Inherited CD19 Deficiency Does Not Impair Plasma Cell Formation or Response to CXCL12.

BACKGROUND: The human CD19 antigen is expressed throughout B cell ontogeny with the exception of neoplastic plasma cells and a subset of normal plasma cells. CD19 plays a role in propagating signals from the B cell receptor and other receptors such as CXCR4 in mature B cells. Studies of CD19-deficient patients have confirmed its function during the initial stages of B cell activation and the production of memory B cells; however, its role in the later stages of B cell differentiation is unclear. OBJECTIVE: Using B cells from a newly identified CD19-deficient individual, we investigated the role of CD19 in the generation and function of plasma cells using an in vitro differentiation model. METHODS: Flow cytometry and long-read nanopore sequencing using locus-specific long-range amplification products were used to screen a patient with suspected primary immunodeficiency. Purified B cells from the patient and healthy controls were activated with CD40L, IL-21, IL-2, and anti-Ig, then transferred to different cytokine conditions to induce plasma cell differentiation. Subsequently, the cells were stimulated with CXCL12 to induce signalling through CXCR4. Phosphorylation of key downstream proteins including ERK and AKT was assessed by Western blotting. RNA-seq was also performed on in vitro differentiating cells. RESULTS: Long-read nanopore sequencing identified the homozygous pathogenic mutation c.622del (p.Ser208Profs*19) which was corroborated by the lack of CD19 cell surface staining. CD19-deficient B cells that are predominantly naïve generate phenotypically normal plasma cells with expected patterns of differentiation-associated genes and normal levels of CXCR4. Differentiated CD19-deficient cells were capable of responding to CXCL12; however, plasma cells derived from naïve B cells, both CD19-deficient and sufficient, had relatively diminished signaling compared to those generated from total B cells. Additionally, CD19 ligation on normal plasma cells results in AKT phosphorylation. CONCLUSION: CD19 is not required for generation of antibody-secreting cells or the responses of these populations to CXCL12, but may alter the response other ligands that require CD19 potentially affecting localization, proliferation, or survival. The observed hypogammaglobulinemia in CD19-deficient individuals is therefore likely attributable to the lack of memory B cells.

Trial of antibiotic restraint in presumed pneumonia: A Surgical Infection Society multicenter pilot.

BACKGROUND: Pneumonia is the most common intensive care unit-acquired infection in the trauma and emergency general surgery population. Despite guidelines urging rapid antibiotic use, data supporting immediate antibiotic initiation in cases of suspected infection are limited. Our hypothesis was that a protocol of specimen-initiated antibiotic initiation would have similar compliance and outcomes to an immediate initiation protocol. METHODS: We devised a pragmatic cluster-randomized crossover pilot trial. Four surgical and trauma intensive care units were randomized to either an immediate initiation or specimen-initiated antibiotic protocol for intubated patients with suspected pneumonia and bronchoscopically obtained cultures who did not require vasopressors. In the immediate initiation arm, antibiotics were started immediately after the culture regardless of patient status. In the specimen-initiated arm, antibiotics were delayed until objective Gram stain or culture results suggested infection. Each site participated in both arms after a washout period and crossover. Outcomes were protocol compliance, all-cause 30-day mortality, and ventilator-free alive days at 30 days. Standard statistical techniques were applied. RESULTS: A total of 186 patients had 244 total cultures, of which only the first was analyzed. Ninety-three patients (50%) were enrolled in each arm, and 94.6% were trauma patients (84.4% blunt trauma). The median age was 50.5 years, and 21% of the cohort was female. There were no differences in demographics, comorbidities, sequential organ failure assessment, Acute Physiology and Chronic Health Evaluation II, or Injury Severity Scores. Antibiotics were started significantly later in the specimen-initiated arm (0 vs. 9.3 hours; p < 0.0001) with 19.4% avoiding antibiotics completely for that episode. There were no differences in the rate of protocol adherence, 30-day mortality, or ventilator-free alive days at 30 days. CONCLUSION: In this cluster-randomized crossover trial, we found similar compliance rates between immediate and specimen-initiated antibiotic strategies. Specimen-initiated antibiotic protocol in patients with a suspected hospital-acquired pneumonia did not result in worse clinical outcomes compared with immediate initiation. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level II.

Identification of a novel MAGT1 mutation supports a diagnosis of XMEN disease.

XMEN (X-linked immunodeficiency with magnesium defect) is caused by loss-of-function mutations in MAGT1 which is encoded on the X chromosome. The disorder is characterised by CD4 lymphopenia, severe chronic viral infections and defective T-lymphocyte activation. XMEN patients are susceptible to Epstein-Barr virus infections and persistently low levels of intracellular Mg2+. Here we describe a patient that presented with multiple recurrent infections and a subsequent diffuse B-cell lymphoma. Molecular genetic analysis by exome sequencing identified a novel hemizygous MAGT1 nonsense mutation c.1005T>A (NM_032121.5) p.(Cys335*), confirming a diagnosis of XMEN deficiency. Follow-up immunophenotyping was performed by antibody staining and flow cytometry; proliferation was determined by 3H-thymidine uptake after activation by PHA and anti-CD3. Cytotoxic natural killer cell activity was assessed with K562 target cells using the NKTESTTM assay. While lymphocyte populations were superficially intact, B cells were largely naive with a reduced memory cell compartment. Translated NKG2D was absent on both NK and T cells in the proband, and normally expressed in the carrier mother. In vitro NK cell activity was intact in both the proband and his mother. This report adds to the growing number of identified XMEN cases, raising awareness of a, still rare, X-linked immunodeficiency.

Adenoviral Respiratory Infection-Associated Mortality in Children: A Retrospective Case Series.

Viral respiratory infections are a leading cause of illness and hospitalization in young children worldwide. Case fatality rates in pediatric patients with adenoviral lower respiratory tract infection requiring intensive care unit (ICU) admission have been reported between 7 and 22%. We investigated the demographics and clinical characteristics in pediatric mortalities associated with adenoviral respiratory infection at 12 academic children's hospitals in the United States. There were 107 mortality cases included in our study, 73% of which had a chronic medical condition. The most common chronic medical condition was immunocompromised state in 37 cases (35%). The incidences of pediatric acute respiratory distress syndrome (78%) and multiple organ dysfunction syndrome (94%) were profound. Immunocompetent cases were more likely to receive mechanical ventilation within the first hour of ICU admission (60 vs. 14%, p < 0.001) and extracorporeal membrane oxygenation (27 vs. 5%, p = 0.009), and less likely to receive continuous renal replacement therapy (20 vs. 49%, p = 0.002) or have renal dysfunction (54 vs. 78%, p = 0.014) as compared with immunocompromised cases. Immunocompromised cases were more likely to have bacteremia (57 vs. 16%, p < 0.001) and adenoviremia (51 vs. 17%, p < 0.001) and be treated with antiviral medications (81 vs. 26%, p < 0.001). We observed a high burden of nonrespiratory organ system dysfunction in a cohort of pediatric case fatalities with adenoviral respiratory infection. The majority of cases had a chronic medical condition associated with an increased risk of complications from viral respiratory illness, most notably immunocompromised state. Important treatment differences were noted between immunocompromised and immunocompetent cases.

Long-read nanopore DNA sequencing can resolve complex intragenic duplication/deletion variants, providing information to enable preimplantation genetic diagnosis.

BACKGROUND: The adoption of massively parallel short-read DNA sequencing methods has greatly expanded the scope and availability of genetic testing for inherited diseases. Indeed, the power of these methods has encouraged the integration of whole genome sequencing, the most comprehensive single approach to genomic analysis, into clinical practice. Despite these advances, diagnostic techniques that incompletely resolve the precise molecular boundaries of pathogenic sequence variants continue to be routinely deployed. This can present a barrier for certain prenatal diagnostic approaches. For example, the pre-referral workup for couples seeking preimplantation genetic diagnosis requires intragenic dosage variants to be characterised at nucleotide resolution. OBJECTIVE: We sought to assess the use of long-read nanopore sequencing to rapidly characterise an apparent heterozygous RB1 exon 23 deletion that was initially identified by multiplex ligation-dependent probe amplification (MLPA), in a patient with bilateral retinoblastoma. METHODS: Target enrichment was performed by long-range polymerase chain reaction (PCR) amplification prior to Flongle sequencing on a MinION long-read sequencer. RESULTS: Characterisation of the deletion breakpoint included an unexpected 85-bp insertion which duplicated RB1 exon 24 (and was undetected by MLPA). The long-read sequence permitted design of a multiplex PCR assay, which confirmed that the mutation arose de novo. CONCLUSION: Our experience demonstrates the diagnostic utility of long-read technology for the precise characterisation of structural variants, and highlights how this technology can be efficiently deployed to enable onward referral to reproductive medicine services.

Molecular diagnoses in the congenital malformations caused by ciliopathies cohort of the 100,000 Genomes Project.

BACKGROUND: Primary ciliopathies represent a group of inherited disorders due to defects in the primary cilium, the 'cell's antenna'. The 100,000 Genomes Project was launched in 2012 by Genomics England (GEL), recruiting National Health Service (NHS) patients with eligible rare diseases and cancer. Sequence data were linked to Human Phenotype Ontology (HPO) terms entered by recruiting clinicians. METHODS: Eighty-three prescreened probands were recruited to the 100,000 Genomes Project suspected to have congenital malformations caused by ciliopathies in the following disease categories: Bardet-Biedl syndrome (n=45), Joubert syndrome (n=14) and 'Rare Multisystem Ciliopathy Disorders' (n=24). We implemented a bespoke variant filtering and analysis strategy to improve molecular diagnostic rates for these participants. RESULTS: We determined a research molecular diagnosis for n=43/83 (51.8%) probands. This is 19.3% higher than previously reported by GEL (n=27/83 (32.5%)). A high proportion of diagnoses are due to variants in non-ciliopathy disease genes (n=19/43, 44.2%) which may reflect difficulties in clinical recognition of ciliopathies. n=11/83 probands (13.3%) had at least one causative variant outside the tiers 1 and 2 variant prioritisation categories (GEL's automated triaging procedure), which would not be reviewed in standard 100,000 Genomes Project diagnostic strategies. These include four structural variants and three predicted to cause non-canonical splicing defects. Two unrelated participants have biallelic likely pathogenic variants in LRRC45, a putative novel ciliopathy disease gene. CONCLUSION: These data illustrate the power of linking large-scale genome sequence to phenotype information. They demonstrate the value of research collaborations in order to maximise interpretation of genomic data.

Long-read nanopore sequencing enables accurate confirmation of a recurrent PMS2 insertion-deletion variant located in a region of complex genomic architecture.

Targeted next generation sequencing (NGS) is the predominant methodology for the molecular genetic diagnosis of inherited conditions. In many laboratories, NGS-identified variants are routinely validated using a different method, to minimize the risk of a false-positive diagnosis. This can be particularly important when pathogenic variants are located in complex genomic regions. In this situation, new long-read sequencing technologies have potential advantages over existing alternatives. However, practical examples of their utility for diagnostic purposes remain scant. Here, we report the use of nanopore sequencing to validate a PMS2 mutation refractory to conventional methods. In a patient who presented with colorectal cancer and loss of PMS2 immunostaining, short-read NGS of Lynch syndrome-associated genes identified the recurrent PMS2 insertion-deletion variant, c.736_741delinsTGTGTGTGAAG (p.Pro246Cysfs*3). Confirmation of this variant using bidirectional Sanger sequencing was impeded by an upstream intron 6 poly(T) tract. Using a locus-specific amplicon template, we undertook nanopore long-read sequencing in order to assess the diagnostic accuracy of this platform. Pairwise comparison between a curated benchmark allele (derived from short-read NGS and unidirectional Sanger sequencing) and the consensus nanopore dataset revealed 100% sequence identity. Our experience provides insight into the robustness and ease of deployment of "third-generation" sequencing for accurate characterisation of pathogenic variants.

Assessing the utility of long-read nanopore sequencing for rapid and efficient characterization of mobile element insertions.

Short-read next generation sequencing (NGS) has become the predominant first-line technique used to diagnose patients with rare genetic conditions. Inherent limitations of short-read technology, notably for the detection and characterization of complex insertion-containing variants, are offset by the ability to concurrently screen many disease genes. "Third-generation" long-read sequencers are increasingly being deployed as an orthogonal adjunct technology, but their full potential for molecular genetic diagnosis has yet to be exploited. Here, we describe three diagnostic cases in which pathogenic mobile element insertions were refractory to characterization by short-read sequencing. To validate the accuracy of the long-read technology, we first used Sanger sequencing to confirm the integration sites and derive curated benchmark sequences of the variant-containing alleles. Long-read nanopore sequencing was then performed on locus-specific amplicons. Pairwise comparison between these data and the previously determined benchmark alleles revealed 100% identity of the variant-containing sequences. We demonstrate a number of technical advantages over existing wet-laboratory approaches, including in silico size selection of a mixed pool of amplification products, and the relative ease with which an automated informatics workflow can be established. Our findings add to a growing body of literature describing the diagnostic utility of long-read sequencing.

Cas9-based enrichment and single-molecule sequencing for precise characterization of genomic duplications.

The widespread use of genome-wide diagnostic screening methods has greatly increased the frequency with which incidental (but possibly pathogenic) copy number changes affecting single genes are detected. These findings require validation to allow appropriate clinical management. Deletion variants can usually be readily validated using a range of short-read next-generation sequencing (NGS) strategies, but the characterization of duplication variants at nucleotide resolution remains challenging. This presents diagnostic problems, since pathogenicity cannot generally be assessed without knowing the structure of the variant. We have used a novel Cas9 enrichment strategy, in combination with long-read single-molecule nanopore sequencing, to address this need. We describe the nucleotide-level resolution of two problematic cases, both of whom presented with neurodevelopmental problems and were initially investigated by array CGH. In the first case, an incidental 1.7-kb imbalance involving a partial duplication of VHL exon 3 was detected. This variant was inherited from the patient's father, who had a history of renal cancer at 38 years. In the second case, an incidental ~200-kb de novo duplication that included DMD exons 30-44 was resolved. In both cases, the long-read data yielded sufficient information to enable Sanger sequencing to define the rearrangement breakpoints, and creation of breakpoint-spanning PCR assays suitable for testing of relatives. Our Cas9 enrichment and nanopore sequencing approach can be readily adopted by molecular diagnostic laboratories for cost-effective and rapid characterization of challenging duplication-containing alleles. We also anticipate that in future this method may prove useful for characterizing acquired translocations in tumor cells, and for precisely identifying transgene integration sites in mouse models.

An alternative to array-based diagnostics: a prospectively recruited cohort, comparing arrayCGH to next-generation sequencing to evaluate foetal structural abnormalities.

Molecular diagnostic investigations, following the identification of foetal abnormalities, are routinely performed using array comparative genomic hybridisation (aCGH). Despite the utility of this technique, contemporary approaches for the detection of copy number variation are typically based on next-generation sequencing (NGS). We sought to compare an in-house NGS-based workflow (CNVseq) with aCGH, for invasively obtained foetal samples from pregnancies complicated by foetal structural abnormality. DNA from 40 foetuses was screened using both 8 × 60 K aCGH oligoarrays and low-coverage whole genome sequencing. Sequencer-compatible libraries were combined in a ten-sample multiplex and sequenced using an Illumina HiSeq2500. The mean resolution of CNVseq was 29 kb, compared to 60 kb for aCGH analyses. Four clinically significant, concordant, copy number imbalances were detected using both techniques, however, genomic breakpoints were more precisely defined by CNVseq. This data indicates CNVseq is a robust and sensitive alternative to aCGH, for the prenatal investigation of foetuses with structural abnormalities. Impact statement What is already known about this subject? Copy number variant analysis using next-generation sequencing has been successfully applied to investigations of tumour specimens and patients with developmental delays. The application of our approach, to a prospective prenatal diagnosis cohort, has not hitherto been assessed. What do the results of this study add? Next-generation sequencing has a comparable turnaround time and assay sensitivity to copy number variant analysis performed using array CGH. We demonstrate that having established a next-generation sequencing facility, high-throughput CNVseq sample processing and analysis can be undertaken within the framework of a regional diagnostic service. What are the implications of these findings for clinical practice and/or further research? Array CGH is a legacy technology which is likely to be superseded by low-coverage whole genome sequencing, for the detection of copy number variants, in the prenatal diagnosis of structural abnormalities.

Biallelic Mutations in LRRC56, Encoding a Protein Associated with Intraflagellar Transport, Cause Mucociliary Clearance and Laterality Defects.

Primary defects in motile cilia result in dysfunction of the apparatus responsible for generating fluid flows. Defects in these mechanisms underlie disorders characterized by poor mucus clearance, resulting in susceptibility to chronic recurrent respiratory infections, often associated with infertility; laterality defects occur in about 50% of such individuals. Here we report biallelic variants in LRRC56 (known as oda8 in Chlamydomonas) identified in three unrelated families. The phenotype comprises laterality defects and chronic pulmonary infections. High-speed video microscopy of cultured epithelial cells from an affected individual showed severely dyskinetic cilia but no obvious ultra-structural abnormalities on routine transmission electron microscopy (TEM). Further investigation revealed that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link with IFT was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the cilium during axoneme construction, where it co-localizes with IFT trains and is required for the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying LRRC56-null mutations, or a variant resulting in the p.Leu259Pro substitution corresponding to the p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. Together, our findings confirm that deleterious variants in LRRC56 result in a human disease and suggest that this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility.

Short-Course Antimicrobial Therapy Does Not Increase Treatment Failure Rate in Patients with Intra-Abdominal Infection Involving Fungal Organisms.

BACKGROUND: Fungi frequently are isolated in intra-abdominal infections (IAI). The Study to Optimize Peritoneal Infection Therapy (STOP-IT) recently suggested short-course treatment for patients with IAI. It remains unclear whether the presence of fungi in IAI affects the optimal duration of Antimicrobial therapy. We hypothesized that a shorter treatment course in IAI with fungal organisms would be associated with a higher rate of treatment failure. METHODS: Patients enrolled in the STOP-IT trial were stratified according to the presence or absence of a fungal isolate. They were analyzed as a subgroup based on original randomization to either the control group or an experimental group that received a four-day course of Antimicrobial therapy and by comparison with those without a fungal component to their infection. Descriptive comparisons were performed using a χ2, Fisher exact, or Kruskal-Wallis test as appropriate. The primary outcome was a composite of recurrent IAI, surgical site infection, and death. RESULTS: A total of 411 patients in the study (79%) had available culture data, of which 58 (14%) had positive fungal cultures. The most common organisms were Candida albicans and C. glabrata. The treatment failure rate was equivalent in the experimental and control arms (29.6% vs. 22.6%; p = 0.54). Patients with fungal isolates were more likely to have malignant disease (25.9% vs. 9.6%; p = 0.0004) and coronary artery disease (22% vs. 12%; p = 0.04), but were otherwise similar to those without fungal isolates. Patients with fungal isolates had more hospital days (median 10 vs. 7; p < 0.0001) and more days to resumption of enteral intake (median 5 vs. 3; p = 0.0006), but there was no difference in the composite outcome. CONCLUSIONS: Patients with IAI involving fungal organisms randomized to a shorter course of Antimicrobial therapy had no difference in the rate of treatment failure. These results suggest that the presence of fungi in IAI may not indicate independently the need for a longer course of Antimicrobial therapy.

DNAAF1 links heart laterality with the AAA+ ATPase RUVBL1 and ciliary intraflagellar transport.

DNAAF1 (LRRC50) is a cytoplasmic protein required for dynein heavy chain assembly and cilia motility, and DNAAF1 mutations cause primary ciliary dyskinesia (PCD; MIM 613193). We describe four families with DNAAF1 mutations and complex congenital heart disease (CHD). In three families, all affected individuals have typical PCD phenotypes. However, an additional family demonstrates isolated CHD (heterotaxy) in two affected siblings, but no clinical evidence of PCD. We identified a homozygous DNAAF1 missense mutation, p.Leu191Phe, as causative for heterotaxy in this family. Genetic complementation in dnaaf1-null zebrafish embryos demonstrated the rescue of normal heart looping with wild-type human DNAAF1, but not the p.Leu191Phe variant, supporting the conserved pathogenicity of this DNAAF1 missense mutation. This observation points to a phenotypic continuum between CHD and PCD, providing new insights into the pathogenesis of isolated CHD. In further investigations of the function of DNAAF1 in dynein arm assembly, we identified interactions with members of a putative dynein arm assembly complex. These include the ciliary intraflagellar transport protein IFT88 and the AAA+ (ATPases Associated with various cellular Activities) family proteins RUVBL1 (Pontin) and RUVBL2 (Reptin). Co-localization studies support these findings, with the loss of RUVBL1 perturbing the co-localization of DNAAF1 with IFT88. We show that RUVBL1 orthologues have an asymmetric left-sided distribution at both the mouse embryonic node and the Kupffer's vesicle in zebrafish embryos, with the latter asymmetry dependent on DNAAF1. These results suggest that DNAAF1-RUVBL1 biochemical and genetic interactions have a novel functional role in symmetry breaking and cardiac development.

Increased Sensitivity of Diagnostic Mutation Detection by Re-analysis Incorporating Local Reassembly of Sequence Reads.

BACKGROUND: Diagnostic genetic testing programmes based on next-generation DNA sequencing have resulted in the accrual of large datasets of targeted raw sequence data. Most diagnostic laboratories process these data through an automated variant-calling pipeline. Validation of the chosen analytical methods typically depends on confirming the detection of known sequence variants. Despite improvements in short-read alignment methods, current pipelines are known to be comparatively poor at detecting large insertion/deletion mutations. METHODS: We performed clinical validation of a local reassembly tool, ABRA (assembly-based realigner), through retrospective reanalysis of a cohort of more than 2000 hereditary cancer cases. RESULTS: ABRA enabled detection of a 96-bp deletion, 4-bp insertion mutation in PMS2 that had been initially identified using a comparative read-depth approach. We applied an updated pipeline incorporating ABRA to the entire cohort of 2000 cases and identified one previously undetected pathogenic variant, a 23-bp duplication in PTEN. We demonstrate the effect of read length on the ability to detect insertion/deletion variants by comparing HiSeq2500 (2 × 101-bp) and NextSeq500 (2 × 151-bp) sequence data for a range of variants and thereby show that the limitations of shorter read lengths can be mitigated using appropriate informatics tools. CONCLUSIONS: This work highlights the need for ongoing development of diagnostic pipelines to maximize test sensitivity. We also draw attention to the large differences in computational infrastructure required to perform day-to-day versus large-scale reprocessing tasks.

Characterization and Genomic Localization of a SMAD4 Processed Pseudogene.

Like many clinical diagnostic laboratories, the Yorkshire Regional Genetics Service undertakes routine investigation of cancer-predisposed individuals by high-throughput sequencing of patient DNA that has been target-enriched for genes associated with hereditary cancer. Accurate diagnosis using such reagents requires alertness regarding rare nonpathogenic variants that may interfere with variant calling. In a cohort of 2042 such cases, we identified 5 that initially appeared to be carriers of a 95-bp deletion of SMAD4 intron 6. More detailed analysis indicated that these individuals all carried one copy of a SMAD4 processed gene. Because of its interference with diagnostic analysis, we characterized this processed gene in detail. Whole-genome sequencing and confirmatory Sanger sequencing of junction PCR products were used to show that in each of the 5 cases, the SMAD4 processed gene was integrated at the same position on chromosome 9, located within the last intron of the SCAI gene. This rare polymorphic processed gene therefore reflects the occurrence of a single ancestral retrotransposition event. Compared to the reference SMAD4 mRNA sequence NM_005359.5 (https://www.ncbi.nlm.nih.gov/nucleotide), the 5' and 3' untranslated regions of the processed gene are both truncated, but its open reading frame is unaltered. Our experience leads us to advocate the use of an RNA-seq aligner as part of diagnostic assay quality assurance, since this allows recognition of processed pseudogenes in a comparatively facile automated fashion.

m6aViewer: software for the detection, analysis, and visualization of N6-methyladenosine peaks from m6A-seq/ME-RIP sequencing data.

Recent methods for transcriptome-wide N6-methyladenosine (m6A) profiling have facilitated investigations into the RNA methylome and established m6A as a dynamic modification that has critical regulatory roles in gene expression and may play a role in human disease. However, bioinformatics resources available for the analysis of m6A sequencing data are still limited. Here, we describe m6aViewer-a cross-platform application for analysis and visualization of m6A peaks from sequencing data. m6aViewer implements a novel m6A peak-calling algorithm that identifies high-confidence methylated residues with more precision than previously described approaches. The application enables data analysis through a graphical user interface, and thus, in contrast to other currently available tools, does not require the user to be skilled in computer programming. m6aViewer and test data can be downloaded here: http://dna2.leeds.ac.uk/m6a.

Enhanced diagnostic yield in Meckel-Gruber and Joubert syndrome through exome sequencing supplemented with split-read mapping.

BACKGROUND: The widespread adoption of high-throughput sequencing technologies by genetic diagnostic laboratories has enabled significant expansion of their testing portfolios. Rare autosomal recessive conditions have been a particular focus of many new services. Here we report a cohort of 26 patients referred for genetic analysis of Joubert (JBTS) and Meckel-Gruber (MKS) syndromes, two clinically and genetically heterogeneous neurodevelopmental conditions that define a phenotypic spectrum, with MKS at the severe end. METHODS: Exome sequencing was performed for all cases, using Agilent SureSelect v5 reagents and Illumina paired-end sequencing. For two cases medium-coverage (9×) whole genome sequencing was subsequently undertaken. RESULTS: Using a standard analysis pipeline for the detection of single nucleotide and small insertion or deletion variants, molecular diagnoses were confirmed in 12 cases (4%). Seeking to determine whether our cohort harboured pathogenic copy number variants (CNV), in JBTS- or MKS-associated genes, targeted comparative read-depth analysis was performed using FishingCNV. These analyses identified a putative intragenic AHI1 deletion that included three exons spanning at least 3.4 kb and an intergenic MPP4 to TMEM237 deletion that included exons spanning at least 21.5 kb. Whole genome sequencing enabled confirmation of the deletion-containing alleles and precise characterisation of the mutation breakpoints at nucleotide resolution. These data were validated following development of PCR-based assays that could be subsequently used for "cascade" screening and/or prenatal diagnosis. CONCLUSIONS: Our investigations expand the AHI1 and TMEM237 mutation spectrum and highlight the importance of performing CNV screening of disease-associated genes. We demonstrate a robust increasingly cost-effective CNV detection workflow that is applicable to all MKS/JBTS referrals.

Reducing Accidental Dislodgement of the Percutaneous Endoscopic Gastrostomy: A Prospective Trial of the "SafetyBreak" Device.

BACKGROUND: The percutaneous endoscopic gastrostomy (PEG) is a ubiquitous feeding tube with high rates of accidental dislodgement, with significant morbidity and health care costs. We hypothesized use of a decoupling device is a safe and effective mechanism to reduce dislodgements. STUDY DESIGN: We studied a prospective cohort of 100 patients from an academic center. Enrollment included patients requiring PEG tube placement with follow up extending through an individual's lifetime use of their PEG tube. The primary endpoint was accidental dislodgement of the principally placed PEG tube. The secondary endpoint was time to accidental dislodgement of the PEG tube. RESULTS: All 100 patients received the SafetyBreak device and had complete follow-up. Half of the patients had at least a single episode of device decoupling, indicating prevention of dislodgement of the PEG. Eight patients ultimately had dislodgement, resulting in a significantly lower dislodgement rate when compared with a historical cohort (P = .036) and significantly longer survival of the PEG (log rank = 0.005). When compared with a concurrent cohort (without the device) there was also significantly lower dislodgement rate (P = .03) and a trend toward longer survival of the PEG (log rank = 0.08). CONCLUSIONS: When compared with both a historical and concurrent cohort of patients, the SafetyBreak device reduces accidental dislodgement of PEG tubes. As an increasing number of PEGs are being placed, an increasing number of patients are at risk for dislodgement. The SafetyBreak device is an innovative, economical solution to the problem of accidental dislodgement of the PEG tube.

Towards a Next-Generation Sequencing Diagnostic Service for Tumour Genotyping: A Comparison of Panels and Platforms.

Detection of clinically actionable mutations in diagnostic tumour specimens aids in the selection of targeted therapeutics. With an ever increasing number of clinically significant mutations identified, tumour genetic diagnostics is moving from single to multigene analysis. As it is still not feasible for routine diagnostic laboratories to perform sequencing of the entire cancer genome, our approach was to undertake targeted mutation detection. To optimise our diagnostic workflow, we evaluated three target enrichment strategies using two next-generation sequencing (NGS) platforms (Illumina MiSeq and Ion PGM). The target enrichment strategies were Fluidigm Access Array custom amplicon panel including 13 genes (MiSeq sequencing), the Oxford Gene Technologies (OGT) SureSeq Solid Tumour hybridisation panel including 60 genes (MiSeq sequencing), and an Ion AmpliSeq Cancer Hotspot Panel including 50 genes (Ion PGM sequencing). DNA extracted from formalin-fixed paraffin-embedded (FFPE) blocks of eight previously characterised cancer cell lines was tested using the three panels. Matching genomic DNA from fresh cultures of these cell lines was also tested using the custom Fluidigm panel and the OGT SureSeq Solid Tumour panel. Each panel allowed mutation detection of core cancer genes including KRAS, BRAF, and EGFR. Our results indicate that the panels enable accurate variant detection despite sequencing from FFPE DNA.