High-Throughput Screening for CYP21A1P-TNXA/TNXB Chimeric Genes Responsible for Ehlers-Danlos Syndrome in Patients with Congenital Adrenal Hyperplasia
Many patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency have CAH-X syndrome, a connective tissue dysplasia consistent with hypermobility-type Ehlers-Danlos syndrome due to a contiguous gene deletion involving the adjacent CYP21A2 and TNXB genes. CAH-X syndrome is caused by carrying CYP21A1P-TNXA/TNXB chimeric genes (CAH-X CH-1 and CH-2) on one or more alleles. Genetic analysis is cumbersome due to pseudogene interference. We developed a PCR-based CAH-X high-throughput screening method to assess the copy numbers of TNXB exons 35 and 40; this method is amenable to either real-time quantitativ...
Source: Journal of Molecular Diagnostics - June 20, 2019 Category: Pathology Authors: Qizong Lao, Brittany Brookner, Deborah P. Merke Tags: Regular article Source Type: research

PipeIT: A Singularity Container for Molecular Diagnostic Somatic Variant Calling on the Ion Torrent Next-Generation Sequencing Platform
The accurate identification of somatic mutations has become a pivotal component of tumor profiling and precision medicine. In molecular diagnostics laboratories, somatic mutation analyses on the Ion Torrent sequencing platform are typically performed on the Ion Reporter platform, which requires extensive manual review of the results and lacks optimized analysis workflows for custom targeted sequencing panels. Alternative solutions that involve custom bioinformatics pipelines involve the sequential execution of software tools with numerous parameters, leading to poor reproducibility and portability. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 20, 2019 Category: Pathology Authors: Andrea Garofoli, Viola Paradiso, Hesam Montazeri, Philip M. Jermann, Guglielmo Roma, Luigi Tornillo, Luigi M. Terracciano, Salvatore Piscuoglio, Charlotte K.Y. Ng Tags: Regular article Source Type: research

PipeIT: Singularity Container for Molecular Diagnostic Somatic Variant Calling on Ion Torrent NGS Platform
The accurate identification of somatic mutations has become a pivotal component of tumor profiling and precision medicine. In molecular diagnostics laboratories, somatic mutation analyses on the Ion Torrent sequencing platform are typically performed on the Ion Reporter platform, which requires extensive manual review of the results and lacks optimized analysis workflows for custom targeted sequencing panels. Alternative solutions involving custom bioinformatics pipelines involve the sequential execution of software tools with numerous parameters, leading to poor reproducibility and portability. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 20, 2019 Category: Pathology Authors: Andrea Garofoli, Viola Paradiso, Hesam Montazeri, Philip M. Jermann, Guglielmo Roma, Luigi Tornillo, Luigi M. Terracciano, Salvatore Piscuoglio, Charlotte K.Y. Ng Tags: Regular Article Source Type: research

Validation and Retrospective Clinical Evaluation of a Quantitative 16S rRNA Gene Metagenomic Sequencing Assay for Bacterial Pathogen Detection in Body Fluids
Next-generation sequencing –based 16S rRNA gene metagenomic sequencing (16S MG) technology has tremendous potential for improving diagnosis of bacterial infections given its quantitative capability and culture-independent approach. We validated and utilized a quantitative 16S MG assay to identify and quantify bacterial spec ies in clinical samples from a wide spectrum of infections including meningitis, septic arthritis, brain abscess, intra-abdominal abscess, soft tissue abscess, and pneumonia. Twenty clinical samples were tested and 16S MG identified a total of 34 species, compared to 22 species and three descripti...
Source: Journal of Molecular Diagnostics - June 20, 2019 Category: Pathology Authors: Karissa Culbreath, Suzanne Melanson, James Gale, Justin Baker, Fan Li, Oystein Saebo, Oyvind Kommedal, Deisy Contreras, Omai B. Garner, Shangxin Yang Tags: Regular article Source Type: research

Optimized (pre) Analytic Conditions and Workflow for ddPCR Analysis of cfDNA from Patients Suspected of Lung Carcinoma
For patients suspected of lung carcinoma, the analysis of circulating tumor DNA, obtained by liquid biopsy, has the potential to support cancer diagnosis and guide targeted therapy. To ensure sensitive and reproducible detection of circulating tumor DNA in daily routine practice, a standardized (pre) analytical workflow is required. Plasma was obtained from patients and healthy volunteers. Using the Qiagen Circulating Nucleic Acid kit, six different procedures for the isolation of cell-free DNA (cfDNA) were compared. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 20, 2019 Category: Pathology Authors: Remco de Kock, Birgit Deiman, Raisa Kraaijvanger, Volkher Scharnhorst Tags: Regular Article Source Type: research

High-Throughput Screening for CYP21A1P-TNXA/TNXB Chimeric Genes Responsible for Ehlers Danlos Syndrome in Patients with Congenital Adrenal Hyperplasia
Many patients with congenital adrenal hyperplasia (CAH) due to 21 hydroxylase deficiency have CAH-X syndrome, a connective tissue dysplasia consistent with hypermobility type Ehlers Danlos syndrome due to a contiguous gene deletion involving the adjacent CYP21A2 and TNXB genes. CAH-X syndrome is caused by carrying CYP21A1P-TNXA/TNXB chimeric genes (CAH-X CH-1 and CH-2) on one or more alleles. Genetic analysis is cumbersome due to pseudogene interference. We developed a PCR-based CAH-X high-throughput screening method to assess the copy numbers of TNXB exons 35 and 40 that is amenable to either quantitative PCR or droplet d...
Source: Journal of Molecular Diagnostics - June 20, 2019 Category: Pathology Authors: Qizong Lao, Brittany Brookner, Deborah P. Merke Tags: Regular Article Source Type: research

Detection of Single-Nucleotide Polymorphism Markers of Antimalarial Drug Resistance Directly from Whole Blood
Monitoring of antimalarial resistance is important to prevent its further spread, but the available options for assessing resistance are less than ideal for field settings. Although molecular detection is perhaps the most efficient method, it is also the most complex because it requires DNA extraction and PCR instrumentation. To develop a more deployable approach, we designed new probes, which, when used in combination with an inhibitor-tolerant Taq polymerase, enable single-nucleotide polymorphism genotyping directly from whole blood. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 13, 2019 Category: Pathology Authors: Mindy Leelawong, Nicholas M. Adams, William E. Gabella, David W. Wright, Frederick R. Haselton Tags: Regular article Source Type: research

Multi-Gene Panel Testing of 23,179 Individuals for Hereditary Cancer Risk Identifies Pathogenic Variant Carriers Missed by Current Genetic Testing  Guidelines
Recent advancements in next-generation sequencing have greatly expanded the use of multi-gene panel testing for hereditary cancer risk. Although genetic testing helps guide clinical diagnosis and management, testing recommendations are based on personal and family history of cancer and ethnicity, and many carriers are being missed. Herein, we report the results from 23,179 individuals who were referred for 30-gene next-generation sequencing panel testing for hereditary cancer risk, independent of current testing guidelines —38.7% of individuals would not have met National Comprehensive Cancer Network criteria for gen...
Source: Journal of Molecular Diagnostics - June 11, 2019 Category: Pathology Authors: Cynthia L. Neben, Anjali D. Zimmer, Will Stedden, Jeroen van den Akker, Robert O'Connor, Raymond C. Chan, Elaine Chen, Zheng Tan, Annette Leon, Jack Ji, Scott Topper, Alicia Y. Zhou Tags: Regular articles Source Type: research

Proof of Concept for a Portable Platform for Molecular Diagnosis of Tropical Diseases
Although molecular diagnostics is well established in clinical laboratories, its full potential has not been extended to field settings. Typically, diagnostic real-time quantitative PCR (qPCR) reagents require temperature-controlled transportation and storage. Furthermore, thermocyclers are bulky and fragile, requiring good infrastructure for optimal operation. These major hurdles strongly limit use of molecular-based tests in low-resource scenarios. Herein, Trypanosoma cruzi or Plasmodium spp. DNA were detected with qPCR using commercial equipment (ABI7500 instrument) and a prototype platform comprising a portable device ...
Source: Journal of Molecular Diagnostics - June 4, 2019 Category: Pathology Authors: Rita C.P. Rampazzo, Ana C. Graziani, Keren K. Leite, Jhully A. Surdi, Cheysa A. Biondo, Maykon L.N. Costa, Thiago Jacomasso, Marco Cereda, Marco De Fazio, Marco A. Bianchessi, Otac ílio C. Moreira, Constança Britto, Joana D.N. Costa, Viviane M. Góes, A Tags: Regular article Source Type: research

MammaPrint and BluePrint Molecular Diagnostics using targeted RNA Next-Generation Sequencing technology
Next-generation DNA sequencing (NGS) is rapidly becoming an indispensable tool for genome-directed cancer diagnostics, but next-generation RNA sequencing (RNA-seq) is currently not standardly used in clinical diagnostics for expression assessment. However, multi-gene RNA diagnostic assays are used increasingly in the routine diagnosis of early stage breast cancer. Two of the most widely used tests are currently available only as a central laboratory service, which limits their clinical use. We evaluated the use of RNA-seq as a de-centralized methodology to perform such tests. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 4, 2019 Category: Pathology Authors: Lorenza Mittempergher, Leonie JMJ. Delahaye, Anke Witteveen, Jacob B. Spangler, Fariet Hassenmahomed, Sammy Mee, Soufiane Mahmoudi, Jiang Chen, Simon Bao, Mireille HJ. Snel, Sandra Leidelmeijer, Naomi Besseling, Anne Bergstrom Lucas, Carlos Pab ón-Peña, Tags: Regular Article Source Type: research

Proof of concept for a portable platform for molecular diagnosis of tropical diseases: On-chip ready-to-use qPCR for detection of Trypanosoma cruzi or Plasmodium spp
Although molecular diagnostics is well established in clinical laboratories, its full potential has not been extended to field settings. Typically, diagnostic qPCR reagents require temperature-controlled transportation and storage. Furthermore, thermocyclers are bulky and fragile, requiring good infrastructure for optimal operation. These major hurdles strongly limit the use of molecular-based tests in low-resource scenarios. Here, qPCR was used for detection of Trypanosoma cruzi or Plasmodium spp. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 4, 2019 Category: Pathology Authors: Rita C.P. Rampazzo, Ana C. Graziani, Keren K. Leite, Jhully A. Surdi, Cheysa A. Biondo, Maykon L.N. Costa, Thiago Jacomasso, Marco Cereda, Marco De Fazio, Marco A. Bianchessi, Otac ílio C. Moreira, Constança Britto, Joana D.N. Costa, Viviane M. Góes, A Tags: Regular Article Source Type: research

Resolving MiSeq-generated ambiguities in HLA-DPB1 typing by using the Oxford Nanopore technology
The technical limitations of current next-generation sequencing technologies, combined with an ever-increasing number of human leukocyte antigen (HLA) alleles, forms the basis for the additional ambiguities we encounter in our clinical practice at an increasing rate. HLA-DPB1 characterization, particularly, generates a significant percent of ambiguities (25.5%) posing a challenge for accurate and unambiguous HLA-DPB1 genotyping. Phasing of exonic heterozygous positions between exon 2 and all other downstream exons has been the major cause of ambiguities. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - June 4, 2019 Category: Pathology Authors: Jamie L. Duke, Timothy L. Mosbruger, Deborah Ferriola, Nilesh Chitnis, Taishan Hu, Nikolaos Tairis, David J. Margolis, Dimitri S. Monos Tags: Regular Article Source Type: research

Genome Mining –Based Identification of Identical Multirepeat Sequences in Plasmodium falciparum Genome for Highly Sensitive Real-Time Quantitative PCR Assay and Its Application in Malaria Diagnosis
Malaria continues to impact global health, and developing ultrasensitive methods capable of detecting submicroscopic parasitemia —a challenge that persists in low transmission areas, asymptomatic carriers, and patients showing recrudescence—is vital to achieving malaria eradication. Nucleic acid amplification techniques offer improved analytical sensitivity but are limited by the number of copies of the amplification targ ets. Herein, we perform a novel genome mining approach to identify a pair of identical multirepeat sequences (IMRSs) that constitute 170 and 123 copies in the Plasmodium falciparum genome and ...
Source: Journal of Molecular Diagnostics - May 31, 2019 Category: Pathology Authors: Lolabattu S. Raju, Shwetha Kamath, Manjunatha C. Shetty, Sanghamitra Satpathi, Akshaya K. Mohanty, Susanta K. Ghosh, Nikunja Kolluri, Catherine M. Klapperich, Mario Cabodi, Govindarajan Padmanaban, Viswanathan A. Nagaraj Tags: Regular article Source Type: research

The Optimal Sequencing Depth of Tumor Biopsies for Identifying Clonal Cell Populations
The tumor content of a biopsy and the average depth of coverage are two essential aspects when performing DNA sequencing using next-generation sequencing technologies. The heterogeneous nature of cancer necessitates the identification of distinct clonal cell populations to better understand and treat cancer. Deep sequencing enables researchers to identify these populations, but no consensus on an optimal depth exists for identifying clonal populations. Data from eight deep sequenced oral squamous cell carcinoma biopsies obtained from three stage IV patients, with various degrees of tumor content, were used to randomly down...
Source: Journal of Molecular Diagnostics - May 31, 2019 Category: Pathology Authors: Siavosh Tabatabaeifar, Martin Jakob Larsen, Mads Thomassen, Stine Rosenkilde Larsen, Torben Arvid Kruse, Jens Ahm S ørensen Tags: Regular Article Source Type: research

Genome Mining –Based Identification of Identical Multi-Repeat Sequences (IMRS) in Plasmodium falciparum Genome for Highly Sensitive qPCR Assay and its Application in Malaria Diagnosis
Malaria continues to impact global health, and developing ultra-sensitive methods capable of detecting submicroscopic parasitemia —a challenge that persists in low transmission areas, asymptomatic carriers, and patients showing recrudescence—is vital to achieving malaria eradication. Nucleic acid amplification techniques offer improved analytical sensitivity, but limited by the number of copies of the amplification targets . Here, we perform a novel genome mining approach to identify a pair of identical multi-repeat sequences (IMRS) that constitute 170 and 123 copies in Plasmodium falciparum (Pf) genome, and ex...
Source: Journal of Molecular Diagnostics - May 31, 2019 Category: Pathology Authors: Lolabattu Srinivasa Raju, Shwetha Kamath, Manjunatha Chandana Shetty, Sanghamitra Satpathi, Akshaya Kumar Mohanty, Susanta Kumar Ghosh, Nikunja Kolluri, Catherine M. Klapperich, Mario Cabodi, Govindarajan Padmanaban, Viswanathan Arun Nagaraj Tags: Regular Article Source Type: research

A Rapid Allele-Specific Assay for HLA-A*32:01 to Identify Patients at Risk for Vancomycin-Induced Drug Reaction with Eosinophilia and Systemic Symptoms
Human leukocyte antigen (HLA) alleles have been implicated as risk factors for immune-mediated adverse drug reactions. We recently reported a strong association between HLA-A*32:01 and vancomycin-induced drug reaction with eosinophilia and systemic symptoms (DRESS). Identification of individuals with the risk allele prior to or shortly after the initiation of vancomycin therapy is of great clinical importance to prevent morbidity and mortality, improve drug safety and antibiotic treatment options. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 31, 2019 Category: Pathology Authors: Francois X. Rwandamuriye, Abha Chopra, Katherine C. Konvinse, Linda Choo, Jason A. Trubiano, Christian M. Shaffer, Mark Watson, Simon A. Mallal, Elizabeth J. Phillips Tags: Regular article Source Type: research

Recommendations for Clinical CYP2C9 Genotyping Allele Selection
The goals of the Association for Molecular Pathology Pharmacogenomics (PGx) Working Group of the Association for Molecular Pathology Clinical Practice Committee are to define the key attributes of PGx alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document provides recommendations for a minimum panel of variant alleles (Tier 1) and an extended panel of variant alleles (Tier 2) that will aid clinical laboratories when designing assays for CYP2C9 testing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 7, 2019 Category: Pathology Authors: Victoria M. Pratt, Larisa H. Cavallari, Andria L. Del Tredici, Houda Hachad, Yuan Ji, Ann M. Moyer, Stuart A. Scott, Michelle Whirl-Carrillo, Karen E. Weck Tags: Special article Source Type: research

Detection of Tumor NTRK Gene Fusions to Identify Patients Who May Benefit from Tyrosine Kinase (TRK) Inhibitor Therapy
Chromosomal rearrangements involving the NTRK1, NTRK2, and NTRK3 genes (NTRK genes), which encode the high-affinity nerve growth factor receptor (TRKA), brain-derived neurotrophic factor/neurotrophin-3 (BDNF/NT-3) growth factor receptor (TRKB), and neurotrophin-3 (NT-3) growth factor receptor (TRKC) tyrosine kinases (TRK proteins), act as oncogenic drivers in a broad range of pediatric and adult tumor types. NTRK gene fusions have been shown to be actionable genomic events that are predictive of response to TRK kinase inhibitors, making their routine detection an evolving clinical priority. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 7, 2019 Category: Pathology Authors: Susan J. Hsiao, Ahmet Zehir, Anthony N. Sireci, Dara L. Aisner Tags: Review Source Type: research

Diagnostics Reform and Harmonization of Clinical Laboratory Testing
Developments in diagnostics reform legislation in the United States are occurring at a rapid pace. The framework for future regulatory oversight of clinical laboratory testing is currently under intensive debate among stakeholders that represent patients, providers, laboratories, diagnostic manufacturers, and regulators. The importance of clinical laboratory test standardization is a key component of any plan for regulatory reform. A laboratory-developed test is performed in a specific laboratory setting, often led by clinical laboratory professionals who possess specific expertise for developing and running the test to fi...
Source: Journal of Molecular Diagnostics - May 7, 2019 Category: Pathology Authors: Jeff Schreier, Robert Feeney, Peter Keeling Tags: Perspectives Source Type: research

Recommendations for Clinical CYP2C9 Genotyping Allele Selection: A Joint Recommendation of the Association for Molecular Pathology and College of American Pathologists
The goals of the Association for Molecular Pathology Pharmacogenomics (PGx) Working Group of the Association for Molecular Pathology Clinical Practice Committee are to define the key attributes of PGx alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document provides recommendations for a minimum panel of variant alleles (Tier 1) and an extended panel of variant alleles (Tier 2) that will aid clinical laboratories when designing assays for CYP2C9 testing. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 7, 2019 Category: Pathology Authors: Victoria M. Pratt, Larisa H. Cavallari, Andria L. Del Tredici, Houda Hachad, Yuan Ji, Ann M. Moyer, Stuart A. Scott, Michelle Whirl-Carrillo, Karen E. Weck Tags: Special Article Source Type: research

Detection of tumor NTRK gene fusions to identify patients who may benefit from TRK inhibitor therapy
Chromosomal rearrangements involving the NTRK1, NTRK2, and NTRK3 genes (NTRK gene fusions), which encode the TRKA, TRKB, and TRKC receptor tyrosine kinases, act as oncogenic drivers in a broad range of pediatric and adult tumor types. NTRK gene fusions have been shown to be actionable genomic events that are predictive of response to TRK kinase inhibitors, making their routine detection an evolving clinical priority. In certain exceedingly rare tumor types, NTRK gene fusions may be seen in the overwhelming majority of cases, whereas in a range of common cancers, reported incidences are in the range of 0.1% to 2%. (Source: ...
Source: Journal of Molecular Diagnostics - May 7, 2019 Category: Pathology Authors: Susan J. Hsiao, Ahmet Zehir, Anthony N. Sireci, Dara L. Aisner Tags: Review Source Type: research

Fixation Effects on Variant Calling in a Clinical Resequencing Panel
Formalin fixation is the standard method for the preservation of tissue for diagnostic purposes, including pathologic review and molecular assays. However, this method is known to cause artifacts that can affect the accuracy of molecular genetic test results. We assessed the applicability of alternative fixatives to determine whether these perform significantly better on next-generation sequencing assays, and whether adequate morphology is retained for primary diagnosis, in a prospective study using a clinical-grade, laboratory-developed targeted resequencing assay. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 2, 2019 Category: Pathology Authors: Jeremy D.K. Parker, Shyong Q. Yap, Elizabeth Starks, Jillian Slind, Lucas Swanson, T. Roderick Docking, Megan Fuller, Chen Zhou, Blair Walker, Douglas Filipenko, Wei Xiong, Ahmer A. Karimuddin, P. Terry Phang, Manoj Raval, Carl J. Brown, Aly Karsan Tags: Regular article Source Type: research

Multilaboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements
We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele fractions (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 2, 2019 Category: Pathology Authors: Hua-Jun He, Erica V. Stein, Yves Konigshofer, Thomas Forbes, Farol L. Tomson, Russell Garlick, Emiko Yamada, Tony Godfrey, Toshiya Abe, Koji Tamura, Michael Borges, Michael Goggins, Sandra Elmore, Margaret L. Gulley, Jessica L. Larson, Lando Ringel, Brian Tags: Regular article Source Type: research

Fixation Effects on Variant-Calling in a Clinical Resequencing Panel
Formalin fixation is the standard method for preservation of tissue for diagnostic purposes, including pathological review and molecular assays. However, this method is known to cause artifacts that can affect the accuracy of molecular genetic tests. We assessed the applicability of alternative fixatives to determine whether these perform significantly better on next-generation sequencing assays, and whether adequate morphology is retained for primary diagnosis, in a prospective study using a clinical-grade laboratory-developed targeted resequencing assay. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 2, 2019 Category: Pathology Authors: Jeremy D.K. Parker, Shyong Quin Yap, Elizabeth Starks, Jillian Slind, Lucas Swanson, T. Roderick Docking, Megan Fuller, Chen Zhou, Blair Walker, Douglas Filipenko, Wei Xiong, Ahmer A. Karimuddin, P. Terry Phang, Manoj Raval, Carl J. Brown, Aly Karsan Tags: Regular Article Source Type: research

Multi-Laboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements
We conducted a multi-laboratory assessment to determine the suitability of a new commercially-available reference material with 40 cancer variants in a background of wild type DNA at four different variant allele fractions (VAF): 2%, 0.5%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected both for their clinical importance, and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 2, 2019 Category: Pathology Authors: Hua-Jun He, Erica V. Stein, Yves Konigshofer, Thomas Forbes, Farol L. Tomson, Russell Garlick, Emiko Yamada, Tony Godfrey, Toshiya Abe, Koji Tamura, Michael Borges, Michael Goggins, Sandra Elmore, Margaret L. Gulley, Jessica L. Larson, Lando Ringel, Brian Tags: Regular Article Source Type: research

Promoter hypermethylation of genes encoding for RASSF/Hippo pathway members reveals specific alteration pattern in diffuse gliomas
RASSF/Hippo pathway alterations are poorly characterized in diffuse gliomas. We assayed promoter methylation of LATS1/2, MST1(STK4)/MST2(STK3), RASSF1, RASSF2, Nore1A/RASSF5, RASSF6, and RASSF10 genes in 133 diffuse Grade II-III-IV gliomas, using methylation-specific PCR or PCR coupled to Cobra. RASSF/Hippo pathway was highly silenced in gliomas, particularly RASSF1A (79.4%) and LATS2 (35.9%). Most gliomas (75.2%) exhibited at least hypermethylation for two promoters of the RASSF/Hippo member ’s genes. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 2, 2019 Category: Pathology Authors: Gu énaëlle Levallet, Christian Creveuil, Lien Bekaert, Elodie Péres, Gaëtane Planchard, Sylvie Lecot-Cotigny, Jean-Sébastien Guillamo, Evelyne Emery, Gérard Zalcman, Emmanuèle Lechapt-Zalcman Tags: Regular article Source Type: research

Abstracts of the 2nd Global Congress on Molecular Pathology
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 25, 2019 Category: Pathology Source Type: research

Author Index
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 25, 2019 Category: Pathology Source Type: research

Editorial Board
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 24, 2019 Category: Pathology Source Type: research

Table of Contents
(Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 24, 2019 Category: Pathology Source Type: research

Validation of Extensive Next-Generation Sequencing Method for Monogenic Disorder Analysis on Cell-Free Fetal DNA
During pregnancy, a percentage of the cell-free DNA circulating in the maternal blood is represented by the cell-free fetal DNA (cffDNA), constituting an accessible source for noninvasive prenatal genetic screening. The coexistence of the maternal DNA, the dominant fraction of cell-free DNA, together with the cffDNA component and the scarcity of the cffDNA itself make applying traditional methods of genetics and molecular biology impossible. Next-generation sequencing methods are widely used to study fetal aneuploidies. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 24, 2019 Category: Pathology Authors: Claudio Dello Russo, Anthony Cesta, Salvatore Longo, Maria A. Barone, Antonella Cima, Alvaro Mesoraca, Davide Sparacino, Antonella Viola, Claudio Giorlandino Tags: Technical advance Source Type: research

Adopting High-Resolution Allele Frequencies Substantially Expedites Variant Interpretation in Genetic Diagnostic Laboratories
A cohort of 1,242 individuals tested in a clinical diagnostic laboratory was used to test whether the “Filtering Allele Frequencies” (FAFs)-based framework, recently recommended for MHY7-associated cardiomyopathy, is extendable to 45 cardiomyopathy genes. Statistical analysis revealed a threshold of 0.00164% for the extreme outlier allele frequencies (AFs), based on the gnomAD (exome fraction) t otal AFs of 138 unique pathogenic and likely pathogenic variants; 135 of which (97.8%) had AFs (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 24, 2019 Category: Pathology Authors: Mahdi Ghani, Landry Nfonsam, Erinija Pranckeviciene, Hussein Daoud, Ryan Potter, Caitlin Chisholm, Patricia E. Harper, Audrey Schaffer, Leichelle Little, Elizabeth Sinclair-Bourque, Jean McGowan-Jordan, Amanda Smith, Lucas Bronicki, Olga Jarinova Tags: Regular article Source Type: research

Validation of Extensive Next-Generation Sequencing Method for Monogenic Disorder Analysis on Cell Free Fetal DNA
During pregnancy, a percentage of the cell-free DNA circulating in the maternal blood is represented by the cell-free foetal DNA (cffDNA), constituting an accessible source for non-invasive prenatal genetic screening. The coexistence of the maternal DNA, the dominant fraction of cell-free DNA, together with the cffDNA component, and the scarcity of the cffDNA itself, make applying traditional methods of genetics and molecular biology impossible. Next-generation sequencing methods are widely used to study foetal aneuploidies. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 24, 2019 Category: Pathology Authors: Claudio Dello Russo, Anthony Cesta, Salvatore Longo, Maria Antonietta Barone, Antonella Cima, Alvaro Mesoraca, Davide Sparacino, Antonella Viola, Claudio Giorlandino Tags: Technical Advance Source Type: research

Development of a Novel Next-Generation Sequencing Assay for Carrier Screening in Old Order Amish and Mennonite Populations of Pennsylvania
Genetically isolated populations such as the Old Order Amish and Old Order Mennonite communities have an increased incidence of specific autosomal recessive disorders due to the founder effect. In these populations, robust expanded carrier screening and diagnostic testing have the potential to reduce overall medical costs and improve patient outcomes. A novel next-generation sequencing assay was developed using anchored multiplex PCR technology (ArcherDX) for 162 different genetic syndromes caused by 202 pathogenic variants consisting of 150 single nucleotide changes, 43 small insertion/deletions, and nine large deletions ...
Source: Journal of Molecular Diagnostics - April 24, 2019 Category: Pathology Authors: Erin L. Crowgey, Michael C. Washburn, E. Anders Kolb, Erik G. Puffenberger Tags: Regular article Source Type: research

Multiplex Solid-Phase Melt Curve Analysis for the Point-of-Care Detection of HIV-1 Drug Resistance
A point-of-care HIV-1 genotypic resistance assay that could be performed during a clinic visit would enable care providers to make informed treatment decisions for patients starting therapy or experiencing virological failure on therapy. The main challenge for such an assay is the genetic variability at and surrounding each drug-resistance mutation (DRM). We analyzed a database of diverse global HIV sequences and used thermodynamic simulations to design an array of surface-bound oligonucleotide probe sets with each set sharing distinct 5 ’ and 3’ flanking sequences but having different centrally located nucleot...
Source: Journal of Molecular Diagnostics - April 23, 2019 Category: Pathology Authors: Dana S. Clutter, Gelareh Mazarei, Ruma Sinha, Justen Manasa, Janin Nouhin, Ellen LaPrade, Sara Bolouki, Philip L. Tzou, Jessica Hannita-Hui, Malaya K. Sahoo, Peter Kuimelis, Robert G. Kuimelis, Benjamin A. Pinsky, Gary K. Schoolnik, Arjang Hassibi, Robert Tags: Regular Article Source Type: research

An effective strategy to eliminate inherent cross-contamination in mtDNA next-generation sequencing of multiple samples
In this study, a novel sequencing strategy based on a unique double-barcode design was established. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 23, 2019 Category: Pathology Authors: Chun Yin, Yang Liu, Xu Guo, Deyang Li, Wan Fang, Jin Yang, Feng Zhou, Wancheng Niu, Yongfeng Jia, Hushan Yang, Jinliang Xing Tags: Regular Article Source Type: research

A reference system for BRCA mutation detection based on next-generation sequencing in the Chinese population
This study was performed to establish a reference system for performance evaluation of BRCA genetic testing and variant interpretation, which includes interpretation rules, reference materials (RMs), and a reference database (RD). BRCA1/2 mutations identified in cell lines and clinical cases were selected to establish RMs. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 23, 2019 Category: Pathology Authors: Shoufang Qu, Qiong Chen, Yuting Yi, Kang Shao, Wenxin Zhang, Yin Wang, Jian Bai, Xuchao Li, Zhiyuan Liu, Xiaowen Wang, Ruilin Jing, Yanfang Guan, Xin Yi, Miaoli Yan, Boyang Cao, Feng Chen, Shida Zhu, Xuexi Yang, Yingsong Wu, Jie Huang Tags: Regular article Source Type: research

Analytical validation of a highly sensitive, multiplexed chronic myeloid leukemia monitoring system targeting BCR-ABL1 RNA
This study describes the analytical performance of the QuantideX qPCR BCR-ABL IS Kit, the first Federal Drug Administration –cleared assay designed to monitor BCR-ABL1 fusion transcripts isolated from peripheral blood specimens from patients with chronic myeloid leukemia. This multiplex RT-qPCR assay amplifies both e13a2 and e14a2 major BCR-ABL1 transcripts and the reference target ABL1. The test results are provided i n international scale (IS) values by incorporating armored RNA-based calibrators that have defined IS values tied directly to the WHO BCR-ABL1 Primary Reference Materials, without the necessity of dete...
Source: Journal of Molecular Diagnostics - April 23, 2019 Category: Pathology Authors: Justin T. Brown, Ion J. Beldorth, Walairat Laosinchai-Wolf, Marie E. Fahey, Keri L. Jefferson, Adam K. Ruskin, Jacquelyn J. Roth, Li Cai, Christopher D. Watt, Richard D. Press, Fei Yang, John B. Hedges, Bernard F. Andruss Tags: Regular Article Source Type: research

Clinical validation of a cell-free DNA gene panel
We describe herein the development and clinical validation of a 28 gene cell-free DNA panel that targets the most common genetic alterations in solid tumors. Bioinformatic and variant filtering solutions were developed to improve test sensitivity and specificity. The panel and these tools were used to analyze commercially available controls, allowing establishment of a limit of detection allele frequency cutoff of 0.25%, with 100% (95% CI: 81.5% to 100%) specificity and 89.8% (95% CI: 81.0% to 94.9%) sensitivity. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 23, 2019 Category: Pathology Authors: Ju Cheng, Yi Cao, Allison MacLeay, Jochen K. Lennerz, Aymen Baig, Ryan P. Frazier, Jesse Lee, Krista Hu, Maciej Pacula, Enrique Meneses, Hayley Robinson, Julie M. Batten, Priscilla K. Brastianos, Rebecca S. Heist, Aditya Bardia, Long P. Le, A. John Iafrat Tags: Regular Article Source Type: research

Tumor Heterogeneity Index to Detect Human Epidermal Growth Factor Receptor 2 Amplification by Next-Generation Sequencing
Intratumoral heterogeneity of human epidermal growth factor receptor 2 (HER2) is common in gastric cancer (GC). However, a direct comparison between the results of HER2 immunohistochemistry (IHC) and next-generation sequencing (NGS) –based cancer panel tests has not been explored in GC. We aimed to determine optimal thresholds of HER2 overexpression to be detected by NGS with the data of 168 metastatic GC cases with known expression levels of HER2 by IHC and the copy number alteration of ERBB2 obtained by NGS test by applying tumor heterogeneity index (THI) and receiver operating characteristic curve. (Source: Journa...
Source: Journal of Molecular Diagnostics - April 22, 2019 Category: Pathology Authors: Sangjoon Choi, Jinah Chu, Binnari Kim, Sang Yun Ha, Seung Tae Kim, Jeeyun Lee, Won Ki Kang, Heewon Han, Insuk Sohn, Kyoung-Mee Kim Tags: Regular article Source Type: research

Tumor Heterogeneity Index to Detect HER2 Amplification by Next-Generation Sequencing
Intratumoral heterogeneity of HER2 is common in gastric cancer (GC). However, a direct comparison between the results of HER2 immunohistochemistry (IHC) and next-generation sequencing (NGS) –based cancer panel tests has not been explored in GC. We aimed to determine optimal thresholds of HER2 overexpression to be detected by NGS with the data of 168 metastatic GC cases with known expression levels of HER2 by IHC and the copy number alteration of ERBB2 obtained by NGS test by applying tumor heterogeneity index (THI) and receiver operating characteristic curve. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 22, 2019 Category: Pathology Authors: Sangjoon Choi, Jinah Chu, Binnari Kim, Sang Yun Ha, Seung Tae Kim, Jeeyun Lee, Won Ki Kang, Heewon Han, Insuk Sohn, Kyoung-Mee Kim Tags: Regular article Source Type: research

Tumor Heterogeneity Index to detect HER2 amplification by next-generation sequencing: A direct comparison study with immunohistochemistry
Intratumoral heterogeneity of HER2 is common in gastric cancer (GC). However, a direct comparison between the results of HER2 immunohistochemistry (IHC) and next-generation sequencing (NGS)-based cancer panel tests has not been explored in GC. We aimed to determine optimal thresholds of HER2 overexpression to be detected by NGS with the data of 168 metastatic GC cases with known expression levels of HER2 by IHC and the copy number alteration of ERBB2 obtained by NGS test by applying tumor heterogeneity index (THI) and receiver operating characteristic curve. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - April 22, 2019 Category: Pathology Authors: Sangjoon Choi, Jinah Chu, Binnari Kim, Sang Yun Ha, Seung Tae Kim, Jeeyun Lee, Won Ki Kang, Heewon Han, Insuk Sohn, Kyoung-Mee Kim Tags: Regular Article Source Type: research

Leveraging Human Microbiome Features to Diagnose and Stratify Children with Irritable Bowel Syndrome
Accurate diagnosis and stratification of children with irritable bowel syndrome (IBS) remain challenging. Given the central role of recurrent abdominal pain in IBS, we evaluated the relationships of pediatric IBS and abdominal pain with intestinal microbes and fecal metabolites using a comprehensive clinical characterization and multiomics strategy. Using rigorous clinical phenotyping, we identified preadolescent children (aged 7 to 12 years) with Rome III IBS (n = 23) and healthy controls (n = 22) and characterized their fecal microbial communities using whole-genome shotgun metagenomics and global unbiased fecal metabolo...
Source: Journal of Molecular Diagnostics - April 17, 2019 Category: Pathology Authors: Emily B. Hollister, Numan Oezguen, Bruno P. Chumpitazi, Ruth Ann Luna, Erica M. Weidler, Michelle Rubio-Gonzales, Mahmoud Dahdouli, Julia L. Cope, Toni-Ann Mistretta, Sabeen Raza, Ginger A. Metcalf, Donna M. Muzny, Richard A. Gibbs, Joseph F. Petrosino, M Tags: Regular articles Source Type: research

Practical Bioinformatic DNA-Sequencing Pipeline for Detecting Oncogene Amplification and EGFRvIII Mutational Status in Clinical Glioblastoma Samples
Glioblastoma is a malignant brain tumor with dismal prognosis. Oncogenic mutations in glioblastoma frequently affect receptor tyrosine kinase pathway components that are challenging to quantify because of heterogeneous expression. EGFRvIII, a common oncogenic receptor tyrosine kinase mutant protein in glioblastoma, potentiates tumor malignancy and is an emerging tumor-specific immunotarget, underlining the need for its more accessible and quantitative detection. We used normalized next-generation sequencing data from 117 brain and 371 reference clinical tumor samples to detect focal gene amplifications across the commercia...
Source: Journal of Molecular Diagnostics - April 15, 2019 Category: Pathology Authors: Michael L. Miller, Jessica Tome-Garcia, Aneta Waluszko, Tatyana Sidorenko, Chitra Kumar, Fei Ye, Nadejda M. Tsankova Tags: Regular articles Source Type: research

Panel-Based Nuclear and Mitochondrial Next-Generation Sequencing Outcomes of an Ethnically Diverse Pediatric Patient Cohort with Mitochondrial Disease
We report on a molecular diagnostic study using mitochondrial DNA (mtDNA) and targeted nuclear DNA (nDNA) NGS of an extensive cohort of predominantly sub-Saharan African paediatric patients with clinical and biochemically defined MD. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 11, 2019 Category: Pathology Authors: Maryke Schoonen, Izelle Smuts, Roan Louw, Joanna L. Elson, Etresia van Dyk, Lindi-Maryn Jonck, Richard J.T. Rodenburg, Francois H. van der Westhuizen Tags: Regular article Source Type: research

A new fast phasing method based on haplotype subtraction
We developed a novel phasing approach, based solely on molecules and genotype frequency, and that does not rely on inference of new alleles. We initiated the project because of errors that were detected in the phased 1000 genomes data. The algorithm first combined identical genotypes into clusters and ranked them by descending frequency. Using alleles defined in homozygotes, it combined them to produce expected genotypes that were dismissed, and subtracted them from remaining genotypes to define additional new putative alleles. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 11, 2019 Category: Pathology Authors: Evelina Mocci, Marija Debeljak, Alison P. Klein, James R. Eshleman Tags: Regular Article Source Type: research

Validated Reference Panel from renewable source of genomic DNA available for standardization of blood group genotyping
Extended blood group genotyping is an invaluable tool used for prevention of alloimmunization. Genotyping is particularly suitable when antigens are weak, specific antisera are unavailable, or accurate phenotyping is problematic due to a disease state or recent transfusions. Additionally, genotyping facilitates establishment of mass-scale patient-matched donor databases. However, standardization of genotyping technologies has been hindered by the lack of reference panels. A well-characterized renewable reference panel for standardization of blood group genotyping was developed. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 11, 2019 Category: Pathology Authors: Evgeniya Volkova, Emilia Sippert, Meihong Liu, Teresita Mercado, Gregory A. Denomme, Orieji Illoh, Zhugong Liu, Maria Rios, Collaborative Study Group Tags: Regular Article Source Type: research

Highly Multiplexed Fluorescence in Situ Hybridization for in Situ Genomics
The quantification of changes in gene copy number is critical to our understanding of tumor biology and for the clinical management of cancer patients. DNA fluorescence in situ hybridization is the gold standard method to detect copy number alterations, but it is limited by the number of genes one can quantify simultaneously. To increase the throughput of this informative technique, a fluorescent bar-code system for the unique labeling of dozens of genes and an automated image analysis algorithm that enabled their simultaneous hybridization for the quantification of gene copy numbers were devised. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - March 9, 2019 Category: Pathology Authors: Maristela L. Onozato, Clarence Yapp, Douglas Richardson, Tilak Sundaresan, Varun Chahal, Jesse Lee, James P. Sullivan, Marisa W. Madden, Hyo S. Shim, Mathew Liebers, Quan Ho, Shyamala Maheswaran, Daniel A. Haber, Zongli Zheng, Brian Clancy, Hunter L. Elli Tags: Technical advance Source Type: research

Highly Multiplexed FISH for in Situ Genomics
The quantification of changes in gene copy number is critical to our understanding of tumor biology and for the clinical management of cancer patients. DNA fluorescence in situ hybridization is the gold standard method to detect copy number alterations, but is limited by the number of genes one can quantify simultaneously. To increase the throughput of this informative technique, a fluorescent “barcode” system for the unique labeling of dozens of genes and an automated image analysis algorithm that enabled their simultaneous hybridization for the quantification of gene copy numbers were devised. (Source: Journa...
Source: Journal of Molecular Diagnostics - March 9, 2019 Category: Pathology Authors: Maristela L. Onozato, Clarence Yapp, Douglas Richardson, Tilak Sundaresan, Varun Chahal, Jesse Lee, James P. Sullivan, Marisa W. Madden, Hyo Sup Shim, Mathew Liebers, Quan Ho, Shyamala Maheswaran, Daniel A. Haber, Zongli Zheng, Brian Clancy, Hunter L. Ell Tags: Technical advance Source Type: research

Correction
In the article entitled, “The Development and Validation of Clinical Exome-Based Panels Using ExomeSlicer Considerations and Proof of Concept Using an Epilepsy Panel” (Volume 20, pages 643–652), which appeared in the September 2018 issue of The Journal of Molecular Diagnostics; https://doi.org/10.1016/j.jmoldx.2018.05 .003, there was an error during copyediting which updated the verb tense used in the first three paragraphs of the Results section. This language is meant to be in present tense to reflect general accepted practices, not past tense to reflect actions taken by the authors solely for this manu...
Source: Journal of Molecular Diagnostics - February 28, 2019 Category: Pathology Tags: Correction Source Type: research