Analytical Validation of a Telomerase Reverse Transcriptase (TERT) Promoter Mutation Assay.

CONTEXT: TERT promoter mutated thyroid cancers are associated with a decreased rate of disease free and disease specific survival. High quality analytical validation of a diagnostic test promotes confidence in the results which inform clinical decision making. OBJECTIVE: To demonstrate the analytical validation of the Afirma TERT promoter mutation assay. METHODS: TERT promoter C228T and C250T variant detection in genomic DNA (gDNA) was analyzed by assessing variable DNA input and the limit of detection (LOD) of variant allele frequency (VAF). The negative and positive percent agreement (NPA and PPA) of the Afirma TERT test was examined against a reference primer pair as was the analytical specificity from potential interfering substances (RNA and blood gDNA). Further, the intra-run, inter-run and inter-laboratory reproducibility of the assay were tested. RESULTS: The Afirma TERT test is tolerant to variation in DNA input amount (7-13 ng) and can detect expected positive TERT promoter variants down to 5% VAF LOD at 7ng DNA input with > 95% sensitivity. Both NPA and PPA were 100% against the reference primer pair. The test remains accurate in presence of 20% RNA or 80% blood gDNA for an average patient sample that typically has 30% VAF. The test also demonstrated a 100% confirmation rate when compared with an external NGS-based reference assay executed in a non-Veracyte laboratory. CONCLUSION: The analytical robustness and reproducibility of the Afirma TERT test support its routine clinical use among thyroid nodules with indeterminate cytology that are Afirma GSC suspicious or among Bethesda V/VI nodules.

The matrix in focus: new directions in extracellular matrix research from the 2021 ASMB hybrid meeting.

The extracellular matrix (ECM) is a complex assembly of macromolecules that provides both architectural support and molecular signals to cells and modulate their behaviors. Originally considered a passive mechanical structure, decades of research have since demonstrated how the ECM dynamically regulates a diverse set of cellular processes in development, homeostasis, and disease progression. In September 2021, the American Society for Matrix Biology (ASMB) organized a hybrid scientific meeting, integrating in-person and virtual formats, to discuss the latest developments in ECM research. Here, we highlight exciting scientific advances that emerged from the meeting including (1) the use of model systems for fundamental and translation ECM research, (2) ECM-targeting approaches as therapeutic modalities, (3) cell-ECM interactions, and (4) the ECM as a critical component of tissue engineering strategies. In addition, we discuss how the ASMB incorporated mentoring, career development, and diversity, equity, and inclusion initiatives in both virtual and in-person events. Finally, we reflect on the hybrid scientific conference format and how it will help the ASMB accomplish its mission moving forward.

Computational and experimental characterization of the novel ECM glycoprotein SNED1 and prediction of its interactome.

The extracellular matrix (ECM) is a complex meshwork of proteins and an essential component of multicellular life. We have recently reported the characterization of a novel ECM protein, SNED1, and showed that it promotes breast cancer metastasis and regulates craniofacial development. However, the mechanisms by which it does so remain unknown. ECM proteins exert their functions by binding to cell surface receptors and interacting with other ECM proteins, actions that we can predict using knowledge of protein's sequence, structure, and post-translational modifications. Here, we combined in-silico and in-vitro approaches to characterize the physico-chemical properties of SNED1 and infer its putative functions. To do so, we established a mammalian cell system to produce and purify SNED1 and its N-terminal fragment, which contains a NIDO domain, and demonstrated experimentally SNED1's potential to be glycosylated, phosphorylated, and incorporated into an insoluble ECM. We also determined the secondary and tertiary structures of SNED1 and its N-terminal fragment and obtained a model for its NIDO domain. Using computational predictions, we identified 114 proteins as putative SNED1 interactors, including the ECM protein fibronectin. Pathway analysis of the predicted SNED1 interactome further revealed that it may contribute to signaling through cell surface receptors, such as integrins, and participate in the regulation of ECM organization and developmental processes. Last, using fluorescence microscopy, we showed that SNED1 forms microfibrils within the ECM and partially colocalizes with fibronectin. Altogether, we provide a wealth of information on an understudied yet important ECM protein with the potential to decipher its pathophysiological functions.

Knockout of the gene encoding the extracellular matrix protein SNED1 results in early neonatal lethality and craniofacial malformations.

BACKGROUND: The extracellular matrix (ECM) is a fundamental component of multicellular organisms that orchestrates developmental processes and controls cell and tissue organization. We previously identified the novel ECM protein SNED1 as a promoter of breast cancer metastasis and showed that its level of expression negatively correlated with breast cancer patient survival. Here, we sought to identify the roles of SNED1 during murine development. RESULTS: We generated two novel Sned1 knockout mouse strains and showed that Sned1 is essential since homozygous ablation of the gene led to early neonatal lethality. Phenotypic analysis of the surviving knockout mice revealed a role for SNED1 in the development of craniofacial and skeletal structures since Sned1 knockout resulted in growth defects, nasal cavity occlusion, and craniofacial malformations. Sned1 is widely expressed in embryos, notably by cell populations undergoing epithelial-to-mesenchymal transition, such as the neural crest cells. We further show that mice with a neural-crest-cell-specific deletion of Sned1 survive, but display facial anomalies partly phenocopying the global knockout mice. CONCLUSIONS: Our results demonstrate requisite roles for SNED1 during development and neonatal survival. Importantly, the deletion of 2q37.3 in humans, a region that includes the SNED1 locus, has been associated with facial dysmorphism and short stature.

Genetic and Clinical Heterogeneity in Thirteen New Cases with Aceruloplasminemia. Atypical Anemia as a Clue for an Early Diagnosis.

Aceruloplasminemia is a rare autosomal recessive genetic disease characterized by mild microcytic anemia, diabetes, retinopathy, liver disease, and progressive neurological symptoms due to iron accumulation in pancreas, retina, liver, and brain. The disease is caused by mutations in the Ceruloplasmin (CP) gene that produce a strong reduction or absence of ceruloplasmin ferroxidase activity, leading to an impairment of iron metabolism. Most patients described so far are from Japan. Prompt diagnosis and therapy are crucial to prevent neurological complications since, once established, they are usually irreversible. Here, we describe the largest series of non-Japanese patients with aceruloplasminemia published so far, including 13 individuals from 11 families carrying 13 mutations in the CP gene (7 missense, 3 frameshifts, and 3 splicing mutations), 10 of which are novel. All missense mutations were studied by computational modeling. Clinical manifestations were heterogeneous, but anemia, often but not necessarily microcytic, was frequently the earliest one. This study confirms the clinical and genetic heterogeneity of aceruloplasminemia, a disease expected to be increasingly diagnosed in the Next-Generation Sequencing (NGS) era. Unexplained anemia with low transferrin saturation and high ferritin levels without inflammation should prompt the suspicion of aceruloplasminemia, which can be easily confirmed by low serum ceruloplasmin levels. Collaborative joint efforts are needed to better understand the pathophysiology of this potentially disabling disease.

Functional consequences of transferrin receptor-2 mutations causing hereditary hemochromatosis type 3.

Hereditary hemochromatosis (HH) type 3 is an autosomal recessive disorder of iron metabolism characterized by excessive iron deposition in the liver and caused by mutations in the transferrin receptor 2 (TFR2) gene. Here, we describe three new HH type 3 Spanish families with four TFR2 mutations (p.Gly792Arg, c.1606-8A>G, Gln306*, and Gln672*). The missense variation p.Gly792Arg was found in homozygosity in two adult patients of the same family, and in compound heterozygosity in an adult proband that also carries a novel intronic change (c.1606-8A>G). Two new nonsense TFR2 mutations (Gln306* and Gln672*) were detected in a pediatric case. We examine the functional consequences of two TFR2 variants (p.Gly792Arg and c.1606-8A>G) using molecular and computational methods. Cellular protein localization studies using immunofluorescence demonstrated that the plasma membrane localization of p.Gly792Arg TFR2 is impaired. Splicing studies in vitro and in vivo reveal that the c.1606-8A>G mutation leads to the creation of a new acceptor splice site and an aberrant TFR2 mRNA. The reported mutations caused HH type 3 by protein truncation, altering TFR2 membrane localization or by mRNA splicing defect, producing a nonfunctional TFR2 protein and a defective signaling transduction for hepcidin regulation. TFR2 genotyping should be considered in adult but also in pediatric cases with early-onset of iron overload.