Next-generation Sequencing-based Panel Research Articles

The Genetic Basis of Pericentral Retinitis Pigmentosa-A Form of Mild Retinitis Pigmentosa.

Pericentral retinitis pigmentosa (RP) is an atypical form of RP that affects the near-peripheral retina first and tends to spare the far periphery. This study was performed to further define the genetic basis of this phenotype. We identified a cohort of 43 probands with pericentral RP based on a comprehensive analysis of their retinal phenotype. Genetic analyses of DNA samples from these patients were performed using panel-based next-generation sequencing, copy number variations, and whole exome sequencing (WES). Mutations provisionally responsible for disease were found in 19 of the 43 families (44%) analyzed. These include mutations in RHO (five patients), USH2A (four patients), and PDE6B (two patients). Of 28 putatively pathogenic alleles, 15 (54%) have been previously identified in patients with more common forms of typical RP, while the remaining 13 mutations (46%) were novel. Burden testing of WES data successfully identified HGSNAT as a cause of pericentral RP in at least two patients, suggesting it is also a relatively common cause of pericentral RP. While additional sequencing might uncover new genes specifically associated with pericentral RP, the current results suggest that genetically pericentral RP is not a separate clinical entity, but rather is part of the spectrum of mild RP phenotypes.

Identification of TAZ mutations in pediatric patients with cardiomyopathy by targeted next-generation sequencing in a Chinese cohort

BackgroundBarth syndrome (BTHS) is a rare X-linked recessive disease characterized by cardiomyopathy, neutropenia, skeletal myopathy and growth delay. Early diagnosis and appropriate treatment may improve the prognosis of this disease. The purpose of this study is to determine the role of targeted next-generation sequencing (NGS) in the early diagnosis of BTHS in children with cardiomyopathy.MethodsDuring the period between 2012 and 2015, a gene panel-based NGS approach was used to search for potentially disease-causing genetic variants in all patients referred to our institution with a clinical diagnosis of primary cardiomyopathy. NGS was performed using the Illumina sequencing system.ResultsA total of 180 Chinese pediatric patients (114 males and 66 females) diagnosed with primary cardiomyopathy were enrolled in this study. TAZ mutations were identified in four of the male index patients, including two novel mutations (c.527A > G, p.H176R and c.134_136delinsCC, p.H45PfsX38). All four probands and two additional affected male family members were born at full term with a median birth weight of 2350 g (range, 2000–2850 g). The median age at diagnosis of cardiomyopathy was 3.0 months (range, 1.0–20.0 months). The baseline echocardiography revealed prominent dilation and trabeculations of the left ventricle with impaired systolic function in the six patients, four of which fulfilled the diagnostic criteria of left ventricular noncompaction. Other aspects of their clinical presentations included hypotonia (6/6), growth delay (6/6), neutropenia (3/6) and 3-methylglutaconic aciduria (4/5). Five patients died at a median age of 7.5 months (range, 7.0–12.0 months). The cause of death was heart failure associated with infection in three patients and cardiac arrhythmia in two patients. The remaining one patient survived beyond infancy but had fallen into a persistent vegetative state after suffering from cardiac arrest.ConclusionsThis is the first report of systematic mutation screening of TAZ in a large cohort of pediatric patients with primary cardiomyopathy using the NGS approach. TAZ mutations were found in 4/114 (3.5%) male patients with primary cardiomyopathy. Our findings indicate that the inclusion of TAZ gene testing in cardiomyopathy genetic testing panels may contribute to the early diagnosis of BTHS.

Feasibility and clinical integration of molecular profiling for target identification in pediatric solid tumors.

The role of tumor molecular profiling in directing targeted therapy utilization remains to be defined for pediatric tumors. We aimed to evaluate the feasibility of a sequencing and molecular biology tumor board (MBB) program, and its clinical impact on children with solid tumors. We report on a single-center MBB experience of 60 pediatric patients with a poor prognosis or relapsed/refractory solid tumors screened between October 2014 and November 2015. Tumor molecular profiling was performed with panel-based next-generation sequencing and array comparative genomic hybridization. Mean age was 12 ± 5.7 years (range 0.1-21.5); main tumor types were high-grade gliomas (n = 14), rare sarcomas (n = 9), and neuroblastomas (n = 8). The indication was a poor prognosis tumor at diagnosis for 16 patients and relapsed (n = 26) or refractory disease (n = 18) for the remaining 44 patients. Molecular profiling was feasible in 58 patients. Twenty-three patients (40%) had a potentially actionable finding. Patients with high-grade gliomas had the highest number of targetable alterations (57%). Six of the 23 patients subsequently received a matched targeted therapy for a period ranging from 16 days to 11 months. The main reasons for not receiving targeted therapy were poor general condition (n = 5), pursuit of conventional therapy (n = 6), or lack of pediatric trial (n = 4). Pediatric molecular profiling is feasible, with more than a third of patients being eligible to receive targeted therapy, yet only a small proportion were treated with these therapies. Analysis at diagnosis may be useful for children with very poor prognosis tumsors.

Germline multi-gene hereditary cancer panel testing in an unselected endometrial cancer cohort

AbstractHereditary endometrial carcinoma is associated with germline mutations in Lynch syndrome genes. The role of other cancer predisposition genes is unclear. We aimed to determine the prevalence of cancer predisposition gene mutations in an unselected endometrial carcinoma patient cohort. Mutations in 25 genes were identified using a next-generation sequencing-based panel applied in 381 endometrial carcinoma patients who had undergone tumor testing to screen for Lynch syndrome. Thirty-five patients (9.2%) had a deleterious mutation: 22 (5.8%) in Lynch syndrome genes (three MLH1, five MSH2, two EPCAM-MSH2, six MSH6, and six PMS2) and 13 (3.4%) in 10 non-Lynch syndrome genes (four CHEK2, one each in APC, ATM, BARD1, BRCA1, BRCA2, BRIP1, NBN, PTEN, and RAD51C). Of 21 patients with deleterious mutations in Lynch syndrome genes with tumor testing, 2 (9.5%) had tumor testing results suggestive of sporadic cancer. Of 12 patients with deleterious mutations in MSH6 and PMS2, 10 were diagnosed at age >50 and 8 did not have a family history of Lynch syndrome-associated cancers. Patients with deleterious mutations in non-Lynch syndrome genes were more likely to have serous tumor histology (23.1 vs 6.4%, P=0.02). The three patients with non-Lynch syndrome deleterious mutations and serous histology had mutations in BRCA2, BRIP1, and RAD51C. Current clinical criteria fail to identify a portion of actionable mutations in Lynch syndrome and other hereditary cancer syndromes. Performance characteristics of tumor testing are sufficiently robust to implement universal tumor testing to identify patients with Lynch syndrome. Germline multi-gene panel testing is feasible and informative, leading to the identification of additional actionable mutations.

Panel-Based Population Next-Generation Sequencing for Inherited Retinal Degenerations.

Inherited retinopathies affect approximately two and a half million people globally, yet the majority of affected patients lack clear genetic diagnoses given the diverse range of genes and mutations implicated in these conditions. We present results from a next-generation sequencing study of a large inherited retinal disease patient population, with the goal of providing clear and actionable genetic diagnoses. Targeted sequencing was performed on 539 individuals from 309 inherited retinal disease pedigrees. Causative mutations were identified in the majority (57%, 176/309) of pedigrees. We report the association of many previously unreported variants with retinal disease, as well as new disease phenotypes associated with known genes, including the first association of the SLC24A1 gene with retinitis pigmentosa. Population statistics reporting the genes most commonly implicated in retinal disease in the cohort are presented, as are some diagnostic conundrums that can arise during such studies. Inherited retinal diseases represent an exemplar group of disorders for the application of panel-based next-generation sequencing as an effective tool for detection of causative mutations.

Panel-based NGS Reveals Novel Pathogenic Mutations in Autosomal Recessive Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited progressive retinal dystrophies (RD) characterized by photoreceptor degeneration. RP is highly heterogeneous both clinically and genetically, which complicates the identification of causative genes and mutations. Targeted next-generation sequencing (NGS) has been demonstrated to be an effective strategy for the detection of mutations in RP. In our study, an in-house gene panel comprising 75 known RP genes was used to analyze a cohort of 47 unrelated Spanish families pre-classified as autosomal recessive or isolated RP. Disease-causing mutations were found in 27 out of 47 cases achieving a mutation detection rate of 57.4%. In total, 33 pathogenic mutations were identified, 20 of which were novel mutations (60.6%). Furthermore, not only single nucleotide variations but also copy-number variations, including three large deletions in the USH2A and EYS genes, were identified. Finally seven out of 27 families, displaying mutations in the ABCA4, RP1, RP2 and USH2A genes, could be genetically or clinically reclassified. These results demonstrate the potential of our panel-based NGS strategy in RP diagnosis.

Screening of an Indian cohort with breast and/or ovarian cancer by a next-generation sequencing-based panel to detect a high frequency of mutations.

e12505 Background: Breast and/or ovarian cancer are among the most prevalent forms of hereditary cancers in India causing an increasing societal burden. Early detection and identification of those ...

Lynch syndrome: five unanswered questions.

Lynch syndrome: five unanswered questions.

Next-generation sequencing-based panel testing for myeloid neoplasms.

Our ability to interrogate a broad array of genetic alterations in myeloid neoplasm has increased significantly with the advance in next-generation sequencing (NGS). In addition to morphologic examination, flow cytometry, and cytogenetics, NGS-based testing can add additional information to the diagnostic workup. More than a dozen myeloid-focused NGS-based panels are now available from commercial and academic laboratories. In this review, we examine the content of these panels in the context of our current understanding of driver alterations in myeloid neoplasms. With improved turnaround time, decreasing costs, and an expanding knowledge of the therapeutic and prognostic significance of the detected variants, NGS-based panel testing is likely to play a major role in the management of patients with myeloid neoplasm in the coming decade.

Phenotypic heterogeneity in monogenic diabetes: The clinical and diagnostic utility of a gene panel-based next-generation sequencing approach

Single gene mutations that primarily affect pancreatic β-cell function account for approximately 1–2% of all cases of diabetes. Overlapping clinical features with common forms of diabetes makes diagnosis of monogenic diabetes challenging. A genetic diagnosis often leads to significant alterations in treatment, allows better prediction of disease prognosis and progression, and has implications for family members. Currently, genetic testing for monogenic diabetes relies on selection of appropriate individual genes for analysis based on the availability of often-limited phenotypic information, decreasing the likelihood of making a genetic diagnosis. We thus developed a targeted next-generation sequencing (NGS) assay for the detection of mutations in 36 genes known to cause monogenic forms of diabetes, including transient or permanent neonatal diabetes mellitus (TNDM or PNDM), maturity-onset diabetes of the young (MODY) and rare syndromic forms of diabetes. A total of 95 patient samples were analyzed: 19 with known causal mutations and 76 with a clinically suggestive phenotype but lacking a genetic diagnosis. All previously identified mutations were detected, validating our assay. Pathogenic sequence changes were identified in 19 out of 76 (25%) patients: 7 of 32 (22%) NDM cases, and 12 of 44 (27%) MODY cases. In 2 NDM patients the causal mutation was not expected as consanguinity was not reported and there were no clinical features aside from diabetes. A 3year old patient with NDM diagnosed at 3months of age, who previously tested negative for INS, KCNJ11 and ABCC8 mutations, was found to carry a novel homozygous mutation in EIF2AK3 (associated with Wolcott–Rallison syndrome), a gene not previously suspected because consanguinity, delayed growth, abnormal bone development and hepatic complications had not been reported. Similarly, another infant without a history of consanguinity was found to have a homozygous GCK mutation causing PNDM at birth. This study demonstrates the effectiveness of multi-gene panel analysis in uncovering molecular diagnoses in patients with monogenic forms of diabetes.

Panel-Based Next-Generation Sequencing of Cancer-Relevant Genes As Treatment Decision Support

ABSTRACT Aim: Knowledge about somatic driver mutations in cancer is crucial for decision-making in personalized therapy. Samples with low tumor content cannot be sequenced reliably with traditional methods. We have developed a complete pipeline to sequence more than 550 cancer-relevant genes in parallel using next-generation sequencing of FFPE or frozen tumor tissue. Interpretation of somatic mutations yields a medical report for the oncologist to support treatment decision. Methods: FFPE or frozen tumor sections are macrodissected and DNA is extracted from tumor material and patient's blood. Enrichment for more than 550 cancer-relevant genes is performed using a specially designed custom enrichment kit and the samples are sequenced to a very high depth. Somatic mutations are detected with very high sensitivity by comparison of sequencing data from the tumor and the blood sample. Clinical interpretation of mutations is performed by a team of scientists and medical doctors. A medical report listing actionable mutations, potentially beneficial targeted drugs and contraindications, as well as clinical trials the patient may wish to participate in. Results: Sensitive and specific detection of somatic mutations is achieved with this diagnostic sequencing panel, even in samples with a tumor content as low as 20%. A majority of patient samples contains actionable mutations, allowing personalized treatment. In addition, the panel allows the classification of CUP as metastasis of previous cancer. The analysis is also able to assign the source of metastasis to one of multiple primary tumors, where histology was not informative. In addition, the panel allowed the classification of CUP as metastasis of a previous cancer and assignment of metastases to one of multiple primary tumors, when histology was not informative. Conclusions: Conclusions: Comprehensive molecular profiling using panel-based next-generation sequencing can identify therapeutic targets in tumor patients. This helps to decide on an optimized therapy and to clarify the source of metastases. Early sequencing can also save costs and provide an advantage to the patient by excluding therapies that will likely have no benefit. Disclosure: M. Menzel: Employee of CeGaT; S. Armeanu-Ebinger: Employee of CeGaT; M. Feldhahn: Employee of CeGaT; S. Biskup: Founder and director of CeGaT.

From single-gene to multiplex analysis in lung cancer, challenges and accomplishments: a review of a single institution's experience.

Molecular selection has led to the successful use of novel tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancers (NSCLCs). For instance, mutations in EGFR and translocations and fusions in ALK render tumor cells sensitive to some TKIs, leading to substantial clinical benefits. Molecular testing such as DNA sequencing or fragment analysis following PCR, and evaluation of copy number and gene positioning by FISH, have been developed and used clinically to identify mutations/fusions. Meanwhile, TKIs to target actionable mutations/fusions in several other oncogenes are being evaluated. High-throughput sequencing can provide therapy-predictive information as well as identify novel targetable genomic alterations. In this article, we report our experience enabling single-gene testing, and our evolution to panel-based next-generation sequencing.

Detection of Gene Rearrangements in Targeted Clinical Next-Generation Sequencing

The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers, six KMT2A rearranged leukemias, and 77 ALK/KMT2A rearrangement-negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALK rearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel-based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.

Targeted NGS gene panel identifies mutations in RSPH1 causing primary ciliary dyskinesia and a common mechanism for ciliary central pair agenesis due to radial spoke defects

Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia and sperm dysmotility. PCD is characterized by clinical variability and extensive genetic heterogeneity, associated with different cilia ultrastructural defects and mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for genetic diagnosis which is not yet in routine use. We developed a targeted panel-based NGS pipeline to identify mutations by sequencing of selected candidate genes in 70 genetically undefined PCD patients. This detected loss-of-function RSPH1 mutations in four individuals with isolated central pair (CP) agenesis and normal body laterality, from two unrelated families. Ultrastructural analysis in RSPH1-mutated cilia revealed transposition of peripheral outer microtubules into the ‘empty’ CP space, accompanied by a distinctive intermittent loss of the central pair microtubules. We find that mutations in RSPH1, RSPH4A and RSPH9, which all encode homologs of components of the ‘head’ structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. By high-resolution immunofluorescence we identified a loss of RSPH4A and RSPH9 along with RSPH1 from RSPH1-mutated cilia, suggesting RSPH1 mutations may result in loss of the entire spoke head structure. CP loss is seen in up to 28% of PCD cases, in whom laterality determination specified by CP-less embryonic node cilia remains undisturbed. We propose this defect could arise from instability or agenesis of the ciliary central microtubules due to loss of their normal radial spoke head tethering.

Detection of FLT3 Internal Tandem Duplication in Targeted, Short-Read-Length, Next-Generation Sequencing Data

A recurrent somatic mutation frequently found in cytogenetically normal acute myeloid leukemia (AML) is internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene (FLT3). This mutation is generally detected in the clinical laboratory by PCR and electrophoresis-based product sizing. As the number of clinically relevant somatic mutations in AML increases, it becomes increasingly attractive to incorporate FLT3 ITD testing into multiplex assays for many somatic mutations simultaneously, using next-generation sequencing (NGS). However, the performance of most NGS analysis tools for identifying medium-size insertions such as FLT3 ITD mutations is largely unknown. We used a multigene, targeted NGS assay to obtain deep sequence coverage (>1000-fold) of FLT3 and 26 other genes from 22 FLT3 ITD-positive and 29 ITD-negative specimens to examine the performance of several commonly used NGS analysis tools for identifying FLT3 ITD mutations. ITD mutations were present in hybridization-capture sequencing data, and Pindel was the only tool out of the seven tested that reliably detected these insertions. Pindel had 100% sensitivity (95% CI = 83% to 100%) and 100% specificity (95% CI = 88% to 100%) in our samples; Pindel provided accurate ITD insertion sizes and was able to detect ITD alleles present at estimated frequencies as low as 1%. These data demonstrate that FLT3 ITDs can be reliably detected in panel-based, next-generation sequencing assays.