RESUMEN
RAD51D mutations have been recently identified in breast (BC) and ovarian cancer (OC) families. Although an etiological role in OC appears to be present, the association of RAD51D mutations and BC risk is more unclear. We aimed to determine the prevalence of germline RAD51D mutations in Spanish BC/OC families negative for BRCA1/BRCA2 mutations. We analyzed 842 index patients: 491 from BC/OC families, 171 BC families, 51 OC families and 129 patients without family history but with early-onset BC or OC or metachronous BC and OC. Mutation detection was performed with high-resolution melting, denaturing high-performance liquid chromatography or Sanger sequencing. Three mutations were found in four families with BC and OC cases (0.82%). Two were novel: c.1A>T (p.Met1?) and c.667+2_667+23del, leading to the exon 7 skipping and one previously described: c.674C>T (p.Arg232*). All were present in BC/OC families with only one OC. The c.667+2_667+23del cosegregated in the family with one early-onset BC and two bilateral BC cases. We also identified the c.629C>T (p.Ala210Val) variant, which was predicted in silico to be potentially pathogenic. About 1% of the BC and OC Spanish families negative for BRCA1/BRCA2 are carriers of RAD51D mutations. The presence of several BC mutation carriers, albeit in the context of familial OC, suggests an increased risk for BC, which should be taken into account in the follow-up and early detection measures. RAD51D testing should be considered in clinical setting for families with BC and OC, irrespective of the number of OC cases in the family.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Unión al ADN/genética , Predisposición Genética a la Enfermedad/genética , Neoplasias Ováricas/genética , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , Femenino , Genes BRCA1 , Genes BRCA2 , Mutación de Línea Germinal , Heterocigoto , Humanos , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Linaje , EspañaRESUMEN
Circulating tumor cells (CTCs) are becoming a scientifically recognized indicator of primary tumors and/or metastasis. These cells can now be accurately detected and characterized as the result of technological advances. We analyzed the presence of CTCs in the peripheral blood of patients with metastatic breast cancer by real-time reverse-transcription PCR (RT-qPCR) using a panel of selected genes. The analysis of a single marker, without an EpCAM based enrichment approach, allowed the positive identification of 35% of the metastatic breast cancer patients. The analysis of five genes (SCGB2, TFF1, TFF3, Muc1, KRT20) performed in all the samples increased the detection to 61%. We describe a sensitive, reproducible and easy to implement approach to characterize CTC in patients with metastasic breast cancer.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias de la Mama/sangre , Proteínas de Neoplasias/sangre , Células Neoplásicas Circulantes/metabolismo , ARN Mensajero/sangre , ARN Neoplásico/sangre , Biomarcadores de Tumor/genética , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Expresión Génica , Humanos , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Células Neoplásicas Circulantes/patología , ARN Mensajero/genética , ARN Neoplásico/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
BRCA1 and BRCA2 genes are essential in preserving the integrity of genome, and it is not unambiguously clear whether the heterozygosity status may affect BRCA1 or BRCA2 functions. This may have implications for the clinical management of BRCA1 and BRCA2 mutation carriers both in breast cancer (BC) screening modality and in cancer treatment based on DNA-damaging or DNA-repair-inhibiting drugs. We investigated whether lymphocytes carrying BRCA1 or BRCA2 mutations displayed an increased sensitivity to radiation or mitomycin C (MMC) in vitro treatments. Peripheral blood from 21 BRCA1 mutation carriers (12 with BC and 9 healthy), 24 BRCA2 carriers (13 with BC and 11 healthy), 15 familial BC patients without detected mutation in BRCA1 or BRCA2 and 16 controls without familial history of cancer (5 with BC and 11 healthy) were irradiated or treated with MMC. Chromosomal damage was measured using the cytokinesis-block micronucleus assay. We evaluated micronuclei (MN) and nucleoplasmic bridges (NPBs). The BRCA2 mutation carriers and familial BC patients without detected mutation in BRCA1 or BRCA2 showed less basal NPB than BRCA1 carriers and controls. The BRCA1 (+/-) or BRCA2 (+/-) lymphocytes did not have increased frequencies of MN or NPB after irradiation. In contrast, BRCA2 (+/-) lymphocytes presented higher levels of MN after MMC exposure than BRCA1 carriers and controls. The monoallelic BRCA1 or BRCA2 pathogenic mutations seem not to be associated with an enhanced radiosensitivity. The mutation of one BRCA2 allele conferred an increased sensitivity to MMC, presumably because of the role of this gene in the repair of MMC-induced DNA damage. This finding indicates that the MMC-induced MN analysis could be useful in identifying functional deficiencies of BRCA2 or genes related to BRCA2. Since MMC can be used as an anti-cancer drug, these data may be relevant for the management and follow-up of BRCA2 mutation carriers.
Asunto(s)
Genes BRCA1 , Genes BRCA2 , Linfocitos/efectos de los fármacos , Linfocitos/efectos de la radiación , Micronúcleos con Defecto Cromosómico , Mitomicina/farmacología , Mutación , Adulto , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Femenino , Humanos , Linfocitos/citología , Micronúcleos con Defecto Cromosómico/inducido químicamente , Micronúcleos con Defecto Cromosómico/efectos de la radiación , Persona de Mediana Edad , Nucleoplasminas/análisisRESUMEN
Hemophilia A (HA) is an X-linked bleeding disorder caused by a wide variety of mutations in the factor 8 (F8) gene, leading to absent or deficient factor VIII (FVIII). We analyzed the F8 gene of 267 unrelated Spanish patients with HA. After excluding patients with the common intron-1 and intron-22 inversions and large deletions, we detected 137 individuals with small mutations, 31 of which had not been reported previously. Eleven of these were nonsense, frameshift, and splicing mutations, whereas 20 were missense changes. We assessed the impact of the 20 substitutions based on currently available information about FV and FVIII structure and function relationship, including previously reported results of replacements at these and topologically equivalent positions. Although most changes are likely to cause gross structural perturbations and concomitant cofactor instability, p.Ala375Ser is predicted to affect cofactor activation. Finally, 3 further mutations (p.Pro64Arg, p.Gly494Val, and p.Asp2267Gly) appear to affect cofactor interactions with its carrier protein, von Willebrand factor, with the scavenger receptor low-density lipoprotein receptor-related protein (LRP), and/or with the substrate of the FVIIIapi*FIXa (Xase) complex, factor X. Characterization of these novel mutations is important for adequate genetic counseling in HA families, but also contributes to a better understanding of FVIII structure-function relationship.
Asunto(s)
Factor VIII/genética , Hemofilia A/genética , Modelos Moleculares , Mutación Missense , Sitios de Unión/genética , Codón sin Sentido , Factor IXa/genética , Factor IXa/metabolismo , Factor VIII/metabolismo , Mutación del Sistema de Lectura , Hemofilia A/metabolismo , Humanos , Intrones/genética , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica/genética , Empalme del ARN/genética , España , Relación Estructura-Actividad , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismoRESUMEN
Large deletions of the factorVIII gene account for approximately 5% of severe haemophilia A patients. Although deletions are readily detectable in males, the identification of heterozygosity in possible carriers of these families still constitutes a challenge. In order to identify a deleted allele over the background of the normal allele in these carriers, we developed a rapid real-time quantitative PCR approach by means of LightCycler technology and SYBR green I for monitoring product formation. The method was applied to families with independent deletions (one in exon 14 and the other in exons 23-24) of the Factor VIII gene, thereby allowing a reliable determination of carrier or non-carrier status. The method is extremely versatile and can be adapted to other deletions within the factorVIII gene as well as to other diseases whose molecular pathology consists of deletions or duplications.
Asunto(s)
Secuencia de Bases , Factor VIII/genética , Tamización de Portadores Genéticos/métodos , Pruebas Genéticas/métodos , Hemofilia A/diagnóstico , Eliminación de Secuencia , Benzotiazoles , Diaminas , Exones , Femenino , Hemofilia A/genética , Humanos , Masculino , Compuestos Orgánicos , Linaje , Reacción en Cadena de la Polimerasa , Quinolinas , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND AND OBJECTIVES: The modulation of disease severity in hemophilia A (HA) patients may be related to the co-inheritance of mutations in genes with a known thrombotic effect such as factor V Leiden (FVL) and prothrombin. In the Spanish population, the prothrombin 20210A (PT20210A) allele is the most prevalent genetic risk factor for venous thromboembolism. DESIGN AND METHODS: We investigated the presence of both mutations in a cohort of 265 hemophiliac patients divided into two groups: I) 140 unrelated patients with moderate and mild HA and II) 125 unrelated patients with severe HA (83 carrying an inversion of intron 22). RESULTS: In group I, 4 patients had the FVL (2.8% vs. 2.98% controls) and 5 had the PT20210A (3.6% vs. 6.46% controls). In group II, two patients with inversion had the FVL (1.6%) and PT20210A was found in 10 patients (8%), five of them with inversion of intron 22 without inhibitors. One of these patients had the FVL and PT20210A mutations concomitantly. In the subgroup of patients with inversion who were carriers of the PT20210A, three parameters i.e. spontaneous bleeding (p=0.008), factor VIII utilization (p=0.016) and number of hemophilic arthropathies (p<0.0005) were significantly lower than in a subgroup of 11 age-matched non-PT20210A severe HA patients with inversion and without inhibitors. INTERPRETATION AND CONCLUSIONS: These results indicate that the inheritance of PT20210A could be a protective factor that mitigates the clinical severity of HA.