Association For Molecular Pathology Research Articles

The Somatic Curation and Interpretation Across Laboratories (SOCIAL) Project—Current State of Solid-Tumour Variant Interpretation for Molecular Pathology in Canada

Practices in somatic variant interpretation and classification vary between Canadian clinical molecular diagnostic laboratories, and understanding of current practices and perspectives is limited. To define gaps and future directions, including consensus guideline development, the Somatic Curation and Interpretation Across Laboratories (social) project examined the present state of somatic variant interpretation in Canadian molecular laboratories, including testing volumes and methods, data sources and evidence criteria, and application of published classification guidelines. Individuals who perform somatic variant interpretation in Canadian centres were invited to participate in an online survey. Invitees included laboratory directors (certified as Fellows of the Canadian College of Medical Geneticists or the American College of Medical Geneticists), md or md and phd molecular pathologists, and other phd experts, including phd specialists in variant annotation or bioinformatics. Current testing methods, volumes, and platforms in next-generation sequencing, use of variant annotation resources and evidence criteria, and preference for variant classification schemes were evaluated. Responses were received from 37 participants in 8 provinces. A somatic variant classification scheme jointly supported by the Association for Molecular Pathology (amp), the American Society of Clinical Oncology (asco), and the College of American Pathologists (cap) was used by 47% of respondents; an alternative guideline or a combination of published guidelines was used by 35% of respondents. The remaining 18% did not use a published scheme. Only 41% of respondents used a published scheme without alteration. Although all respondents indicated that there is a need for Canadian laboratories to adopt a somatic variant classification guideline, only 38% of respondents felt that it should be mandatory to adopt the amp/asco/cap-endorsed guideline. Data from the social project identified high variability in current practice, yet strong support for standardization of solid-tumour somatic variant interpretation across Canadian institutions. Aligning classification methods will reduce variation in cross-institutional classification and reporting practices, aiding in consistent practice nationwide.

Comparative analysis of functional assay evidence use by ClinGen Variant Curation Expert Panels

BackgroundThe 2015 American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines for clinical sequence variant interpretation state that “well-established” functional studies can be used as evidence in variant classification. These guidelines articulated key attributes of functional data, including that assays should reflect the biological environment and be analytically sound; however, details of how to evaluate these attributes were left to expert judgment. The Clinical Genome Resource (ClinGen) designates Variant Curation Expert Panels (VCEPs) in specific disease areas to make gene-centric specifications to the ACMG/AMP guidelines, including more specific definitions of appropriate functional assays. We set out to evaluate the existing VCEP guidelines for functional assays.MethodsWe evaluated the functional criteria (PS3/BS3) of six VCEPs (CDH1, Hearing Loss, Inherited Cardiomyopathy-MYH7, PAH, PTEN, RASopathy). We then established criteria for evaluating functional studies based on disease mechanism, general class of assay, and the characteristics of specific assay instances described in the primary literature. Using these criteria, we extensively curated assay instances cited by each VCEP in their pilot variant classification to analyze VCEP recommendations and their use in the interpretation of functional studies.ResultsUnsurprisingly, our analysis highlighted the breadth of VCEP-approved assays, reflecting the diversity of disease mechanisms among VCEPs. We also noted substantial variability between VCEPs in the method used to select these assays and in the approach used to specify strength modifications, as well as differences in suggested validation parameters. Importantly, we observed discrepancies between the parameters VCEPs specified as required for approved assay instances and the fulfillment of these requirements in the individual assays cited in pilot variant interpretation.ConclusionsInterpretation of the intricacies of functional assays often requires expert-level knowledge of the gene and disease, and current VCEP recommendations for functional assay evidence are a useful tool to improve the accessibility of functional data by providing a starting point for curators to identify approved functional assays and key metrics. However, our analysis suggests that further guidance is needed to standardize this process and ensure consistency in the application of functional evidence.

Comprehensive Assessment of BARD1 Messenger Ribonucleic Acid Splicing With Implications for Variant Classification.

Introduction: Case–control analyses have shown BARD1 variants to be associated with up to >2-fold increase in risk of breast cancer, and potentially greater risk of triple negative breast cancer. BARD1 is included in several gene sequencing panels currently marketed for the prediction of risk of cancer, however there are no gene-specific guidelines for the classification of BARD1 variants. We present the most comprehensive assessment of BARD1 messenger RNA splicing, and demonstrate the application of these data for the classification of truncating and splice site variants according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. Methods: Nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and capillary electrophoresis analysis were performed by four laboratories to investigate alternative BARD1 splicing in blood, breast, and fimbriae/ovary related specimens from non-cancer affected tissues. Splicing data were also collated from published studies of nine different tissues. The impact of the findings for PVS1 annotation was assessed for truncating and splice site variants. Results: We identified 62 naturally occurring alternative spliced BARD1 splicing events, including 19 novel events found by next generation sequencing and/or reverse transcription PCR analysis performed for this study. Quantitative analysis showed that naturally occurring splicing events causing loss of clinically relevant domains or nonsense mediated decay can constitute up to 11.9% of overlapping natural junctions, suggesting that aberrant splicing can be tolerated up to this level. Nanopore sequencing of whole BARD1 transcripts characterized 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splicing events. Bioinformatic analysis of ClinVar submitted variants at or near BARD1 splice sites suggest that all consensus splice site variants in BARD1 should be considered likely pathogenic, with the possible exception of variants at the donor site of exon 5. Conclusions: No BARD1 candidate rescue transcripts were identified in this study, indicating that all premature translation-termination codons variants can be annotated as PVS1. Furthermore, our analysis suggests that all donor and acceptor (IVS+/−1,2) variants can be considered PVS1 or PVS1_strong, with the exception of variants targeting the exon 5 donor site, that we recommend considering as PVS1_moderate.

Determination of Pathogenicity of Breast Cancer 1 Gene Variants using the American College of Medical Genetics and Genomics and the Association for Molecular Pathology Guidelines.

ObjectivesMolecular diagnostic laboratories screen for mutations in disease-causing genes in order to confirm a clinical diagnosis. The classification of DNA variants as ‘pathogenic’ or ‘likely pathogenic’ mutations creates a workflow bottleneck, which becomes increasingly challenging as greater number of genes are screened. The classification challenge is also acute if there are conflicting reports regarding pathogenicity and differing classification criteria between laboratories. This study aimed to compare two procedures for the classification of variants in the breast cancer (BRCA)1 gene.MethodsThis bioinformatic study was conducted at LabPLUS, Auckland, New Zealand, from February to June 2017. DNA was extracted from peripheral blood samples of 30 patients and gene library construction was carried out using a commercially available targeted panel for the BRCA1 and BRCA2 genes. The genes were subsequently sequenced and the sequence data analysed. The guidelines published by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) provides a comprehensive framework for the interpretation of variants in genes that are associated with Mendelian disorders. The use of these guidelines were compared to the variant classifications that were achieved by reference to those reported in the BRCA Exchange database.ResultsThe results showed concordance between the two classification protocols for a panel of 30 BRCA1 gene variants, although the transparency in following the ACMG/AMP guidelines provides a diagnostic laboratory with a generalisable approach that allows laboratory-directed revisions to be undertaken in light of new information.ConclusionThe ACMG/AMP-based guidelines were applied to a cohort of patients with BRCA1 gene variants. The use of these guidelines provides a system which creates consistency in variant interpretation and supports subsequent clinical management.

ClinGen Myeloid Malignancy Variant Curation Expert Panel recommendations for germline RUNX1 variants

AbstractStandardized variant curation is essential for clinical care recommendations for patients with inherited disorders. Clinical Genome Resource (ClinGen) variant curation expert panels are developing disease-associated gene specifications using the 2015 American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) guidelines to reduce curation discrepancies. The ClinGen Myeloid Malignancy Variant Curation Expert Panel (MM-VCEP) was created collaboratively between the American Society of Hematology and ClinGen to perform gene- and disease-specific modifications for inherited myeloid malignancies. The MM-VCEP began optimizing ACMG/AMP rules for RUNX1 because many germline variants have been described in patients with familial platelet disorder with a predisposition to acute myeloid leukemia, characterized by thrombocytopenia, platelet functional/ultrastructural defects, and a predisposition to hematologic malignancies. The 28 ACMG/AMP codes were tailored for RUNX1 variants by modifying gene/disease specifications, incorporating strength adjustments of existing rules, or both. Key specifications included calculation of minor allele frequency thresholds, formulating a semi-quantitative approach to counting multiple independent variant occurrences, identifying functional domains and mutational hotspots, establishing functional assay thresholds, and characterizing phenotype-specific guidelines. Preliminary rules were tested by using a pilot set of 52 variants; among these, 50 were previously classified as benign/likely benign, pathogenic/likely pathogenic, variant of unknown significance (VUS), or conflicting interpretations (CONF) in ClinVar. The application of RUNX1-specific criteria resulted in a reduction in CONF and VUS variants by 33%, emphasizing the benefit of gene-specific criteria and sharing internal laboratory data.

Patenting of Human Genes: The United States, Canada and Australia Case Law

The paper is a review of the case law of the United States of America, Canada and Australia, in which an attempt is made to answer the question on possibility of human gene patenting. The paper substantiates the relevance of this issue, examines the ethical aspects of gene patenting. The author analyzes the landmark and most significant cases from the point of view of the development of patent law of foreign countries: Diamond v. Chakrabarty (USA), Association for Molecular Pathology v. Myriad Genetics (USA), Myriad v. Cancer Voices (Australia), The Children’s Hospital of Eastern Ontario (CHEO) v. Transgenomic (Canada). In the analysis, the author gives special attention to the arguments and conclusions of judicial institutions regarding the patentability of human genes. A conclusion is drawn regarding the continuity and possible harmonization of legislation and judicial practices of both the states mentioned in the paper and countries that have just embarked on the development of biomedical technologies.

MS02.03 Achieving Effective Lung Cancer Genotyping While Balancing Constrained Resources

Modern oncologic practice for patients with non-small cell lung carcinoma (NSCLC) demands real time data for an increasing numbers of tumor biomarkers. As a result, laboratories are embracing panel-based approaches to tumor molecular profiling, a trend that is facilitated by the adoption of next generation sequencing (NGS) assays. NGS assays may be designed for highly sensitive and focused detection of hotspot mutations (typically by amplicon sequencing) or broader, more comprehensive profiling for detection of a wide variety of alterations in oncogenes and tumor suppressor genes (typically by hybrid capture technology). There are benefits and drawbacks to both approaches. In general, amplicon sequencing offers speed and sensitivity but limited scope and a propensity for PCR-related artifacts that may adversely affect detection of certain mutation types. In contrast, hybrid capture techniques offer tremendous breadth, enabling capture of 100s of genes to whole genomes, but are limited by sensitivity and longer turnaround time.1 Preanalytic steps including sample acquisition, pathologist review to confirm sample adequacy, and nucleic acid extraction often comprise the majority of the turnaround time required to complete focused tumor tissue molecular profiling. Some assays have been optimized for turnaround times of just a few hours- these assays bypass the separate nucleic acid extraction step,2 but are limited by the few number of mutational targets detected, ultimately requiring the use of multiple simultaneous or sequential focused panels.3 This practice, while perhaps the optimal current model from the standpoint of patient care, may be financially prohibitive for many laboratories. This approach additionally requires careful stewardship of patient tissues, as the use of multiple competing assays and may lead to exhaustion of tumor tissue and incomplete tumor molecular profiling. For patients with limited tissue, a considered plan incorporating input from the treating physician, surgical pathologist, and molecular laboratorian should developed early in order to guide assay priority and ensure adequate tissue is available to confirm any unexpected or contradictory results. When the turnaround time for comprehensive large panel testing is clinically acceptable, this approach may be theoretically more cost effective and in most cases will deliver information for essential and emerging biomarkers.4 It is important to keep in mind, however, that even comprehensive DNA-based assays may lack optimal sensitivity for certain structural variants (fusions, large insertion-deletion events), and it may be necessary to couple these tests with focused RNA-based panels optimized for transcript fusion detection.5 1. Rizzo JM, Buck MJ. Key principles and clinical applications of "next-generation" DNA sequencing. Cancer Prev Res (Phila). 2012;5(7):887-900. 2. Ilie M, Butori C, Lassalle S, et al. Optimization of EGFR mutation detection by the fully-automated qPCR-based Idylla system on tumor tissue from patients with non-small cell lung cancer. Oncotarget. 2017;8(61):103055-103062. 3. Lindeman NI, Cagle PT, Aisner DL, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Mol Diagn. 2018;20(2):129-159. 4. Sireci AN. Single Genes, Panels, and Next-Generation Sequencing Platforms: A Financial Perspective. Arch Pathol Lab Med. 2018;142(7):790-791. 5. Benayed R, Offin M, Mullaney K, et al. High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden. Clin Cancer Res. 2019. amplicon sequencing, hybrid capture, turnaround time

ES07.04 Predictive Molecular Testing on Small Biopsy Samples

ES07.04 Predictive Molecular Testing on Small Biopsy Samples

Pathogenic APC Variants in Latvian Familial Adenomatous Polyposis Patients.

Background and objectives: Familial adenomatous polyposis is one of the APC-associated polyposis conditions described as genetically predetermined colorectal polyposis syndrome with a variety of symptoms. The purpose of this study was to determine sequence variants of the APC gene in patients with familial adenomatous polyposis (FAP) phenotype and positive or negative family history. Materials and Methods: Eight families with defined criteria of adenomatous polyposis underwent molecular genetic testing. Coding regions and flanking intron regions of the APC gene were analyzed by Sanger sequencing. Results: Eight allelic variants of the APC gene coding sequence were detected. All allelic variants of the APC gene were predicted to be pathogenic based on criteria according to the “Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology” (2015), four of them c.1586_1587insAT, c.2336delT, c.3066_3067insGA, and c.4303_4304insC, were considered novel. Conclusions: The timely molecular genetic analysis of APC germline variants and standardized interpretation of the pathogenicity of novel allelic variants has a high impact on choice for treatment, cancer prevention, and family genetic counseling.

Variant Interpretation for Cancer (VIC): a computational tool for assessing clinical impacts of somatic variants

BackgroundClinical laboratories implement a variety of measures to classify somatic sequence variants and identify clinically significant variants to facilitate the implementation of precision medicine. To standardize the interpretation process, the Association for Molecular Pathology (AMP), American Society of Clinical Oncology (ASCO), and College of American Pathologists (CAP) published guidelines for the interpretation and reporting of sequence variants in cancer in 2017. These guidelines classify somatic variants using a four-tiered system with ten criteria. Even with the standardized guidelines, assessing clinical impacts of somatic variants remains to be tedious. Additionally, manual implementation of the guidelines may vary among professionals and may lack reproducibility when the supporting evidence is not documented in a consistent manner.ResultsWe developed a semi-automated tool called “Variant Interpretation for Cancer” (VIC) to accelerate the interpretation process and minimize individual biases. VIC takes pre-annotated files and automatically classifies sequence variants based on several criteria, with the ability for users to integrate additional evidence to optimize the interpretation on clinical impacts. We evaluated VIC using several publicly available databases and compared with several predictive software programs. We found that VIC is time-efficient and conservative in classifying somatic variants under default settings, especially for variants with strong and/or potential clinical significance. Additionally, we also tested VIC on two cancer-panel sequencing datasets to show its effectiveness in facilitating manual interpretation of somatic variants.ConclusionsAlthough VIC cannot replace human reviewers, it will accelerate the interpretation process on somatic variants. VIC can also be customized by clinical laboratories to fit into their analytical pipelines to facilitate the laborious process of somatic variant interpretation. VIC is freely available at https://github.com/HGLab/VIC/.

One NF1 Mutation may Conceal Another.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance but high variable expressivity. NF1 is caused by loss-of-function mutations in the NF1 gene, a negative regulator of the RAS-MAPK pathway. The NF1 gene has one of the highest mutation rates in human disorders, which may explain the outbreak of independent de novo variants in the same family. Here, we report the co-occurrence of pathogenic variants in the NF1 and SPRED1 genes in six families with NF1 and Legius syndrome, using next-generation sequencing. In five of these families, we observed the co-occurrence of two independent NF1 variants. All NF1 variants were classified as pathogenic, according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines. In the sixth family, one sibling inherited a complete deletion of the NF1 gene from her mother and carried a variant of unknown significance in the SPRED1 gene. This variant was also present in her brother, who was diagnosed with Legius syndrome, a differential diagnosis of NF1. This work illustrates the complexity of molecular diagnosis in a not-so-rare genetic disease.

Short QT Syndrome: A Comprehensive Genetic Interpretation and Clinical Translation of Rare Variants.

Short QT syndrome, one of the most lethal entities associated with sudden cardiac death, is a rare genetic disease characterized by short QT intervals detected by electrocardiogram. Several genetic variants are causally linked to the disease, but there has yet to be a comprehensive analysis of variants among patients with short QT syndrome. To fill this gap, we performed an exhaustive study of variants currently catalogued as deleterious in short QT syndrome according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Analysis of the 32 variants described in the literature determined that only nine (28.12%) have a conclusive pathogenic role. All definitively pathogenic variants are located in KCNQ1, KCNH2, or KCNJ2; three genes encoding potassium channels. Other variants located in genes encoding calcium or sodium channels are associated with electrical alterations concomitant with shortened QT intervals but do not guarantee a diagnosis of short QT syndrome. We recommend caution regarding previously reported variants classified as pathogenic. An exhaustive re-analysis is necessary to clarify the role of each variant before routinely translating genetic findings to the clinical setting.

"Somatic" and "pathogenic" - is the classification strategy applicable in times of large-scale sequencing?

In the early days of sequencing only a small number of bases was evaluated because of the labor-intensive nature of the procedure. Genes were identified to play a role in the pathogenesis of neoplasms in animal models and cell lines. Subsequently, these mutations were analyzed in samples from patients and their impact on prognosis was evaluated. The list of examples is long: e.g. TP53 was found to be universally mutated across cancers,1 and NPM1 is now among the most frequently analyzed genes in acute myeloid leukemia2 and the mutation defines its own acute myeloid leukemia subtype in the current World Health Organization classification.3 High-throughput sequencing has changed the landscape. It is now possible to sequence a huge number of genes up to exomes and even whole genomes in a comparably short time at affordable cost. The challenge is no longer the sequencing, but rather the evaluation of the results and the interpretation of their impact on diagnosis, prognosis or therapeutic decisions. This has led to some major changes in the way we view sequencing data. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) recommended changing the terms mutation and polymorphism to “variant”. Variants are then further subdivided into five categories depending on the likelihood of their association with the disease.4 The definition was designed for hereditary diseases and therefore addresses germline variants. The vast majority of genetic events in cancer are somatic.5 Acquired variants represent potential drug targets or biomarkers. Testing a sample, e.g. of colon cancer, for mutations is frequently performed by comparing variants from a biopsy to those in leukocytes as reference to identify only somatic variants (tumor-normal comparison).6,7 The classical tumor-normal workflow is challenging in studies with large or historic cohorts because of additional sequencing costs, or limited availability of reference material. Leukemia represents another challenge, since blood cannot be used as the reference material. In addition, the growing knowledge of genetic complexity and tumor heterogeneity challenges the historic binary variant classifications (mutation, polymorphism) in the somatic field as well (Figure 1). Open in a separate window Figure 1. Clinical questions for current variant classifications. The complexity of variant classification challenges the terms “mutation” and “polymorphism”, which were clearly associated with clinical relevance in the past. The classification, interpretation and consequences of each variant depend on the context. The identification of somatic changes proves clonality, and somatic aberrations qualify as markers of minimal/measurable residual disease. Other clinical issues are the availability of targeted therapies for mutations and the World Health Organization classification, which genetically defines certain entities. Finally, inherited variants are not only discussed in the context of familial predisposition, but are also relevant if family members are considered as stem cell donors. AlloSCT: allogeneic stem cell transplantation; MRD: minimal/measurable residual disease; WHO: World Health Organization.

Erratum: Challenges and Considerations in Sequence Variant Interpretation for Mendelian Disorders.

This corrects the article on p. 421 in vol. 39, PMID: 31037860.

Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection.

IntroductionComprehensive genetic cancer profiling using circulating tumor DNA has enabled the detection of National Comprehensive Cancer Network (NCCN) guideline-recommended somatic alterations from a single, non-invasive blood draw. However, reliably detecting somatic variants at low variant allele fractions (VAFs) remains a challenge for next-generation sequencing (NGS)-based tests. We have developed the single-molecule sequencing (SMSEQ) platform to address these challenges.MethodsThe OncoLBx assay utilizes the SMSEQ platform to optimize cell-free DNA extraction and library preparation with variant type-specific calling algorithms to improve sensitivity and specificity. OncoLBx is a pan-cancer panel for solid tumors targeting 75 genes and five microsatellite sites analyzing five classes of NCCN-recommended somatic variants: single-nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), fusions and microsatellite instability (MSI). Circulating DNA was extracted from plasma, followed by library preparation using SMSEQ. Analytical validation was performed according to recently published American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines and established the limit of detection (LOD), sensitivity, specificity, accuracy and reproducibility using 126 gold-standard reference samples, healthy donor samples verified by whole-exome sequencing by an external College of American Pathologists (CAP) reference lab and cell lines with known variants. Results were analyzed using a locus-specific modeling algorithm.ResultsWe have demonstrated that OncoLBx detects VAFs of ≥ 0.1% for SNVs and indels, ≥ 0.5% for fusions, ≥ 4.5 copies for CNVs and ≥ 2% for MSI, with all variant types having specificity ≥ 99.999%. Diagnostic performance of paired samples displays 80% sensitivity and > 99.999% clinical specificity. Clinical utility and performance were assessed in 416 solid tumor samples. Variants were detected in 79% of samples, for which 87.34% of positive samples had available targeted therapy.Electronic supplementary materialThe online version of this article (10.1007/s40291-019-00406-0) contains supplementary material, which is available to authorized users.

The Effects of Myriad and Mayo on Molecular Test Development in the US and Europe: Interviews from the Frontline

US Supreme Court decisions in Mayo Collaborative Services v. Prometheus Laboratories and Association for Molecular Pathology v. Myriad Genetics Inc. caused US and European law on what is patentable subject matter to diverge significantly. Both cases related to molecular tests and changed decades of patent practice. Whether the decisions adversely affect the development of molecular tests in the United States and Europe has been a matter of much speculation but limited empirical investigation. This interview-based study has three main findings. First, Myriad and Mayo have negatively affected the development of some molecular tests. Notably, half of the US university technology-transfer offices interviewed decided not to develop tests, and many other organizations have found the legal uncertainty following the cases problematic. Second, small patent-precarious organizations—those that rely heavily on patents for competitive advantages, such as technology-transfer offices—have been the most affected because patent protection is now often weaker and more difficult

Increased diagnostic yield by reanalysis of data from a hearing loss gene panel

BackgroundCongenital hearing loss affects approximately 1–2 infants out of every 1000, with 50% of the cases resulting from genetic factors. Targeted gene panels have been widely used for genetic diagnosis of hearing loss. This study aims to reveal new diagnoses via reanalyzing historical data of a multigene panel, and exam the reasons for new diagnoses.MethodsA total of 210 samples were enlisted, including clinical reports and sequencing data of patients with congenital/prelingual hearing loss who were referred to clinical genetic testing from October 2014 to June 2017. All variants listed on the original clinical reports were reinterpreted according to the standards and guidelines recommended by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). Expanded analysis of raw data were performed in undiagnosed cases.ResultsRe-analysis resulted in nine new diagnoses, improving the overall diagnostic rate from 39 to 43%. New diagnoses were attributed to newly published clinical evidence in the literature, adoption of new interpretation guidelines and expanded analysis range.ConclusionThis work demonstrates benefits of reanalysis of targeted gene panel data, indicating that periodical reanalysis should be performed in clinical practice.

Challenges and Considerations in Sequence Variant Interpretation for Mendelian Disorders.

In 2015, the American College of Medical Genetics and Genomics (ACMG), together with the Association for Molecular Pathology (AMP), published the latest guidelines for the interpretation of sequence variants, which have been widely adopted into clinical practice. Despite these standardized efforts, the degrees of subjectivity and uncertainty allowed by the guidelines can lead to inconsistent variant classification across clinical laboratories, making it difficult to assess the pathogenicity of identified variants. We describe the critical elements of variant interpretation processes and potential pitfalls through practical examples and provide updated information based on a review of recent literature. The variant classification we describe is meant to be applicable to sequence variants for Mendelian disorders, whether identified by single-gene tests, multi-gene panels, exome sequencing, or genome sequencing. Continuing efforts to improve the reproducibility and objectivity of sequence variant interpretation across individuals and laboratories are needed.

Combined pituitary hormone deficiency caused by PROP1 mutations: update 20 years post-discovery.

The first description of patients with combined pituitary hormone deficiencies (CPHD) caused by PROP1 mutations was made 20 years ago. Here we updated the clinical and genetic characteristics of patients with PROP1 mutations and summarized the phenotypes of 14 patients with 7 different pathogenic PROP1 mutations followed at the Hospital das Clínicas of the University of Sao Paulo. In addition to deficiencies in GH, TSH, PRL and gonadotropins some patients develop late ACTH deficiency. Therefore, patients with PROP1 mutations require permanent surveillance. On magnetic resonance imaging, the pituitary stalk is normal, and the posterior lobe is in the normal position. The anterior lobe in patients with PROP1 mutations is usually hypoplastic but may be normal or even enlarged. Bi-allelic PROP1 mutations are currently the most frequently recognized genetic cause of CPHD worldwide. PROP1 defects occur more frequently among offspring of consanguineous parents and familial cases, but they also occur in sporadic cases, especially in countries in which the prevalence of PROP1 mutations is relatively high. We classified all reported PROP1 variants described to date according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines: 29 were pathogenic, 2 were likely pathogenic, and 2 were of unknown significance. An expansion of the phenotype of patients with PROP1 mutations was observed since the first description 20 years ago: variable anterior pituitary size, different pathogenic mutations, and late development of ACTH deficiency. PROP1 mutations are the most common cause of autosomal recessive CPHD with a topic posterior pituitary lobe. Arch Endocrinol Metab. 2019;63(2):167-74.

Corrigendum.

Corrigendum.