Targeted Resequencing of Putative Growth-Related Genes Using Whole Exome Sequencing in Patients with Severe Primary IGF-I Deficiency

Background/Aims: To elucidate the genetic causes of severe primary insulin-like growth factor-I deficiency (SPIGFD) by systematic, targeted, next-generation sequencing (NGS)-based resequencing of growth-related genes. Methods: Clinical phenotyping followed by NGS in 17 families including 6 affected sib pairs. Results: We identified disease-causing, heterozygous, de novo variants in HRAS (p.Gly13Cys) and FAM111A (p.Arg569His) in 2 male patients with syndromic SPIGFD. A previously described homozygous GHR nonsense variant was detected in 2 siblings of a consanguineous family (p.Glu198*). Furthermore, we identified an inherited novel variant in the IGF2 gene (p.Arg156Cys) of a maternally imprinted gene in a less severely affected father and his affected daughter. We detected 2 other novel missense variants in SH2B1 and SOCS2, both were inherited from an unaffected parent. Conclusions: Screening of growth-related genes using NGS-based, large-scale, targeted resequencing identified disease-causing variants in HRAS, FAM111A, and GHR. Considering the increased risk of subjects with HRAS mutations for neoplasms, close clinical monitoring and a thorough discussion of the risk/benefit ratio of the treatment with recombinant IGF-I is mandatory. Segregation analysis proved to be critical in the interpretation of potential SPIGFD-associated gene variations.

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Digenic Inheritance of LAMA4 and MYH7 Mutations in Patient with Infantile Dilated Cardiomyopathy

Medicina ◽

10.3390/medicina55010017 ◽

2019 ◽

Vol 55 (1) ◽

pp. 17 ◽

Cited By ~ 1

Author(s):

Atiyeh M Abdallah ◽

S. Justin Carlus ◽

Abdulhadi H Al-Mazroea ◽

Mohammad Alluqmani ◽

Yousef Almohammadi ◽

...

Keyword(s):

Dilated Cardiomyopathy ◽

Systolic Function ◽

Left Ventricular ◽

Dose Effect ◽

Family Management ◽

Ventricular Contractility ◽

Targeted Next Generation Sequencing ◽

Digenic Inheritance ◽

Novel Variant ◽

Next Generation Sequencing Ngs

Background and objectives: Dilated cardiomyopathy (DCM) is a rare cardiac disease characterised by left ventricular enlargement, reduced left ventricular contractility, and impaired systolic function. Childhood DCM is clinically and genetically heterogenous and associated with mutations in over 100 genes. The aim of this study was to identify novel variations associated with infantile DCM. Materials and Methods: Targeted next generation sequencing (NGS) of 181 cardiomyopathy-related genes was performed in three unrelated consanguineous families from Saudi Arabia. Variants were confirmed and their frequency established in 50 known DCM cases and 80 clinically annotated healthy controls. Results: The three index cases presented between 7 and 10 months of age with severe DCM. In Family A, there was digenic inheritance of two heterozygous variants: a novel variant in LAMA4 (c.3925G > A, p.Asp1309Asn) and a known DCM mutation in MYH7 (c.2770G > A; p.Glu924Lys). The LAMA4 p.Asp1309Asn variant was predicted to be likely pathogenic according to international guidelines. The other two families had no identifiable potentially deleterious variants. Conclusions: Inheritance of two genetic variants may have a synergistic or dose effect to cause severe DCM. We report of a novel p.Asp1309Asn variation associated with DCM. Targeted NGS is useful in the molecular diagnosis of DCM and to guide whole-family management and counselling.

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De Novo PTEN Mutation in a Young Boy with Cutaneous Vasculitis

Case Reports in Pediatrics ◽

10.1155/2017/9682803 ◽

2017 ◽

Vol 2017 ◽

pp. 1-4 ◽

Cited By ~ 3

Author(s):

Angela Mauro ◽

Ebun Omoyinmi ◽

Neil James Sebire ◽

Angela Barnicoat ◽

Paul Brogan

Keyword(s):

De Novo ◽

Cowden Syndrome ◽

Cancer Surveillance ◽

Cutaneous Vasculitis ◽

De Novo Mutation ◽

Proteus Syndrome ◽

Targeted Next Generation Sequencing ◽

Tensin Homolog ◽

Increased Risk ◽

Risk Of Malignancy

Phosphatase and tensin homolog (PTEN) is the protein encoded by the PTEN gene (10q23.3). PTEN mutations are related to a variety of rare diseases referred to collectively as PTEN hamartoma tumor syndromes (PHTS), which include Cowden Syndrome, Bannayan-Riley-Ruvalcaba syndrome, Proteus Syndrome, and Proteus-like syndrome. These diseases are associated with an increased risk of malignancy and for this reason an accurate and early diagnosis is essential in order to institute cancer surveillance. PTEN is a regulator of growth and homeostasis in immune system cells, although there are limited data describing immune dysregulation caused by PTEN mutations. We describe a case of PHTS syndrome caused by a de novo mutation in PTEN detected using a targeted next generation sequencing (NGS) gene panel which was instigated for workup of cutaneous vasculitis. We highlight the diagnostic utility of this approach and that mutations in PTEN may be associated with immune-dysregulatory features such as vasculitis in young children.

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Ethical and public policy challenges for pharmacogenomics

Dialogues in Clinical Neuroscience ◽

10.31887/dcns.2014.16.4/egershon ◽

2014 ◽

Vol 16 (4) ◽

pp. 567-574 ◽

Cited By ~ 5

Keyword(s):

Psychiatric Disorders ◽

Large Scale ◽

Ethical Issues ◽

De Novo ◽

Correct Choice ◽

Genomic Research ◽

Drug Metabolizing Enzyme ◽

Increased Risk ◽

Number Variation ◽

Metabolizing Enzyme

It is timely to consider the ethical and social questions raised by progress in pharmacogenomics, based on the current importance of pharmacogenomics for avoidance of predictable side effects of drugs, and for correct choice of medications in certain cancers. It has been proposed that the entire population be genotyped for drug-metabolizing enzyme polymorphisms, as a measure that would prevent many untoward and dangerous drug reactions. Pharmacologic treatment targeting based on genomics of disease can be expected to increase greatly in the coming years. Policy and ethical issues exist on consent for large-scale genomic pharmacogenomic data collection, public vs corporate ownership of genomic research results, testing efficacy and safety of drugs used for rare genomic indications, and accessibility of treatments based on costly research that is applicable to relatively few patients. In major psychiatric disorders and intellectual deficiency, rare and de novo deletion or duplication of chromosomal segments (copy number variation), in the aggregate, are common causes of increased risk. This implies that the policy problems of pharmacogenomics will be particularly important for the psychiatric disorders.

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The Genotype and Phenotype of Proline-Rich Transmembrane Protein 2 Associated Disorders in Chinese Children

Frontiers in Pediatrics ◽

10.3389/fped.2021.676616 ◽

2021 ◽

Vol 9 ◽

Author(s):

Han-yu Luo ◽

Ling-ling Xie ◽

Si-qi Hong ◽

Xiu-juan Li ◽

Mei Li ◽

...

Keyword(s):

De Novo ◽

Transmembrane Protein ◽

Chinese Children ◽

Gene Deletions ◽

Targeted Next Generation Sequencing ◽

Mild Developmental Delay ◽

Good Treatment Response ◽

Infantile Epilepsy ◽

Next Generation Sequencing Ngs

Objectives: To study the genetic and clinical characteristics of Chinese children with pathogenic proline-rich transmembrane protein 2 (PRRT2) gene-associated disorders.Methods: Targeted next generation sequencing (NGS) was used to identify pathogenic PRRT2 variations in Chinese children with epilepsy and/or kinesigenic dyskinesia. Patients with confirmed PRRT2-associated disorders were monitored and their clinical data were analyzed.Results: Forty-four patients with pathogenic PRRT2 variants were recruited. Thirty-five of them (79.5%) had heterozygous mutations, including 30 frameshifts, three missenses, one nonsense, and one splice site variant. The c.649dupC was the most common variant (56.8%). Eight patients (18.2%) showed whole gene deletions, and one patient (2.3%) had 16p11.2 microdeletion. Thirty-four cases (97.1%) were inherited and one case (2.9%) was de novo. Forty patients were diagnosed with benign familial infantile epilepsy (BFIE), two patients had paroxysmal kinesigenic dyskinesia (PKD) and two had infantile convulsions and choreoathetosis (ICCA). Patients with whole gene deletions had a later remission than patients with heterozygous mutations (13.9 vs. 7.1 months, P = 0.001). Forty-two patients were treated with antiseizure medications (ASMs). At last follow-up, 35 patients, including one who did not receive therapy, were asymptomatic, and one patient without ASMs died of status epilepticus at 12 months of age. One patient developed autism, and one patient showed mild developmental delay/intellectual disability.Conclusion: Our data suggested that patients with whole gene deletions could have more severe manifestations in PRRT2-associated disorders. Conventional ASMs, especially Oxcarbazepine, showed a good treatment response.

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Clinico-pathological and genomic features of NRAS- or HRAS-mutated non-small cell lung cancer (NSCLC) identified in large-scale genomic screening project (LC-SCRUM-Asia).

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.9054 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 9054-9054

Author(s):

Yutaro Tamiya ◽

Shingo Matsumoto ◽

Takaya Ikeda ◽

Kiyotaka Yoh ◽

Terufumi Kato ◽

...

Keyword(s):

Lung Cancer ◽

Large Scale ◽

Patient Characteristics ◽

Kras Mutations ◽

Targeted Next Generation Sequencing ◽

Mutation Burden ◽

Genomic Screening ◽

Frequent Variant ◽

Nsclc Patients ◽

Next Generation Sequencing Ngs

9054 Background: RAS ( KRAS, NRAS and HRAS) is a targetable oncogene family in several cancers, including NSCLC, and the clinical development of various RAS-targeted therapies are ongoing. However, the clinical relevance of uncommon RAS mutations, such as NRAS and HRAS mutations, in NSCLC patients (pts) remains unclear. Methods: In a large-scale genomic screening project (LC-SCRUM-Asia), we have prospectively analyzed lung cancer pts for genomic alterations by a targeted next-generation sequencing (NGS) system, Oncomine Comprehensive Assay. We evaluated clinico-pathological and genomic characteristics in NRAS- or HRAS-mutated NSCLC pts comparing with those in KRAS-mutated pts based on the LC-SCRUM-Asia database. Results: Since March 2015 to December 2020, 9131 NSCLC pts were enrolled in the LC-SCRUM-Asia, and 8374 of them (92%) were successfully analyzed by NGS. The RAS mutation frequencies were 1134 KRAS (14%), 50 NRAS (0.6%), and 15 HRAS (0.2%). The most frequent variant of NRAS and HRAS mutations was Q61X (78%) and G13X (80%), respectively, whereas that of KRAS was G12X (84%). Patient characteristics were summarized in Table. Male was significantly frequent in NRAS- than in KRAS-group (p=0.03), and smokers were frequent in all the three groups (overall, 79%). The majority of NRAS (70%) and KRAS mutations (89%) were detected in adenocarcinoma (Ad), whereas 60% of HRAS mutations were in squamous cell carcinoma (Sq). Tumor mutation burden (TMB) was significantly higher in NRAS-mutated tumors than in KRAS-mutated tumors (p=0.03). Concomitant TP53 mutations were significantly frequent in HRAS-mutated pts than in KRAS-mutated pts (53% vs. 30%, p=0.05), and STK11 mutations were also tended to be frequent in HRAS-mutated pts than in KRAS-mutated pts (20 vs. 7%, p=0.10). Therapeutic efficacy of PD-1/PD-L1 inhibitors was not different among the three groups in the current follow-up data, but HRAS-mutated tumors did not respond to PD-1/PD-L1 inhibitors (response rate, 0%; median PFS, 1.6 months). Conclusions: NRAS- or HRAS-mutated NSCLCs were different from KRAS-mutated NSCLCs in clinico-pathological and genomic profiles. In particular, the immunotherapies were not effective for HRAS-mutated NSCLCs.[Table: see text]

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Quick and efficient approach to develop genomic resources in orphan species: application in Lavandula angustifolia

10.1101/381400 ◽

2018 ◽

Author(s):

Berline Fopa Fomeju ◽

Dominique Brunel ◽

Aurélie Bérard ◽

Jean-Baptiste Rivoal ◽

Philippe Gallois ◽

...

Keyword(s):

Large Scale ◽

De Novo ◽

Rapid Development ◽

Genetic Distances ◽

Lavandula Angustifolia ◽

Alternative Medicines ◽

Snp Development ◽

High Level ◽

The Cost ◽

Next Generation Sequencing Ngs

AbstractNext-Generation Sequencing (NGS) technologies, by reducing the cost and increasing the throughput of sequencing, have opened doors of research efforts to generate genomic data to a range of previously poorly studied species. In this study, we proposed a method for the rapid development of a large scale molecular resources for orphan species. We studied as an example Lavandula angustifolia, a perennial sub-shrub plant native from the Mediterranean region and whose essential oil have numerous applications in cosmetics, pharmaceuticals, and alternative medicines.We first built a ‘Maillette’ reference Unigene, compound of coding sequences, thanks to de novo RNA-seq assembly. Then, we reconstructed the complete genes sequences (with exons and introns) using a transcriptome-guided DNA-seq assembly approach in order to maximize the possibilities of finding polymorphism between genetically close individuals. Finally, we used these resources for SNP mining within a collection of 16 lavender clones and tested the SNP within the scope of a phylogeny analysis. We obtained a cleaned reference of 8, 030 functionally annotated ‘genes’ (in silico annotation). We found up to 400K polymorphic sites, depending on the genotype analyzed, and observed a high SNP frequency (mean of 1 SNP per 90 bp) and a high level of heterozygosity (more than 60% of heterozygous SNP per genotype). We found similar genetic distances between pairs of clones, related to the out-crossing nature of the species, the restricted area of cultivation and the clonal propagation of the varieties.The method propose is transferable to other orphan species, requires little bioinformatics resources and can be realized within a year. This is the first reported large-scale SNP development on Lavandula angustifolia. All this data provides a rich pool of molecular resource to explore and exploit biodiversity in breeding programs.

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TnClone: high-throughput clonal analysis using Tn5-mediated library construction and de novo assembly

10.1101/367045 ◽

2018 ◽

Author(s):

Byungjin Hwang ◽

Sunghoon Heo ◽

Namjin Cho ◽

Duhee Bang

Keyword(s):

Sanger Sequencing ◽

Large Scale ◽

De Novo ◽

Low Frequency ◽

Clonal Analysis ◽

Hands On ◽

The Cost ◽

Next Generation Sequencing Ngs ◽

User Friendly ◽

Analysis Platform

ABSTRACTA typical molecular cloning procedure requires Sanger sequencing for validation, which becomes cost-prohibitive and labour-intensive for large-scale clonal analysis of genotype-phenotype studies. Here we present a Tn5-mediated clonal analysis platform TnClone, which uses next-generation sequencing (NGS) to rapidly and cost-effectively analyze a large number of clones. We also developed a user-friendly graphical user interface and have provided general guidelines for conducting validation experiments. Using TnClone, we achieved more than 20-fold cost reduction compared with the cost incurred using conventional Sanger sequencing and detected low-frequency mutant clones (~10%) in mixed samples. We tested our programme and achieved 99.4% sensitivity. Our platform provides rapid turnaround with minimal hands-on time for secondary evaluation as NGS technology continues to evolve.

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Genetic Variants and Tumor Immune Microenvironment: Clues for Targeted Therapies in Inflammatory Breast Cancer (IBC)

International Journal of Molecular Sciences ◽

10.3390/ijms22168924 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8924

Author(s):

Yulan Gong ◽

Rajeswari Nagarathinam ◽

Maria F. Arisi ◽

Lorenzo Gerratana ◽

Jennifer S. Winn ◽

...

Keyword(s):

Breast Cancer ◽

Inflammatory Breast Cancer ◽

Tumor Tissue ◽

Tumor Infiltrating Lymphocytes ◽

Parp Inhibitors ◽

Targeted Next Generation Sequencing ◽

Increased Risk ◽

Brca1 Brca2 ◽

Genetic Profiles ◽

Next Generation Sequencing Ngs

To better understand the etiology of inflammatory breast cancer (IBC) and identify potential therapies, we studied genomic alterations in IBC patients. Targeted, next-generation sequencing (NGS) was performed on cell-free DNA (cfDNA) (n = 33) and paired DNA from tumor tissues (n = 29) from 32 IBC patients. We confirmed complementarity between cfDNA and tumor tissue genetic profiles. We found a high incidence of germline variants in IBC patients that could be associated with an increased risk of developing the disease. Furthermore, 31% of IBC patients showed deficiencies in the homologous recombination repair (HRR) pathway (BRCA1, BRCA2, PALB2, RAD51C, ATM, BARD1) making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. We also characterized the tumor-infiltrating lymphocytes (TILs) in tumor tissue biopsies by studying several markers (CD4, CD8, FoxP3, CD20, PD-1, and PD-L1) through immunohistochemistry (IHC) staining. In 7 of 24 (29%) patients, tumor biopsies were positive for PD-L1 and PD-1 expression on TILs, making them sensitive to PD-1/PD-L1 blocking therapies. Our results provide a rationale for considering PARP inhibitors and PD-1/PDL1 blocking immunotherapy in qualifying IBC patients.

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Target-Templated de novo Design of Macrocyclic D-/L-Peptides: Inhibitors of the PD-1/PD-L1 Interaction

10.26434/chemrxiv.11663337.v3 ◽

2020 ◽

Author(s):

Salvador Guardiola ◽

Monica Varese ◽

Xavier Roig ◽

Jesús Garcia ◽

Ernest Giralt

Keyword(s):

Protein Interactions ◽

Cyclic Peptides ◽

General Framework ◽

Large Scale ◽

De Novo ◽

Inhibitory Effect ◽

Original Text ◽

Protein Protein Interactions ◽

Retraction Notice ◽

Pharmaceutical Properties

NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above. ------------------------------------------------------------------------ Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-i3 and PD-i6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our de novo design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.

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