An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge

BACKGROUND: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data was donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. RESULTS: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. CONCLUSIONS: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups

Intracellular Calcium Disturbances Induced by Arsenic and Its Methylated Derivatives in Relation to Genomic Damage and Apoptosis Induction

Arsenic and its methylated derivatives are contaminants of air, water, and food and are known as toxicants and carcinogens. Arsenic compounds are also being used as cancer chemotherapeutic agents. In humans, inorganic arsenic is metabolically methylated to mono-, di-, and trimethylated forms. Recent findings suggest that the methylation reactions represent a toxification rather than a detoxification pathway. In recent years, the correlation between arsenic exposure, cytotoxicity and genotoxicity, mutagenicity, and tumor promotion has been established, as well as the association of arsenic exposure with perturbation of physiologic processes, generation of reactive oxygen species, DNA damage, and apoptosis induction. Trivalent forms of arsenic have been found to induce apoptosis in several cellular systems with involvement of membrane-bound cell death receptors, activation of caspases, release of calcium stores, and changes of the intracellular glutathione level. It is well known that calcium ion deregulation plays a critical role in apoptotic cell death. A calcium increase in the nuclei might lead to toxic effects in the cell. In this review, we highlight the relationship between induced disturbances of calcium homeostasis, genomic damage, and apoptotic cell death caused by arsenic and its organic derivatives

An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge

There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. RESULTS: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. CONCLUSIONS: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups

Genetic Testing for the Diagnosis of Hemoglobinopathies Has Very High Clinical Utility

Abstract Background: The hemoglobinopathies are a genetically complex group of blood disorders that includes sickle cell disease (SCD), thalassemia, and other structural and/or functional hemoglobin (Hb) variants. For decades, the mainstay of diagnostic testing for hemoglobinopathies has been Hb separation techniques (e.g., electrophoresis, chromatography), sometimes paired with functional assays of Hb (e.g., solubility, instability, oxygen affinity). Such protein-based testing cannot detect or adequately differentiate a number of clinically significant hemoglobinopathies. Nevertheless, most clinicians continue to rely primarily on protein-based diagnostic methods despite the availability of genetic testing. The reasons for this are not fully understood, but include concerns about the cost of genetic testing and the perception that protein-based methods are usually sufficient for clinical care. Objective: To determine the clinical utility of genetic testing for the diagnosis of hemoglobinopathies by quantifying how often a suspected clinical diagnosis is changed, clarified or excluded by genetic testing. Methods: We reviewed the results of 500 sequential orders for clinical genetic testing for known or suspected hemoglobinopathies that were performed and interpreted at Cincinnati Children's Hospital Medical Center between 1/2013 and 5/2015. This comprehensive genetic testing service is a collaboration between the divisions of Hematology and Human Genetics. For this analysis we reviewed orders for copy number variation analysis (CNV) of the α-globin gene cluster, sequencing of the α-globin genes (HBA1, HBA2), CNV of the β-globin gene cluster, and/or sequencing of the β-globin genes (HBB). An order for testing could include any combination of these 4 tests. We compared the stated indications and suspected diagnoses for testing provided by the ordering physician with the final results of the genetic testing. For each order, the results of this comparison (suspected diagnosis vs. genetic diagnosis) were classified into 1 of 4 mutually exclusive groups: (1) suspected diagnosis confirmed, (2) suspected diagnosis excluded, (3) suspected diagnosis clarified, or (4) new or unexpected diagnosis. The category "suspected diagnosis clarified" includes cases where the overall suspected diagnosis was confirmed, but additional clinically meaningful genetic data was also uncovered (e.g., homozygous sickle cell anemia was confirmed, but co-inherited α-globin gene deletions were also identified). Results: 500 serial genetic testing panels were performed on 475 unique individuals (7 specimens were from an embryo or fetus). These 500 panels included 1-4 individual tests: HBB sequencing in 423, α-globin CNV in 475, β-globin CNV in 345, and HBA1/2 sequencing in 164. The final diagnosis was a specific genotype of SCD (± α-thalassemia and/or gene-deletion HPFH) in 234; thalassemia (α, β, or δβ) in 139; Hb S trait (± α-thalassemia) in 18; Hb C trait or disease (± α-thalassemia) in 13; Hb E trait or disease (± α-thalassemia) in 9; another named or novel Hb variant in 22; and normal Hb in 65. Overall, the suspected diagnosis was confirmed in 246 (49.2%), clarified in 156 (31.2%), excluded in 77 (15.4%), and in 21 (4.2%) a new or unsuspected diagnosis was made. For patients with a suspected diagnosis of SCD, the diagnosis was mostly confirmed (66.7%) and never excluded by genetic testing, but it was clarified in 31.6%, and an unsuspected genotype of SCD was identified in 1.8%. For patients with a suspected diagnosis other than SCD, the diagnosis was confirmed in 34%, clarified in 30.6%, excluded in 29.1%, and an unsuspected genotype was identified in 6.4%. Not considering the clinical utility of the "suspected diagnosis clarified" category, a suspected diagnosis was excluded or determined to be a new or unsuspected diagnosis in 98/500 (19.6%) of all cases. Additionally, follow-up genetic testing was recommended for 31/500 (6.2%). Conclusion: In half (50.8%) of all cases, genetic testing provided new or additional diagnostic information that was not apparent on protein-based diagnostic methods. Genetic testing excluded or substantially changed a suspected diagnosis in one-quarter (19.6%) of all cases. Therefore, genetic testing for hemoglobinopathies has very high clinical utility, and we propose that it should be considered a standard of care for all patients with known or suspected hemoglobinopathies. Disclosures Quinn: Amgen: Research Funding; Eli Lilly: Research Funding; MAST Therapeutics: Research Funding; Glycomimetics: Research Funding; Silverlake Research: Consultancy. Begtrup:GeneDx: Employment. </jats:sec

Dimethylarsinic acid as an effective compound on hematologic malignancies in vitro

Arsenic agents have been used in ancient Chinese medicine for several diseases. Among these agents. Arsenic trioxide has recently been reported as an effective agent for the therapy of relapse and refractory Acute Promyelocytic Leukemia. Several clinical phase trials to determine its efficacy on other hematological disorders including other types of leukemia, lymphoma,and multiple myeloma are being performed at different centers. Although the results look favorable, toxic side effects of arsenic trioxide is a problem. Unlike inorganic arsenic agents like arsenic trioxide, no deaths or serious cases of toxicity due to organoarsenicals have been reported in the literature. Therefore, we tested the effect of organic arsenic compound Dimethylarsinic Acid (DMAA) on both cancer cells and normal progenitor cells of bone marrow. We used colony formation assay and Flow Cytometry to determine its effect. DMAA inhibited colony formation on all cell lines tested. The leukemia cell lines NB4 (Acute Promyelocytic Leukemia), HL60 (AML3), KBM3 (Acute Myelomonocytic Leukemia; AML-4), AML-3, KBM5 (Chronic Myelocytic Leukemia-Blast Crisis), K562 (Chronic Myelocytic Leukemia-Blast Crisis) were responsive to DMAA as the same as or higher than their response to arsenic trioxide. The dose used for arsenic trioxide in our experiments was 1 -2 uM, the clinically relevant dose of this agent. Effective DMAA dose ranged from 0.5 mM to 2mM depending on cell line tested. Multiple myeloma cell lines RPMI 8226, ARK, CAG were also affected by DMAA much more than the inhibition shown by arsenic trioxide on these cells. Since the LD50 value of DMAA is much higher than arsenic trioxide, we were not concerned about its higher effective dose whether it would not be clinically relevant. DMAA was more effective than arsenic trioxide also in inducing apoptosis which is assessed by annexin V, at all cell lines tested, while it did not have significant killing effect on normal progenitor cells of bone marrow. Further studies are to be done on other types of h\ue9matologie malignancies. According to our in vitro results, DMAA has same or higher effect than arsenic trioxide on caricer cells while sparing normal hematopoetic progenitor cells of bone marrow. We conclude that after confirming its specific toxicity by in vivo animal studies. Dimethylarsinic Acid can be tried in clinical phase studies for the treatment of h\ue9matologie malignancies

A systematic approach to assessing the clinical significance of genetic variants

Molecular genetic testing informs diagnosis, prognosis, and risk assessment for patients and their family members. Recent advances in low-cost, high-throughput DNA sequencing and computing technologies have enabled the rapid expansion of genetic test content, resulting in dramatically increased numbers of DNA variants identified per test. To address this challenge, our laboratory has developed a systematic approach to thorough and efficient assessments of variants for pathogenicity determination. We first search for existing data in publications and databases including internal, collaborative and public resources. We then perform full evidence-based assessments through statistical analyses of observations in the general population and disease cohorts, evaluation of experimental data from in vivo or in vitro studies, and computational predictions of potential impacts of each variant. Finally, we weigh all evidence to reach an overall conclusion on the potential for each variant to be disease-causing. In this report, we highlight the principles of variant assessment, address the caveats and pitfalls, and provide examples to illustrate the process. By sharing our experience and providing a framework for variant assessment, including access to a freely available customizable tool, we hope to help move towards standardized and consistent approaches to variant assessment

ClinGen expert clinical validity curation of 164 hearing loss gene-disease pairs

Item does not contain fulltextPURPOSE: Proper interpretation of genomic variants is critical to successful medical decision making based on genetic testing results. A fundamental prerequisite to accurate variant interpretation is the clear understanding of the clinical validity of gene-disease relationships. The Clinical Genome Resource (ClinGen) has developed a semiquantitative framework to assign clinical validity to gene-disease relationships. METHODS: The ClinGen Hearing Loss Gene Curation Expert Panel (HL GCEP) uses this framework to perform evidence-based curations of genes present on testing panels from 17 clinical laboratories in the Genetic Testing Registry. The HL GCEP curated and reviewed 142 genes and 164 gene-disease pairs, including 105 nonsyndromic and 59 syndromic forms of hearing loss. RESULTS: The final outcome included 82 Definitive (50%), 12 Strong (7%), 25 Moderate (15%), 32 Limited (20%), 10 Disputed (6%), and 3 Refuted (2%) classifications. The summary of each curation is date stamped with the HL GCEP approval, is live, and will be kept up-to-date on the ClinGen website ( https://search.clinicalgenome.org/kb/gene-validity ). CONCLUSION: This gene curation approach serves to optimize the clinical sensitivity of genetic testing while reducing the rate of uncertain or ambiguous test results caused by the interrogation of genes with insufficient evidence of a disease link

Prevalence of disease-causing mutations in families with autosomal dominant retinitis pigmentosa : A screen of known genes in 200 families

PURPOSE: To survey families with clinical evidence of autosomal dominant retinitis pigmentosa (adRP) for mutations in genes known to cause adRP. METHODS: Two hundred adRP families, drawn from a cohort of more than 400 potential families, were selected by analysis of pedigrees. Minimum criteria for inclusion in the adRP cohort included either evidence of at least three generations of affected individuals or two generations with evidence of male-to-male transmission. Probands from each family were screened for mutations in 13 genes known to cause adRP: CA4, CRX, FSCN2, IMPDH1, NRL, PRPF3 (RP18), PRPF8 (RP13), PRPF31 (RP11), RDS, RHO, ROM1, RP1, and RP9. Families without mutations in autosomal genes and in which an X-linked mode of inheritance could not be excluded were tested for mutations in ORF 15 of X-linked RPGR. Potentially pathogenic variants were evaluated based on a variety of genetic and computational criteria, to confirm or exclude pathogenicity. RESULTS: A total of 82 distinct, rare (nonpolymorphic) variants were detected among the genes tested. Of these, 57 are clearly pathogenic based on multiple criteria, 10 are probably pathogenic, and 15 are probably benign. In the cohort of 200 families, 94 (47%) have one of the clearly pathogenic variants and 10 (5%) have one of the probably pathogenic variants. One family (0.5%) has digenic RDS-ROM1 mutations. Two families (1%) have a pathogenic RPGR mutation, indicating that families with apparent autosomal transmission of RP may actually have X-linked genetic disease. Thus, 107 families (53.5%) have mutations in known genes, leaving 93 whose underlying cause is still unknown. CONCLUSIONS: Together, the known adRP genes account for retinal disease in approximately half of the families in this survey, mostly Americans of European origin. Among the adRP genes, IMPDH1, PRPF8, PRPF31, RDS, RHO, and RP1 each accounts for more than 2% of the total; CRX, PRPF3, and RPGR each accounts for roughly 1%. Disease-causing mutations were not found in CA4, FSCN2, NRL, or RP9. Because some mutations are frequent and some regions are more likely to harbor mutations than others, more than two thirds of the detected mutations can be found by screening less than 10% of the total gene sequences. Among the remaining families, mutations may lie in regions of known genes that were not tested, mutations may not be detectable by PCR-based sequencing, or other loci may be involved