The Drosophila Melanogaster RAD54 Homolog, DmRAD54, Is Involved in the Repair of Radiation Damage and Recombination

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRAD54-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X- ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Characterization of the Crumbs homolog 2 (CRB2) gene and analysis of its role in retinitis pigmentosa and Leber congenital amaurosis.

Purpose: Mutations in the Crumbs homolog 1 (CRB1) gene cause autosomal recessive retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Database searches reveal two other Crumbs homologs on chromosomes 9q33.3 and 19p13.3. The purpose of this study was to characterize the Crumbs homolog 2 (CRB2) gene on 9q33.3, to analyze its expression pattern, and to determine whether mutations in CRB2 are associated with RP and LCA. Methods: The CRB2 mRNA and its expression pattern in human tissues were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The cellular expression of Crb2 in the mouse eye was determined by mRNA in situ hybridizations. The open reading frame and splice junctions of CRB2 were analyzed for mutations by single-strand conformation analysis and direct nucleotide sequencing in 85 RP patients and 79 LCA patients. Results: The CRB2 gene consists of 13 exons and encodes a 1285 amino acid transmembrane protein. CRB2 is mainly expressed in retina, brain, and kidney. In mouse retina Crb2 expression was detected in all cell layers. Mutation analysis of the CRB2 gene revealed 11 sequence variants leading to an amino acid substitution. Three of them were not identified in control individuals and affect conserved amino acid residues. However, the patients that carry these sequence variants do not have a second sequence variant on the other allele, excluding autosomal recessive inheritance of CRB2 sequence variants as a cause of their disease. Conclusions: This study shows that CRB2 sequence variants are not a common cause of autosomal recessive RP and LCA. It is possible that a more complex clinical phenotype is associated with the loss or altered function of CRB2 in humans due to its expression in tissues other than the retina

Characterization of the Crumbs homolog 2 (CRB2) gene and analysis of its role in retinitis pigmentosa and Leber congenital amaurosis.

Purpose: Mutations in the Crumbs homolog 1 (CRB1) gene cause autosomal recessive retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Database searches reveal two other Crumbs homologs on chromosomes 9q33.3 and 19p13.3. The purpose of this study was to characterize the Crumbs homolog 2 (CRB2) gene on 9q33.3, to analyze its expression pattern, and to determine whether mutations in CRB2 are associated with RP and LCA. Methods: The CRB2 mRNA and its expression pattern in human tissues were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The cellular expression of Crb2 in the mouse eye was determined by mRNA in situ hybridizations. The open reading frame and splice junctions of CRB2 were analyzed for mutations by single-strand conformation analysis and direct nucleotide sequencing in 85 RP patients and 79 LCA patients. Results: The CRB2 gene consists of 13 exons and encodes a 1285 amino acid transmembrane protein. CRB2 is mainly expressed in retina, brain, and kidney. In mouse retina Crb2 expression was detected in all cell layers. Mutation analysis of the CRB2 gene revealed 11 sequence variants leading to an amino acid substitution. Three of them were not identified in control individuals and affect conserved amino acid residues. However, the patients that carry these sequence variants do not have a second sequence variant on the other allele, excluding autosomal recessive inheritance of CRB2 sequence variants as a cause of their disease. Conclusions: This study shows that CRB2 sequence variants are not a common cause of autosomal recessive RP and LCA. It is possible that a more complex clinical phenotype is associated with the loss or altered function of CRB2 in humans due to its expression in tissues other than the retina

Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency (CMMRD) Syndrome

Monoallelic PMS2 germline mutations cause 5-15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional MMR deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA and RNA-based strategies are applied to overcome problematic PMS2 mutation analysis due to the presence of pseudogenes and frequent gene conversion events. Here, we determined PMS2 mutation detection yield and mutation spectrum in a nationwide cohort of 396 probands. Furthermore, we studied concordance between tumor IHC/ MSI (immunohistochemistry/ microsatellite-instability) profile and mutation carrier state. Overall, we found 52 different pathogenic PMS2 variants explaining 121 Lynch syndrome and nine CMMRD patients. In vitro MMR assays suggested pathogenicity for three missense variants. Ninety-one PMS2 mutation carriers (70%) showed isolated loss of PMS2 in their tumors, for 31 (24%) no or inconclusive IHC was available, and eight carriers (6%) showed discordant IHC (presence of PMS2 or loss of both MLH1 and PMS2). Ten cases with isolated PMS2 loss (10%; 10/97) harbored MLH1 mutations. We confirmed that recently improved mutation analysis provides a high yield of PMS2 mutations in patients with isolated loss of PMS2 expression. Application of universal tumor pre-screening methods will however miss some PMS2 germline mutation carriers. This article is protected by copyright. All rights reserved

A functional assay-based procedure to classify mismatch repair gene variants in Lynch syndrome

PURPOSE: To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cell-free in vitro MMR activity (CIMRA) assay. Here, we calibrate and validate the assay, enabling its integration with in silico and clinical data. METHODS: Two sets of previously classified MLH1 and MSH2 variants were selected from a curated MMR gene database, and their biochemical activity determined by the CIMRA assay. The assay was calibrated by regression analysis followed by symmetric cross-validation and Bayesian integration with in silico predictions of pathogenicity. CIMRA assay reproducibility was assessed in four laboratories. RESULTS: Concordance between the training runs met our prespecified validation criterion. The CIMRA assay alone correctly classified 65% of variants, with only 3% discordant classification. Bayesian integration with in silico predictions of pathogenicity increased the proportion of correctly classified variants to 87%, without changing the discordance rate. Interlaboratory results were highly reproducible. CONCLUSION: The CIMRA assay accurately predicts pathogenic and benign MMR gene variants. Quantitative combination of assay results with in silico analysis correctly classified the majority of variants. Using this calibration, CIMRA assay results can be integrated into the diagnostic algorithm for MMR gene variants. KEYWORDS: Lynch syndrome; assay calibration; functional assay; variant classification; variants of uncertain significanc