Montserrat Domènech

Allelic variants of the thiopurine s-methyltranferase deficiency in patients with ulcerative colitis and in healthy controls

OBJECTIVE:Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the inactivation of mercaptopurine, azathioprine, and thioguanine. The genetic polymorphisms in the TPMT gene that regulate TPMT activity are inherited as an autosomal recessive trait and patients with genetically determined low levels of TPMT activity develop severe myelosupression when treated with standard doses of the above-mentioned drugs. We have analyzed the frequencies of the allelic variants of the TPMT gene in a white European population of healthy blood donors from Spain and The Netherlands, and in a group of patients suffering from ulcerative colitis (UC) with a similar genetic background.METHODS:Two hundred and thirteen unrelated healthy individuals (HC) and 146 UC patients were typed for the polymorphic sites at positions 460 (G → A) and 719 (A → G) of the TPMT gene using specific polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) methods.RESULTS:There were no significant differences between the allele frequencies observed in the group of UC patients and those of the control group (10% of cases were heterozygous carriers of a TPMT mutant allele). The most frequent mutant allele in both UC and HC groups was TPMT3A (A460→ G719) (60% of carriers). TPMT3B (A460→ A719) and TPMT3C (G460→ G719) alleles were more often found in our study than in previously reported studies, reflecting the different genetic backgrounds of the European populations analyzed.CONCLUSIONS:Genotyping methods provide a simple and reliable screening to identify patients with a high risk of developing severe bone marrow toxicity if treated with thiopurine drugs. In UC patients, TPMT genotype should be determined before the initiation of azathioprine therapy.

Identification of the 185delAG BRCA1 mutation in a Spanish Gypsy population

Abstract The 185delAG BRCA1 deletion occurs with a high frequency in Ashkenazi Jews. We detected this mutation in two Spanish Gypsy women (the only Gypsy participants) in an extensive study of 90 high-risk families and 160 women with early-onset breast cancer. One of these Gypsy women belonged to a high-risk family and the other had had early-onset breast cancer. The mutation was also detected in 1 out of 25 Gypsy samples unrelated to breast cancer. All the samples with the mutation shared the marker alleles present in Jewish samples with 185delAG. This is the first report of this mutation in a non-Jewish well-defined ethnic population. According to these findings the carrier frequency of this mutation in Gypsy individuals could be several times higher than that of the general population, and this should be taken into consideration in genetic screening for cancer in Gypsy populations.

Modifier genes in haemophilia: their expansion in the human genome

Mutations in factor VIII and IX genes have a determinant effect on the severity of haemophilia. Modulation of clinical manifestations depends on other genetic factors, including modifier genes. In the context of haemophilia, such genes could be the ones involved in thrombophilia. Factor V Leiden and prothrombin 20210A were studied as possible phenotypic modifiers. Inhibitor development after therapeutic factor replacement depends on the type of mutation and on the genetic factors related to the immune response of each patient. The study of all these variants in haemophiliacs constitutes an important step in prevention, prognosis and therapeutic alternatives of the disease.

Prevalence of 2314delG mutation in Spanish patients with Usher syndrome type II (USH2)

The Usher syndrome (USH) is a group of autosomal recessive diseases characterized by congenital sensorineural hearing loss and retinitis pigmentosa. Three clinically distinct forms of Usher syndrome have so far been recognized and can be distinguished from one another by assessing auditory and vestibular function. Usher syndrome type II (USH2) patients have congenital moderate-to-severe nonprogressive hearing loss, retinitis pigmentosa, and normal vestibular function. Genetic linkage studies have revealed genetic heterogeneity among the three types of USH, with the majority of USH2 families showing linkage to the USH2A locus in 1q41. The USH2A gene (MIM 276901) has been identified: three mutations, 2314delG, 2913delG, and 4353-54delC, were initially reported in USH2A patients, the most frequent of which is the 2314delG mutation. It has been reported that this mutation can give rise to typical and atypical USH2 phenotypes. USH2 cases represent 62% of all USH cases in the Spanish population, and 95% of these cases have provided evidence of linkage to the USH2A locus. In the present study, the three reported mutations were analyzed in 59 Spanish families with a diagnosis of USH type II. The 2314delG was the only mutation identified in our population: it was detected in 25% of families and 16% of USH2 chromosomes analyzed. This study attempts to estimate the prevalence of this common mutation in a homogeneous Spanish population.

BRCA2 mutation analysis of 87 Spanish breast/ovarian cancer families

BACKGROUND It is estimated that about 5% -10% of breast cancer (BC) cases is due to inherited predisposition. Early works reported that 45%-50% of site-specific BC families had BRCA1 mutations and 25%-35% BRCA2 mutations. However, these percentages could have been overestimated and likely vary among the populations studied. PATIENTS AND METHODS We analysed the BRCA2 gene in 87 Spanish breast/ovarian cancer families in which the BRCA1 mutation screening was negative. RESULTS We detected 15 (17.2%) disease-causing mutations and 11 polymorphisms and unclassified variants. Four mutations were recurrent, and five were novel. Seven (47%) mutations were found in site-specific female BC families, five (33%) in families with OC cases, and three (20%) mutations in families with male BC cases. There was incomplete penetrance of the mutations in some families, and considerable phenotypic variations with respect to the age of diagnosis and cancer types. CONCLUSIONS The percentage of mutations detected reinforces the possibility that some of these families have mutations in genes other than BRCA1 or BRCA2 that confer lower BC risks.

Aplasia medular tras administración de azatioprina: papel del polimorfismo genético de la tiopurina metiltransferasa

Fundamento La azatioprina es un farmaco inmunodepresor ampliamente utilizado en el tratamiento de diversos procesos autoinmunes. Los efectos adversos del tratamiento estan en relacion con la actividad de la tiopurina metiltransferasa (TPMT), enzima que interviene en el metabolismo de la azatioprina. La existencia de variantes alelicas del gen que codifica dicha enzima permite clasificar a los pacientes en tres grupos: de riesgo leve, moderado y alto de padecer una mielodepresion tras la administracion de azatioprina. Pacientes y metodo Se ha realizado el estudio de las variantes alelicas del gen de la TPMT en las posiciones 460 y 719 mediante reaccion en cadena de la polimerasa y digestion con enzimas de restriccion, en un enfermo con enfermedad de Crohn que presento aplasia tras la administracion de azatioprina, asi como de sus familiares directos disponibles. Resultados En la muestra del caso en estudio se identifico la variante alelica mas frecuente del gen de la TPMT asociada a una actividad enzimatica disminuida. La madre del paciente, asi como su hermana, tambien presentaron esta variante. Conclusiones La identificacion genotipica de las variantes alelicas del gen TPMT es un metodo eficaz para identificar a los pacientes con riesgo leve, moderado o grave de padecer una mielo-depresion tras la administracion de azatioprina.

Conditions for single-strand conformation polymorphism (SSCP) analysis of BRCA1 gene using an automated electrophoresis unit.

Abstract The single-strand conformation polymorphism procedure has been applied in routine testing for hereditary diseases and cancer. However, temperature, running time, gel composition, fragment length, etc. can influence its sensitivity. Mutation detection in the clinical setting depends on the development of automated technology, especially for large genes such as the breast cancer gene BRCA1. We analysed DNA samples with BRCA1 mutations in an automated system (GenePhor System; Amersham-Pharmacia Biotech, Uppsala, Sweden). The concentrations of DNA template and PCR primers, the effect of chilling after denaturation, and the temperature and time of the electrophoresis were investigated. All band-shifts were detected by electrophoresis at 5 °C for 2 h 15 min. Concentrations of DNA and samples used in the PCR did not affect the SSCP pattern, but chilling the PCR product in ice after denaturation was required. The type and position of mutation in the fragments did not influence the probability of a mobility shift, although SSCP analysis was more sensitive for fragments shorter than 350 bp. This automated SSCP method meets the requirements of fast turnaround and sensitivity and can be readily adapted to the screening of large genes such as BRCA1.

Análisis del haplotipo en portadores de la mutación 6857delAA en el gen BRCA2 en 4 familias con cáncer de mama u ovario hereditario

Fundamento y objetivo: Entre un 5 y un 10% de los canceres de mama son de caracter hereditario y se conocen en la actualidad 2 genes principalmente responsables: BRCA1 y BRCA2. Pacientes y metodo: En el estudio genetico de pacientes con antecedentes familiares de cancer de mama/ovario hereditario realizado en 2 centros, en Espana y Chile, se identifico la mutacion 6857delAA en el gen BRCA2 de 3 familias espanolas y de una chilena. Se realizo el analisis del haplotipo en los portadores mediante 5 microsatelites que rodean el gen y abarcan una region de 6 cM: cen-D13S260, D13S1698 (BRCA2), D13S171, D13S310 y D13S267-tel. Resultados: Dos familias comparten la variante alelica de los 5 microsatelites estudiados. Los marcadores D13S260 y D13S267 difieren cada uno en una familia. No se identifico el haplotipo comun en los no portadores de la mutacion y su frecuencia fue menor del 10% en los cromosomas control. Conclusiones: Los resultados apuntan a la existencia de un ancestro comun con la mutacion, originada en el area de Cataluna. Teniendo en cuenta los movimientos migratorios desde Espana a Latinoamerica, la busqueda en estos paises de mutaciones aparecidas recurrentemente en Espana puede favorecer un analisis mas racional, efectivo y de menor coste en dichos genes.

Exclusion of mosaicism in Spanish haemophilia A families with inversion of intron 22.

Summary.  Inversion of intron 22, the most frequent mutation event in haemophilia A (HA), was tested in our HA families to diagnose the females at risk of being carriers, to trace the origin of the mutation and to investigate the presence of germinal or somatic mosaicism. A total of 166 females belonging to 54 families with inversion, were analysed. All but one of the mothers tested were carriers and the inversion originated almost exclusively in male germ cells. Somatic or germline mosaicisms were excluded in 53 of these women and in 20 grandfathers, suggesting that such mosaicisms may be a rare event in families with inversion of intron 22.

Evaluation of two strategies for the interpretation of tumour markers in pleural effusions

BackgroundPleural effusions present a diagnostic challenge. Approximately 20% are associated with cancer and some 50% require invasive procedures to perform diagnosis. Determination of tumour markers may help to identify patients with malignant effusions. Two strategies are used to obtain high specificity in the differential diagnosis of malignant pleural effusions: a) high cut-off, and b) fluid/serum (F/S) ratio and low cut-off. The aim of this study is to compare these two strategies and to establish whether the identification of possible false positives using benign biomarkers – ADA, CRP and % of polymorphonuclear cells – improves diagnostic accuracy.MethodsWe studied 402 pleural effusions, 122 of them malignant. Benign biomarkers were determined in pleural fluid, and CEA, CA72-4, CA19-9 and CA15-3 in pleural fluid and serum.ResultsEstablishing a cut-off value for each TM for a specificity of 100%, a joint sensitivity of 66.5% was obtained. With the F/S strategy and low cut-off points, sensitivity was 77% and specificity 98.2%, Subclassifying cases with negative benign biomarkers, both strategies achieved a specificity of 100%; sensitivity was 69.9% for single determination and 80.6% for F/S ratio.ConclusionsThe best interpretation of TM in the differential diagnosis of malignant pleural effusions is obtained using the F/S ratio in the group with negative benign biomarkers.