Cristina Miner

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.

High proportion of novel mutations of BRCA1 and BRCA2 in breast/ovarian cancer patients from Castilla-León (central Spain)

AbstractA total of 264 unrelated breast/ovarian cancer patients and 45 healthy individuals with familial antecedents referred for genetic testing were scanned for germ-line mutations in BRCA1 and BRCA2 by conformation-sensitive gel electrophoresis (CSGE) and heteroduplex analysis by capillary array electrophoresis (HA-CAE). We detected 101 distinct mutations (41 in BRCA1 and 60 in BRCA2); ten of them have not been previously reported. These mutations were c.2411_2429dup19, c.2802_2805delCAAA and c.5294A>G (p.E1725E) of BRCA1; and c.667C>T (p.Q147X), c.2683C>T (p.Q819X), c.5344_5347delAATA, c.5578_5579delAA;insT, c.8260_8261insGA, c.744+14C>T and c.8099A>G (p.Y2624C) of BRCA2. Twenty-four different mutations, including seven of the new mutations (five frameshift and two nonsense), were classified as pathogenic. These 24 alterations were found in 39 families (12.6% of all families). A remarkable proportion of deleterious mutations were found in BRCA2: 25 families carried a mutation in BRCA2 (BRCA2+; 64.1%) compared with 14 families BRCA1+ (35.9%). The highest incidences of deleterious mutations were found in families with three or more cases of site-specific breast cancer (BC) (27.4%) and families with BC and ovarian cancer (22.2%). Finally, four recurrent mutations, 3036_3039delACAA, c.5374_5377delTATG of BRCA2, as well as c.5272-1G>A and c.5242C>A (p.A1708E) of BRCA1, accounted for 44% of all of the deleterious mutations.

THE VARIANT E233G OF THE RAD51D GENE COULD BE A LOW-PENETRANCE ALLELE IN HIGH-RISK BREAST CANCER FAMILIES WITHOUT BRCA1/2 MUTATIONS

Six SNPs have been detected in the DNA repair genes RAD51C and RAD51D, not previously characterized. The novel variant E233G in RAD51D is more highly represented in high‐risk, site‐specific, familial breast cancer cases that are not associated with the BRCA1/2 genes, with a frequency of 5.74% (n = 174) compared to a control population (n = 567) and another subset of breast cancer patients (n = 765) with a prevalence of around 2% only (comparison to controls, OR = 2.6, 95% CI 1.12–6.03; p < 0.021). We found that the immunohistochemical profile detected in available tumors from these patients differs slightly from those described in non‐BRCA1/2 tumors. Finally, the structural prediction of the putative functional consequence of this change indicates that it can diminish protein stability and structure. This suggests a role for E233G as a low‐penetrance susceptibility gene in the specific subgroup of high‐risk familial breast cancer cases that are not related to BRCA1/2.

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.

Inositol phospho-oligosaccharide stimulates cell proliferation in the early developing inner ear

The ability of an inositol phospho-oligosaccharide (POS) to mimic the mitogenic effects of nerve growth factor (NGF) and insulin on the early development of the inner ear was investigated. POS (10 microM) stimulated the incorporation of [3H]thymidine into the cochleovestibular ganglion by 3.9-fold. NGF (50 ng/ml) stimulation was 4.7-fold. POS and NGF showed no additivity. Cells induced to proliferate by POS overlapped with those expressing NGF receptors. POS, like insulin, potentiated the mitogenic effect of bombesin on the otic vesicle epithelium. DNA synthesis in the presence of bombesin (100 nM) plus POS (10 microM) was increased by 6.4-fold. POS stimulation was not additive with insulin. The results suggest that POS may play a role in growth factor regulation of cell proliferation during embryonic development.

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.

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.

Myb p75 oncoprotein is expressed in developing otic and epibranchial placodes.

The c-myb proto-oncogene encodes a transcriptional regulatory protein which is highly conserved throughout evolution. Myb has been considered to be normally restricted to hematopoietic tissues, but there are indications that this might not always be the case. The present work shows the expression of a p75 Myb oncoprotein in the otic vesicle and epibranchial placodes of the early chick embryo. Expression was sequential and followed the same time course as the formation of placode-derived cranial ganglia. The results suggest a potential role for c-myb in regulation of placode development and neurogenesis.