Design and Validate of Next-Generation Sequencing Panel for Inherited Platelet Disorders

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4210-4210 ◽  
Author(s):  
Jose Maria Bastida ◽  
Mónica del Rey ◽  
Rocío Benito ◽  
Isabel Sanchez-Guiu ◽  
Susana Riesco ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Candidate Genes ◽  
Genetic Diagnosis ◽  
Diagnostic Strategy ◽  
Severe Thrombocytopenia ◽  
Sequencing Analysis ◽  
Next Generation ◽  
Validation Process ◽  
Platelet Disorders ◽  
Generation Sequencing

Abstract Introduction The inherited platelet disorders (IPD) are a heterogeneous group of rare diseases including quantitative and/or qualitative platelet defects. Classically, patients with IPD are first functionally tested to know the possible defect before sequencing a single or a few genes. Phenotipyc diagnostic of IPD often requires light transmission aggregometry, quantitative analysis of receptors by flow cytometry and fluorescence and electron microscopy. This diagnostic strategy is complex, poorly standardised and time consuming. In addition, the phenotype can seldom guide the singles candidates genes for conventional Sanger squencing. Therefore, many patients remain without a accurate diagnosis of their IPD. Next generation sequencing (NGS) enables the simultaneous analysis of large groups of candidate genes in IPD and may be useful for rapid genetic diagnosis. The aim of this study was to design and validate a NGS panel for IPD. Patients & Methods We describe a strategy for rapid genetic diagnosis of IPD with Illumina sequencing of 60 candidates genes previously associated with IPD (table1). The baits were designed to tile 400 kb of gDNA sequence corresponding to the exons and splice sites in all known transcripts of the candidate genes identified. The bait library was tested by enriching the candidate IPD genes from 50 ng DNA obtained and sequencing by Nextera Rapid Custom Enrichment system. Results were analysed by Variant Studio system and Sequencing Analysis Viewer. A total of 21 patients were studied. For the validation process, DNA samples of 9 unrelated patients with IPD and their mutation known were used: two patients with Glanzmann Thrombasthenia (ITGA2B, p.Ala989Thr, p.Val982Met and p.Glu538Stop; ITGB3, p.Leu222Pro and p.Tyr216Cys), one Hermansky-Pudlak Sd. (HPS1, p.Glu204 Stop), another with Bernard-Soulier Sd. (GPIX, p.Phe71Stop), a case of Congenital Amegakaryocytic Thrombocytopenia (MPL, p.Arg102Cys), and 2 patients with Chediak Higashi Sd. (LYST, p.Gly3725Arg and p.Cys258Arg). Once validated, the NGS panel was used for genetic diagnostic of 8 patients with suspected IPD. Results Eleven mutations, previously identified in another center by conventional sequencing, were detected by our panel NGS (100% success in the validation process). We then tested this strategy for patients with suspected of IPD without diagnosis: I. a 13 years old girl with agenesis of the corpus callosum, facial dysmorphia, renal agenesis and thrombocytopenia was diagnosed of Thrombocytopenia FLNA-related and Periventricular Nodular Heterotopia (PNHV)[mutation in the FLNA was detected (p.Thr1232Ile)]. II. A two years old patient with severe thrombocytopenia and recurrent infections was diagnosed of Wiskott-Aldrich Sd (WAS, p.Arg268Gly fs Stop40). III. A patient with deafness, macrothrombocytopenia, and Döhle bodies was diagnosed by MYH9 deletion (MYH9; p.Asp1925Thr fs Stop23). IV. Six members of a family (2 of them with symptoms of mucocutaneous bleeding, and macrothrombocytopenia), in which an insertion in NBAL2 (p.Gly1142Arg fs Stop49) gene was found. Therefore, Gray Platelet Sd was diagnosed. Moreover, one patient with “aspirin-like syndrome” showed a P2RY12 mutation (p.Val279Met). Finally, mother and son with mild Hemophilia A (F8; p.Gln2208Arg) were detected. Conclusions This NGS panel enables a rapid genetic diagnostic of IPD. The use of NGS-based strategy is a feasible tool for the diagnosis of IPD that could be added to the screening of these disorders. Five mutations have not previously been described in the literature. Table 1: Sixty candidates' genes previously associated with IPD: Inherited Platelet Disorders Genes = 60 Cytoskeletal Assembly and Structural Proteins GP1BA, GP1BB, GP5, A2M, GP9, VWF, ITGA2, ITGA2B, ITGB3, ABCA1, ANO6,FERMT3, ACTN1, MASTL Disorders of agonist platelet receptors P2RX1, P2RY1, P2RY12, TBXA2R, TBXAS1, ADRA2A, GP6, CD36 o GP4, DTNBP1 Disorders signal transduction GNAI3, GNAQ, GNAS, PLA2G7, PLCB2PTS, GGCX, DPAGT1, DHCR24 Disorders of platelet granules NBEAL2, GFI1B, PLAU, HPS1, HPS3, HPS4, HPS5, HPS6, LYST, MLPH, BLOC1S3, BLOC1S6, AP3B1, VIPAS39, VPS33B, RAB27A, MYO5A, USF1 Thrombocytopenias and syndromes WAS, MYH9, FLNA, FLI1, STIM1, HOXA11, ANKRD26, MPL, RBM8A RUNX1, GATA1 Disclosures No relevant conflicts of interest to declare.

Clinical Next Generation Sequencing to Identify Novel Platelet Disorders

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-38-SCI-38 ◽  
Author(s):  
Kathleen Freson
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Gene List ◽  
Genetic Diagnosis ◽  
Next Generation ◽  
Disease Etiology ◽  
Human Phenotype ◽  
Pathogenic Variants ◽  
Platelet Disorders ◽  
Generation Sequencing

Abstract Inherited platelet disorders (IPDs) comprise a heterogeneous group of disorders with a complex genetic etiology, characterized by impairments in platelet formation, morphology and function. Since the implementation of next generation sequencing (NGS) in 2009, the gene list for diagnosis of IPDs rapidly expanded from 39 to 53 genes. A diagnostic high-throughput targeted NGS platform (referred to as ThromboGenomics; www.thrombogenomics.org.uk) was very recently described as an affordable DNA-based test of 76 genes to diagnose patients 'suspected of having a known inherited platelet, thrombotic or bleeding disorder' (BPD). When the phenotype is strongly indicative of the presence of a particular disease etiology but the variants are unknown, sensitivity remains high (>90% based on 61 samples) while patients included with an uncertain disease such as delta storage pool disease, mostly receive no genetic diagnosis (only 10% a genetic diagnosis was obtained). Such IPDs should be included in gene discovery NGS programs such as the BRIDGE-BPD2 study. For this study, whole genome sequencing results of the DNA samples of nearly 1000 probands with uncharacterized IPDs, analyzed using assigned Human Phenotype Ontology (HPO) terms have helped to identify pathogenic variants in almost 20% of cases. New clustering algorithms to group cases with similar phenotypes have been used to identify two novel IPD genes (DIAPH1 and SRC2) and several putative ones. Still many IPD patients don't receive a genetic diagnosis. A majority of cases either harbor pathogenic variants in unknown genes or in regulatory regions or are the result of a digenic mode of inheritance. NGS combined with data from RNA-seq, ChIP-seq, gene regulatory network analysis, epigenome, proteomics and mouse knock-out studies amongst others will also help explore the non-coding regulatory space and gene-gene interactions. Given the existence of many non-pathogenic variants in any individual's genome, the main challenge faced by researchers when interpreting NGS data of an IPD case is determining which variants are causing the disorder.3Interpreting the functional consequences of novel rare variants is not easy and it is extremely important to apply rigorous standards when assigning pathogenicity. Clinical genomic data are the same as other complex medical data and should be interpreted by a multidisciplinary team comprising typically a statistical geneticist, clinical geneticist, and genetic counselors, who have the skills to interpret these results in the context of the test methodology, the theoretical background of genetics, Bayesian reasoning, and a myriad of other factors. 1. Simeoni I, Stephens JC, Hu F, et al. A comprehensive high-throughput sequencing test for the diagnosis of inherited bleeding, thrombotic and platelet disorders. Blood. 2016; 127: 279. 2. Turro E, Greene D, Wijgaerts A, et al. A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies. Sci Transl Med. 2016;8:328. 3. Lentaigne C, Freson K, Laffan MA, et al. Inherited platelet disorders: towards DNA-based diagnosis. Blood. 2016; 127: 2814. Disclosures No relevant conflicts of interest to declare.

scholarly journals Germline Mutation in KIF1Bβ Gene Associated with Loss of Heterozygosity: Usefulness of Next-Generation Sequencing in the Genetic Screening of Patients with Pheochromocytoma

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Giuseppina De Filpo ◽  
Elisa Contini ◽  
Viola Serio ◽  
Andrea Valeri ◽  
Massimiliano Chetta ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Loss Of Heterozygosity ◽  
Germline Mutation ◽  
In Silico ◽  
Genetic Diagnosis ◽  
Sequencing Analysis ◽  
Next Generation ◽  
Abdominal Lymph Node ◽  
In Silico Studies ◽  
Generation Sequencing

The genetic approach of pheochromocytomas and paragangliomas has changed in the last two decades. Nowadays, we know that more than 40% of patients have a germline mutation in one of the susceptibility genes identified to date. Our aim is to underline how genetic diagnosis by next-generation sequencing (NGS) can improve the management of patients affected by pheochromocytomas and paragangliomas in our routine diagnostic screening. We reported a case presentation and next-generation sequencing analysis supported by in silico studies and evaluation of mitochondrial status in KIF1Bβ tissue. A 46-year-old male affected by a left secreting pheochromocytoma underwent surgery in 2017. After surgery, the normetanephrine levels decreased very slowly and a suspected abdominal lymph node was detected. We found a novel germline KIF1Bβ gene mutation, c.4052C > T, p. Pro1351Leu associated with tumor loss of heterozygosity, and resulted likely-pathogenetic by in silico studies. This mutation was also associated with an increased number of mitochondria through the electron microscopy compared with wild-type tissues as suggestive for mitochondria neoformation compensatory to the mitochondrial autophagic figures observed. Our results underline the usefulness of next-generation sequencing in the presence of multiple tumor predisposition genes and how, at the same time, its use may result challenging for the clinicians. To date, performing the genetic analysis according to the latest Consensus Statement is mandatory in patients affected by PHEO/PGL.

Identification of a novel c.3080delC JAG1 gene mutation associated with Alagille syndrome by whole-exome sequencing (Preprint)

2021 ◽  
Author(s):  
Dr. Deepak Panwar ◽  
Dr. Kumar Gautam Singh ◽  
Ms. Shruti Mathur ◽  
Mr. Bhagwati Prasad ◽  
Ms. Anita Joshi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Heart Defects ◽  
Genetic Diagnosis ◽  
Alagille Syndrome ◽  
Sequencing Analysis ◽  
Next Generation ◽  
Whole Exome ◽  
Generation Sequencing

BACKGROUND Alagille syndrome is an autosomal dominant disorder associated with variable clinical phenotypic features including cholestasis, congenital heart defects, vertebral defects, and dysmorphic facies. OBJECTIVE Whole-exome sequencing (WES) has become technically feasible due to the recent advances in next-generation sequencing technologies, therefore offering new opportunities for mutations/genes identification. METHODS Next-generation sequencing (NGS) - Whole-exome sequencing was used to identify pathogenic variants of the proband. In this paper, we have uncovered a novel JAG1 mutation associated with Alagille syndrome in a 5 years old girl presented with conjugated hyperbilirubinemia and infantile cholestasis. RESULTS The exome sequencing analysis revealed the presence of a novel JAG1 heterozygous c.3080delC variant in exon 25. The detected mutation determines a stop codon (p.P1027RfsTer9) in the gene sequence, encoding a truncated protein. Our exome observations were confirmed through Sanger sequencing as well. CONCLUSIONS Here, we report a case of a patient diagnosed with Alagille syndrome, and our finding emphasis the detection of novel JAG1 mutation associated with Alagille syndrome variants thereby, establishing the genetic diagnosis of the disease. CLINICALTRIAL N/A

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