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Next-generation sequencing identifies rare variants associated with Noonan syndrome.

ABSTRACT: Noonan syndrome (NS) is a relatively common genetic disorder, characterized by typical facies, short stature, developmental delay, and cardiac abnormalities. Known causative genes account for 70-80% of clinically diagnosed NS patients, but the genetic basis for the remaining 20-30% of cases is unknown. We performed next-generation sequencing on germ-line DNA from 27 NS patients lacking a mutation in the known NS genes. We identified gain-of-function alleles in Ras-like without CAAX 1 (RIT1) and mitogen-activated protein kinase kinase 1 (MAP2K1) and previously unseen loss-of-function variants in RAS p21 protein activator 2 (RASA2) that are likely to cause NS in these patients. Expression of the mutant RASA2, MAP2K1, or RIT1 alleles in heterologous cells increased RAS-ERK pathway activation, supporting a causative role in NS pathogenesis. Two patients had more than one disease-associated variant. Moreover, the diagnosis of an individual initially thought to have NS was revised to neurofibromatosis type 1 based on an NF1 nonsense mutation detected in this patient. Another patient harbored a missense mutation in NF1 that resulted in decreased protein stability and impaired ability to suppress RAS-ERK activation; however, this patient continues to exhibit a NS-like phenotype. In addition, a nonsense mutation in RPS6KA3 was found in one patient initially diagnosed with NS whose diagnosis was later revised to Coffin-Lowry syndrome. Finally, we identified other potential candidates for new NS genes, as well as potential carrier alleles for unrelated syndromes. Taken together, our data suggest that next-generation sequencing can provide a useful adjunct to RASopathy diagnosis and emphasize that the standard clinical categories for RASopathies might not be adequate to describe all patients.

SUBMITTER: Chen PC

PROVIDER: S-EPMC4128129 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Next-generation sequencing identifies rare variants associated with Noonan syndrome.

Chen Peng-Chieh PC Yin Jiani J Yu Hui-Wen HW Yuan Tao T Fernandez Minerva M Yung Christina K CK Trinh Quang M QM Peltekova Vanya D VD Reid Jeffrey G JG Tworog-Dube Erica E Morgan Margaret B MB Muzny Donna M DM Stein Lincoln L McPherson John D JD Roberts Amy E AE Gibbs Richard A RA Neel Benjamin G BG Kucherlapati Raju R

Proceedings of the National Academy of Sciences of the United States of America 20140721 31

Noonan syndrome (NS) is a relatively common genetic disorder, characterized by typical facies, short stature, developmental delay, and cardiac abnormalities. Known causative genes account for 70-80% of clinically diagnosed NS patients, but the genetic basis for the remaining 20-30% of cases is unknown. We performed next-generation sequencing on germ-line DNA from 27 NS patients lacking a mutation in the known NS genes. We identified gain-of-function alleles in Ras-like without CAAX 1 (RIT1) and ...[more]

PMID: 25049390

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Project description:BackgroundNoonan syndrome is an autosomal dominant developmental disorder with a high phenotypic variability, which shares clinical features with other rare conditions, including LEOPARD syndrome, cardiofaciocutaneous syndrome, Noonan-like syndrome with loose anagen hair, and Costello syndrome. This group of related disorders, so-called RASopathies, is caused by germline mutations in distinct genes encoding for components of the RAS-MAPK signalling pathway. Due to high number of genes associated with these disorders, standard diagnostic testing requires expensive and time consuming approaches using Sanger sequencing. In this study we show how targeted Next Generation Sequencing (NGS) technique can enable accurate, faster and cost-effective diagnosis of RASopathies.MethodsIn this study we used a validation set of 10 patients (6 positive controls previously characterized by Sanger-sequencing and 4 negative controls) to assess the analytical sensitivity and specificity of the targeted NGS. As second step, a training set of 80 enrolled patients with a clinical suspect of RASopathies has been tested. Targeted NGS has been successfully applied over 92% of the regions of interest, including exons for the following genes: PTPN11, SOS1, RAF1, BRAF, HRAS, KRAS, NRAS, SHOC, MAP2K1, MAP2K2, CBL.ResultsAll expected variants in patients belonging to the validation set have been identified by targeted NGS providing a detection rate of 100%. Furthermore, all the newly detected mutations in patients from the training set have been confirmed by Sanger sequencing. Absence of any false negative event has been excluded by testing some of the negative patients, randomly selected, with Sanger sequencing.ConclusionHere we show how molecular testing of RASopathies by targeted NGS could allow an early and accurate diagnosis for all enrolled patients, enabling a prompt diagnosis especially for those patients with mild, non-specific or atypical features, in whom the detection of the causative mutation usually requires prolonged diagnostic timings when using standard routine. This approach strongly improved genetic counselling and clinical management.

Project description:BACKGROUND: Multiple genes have been implicated by association studies in altering inflammatory bowel disease (IBD) predisposition. Paediatric patients often manifest more extensive disease and a particularly severe disease course. It is likely that genetic predisposition plays a more substantial role in this group. OBJECTIVE: To identify the spectrum of rare and novel variation in known IBD susceptibility genes using exome sequencing analysis in eight individual cases of childhood onset severe disease. DESIGN: DNA samples from the eight patients underwent targeted exome capture and sequencing. Data were processed through an analytical pipeline to align sequence reads, conduct quality checks, and identify and annotate variants where patient sequence differed from the reference sequence. For each patient, the entire complement of rare variation within strongly associated candidate genes was catalogued. RESULTS: Across the panel of 169 known IBD susceptibility genes, approximately 300 variants in 104 genes were found. Excluding splicing and HLA-class variants, 58 variants across 39 of these genes were classified as rare, with an alternative allele frequency of <5%, of which 17 were novel. Only two patients with early onset Crohn's disease exhibited rare deleterious variations within NOD2: the previously described R702W variant was the sole NOD2 variant in one patient, while the second patient also carried the L1007 frameshift insertion. Both patients harboured other potentially damaging mutations in the GSDMB, ERAP2 and SEC16A genes. The two patients severely affected with ulcerative colitis exhibited a distinct profile: both carried potentially detrimental variation in the BACH2 and IL10 genes not seen in other patients. CONCLUSION: For each of the eight individuals studied, all non-synonymous, truncating and frameshift mutations across all known IBD genes were identified. A unique profile of rare and potentially damaging variants was evident for each patient with this complex disease.

Project description:An erythrocytosis is present when the red blood cell mass is increased, demonstrated as elevated hemoglobin and hematocrit in the laboratory evaluation. Congenital predispositions for erythrocytosis are rare, with germline variants in several genes involved in oxygen sensing (VHL, EGLN1, and EPAS1), signaling for hematopoietic cell maturation (EPOR and EPO), and oxygen transfer (HBB, HBA1, HBA2, and BPGM) that were already associated with the eight congenital types (ECYT1-8). Screening for variants in known congenital erythrocytosis genes with classical sequencing approach gives a correct diagnosis for only up to one-third of the patients. The genetic background of erythrocytosis is more heterogeneous, and additional genes involved in erythropoiesis and iron metabolism could have a putative effect on the development of erythrocytosis. This study aimed to detect variants in patients with yet unexplained erythrocytosis using the next-generation sequencing (NGS) approach, targeting genes associated with erythrocytosis and increased iron uptake and implementing the diagnostics of congenital erythrocytosis in Slovenia. Selected 25 patients with high hemoglobin, high hematocrit, and no acquired causes were screened for variants in the 39 candidate genes. We identified one pathogenic variant in EPAS1 gene and three novel variants with yet unknown significance in genes EPAS1, JAK2, and SH2B3. Interestingly, a high proportion of patients were heterozygous carriers for two variants in HFE gene, otherwise pathogenic for the condition of iron overload. The association between the HFE variants and the development of erythrocytosis is not clearly understood. With a targeted NGS approach, we determined an actual genetic cause for the erythrocytosis in one patient and contributed to better management of the disease for the patient and his family. The effect of variants of unknown significance on the enhanced production of red blood cells needs to be further explored with functional analysis. This study is of great significance for the improvement of diagnosis of Slovenian patients with unexplained erythrocytosis and future research on the etiology of this rare hematological disorder.

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Next-generation sequencing identifies rare variants associated with Noonan syndrome.

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Next-generation sequencing identifies rare variants associated with Noonan syndrome.

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