Ana Peixoto

The c.156_157insAlu BRCA2 rearrangement accounts for more than one-fourth of deleterious BRCA mutations in northern/central Portugal

We evaluated the contribution of an Alu insertion in BRCA2 exon 3 (c.156_157insAlu) to inherited predisposition to breast/ovarian cancer in 208 families originated mostly from northern/central Portugal. We identified the c.156_157insAlu BRCA2 mutation in 14 families and showed that it accounts for more that one-fourth of deleterious BRCA1/BRCA2 mutations in breast/ovarian cancer families originated from this part of the country. This mutation originates BRCA2 exon 3 skipping and we demonstrated its pathogenic effect by showing that the BRCA2 full length transcript is derived only from the wild type allele in carriers, that it is absent in 262 chromosomes from healthy blood donors, and that it co-segregates with the disease. Polymorphic microsatellite markers were used for haplotype analysis in three informative families. In two of the three families one haplotype was shared for all but two markers, whereas in the third family all markers telomeric to BRCA2 differed from that observed in the other two. Although the c.156_157insAlu BRCA2 mutation has so far only been identified in Portuguese breast/ovarian cancer families, screening of this rearrangement in other populations will allow evaluation of whether or not it is a population-specific founder mutation and a more accurate estimation of its distribution and age.

BRCA1 and BRCA2 germline mutational spectrum and evidence for genetic anticipation in Portuguese breast/ovarian cancer families

We present the first characterisation of the mutational spectrum of the entire coding sequences and exon–intron boundaries of the BRCA1 and BRCA2 genes as well as large BRCA1 rearrangements in Portuguese families with inherited predisposition to breast/ovarian cancer. Of the 100 probands studied, pathogenic mutations were identified in 22 (24.7%) of 89 breast and/or ovarian cancer families with more than one affected member (15 in BRCA1 and seven in BRCA2), but in none of the 11 patients without family history of cancer. One (6.7%) of the BRCA1 mutations is a large deletion involving exons 11–15. Seven pathogenic point mutations are novel: 2088C>T, 2156delinsCC, and 4255_4256delCT in BRCA1 and 4608_4609delTT, 5036delA, 5583_5584insT, and 8923C>T in BRCA2. The novel 2156delinsCC was identified in three probands from different families and probably represents a founder mutation in our population. We also found a previously reported 3450_3453del4 mutation in three unrelated patients. In addition to the 22 pathogenic mutations, we identified 19 missense mutations of uncertain pathogenic significance, three of them (5241G>C in BRCA1 and IVS6+13C>T and 3731T>C in BRCA2) previously undescribed. The percentage of cases with truncating mutations in BRCA1 and BRCA2 was higher in breast/ovarian cancer (37.0%, mostly BRCA1) and male breast cancer (40%, all BRCA2) families than in families with only female breast cancer (17.5%). Interestingly, we found evidence for genetic anticipation regarding age at diagnosis of both breast and ovarian cancer in those families presenting affected members in more than one generation. These findings should be taken into consideration while planning screening and prophylactic measures in families with inherited predisposition to breast and ovarian cancer.

Comparison of methodologies for KRAS mutation detection in metastatic colorectal cancer

Cetuximab and panitumumab are two monoclonal antibodies targeting the epidermal growth factor receptor that have been approved for treatment of metastatic colorectal cancer. Recent clinical trials found an association between KRAS mutation status and resistance to anti-epidermal growth factor receptor therapy, leading to the recommendation to perform KRAS mutation analysis before cetuximab or panitumumab treatment. This study was designed to compare and evaluate the efficacy of four different methodologies--high resolution melting, Sanger sequencing, DxS kit, and SNaPshot--for KRAS mutation detection in a clinical setting. In total, 372 samples from patients with metastatic colorectal cancer were analyzed by high resolution melting and SNaPshot, with 184 of those being further analyzed by Sanger sequencing and 188 with the DxS kit. Sensitivities were compared after consensus findings were determined by the presence of the same result in two of the three methodologies used in each case. The frequency of KRAS codon 12 and 13 mutations in our population was 43.5%, and a discordant finding was observed in 22 samples. Comparing to Sanger sequencing, significantly more consensus mutations were detected by the DxS kit (P=0.0139), high resolution melting (P=0.0004), and SNaPshot (P=0.00001), but no statistically significant differences were found among the three methodologies with higher sensitivity.

High resolution melting analysis of KRAS, BRAF and PIK3CA in KRAS exon 2 wild-type metastatic colorectal cancer

BackgroundKRAS is an EGFR effector in the RAS/RAF/ERK cascade that is mutated in about 40% of metastatic colorectal cancer (mCRC). Activating mutations in codons 12 and 13 of the KRAS gene are the only established negative predictors of response to anti-EGFR therapy and patients whose tumors harbor such mutations are not candidates for therapy. However, 40 to 60% of wild-type cases do not respond to anti-EGFR therapy, suggesting the involvement of other genes that act downstream of EGFR in the RAS-RAF-MAPK and PI3K-AKT pathways or activating KRAS mutations at other locations of the gene.MethodsDNA was obtained from a consecutive series of 201 mCRC cases (FFPE tissue), wild-type for KRAS exon 2 (codons 12 and 13). Mutational analysis of KRAS (exons 3 and 4), BRAF (exons 11 and 15), and PIK3CA (exons 9 and 20) was performed by high resolution melting (HRM) and positive cases were then sequenced.ResultsOne mutation was present in 23.4% (47/201) of the cases and 3.0% additional cases (6/201) had two concomitant mutations. A total of 53 cases showed 59 mutations, with the following distribution: 44.1% (26/59) in KRAS (13 in exon 3 and 13 in exon 4), 18.6% (11/59) in BRAF (two in exon 11 and nine in exon 15) and 37.3% (22/59) in PIK3CA (16 in exon 9 and six in exon 20). In total, 26.4% (53/201) of the cases had at least one mutation and the remaining 73.6% (148/201) were wild-type for all regions studied. Five of the mutations we report, four in KRAS and one in BRAF, have not previously been described in CRC. BRAF and PIK3CA mutations were more frequent in the colon than in the sigmoid or rectum: 20.8% vs. 1.6% vs. 0.0% (P=0.000) for BRAF and 23.4% vs. 12.1% vs. 5.4% (P=0.011) for PIK3CA mutations.ConclusionsAbout one fourth of mCRC cases wild-type for KRAS codons 12 and 13 present other mutations either in KRAS, BRAF, or PIK3CA, many of which may explain the lack of response to anti-EGFR therapy observed in a significant proportion of these patients.

Feasibility of differential diagnosis of kidney tumors by comparative genomic hybridization of fine needle aspiration biopsies.

The association of a genetic analysis that could improve the diagnostic accuracy of renal cell tumors in biopsy samples would allow better‐informed therapeutic decisions. We performed comparative genomic hybridization (CGH) on an ex vivo fine‐needle aspiration (FNA) biopsy and a tumor fragment obtained from 75 patients consecutively diagnosed with renal tumors and subjected to radical nephrectomy. The pattern of genomic changes by CGH was used blindly to classify the renal tumors and the genetic findings were subsequently compared with the histopathologic diagnosis. In particular cases, including in two carcinomas with morphologically distinct tumor areas, we performed FISH with several locus‐specific probes, and looked for VHL point mutations, exonic rearrangements, or promoter methylation. CGH was successful in 82.7% FNA biopsies and in 96% tumor fragments, with the former allowing genetic diagnosis in 75% of renal cell tumors. The genetic and the initial histological classification differed in two renal neoplasias, but the genetic diagnosis was confirmed after review. The genetic pattern correctly diagnosed 93.5% of clear cell renal cell carcinomas (RCC), 61.5% of chromophobe RCC, 100% of papillary RCC, and 14.3% of oncocytomas, with the negative predictive value being 93.9, 90.7, 100, and 90.2%, respectively. The positive predictive value and specificity of copy number profiles was 100%. We demonstrate that genetic diagnosis by CGH on FNA biopsies can improve differential diagnosis in patients with kidney tumors.

Li-Fraumeni-like syndrome associated with a large BRCA1 intragenic deletion.

BackgroundLi-Fraumeni (LFS) and Li-Fraumeni-like (LFL) syndromes are associated to germline TP53 mutations, and are characterized by the development of central nervous system tumors, sarcomas, adrenocortical carcinomas, and other early-onset tumors. Due to the high frequency of breast cancer in LFS/LFL families, these syndromes clinically overlap with hereditary breast cancer (HBC). Germline point mutations in BRCA1, BRCA2, and TP53 genes are associated with high risk of breast cancer. Large rearrangements involving these genes are also implicated in the HBC phenotype.MethodsWe have screened DNA copy number changes by MLPA on BRCA1, BRCA2, and TP53 genes in 23 breast cancer patients with a clinical diagnosis consistent with LFS/LFL; most of these families also met the clinical criteria for other HBC syndromes.ResultsWe found no DNA copy number alterations in the BRCA2 and TP53 genes, but we detected in one patient a 36.4 Kb BRCA1 microdeletion, confirmed and further mapped by array-CGH, encompassing exons 9–19. Breakpoints sequencing analysis suggests that this rearrangement was mediated by flanking Alu sequences.ConclusionThis is the first description of a germline intragenic BRCA1 deletion in a breast cancer patient with a family history consistent with both LFL and HBC syndromes. Our results show that large rearrangements in these known cancer predisposition genes occur, but are not a frequent cause of cancer susceptibility.

A new insight into fragile X syndrome among Basque population.

The expansion of a trinucleotide repeat [CGG]n located in the FMR1 X‐linked gene is the main cause of fragile X syndrome, the most common form of inherited mental retardation. We have analyzed the factors known, to date, to influence the instability of the repeat in 158 normal X chromosomes from the Spanish Basque population. These factors included length of the repeat, AGG interspersion pattern, length of uninterrupted CGG and DXS548‐FRAXAC1 markers associated haplotype. Previous investigations on Basques showed an absence of this disorder among mentally retarded individuals that was likely due to a low prevalence of large CGG alleles and the presence of AGG interruptions on them. The present report suggests that, although the frequency of large alleles is low and they do maintain AGG interruptions, different mutational pathways that might lead to fragile X syndrome could be occurring among Basques. These pathways mainly include alleles with internal sequences 9 + 9 + n and 9 + 12 + 9 that show fragile X associated haplotypes. Besides, the lack of the most proximal AGG interruption, proposed recently as a novel factor involved in CGG repeat instability, was highly identified among alleles with long pure CGG tracts, which showed an internal sequence n + 9. The data suggest that, despite the lower incidence of large alleles, the prevalence of potentially unstable alleles among Basques is similar to that of other Caucasian populations and that these alleles could become fragile X chromosomes.

A novel exonic rearrangement affecting MLH1 and the contiguous LRRFIP2 is a founder mutation in Portuguese Lynch syndrome families

Purpose: Although Lynch syndrome is characterized by marked genetic heterogeneity, some specific mutations are observed at high frequency in well-defined populations or ethnic groups due to founder effects.Methods: Genomic breakpoint identification, haplotype analysis, and mutation age determination were performed in 14 unrelated patients and 95 family members presenting the same MLH1 exonic rearrangement, among a series of 84 Lynch syndrome families with germline mutations in MLH1, MSH2, or MSH6.Results: All 14 probands harbored an identical deletion, comprising exons 17–19 of the MLH1 gene and exons 26–29 of the LRRFIP2 gene, corresponding to the MLH1 mutation c.1896 + 280_oLRRFIP2:c.1750-678del. This mutation represents 17% of all deleterious mismatch repair mutations in our series. Haplotype analysis showed a conserved region of approximately 1 Mb, and the mutation age was estimated to be 283 ± 78 years. All 14 families are originated from the Porto district countryside.Conclusion: We have identified a novel MLH1 exonic rearrangement that is a common founder mutation in Lynch syndrome families, indicating that screening for this rearrangement as a first step may be cost-effective during genetic testing of Lynch syndrome suspects of Portuguese ancestry, especially those originating from the Porto district.

CSF1R copy number changes, point mutations, and RNA and protein overexpression in renal cell carcinomas

Renal cell carcinomas comprise a heterogeneous group of tumors. Of these, 80% are clear cell renal cell carcinomas, which are characterized by loss of 3p, often with concomitant gain of 5q22qter. Although VHL is considered the main target gene of the 3p deletions, none has been identified as the relevant target gene for the 5q gain. We have studied 75 consecutive kidney tumors and paired normal kidney samples to evaluate at the genomic and expression levels the tyrosine kinase genes CSF1R and PDGFRB as potential targets in this region. Our findings show that RNA expression of CSF1R, but not of PDGFRB, was significantly higher in clear cell renal cell carcinomas than in normal tissue samples, something that was corroborated at the protein level by immunohistochemistry. The CSF1R staining pattern in clear cell renal cell carcinomas was clearly different from that observed in other renal cell carcinomas, suggesting its potential usefulness in differential diagnosis. FISH analysis demonstrated whole chromosomal gain and relative CSF1R/PDGFRB copy number gain in clear cell renal cell carcinomas, which might contribute to CSF1R overexpression. Finally, one polymorphism and two novel mutations were identified in CSF1R in clear cell renal cell carcinoma patients. Our data allow us to conclude that CSF1R plays a relevant role in clear cell renal cell carcinoma carcinogenesis and raise the possibility that CSF1R may represent a future valuable therapeutic target in these patients.

Genomic characterization of two large Alu-mediated rearrangements of the BRCA1 gene

To determine whether a large genomic rearrangement is actually novel and to gain insight about the mutational mechanism responsible for its occurrence, molecular characterization with breakpoint identification is mandatory. We here report the characterization of two large deletions involving the BRCA1 gene. The first rearrangement harbored a 89 664-bp deletion comprising exon 7 of the BRCA1 gene to exon 11 of the NBR1 gene (c.441+1724_oNBR1:c.1073+480del). Two highly homologous Alu elements were found in the genomic sequences flanking the deletion breakpoints. Furthermore, a 20-bp overlapping sequence at the breakpoint junction was observed, suggesting that the most likely mechanism for the occurrence of this rearrangement was nonallelic homologous recombination. The second rearrangement fully characterized at the nucleotide level was a BRCA1 exons 11–15 deletion (c.671-319_4677-578delinsAlu). The case harbored a 23 363-bp deletion with an Alu element inserted at the breakpoints of the deleted region. As the Alu element inserted belongs to a still active AluY family, the observed rearrangement could be due to an insertion-mediated deletion mechanism caused by Alu retrotransposition. To conclude, we describe the breakpoints of two novel large deletions involving the BRCA1 gene and analysis of their genomic context allowed us to gain insight about the respective mutational mechanism.