GENETICS IN ENDOCRINOLOGY: Genetic diagnosis of endocrine diseases by NGS: novel scenarios and unpredictable results and risks

The technological advancements in genetics produced a profound impact on the research and diagnostics of non-communicable diseases. The availability of next-generation sequencing (NGS) allowed the identification of novel candidate genes but also an in-depth modification of the understanding of the architecture of several endocrine diseases. Several different NGS approaches are available allowing the sequencing of several regions of interest or the whole exome or genome (WGS, WES or targeted NGS), with highly variable costs, potentials and limitations that should be clearly known before designing the experiment. Here, we illustrate the NGS scenario, describe the advantages and limitations of the different protocols and review some of the NGS results obtained in different endocrine conditions. We finally give insights on the terminology and requirements for the implementation of NGS in research and diagnostic labs.

Download Full-text

Next Generation Sequencing Identifies Five Novel Mutations in Lebanese Patients with Bardet–Biedl and Usher Syndromes

Genes ◽

10.3390/genes10121047 ◽

2019 ◽

Vol 10 (12) ◽

pp. 1047 ◽

Cited By ~ 1

Author(s):

Lama Jaffal ◽

Wissam H Joumaa ◽

Alexandre Assi ◽

Charles Helou ◽

George Cherfan ◽

...

Keyword(s):

Next Generation Sequencing ◽

Usher Syndrome ◽

Bioinformatic Analysis ◽

Next Generation ◽

Novel Mutations ◽

Pathogenic Variants ◽

Whole Exome ◽

Targeted Ngs ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Aim: To identify disease-causing mutations in four Lebanese families: three families with Bardet–Biedl and one family with Usher syndrome (BBS and USH respectively), using next generation sequencing (NGS). Methods: We applied targeted NGS in two families and whole exome sequencing (WES) in two other families. Pathogenicity of candidate mutations was evaluated according to frequency, conservation, in silico prediction tools, segregation with disease, and compatibility with inheritance pattern. The presence of pathogenic variants was confirmed via Sanger sequencing followed by segregation analysis. Results: Most likely disease-causing mutations were identified in all included patients. In BBS patients, we found (M1): c.2258A > T, p. (Glu753Val) in BBS9, (M2): c.68T > C; p. (Leu23Pro) in ARL6, (M3): c.265_266delTT; p. (Leu89Valfs*11) and (M4): c.880T > G; p. (Tyr294Asp) in BBS12. A previously known variant (M5): c.551A > G; p. (Asp184Ser) was also detected in BBS5. In the USH patient, we found (M6): c.188A > C, p. (Tyr63Ser) in CLRN1. M2, M3, M4, and M6 were novel. All of the candidate mutations were shown to be likely disease-causing through our bioinformatic analysis. They also segregated with the corresponding phenotype in available family members. Conclusion: This study expanded the mutational spectrum and showed the genetic diversity of BBS and USH. It also spotlighted the efficiency of NGS techniques in revealing mutations underlying clinically and genetically heterogeneous disorders.

Download Full-text

New Challenges in Tumor Mutation Heterogeneity in Advanced Ovarian Cancer by a Targeted Next-Generation Sequencing (NGS) Approach

Cells ◽

10.3390/cells8060584 ◽

2019 ◽

Vol 8 (6) ◽

pp. 584 ◽

Cited By ~ 10

Author(s):

Marica Garziera ◽

Rossana Roncato ◽

Marcella Montico ◽

Elena De Mattia ◽

Sara Gagno ◽

...

Keyword(s):

Ovarian Cancer ◽

Next Generation Sequencing ◽

Residual Disease ◽

Advanced Ovarian Cancer ◽

Genetic Profile ◽

Next Generation ◽

Platinum Based Chemotherapy ◽

Targeted Ngs ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Next-generation sequencing (NGS) technology has advanced knowledge of the genomic landscape of ovarian cancer, leading to an innovative molecular classification of the disease. However, patient survival and response to platinum-based treatments are still not predictable based on the tumor genetic profile. This retrospective study characterized the repertoire of somatic mutations in advanced ovarian cancer to identify tumor genetic markers predictive of platinum chemo-resistance and prognosis. Using targeted NGS, 79 primary advanced (III–IV stage, tumor grade G2-3) ovarian cancer tumors, including 64 high-grade serous ovarian cancers (HGSOCs), were screened with a 26 cancer-genes panel. Patients, enrolled between 1995 and 2011, underwent primary debulking surgery (PDS) with optimal residual disease (RD < 1 cm) and platinum-based chemotherapy as first-line treatment. We found a heterogeneous mutational landscape in some uncommon ovarian histotypes and in HGSOC tumor samples with relevance in predicting platinum sensitivity. In particular, we identified a poor prognostic signature in patients with HGSOC harboring concurrent mutations in two driver actionable genes of the panel. The tumor heterogeneity described, sheds light on the translational potential of targeted NGS approach for the identification of subgroups of patients with distinct therapeutic vulnerabilities, that are modulated by the specific mutational profile expressed by the ovarian tumor.

Download Full-text

Pushing the limits of preimplantation genetic diagnosis (PGD): a Karyomapping and Next Generation Sequencing (NGS) stress test

Fertility and Sterility ◽

10.1016/j.fertnstert.2015.07.881 ◽

2015 ◽

Vol 104 (3) ◽

pp. e281

Author(s):

M.R. Lindeman ◽

E. Czuprenski ◽

J. Kitchen ◽

T.K. McWilliams ◽

M. Wyatt ◽

...

Keyword(s):

Next Generation Sequencing ◽

Preimplantation Genetic Diagnosis ◽

Stress Test ◽

Genetic Diagnosis ◽

Next Generation ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Download Full-text

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

Blood ◽

10.1182/blood.v124.21.4210.4210 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4210-4210 ◽

Cited By ~ 1

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.

Download Full-text

Progressive Myoclonus Epilepsies

Neurology Genetics ◽

10.1212/nxg.0000000000000641 ◽

2021 ◽

Vol 7 (6) ◽

pp. e641

Author(s):

Laura Canafoglia ◽

Silvana Franceschetti ◽

Antonio Gambardella ◽

Pasquale Striano ◽

Anna Teresa Giallonardo ◽

...

Keyword(s):

Late Onset ◽

Diagnostic Yield ◽

Homozygosity Mapping ◽

Genetic Origin ◽

Pathogenic Variants ◽

Whole Exome ◽

Progressive Ataxia ◽

Targeted Ngs ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Background and ObjectivesTo assess the current diagnostic yield of genetic testing for the progressive myoclonus epilepsies (PMEs) of an Italian series described in 2014 where Unverricht-Lundborg and Lafora diseases accounted for ∼50% of the cohort.MethodsOf 47/165 unrelated patients with PME of indeterminate genetic origin, 38 underwent new molecular evaluations. Various next-generation sequencing (NGS) techniques were applied including gene panel analysis (n = 7) and/or whole-exome sequencing (WES) (WES singleton n = 29, WES trio n = 7, and WES sibling n = 4). In 1 family, homozygosity mapping was followed by targeted NGS. Clinically, the patients were grouped in 4 phenotypic categories: “Unverricht-Lundborg disease-like PME,” “late-onset PME,” “PME plus developmental delay,” and “PME plus dementia.”ResultsSixteen of 38 (42%) unrelated patients reached a positive diagnosis, increasing the overall proportion of solved families in the total series from 72% to 82%. Likely pathogenic variants were identified in NEU1 (2 families), CERS1 (1 family), and in 13 nonfamilial patients in KCNC1 (3), DHDDS (3), SACS, CACNA2D2, STUB1, AFG3L2, CLN6, NAXE, and CHD2. Across the different phenotypic categories, the diagnostic rate was similar, and the same gene could be found in different phenotypic categories.DiscussionThe application of NGS technology to unsolved patients with PME has revealed a collection of very rare genetic causes. Pathogenic variants were detected in both established PME genes and in genes not previously associated with PME, but with progressive ataxia or with developmental encephalopathies. With a diagnostic yield >80%, PME is one of the best genetically defined epilepsy syndromes.

Download Full-text

Characterizing the pharmacogenome using molecular inversion probes for targeted next-generation sequencing

Pharmacogenomics ◽

10.2217/pgs-2019-0057 ◽

2019 ◽

Vol 20 (14) ◽

pp. 1005-1020 ◽

Cited By ~ 2

Author(s):

Oscar Suzuki ◽

Olivia M Dong ◽

Rachel M Howard ◽

Tim Wiltshire

Keyword(s):

Next Generation Sequencing ◽

Control Cell ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Technical Performance ◽

Targeted Ngs ◽

Molecular Inversion Probes ◽

Next Generation Sequencing Ngs ◽

Molecular Inversion Probe ◽

Generation Sequencing

Aim: This study assesses the technical performance and cost of a targeted next-generation sequencing (NGS) multigene pharmacogenetic (PGx) test. Materials & methods: A genetic test was developed for 21 PGx genes using molecular inversion probes to generate library fragments for NGS. Performance of this test was assessed using 53 unique reference control cell lines from the Genetic Testing Reference Materials Coordination Program (GeT-RM). Results: 93.7% of variants were successfully called and the repeatability rate was 99.9%. Reference calls were available for 78.4% of diplotype calls resulting from PGx testing, and concordance for the test was 85.7%. Cost per sample was $32–$56. Conclusion: A targeted NGS assay using molecular inversion probe technology is able to characterize the pharmacogenome efficiently.

Download Full-text

Current Challenges and Implications of Proteogenomic Approaches in Prostate Cancer

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200722112450 ◽

2020 ◽

Vol 20 (22) ◽

pp. 1968-1980

Author(s):

Nidhi Shukla ◽

Narmadhaa Siva ◽

Babita Malik ◽

Prashanth Suravajhala

Keyword(s):

Prostate Cancer ◽

Next Generation Sequencing ◽

Candidate Genes ◽

Next Generation ◽

Recent Past ◽

Next Generation Sequencing Ngs ◽

Ngs Data ◽

Omic Data ◽

Generation Sequencing

In the recent past, next-generation sequencing (NGS) approaches have heralded the omics era. With NGS data burgeoning, there arose a need to disseminate the omic data better. Proteogenomics has been vividly used for characterising the functions of candidate genes and is applied in ascertaining various diseased phenotypes, including cancers. However, not much is known about the role and application of proteogenomics, especially Prostate Cancer (PCa). In this review, we outline the need for proteogenomic approaches, their applications and their role in PCa.

Download Full-text

Next-Generation Sequencing (NGS) in CMML, MDS and AML Detects Molecular Mutations in Oncogenes and Allows the Identification of Balanced Chromosomal Abnormalities with Extraordinary Sensitivity and Specificity.

Blood ◽

10.1182/blood.v114.22.144.144 ◽

2009 ◽

Vol 114 (22) ◽

pp. 144-144

Author(s):

Vera Grossmann ◽

Alexander Kohlmann ◽

Claudia Haferlach ◽

Hans-Ulrich Klein ◽

Martin Dugas ◽

...

Keyword(s):

Bone Marrow ◽

Next Generation Sequencing ◽

Candidate Genes ◽

Point Mutations ◽

Diagnostic Methods ◽

Next Generation ◽

Coding Regions ◽

Equity Ownership ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Abstract 144 PicoTiterPlate (PTP) pyrosequencing allows the detection of low-abundance oncogene aberrations in complex samples even with low tumor content. Here, we compared deep sequencing data of two Next-Generation Sequencing (NGS) assays to detect molecular mutations using a PCR-based strategy and, in addition, to uncover inversions, translocations, and insertions in a targeted sequence enrichment workflow (454 Life Sciences, Roche Diagnostics Corporation, Branford, CT). First, we studied 95 patients (CMML, n=81; AML, n=6; MDS, n=3; MPS, n=3; ET, n=2) using the amplicon approach and investigated seven candidate genes with relevance in oncogenesis of myeloid malignancies: TET2, RUNX1, JAK2, MPL, KRAS, NRAS, and CBL. 43 primer pairs were designed to cover the complete coding regions of TET2, RUNX1 (beta isoform), and hotspot regions of the latter genes. In total, 4128 individual PCR reactions were performed with DNA isolated from bone marrow mononuclear cells, followed by product purification, fluorometric quantitation, and equimolar pooling of the corresponding 43 amplicon products to generate one single sequence library per patient. For sequencing, a 454 8-lane PTP was used applying standard FLX chemistry and representing one patient per lane. The median number of base pairs sequenced per patient was 9.23 Mb. For each amplicon a median of 840 reads was generated (coverage range: 485–1929 reads). As initial proof-of-concept analysis 27 of the 95 patients with known mutations (n=32) as detected by conventional sequencing or melting curve analyses were investigated (range of cells carrying the respective mutation: 1.1% for JAK2 V617F to 98.14% for TET2 C1464X). In all cases, 454 NGS confirmed results from routine diagnostic methods (GS Amplicon Variant Analyzer software version 2.0.01). We then investigated the remaining 69 CMML patients: In median, 2 variances (range 1–8 variances), i.e. differences in comparison to the reference sequence, per patient were detected. These variances included both point mutations in all candidate genes and large deletions (12-19 bp) in CBL, RUNX1, and TET2. Only 20/81 patients of the CMML-cohort (24.69%) were without any detectable mutation. Secondly, in a cohort of six AML bone marrow specimens a custom NimbleGen array (385K format; Madison, WI) was used to perform a targeted DNA sequence enrichment procedure. In total, capture probes spanning 1.91 Mb were designed to represent all coding regions of 92 target genes (1559 exons) with relevance in hematological malignancies (e.g. KIT, NF1, TP53, BCR, ABL1, NPM1, or FLT3). In addition, the complete genomic regions were targeted for RUNX1, CBFB, and MLL. For sequencing, 454 Titanium chemistry was applied, loading three patients per lane on a 2-lane PTP including three molecular identifiers (MIDs) each. Data analysis was performed using the GS Reference Mapper software version 2.0.01. For the enrichment assay, the median enrichment of the targeted genomic loci was 207-fold, as assessed by ligation-mediated LM-PCR. Overall, 1,098,132 reads were generated in the two lanes, yielding a total sequence length of 386,097,740 bases. In median, 96.52% of the sequenced bases mapped against the human genome, and 66.0% were derived from the customized NimbleGen array capture probes, resulting in a median coverage of 18.7-fold . With this method it was possible to detect and confirm point mutations (KIT, FLT3-TKD, and KRAS) and insertions (FLT3-ITD). Moreover, by capturing chimeric DNA fragments and generating reads mapping to both fusion partners this approach detected balanced aberrations, i.e. inv(16)(p13q22) and the translocations t(8;21)(q22;q22) or t(9;11)(p22;q23). In conclusion, both assays to specifically sequence targeted regions with oncogenic relevance on a NGS platform demonstrated promising results and are feasible. The amplicon approach is more suitable for detection of mutations in a routine setting and is ideally suited for large genes such as TET2, ATM, and NF1, which are labor-intensive to sequence conventionally. The array-based capturing assay is characterized by a complex and time-consuming workflow with low-throughput. However, the ability to detect balanced genomic aberrations which are detectable thus far only by cytogenetics and FISH has the potential to become an important diagnostic assay, especially in tumors in which cytogenetics can not be applied successfully. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Kazak:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Download Full-text

Genetic landscape of pediatric movement disorders and management implications

Neurology Genetics ◽

10.1212/nxg.0000000000000265 ◽

2018 ◽

Vol 4 (5) ◽

pp. e265 ◽

Cited By ~ 8

Author(s):

Dawn Cordeiro ◽

Garrett Bullivant ◽

Komudi Siriwardena ◽

Andrea Evans ◽

Jeff Kobayashi ◽

...

Keyword(s):

Next Generation Sequencing ◽

Movement Disorder ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Movement Disorders ◽

Genetic Diagnosis ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Whole Exome ◽

Generation Sequencing

ObjectiveTo identify underlying genetic causes in patients with pediatric movement disorders by genetic investigations.MethodsAll patients with a movement disorder seen in a single Pediatric Genetic Movement Disorder Clinic were included in this retrospective cohort study. We reviewed electronic patient charts for clinical, neuroimaging, biochemical, and molecular genetic features. DNA samples were used for targeted direct sequencing, targeted next-generation sequencing, or whole exome sequencing.ResultsThere were 51 patients in the Pediatric Genetic Movement Disorder Clinic. Twenty-five patients had dystonia, 27 patients had ataxia, 7 patients had chorea-athetosis, 8 patients had tremor, and 7 patients had hyperkinetic movements. A genetic diagnosis was confirmed in 26 patients, including in 20 patients with ataxia and 6 patients with dystonia. Targeted next-generation sequencing panels confirmed a genetic diagnosis in 9 patients, and whole exome sequencing identified a genetic diagnosis in 14 patients.ConclusionsWe report a genetic diagnosis in 26 (51%) patients with pediatric movement disorders seen in a single Pediatric Genetic Movement Disorder Clinic. A genetic diagnosis provided either disease-specific treatment or effected management in 10 patients with a genetic diagnosis, highlighting the importance of early and specific diagnosis.

Download Full-text