. First, we found and functionally validated five heterozygous variants in NUS1 and DHDDS and a homozygous variant in ALG10, with no previous disease associations. All three genes are involved in dolichol-dependent protein glycosylation, a pathway not previously implicated in PME. Second, we independently validate SEMA6B as a dominant PME gene in two unrelated individuals. Third, in five families, we identified variants in established PME genes; three with intronic or copy-number changes (CLN6, GBA, NEU1) and two very rare causes (ASAH1, CERS1). Fourth, we found a group of genes usually associated with developmental and epileptic encephalopathies, but here, remarkably, presenting as PME, with or without prior developmental delay. Our systematic analysis of these cases suggests that the small residuum of unsolved cases will most likely be a collection of very rare, genetically heterogeneous etiologies.

Keywords: dolichol-dependent glycosylation; epilepsy genetics; progressive myoclonus epilepsy; whole-exome sequencing.

Figure 1

Proportion of all 78 unrelated individuals with PME with (solved) and without (unsolved) likely pathogenic variants by clinical group

Figure 2

PME-associated genes (n = 18) with high and moderate confidence variants detected in our cohort of 78 unrelated individuals with PME that had previous extensive genetic investigations The number of unrelated individuals with variants in each gene is shown in parentheses with the known primary function/pathway of each gene also listed. See subjects and methods for criteria followed when classifying variants as high versus moderate confidence. ^∗Functionally validated genes in this study.

Figure 3

The NUS1 and DHDDS variants cause defects in protein glycosylation due to reduced cisPTase activity in patient-derived fibroblast cell lines (A) Reduced microsomal cisPTase activity in isolated membranes from PME (NUS1: PME1, PME2 and DHDDS: PME71, PME27) compared to control (C) fibroblasts. ^∗∗p < 0.005, ^∗∗∗p < 0.001. Data presented as mean ± SEM of at least three independent measurements. (B) Affected protein glycosylation in PME fibroblasts. Western blot analysis of NUS1, DHDDS, LAMP1, and ICAM1 levels. HSP90 was used as loading control. (C) Increased cholesterol accumulation in PME fibroblasts. Filipin staining and quantitative representation from PME and control cells. U18666A was used as a positive control for inhibition of cholesterol trafficking. ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.001, a.u., arbitrary units. Data are representative of at least three experiments.

Figure 4

The ALG10 frameshift mutation causes defects in protein N-glycosylation due to a predicted defect in alpha-1,2-glucosyltransferase activity (A) Affected protein glycosylation in fibroblasts carrying the ALG10 and ALG10B variants. Western blot analysis of ICAM1 and LAMP1 expression. HSP90 was used as loading control. (B) Protein N-glycosylation of CPY shows multiple hypo-glycosylated bands in a yeast alg10 deletion strain transformed with mutated human (h) ALG10 (hALG10fs) or empty vector. N-glycosylation deficiency is rescued when transformed with either wild-type yeast ALG10 (yALG10), hALG10, or hALG10B.

Figure 5

Gene co-expression matrix for 33 known (black) and candidate (gray) PME genes Pairwise Spearman correlations between genes shown, based on 524 samples from 42 individuals from the BrainSpan resource. Genes are ordered and grouped with hierarchical clustering, using the median linkage method.