A comprehensive Triple Repeat Primed PCR (TR-PCR) and a Long-Range PCR (LR-PCR) agarose-based assay for improved genotyping of GAA repeats in Friedreich ’s Ataxia.
Friedreich ’s ataxia (FRDA) is a rare autosomal recessive, neuro-muscular degenerative disease caused by an expansion of a trinucleotide (GAA) repeat in intron 1 of the FXN gene. It is common in the Caucasian population, characterized by progressive gait and limb ataxia, lack of tendon reflexes in the legs, loss of position sense, and hypertrophic cardiomyopathy. Detection and genotyping of the trinucleotide repeat length is important for the diagnosis and prognosis of the disease. A two-tier genotyping assay with an improved triple-repeat primed PCR (TR-PCR) for alleles 200 GAA repeats ( ± 50 repeats) is described. (So...
Source: Journal of Molecular Diagnostics - May 17, 2022 Category: Pathology Authors: Mohamed Jama, Rebecca L. Margraf, Ping Yu, N. Scott Reading, Pinar Bayrak-Toydemir Tags: Regular Article Source Type: research

Long-Reads Sequencing Strategy to Localize Variants in TTN Repeated Domains
Titin protein is responsible for muscle elasticity. The TTN gene, composed of 364 exons, is subjected to extensive alternative splicing and leads to different isoforms expressed in skeletal and cardiac muscle. Variants in TTN are responsible for myopathies with a wide phenotypic spectrum and autosomal dominant or recessive transmission. The I-band coding domain, highly subject to alternative splicing, contains a three-zone block of repeated sequences with 99% homology. Sequencing and localization of variants in these areas are complex when using short-reads sequencing, a second-generation sequencing technique. (Source: Jou...
Source: Journal of Molecular Diagnostics - May 14, 2022 Category: Pathology Authors: Aur élien Perrin, Charles Van Goethem, Corinne Thèze, Jacques Puechberty, Thomas Guignard, Bérénice Lecardonnel, Delphine Lacourt, Corinne Métay, Arnaud Isapof, Sandra Whalen, Ana Ferreiro, Marie-Christine Arne-Bes, Susana Quijano-Roy, Juliette Necto Tags: Technical advance Source Type: research

Long reads sequencing strategy to localize variants in TTN repeated domains
Titin protein is responsible of muscle elasticity. The TTN gene, composed of 364 exons is subjected to extensive alternative splicing and leads to different isoforms expressed in skeletal and cardiac muscle. Variants in TTN are responsible for myopathies with a wide phenotypic spectrum and autosomal dominant or recessive transmission. The I-band coding domain, highly subject to alternative splicing, contains a three-zone block of repeated sequences with 99% homology. Sequencing and localization of variants in these areas are complex when using short reads sequencing, a second-generation sequencing technique. (Source: Journ...
Source: Journal of Molecular Diagnostics - May 14, 2022 Category: Pathology Authors: Aur élien Perrin, Charles Van Goethem, Corinne Thèze, Jacques Puechberty, Thomas Guignard, Bérénice Lecardonnel, Delphine Lacourt, Corinne Métay, Arnaud Isapof, Sandra Whalen, Ana Ferreiro, Marie-Christine Arne-Bes, Susana Quijano-Roy, Juliette Necto Tags: Technical Advance Source Type: research

A Novel Targeted Amplicon Next-Generation Sequencing Gene Panel for the Diagnosis of Common Variable Immunodeficiency Has a High Diagnostic  Yield
With the advent of next-generation sequencing (NGS), monogenic forms of common variable immunodeficiency (CVID) have been increasingly described. Our study aimed to identify disease-causing variants in a Western Australian CVID cohort using a novel targeted NGS panel. Targeted amplicon NGS was performed on 22 unrelated subjects who met the formal European Society for Immunodeficiencies –Pan-American Group for Immunodeficiency diagnostic criteria for CVID and had at least one of the following additional criteria: disease onset at age (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 12, 2022 Category: Pathology Authors: William Kermode, Dianne De Santis, Linh Truong, Erika Della Mina, Sam Salman, Grace Thompson, David Nolan, Richard Loh, Dominic Mallon, Andrew Mclean-Tooke, Mina John, Stuart G. Tangye, Michael O'Sullivan, Lloyd J. D'Orsogna Tags: Regular article Source Type: research

MET Exon 14 Skipping Mutations: Essential Considerations for Current Management of Non –Small Cell Lung Cancer
Genetic alterations in MET that lead to exclusion of exon 14 from the transcript are now targetable in non –small cell lung cancer (NSCLC) after recent drug approvals for this indication. However, this class of alteration is much more diverse than activating mutations in other targetable oncogenes, which presents challenges for clinical detection by molecular diagnostic assays. For this reason, MET exo n 14 (METex14) skipping was highlighted in the Association for Molecular Pathology Emerging and Evolving Biomarkers webinar series (https://www.amp.org/education/emerging-and-evolving-biomarkers, last accessed April 26, 20...
Source: Journal of Molecular Diagnostics - May 9, 2022 Category: Pathology Authors: Kurtis D. Davies, Lauren L. Ritterhouse, Anthony N. Snow, Nikoletta Sidiropoulos Tags: Practice advances Source Type: research

MET exon 14 Skipping Mutations: Essential Considerations for Current Management of Non –Small-Cell Lung Cancer
Genetic alterations in MET that lead to exclusion of exon 14 from the transcript are now ‘targetable’ in non-small cell lung cancer (NSCLC) following recent drug approvals for this indication. However, this class of alteration is much more diverse than activating mutations in other targetable oncogenes, which presents challenges for clinical detection by molecular diagnostic assays. For this reason, MET exon 14 (METex14) skipping was highlighted in the Association for Molecular Pathology Emerging and Evolving Biomarkers webinar series (https://www.amp.org/education/emerging-and-evolving-biomarkers/, date of last access...
Source: Journal of Molecular Diagnostics - May 9, 2022 Category: Pathology Authors: Kurtis D. Davies, Lauren L. Ritterhouse, Anthony N. Snow, Nikoletta Sidiropoulos Tags: Practice Advances Source Type: research

RNA-based targeted gene sequencing improves the diagnostic yield of mutant detection in chronic myeloid leukemia
Mutation detection is increasingly used for the management of hematological malignancies. Prior whole transcriptome and whole exome sequencing studies using total RNA and DNA identified diverse mutation types in cancer-related genes associated with treatment failure in patients with chronic myeloid leukemia (CML). Variants included single nucleotide variants and small insertions/deletions, plus fusion transcripts and partial or whole gene deletions. The hypothesis that all of these mutation types could be detected by a single cost-effective hybridization capture next-generation sequencing method using total RNA was assesse...
Source: Journal of Molecular Diagnostics - May 9, 2022 Category: Pathology Authors: Naranie Shanmuganathan, Carol Wadham, Daniel Thomson, Nur Hezrin Shahrin, Chloe Vignaud, Vanessa Obourn, Shalini Chaturvedi, Feng Yang, Jinghua Feng, Verity Saunders, Chung H. Kok, David Yeung, Rob M. King, Rosalie R. Kenyon, Ming Lin, Paul Wang, Hamish S Tags: Regular Article Source Type: research

NTRK Gene Fusion Detection in a Pan-Cancer Setting using the Idylla GeneFusion Assay
In this study, performance of the fully automated Idylla GeneFusion Assay was assessed in a set of clinically relevant cancer types, including glioblastoma, non-small cell lung cancer, MSI-positive colorectal cancer, and thyroid carcinoma. Analysis with the Idylla GeneFusion Assay revealed significant differences in baseline RNA expression profile between the different cancer types, which corresponded to both literature and pan-TRK IHC staining. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 8, 2022 Category: Pathology Authors: Laure Sorber, Bieke Van Dorst, Ellen Bellon, Karen Zwaenepoel, Suzan Lambin, Koen De Winne, Filip Lardon, Patrick Pauwels, Vasiliki Siozopoulou Tags: Regular Article Source Type: research

Third-Generation Cytogenetic Analysis
Copy number variants (CNVs) play important roles in the pathogenesis of several genetic syndromes. Traditional and molecular karyotyping are considered the first-tier diagnostic tests to detect macroscopic and cryptic deletions/duplications. However, their time-consuming and laborious experimental protocols protract diagnostic times from 3 to 15 days. Nanopore sequencing has the ability to reduce time to results for the detection of CNVs with the same resolution of current state-of-the-art diagnostic tests. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 5, 2022 Category: Pathology Authors: Pamela Magini, Alessandra Mingrino, Barbara Gega, Gianluca Mattei, Roberto Semeraro, Davide Bolognini, Patrizia Mongelli, Laura Desiderio, Maria Carla Pittalis, Tommaso Pippucci, Alberto Magi Tags: Technical advance Source Type: research

Discordance between Immunohistochemistry and ERBB2 mRNA to Determine HER2 Low Status for Breast Cancer
Novel HER2-directed antibody-drug conjugates have demonstrated efficacy in HER2-low expressing breast cancers, which are currently defined as those with immunohistochemistry (IHC) scores of 1+ or 2+ with a negative in situ hybridization assay. However, current HER2 testing methods are designed to identify HER2-amplified tumors with high expression levels. The true definition of HER2-low expressing breast cancers remains controversial. Using quantitative molecular analysis of breast cancers based on RNA expression, the dynamic range of HER2 expression exceeds that detected by in situ IHC approaches. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 5, 2022 Category: Pathology Authors: Keying Xu, Jane Bayani, Elizabeth Mallon, Gregory R. Pond, Tammy Piper, Annette Hasenburg, Christos J. Markopoulos, Luc Dirix, Caroline M. Seynaeve, Cornelis J.H. van de Velde, Daniel W. Rea, John M.S. Bartlett Tags: Regular article Source Type: research

Discordance between IHC and ERBB2 mRNA to Determine HER2 Low Status for Breast Cancer
Novel HER2-directed antibody-drug conjugates (ADC) have demonstrated efficacy in HER2-low expressing breast cancers, which are currently defined as those with IHC scores of 1+ or 2+ with a negative ISH assay. However, current HER2 testing methods are designed to identify HER2 amplified tumors with “high” expression levels. The true definition of “HER2-low” expressing breast cancers remains controversial. Using quantitative molecular analysis of breast cancers based on RNA expression, the dynamic range of HER2 expression exceeds that detected by in situ IHC approaches. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 5, 2022 Category: Pathology Authors: Keying Xu, Jane Bayani, Elizabeth Mallon, Gregory R. Pond, Tammy Piper, Annette Hasenburg, Christos J. Markopoulos, Luc Dirix, Caroline M. Seynaeve, Cornelis J.H. van de Velde, Daniel W. Rea, John MS. Bartlett Tags: Regular Article Source Type: research

Third Generation Cytogenetic Analysis (TGCA): diagnostic application of long-read sequencing.
Copy number variants (CNVs) play important roles in the pathogenesis of several genetic syndromes. Traditional and molecular karyotyping are considered the first-tier diagnostic tests to detect macroscopic and cryptic deletions/duplications. However, their time-consuming and laborious experimental protocols protract diagnostic times from three to fifteen days. Nanopore sequencing have the ability to reduce time to results for the detection of CNVs wi th the same resolution of current state-of-the-art diagnostic tests. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 5, 2022 Category: Pathology Authors: Pamela Magini, Alessandra Mingrino, Barbara Gega, Gianluca Mattei, Roberto Semeraro, Davide Bolognini, Patrizia Mongelli, Laura Desiderio, Maria Carla Pittalis, Tommaso Pippucci, Alberto Magi Tags: Technical Advance Source Type: research

RT-PCR and Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry Diagnostic Target Performance Reflects Circulating Severe Acute Respiratory Syndrome Coronavirus 2 Variant Diversity in New York City
As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate, multiple variants of concern have emerged. New variants pose challenges for diagnostic platforms because sequence diversity can alter primer/probe-binding sites (PBSs), causing false-negative results. The Agena MassARRAY SARS-CoV-2 Panel (Agena Bioscience) uses RT-PCR and mass spectrometry to detect five multiplex targets across N and ORF1ab genes. Herein, we use a data set of 256 SARS-CoV-2 –positive specimens collected between April 11, 2021, and August 28, 2021, to evaluate target performance with paired sequencing data. (Source: J...
Source: Journal of Molecular Diagnostics - May 4, 2022 Category: Pathology Authors: Matthew M. Hernandez, Radhika Banu, Ana S. Gonzalez-Reiche, Brandon Gray, Paras Shrestha, Liyong Cao, Feng Chen, Huanzhi Shi, Ayman Hanna, Juan David Ram írez, Adriana van de Guchte, Robert Sebra, Melissa R. Gitman, Michael D. Nowak, Carlos Cordon-Cardo, Tags: Regular article Source Type: research

RT-PCR/MALDI-TOF diagnostic target performance reflects circulating SARS-CoV-2 variant diversity in New York City
As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate, multiple variants of concern (VOC) have emerged. New variants pose challenges for diagnostic platforms since sequence diversity can alter primer/probe binding sites (PBS), causing false-negative results. The Agena MassARRAY SARS-CoV-2 Panel utilizes reverse-transcription polymerase chain reaction and mass-spectrometry to detect five multiplex targets across N and ORF1ab genes. Herein, we utilize a dataset of 256 SARS-CoV-2-positive specimens collected between April 11, 2021-August 28, 2021 to evaluate target performance with paired sequ...
Source: Journal of Molecular Diagnostics - May 4, 2022 Category: Pathology Authors: Matthew M. Hernandez, Radhika Banu, Ana S. Gonzalez-Reiche, Brandon Gray, Paras Shrestha, Liyong Cao, Feng Chen, Huanzhi Shi, Ayman Hanna, Juan David Ram írez, Adriana van de Guchte, Robert Sebra, Melissa R. Gitman, Michael D. Nowak, Carlos Cordon-Cardo, Tags: Regular Article Source Type: research

Farewell with Gratitude
Communicating effectively with peers and colleagues is an essential component of professional development. Publishing one's work in medical and scientific journals enables these communities to evaluate the findings for themselves. These published reports contribute knowledge to the field, bring value to the author's insights and recognition of expertise among peers, and may foster collaborations to expand these perspectives. Journals disseminate this documentation of practical experiences to increase the understanding of the basis of clinical disease and support decisions for patient care. (Source: Journal of Molecular Diagnostics)
Source: Journal of Molecular Diagnostics - May 1, 2022 Category: Pathology Authors: Barbara A. Zehnbauer Tags: Editorial Source Type: research