Human Mutation

doi: 10.1002/humu.22322pmid: 23554237

Massively parallel (“next generation”) DNA sequencing (NGS) has quickly become the method of choice for seeking pathogenic mutations in rare uncharacterized monogenic diseases. Typically, before DNA sequencing, protein‐coding regions are enriched from patient genomic DNA, representing either the entire genome (“exome sequencing”) or selected mapped candidate loci. Sequence variants, identified as differences between the patient's and the human genome reference sequences, are then filtered according to various quality parameters. Changes are screened against datasets of known polymorphisms, such as dbSNP and the 1000 Genomes Project, in the effort to narrow the list of candidate causative variants. An increasing number of commercial services now offer to both generate and align NGS data to a reference genome. This potentially allows small groups with limited computing infrastructure and informatics skills to utilize this technology. However, the capability to effectively filter and assess sequence variants is still an important bottleneck in the identification of deleterious sequence variants in both research and diagnostic settings. We have developed an approach to this problem comprising a user‐friendly suite of programs that can interactively analyze, filter and screen data from enrichment‐capture NGS data. These programs (“Agile Suite”) are particularly suitable for small‐scale gene discovery or for diagnostic analysis.

doi: 10.1002/humu.22329pmid: 23568759

The dihydropyrimidinase‐like 3 (DPYSL3) or Collapsin Response Mediator Protein 4a (CRMP4a) expression is modified in neurodegeneration and is involved in several ALS‐associated pathways including axonal transport, glutamate excitotoxicity, and oxidative stress. The objective of the study was to analyze CRMP4 as a risk factor for ALS. We analyzed the DPYSL3/CRMP4 gene in French ALS patients (n = 468) and matched‐controls (n = 394). We subsequently examined a variant in a Swedish population (184 SALS, 186 controls), and evaluated its functional effects on axonal growth and survival in motor neuron cell culture. The rs147541241:A>G missense mutation occurred in higher frequency among French ALS patients (odds ratio = 2.99) but the association was not confirmed in the Swedish population. In vitro expression of mutated DPYSL3 in motor neurons reduced axonal growth and accelerated cell death compared with wild type protein. Thus, the association between the rs147541241 variant and ALS was limited to the French population, highlighting the geographic particularities of genetic influences (risks, contributors). The identified variant appears to shorten motor neuron survival through a detrimental effect on axonal growth and CRMP4 could act as a key unifier in transduction pathways leading to neurodegeneration through effects on early axon development.

doi: 10.1002/humu.22328pmid: 23559163

Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%–25% of BrS patients carry genetic defects that cause loss‐of‐function of the voltage‐gated cardiac sodium channel. Thus, 70%–75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium β2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (INa) from cells coexpressing Nav1.5 and wild‐type (β2WT) or mutant (β2D211G) β2 subunits. Our electrophysiological analysis showed a 39.4% reduction in INa density when Nav1.5 was coexpressed with the β2D211G. Single channel analysis showed that the mutation did not affect the Nav1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that β2D211G decreases Nav1.5 cell surface expression. The effect of the mutant β2 subunit on the INa strongly suggests that SCN2B is a new candidate gene associated with BrS.

doi: 10.1002/humu.22316pmid: 23504699

Data sharing is essential for a better understanding of genetic disorders. Good phenotype coding plays a key role in this process. Unfortunately, the two most widely used coding systems in medicine, ICD‐10 and SNOMED‐CT, lack information necessary for the detailed classification and annotation of rare and genetic disorders. This prevents the optimal registration of such patients in databases and thus data‐sharing efforts. To improve care and to facilitate research for patients with metabolic disorders, we developed a new coding system for metabolic diseases with a dedicated group of clinical specialists. Next, we compared the resulting codes with those in ICD and SNOMED‐CT. No matches were found in 76% of cases in ICD‐10 and in 54% in SNOMED‐CT. We conclude that there are sizable gaps in the SNOMED‐CT and ICD coding systems for metabolic disorders. There may be similar gaps for other classes of rare and genetic disorders. We have demonstrated that expert groups can help in addressing such coding issues. Our coding system has been made available to the ICD and SNOMED‐CT organizations as well as to the Orphanet and HPO organizations for further public application and updates will be published online (www.ddrmd.nl and www.cineas.org).

doi: 10.1002/humu.22326pmid: 23553787

In skeletal muscle, excitation–contraction (EC) coupling is the process whereby the voltage‐gated dihydropyridine receptor (DHPR) located on the transverse tubules activates calcium release from the sarcoplasmic reticulum by activating ryanodine receptor (RyR1) Ca2+ channels located on the terminal cisternae. This subcellular membrane specialization is necessary for proper intracellular signaling and any alterations in its architecture may lead to neuromuscular disorders. In this study, we present evidence that patients with recessive RYR1‐related congenital myopathies due to primary RyR1 deficiency also exhibit downregulation of the alfa 1 subunit of the DHPR and show disruption of the spatial organization of the EC coupling machinery. We created a cellular RyR1 knockdown model using immortalized human myoblasts transfected with RyR1 siRNA and confirm that knocking down RyR1 concomitantly downregulates not only the DHPR but also the expression of other proteins involved in EC coupling. Unexpectedly, this was paralleled by the upregulation of inositol‐1,4,5‐triphosphate receptors; functionally however, upregulation of the latter Ca2+ channels did not compensate for the lack of RyR1‐mediated Ca2+ release. These results indicate that in some patients, RyR1 deficiency concomitantly alters the expression pattern of several proteins involved in calcium homeostasis and that this may influence the manifestation of these diseases.

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is mostly composed of an asymmetric form in which three tetramers of catalytic AChE subunits are linked to a triple helical collagen Q (ColQ). Mutations in COLQ cause endplate AChE deficiency. We report three patients with endplate AChE deficiency with five recessive COLQ mutations. Sedimentation profiles showed that p.Val322Asp and p.Arg227X, but not p.Cys444Tyr, p.Asp447His, or p.Arg452Cys, inhibit formation of triple helical ColQ. In vitro overlay of mutant ColQ‐tailed AChE on muscle sections of Colq−/− mice revealed that p.Cys444Tyr, p.Asp447His, and p.Arg452Cys in the C‐terminal domain (CTD) abrogate anchoring ColQ‐tailed AChE to the NMJ. In vitro plate‐binding assay similarly demonstrated that the three mutants inhibit binding of ColQ‐tailed AChE to MuSK. We also confirmed the pathogenicity of p.Asp447His by treating Colq−/− mice with adeno‐associated virus serotype 8 carrying mutant COLQ‐p.Asp447His. The treated mice showed no improvement in motor functions and no anchoring of ColQ‐tailed AChE at the NMJ. Electroporation of mutant COLQ harboring p.Cys444Tyr, p.Asp447His, and p.Arg452Cys into anterior tibial muscles of Colq−/− mice similarly failed to anchor ColQ‐tailed AChE at the NMJ. We proved that the missense mutations in ColQ–CTD cause endplate AChE deficiency by compromising ColQ–MuSK interaction at the NMJ.