Allele-specific suppression in C. elegans reveals details of EMS mutagenesis and a possible moonlighting interaction between the vesicular acetylcholine transporter and ERD2 receptors

A missense mutant, unc-17(e245), which affects the Caenorhabditis elegans vesicular acetylcholine transporter UNC-17, has a severe uncoordinated phenotype, allowing efficient selection of dominant suppressors that revert this phenotype to wild-type. Such selections permitted isolation of numerous suppressors after EMS (ethyl methanesulfonate) mutagenesis, leading to demonstration of delays in mutation fixation after initial EMS treatment, as has been shown in T4 bacteriophage but not previously in eukaryotes. Three strong dominant extragenic suppressor loci have been defined, all of which act specifically on allele e245, which causes a G347R mutation in UNC-17. Two of the suppressors (sup-1 and sup-8/snb-1) have previously been shown to encode synaptic proteins able to interact directly with UNC-17. We found that the remaining suppressor, sup-2, corresponds to a mutation in erd-2.1, which encodes an endoplasmic reticulum retention protein; sup-2 causes a V186E missense mutation in transmembrane helix 7 of ERD-2.1. The same missense change introduced into the redundant paralogous gene erd-2.2 also suppressed unc-17(e245). Suppression presumably occurred by compensatory charge interactions between transmembrane helices of UNC-17 and ERD-2.1 or ERD-2.2, as previously proposed in work on suppression by SUP-1(G84E) or SUP-8(I97D)/synaptobrevin. erd-2.1(V186E) homozygotes were fully viable, but erd-2.1(V186E); erd-2.2(RNAi) exhibited synthetic lethality (like erd-2.1(RNAi); erd-2.2(RNAi)), indicating that the missense change in ERD-2.1 impairs its normal function in the secretory pathway but may allow it to adopt a novel moonlighting function as an unc-17 suppressor.

Functional analysis of TLK2 variants and their proximal interactomes implicates impaired kinase activity and chromatin maintenance defects in their pathogenesis

IntroductionThe Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations in TLK2 were recently associated with ‘Mental Retardation Autosomal Dominant 57’ (MRD57, MIM# 618050), a neurodevelopmental disorder characterised by a highly variable phenotype, including mild-to-moderate intellectual disability, behavioural abnormalities, facial dysmorphisms, microcephaly, epilepsy and skeletal anomalies.MethodsWe re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape of TLK2 and analysed the effects of p.(Asp551Gly) and a previously reported missense variant (c.1850C>T; p.(Ser617Leu)) on TLK2 interactions, localisation and activity.ResultsWe identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652A>G; p.(Asp551Gly)) or a 39 kb deletion encompassing TLK2, and one was familial with three affected siblings who inherited a nonsense change from an affected mother (c.1423G>T; p.(Glu475Ter)). The clinical phenotypes were consistent with those of previously reported cases. The tested mutations strongly impaired TLK2 kinase activity. Proximal interactions between TLK2 and other factors implicated in neurological disorders, including CHD7, CHD8, BRD4 and NACC1, were identified. Finally, we demonstrated a more relaxed chromatin state in lymphoblastoid cells harbouring the p.(Asp551Gly) variant compared with control cells, conferring susceptibility to DNA damage.ConclusionOur study identified novel TLK2 pathogenic variants, confirming and further expanding the MRD57-related phenotype. The molecular characterisation of missense variants increases our knowledge about TLK2 function and provides new insights into its role in neurodevelopmental disorders.

A novel case of congenital spinocerebellar ataxia 5: further support for a specific phenotype associated with the p.(Arg480Trp) variant in SPTBN2

A 4-year-old girl was referred to the geneticist with a history of ataxia associated with intention tremor of the hands, strabismus and hypermetropy. Her symptoms presented about 2 years earlier with inability to walk unaided and lower limbs hypotonia. Cognitive functions were normal. Brain MRI showed a cerebellar and vermian hypoplasia with enlargement of both the cerebrospinal fluid spaces and the IV brain ventricle. Family history was unremarkable. A genetic screening using a 42-gene panel for hereditary ataxia/spastic paraparesis identified a de novo c.1438C>T - p.(Arg480Trp) missense change in the SPTBN2 gene (NM_006946.2). This variant is reported to be associated with congenital ataxia, later evolving into ataxia and intellectual disability. This case further supports the existence of a specific SPTBN2 p.(Arg480Trp)-associated phenotype, with a de novo recurrence of this variant in the heterozygous state.

De novo CTBP1 variant is associated with decreased mitochondrial respiratory chain activities

Objective:To determine the genetic etiology of a young woman presenting an early-onset, progressive neurodegenerative disorder with evidence of decreased mitochondrial complex I and IV activities in skeletal muscle suggestive of a mitochondrial disorder.Methods:A case report including diagnostic workup, whole-exome sequencing of the affected patient, filtering, and prioritization of candidate variants assuming a suspected autosomal recessive mitochondrial disorder and segregation studies.Results:After excluding candidate variants for an autosomal recessive mitochondrial disorder, re-evaluation of rare and novel heterozygous variants identified a recently reported, recurrent pathogenic heterozygous CTBP1 missense change (c.991C>T, p.Arg331Trp), which was confirmed to have arisen de novo.Conclusions:We report the fifth known patient harboring a recurrent pathogenic de novo c.991C>T p.(Arg331Trp) CTBP1 variant, who was initially suspected to have an autosomal recessive mitochondrial disorder. Inheritance of suspected early-onset mitochondrial disease could wrongly be assumed to be autosomal recessive. Hence, this warrants continued re-evaluation of rare and novel heterozygous variants in patients with apparently unsolved suspected mitochondrial disease investigated using next-generation sequencing.

MS2 Lysis of Escherichia coli Depends on Host Chaperone DnaJ

ABSTRACT The L protein of the single-stranded RNA phage MS2 causes lysis of Escherichia coli without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (insensitivity to L lysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the dnaJ P330Q mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the dnaJ P330Q mutant was recessive and could be suppressed by L overcomes d na J (Lodj ) alleles selected for restoration of lysis. All three Lodj alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ∼20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the Lodj truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of ϕX174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions. IMPORTANCE Small single-stranded nucleic acid lytic phages (Microviridae and Leviviridae) lyse their host by expressing a single “protein antibiotic.” The protein antibiotics from two out of three prototypic small lytic viruses have been shown to inhibit two different steps in the conserved PG biosynthesis pathway. However, the molecular basis of lysis caused by L, the lysis protein of the third prototypic virus, MS2, is unknown. The significance of our research lies in the identification of DnaJ as a chaperone in the MS2 L lysis pathway and the identification of the minimal lytic domain of MS2 L. Additionally, our research highlights the importance of the highly conserved P330 residue in the C-terminal domain of DnaJ for specific protein interactions.

Assessment of Mutation Status in a Large Series of Patients with Cytopenias with Normal Karyotypes and without Increased Blasts

Background: Myelodysplastic syndromes (MDS) are clonal stem cell disorders that can variably affect the erythroid, myeloid, and megakaryocytic hematopoietic lineages producing persistent cytopenias. Distinguishing MDS without increased blasts from immune-mediated cytopenias is difficult as criteria for dysplasia lack specificity and karyotypic changes are frequently absent. Given the need for better diagnostic assays to identify the clonal hematopoietic populations characteristic of MDS, we examined mutation status of 8 genes that are commonly mutated in the early stages of MDS. We profiled a group of 505 samples from patients with cytopenia(s) that were clinically and/or morphologically suspicious for MDS but had no increase in blasts and normal karyotypes. Methods: All 505 samples analyzed met these criteria: 1) clinical suspicion of MDS; 2) concurrent complete blood count with cytopenia(s); 3) bone marrow biopsy without increase in blasts; 4) normal karyotype (ie normal diploid, constitutional changes or –Y). In 170 cases, a FISH panel for 6 MDS changes was also performed and was negative. DNA was extracted from bone marrow aspirate (n=461) or blood (n =44). Sequencing of EZH2, TET2, exon 12 of ASXL1, JAK2 codon 617 and mutation hotspots in IDH1, IDH2, KRAS and NRAS was performed on the Ion Torrent PGM platform and analyzed using SeqNext software. To exclude artifacts and constitutional and low-level background alterations, a positive result required confirmation by Sanger sequencing (or pyrosequencing) and was defined as frameshift change, nonsense or previously characterized missense change, or novel missense change with high mutation probability based on level and structural features. All single nucleotide polymorphisms (SNPs) seen in normal controls or public databases were excluded. Ninety-two cases were subsequently analyzed for mutations in 21 additional MDS-associated genes using a TruSeq assay on the MiSeq platform. Results: Among 505 cases, M:F ratio was 1.02; 90.9% of patients were ≥50 years old. The frequency of MDS-associated mutations in cytopenic samples that had normal karyotype was 19.6% (99/505); mutations were identified in TET2 (63 cases), ASXL1 (36), IDH1 or IDH2 (8), NRAS (5), EZH2 (4), JAK2 V617F (4) and KRAS (1). The lowest mutation rate was in samples with isolated neutropenia (8.8%) with any cases showing neutropenia having an unusually high frequency of IDH1 or IDH2 mutation (7/39, 17.9%); other CBC parameters did not correlate significantly with incidence or type of mutation (Table). Multiple mutations were seen in 40 (7.9%) samples, with dual TET2 and ASXL1 mutation in 13. Among cases with several mutations, unequivocal subclonal mutation changes were seen in 17. Among 69 cases with mutations detected by the 8-gene panel, a larger sequencing panel detected additional mutations in 49 (71%), most commonly SRSF2 (9) and RUNX1 (3). 6/23 cases (26.1%) that were negative in the 8-gene panel had MDS-associated mutation(s) identified in the larger panel. Table. Frequency of mutations (mut) by cytopenia. CBC finding Number of cases Any mutation % of all cases TET2 mut % of mut cases ASXL1 mut % of mut cases All cases 505 99 19.6 63 63.6 36 36.4 Pancytopenia 91 17 18.7 13 76.5 4 23.5 2 cytopenias 166 37 22.3 23 62.2 18 48.6 Anemia only 175 36 20.6 19 52.8 13 36.1 Macrocytic anemia 55 8 14.5 4 50.0 3 37.5 Thrombocytopenia only 39 6 15.4 6 100.0 1 16.7 Neutropenia only 34 3 8.8 2 66.7 1 33.3 Conclusions: Using a targeted 8-gene panel, we identified MDS-associated mutations in approximately 20% of samples suspicious for MDS based on cytopenia(s) but with normal karyotype and no increase in blasts. In this community-based, unselected series, mutation detection rate was largely independent of hematologic pattern. Mutation density and subclonal complexity were relatively low even using a larger panel of genes. The high detection rate illustrates the enhanced clinical utility of targeted sequencing panels for identifying clonal alterations in early/low-grade MDS. Further work is needed to delineate the prognostic significance of mutations identified in minimally atypical cytopenic bone marrow samples. Disclosures Lee: Quest Diagnostics: Employment. Wang:Quest Diagnostics: Employment. Maiese:AmeriPath: Employment. Arvai:Quest Diagnostics: Employment. Pan:Quest Diagnostics: Employment. Mehta:Quest Diagnostics: Employment. Caruthers:Quest Diagnostics: Employment. Gersen:AmeriPath: Employment. Billouin-Frazier:Quest Diagnostics: Employment. Racke:Quest Diagnostics: Employment. Jones:Quest Diagnostics: Employment.

Long-term balancing selection in LAD1 maintains a missense trans-species polymorphism in humans, chimpanzees and bonobos

Balancing selection maintains advantageous genetic and phenotypic diversity in populations. When selection acts for long evolutionary periods selected polymorphisms may survive species splits and segregate in present-day populations of different species. Here, we investigate the role of long-term balancing selection in the evolution of protein-coding sequences in the Homo-Pan clade. We sequenced the exome of 20 humans, 20 chimpanzees and 20 bonobos and detected eight coding trans-species polymorphisms (trSNPs) that are shared among the three species and have segregated for approximately 14 million years of independent evolution. While the majority of these trSNPs were found in three genes of the MHC cluster, we also uncovered one coding trSNP (rs12088790) in the gene LAD1. All these trSNPs show clustering of sequences by allele rather than by species and also exhibit other signatures of long-term balancing selection, such as segregating at intermediate frequency and lying in a locus with high genetic diversity. Here we focus on the trSNP in LAD1, a gene that encodes for Ladinin-1, a collagenous anchoring filament protein of basement membrane that is responsible for maintaining cohesion at the dermal-epidermal junction; the gene is also an autoantigen responsible for linear IgA disease. This trSNP results in a missense change (Leucine257Proline) and, besides altering the protein sequence, is associated with changes in gene expression of LAD1.