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To provide precise age-specific risk estimates of cancers other than female breast and ovarian cancers associated with pathogenic variants (PVs) inWe used data from 3,184In addition to female breast and ovarian cancers

Background Gene panel testing by massive parallel sequencing has increased the diagnostic yield but also the number of variants of uncertain significance. Clinical interpretation of genomic data requires expertise for each gene and disease. Heterozygous ATM pathogenic variants increase the risk of cancer, particularly breast cancer. For this reason, ATM is included in most hereditary cancer panels. It is a large gene, showing a high number of variants, most of them of uncertain significance. Hence, we initiated a collaborative effort to improve and standardize variant classification for the ATM gene. Methods Six independent laboratories collected information from 766 ATM variant carriers harboring 283 different variants. Data were submitted in a consensus template form, variant nomenclature and clinical information were curated, and monthly team conferences were established to review and adapt American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria to ATM, which were used to classify 50 representative variants. Results Amid 283 different variants, 99 appeared more than once, 35 had differences in classification among laboratories. Refinement of ACMG/AMP criteria to ATM involved specification for twenty-one criteria and adjustment of strength for fourteen others. Afterwards, 50 variants carried by 254 index cases were classified with the established framework resulting in a consensus classification for all of them and a reduction in the number of variants of uncertain significance from 58% to 42%. Conclusions Our results highlight the relevance of data sharing and data curation by multidisciplinary experts to achieve improved variant classification that will eventually improve clinical management., FEDER funds-a way to build Europe PI19/00553 PI16/00563 PI16/01898 SAF2015-68016-R, Generalitat de Catalunya 2017SGR1282 2017SGR496, CERCA Program: Government of Catalonia, Xunta de Galicia, Instituto de Salud Carlos III. AES PI19/00340, Spanish Government SAF2016-80255-R, European Commission EFA086/15, Instituto de Salud Carlos III European Commission

© 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc. The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early-onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5′ noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency < 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.-287C>T and PAX5 binding to BRCA2:c.-296C>T. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC.

BACKGROUND: BRCA1 and BRCA2 are the two principal tumour suppressor genes associated with inherited high risk of breast and ovarian cancer. Genetic testing of BRCA1/2 will often reveal one or more sequence variants of uncertain clinical significance, some of which may affect normal splicing patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation or defects in gene function. To understand which novel splicing events are associated with splicing mutations and which are part of the normal BRCA2 splicing repertoire, a study was undertaken by members of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium to characterise the spectrum of naturally occurring BRCA2 mRNA alternate-splicing events.METHODS: mRNA was prepared from several blood and breast tissue-derived cells and cell lines by contributing ENIGMA laboratories. cDNA representing BRCA2 alternate splice sites was amplified and visualised using capillary or agarose gel electrophoresis, followed by sequencing.RESULTS: We demonstrate the existence of 24 different BRCA2 mRNA alternate-splicing events in lymphoblastoid cell lines and both breast cancer and non-cancerous breast cell lines.CONCLUSIONS: These naturally occurring alternate-splicing events contribute to the array of cDNA fragments that may be seen in assays for mutation-associated splicing defects. Caution must be observed in assigning alternate-splicing events to potential splicing mutations.

For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies.

A recent analysis using family history weighting and co-observation classification modeling indicated that BRCA1 c.594-2A>C (IVS9-2A>C), previously described to cause exon 10 skipping (a truncating alteration), displays characteristics inconsistent with those of a high risk pathogenic BRCA1 variant. We used large-scale genetic and clinical resources from the ENIGMA, CIMBA and BCAC consortia to assess pathogenicity of c.594-2A>C. The combined odds for causality considering case-control, segregation, and breast tumor pathology information was 3.23x10-8. Our data indicate that c.594-2A>C is always in cis with c.641A>G.The spliceogenic effect of c.[594-2A>C;641A>G] was characterized using RNA analysis of human samples and splicing minigenes. As expected, c.[594-2A>C; 641A>G] caused exon 10 skipping, albeit not due to c.594-2A>C impairing the acceptor site but rather by c.641A>G modifying exon 10 splicing regulatory element(s). Multiple blood-based RNA assays indicated that the variant allele did not produce detectable levels of full-length transcripts, with a per allele BRCA1 expression profile comprised of ?70-80% truncating transcripts, and ?20-30% of in-frame ?9,10 transcripts predicted to encode a BRCA1 protein with tumor suppression function.We confirm that BRCA1c.[594-2A>C;641A>G] should not be considered a high-risk pathogenic variant. Importantly, results from our detailed mRNA analysis suggest that BRCA-associated cancer risk is likely not markedly increased for individuals who carry a truncating variant in BRCA1 exons 9 or 10, or any other BRCA1 allele that permits 20-30% of tumor suppressor function. More generally, our findings highlight the importance of assessing naturally occurring alternative splicing for clinical evaluation of variants in disease-causing genes.

Loss-of-function germline mutations in BRCA1 (MIM #113705) confer markedly increased risk of breast and ovarian cancer. The full-length transcript codifies for a protein involved in DNA repair pathways and cell-cycle checkpoints. Several BRCA1 splicing isoforms have been described in public domain databases, but the physiological role (if any) of BRCA1 alternative splicing remains to be established. An accurate description of 'naturally occurring' alternative splicing at this locus is a prerequisite to understand its biological significance. However, a systematic analysis of alternative splicing at the BRCA1 locus is yet to be conducted. Here, the Evidence-Based Network for the Interpretation of Germ-Line Mutant Alleles consortium combines RT-PCR, exon scanning, cloning, sequencing and relative semi-quantification to describe naturally occurring BRCA1 alternative splicing with unprecedented resolution. The study has been conducted in blood-related RNA sources, commonly used for clinical splicing assays, as well as in one healthy breast tissue. We have characterized a total of 63 BRCA1 alternative splicing events, including 35 novel findings. A minimum of 10 splicing events (Delta 1Aq, Delta 5, Delta 5q, Delta 8p, Delta 9, Delta(9,10), Delta 9_11, Delta 11q, Delta 13p and Delta 14p) represent a substantial fraction of the full-length expression level (ranging from 5 to 100%). Remarkably, our data indicate that BRCA1 alternative splicing is similar in blood and breast, a finding supporting the clinical relevance of blood-based in vitro splicing assays. Overall, our data suggest an alternative splicing model in which most non-mutually exclusive alternative splicing events are randomly combined into individual mRNA molecules to produce hundreds of different BRCA1 isoforms.

In the Iberian Peninsula, which includes mainly Spain and Portugal, large genomic rearrangements (LGRs) of BRCA1 and BRCA2 have respectively been found in up to 2.33% and 8.4% of families with hereditary breast and/or ovarian cancer (HBOC) that lack point mutations and small indels. In Galicia (Northwest Spain), the spectrum and frequency of BRCA1/ BRCA2 point mutations differs from the rest of the Iberian populations. However, to date there are no Galician frequency reports of BRCA1/BRCA2 LGRs. Here we used multiplex ligation-dependent probe amplification (MLPA) to screen 651 Galician index cases (out of the 830 individuals referred for genetic analysis) without point mutations or small indels. We identified three different BRCA1 LGRs in four families. Two of them have been previously classified as pathogenic LGRs: the complete deletion of BRCA1 (identified in two unrelated families) and the deletion of exons 1 to 13. We also identified the duplication of exons 1 and 2 that is a LGR with unknown pathogenicity. Determination of the breakpoints of the BRCA1 LGRs using CNV/SNP arrays and sequencing identified them as NG_005905.2:g.70536_180359del, NG_005905.2:g.90012_97270dup, and NC_000017.10:g.41230935_41399840delinsAluSx1, respectively; previous observations of BRCA1 exon1-24del, exon1-2dup, and exon1-13del LGRs have not characterized them in such detail. All the BRCA1 LGRs arose from unequal homologous recombination events involving Alu elements. We also detected, by sequencing, one BRCA2 LGR, the Portuguese founder mutation c.156_157insAluYa5. The low frequency of BRCA1 LGRs within BRCA1 mutation carriers in Galicia (2.34%, 95% CI: 0.61–7.22) seems to differ from the Spanish population (9.93%, 95% CI: 6.76– 14.27, P-value = 0.013) and from the rest of the Iberian population (9.76%, 95% CI: 6.69–13.94, P-value = 0.014). This work was supported by grants to AV from the Xunta de Galicia (10 PXIB 9101 297 PR) and the Fundación Mutua Madrileña. LF is supported by the Isabel Barreto programme of the Xunta de Galicia and the European Social Fund SI

Splicing assays are commonly undertaken in the clinical setting to assess the clinical relevance of sequence variants in disease predisposition genes. A 5-tier classification system incorporating both bioinformatic and splicing assay information was previously proposed as a method to provide consistent clinical classification of such variants. Members of the ENIGMA Consortium Splicing Working Group undertook a study to assess the applicability of the scheme to published assay results, and the consistency of classifications across multiple reviewers. Splicing assay data were identified for 235 BRCA1 and 176 BRCA2 unique variants, from 77 publications. At least six independent reviewers from research and/or clinical settings comprehensively examined splicing assay methods and data reported for 22 variant assays of 21 variants in four publications, and classified the variants using the 5-tier classification scheme. Inconsistencies in variant classification occurred between reviewers for 17 of the variant assays. These could be attributed to a combination of ambiguity in presentation of the classification criteria, differences in interpretation of the data provided, nonstandardized reporting of results, and the lack of quantitative data for the aberrant transcripts. We propose suggestions for minimum reporting guidelines for splicing assays, and improvements to the 5-tier splicing classification system to allow future evaluation of its performance as a clinical tool.

Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of collaboration between laboratories, and across disciplines, to collate and interpret information from clinical testing laboratories to consolidate patient management.

Biallelic inactivation of ATM gene causes the rare autosomal recessive disorder Ataxia-telangiectasia (A-T). Female relatives of A-T patients have a two-fold higher risk of developing breast cancer (BC) compared with the general population. ATM mutation carrier identification is laborious and expensive, therefore, a more rapid and directed strategy for ATM mutation profiling is needed. We designed a case-control study to determine the prevalence of 32 known ATM mutations causing A-T in Spanish population in 323 BRCA1/BRCA2 negative hereditary breast cancer (HBC) cases and 625 matched Spanish controls. For the detection of the 32 ATM mutations we used the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique. We identified one patient carrier of the c.8264_8268delATAAG ATM mutation. This mutation was not found in the 625 controls. These results suggest a low frequency of these 32 A-T causing mutations in the HBC cases in our population. Further case-control studies analyzing the entire coding and flanking sequences of the ATM gene are warranted in Spanish BC patients to know its implication in BC predisposition.

Last decade, the detection of the CHEK2 c.1100delC mutation allowed for the identification of CHEK2 as a lowpenetrance breast cancer (BC) susceptibility gene [1]. Since then, numerous articles have been published regarding c.1100delC frequency among European hereditary breast cancer (HBC) patients negative for BRCA1/2 (summarized in Desrichard et al. [2]). It seems that c.1100delC shows a Northwest–Southeast frequency gradient in Europe (Fig. 1), but inconsistent results have been reported in Spain. Thus, Osorio et al. [3] fail to detect this mutation in a Spanish cohort comprising 400 non-BRCA1/2 HBC cases and 400 selected controls. On the contrary, Martinez-Bouzas and colleagues [4] detect a 0.93 % prevalence among 214 Basque Country (North Spain) HBC non-BRCA1/2 patients. Similarly, Gutierrez-Enriquez and colleagues [5] report 0.3 % frequency in 331 non-BRCA1/2 HBC families from Basque Country and Catalonia (Northeast Spain). Multiplex ligation-dependent probe amplification (MLPA) routine assays for BRCA1/2 large genomic rearrangements (LGRS), which includes a probe for CHEK2 c.1100delC mutation, led us to identify two (0.36 %) c.1100delC unrelated heterozygous carriers out of 552 Galician HBC families without BRCA1/2 point mutations. As there were no reports on CHEK2 c.1100delC mutation in our population, we screened 1,121 Galician individuals without personal history of cancer, and found one carrier (1/1,121; 0.09 %). Differences between cases and controls were not statistically significant (Fisher exact test p value = 0.23). To our knowledge, this is the largest c.1100delC mutation screening study performed on a Spanish cohort. Our study shows that CHEK2 c.1100delC is present in Galician (Northwest Spain) population and shed light into its distribution in Spanish non-BRCA1/2 HBC patients, pointing to a population substructure for CHEK2, as previously described for other genes [6–8]. Recent reports on non-BRCA1/2 HBC families calculate that c.1100delC heterozygous and homozygous carriers’ odds ratio for BC is 3.29–4.04 [2, 9] and 101.34 [9], respectively. Lifetime BC risk for carriers with an affected first-degree relative has been estimated to be 25 %, but might be higher if the woman has multiple affected relatives [10]. For a carrier with unilateral breast cancer, the risk of a second primary breast cancer is 1 % per year [10]. Moreover, it seems that this mutation is a predictor of adverse prognosis and poor survival (reviewed in Narod et al. [10]). Given this, it is highly recommended to include the screening of CHEK2 c.1100delC mutation in HBC families from populations where this mutation has been described [10]. In our study, we demonstrate the presence of c.1100delC mutation in Galician (Northwest Spain) HBC families, and confirmed the utility of genotyping c.1100delC during the routine screening for BRCA1/2 LGRs, as proposed for CHEK2 low-frequency populations [5]. L. Fachal A. Blanco A. Carracedo A. Vega (&) Fundacion Publica Galega de Medicina Xenomica-SERGAS, Grupo de Medicina Xenomica-USC, CIBERER, IDIS, Santiago de Compostela, Spain e-mail: ana.vega@usc.es

This work investigated the location on the parasite of Trichinella antigens recognized by the mouse immune system and the question as to which of them bear the epitope phosphorylcholine (PC). Wheatley's trichrome stain (initially developed for faecal smears) proved to be excellent for visualization of Trichinella structures, enabling four types of stichocyte to be distinguished. By applying this stain on infected muscle sections after immunocytochemistry using (a) anti-PC BH8 monoclonal antibodies, (b) serum from mice that had been infected twice in the presence of 0.05% thiabendazole (to prevent reproduction by adult females) and then bled on day 7 post-reinfection, (c) serum from infected mice that were bled on day 14 postinfection, or (d) serum from infected mice that were bled on day 42 postinfection, we found (1) that PC is an abundant structural epitope on the hypodermis/muscle, genital primordium and intestinal tract but is absent from the cuticle and stichosome; (2) that the principle secretory cells of adult worms are delta- and beta-stichocytes, whereas those of migrating and encysted L1 larvae are alpha-stichocytes; and (3) that Trichinella antigens recognized in the encysted phase of the parasite's life cycle are present in parasitized myofibres in the sarcoplasmic matrix and in the nucleoplasm of hypertrophic nuclei. The significance of these findings is discussed.

These investigations show the immunosuppressive effects of Clofibrate (CPIB) and hydrocortisone on the intestinal cycle of Trichinella nelsoni. They demonstrate an effect of CPIB similar to that of hydrocortisone in the establishment of the muscular and intestinal phase of this parasite. A higher retention rate of intestinal adult worms was observed in hydrocortisone-treated mice. These facts are consistent with the idea that CPIB can act on diverse immune cells, involving both thymus-dependent and thymus-independent antigens.

Phosphorylcholine (PC), an immunodominant component of the cell wall of certain bacteria, fungi and nematodes, is known to induce low anti-PC antibody levels during natural infection byTrichinella spiralis. This article reports a study in which spleen cells from BCF1 mice infected withTrichinellasp. larvae were found to produce large numbers of direct haemolytic plaques in response to PC conjugated to sheep red blood cells (SRBC) after muscle-encysted larvae had been killed by treatment with mebendazole. Inhibition of the response by PC-chloride, immunodiffusion and immunoelectrophoretic studies with the anti-PC IgA (TEPC-15) and anti-idiotype T15 serum assays showed the plaque-forming cell (PFC) response to be specific for PC. The absence of haemolytic plaques when unconjugated SRBC or TNP-SRBC were used as indicator cells ruled out involvement of a polyclonal response. Greatest anti-PC PFC response was found to be associated with a microsomal fraction designated FCpl, a particulate fraction behaving as a thymus-dependent antigen. The FCpl fractions from all four strains ofTrichinellaemployed induced anti-PC PFC responses when injected into mice. These results suggest that FCpl is a suitable antigen for use in detailed studies of immune responses toTrichinellaand related parasites.