Next-generation sequencing-based genome diagnostics across clinical genetics centers: Implementation choices and their effects

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care

The human EKC/KEOPS complex is recruited to Cullin2 ubiquitin ligases by the human tumour antigen PRAME.

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Genetic analysis of plasmid-encoded mcr-1 resistance in Enterobacteriaceae derived from poultry meat in the Netherlands

BACKGROUND: Colistin is classified as the highest priority and critically important antimicrobial for human medicine by WHO as it is the last resort agent for treatment of carbapenem-resistant Enterobacteriaceae in humans. Additional research is necessary to elucidate the genetic structure of mcr-1 resistance genes, commonly found on plasmids, using WGS. OBJECTIVES: To map and compare the genetic characteristics of 35 mcr-1-mediated colistin-resistant Enterobacteriaceae isolated from chicken meat to highlight the genetic variation of the mcr-1-containing plasmids. METHODS: Sequencing was performed using Illumina HiSeq2500, Novaseq6000 and ONT’s GridION. GridION data was locally basecalled and demultiplexed using ONT’s Albacore 2.3.4 followed by Porechop 2.3. Quality filtering was performed using Filtlong 2.0. Hybrid Assembly was performed using Unicycler 4.7. Plasmids were compared with reference sequences in plasmid-RefSeq and pATLAS. RESULTS: A total of 35 mcr-1 positive Enterobacteriaceae were investigated, which resulted in 34 qualitatively robust hybrid assemblies of 2 Klebsiella pneumoniae and 32 Escherichia coli. mcr-1.1 was present in 33/34 isolates. One isolate contained an mcr-1.1-like resistance gene, due to a deletion of one codon. Two mcr-1.1 genes were located on the chromosome, while the majority of the mcr-1 genes were found on IncX4 type plasmids (n = 19). Almost all plasmids identified in this study were highly similar to plasmids found in human-derived strains. CONCLUSIONS: The mcr-1.1-containing plasmids from retail chicken show high sequence similarity to human mcr-1.1 plasmids, suggesting that this may be a contributor to the presence of colistin resistance in humans

PRAME-interacting proteins identified by Mass Spectrometry of purified complexes from K562 and HeLaS3 cells.

<p>PRAME-interacting proteins identified by Mass Spectrometry of purified complexes from K562 and HeLaS3 cells.</p

Organization of human EKC complex.

<p>SF21 cells were co-infected with baculoviruses expressing the indicated proteins. Anti-FLAG immunoprecipitations were performed, and total cell lysates and eluates were analyzed by western blotting. Interactions detected are summarized in the diagram on the right. Panels showing anti-HA immunoblots are from the same exposure of the same blot, as are the panels showing anti-cMyc immunoblots. Asterisk indicates light chains of the antibodies used in the immunoprecipitation.</p

PRAME recruits Cul2-EloBC ligases to EKC.

<p>(A) PRAME does not require an intact BC-box to interact with EKC components. Coimmunoprecipitation assays as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042822#pone-0042822-g001" target="_blank">Fig.1A</a> with wild type and BC-box mutant M2 PRAME. (B) PRAME bridges Cullin2 ligases to EKC complex. Immunoblot analysis as in Fig. 2 of TAG-OSGEP or TAG-LAGE3 immunoprecipitates with or without knock down of endogenous PRAME. Asterisk indicates heavy chains of the antibodies used in the immunoprecipitation. (C) Models of the protein complexes architecture.</p

OSGEP interacts with PRAME and Cul2 ubiquitin complex components.

<p>OSGEP interacts with PRAME and Cul2-EloBC ligases. Immunoblot analysis of TAG-PRAME and TAG-OSGEP protein complexes purified from K562 cells to verify the mass spectrometry data. Mock purification was performed on wild type cells. 0.8% of input and 33% of IP were separated on NuPage 4–12% gels. Tagged proteins were detected with mouse HA antibody (Covance, top panel); endogenous PRAME and TAG-PRAME were detected in the second panel with affinity-purified PRAME antibody after staining for Cul2; the other proteins were detected as indicated. Asterisks indicate protein A that dissociated from the beads after elution.</p

PRAME interacts with OSGEP and LAGE3.

<p>(A) Coimmunoprecipitation assays verify the interaction of PRAME with the EKC subunits OSGEP and LAGE3. Constructs expressing the indicated proteins were transiently transfected in 293T cells. Anti-TAG immunoprecipitations were performed with rabbit HA antibody (Abcam). TAG-PRAME was detected with monoclonal anti-HA (Covance) and TTE-tagged proteins with monoclonal BB2 (Diagenode) which recognizes the TY1 tag. The lower panel shows a scheme of the tagged proteins used (tags and coding sequences are not on scale). (B-C) PRAME directly interacts with EKC complex through OSGEP and LAGE3. SF21 cells were co-infected with baculoviruses expressing the indicated proteins. Anti-FLAG immunoprecipitations were performed, and total cell lysates and eluates were analyzed by western blotting. Panels showing anti-HA immunoblots are from the same exposure of the same blot, as are the panels showing anti-cMyc immunoblots. Asterisks indicate light chains of the antibodies used in the immunoprecipitation.</p

OSGEP protein levels are modulated by protein-protein interactions.

<p>(A) Multiple OSGEP moieties are present in the same complex. Constructs expressing the indicated proteins were transiently transfected in 293T cells. Total cell lysates and FLAG eluates were analyzed by western blotting. A plasmid expressing GFP was cotransfected to control for the transfection efficiency. (B) OSGEP levels are affected by protein-protein interactions. 293T cells were transfected with constructs expressing FLAG-OSGEP and the proteins indicated. The bar graph shows FLAG-OSGEP levels quantified by immunoblot with the Odyssey system (values are the average of two independent experiments). A plasmid expressing GFP was cotransfected to control for the transfection efficiency. (C) and (D) LAGE3-OSGEP interface mutants decrease OSGEP protein levels. Transient transfections in 293T cells with the mutant constructs indicated and immunoblot by Odyssey of total cell lysates. Graphs report intensities of FLAG-OSGEP quantified with Odyssey (A.U., arbitrary units).</p