Consensus interpretation of the p.Met34Thr and p.Val37Ile variants in GJB2 by the ClinGen Hearing Loss Expert Panel.

PURPOSE: Pathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants. METHODS: The ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case-control statistical analyses were performed. RESULTS: The panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared with population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants. CONCLUSION: Resolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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

PURPOSE: 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.

Reporting of Clinical Genome Sequencing Results.

High-throughput sequencing and high-performance computing technologies have become powerful tools in clinical genetic diagnosis of hereditary disorders and genetic screening of healthy individuals to provide information for the diagnosis, treatment, and prevention of diseases or impairment and assessment of health. For patients with undiagnosed disorders, including many rare disorders, the whole-genome sequencing (WGS) test may end the diagnostic odyssey, ultimately guiding clinical care for them and their families. A clinical WGS test relies on high-quality genome-sequencing data as well as sophisticated data-interpretation approaches. Results are returned to the ordering physician in a concise report featuring an overall test result and in-depth phenotype-driven interpretation of the known or plausible genetic explanation of test indications. Patients have the option to decide whether the report should include secondary and incidental findings. Protocols and templates for reporting clinical WGS results and supplementary information are described in this article. © 2018 by John Wiley & Sons, Inc.

Inherited CHST11/MIR3922 deletion is associated with a novel recessive syndrome presenting with skeletal malformation and malignant lymphoproliferative disease.

Glycosaminoglycans (GAGs) such as chondroitin are ubiquitous disaccharide carbohydrate chains that contribute to the formation and function of proteoglycans at the cell membrane and in the extracellular matrix. Although GAG-modifying enzymes are required for diverse cellular functions, the role of these proteins in human development and disease is less well understood. Here, we describe two sisters out of seven siblings affected by congenital limb malformation and malignant lymphoproliferative disease. Using Whole-Genome Sequencing (WGS), we identified in the proband deletion of a 55 kb region within chromosome 12q23 that encompasses part of CHST11 (encoding chondroitin-4-sulfotransferase 1) and an embedded microRNA (MIR3922). The deletion was homozygous in the proband but not in each of three unaffected siblings. Genotyping data from the 1000 Genomes Project suggest that deletions inclusive of both CHST11 and MIR3922 are rare events. Given that CHST11 deficiency causes severe chondrodysplasia in mice that is similar to human limb malformation, these results underscore the importance of chondroitin modification in normal skeletal development. Our findings also potentially reveal an unexpected role for CHST11 and/or MIR3922 as tumor suppressors whose disruption may contribute to malignant lymphoproliferative disease.

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 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.

LDOC1 mRNA is differentially expressed in chronic lymphocytic leukemia and predicts overall survival in untreated patients.

We previously identified LDOC1 as one of the most significantly differentially expressed genes in untreated chronic lymphocytic leukemia (CLL) patients with respect to the somatic mutation status of the immunoglobulin heavy-chain variable region genes. However, little is known about the normal function of LDOC1, its contribution to the pathophysiology of CLL, or its prognostic significance. In this study, we have investigated LDOC1 mRNA expression in a large cohort of untreated CLL patients, as well as in normal peripheral blood B-cell (NBC) subsets and primary B-cell lymphoma samples. We have confirmed that LDOC1 is dramatically down-regulated in mutated CLL cases compared with unmutated cases, and have identified a new splice variant, LDOC1S. We show that LDOC1 is expressed in NBC subsets (naive > memory), suggesting that it may play a role in normal B-cell development. It is also expressed in primary B-cell lymphoma samples, in which its expression is associated with somatic mutation status. In CLL, we show that high levels of LDOC1 correlate with biomarkers of poor prognosis, including cytogenetic markers, unmutated somatic mutation status, and ZAP70 expression. Finally, we demonstrate that LDOC1 mRNA expression is an excellent predictor of overall survival in untreated CLL patients.

MER1, a novel organic arsenic derivative, has potent PML-RARalpha-independent cytotoxic activity against leukemia cells.

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is highly effective against PML-RARalpha-positive leukemia but much less against other hematological malignancies. We synthesized an organic arsenic derivative (OAD), S-dimethylarsino-thiosuccinic acid (MER1), which offers a superior toxicity profile and comparable in vitro activity relative to ATO. In Swiss Webster mice, maximally-tolerated cumulative dose of MER1 when given i.v. for 5 days was 100 mg/kg/d. We demonstrated that MER1 induced apoptosis and dose- and time-dependent inhibition of survival and growth in a panel of myeloid leukemia cell lines. Unlike ATO, this activity was independent of PML-RARalpha status and was not associated with induction of myeloid maturation. In NB4 and HL60 cells, MER1 and ATO induced caspase activation and dissipation of mitochondrial transmembrane potential. At the same time, MER1 induced generation of reactive oxygen species (ROS) and cell cycle arrest in G2/M phase and proved to be more potent than ATO at inducing apoptosis. ROS generation and intracellular glutathione levels were key modulators of MER1-induced cytotoxicity as evidenced by abrogation of apoptosis in myeloid leukemia cell lines pretreated with the disulfide bond-reducing agent dithiothreitol or the radical scavenger N-acetyl-L-cysteine. Collectively, these data indicate that MER1 induces apoptosis in PML-RARalpha-positive and -negative myeloid leukemia cells by enhancing oxidative stress. This agent, therefore, combines low in vivo toxicity with formidable in vitro pro-apoptotic ROS-mediated activity, and may represent a novel OAD suitable for clinical development against a variety of hematological malignancies.

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.

Bone marrow purging studies in acute myelogenous leukemia using the recombinant anti-CD33 immunotoxin HuM195/rGel.

This study was designed to determine the effect of immunotoxin HuM195/rGel on normal human bone marrow before clinical purging. HuM195/rGel is composed of the recombinant plant toxin gelonin (rGel) chemically coupled to the anti-CD33 human chimeric antibody HuM195. The CD33 antigen is of significant interest as a target for therapy of acute myelogenous leukemia because it is present in leukemic blasts of most patients but absent in the earliest progenitor bone marrow cells. HuM195/rGel was optimally cytotoxic to acute myelogenous leukemia HL60 cells with 24 hours of exposure. We developed an in vivo purging model by mixing mobilized peripheral blood progenitor cells with HL60 cells to simulate a remission in bone marrow. Cells were treated with 10 nmol/L of HuM195/rGel either with or without exposure to freeze/thaw procedure, which has been reported to act synergistically with HuM195/rGel to produce cytotoxic effect. When clonogenic cell recovery rates were determined, HuM195/rGel alone did not affect normal peripheral blood progenitor cells, whereas HuM195/rGel plus freeze/thaw provided 2 logs of tumor cell elimination in our purging model. We also observed similar results under conditions used in the transplantation setting. We concluded that for acute myelogenous leukemia blasts expressing CD33, HuM195/rGel could be useful as a purging reagent for autologous transplantation.

In vitro activity of dimethylarsinic acid against human leukemia and multiple myeloma cell lines.

PURPOSE: Arsenic trioxide (As(2)O(3)), an inorganic arsenic compound, has recently been approved for the treatment of relapsed or refractory acute promyelocytic leukemia. However, systemic toxicity associated with As(2)O(3) treatment remains a problem. Inorganic arsenic is detoxified in vivo by methylation reactions into organic arsenic compounds that are less toxic. METHODS AND RESULTS: We investigated the antiproliferative and cytotoxic activity of dimethylarsinic acid (DMAA), an organic arsenic derivative and major metabolic by-product of As(2)O(3), against a panel of eight leukemia and multiple myeloma cell lines. As(2)O(3) was tested in comparison. In clonogenic assay, the average concentration of DMAA that suppressed cell colony growth by 50% was 0.5-1 m M, while for As(2)O(3) it was on average 1-2 microM. At those concentrations DMAA and As(2)O(3) had significantly less effect on colony growth of normal progenitor cells. Cytotoxic doses of DMAA and As(2)O(3) in 3-day trypan blue dye exclusion assay experiments were similar to doses effective in clonogenic assay. Assessment of apoptosis by annexin V assay revealed a high rate of apoptosis in all cell lines treated with DMAA and As(2)O(3), but significantly less effect on normal progenitor cells. DMAA, unlike As(2)O(3), had no effect on the maturation of leukemic cells. CONCLUSIONS: DMAA exerts differential antiproliferative and cytotoxic activity against leukemia and multiple myeloma cells, with no significant effect on normal progenitor cells. However, concentrations of DMAA needed to achieve such efficacy are up to 1000 times those of As(2)O(3). Evaluation of novel organic arsenic that would combine the high efficacy of As(2)O(3) and the low toxicity of DMAA is warranted.