Lucía Pérez-Cabornero

A High Proportion of DNA Variants of BRCA1 and BRCA2 Is Associated with Aberrant Splicing in Breast/Ovarian Cancer Patients

Purpose: Most BRCA1/2 mutations are of unknown clinical relevance. An increasing amount of evidence indicates that there can be deleterious effects through the disruption of the splicing process. We have investigated the effect of aberrant splicing of BRCA1/2 on hereditary breast/ovarian cancer (HBOC). Experimental Design: DNA variants were analyzed with splicing prediction programs to select putative splicing mutations. Splicing assays of 57 genetic variants were done by lymphocyte reverse transcription-PCR and/or hybrid minigenes in HeLa and nontumor breast epithelial cells. Results: Twenty-four BRCA1/2 variants of Spanish HBOC patients were bioinformatically preselected. Functional assays showed that 12 variants induced anomalous splicing patterns, 6 of which accounted for 58.5% of BRCA1 families. To further evaluate the defective splicing of BRCA1/2, we analyzed 31 Breast Cancer Information Core Database (BIC) and two artificial variants that were generated by mutagenesis. Sixteen variants induced different degrees of aberrant splicing. Altogether, anomalous splicing was caused by 28 BRCA1/2 variants of all types, indicating that any DNA change can disrupt pre-mRNA processing. We show that a wide range of regulatory elements can be involved, including the canonical and cryptic splice sites, the polypyrimidine tract, and splicing enhancers/silencers. Twenty mutations were predicted to truncate the BRCA proteins and/or to delete essential domains, thus supporting a role in HBOC. Conclusions: An important fraction of DNA variants of BRCA1/2 presents splicing aberrations that may represent a relevant disease-causing mechanism in HBOC. The identification of splicing disruptions by functional assays is a valuable tool to discriminate between benign polymorphisms and pathogenic mutations. Clin Cancer Res; 16(6); 1957–67

Heteroduplex analysis by capillary array electrophoresis for rapid mutation detection in large multiexon genes

Heteroduplex analysis (HA) has proven to be a robust tool for mutation detection. HA by capillary array electrophoresis (HA-CAE) was developed to increase throughput and allow the scanning of large multiexon genes in multicapillary DNA sequencers. HA-CAE is a straightforward and high-throughput technique to detect both known and novel DNA variants with a high level of sensitivity and specificity. It consists of only three steps: multiplex-PCR using fluorescently labeled primers, heteroduplex formation and electrophoresis in a multicapillary DNA sequencer. It allows, e.g., the complete coding and flanking intronic sequences of BRCA1 and BRCA2 genes from two patients (approximately 25 kb each) to be scanned in a single run of a 16-capillary sequencer, and has enabled us to detect 150 different mutations to date (both single nucleotide substitutions, or SNSs, and small insertions/deletions). Here, we describe the protocol developed in our laboratory to scan BRCA1, BRCA2, MLH1, MSH2 and MSH6 genes using an ABI3130XL sequencer. This protocol could be adapted to other instruments or to the study of other large multiexon genes and can be completed in 7–8 h.

MLH1 Founder Mutations with Moderate Penetrance in Spanish Lynch Syndrome Families

The variants c.306+5G>A and c.1865T>A (p.Leu622His) of the DNA repair gene MLH1 occur frequently in Spanish Lynch syndrome families. To understand their ancestral history and clinical effect, we performed functional assays and a penetrance analysis and studied their genetic and geographic origins. Detailed family histories were taken from 29 carrier families. Functional analysis included in silico and in vitro assays at the RNA and protein levels. Penetrance was calculated using a modified segregation analysis adjusted for ascertainment. Founder effects were evaluated by haplotype analysis. The identified MLH1 c.306+5G>A and c.1865T>A (p.Leu622His) variants are absent in control populations and segregate with the disease. Tumors from carriers of both variants show microsatellite instability and loss of expression of the MLH1 protein. The c.306+5G>A variant is a pathogenic mutation affecting mRNA processing. The c.1865T>A (p.Leu622His) variant causes defects in MLH1 expression and stability. For both mutations, the estimated penetrance is moderate (age-cumulative colorectal cancer risk by age 70 of 20.1% and 14.1% for c.306+5G>A and of 6.8% and 7.3% for c.1865T>A in men and women carriers, respectively) in the lower range of variability estimated for other pathogenic Spanish MLH1 mutations. A common haplotype was associated with each of the identified mutations, confirming their founder origin. The ages of c.306+5G>A and c.1865T>A mutations were estimated to be 53 to 122 and 12 to 22 generations, respectively. Our results confirm the pathogenicity, moderate penetrance, and founder origin of the MLH1 c.306+5G>A and c.1865T>A mutations. These findings have important implications for genetic counseling and molecular diagnosis of Lynch syndrome.

Comprehensive splicing functional analysis of DNA variants of the BRCA2 gene by hybrid minigenes.

IntroductionThe underlying pathogenic mechanism of a large fraction of DNA variants of disease-causing genes is the disruption of the splicing process. We aimed to investigate the effect on splicing of the BRCA2 variants c.8488-1G > A (exon 20) and c.9026_9030del (exon 23), as well as 41 BRCA2 variants reported in the Breast Cancer Information Core (BIC) mutation database.MethodsDNA variants were analyzed with the splicing prediction programs NNSPLICE and Human Splicing Finder. Functional analyses of candidate variants were performed by lymphocyte RT-PCR and/or hybrid minigene assays. Forty-one BIC variants of exons 19, 20, 23 and 24 were bioinformatically selected and generated by PCR-mutagenesis of the wild type minigenes.ResultsLymphocyte RT-PCR of c.8488-1G > A showed intron 19 retention and a 12-nucleotide deletion in exon 20, whereas c.9026_9030del did not show any splicing anomaly. Minigene analysis of c.8488-1G > A displayed the aforementioned aberrant isoforms but also exon 20 skipping. We further evaluated the splicing outcomes of 41 variants of four BRCA2 exons by minigene analysis. Eighteen variants presented splicing aberrations. Most variants (78.9%) disrupted the natural splice sites, whereas four altered putative enhancers/silencers and had a weak effect. Fluorescent RT-PCR of minigenes accurately detected 14 RNA isoforms generated by cryptic site usage, exon skipping and intron retention events. Fourteen variants showed total splicing disruptions and were predicted to truncate or eliminate essential domains of BRCA2.ConclusionsA relevant proportion of BRCA2 variants are correlated with splicing disruptions, indicating that RNA analysis is a valuable tool to assess the pathogenicity of a particular DNA change. The minigene system is a straightforward and robust approach to detect variants with an impact on splicing and contributes to a better knowledge of this gene expression step.

Frequency of rearrangements in lynch syndrome cases associated with MSH2: Characterization of a new deletion involving both EPCAM and the 5′ part of MSH2

Lynch syndrome is caused by germline mutations in MSH2, MLH1, MSH6, and PMS2 mismatch repair genes and leads to a high risk of colorectal and endometrial cancer. It was recently shown that constitutional 3′ end deletions of EPCAM could cause Lynch syndrome in tissues with MSH2 deficiency. We aim to establish the spectrum of mutations in MSH2-associated Lynch syndrome cases and their clinical implications. Probands from 159 families suspected of having Lynch syndrome were enrolled in the study. Immunohistochemistry and microsatellite instability (MSI) analyses were used on the probands of all families. Eighteen cases with MSH2 loss were identified: eight had point mutations in MSH2. In 10 Lynch syndrome families without MSH2 mutations, EPCAM-MSH2genomic rearrangement screening was carried out with the use of multiplex ligation–dependent probe amplification and reverse transcriptase PCR. We report that large germline deletions, encompassing one or more exons of the MSH2 gene, cosegregate with the Lynch syndrome phenotype in 23% (8 of 35) of MSI families tested. A new combined deletion EPCAM-MSH2 was identified and characterized by break point analysis, encompassing from the 3′ end region of EPCAM to the 5′ initial sequences of the MSH2 (c.859-1860_MSH2:646-254del). EPCAM-MSH2 fusion transcript was isolated. The tumors of the carriers show high-level MSI and MSH2 protein loss. The clinical correlation provided evidence that the type of mutation and the extension of the deletions involving the MSH2 gene could have different implications in cancer predisposition. Thus, the identification of EPCAM-MSH2 rearrangements and their comprehensive characterization should be included in the routine mutation screening protocols for Lynch syndrome. Cancer Prev Res; 4(10); 1556–62. ©2011 AACR.

A new strategy to screen MMR genes in Lynch Syndrome: HA-CAE, MLPA and RT-PCR

AIMS Hereditary non-polyposis colon cancer (HNPCC) is an autosomal dominant disorder that is genetically heterogeneous because of underlying mutations in mismatch repair (MMR) genes, primarily MLH1, MSH2 and MSH6. One challenge to correctly diagnose HNPCC is that the large size of the causative genes makes identification of mutations both labour intensive and expensive. METHODS Our heteroduplex analysis by capillary array electrophoresis (HA-CAE) method, previously developed to increase the throughput and allow other multi-exon genes to be scanned, has been adapted for MMR genes. The altered peak patterns were then sequenced. Furthermore, the mutational scanning was completed using the Multiplex Ligation-Dependent Probe Amplification (MLPA) test in all negative HA-CAE cases, and these results were confirmed by RT-PCR. RESULTS We studied 216 individuals belonging to 100 unrelated families that met the Amsterdam I/II criteria for HNPCC. We detected 40 different variants that are classified as follows: 8 (20%) deleterious mutation, 8 (20%) unknown pathogenic significance variants and 24 (60%) coding and intronic sequence variants. Pathogenic mutations were detected in 12% of the families and about 42% of these had a deletion variant. Unknown pathogenic significance variants (UVs) affected 13% of the families. We also found 12.5% of novel polymorphisms in the rest of the variants. CONCLUDING: In short, using a combined method that includes HA-CAE, MLPA and RT-PCR, it is possible to detect the entire mutational spectrum of MMR genes. Twenty percent of the mutations found in the three genes have not been reported before. Relatives at risk will be offered predictive molecular analysis with potential exclusion of non-carriers of mutations.

Characterization of New Founder Alu-Mediated Rearrangements in MSH2 Gene Associated with a Lynch Syndrome Phenotype

It has been reported that large genomic deletions in the MLH1 and MSH2 genes are a frequent cause of Lynch syndrome in certain populations. Here, a cohort has been screened and two new founder rearrangements have been found in the MSH2 gene. These mutations have been characterized by break point determination, haplotype analysis, and genotype–phenotype correlation. Mutations have been identified in the MLH1, MSH2, and MSH6 genes in 303 subjects from 160 suspected Lynch syndrome unrelated families. All subjects were tested using heteroduplex analysis by capillary array electrophoresis. Multiplex ligation-dependent probe amplification was used to detect rearrangements in mutation-negative index patients and confirmed by reverse transcriptase PCR. The break point of the deletions was further characterized by the array comparative genomic hybridization method. Immunohistochemical staining and microsatellite instability were studied in tumor samples. Hereditary nonpolyposis colorectal cancer–related phenotypes were evaluated. More than 16% (24 of 160) of the families had pathogenic mutations (8 MLH1, 15 MSH2, and 1 MSH6). Twelve of these families (50%) are carriers of a novel mutation. Seven of the 15 positive MSH2 families (47%) are carriers of a rearrangement. The exon 7 deletion and exon 4 to 8 deletion of MSH2 are new founder mutations. The segregation of a common haplotype, a similar phenotype, and anticipation effects were observed in these families. These findings will greatly simplify the diagnosis, counseling, and clinical care in suspected Lynch syndrome families and not just in specific geographic areas, so wide distribution may be explained by migration patterns. Cancer Prev Res; 4(10); 1546–55. ©2011 AACR.

BRCA1 5272-1G>A and BRCA2 5374delTATG are founder mutations of high relevance for genetic counselling in breast/ovarian cancer families of Spanish origin.

Infante M, Durán M, Acedo A, Pérez‐Cabornero L, Sanz DJ, García‐González M, Beristain E, Esteban‐Cardeñosa E, de la Hoya M, Teulé A, Vega A, Tejada M‐I, Lastra E, Miner C, Velasco EA. BRCA1 5272‐1G>A and BRCA2 5374delTATG are founder mutations of high relevance for genetic counselling in breast/ovarian cancer families of Spanish origin.

Evaluating the Effect of Unclassified Variants Identified in MMR Genes Using Phenotypic Features, Bioinformatics Prediction, and RNA Assays

Lynch syndrome is caused by mutations in one of the mismatch-repair system (MMR) genes. A major difficulty in diagnosis and management of Lynch syndrome is the existence of unclassified genetic variants (UVs) with unknown clinical significance, especially mutations with new descriptions and missense-type nucleotide substitutions. We evaluated the pathogenicity of 20 such mutations (6 in MLH1, 4 in MSH2, and 7 in MSH6) found in Spanish patients suspected of Lynch syndrome. The UVs were tested for evidence of MMR defect in tumor samples and were evaluated for co-occurrence with a pathogenic mutation, the cosegregation of the variant with the disease; where sufficient data were available, in silico resources at the protein level and mRNA analysis were used to assess the putative effect on the splicing mechanism. To evaluate the frequency of these UVs in the general population, a case--control study was also performed. Five variants were identified with similar frequencies in both cases and controls, suggesting a nonpathogenic effect in patients. In contrast, abnormal splicing mutations were detected in a high proportion of patients [3/20 (15%)]. In this study, we classified 15 of the 20 UVs: six variants with strong evidence of pathogenicity and nine variants that should be considered neutral variants. Clinical significance of the other five remains unknown.

Incidence of −93 MLH1 promoter polymorphism in familial and sporadic colorectal cancer

The MLH1 promoter contains a common single nucleotide polymorphism (−93 guanine > adenine) located in an essential region for maximum transcriptional activity. This has been associated with an increased risk of microsatellite instability (MSI) colorectal cancer. The aim of the study was to compare the distribution of MLH1 −93G>A genotypes between patients with familial colon cancer, sporadic colon cancer and healthy subjects.