Predictors for a dementia gene mutation based on gene-panel next-generation sequencing of a large dementia referral series.

Next-generation genetic sequencing (NGS) technologies facilitate the screening of multiple genes linked to neurodegenerative dementia, but there are few reports about their use in clinical practice. Which patients would most profit from testing, and information on the likelihood of discovery of a causal variant in a clinical syndrome, are conspicuously absent from the literature, mostly for a lack of large-scale studies. We applied a validated NGS dementia panel to 3241 patients with dementia and healthy aged controls; 13,152 variants were classified by likelihood of pathogenicity. We identified 354 deleterious variants (DV, 12.6% of patients); 39 were novel DVs. Age at clinical onset, clinical syndrome and family history each strongly predict the likelihood of finding a DV, but healthcare setting and gender did not. DVs were frequently found in genes not usually associated with the clinical syndrome. Patients recruited from primary referral centres were compared with those seen at higher-level research centres and a national clinical neurogenetic laboratory; rates of discovery were comparable, making selection bias unlikely and the results generalisable to clinical practice. We estimated penetrance of DVs using large-scale online genomic population databases and found 71 with evidence of reduced penetrance. Two DVs in the same patient were found more frequently than expected. These data should provide a basis for more informed counselling and clinical decision making.

The SLE variant Ala71Thr of BLK severely decreases protein abundance and binding to BANK1 through impairment of the SH3 domain function.

The B-lymphocyte kinase (BLK) gene is associated genetically with several human autoimmune diseases including systemic lupus erythematosus. We recently described that the genetic risk is given by two haplotypes: one covering several strongly linked single-nucleotide polymorphisms within the promoter of the gene that correlated with low transcript levels, and a second haplotype that includes a rare nonsynonymous variant (Ala71Thr). Here we show that this variant, located within the BLK SH3 domain, is a major determinant of protein levels. In vitro analyses show that the 71Thr isoform is hyperphosphorylated and promotes kinase activation. As a consequence, BLK is ubiquitinated, its proteasomal degradation enhanced and the average life of the protein is reduced by half. Altogether, these findings suggest that an intrinsic autoregulatory mechanism previously unappreciated in BLK is disrupted by the 71Thr substitution. Because the SH3 domain is also involved in protein interactions, we sought for differences between the two isoforms in trafficking and binding to protein partners. We found that binding of the 71Thr variant to the adaptor protein BANK1 is severely reduced. Our study provides new insights on the intrinsic regulation of BLK activation and highlights the dominant role of its SH3 domain in BANK1 binding.

The dual effect of the lupus-associated polymorphism rs10516487 on BANK1 gene expression and protein localization.

Numerous loci have been found genetically associated with complex diseases, but only in a few cases has the functional variant and the molecular mechanism behind it been identified. Recently, the association of the BANK1 gene with systemic lupus erythematosus (SLE) was described. Here, we investigated the role of the associated polymorphisms on gene function and found that SNP rs17266594 located in the branch point consensus sequence has negligible effect on splicing or gene expression. The non-synonymous SNP rs10516487 located in exon 2 influenced splicing efficiency by creating an exonic splicing enhancer site for the SRp40 factor. Further, this same SNP generates protein isoforms with differential and measurable self-association properties. The full-length protein isoform containing the R61 variant forms larger protein scaffold complexes in the cell cytoplasm compared with the protective BANK1-61H variant. We also observed that, contrary to the full-length isoforms, the short Δ2 isoform of BANK1 displays a homogeneous cytoplasmic distribution, underscoring the potential role of the exon 2-coded protein domain in the scaffolding function of BANK1. We provide evidence that the non-synonymous SNP rs10516487 (G>A; R61H) shows a dual nature by first, influencing mRNA splicing and consequently the quantity of protein, and, second, by producing a risk variant-containing protein isoform with increased potential for multimerization.