Phenotypic Characterization of EIF2AK4 Mutation Carriers in a Large Cohort of Patients Diagnosed Clinically With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene (EIF2AK4) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH. METHODS: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured. RESULTS: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% [interquartile range, 30%-35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival. CONCLUSIONS: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation

Telomerecat: A ploidy-agnostic method for estimating telomere length from whole genome sequencing data.

Telomere length is a risk factor in disease and the dynamics of telomere length are crucial to our understanding of cell replication and vitality. The proliferation of whole genome sequencing represents an unprecedented opportunity to glean new insights into telomere biology on a previously unimaginable scale. To this end, a number of approaches for estimating telomere length from whole-genome sequencing data have been proposed. Here we present Telomerecat, a novel approach to the estimation of telomere length. Previous methods have been dependent on the number of telomeres present in a cell being known, which may be problematic when analysing aneuploid cancer data and non-human samples. Telomerecat is designed to be agnostic to the number of telomeres present, making it suited for the purpose of estimating telomere length in cancer studies. Telomerecat also accounts for interstitial telomeric reads and presents a novel approach to dealing with sequencing errors. We show that Telomerecat performs well at telomere length estimation when compared to leading experimental and computational methods. Furthermore, we show that it detects expected patterns in longitudinal data, repeated measurements, and cross-species comparisons. We also apply the method to a cancer cell data, uncovering an interesting relationship with the underlying telomerase genotype

Comprehensive Cancer-Predisposition Gene Testing in an Adult Multiple Primary Tumor Series Shows a Broad Range of Deleterious Variants and Atypical Tumor Phenotypes.

Multiple primary tumors (MPTs) affect a substantial proportion of cancer survivors and can result from various causes, including inherited predisposition. Currently, germline genetic testing of MPT-affected individuals for variants in cancer-predisposition genes (CPGs) is mostly targeted by tumor type. We ascertained pre-assessed MPT individuals (with at least two primary tumors by age 60 years or at least three by 70 years) from genetics centers and performed whole-genome sequencing (WGS) on 460 individuals from 440 families. Despite previous negative genetic assessment and molecular investigations, pathogenic variants in moderate- and high-risk CPGs were detected in 67/440 (15.2%) probands. WGS detected variants that would not be (or were not) detected by targeted resequencing strategies, including low-frequency structural variants (6/440 [1.4%] probands). In most individuals with a germline variant assessed as pathogenic or likely pathogenic (P/LP), at least one of their tumor types was characteristic of variants in the relevant CPG. However, in 29 probands (42.2% of those with a P/LP variant), the tumor phenotype appeared discordant. The frequency of individuals with truncating or splice-site CPG variants and at least one discordant tumor type was significantly higher than in a control population (χ2 = 43.642; p ≤ 0.0001). 2/67 (3%) probands with P/LP variants had evidence of multiple inherited neoplasia allele syndrome (MINAS) with deleterious variants in two CPGs. Together with variant detection rates from a previous series of similarly ascertained MPT-affected individuals, the present results suggest that first-line comprehensive CPG analysis in an MPT cohort referred to clinical genetics services would detect a deleterious variant in about a third of individuals.JW is supported by a Cancer Research UK Cambridge Cancer Centre Clinical Research Training Fellowship. Funding for the NIHR BioResource – Rare diseases project was provided by the National Institute for Health Research (NIHR, grant number RG65966). ERM acknowledges support from the European Research Council (Advanced Researcher Award), NIHR (Senior Investigator Award and Cambridge NIHR Biomedical Research Centre), Cancer Research UK Cambridge Cancer Centre and Medical Research Council Infrastructure Award. The University of Cambridge has received salary support in respect of EM from the NHS in the East of England through the Clinical Academic Reserve. The views expressed are those of the authors and not necessarily those of the NHS or Department of Health. DGE is an NIHR Senior Investigator and is supported by the all Manchester NIHR Biomedical Research Centre

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in $\textit{CHM}$ in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.This work was supported by The National Institute for Health Research England (NIHR) for the NIHR BioResource – Rare Diseases project (grant number RG65966). The Moorfields Eye Hospital cohort of patients and clinical and imaging data were ascertained and collected with the support of grants from the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital, National Health Service Foundation Trust, and UCL Institute of Ophthalmology, Moorfields Eye Hospital Special Trustees, Moorfields Eye Charity, the Foundation Fighting Blindness (USA), and Retinitis Pigmentosa Fighting Blindness. M.M. is a recipient of an FFB Career Development Award. E.M. is supported by UCLH/UCL NIHR Biomedical Research Centre. F.L.R. and D.G. are supported by Cambridge NIHR Biomedical Research Centre

Publisher Correction: Telomerecat: A ploidy-agnostic method for estimating telomere length from whole genome sequencing data.

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Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Implementation of a Standardized HIPEC Protocol Improves Outcome for Peritoneal Malignancy

Experience with Cytoreductive Surgery (CRS) and Hyperthermic Intraperitoneal Chemotherapy (HIPEC) in a pioneer hospital resulted in a treatment protocol that has become the standard in the Netherlands. Outcome of CRS and HIPEC was reviewed to assure differences between the pioneer phase and the period wherein the Dutch HIPEC protocol was clinically implemented. The first consecutive 100 CRS and HIPEC procedures performed in the Netherlands were included as pioneer cohort (1995-1999). Two-hundred and seventy-two procedures that were performed in three participating HIPEC centres after the implementation of the Dutch HIPEC protocol were included as the implementation cohort (2005-2012). Another 100 recent patients of the first centre were included as a control group (2009-2011). Indications for the CRS and HIPEC treatment were peritoneal carcinomatosis (PC) from colorectal carcinoma and pseudomyxoma peritonei (PMP). Of the 472 included procedures, 327 (69 %) procedures were performed for PC from colorectal carcinoma and 145 for PMP (31 %). Compared with the implementation phase, the pioneer phase was characterized by more affected abdominal regions (mean 4.3 vs. 3.5, p <0.001), more resections (mean 3.8 vs. 3.4, p <0.001), less macroscopic radical cytoreductions (66 vs. 86 %, p <0.001) and more patients with major morbidity (grade III-V) (64 vs. 32 %, p <0.001). Other determinants of morbidity were high tumour load and multiple organ resections. Outcome of the implementation phase was similar to the control group. This study determined that outcome had improved ever since the Dutch HIPEC protocol has been implemented based on completeness of cytoreduction and decreasing morbidity

Long-Term Oncological Outcomes of Papillary Thyroid Cancer and Follicular Thyroid Cancer in Children: A Nationwide Population-Based Study

Introduction: Pediatric thyroid carcinoma is a rare malignancy and data on long-term oncological outcomes are sparse. The aim of this study was to describe the long-term oncological outcomes of pediatric papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) in a national cohort, and to identify risk factors for recurrence. Methods: We conducted a nationwide, retrospective cohort study, in which we combined two national databases. Patients aged <18 years, diagnosed with PTC or FTC in the Netherlands between 2000 and 2016, were included. pT-stage, pN-stage, multifocality and angioinvasion were included in a Cox-regression analysis for the identification of risk factors for recurrence. Results: 133 patients were included: 110 with PTC and 23 with FTC. Patients with PTC most often presented with pT2 tumors (24%) and pN1b (45%). During a median follow-up of 11.3 years, 21 patients with PTC developed a recurrence (19%). Nineteen recurrences were regional (91%) and 2 were pulmonary (9%). No risk factors for recurrence could be determined. One patient who developed pulmonary recurrence died two years later. Cause of death was not captured. Patients with FTC most often presented with pT2 tumors (57%). One patient presented with pN1b (4%). In 70%, no lymph nodes were collected. None of the patients with FTC developed a recurrence or died. Conclusion: Pediatric PTC and FTC are two distinct diseases. Recurrence in pediatric PTC is common, but in FTC it is not. Survival for both pediatric PTC and FTC is very good

Cytoreduction and HIPEC in The Netherlands:Nationwide Long-term Outcome Following the Dutch Protocol

<p>Purpose. This nationwide study evaluated results of cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) for peritoneal metastasis of colorectal origin in the Netherlands following a national protocol.</p><p>Methods. In a multi-institutional study prospective databases of patients with peritoneal carcinomatosis (PC) from colorectal cancer and pseudomyxoma peritonei (PMP) treated according to the Dutch HIPEC protocol, a uniform approach for the CRS and HIPEC treatment, were reviewed. Primary end point was overall survival and secondary end points were surgical outcome and progression- free survival.</p><p>Results. Nine-hundred sixty patients were included; 660 patients (69 %) were affected by PC of colorectal carcinoma and the remaining suffered from PMP (31 %). In 767 procedures (80 %), macroscopic complete cytoreduction was achieved. Three-hundred and thirty one patients had grade III-V complications (34 %). Thirty-two patients died perioperatively (3 %). Median length of hospital stay was 16 days (range 0-166 days). Median follow-up period was 41 months (95 % confidence interval (CI), 36-46 months). Median progression-free survival was 15 months (95 % CI 13-17 months) for CRC patients and 53 months (95 % CI 40-66 months) for PMP patients. Overall median survival was 33 (95 % CI 28-38 months) months for CRC patients and 130 months (95 % CI 98-162 months) for PMP patients. Three- and five-year survival rates were 46 and 31 % respectively in case of CRC patients and 77 and 65 % respectively in case of PMP patients.</p><p>Conclusions. The results underline the safety and efficacy of cytoreduction and HIPEC for PC from CRC and PMP. It is assumed the uniform Dutch HIPEC protocol was beneficial.</p>