Additional file 1: of Identification of genetic variants for clinical management of familial colorectal tumors

Table S1. Primers used in the pCAS2 minigene splicing assay. (DOCX 15 kb

Additional file 1: of Genetic variants of prospectively demonstrated phenocopies in BRCA1/2 kindreds

The concentration in a 10 ml PCR was 1xThermopol Reaction Buffer with 2 mM MgS04, 0.3 μM “reverse” primers, 0.15 μM “forward” primer, 0.1 μM, 6-Carboxyfluorescein-GC clamp primer, 600 μM dNTP, 100 μg Bovine Serum Albumine (Sigma-Aldrich, Oslo, Norway) and 0.75 U Taq DNA polymerase. Plates were sealed with two strips of electrical tape (Clas Ohlson, Oslo, Norway). The temperature cycling was repeated 35 times; 94 °C for 30 s, annealing temperature held for 30 s and extension at 72 °C for 60 s (Eppendorf Mastercycler ep gradient S (Eppendorf, Hamburg, Germany)). Table S1. primers used to amplify PCR product to be analysed by cycling temperature capillary electrophoresis. (DOCX 16 kb

<i>ERCC2</i> allele frequencies (%) in BC/OC patients and corresponding control cohorts.

<p>The allele frequency is counted on the basis of sample size (in brackets) and number of observed cases (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006248#pgen.1006248.t001" target="_blank">Table 1</a>) with hetero- and homozygosity.</p

Domain structure and modeling of the ERCC2 mutations.

<p>(A) Mutations in the XPD/ERCC2 protein domains. The diagram shows the ERCC2 protein with the four XPD domains shown as HD1 (blue), HD2 (green), FeS (Orange) and Arch (purple). The human enzyme has a C-terminal (grey) extension (CTE) that probably forms an interaction surface with the p44 protein. Disease-relevant <i>ERCC2</i> mutation sites are indicated in boxes (blue or red frame: missense or truncating mutation, respectively; fillings: light-gray, cases with breast cancer (BC); pink, case with ovarian cancer only (OC); dark-gray: cases with either breast- or ovarian cancer (BC or OC); dark-green, patients with both breast- and ovarian cancer (BC + OC)). Numbers in brackets indicate recurrent mutations. (B) Structural placement of mutations on a C-alpha trace model of human ERCC2. The residues targeted by HBOC-causing mutations are represented as space-filled red spheres. Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) disease causing mutations sites as reported in ClinVar are shown in yellow and black spheres. Missense variants at residue position 423, 461, 487, 568, 461 and 722 have been found in both BC/OC as well as XP (red-yellow spheres) and TTD (red-black spheres) patients.</p

<i>ERCC2</i> frameshift mutation c.1703_1704delTT (p.Phe568fs) in familial breast and ovarian cancer pedigrees.

<p>Individuals with breast cancer (BC), ovarian cancer (OC) or both (BC, OC) are shown as circles filled in black. Individuals tested positive for the familial mutation are indicated in detail; those with WT (wild-type) have been tested negative. All affected individuals with BC or OC not tested for germline mutations in ERCC2 were either deceased or refused testing. (A) German, (B) Lithuanian and (C-E) Czech pedigrees.</p

Nucleotide excision repair (NER) capacity and Transcriptional activity of breast cancer associated XPD/ERCC2 variants.

<p>(A) Several XPD/ERCC2 variants cloned into an expression vector were analyzed regarding to complementation of <i>ERCC2</i>-defective XP6BE cells overexpressing the NER-deficient R601W XPD mutant [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006248#pgen.1006248.ref015" target="_blank">15</a>] (normalization for overexpression artifacts). Black bars indicate the mean relative repair capacity (in %, WT-XPD was set to 100%) of an UV irradiated firefly luciferase reporter gene plasmid (UVC 1000 J/m²) obtained by host cell reactivation (n>6 in triplicates). Red lines mark the range between DNA-repair levels of empty vector, i.e. residual repair activity of the cells, and WT-XPD, i.e. 100% repair capacity. (B) Dominant modulation of firefly luciferase reporter gene expression (without irradiation) via overexpression of XPD/ERCC2 BC/OC-associated variants was estimated in the transcriptionally-proficient but repair-deficient XPD/ERCC2-defective XP6BE cells. Black bars indicate the mean relative reporter gene expression (in %, empty vector control was set to 100%), obtained by CMV-promotor driven basal transcription (n>6 in triplicates). Error bars indicate the standard error of the mean. Significance levels were calculated, after pairwise testing for normal distribution of the values, using appropriate statistical tests for comparison of two groups (T-Test or U-Test, # = reference group, *** = p<0.001, ** = p<0.01, * = p<0.05, n.s. = not significant). (C) Additional characteristics of the mutations tested for repair efficiency and transcriptional activity.</p

Additional file 1 of Colorectal cancer incidences in Lynch syndrome: a comparison of results from the prospective lynch syndrome database and the international mismatch repair consortium

Additional file 1

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.</p