Next generation sequencing panel as an effective approach to genetic testing in patients with a highly variable phenotype of neuromuscular disorders.

Neuromuscular disorders (NMDs) include a wide range of diseases affecting the peripheral nervous system. The genetic diagnoses are increasingly obtained with using the next generation sequencing (NGS). We applied the custom-design targeted NGS panel including 89 genes, together with genotyping and multiplex ligation-dependent probe amplification (MLPA) to identify a genetic spectrum of NMDs in 52 Polish patients. As a result, the genetic diagnosis was determined by NGS panel in 29 patients so its diagnostic utility is estimated at 55.8%. The most pathogenic variants were found in CLCN1, followed by CAPN3, SCN4A, and SGCA genes. Genotyping of myotonic dystrophy type 1 and 2 (DM1 and DM2) as a secondary approach has been performed. The co-occurrence of CAPN3 and CNBP mutations in one patient as well as DYSF and CNBP mutations in another suggests possibly more complex inheritance as well as expression of a phenotype. In 7 individuals with single nucleotide variant found in NGS testing, the MLPA of the CAPN3 gene was performed detecting the deletion encompassing exons 2-8 in the CAPN3 gene in one patient, confirming recessive limb-girdle muscular dystrophy type 1 (LGMDR1). Thirty patients obtained a genetic diagnosis (57.7%) after using NGS testing, genotyping and MLPA analysis. The study allowed for the identification of 27 known and 4 novel pathogenic/likely pathogenic variants and variants of uncertain significance (VUS) associated with NMDs.In conclusion, the diagnostic approach with diverse molecular techniques enables to broaden the mutational spectrum and maximizes the diagnostic yield. Furthermore, the co-occurrence of DM2 and LGMD has been detected in 2 individuals.

Paired comparison of the analytical performance between the Oncomine™ Comprehensive Assay v3 and whole-exome sequencing of ovarian cancer tissue.

BACKGROUND: Next-generation sequencing (NGS) has been implemented in clinical oncology as a personalized medicine tool to identify targetable genetic alterations and to guide treatment decisions. However, the optimal NGS test strategy and target genes for clinical use are still being discussed. The aim was to compare the performance of the Oncomine™ Comprehensive Assay v3 (OCAv3) (targeted gene panel) and whole-exome sequencing (WES) to investigate somatic single and multiple nucleotide variants and small indels in ovarian cancer patients. METHODS AND RESULTS: Genomic DNA was isolated from fresh frozen samples of five high-grade serous (HGSC) and three clear cell ovarian (oCCC) cancer patients. Exome sequencing libraries were prepared by using the Ion AmpliSeq Exome RDY kit, whereas libraries for OCAv3 were prepared using by Ion AmpliSeq™ Library Kit Plus. Sequencing was performed using the Ion S5XL System (Thermo Fisher Scientific). When including only variants classified as pathogenic, likely pathogenic or unknown significance based on ClinVar database verdicts and comparing overlapping regions covered both by the OCAv3 assay and WES, 23 variants were detected by both assays. However, OCAv3 detected additionally two variants: ARID1A: p.Gln563Ter and TP53: p.Ser261ValfsTer84 that have not passed WES filtering criteria due to low coverage. CONCLUSIONS: With the present treatment possibilities, OCAv3 panel testing provided higher diagnostic yield due to better coverage. Our study emphasizes that WES, although offering the potential to identify novel findings in genes not covered by OCAv3, might overlook variants in genes relevant for OC.

Clinical and genetic spectra of 1550 index patients with hereditary spastic paraplegia.

Hereditary spastic paraplegia refers to rare genetic neurodevelopmental and/or neurodegenerative disorders in which spasticity due to length-dependent damage to the upper motor neuron is a core sign. Their high clinical and genetic heterogeneity makes their diagnosis challenging. Multigene panels allow a high-throughput targeted analysis of the increasing number of genes involved using next-generation sequencing. We report here the clinical and genetic results of 1550 index cases tested for variants in a panel of hereditary spastic paraplegia related genes analysed in routine diagnosis. A causative variant was found in 475 patients (30.7%) in 35/65 screened genes. SPAST and SPG7 were the most frequently mutated genes, representing 142 (9.2%) and 75 (4.8%) index cases of the whole series, respectively. KIF1A, ATL1, SPG11, KIF5A and REEP1 represented more than 1% (>17 cases) each. There were 661 causative variants (382 different ones) and 30 of them were structural variants. This large cohort allowed us to obtain an overview of the clinical and genetic spectrum of hereditary spastic paraplegia in clinical practice. Because of the wide phenotypic variability, there was no very specific sign that could predict the causative gene, but there were some constellations of symptoms that were found often related to specific subtypes. Finally, we confirmed the diagnostic effectiveness of a targeted sequencing panel as a first-line genetic test in hereditary spastic paraplegia. This is a pertinent strategy because of the relative frequency of several known genes (i.e. SPAST, KIF1A) and it allows identification of variants in the rarest involved genes and detection of structural rearrangements via coverage analysis, which is less efficient in exome datasets. It is crucial because these structural variants represent a significant proportion of the pathogenic hereditary spastic paraplegia variants (∼6% of patients), notably for SPAST and REEP1. In a subset of 42 index cases negative for the targeted multigene panel, subsequent whole-exome sequencing allowed a theoretical diagnosis yield of ∼50% to be reached. We then propose a two-step strategy combining the use of a panel of genes followed by whole-exome sequencing in negative cases.

Design of a targeted next-generation DNA sequencing panel for pediatric T-cell lymphoblastic lymphoma to unravel biology and optimize treatment.

Low incidence and molecular heterogeneity of pediatric T-cell lymphoblastic lymphoma (T-LBL) require an international, large-scale effort to identify novel clinical biomarkers. The ongoing international clinical trial LBL2018 (NCT04043494) represents an ideal opportunity to implement a common analytic approach. Targeted next-generation sequencing is well-suited for this purpose; however, selection of relevant target genes for T-LBL remains subject of ongoing debates. Our group has recently designed and evaluated a first target panel of 80 candidate genes for T-LBL. The present study aimed at developing a novel optimized gene panel for large-scale application and to promote an international agreement on a common core panel. Small sequence variants obtained from our former study were systematically analyzed and classified with regards to pathogenic relevance, to prioritize candidate genes. Additional genes were curated from literature and online databases for a more comprehensive analysis of relevant functions and signaling pathways. The new target panel TGP-T-LBL entails 84 candidate genes which are key actors in NOTCH, PI3K-AKT, JAK-STAT, RAS signaling, epigenetic regulation, transcription, DNA repair, cell cycle regulation, and ribosomal function. From our former gene panel, 35 out of 80 candidate genes were selected for the novel panel. Forty-six out of 84 genes are currently being analyzed in the ongoing international trial LBL2018. Exploratory analysis of prognostic relevance on mutation-level suggested a potential association of PIK3CA variants c.1624G>A(p.Glu542Lys) and c.1633G>A(p.Glu545Lys) to occurrence of relapse, emphasizing particular relevance of mutation analysis in PI3K-AKT signaling. Our approach promotes comprehensive and clinically relevant mutational profiling of pediatric T-LBL.

Consistent count region-copy number variation (CCR-CNV): an expandable and robust tool for clinical diagnosis of copy number variation at the exon level using next-generation sequencing data.

PURPOSE: Despite the importance of exonic copy number variations (CNVs) in human genetic diseases, reliable next-generation sequencing-based methods for detecting them are unavailable. We developed an expandable and robust exonic CNV detection tool called consistent count region (CCR)-CNV. METHODS: In total, about 1000 samples of the truth set were used for validating CCR-CNV. We compared CCR-CNV performance with 2 well-known CNV tools. Finally, to overcome the limitations of CCR-CNV, we devised a combined approach. RESULTS: The mean sensitivity and specificity of CCR-CNV alone were above 95%, which was superior to that of other CNV tools, such as DECoN and Atlas-CNV. However, low covered region and positive predictive value and high false discovery rate act as obstacles to its use in clinical settings. The combined approach showed much improved performance than CCR-CNV alone. CONCLUSION: In this study, we present a novel diagnostic tool that allows the identification of exonic CNVs with high confidence using various reagents and clinical next-generation sequencing platforms. We validated this method using the largest multiple ligation-dependent probe amplification-confirmed data set, including sufficient copy normal control data. The approach, combined with existing CNV tools, allows the implementation of CCR-CNV in clinical settings.

Analytical and Clinical Validation of Cell-Free Circulating Tumor DNA Assay for the Estimation of Tumor Mutational Burden.

BACKGROUND: Ultra-deep sequencing to detect low-frequency mutations in circulating tumor-derived DNA (ctDNA) increases the diagnostic value of liquid biopsy. The demand for large ctDNA panels for comprehensive genomic profiling and tumor mutational burden (TMB) estimation is increasing; however, few ctDNA panels for TMB have been validated. Here, we designed a ctDNA panel with 531 genes, named TMB500, along with a technical and clinical validation. METHODS: Synthetic reference cell-free DNA materials with predefined allele frequencies were sequenced in a total of 92 tests in 6 batches to evaluate the precision, linearity, and limit of detection of the assay. We used clinical samples from 50 patients with various cancers, 11 healthy individuals, and paired tissue samples. Molecular barcoding and data analysis were performed using customized pipelines. RESULTS: The assay showed high precision and linearity (coefficient of determination, r2 =0.87) for all single nucleotide variants, with a limit of detection of 0.24%. In clinical samples, the TMB500 ctDNA assay detected most variants present and absent in tissues, showing that ctDNA could assess tumor heterogeneity in different tissues and metastasis sites. The estimated TMBs correlated well between tissue and blood, except in 4 cases with extreme heterogeneity that showed very high blood TMBs compared to tissue TMBs. A pilot evaluation showed that the TMB500 assay could be used for disease monitoring. CONCLUSIONS: The TMB500 assay is an accurate and reliable ctDNA assay for many clinical purposes. It may be useful for guiding the treatment of cancers with diverse genomic profiles, estimating TMB in immune therapy, and disease monitoring.

Cost-Effectiveness of Tumor Genomic Profiling to Guide First-Line Targeted Therapy Selection in Patients With Metastatic Lung Adenocarcinoma.

OBJECTIVES: A cost-effectiveness analysis comparing comprehensive genomic profiling (CGP) of 10 oncogenes, targeted gene panel testing (TGPT) of 4 oncogenes, and no tumor profiling over the lifetime for patients with metastatic lung adenocarcinoma from the Centers for Medicare and Medicaid Services' perspective was conducted. METHODS: A decision analytic model used 10 000 hypothetical Medicare beneficiaries with metastatic lung adenocarcinoma to simulate outcomes associated with CGP (ALK, BRAF, EGFR, ERBB2, MET, NTRK1, NTRK2, NTRK3, RET, ROS1), TGPT (ALK, BRAF, EGFR, ROS1), and no tumor profiling (no genes tested). First-line targeted cancer-directed therapies were assigned if actionable gene variants were detected; otherwise, nontargeted cancer-directed therapies were assigned. Model inputs were derived from randomized trials (progression-free survival, adverse events), the Veterans Health Administration and Medicare (drug costs), published studies (nondrug cancer-related management costs, health state utilities), and published databases (actionable variant prevalences). Costs (2019 US$) and quality-adjusted life-years (QALYs) were discounted at 3% per year. Probabilistic sensitivity analyses used 1000 Monte Carlo simulations. RESULTS: No tumor profiling was the least costly/person ($122 613 vs $184 063 for TGPT and $188 425 for CGP) and yielded the least QALYs/person (0.53 vs 0.73 for TGPT and 0.74 for CGP). The costs per QALY gained and corresponding 95% confidence interval were $310 735 ($278 323-$347 952) for TGPT vs no tumor profiling and $445 545 ($322 297-$572 084) for CGP vs TGPT. All probabilistic sensitivity analysis simulations for both comparisons surpassed the willingness-to-pay threshold ($150 000 per QALY gained). CONCLUSION: Compared with no tumor profiling in patients with metastatic lung adenocarcinoma, tumor profiling (TGPT, CGP) improves quality-adjusted survival but is not cost-effective.

Study of ten causal genes in Turkish patients with clinically suspected maturity-onset diabetes of the young (MODY) using a targeted next-generation sequencing panel.

BACKGROUND: Maturity-onset diabetes of the young (MODY), which is the most common cause of monogenic diabetes, has an autosomal dominant pattern of inheritance and exhibits marked clinical and genetic heterogeneity. The aim of the current study was to investigate molecular defects in patients with clinically suspected MODY using a next-generation sequencing (NGS)-based targeted gene panel. METHODS: Candidate patients with clinical suspicion of MODY and their parents were included in the study. Molecular genetic analyses were performed on genomic DNA by using NGS. A panel of ten MODY-causal genes involving GCK, HNF1A, HNF1B, HNF4A, ABCC8, CEL, INS, KCNJ11, NEUROD1, PDX1 was designed and subsequently implemented to screen 40 patients for genetic variants. RESULTS: Ten different pathogenic or likely pathogenic variants were identified in MODY-suspected patients, with a diagnostic rate of 25%. Three variants of uncertain significance were also detected in the same screen. A novel pathogenic variant in the gene HNF1A (c.505_506delAA [p.Lys169AlafsTer18]) was described for the first time in this report. Intriguingly, we were able to detect variants associated with rare forms of MODY in our study population. CONCLUSIONS: Our results suggest that in heterogenous diseases such as MODY, NGS analysis enables accurate identification of underlying molecular defects in a timely and cost-effective manner. Although MODY accounts for 2-5% of all diabetic cases, molecular genetic diagnosis of MODY is necessary for optimal long-term treatment and prognosis as well as for effective genetic counseling.

Extended endocrine therapy in breast cancer: A basket of length-constraint feature selection metaheuristics to balance Type I against Type II errors.

Extended endocrine therapy beyond 5 years is of major concern to ER + breast cancer survivors. However, it might be unsuitable to apply routinely used genomic tests designed for early recurrence risks to distant recurrence within 10 years in extended treatment context. These tests initially aim at high sensitivities with Type I errors much higher than Type II. Having lower positive predictive values (PPVs), these tests can bring many false positives who might not need further treatment options to avoid adversely affecting quality of life. Alternatively, we proposed a top-down approach to the raised issues. We built 149 targeted genes from four genomic tests upon 381 ER-positive node-negative patients with either metastasis free beyond 10 years (n = 202) or metastasis within 10 years (n = 179). By a basket of SVM-wrapped length-constraint feature selection (LCFS), we discovered four genomic SVMs that traded off Type I against Type II errors. Two independent cohorts were used to validate disease outcome predictions. A 36-gene SVM balanced sensitivities with PPVs at good levels: 74% vs 76% on 10-fold cross validation (n = 347) and 75% vs 71% on a test set (n = 34). Neither Oncotype DX RS (cutoff = 18, 31, 60.97) nor PAM50 ROR-S (cutoff = 29, 53, 61.18) could. Independent cohorts showed the 36-gene SVM predicted disease free survival (n = 136, HR = 2.59; 95% CI, 1.4-4.8) and disease specific survival (n = 127, HR = 4.06; 95% CI, 1.63-10.11) better than RS (DFS, HR = 2.15; DSS, HR = 3.86) and ROR-S (DFS, HR = 2.29; DSS, HR = 2.76). The case study demonstrated how we identified a genomic test to balance Type I against Type II errors for risk stratification. The top-down approach centered around the LCFS-metaheuristics basket is a generic methodology for clinical decision-making and quality of life using targeted profiling data where the number of dimensions (p) is smaller than the number of samples (n).

Primary Immunodeficiencies in India: Molecular Diagnosis and the Role of Next-Generation Sequencing.

Primary immunodeficiency diseases (PIDs) are a group of clinically and genetically heterogeneous disorders showing ethnic and geographic diversities. Next-generation sequencing (NGS) is a comprehensive tool to diagnose PID. Although PID is common in India, data on the genetic spectrum of PIDs are limited due to financial restrictions. The study aims to characterize the clinical and genetic spectrum of PID patients in India and highlight the importance of a cost-effective targeted gene panel sequencing approach for PID in a resource-limited setting. The study includes 229 patients with clinical and laboratory features suggestive of PIDs. Mutation analysis was done by Sanger sequencing and NGS targeting a customized panel of genes. Pathogenic variants were identified in 97 patients involving 42 different genes with BTK and IL12RB1 being the most common mutated genes. Autosomal recessive and X-linked recessive inheritance were seen in 51.6% and 23.7% of patients. Mendelian susceptibility to mycobacterial diseases (MSMD) and IL12RB1 mutations was more common in our population compared to the Western world and the Middle East. Two patients with hypomorphic RAG1 mutations and one female with skewed CYBB mutation were also identified. Another 40 patients had variants classified as variants of uncertain significance (VUS). The study shows that targeted NGS is an effective diagnostic strategy for PIDs in countries with limited diagnostic resources. Molecular diagnosis of PID helps in genetic counseling and to make therapeutic decisions including the need for a stem cell transplantation.

Expanding the clinical phenotype of RASopathies in 38 Turkish patients, including the rare LZTR1, RAF1, RIT1 variants, and large deletion in NF1.

RASopathies are a group of disorders caused by pathogenic variants in the genes encoding Ras/mitogen-activated protein kinase pathway and share overlapping clinical and molecular features. This study is aimed to describe the clinical and molecular features of 38 patients with RASopathies. Sanger or targeted next-generation sequencing of related genes and multiplex ligation-dependent-probe amplification analysis for NF1 were performed. The pathogenic variant detection rate was 94.4%. While PTPN11 was responsible for 50% of 18 patients with Noonan syndrome (NS), SOS1, LZTR1, RIT1, and RAF1 were responsible for the remaining 27.8%, 11.1%, 5.5%, and 5.5%, respectively. Three variants in LZTR1 were novel, of which two were identified in the compound heterozygous state in a patient with intellectual disability and hypertrophic cardiomyopathy, whereas the third variant was found in the heterozygous state in a patient with pulmonary stenosis and normal intelligence. We described pyloric stenosis, knee dislocation, and cleft palate in patients with SOS1, RIT1, and RAF1 variants, respectively, that was not previously reported. We detected a PTPN11 variant in three patients from same family with NS with multiple lentigines. BRAF and MAP2K2 variants were found in eight patients with Cardiofaciocutaneous syndrome. Two variants in HRAS were detected in two Costello syndrome patients, one with a mild and the other with a severe phenotype. While large NF1 deletions were identified in four Neurofibromatosis-NS patients with intellectual disability, intelligence was normal in one patient with missense variant. In conclusion, this study provided three novel variants in LZTR1 and expanded the clinical phenotype of rare RASopathies.

Targeted broad-based genetic testing by next-generation sequencing informs diagnosis and facilitates management in patients with kidney diseases.

BACKGROUND: The clinical diagnosis of genetic renal diseases may be limited by the overlapping spectrum of manifestations between diseases or by the advancement of disease where clues to the original process are absent. The objective of this study was to determine whether genetic testing informs diagnosis and facilitates management of kidney disease patients. METHODS: We developed a comprehensive genetic testing panel (KidneySeq) to evaluate patients with various phenotypes including cystic diseases, congenital anomalies of the kidney and urinary tract (CAKUT), tubulointerstitial diseases, transport disorders and glomerular diseases. We evaluated this panel in 127 consecutive patients ranging in age from newborns to 81 years who had samples sent in for genetic testing. RESULTS: The performance of the sequencing pipeline for single-nucleotide variants was validated using CEPH (Centre de'Etude du Polymorphism) controls and for indels using Genome-in-a-Bottle. To test the reliability of the copy number variant (CNV) analysis, positive samples were re-sequenced and analyzed. For patient samples, a multidisciplinary review board interpreted genetic results in the context of clinical data. A genetic diagnosis was made in 54 (43%) patients and ranged from 54% for CAKUT, 53% for ciliopathies/tubulointerstitial diseases, 45% for transport disorders to 33% for glomerulopathies. Pathogenic and likely pathogenic variants included 46% missense, 11% nonsense, 6% splice site variants, 23% insertion-deletions and 14% CNVs. In 13 cases, the genetic result changed the clinical diagnosis. CONCLUSION: Broad genetic testing should be considered in the evaluation of renal patients as it complements other tests and provides insight into the underlying disease and its management.

The odyssey of complex neurogenetic disorders: From undetermined to positive.

The genetic and phenotypic heterogeneity of neurogenetic diseases forces patients and their families into a "diagnostic odyssey." An increase in the variability of genetic disorders and the corresponding gene-disease associations suggest the need to periodically re-evaluate the significance of variants of undetermined pathogenicity. Here, we report the diagnostic and clinical utility of Targeted Gene Panel Sequencing (TGPS) and Whole Exome Sequencing (WES) in 341 patients with suspected neurogenetic disorders from centers in Buenos Aires and Cincinnati over the last 4 years, focusing on the usefulness of reinterpreting variants previously classified as of uncertain significance. After a mean of ±2years (IC 95:0.73-3.27), approximately 30% of the variants of uncertain significance were reclassified as pathogenic. The use of next generation sequencing methods has facilitated the identification of both germline and mosaic pathogenic variants, expanding the diagnostic yield. These results demonstrate the high clinical impact of periodic reanalysis of undetermined variants in clinical neurology.

Gene and Variant Annotation for Mendelian Disorders in the Era of Advanced Sequencing Technologies.

Comprehensive annotations of genetic and noncoding regions and corresponding accurate variant classification for Mendelian diseases are the next big challenge in the new genomic era of personalized medicine. Progress in the development of faster and more accurate pipelines for genome annotation and variant classification will lead to the discovery of more novel disease associations and candidate therapeutic targets. This ultimately will facilitate better patient recruitment in clinical trials. In this review, we describe the trends in research at the intersection of basic and clinical genomics that aims to increase understanding of overall genomic complexity, complex inheritance patterns of disease, and patient-phenotype-specific genomic associations. We describe the emerging field of translational functional genomics, which integrates other functional "-omics" approaches that support next-generation sequencing genomic data in order to facilitate personalized diagnostics, disease management, biomarker discovery, and medicine. We also discuss the utility of this integrated approach for diagnostic clinics and medical databases and its role in the future of personalized medicine.

Atlas-CNV: a validated approach to call single-exon CNVs in the eMERGESeq gene panel.

PURPOSE: To provide a validated method to confidently identify exon-containing copy-number variants (CNVs), with a low false discovery rate (FDR), in targeted sequencing data from a clinical laboratory with particular focus on single-exon CNVs. METHODS: DNA sequence coverage data are normalized within each sample and subsequently exonic CNVs are identified in a batch of samples, when the target log2 ratio of the sample to the batch median exceeds defined thresholds. The quality of exonic CNV calls is assessed by C-scores (Z-like scores) using thresholds derived from gold standard samples and simulation studies. We integrate an ExonQC threshold to lower FDR and compare performance with alternate software (VisCap). RESULTS: Thirteen CNVs were used as a truth set to validate Atlas-CNV and compared with VisCap. We demonstrated FDR reduction in validation, simulation, and 10,926 eMERGESeq samples without sensitivity loss. Sixty-four multiexon and 29 single-exon CNVs with high C-scores were assessed by Multiplex Ligation-dependent Probe Amplification (MLPA). CONCLUSION: Atlas-CNV is validated as a method to identify exonic CNVs in targeted sequencing data generated in the clinical laboratory. The ExonQC and C-score assignment can reduce FDR (identification of targets with high variance) and improve calling accuracy of single-exon CNVs respectively. We propose guidelines and criteria to identify high confidence single-exon CNVs.

Clinical, immunologic, and genetic spectrum of 696 patients with combined immunodeficiency.

BACKGROUND: Combined immunodeficiencies (CIDs) are diseases of defective adaptive immunity with diverse clinical phenotypes. Although CIDs are more prevalent in the Middle East than Western countries, the resources for genetic diagnosis are limited. OBJECTIVES: This study aims to characterize the categories of patients with CIDs in Iran clinically and genetically. METHODS: Clinical and laboratory data were obtained from 696 patients with CIDs. Patients were subdivided into those with syndromic (344 patients) and nonsyndromic (352 patients) CIDs. Targeted DNA sequencing was performed on 243 (34.9%) patients. RESULTS: The overall diagnostic yield of the 243 sequenced patients was 77.8% (189 patients). The clinical diagnosis of hyper-IgE syndrome (P < .001), onset of disease at greater than 5 years (P = .02), and absence of multiple affected family members (P = .04) were significantly more frequent in the patients without a genetic diagnosis. An autosomal recessive disease was found in 62.9% of patients, reflecting the high rate of consanguinity in this cohort. Mutations impairing VDJ recombination and DNA repair were the most common underlying causes of CIDs. However, in patients with syndromic CIDs, autosomal recessive mutations in ataxia-telangiectasia mutated (ATM), autosomal dominant mutations in signal transducer and activator of transcription 3 (STAT3), and microdeletions in 22q11.21 were the most commonly affected genomic loci. Patients with syndromic CIDs had a significantly lower 5-year survival rate rather than those with nonsyndromic CIDs. CONCLUSIONS: This study provides proof of principle for the application of targeted next-generation sequencing panels in countries with limited diagnostic resources. The effect of genetic diagnosis on clinical care requires continued improvements in therapeutic resources for these patients.

Clinical and genetic features of congenital dyserythropoietic anemia (CDA).

INTRODUCTION: Congenital dyserythropoietic anemias (CDA) are characterized by hyporegenerative anemia with inadequate reticulocyte values, ineffective erythropoiesis, and hemolysis. Distinctive morphology of bone marrow erythroblasts and identification of causative genes allow classification into 4 types caused by variants in CDAN1, c15orf41, SEC23B, KIF23, and KLF1 genes. OBJECTIVE: Identify pathogenic variants in CDA patients. METHODS: Massive parallel sequencing with a targeted gene panel, Sanger sequencing, Comparative Genome Hybridization (CGH), and in silico predictive analysis of pathogenicity. RESULTS: Pathogenic variants were found in 21 of 53 patients studied from 44 unrelated families. Six variants were found in CDAN1: two reported, p.Arg714Trp and p.Arg725Trp and, four novel, p.Arg623Trp, p.Arg946Trp, p.Phe1125Ser and p.Ser1227Gly. Twelve variants were found in SEC23B: seven reported, p.Arg14Trp, p.Glu109Lys, p.Arg217Ter, c.835-2A>G, p.Arg535Ter, p.Arg550Ter and p.Arg718Ter and, five novel, p.Val164Leu, p.Arg190Gln, p.Gln521Ter, p.Arg546Trp, and p.Arg611Gln. The variant p.Glu325Lys in KLF1 was found in one patient and p.Tyr365Cys in ALAS2 in an other. Moreover, we identified genomic rearrangements by CGH in some SEC23B-monoallelic patients. CONCLUSIONS: New technologies for genetic studies will help to find variants in other genes, in addition to those known, that contribute to or modulate the CDA phenotype or support the correct diagnosis.

GATK hard filtering: tunable parameters to improve variant calling for next generation sequencing targeted gene panel data.

BACKGROUND: NGS technology represents a powerful alternative to the standard Sanger sequencing in the context of clinical setting. The proprietary software that are generally used for variant calling often depend on preset parameters that may not fit in a satisfactory manner for different genes. GATK, which is widely used in the academic world, is rich in parameters for variant calling. However the self-adjusting parameter calibration of GATK requires data from a large number of exomes. When these are not available, which is the standard condition of a diagnostic laboratory, the parameters must be set by the operator (hard filtering). The aim of the present paper was to set up a procedure to assess the best parameters to be used in the hard filtering of GATK. This was pursued by using classification trees on true and false variants from simulated sequences of a real dataset data. RESULTS: We simulated two datasets, with different coverages, including all the sequence alterations identified in a real dataset according to their observed frequencies. Simulated sequences were aligned with standard protocols and then regression trees were built up to identify the most reliable parameters and cutoff values to discriminate true and false variant calls. Moreover, we analyzed flanking sequences of region presenting a high rate of false positive calls observing that such sequences present a low complexity make up. CONCLUSIONS: Our results showed that GATK hard filtering parameter values can be tailored through a simulation study based-on the DNA region of interest to ameliorate the accuracy of the variant calling.

Seeking the driver in tumours with apparent normal molecular profile on comparative genomic hybridization and targeted gene panel sequencing: what is the added value of whole exome sequencing?

BACKGROUND: Molecular tumour profiling technologies have become increasingly important in the era of precision medicine, but their routine use is limited by their accessibility, cost, and tumour material availability. It is therefore crucial to assess their relative added value to optimize the sequence and combination of such technologies. PATIENTS AND METHODS: Within the MOSCATO-01 trial, we investigated the added value of whole exome sequencing (WES) in patients that did not present any molecular abnormality on array comparative genomic hybridization (aCGH) and targeted gene panel sequencing (TGPS) using cancer specific panels. The pathogenicity potential and actionability of mutations detected on WES was assessed. RESULTS: Among 420 patients enrolled between December 2011 and December 2013, 283 (67%) patients were analysed for both TGPS and aCGH. The tumour sample of 25 (8.8%) of them presented a flat (or low-dynamic) aCGH profile and no pathogenic mutation on TGPS. We selected the first eligible 10 samples-corresponding to a heterogeneous cohort of different tumour types-to perform WES. This allowed identifying eight mutations of interest in two patients: FGFR3, PDGFRB, and CREBBP missense single-nucleotide variants (SNVs) in an urothelial carcinoma; FGFR2, FBXW7, TP53, and MLH1 missense SNVs as well as an ATM frameshift mutation in a squamous cell carcinoma of the tongue. The FGFR3 alteration had been previously described as an actionable activating mutation and might have resulted in treatment by an FGFR inhibitor. CREBBP and ATM alterations might also have suggested a therapeutic orientation towards epigenetic modifiers and ataxia-telangectasia and Rad3-related inhibitors, respectively. CONCLUSION: The therapeutic added value of performing WES on tumour samples that do not harbour any genetic abnormality on TGPS and aCGH might be limited and variable according to the histotype. Alternative techniques, including RNASeq and methylome analysis, might be more informative in selected cases.

Performant Mutation Identification Using Targeted Next-Generation Sequencing of 14 Thoracic Aortic Aneurysm Genes.

At least 14 causative genes have been identified for both syndromic and nonsyndromic forms of thoracic aortic aneurysm/dissection (TAA), an important cause of death in the industrialized world. Molecular confirmation of the diagnosis is increasingly important for gene-tailored patient management but consecutive, conventional molecular TAA gene screening is expensive and labor-intensive. To circumvent these problems, we developed a TAA gene panel for next-generation sequencing of 14 TAA genes. After validation, we applied the assay to 100 Marfan patients. We identified 90 FBN1 mutations, 44 of which were novel. In addition, Multiplex ligation-dependent probe amplification identified large deletions in six of the remaining samples, whereas false-negative results were excluded by Sanger sequencing of FBN1, TGFBR1, and TGFBR2 in the last four samples. Subsequently, we screened 55 syndromic and nonsyndromic TAA patients. We identified causal mutations in 15 patients (27%), one in each of the six following genes: ACTA2, COL3A1, TGFBR1, MYLK, SMAD3, SLC2A10 (homozygous), two in NOTCH1, and seven in FBN1. We conclude that our approach for TAA genetic testing overcomes the intrinsic hurdles of consecutive Sanger sequencing of all candidate genes and provides a powerful tool for the elaboration of clinical phenotypes assigned to different genes.