Custom pyrosequencing assay to detect short BRAF deletions in Langerhans cell histiocytic lesions

2020 ◽  
pp. jclinpath-2020-206974
Author(s):  
Fanélie Jouenne ◽  
Aurélie Sadoux ◽  
Gwenaël Lorillon ◽  
Baptiste Louveau ◽  
Emmanuelle Bugnet ◽  
...  
Keyword(s):  
Kappa Coefficient ◽  
Langerhans Cell ◽  
Neoplastic Disease ◽  
Gene Deletions ◽  
Activating Mutations ◽  
Pyrosequencing Assay ◽  
Whole Exome ◽  
Good Concordance ◽  
Protein Kinase Signalling ◽  
Generation Sequencing

Langerhans cell histiocytosis (LCH) is a rare inflammatory myeloid neoplastic disease driven by activating mutations in the mitogen-activating protein kinase signalling pathway, including the BRAFV600E mutation and BRAF deletions (BRAFdel). Next-generation sequencing and whole exome sequencing (WES) are valuable and powerful approaches for BRAFdel identification, but these techniques are costly and time consuming. Pyrosequencing is an alternative method that has the potential to rapidly and reliably identify gene deletions. We developed a custom pyrosequencing assay to detect the exon-12 BRAFdel in 18 biopsies from adult patients with LCH, which were all genotyped in parallel using Sanger sequencing and WES. A BRAFdel was detected in 7/18 (39%), 6/18 (33%) and 3/18 (17%) LCH lesions using WES, pyrosequencing and Sanger, respectively, with good concordance between the WES and pyrosequencing results (Kappa-coefficient=0.88). Therefore, our pyrosequencing assay is reliable and useful for detecting BRAFdel, particularly in BRAFV600E-negative LCH lesions, for which targeted treatment is indicated.

Kindler's Syndrome with Recurrent Neutropenia: Report of Two Cases from Saudi Arabia

2020 ◽  
Author(s):  
Yousef Binamer ◽  
Muzamil A. Chisti
Keyword(s):  
Autosomal Recessive ◽  
Common Mechanism ◽  
Sequencing Analysis ◽  
Loss Of Function ◽  
Skin Atrophy ◽  
Whole Exome ◽  
Infancy And Childhood ◽  
Genetics And Genomics ◽  
New Feature ◽  
Generation Sequencing

AbstractKindler syndrome (KS) is a rare photosensitivity disorder with autosomal recessive mode of inheritance. It is characterized by acral blistering in infancy and childhood, progressive poikiloderma, skin atrophy, abnormal photosensitivity, and gingival fragility. Besides these major features, many minor presentations have also been reported in the literature. We are reporting two cases with atypical features of the syndrome and a new feature of recurrent neutropenia. Whole exome sequencing analysis was done using next-generation sequencing which detected a homozygous loss-of-function (LOF) variant of FERMT1 in both patients. The variant is classified as a pathogenic variant as per the American College of Medical Genetics and Genomics guidelines. Homozygous LOF variants of FERMT1 are a common mechanism of KS and as such confirm the diagnosis of KS in our patients even though the presentation was atypical.

scholarly journals Frequency detection of BRAF V600E mutation in a cohort of pediatric langerhans cell histiocytosis patients by next-generation sequencing

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Shunqiao Feng ◽  
Lin Han ◽  
Mei Yue ◽  
Dixiao Zhong ◽  
Jing Cao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Langerhans Cell Histiocytosis ◽  
Mutation Screening ◽  
Langerhans Cell ◽  
Braf V600e ◽  
P Value ◽  
Type I ◽  
Next Generation ◽  
Mutation Status ◽  
Generation Sequencing

Abstract Background Langerhans cell histiocytosis (LCH) is a rare neoplastic disease that occurs in both children and adults, and BRAF V600E is detected in up to 64% of the patients. Several studies have discussed the associations between BRAF V600E mutation and clinicopathological manifestations, but no clear conclusions have been drawn regarding the clinical significance of the mutation in pediatric patients. Results We retrieved the clinical information for 148 pediatric LCH patients and investigated the BRAF V600E mutation using next-generation sequencing alone or with droplet digital PCR. The overall positive rate of BRAF V600E was 60/148 (41%). The type of sample (peripheral blood and formalin-fixed paraffin-embedded tissue) used for testing was significantly associated with the BRAF V600E mutation status (p-value = 0.000 and 0.000). The risk of recurrence declined in patients who received targeted therapy (p-value = 0.006; hazard ratio 0.164, 95%CI: 0.046 to 0.583). However, no correlation was found between the BRAF V600E status and gender, age, stage, specific organ affected, TP53 mutation status, masses close to the lesion or recurrence. Conclusions This is the largest pediatric LCH study conducted with a Chinese population to date. BRAF V600E in LCH may occur less in East Asian populations than in other ethnic groups, regardless of age. Biopsy tissue is a more sensitive sample for BRAF mutation screening because not all of circulating DNA is tumoral. Approaches with low limit of detection or high sensitivity are recommended for mutation screening to avoid type I and II errors.

scholarly journals A simple method to estimate the in-house limit of detection for genetic mutations with low allele frequencies in whole-exome sequencing analysis by next-generation sequencing

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Takumi Miura ◽  
Satoshi Yasuda ◽  
Yoji Sato
Keyword(s):  
Next Generation Sequencing ◽  
Allele Frequency ◽  
Somatic Mutations ◽  
Limit Of Detection ◽  
Allele Frequencies ◽  
Genetic Mutations ◽  
Sequencing Data ◽  
Simple Method ◽  
Whole Exome ◽  
Generation Sequencing

Abstract Background Next-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research. Particularly, the clinical utility of NGS in detecting mutations associated with disease risk has contributed to the development of effective therapeutic strategies. Recently, comprehensive analysis of somatic genetic mutations by NGS has also been used as a new approach for controlling the quality of cell substrates for manufacturing biopharmaceuticals. However, the quality evaluation of cell substrates by NGS largely depends on the limit of detection (LOD) for rare somatic mutations. The purpose of this study was to develop a simple method for evaluating the ability of whole-exome sequencing (WES) by NGS to detect mutations with low allele frequency. To estimate the LOD of WES for low-frequency somatic mutations, we repeatedly and independently performed WES of a reference genomic DNA using the same NGS platform and assay design. LOD was defined as the allele frequency with a relative standard deviation (RSD) value of 30% and was estimated by a moving average curve of the relation between RSD and allele frequency. Results Allele frequencies of 20 mutations in the reference material that had been pre-validated by droplet digital PCR (ddPCR) were obtained from 5, 15, 30, or 40 G base pair (Gbp) sequencing data per run. There was a significant association between the allele frequencies measured by WES and those pre-validated by ddPCR, whose p-value decreased as the sequencing data size increased. By this method, the LOD of allele frequency in WES with the sequencing data of 15 Gbp or more was estimated to be between 5 and 10%. Conclusions For properly interpreting the WES data of somatic genetic mutations, it is necessary to have a cutoff threshold of low allele frequencies. The in-house LOD estimated by the simple method shown in this study provides a rationale for setting the cutoff.

Activation of the MAPK pathway mediates resistance to PI3K inhibitors in chronic lymphocytic leukemia (cll)

Blood ◽  
2021 ◽  
Author(s):  
Ishwarya Murali ◽  
Siddha Kasar ◽  
Aishath Naeem ◽  
Svitlana Tyekucheva ◽  
Jasneet Kaur Khalsa ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Mapk Pathway ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Erk Activation ◽  
Activating Mutations ◽  
Erk Phosphorylation ◽  
Whole Exome ◽  
Erk Inhibition

Inhibitors of Bruton's tyrosine kinase (BTKi) and phosphatidylinositol 3-kinase delta (PI3Kδi) that target the B cell receptor (BCR) signaling pathway have revolutionized the treatment of chronic lymphocytic leukemia (CLL). While mutations associated with resistance to BTK inhibitors have been identified, limited data are available on mechanisms of resistance to PI3Kδi. Here we present findings from longitudinal whole-exome sequencing of multiply relapsed CLL patients (Ncases=28) enrolled in PI3Ki trials. The non-responder subgroup was characterized by baseline activating mutations in MAP2K1, BRAF and KRAS in 60% of patients. PI3Kδ inhibition failed to inhibit ERK phosphorylation (pERK) in non-responder CLL cells with and without mutations, while treatment with MEKi rescued ERK inhibition. Overexpression of MAP2K1 mutants in vitro led to increased basal and inducible pERK and resistance to idelalisib. These data demonstrate that MAPK/ERK activation plays a key role in resistance to PI3Kδi in CLL and provide rationale for combination therapy with PI3Kδ and ERK inhibitors.

scholarly journals Whole exome sequencing in 17 consanguineous Iranian pedigrees expands the mutational spectrum of inherited retinal dystrophies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Atta Ur Rehman ◽  
Neda Sepahi ◽  
Nicola Bedoni ◽  
Zeinab Ravesh ◽  
Arash Salmaninejad ◽  
...  
Keyword(s):  
Disease Genes ◽  
Homozygous State ◽  
Traditional Practice ◽  
Genomic Variants ◽  
Mutational Spectrum ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
Whole Exome ◽  
Human Disorders ◽  
Generation Sequencing

AbstractInherited retinal dystrophies (IRDs) constitute one of the most heterogeneous groups of Mendelian human disorders. Using autozygome-guided next-generation sequencing methods in 17 consanguineous pedigrees of Iranian descent with isolated or syndromic IRD, we identified 17 distinct genomic variants in 11 previously-reported disease genes. Consistent with a recessive inheritance pattern, as suggested by pedigrees, variants discovered in our study were exclusively bi-allelic and mostly in a homozygous state (in 15 families out of 17, or 88%). Out of the 17 variants identified, 5 (29%) were never reported before. Interestingly, two mutations (GUCY2D:c.564dup, p.Ala189ArgfsTer130 and TULP1:c.1199G > A, p.Arg400Gln) were also identified in four separate pedigrees (two pedigrees each). In addition to expanding the mutational spectrum of IRDs, our findings confirm that the traditional practice of endogamy in the Iranian population is a prime cause for the appearance of IRDs.

scholarly journals Genome-wide characterization of copy number variations in the host genome in genetic resistance to Marek's disease using next generation sequencing

2020 ◽  
Author(s):  
Hao Bai ◽  
Yanghua He ◽  
Yi Ding ◽  
Huanmin Zhang ◽  
Jilan Chen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Copy Number ◽  
Marek's Disease ◽  
Neoplastic Disease ◽  
Marek’S Disease ◽  
Next Generation ◽  
Qrt Pcr ◽  
Genome Wide ◽  
A Genome ◽  
Generation Sequencing

Abstract Background: Marek’s disease (MD) is a highly neoplastic disease primarily affecting chickens, and remains as a chronic infectious disease that threatens the poultry industry. Copy number variation (CNV) has been examined in many species and is recognized as a major source of genetic variation that directly contributes to phenotypic variation such as resistance to infectious diseases. Two highly inbred chicken lines 63 (MD-resistant) and 72 (MD-susceptible), as well as their F1 generation and six recombinant congenic strains (RCSs) with varied susceptibility to MD, are considered as ideal models to identify the complex mechanisms of genetic and molecular resistance to MD.Results: In the present study, to unravel the potential genetic mechanisms underlying resistance to MD, we performed a genome-wide CNV detection using next generation sequencing on the inbred chicken lines with the assistance of CNVnator. As a result, a total of 1,649 CNV regions (CNVRs) were successfully identified after merging all the nine datasets, of which 90 CNVRs were overlapped across all the chicken lines. Within these shared regions, 1,360 harbored genes were identified. In addition, 55 and 44 CNVRs with 62 and 57 harbored genes were specifically identified in line 63 and 72, respectively. Bioinformatics analysis showed that the nearby genes were significantly enriched in 36 GO terms and 6 KEGG pathways including JAK/STAT signaling pathway. Ten CNVRs (nine deletions and one duplication) involved in 10 disease-related genes were selected for validation by using qRT-PCR, all of which were successfully confirmed. Finally, qRT-PCR was also used to validate two deletion events in line 72 that were definitely normal in line 63. One high-confidence gene, IRF2 was identified as the most promising candidate gene underlying resistance and susceptibility to MD in view of its function and overlaps with data from previous study.Conclusions: Our findings provide valuable insights for understanding the genetic mechanism of resistance to MD and the identified gene and pathway could be considered as the subject of further functional characterization.

Detection of a novel gene mutation, ERBB2 exon 20 insertion, in bronchial adenoma using next-generation sequencing and a review of the literature

2020 ◽  
Author(s):  
Qian Wu ◽  
Lin Li ◽  
Ke Zheng ◽  
Yuan Tang ◽  
Lili Jiang
Keyword(s):  
Computed Tomography ◽  
Next Generation Sequencing ◽  
Biological Behavior ◽  
Molecular Characteristics ◽  
Neoplastic Disease ◽  
Next Generation ◽  
Computed Tomography Images ◽  
Exon 20 ◽  
Generation Sequencing ◽  
Bronchial Adenoma

Abstract Objectives: Ciliated muconodular papillary tumor (CMPT) is a rare peripheral lung tumor and is a subtype of bronchial adenoma (BA). Although recent studies have suggested that BA is a neoplastic disease, the complete histogenesis of BA is not fully understood and molecular data are limited. Methods: We examined the clinicopathological features of four patients with BA and performed immunohistochemical analysis and next-generation sequencing to characterize the molecular features of BA. A review of the previous literature was also undertaken to comprehensively conclude the molecular characteristics of this disease. Results: From previous studies and the present study, 99 BA /CMPT cases have been reported to date, with most of the patients from East Asia (77/99, 77.8%). The median age was 64 years old and the ages ranged from 19 to 84 years. The proportion of males and females was close, being approximately 1:1.3. From the computed tomography images, the BA /CMPT tumor usually presented as a peripheral solid mass, part-solid nodules, or ground-glass opaque with an irregular border and occasional central cavities. ERBB2, EGFR, BRAF, and AKT1 mutations were found on the computed tomography images of the BAs. To the best of our knowledge, this is the first study to report about ERBB2 exon 20 insertion in BA. Conclusion: BA /CMPT is a rare pulmonary disease that mainly affects elderly Asian patients. Many abnormal molecular changes were found, which confirmed the neoplastic nature of BA /CMPT. However, it also added to the debate regarding the biological behavior of BA /CMPT.

Childhood Histiocytoses

2020 ◽  
pp. 162-168
Author(s):  
Maurizio Aricò ◽  
Cor van den Bos ◽  
Sheila Weitzman
Keyword(s):  
Targeted Therapy ◽  
Langerhans Cell Histiocytosis ◽  
Langerhans Cell ◽  
Clinical Spectrum ◽  
Juvenile Xanthogranuloma ◽  
Erk Pathway ◽  
Therapeutic Approaches ◽  
Activating Mutations ◽  
Rosai Dorfman Disease ◽  
Genetic Abnormalities

This chapter summarizes the clinical spectrum of the histiocytic disorders—Langerhans cell histiocytosis (LCH), haemophagocytic lymphohistiocytosis (HLH), and some uncommon histiocytic disorders, including juvenile xanthogranuloma (JXG) and Rosai–Dorfman disease—as well as the current diagnostic and therapeutic approaches in these diseases. Multiple activating mutations in the RAS–RAF–MEK–ERK pathway have recently been described in LCH. Their role in the pathophysiology of the disorder and in targeted therapy is reviewed. This chapter explains the differences between primary and secondary HLH, and reviews the genetic abnormalities playing a role in both forms of HLH.

scholarly journals Disseminated non-Langerhans cell histiocytosis with an IRF2BP2-NTRK1 gene fusion identified by next-generation sequencing

2020 ◽  
Vol 6 (11) ◽  
pp. 1156-1158
Author(s):  
Warren H. Chan ◽  
Aatman Shah ◽  
Gordon Bae ◽  
Caely Hambro ◽  
Beth A. Martin ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Langerhans Cell Histiocytosis ◽  
Gene Fusion ◽  
Langerhans Cell ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals Proposal for Modification of Cahan's Criteria Utilizing Molecular Genetic Analyses for Cases without Baseline Histopathology: A Unique Method Applicable to Primary Radiosurgery

2018 ◽  
Vol 80 (01) ◽  
pp. 010-017
Author(s):  
Aaron Rusheen ◽  
James Smadbeck ◽  
Lisa Schimmenti ◽  
Eric Klee ◽  
Michael Link ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Vestibular Schwannoma ◽  
Radiation Treatment ◽  
Molecular Genetic ◽  
Secondary Malignancy ◽  
Whole Exome ◽  
Mate Pair ◽  
Generation Sequencing

Background Cahan's criteria have been utilized since 1948 to establish causality between prior radiation treatment and the development of secondary malignancy. One major criterion specifies that histological and radiographic evidence collected before and after radiation treatment must confirm separate tumor types; however, pretreatment biopsy is rarely obtained prior to radiosurgery for vestibular schwannoma and many other skull base and cranial lesions. Therefore, in these cases Cahan's criteria cannot be validly applied. Objective This article proposes an update to Cahan's criteria using modern molecular genetic analysis for cases lacking baseline histopathology. Methods Mate-pair sequencing and whole exome sequencing of a cerebellopontine angle undifferentiated high-grade pleomorphic sarcoma (UHGPS) that developed after stereotactic radiosurgery of a presumed benign vestibular schwannoma. Results Mate-pair sequencing and whole exome sequencing of the sarcoma revealed complex chromosomal aberrations. Notably, the tumor contained a deletion in the NF2 gene at 22q12 and an in-frame deletion on exon 5 of the remaining copy of NF2. Biallelic events impacting NF2 are atypical for UHGPS but are characteristic for vestibular schwannoma. These findings help support the conclusion that the UHGPS arose from a benign vestibular schwannoma all along. Conclusions Next-generation sequencing can be successfully applied to a radiation-induced sarcoma when both the original and malignant tumors harbor separate signature genetic markers. As our understanding of the genetic profile of various tumors expand, we believe that next-generation sequencing and other genomic tools will play an increasingly important role in establishing causality between radiation and the development of secondary malignancy.

Sign in / Sign up

Export Citation Format

Share Document