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In this report we described the case of a BRCA1/2 (BRCA) molecular testing performed on tumor sample in a High Grade Serous Ovarian Cancer (HGSOC) patient with two different Next Generation Tumor Sequencing (NGTS) pipelines. The two clinical reports leaded to apparently different BRCA status, providing important foods for thought. After NGTS, the gene sequencing information (i.e., reads) are aligned to the reference gene sequences obtained from public databases, in order to provide an uniform nomenclature for unambiguous variant designation. However, the criteria adopted for variant reporting in tissue test are not always univocal. Particularly, this is the case of rare and unclassified BRCA variants for which the molecular evaluation may be a relevant challenge. Here we described a BRCA1 unclassified variant that may be re-evaluated in the context of alternative BRCA1 transcripts due to its different biological effect. We underlined that an in-depth knowledge of BRCA testing is mandatory for its appropriate use.

Clinical mutation screening of the cancer susceptibility genes BRCA1 and BRCA2 generates many unclassified variants (UVs). Most of these UVs are either rare missense substitutions or nucleotide substitutions near the splice junctions of the protein coding exons. Previously, we developed a quantitative method for evaluation of BRCA gene UVs—the “integrated evaluation”—that combines a sequence analysis‐based prior probability of pathogenicity with patient and/or tumor observational data to arrive at a posterior probability of pathogenicity. One limitation of the sequence analysis‐based prior has been that it evaluates UVs from the perspective of missense substitution severity but not probability to disrupt normal mRNA splicing. Here, we calibrated output from the splice‐site fitness program MaxEntScan to generate spliceogenicity‐based prior probabilities of pathogenicity for BRCA gene variants; these range from 0.97 for variants with high probability to damage a donor or acceptor to 0.02 for exonic variants that do not impact a splice junction and are unlikely to create a de novo donor. We created a database http://priors.hci.utah.edu/PRIORS/ that provides the combined missense substitution severity and spliceogenicity‐based probability of pathogenicity for BRCA gene single‐nucleotide substitutions. We also updated the BRCA gene Ex‐UV LOVD, available at http://hci‐exlovd.hci.utah.edu, with 77 re‐evaluable variants.

A survey of viruses affecting grapevine in the wine regions of the Western Cape Province in South Africa was conducted. The survey determined the relative abundance of five different Grapevine leafroll-associated virus 3 (GLRaV-3) variants. Virus profiles were also determined for individual vines. A total of 315 plants were sampled and analysed over two growing seasons. Five GLRaV-3 variants were detected as either single or mixed infections, with GLRaV-3 variant groups II and VI being the most prominent as single infections and in combinations with each other and other variants. An analysis of the distribution of variants per region showed that single infections of variant groups II and VI occurred predominantly in certain regions, and were equally distributed in the red and white cultivars studied. The distribution of a recently identified, unclassified variant of GLRaV-3 (represented by isolates GH24 and GTG10) was included in the study. The overall analysis showed that infection with variant groups II and VI were the most abundant among the samples with 49.8 and 47.6 %, respectively, followed by variant group I, variants similar to isolate GH24 and variant group III with 16.2, 13.3 and 2.5 % infection, respectively. Mixed infections, representing 36 virus combinations, were found in 251 plants. The most abundant virus combination was GLRaV-3 with Grapevine virus E (GVE), found in 28 % of the plants. GLRaV-3 was the predominant virus detected in the samples with a frequency of 80 % detection, followed by GVE (57.4 %), Grapevine rupestris stem pitting-associated virus (GRSPaV) (36.8 %), Grapevine virus A (GVA) (19.3 %), Grapevine virus F (GVF) (16.25 %), Grapevine leafroll-associated virus 2 (GLRaV-2) (8.25 %), Grapevine leafroll-associated virus 1 (GLRaV-1) (1.58 % infection) and Grapevine leafroll-associated virus 4 (GLRaV-4 like) (0.6 %). Most of the plants tested were infected with multiple viruses. The complexity of virus populations detected in this study, highlights the need for detection methods able to identify all viruses and their variants in vineyards. The information generated in this study will assist in the development of reliable detection assays that will benefit the monitoring of disease spread and aid in the efficient management of Grapevine leafroll disease (GLD).

Unclassified variants (UV) of BRCA1 can affect normal pre-mRNA splicing. Here, we investigate the UV c.693G>A, a “silent” change in BRCA1 exon 11, which we have found induces aberrant splicing in patient carriers and in vitro. Using a minigene assay, we show that the UV c.693G>A has a strong effect on the splicing isoform ratio of BRCA1. Systematic site-directed mutagenesis of the area surrounding the nucleotide position c.693G>A induced variable changes in the level of exon 11 inclusion/exclusion in the mRNA, pointing to the presence of a complex regulatory element with overlapping enhancer and silencer functions. Accordingly, protein binding analysis in the region detected several splicing regulatory factors involved, including SRSF1, SRSF6 and SRSF9, suggesting that this sequence represents a composite regulatory element of splicing (CERES).

Variants that disrupt the translation initiation sequences in cancer predisposition genes are generally assumed to be deleterious. However, few studies have validated these assumptions with functional and clinical data. Two cancer syndrome gene variants likely to affect native translation initiation were identified by clinical genetic testing: MLH1:c.1A>G p.(Met1?) and BRCA2:c.67+3A>G. In vitro GFP-reporter assays were conducted to assess the consequences of translation initiation disruption on alternative downstream initiation codon usage. Analysis of MLH1:c.1A>G p.(Met1?) showed that translation was mostly initiated at an in-frame position 103 nucleotides downstream, but also at two ATG sequences downstream. The protein product encoded by the in-frame transcript initiating from position c.103 showed loss of in vitro mismatch repair activity comparable to known pathogenic mutations. BRCA2:c.67+3A>G was shown by mRNA analysis to result in an aberrantly spliced transcript deleting exon 2 and the consensus ATG site. In the absence of exon 2, translation initiated mostly at an out-of-frame ATG 323 nucleotides downstream, and to a lesser extent at an in-frame ATG 370 nucleotides downstream. Initiation from any of the downstream alternative sites tested in both genes would lead to loss of protein function, but further clinical data is required to confirm if these variants are associated with a high cancer risk. Importantly, our results highlight the need for caution in interpreting the functional and clinical consequences of variation that leads to disruption of the initiation codon, since translation may not necessarily occur from the first downstream alternative start site, or from a single alternative start site.

We propose an addition to the Snosek et al. classification to include a subtype variant of sternalis muscle: mixed type and triple subtype. Dissection of the anterior thorax of a 96-year-old female cadaver revealed bilateral sternalis muscles with an undocumented variant of the right sternalis muscle. The left sternalis muscle presented as a simple type—left single using the Snosek et al. classification scheme. The right sternalis muscle revealed a previously undocumented classification type. It consisted of three bellies and two heads, with the lateral head formed by two converging bellies and the medial head formed from the superficial medial belly. The unique presentation of right sternalis muscle can be classified by expanding the Snosek et al. classification scheme to include triple-bellied subtypes. This presentation is classified as a mixed type—right triple, with single bicipital converging and single bicipital diverging. Documentation of sternalis muscle variations can prevent misdiagnoses within the anterior thorax.

CHARGE syndrome is a rare autosomal dominant disease that is typically caused by heterozygous CHD7 mutations. A de novo variant in a CHD7 splicing acceptor site (NM_017780.3:c.7165-4A>G) was identified in a Japanese boy with CHARGE syndrome. This variant has been considered to be an "unclassified variant" due to its position outside the consensus splicing sites. In this study, abnormal splicing derived from this known variant was confirmed by cDNA sequencing.

Introduction A sleeve fracture classically describes an avulsion of cartilage or periosteum with or without osseous fragments and usually occurs at the inferior margin of the patella. Tibial tubercle sleeve fractures in the skeletally immature are extremely rare. Case Presentation In this report the authors describe a 12-year-old boy with no systemic disease and no steroid use who sustained bilateral proximal tibial sleeve fractures whilst playing football. Both ruptures were associated with rupture of the medial patellofemoral ligament and tear of the medial retinaculum. Treatment was performed with primary end-to-end repair, reinforcement with bone anchors and cerclage wires with an excellent outcome. Conclusions We feel this rare, currently unclassified variant of a tibial tubercle avulsion fracture should be recognised and consideration taken to adding it to existing classification systems.

A 23-year-old woman with metastatic phaeochromocytoma was found to have a previously unclassified variant in the von Hippel Lindau disease gene (c.361G>C). We use this case to highlight the issue of unclassified single nucleotide variants and the approaches to help predict whether they are disease causing or neutral. With increasing use of genetic testing, and widespread clinical use of next-generation sequencing around the corner, this issue is likely to become more prominent.

International audience; With the increased number of identified nucleotide sequence variations in genes, the current challenge is to classify them as disease causing or neutral. These variants of unknown clinical significance can alter multiple processes, from gene transcription to RNA splicing or protein function. Using an approach combining several in silico tools, we identified some exons presenting weaker splicing motifs than other exons in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. These exons exhibit higher rates of basal skipping than exons harboring no identifiable weak splicing signals using minigene assays. We then screened 19 described mutations in three different exons, and identified exon-skipping substitutions. These substitutions induced higher skipping levels in exons having one or more weak splicing motifs. Indeed, this level remained under 2% for exons with strong splicing motifs and could reach 40% for exons having at least one weak motif. Further analysis revealed a functional exon splicing enhancer within exon 3 that was associated with the SR protein SF2/ASF and whose disruption induced exon skipping. Exon skipping was confirmed in vivo in two nasal epithelial cell brushing samples. Our approach, which point out exons with some splicing signals weaknesses, will help spot splicing mutations of clinical relevance.

After completion of Human Genome Project (HGP) in 2003, as well as the new technology development in genomic research, the most accurate genetics blueprint of human is available. Researchers started to dissect and understand the genetic map of the human species. As a consequence, analyses of novel or unclassified genetic variations become increasingly important in translational medicine. One of the medical specialties in modern medicine is clinical genetics, which is overseen by the American Board of Medical Genetics (ABMG). In 2008, ABMG published a guideline for interpretation of new variants using ACMG Standards and Guidelines (Richards et al. Genet Med 10:294-300, 2008). In this chapter, we provide updated procedures of evaluating different databases, computational tools, and structural analysis methods that we currently utilize to assist in clinical interpretation.

As genetic testing for predisposition to human diseases has become an increasingly common practice in medicine, the need for clear interpretation of the test results is apparent. However, for many disease genes, including the breast cancer susceptibility genes BRCA1 and BRCA2, a significant fraction of tests results in the detection of a genetic variant for which disease association is not known. The finding of an "unclassified" variant (UV)/variant of uncertain significance (VUS) complicates genetic test reporting and counseling. As these variants are individually rare, a large collaboration of researchers and clinicians will facilitate studies to assess their association with cancer predisposition. It was with this in mind that the ENIGMA consortium (www.enigmaconsortium.org) was initiated in 2009. The membership is both international and interdisciplinary, and currently includes more than 100 research scientists and clinicians from 19 countries. Within ENIGMA, there are presently six working groups focused on the following topics: analysis, clinical, database, functional, tumor histopathology, and mRNA splicing. ENIGMA provides a mechanism to pool resources, exchange methods and data, and coordinately develop and apply algorithms for classification of variants in BRCA1 and BRCA2. It is envisaged that the research and clinical application of models developed by ENIGMA will be relevant to the interpretation of sequence variants in other disease genes. Hum Mutat 00:1-6, 2011. © 2011 Wiley Periodicals, Inc.

International audience; Several unclassified variants (UV) of can be deleterious by affecting normal pre-mRNA splicing. Here, we investigated the consequences at the mRNA level of the frequently encountered c.5242C>A UV in exon 18. We show that the c.5242C>A variant induces skipping of exon 18 in UV carriers and in vitro. This alteration predicted to disrupt the first BRCT domain of . We show that two splicing repressors, hnRNP A1 and hnRNP H/F, display a significant preference toward binding with the mutated exon 18 and assemble into a protein complex. Sequence analysis of the region surrounding the c.5242C>A change reveals the presence of hnRNP A1 and hnRNP H/F binding sites, which are modified by several UVs. Mutation of these sites alters the RNA binding ability of both splicing regulators. In conclusion, our work supports the model of the pathogenicity of the c.5242C>A variant that induces exon skipping by creating a sequence with silencer properties. We propose that other UVs in exon 18 interfere with splicing complex assembly by perturbing the binding of hnRNP A1 and hnRNP H/F to their respective -elements. RNA analysis is therefore necessary for the assessment of the consequences of UVs on splicing of disease-associated genes and to enable adequate genetic counseling for breast/ovarian cancer families.

Inherited predisposition to disease is often linked to reduced activity of a disease associated gene product. Thus, quantitation of the influence of inherited variants on gene function can potentially be used to predict the disease relevance of these variants. While many disease genes have been extensively characterized at the functional level, few assays based Oil functional properties of the encoded proteins have been established for the purpose of predicting the contribution of rare inherited variants to disease. Much of the difficulty in establishing predictive functional assays stems from the technical complexity of the assays. However, perhaps the most challenging aspect of functional assay development for Clinical testing, purposes is the absolute requirement for validation of the sensitivity and specificity of the assays and the determination of positive predictive values (PPVs) and negative predictive values (NPVs) of the assays relative to a "gold standard" measure of disease predisposition. In this commentary, we provide examples of some of the functional assays tinder development for several cancer predisposition genes (BRCA1, BRCA2, CDKN2A, and mismatch repair [MMR] genes MLH1, MSH2, MSH6, and PMS2) and present a detailed review of the issues associated with functional assay development. We conclude that validation is paramount for all assays that will be used for clinical interpretation of inherited variants of any gene, but note that in certain circumstances information derived from incompletely validated assays may be valuable for classification of variants for clinical purposes when used to supplement data derived front other sources. Hunt Mutat 29(11), 1314-1326, 2008. (C) 2008 Wiley-Liss, Inc.

Objective: Unclassified variants (UVs, variants of uncertain clinical significance) are found in 13% of all BRCA1/2 mutation analyses. Little is known about the counsellees' recall and interpretation of a UV, and its psychosocial/medical impact. Method: Retrospective semi-structured interviews with open questions and five-point Likert scales were carried out in 24 counsellees who received a UV result 3 years before (sd=1.9). Results: Sixty-seven percent (16/24) recalled the UV result as a non-informative DNA result; 29% recalled a pathogenic result. However, 79% of all counsellees interpreted the UV result as a genetic predisposition for cancer. Variations in recall and interpretation were unexplained by demographics, cancer history of themselves and relatives, and communication aspects of UV disclosure. Sixty-seven percent perceived genetic counselling as completed, whereas 71% expected to receive new DNA information. Although most counsellees reported that UV disclosure had changed their lives in general little, one in three counsellees reported large changes in specific life domains, especially in surveillance behavior and medical decisions. Ten out of 19 participants who interpreted the UV as pathogenic had undergone preventive surgery against none of the 5 counsellees who interpreted the UV as non-informative. Conclusion: Counsellors and researchers need to address discrepancies between the counsellees' factual recall and their subjective interpretation of non-informative BRCA1/2-test results. Copyright © 2007 John Wiley & Sons, Ltd.

Purpose To obtain and compare the probabilities of finding a mutation in the BRCA1 or BRCA2 genes, the clinical features, and the family history among patients with an unclassified variant (UV) and those with a pathogenic mutation. Patients and Methods The study included 70 patients: 24 with a UV (BRCA1, n = 4; BRCA2, n = 19; both, n = 1), and 46 with a mutation (BRCA1, n = 32; BRCA2, n = 14). Two of the UVs were novel variants; the rest had been reported previously as UVs. Probabilities of finding a mutation were retrospectively obtained using BRCAPRO and Myriad II programs. Results The probability to detect a mutation was significantly lower in the group of patients with a UV than in those with a mutation (BRCAPRO [mean ± standard deviation], 0.297 ± 0.312 v 0.627 ± 0.315, P = .001; and Myriad II, 0.124 ± 0.090 v 0.283 ± 0.176, P = .001, respectively). Independent predictive factors of finding either a UV or a mutation were number of affected relatives (2.9 ± 1.4 v 4.0 ± 1.9; P = .039) and number of tumors among relatives (3.3 ± 1.4 v 4.4 ± 1.8; P = .031), respectively. Conclusion The combined data about the predictive models show significant differences between both groups. Individual probabilities can be regarded as a help to guide the clinical management of patients with a UV in those genes. However, a definitive conclusion about the pathogenicity of a UV can not be obtained from the clinical features alone, but only in combination with biochemical and epidemiologic data.

Locus-specific databases (LSDBs) are curated collections of sequence variants in genes associated with disease. LSDBs of cancer-related genes often serve as a critical resource to researchers, diagnostic laboratories, clinicians, and others in the cancer genetics community. LSDBs are poised to play an important role in disseminating clinical classification of variants. The IARC Working Group on Unclassified Genetic Variants has proposed a new system of five classes of variants in cancer susceptibility genes. However, standards are lacking for reporting and analyzing the multiple data types that assist in classifying variants. By adhering to standards of transparency and consistency in the curation and annotation of data, LSDBs can be critical for organizing our understanding of how genetic variation relates to disease. In this article we discuss how LSDBs can accomplish these goals, using existing databases for BRCA1, BRCA2, MSH2, MLH1, TP53, and CDKN2A to illustrate the progress and remaining challenges in this field. We recommend that: 1) LSDBs should only report a conclusion related to pathogenicity if a consensus has been reached by an expert panel. 2) The system used to classify variants should be standardized. The Working Group encourages use of the five class system described in this issue by Plon and colleagues. 3) Evidence that supports a conclusion should be reported in the database, including sources and criteria used for assignment. 4) Variants should only be classified as pathogenic if more than one type of evidence has been considered. 5) All instances of all variants should be recorded. Hum Mutat 29(11), 1273-1281, 2008. Published 2008 Wiley-Liss, Inc.

Results 47 surveys were collected, representing one-third of searches performed in the data collection period. It was estimated that each laboratory encountered 150 genetic tests each year that required information on an unclassified variant to be searched for and reported on. From this the activity of searching and reporting analysed was calculated at 20 weeks’ effort across the state of Victoria each year. Differences were observed in times reported (Table 1). This was attributable to the level of automation of the two processes: Conclusion The study found that if all laboratories could benefit from the automated process, the 20 weeks’ effort to search for and report on unclassified variants found by genetic testing for familial bowel and breast cancer could be reduced to 5 weeks. This study supports the view that the development of a common repository approach would have significant economies of scale over repositories being developed by laboratories for individual diseases, particularly as genetic testing becomes more common.