The 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.
DNA 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.
Lymphocyte 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.
A 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.
Background Mutations in the
gene explain abnormally long telomeres and multiple tumors including cardiac angiosarcomas (CAS). However, the link between long telomeres and tumorigenesis is poorly ...understood. Methods and Results Here, we have studied the somatic landscape of 3 different angiosarcoma patients with mutations in the
gene to further investigate this tumorigenesis process. In addition, the genetic landscape of 7 CAS patients without mutations in the
gene has been studied. Patients with CAS and nonfunctional
did not repress ATR (ataxia telangiectasia RAD3-related)-dependent DNA damage signaling and showed a constitutive increase of cell cycle arrest and somatic activating mutations in the VEGF (vascular endothelial growth factor)/angiogenesis pathway (
gene). The same observation was made in
mutation carriers with tumors different from CAS and also in CAS patients without mutations in the
gene but with mutations in other genes involved in DNA damage signaling. Conclusions Inhibition of POT1 function and damage-response malfunction activated DNA damage signaling and increased cell cycle arrest as well as interfered with apoptosis, which would permit acquisition of somatic mutations in the VEGF/angiogenesis pathway that drives tumor formation. Therapies based on the inhibition of damage signaling in asymptomatic carriers may diminish defects on cell cycle arrest and thus prevent the apoptosis deregulation that leads to the acquisition of driver mutations.
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).
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.
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.
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.
Background
Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by heterozygous mutations in mismatch repair (MMR) genes. Approximately 85 % of genetically defined HNPCC patients have germline ...mutations in
MLH1
and
MSH2
. HNPCC patients are at increased risk of developing extracolonic cancers. The early age of onset, predominantly right-sided colon cancers, and synchronous and metachronous cancers are other features of the syndrome. HNPCC shows heterogeneous clinical phenotypes, and differences in gene mutation frequencies have been observed in some countries. Several investigators have tried to correlate the phenotype with the affected gene.
Methods
A total of 46 individuals from 22 unrelated families, of the 264 families fulfilling the inclusion criteria, with deleterious mutations in
MLH1
,
MSH2
,
or MSH6
genes were identified. We evaluated these clinicopathological features in their relation to different genetic parameters (gene mutated, type of mutation, or alteration of the MMR system in high-risk families) in order to establish a relationship between the phenotype and the genotype in our series.
Results
The phenotype of the disease seems not to be influenced by the type of mutation, but rather by the mutated gene. The presence of multiple tumors is associated with mutations in the MSH2 gene. The mean age at diagnosis of the first colorectal cancer (CRC) was almost identical in families with mutations in MLH1 and MSH2, about 50 years of age, but this age may increase by almost 10 years for MSH6 mutation carriers.
Conclusion
The identification of genotype–phenotype correlations could provide a more specific surveillance program focused on the individualized risk.
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EMUNI, NUK, SBMB, SBNM, UL, UPUK
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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.
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.
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.
The mutation spectrum of BRCA1 and BRCA2 presents a wide range of unique mutations in breast/ovarian cancer patients but recurrent mutations with founder effects have also been described. BRCA2 ...5344delAATA and 9538delAA are recurrent mutations in Castilla-León (Spain) representing 10.6% of BRCA2 positive families. By genotyping eleven chromosome 13 markers (4.3 Mb) we demonstrate that each mutation shows core haplotypes of 1.66 and 0.87 Mb, respectively, supporting a common ancestor in Castilla-León. Furthermore, both mutations are associated with earlier onset of breast cancer (5344delAATA: 37.4 years, P = 0.033; 9538delAA: 39.4 years, P = 0.008). The identification of founder effects improves the genetic screening strategy to be followed and facilitates the clinical management of asymptomatic carriers
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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