[ca] En les ultimes decades el diagnostic prenatal ha estat aplicat en parelles amb un risc elevat de transmetre una malaltia genetica a la seva descendencia. Aquesta tecnica implica que en cas de detectar un fetus afectat, els pares tenen dues alternatives: seguir endavant amb l'embaras (malgrat l'afectacio del fetus) o l'avortament terapeutic. Mes recentment, les tecniques de reproduccio assistida han permes una aproximacio molt mes primerenca que el prenatal, l'anomenat diagnostic genetic preimplantacional (DGP), que es basa en l'estudi de biopsies d'embrions, les quals ens permeten seleccionar unicament els embrions sans per a la seva transferencia a l'uter matern. El DGP requereix de fecundacio in vitro i pot ser aplicat en dos estadis diferents, en el primer corpuscle polar d'oocits madurs o en blastomers d'embrions. Per l'estudi d'anomalies cromosomiques en parelles portadores de reorganitzacions o en dones d'edat avansada s'apliquen tecniques d'hibridacio in situ fluorescent (FISH). En el cas de parelles portadores de malalties genetiques l'analisi es realitza basicament amb PCR (reaccio en cadena de la polimerasa). Les malalties genetiques estan causades per mutacions al seu gen responsable, com per exemple el gen CFTR de la Fibrosi Quistica (FQ). La FQ es la malaltia genetica autosomica recessiva mes frequent i afecta a un de cada 2500 nounats. Es caracteritza per un augment d'ions clorur en la suor i per obstruccio pulmonar i pancreatica. Un altre exemple de malaltien monogeniques serien les Hemofilies A i B causades per mutacions als gens dels factors de coagulacio FVIII i FIX. Les Hemofilies son les coagulopaties congenites de mes impacte clinic i social i es deuen a l'alteracio o l'absencia d'activitat del factor de coagulacio pel que codifiquen. Presenten una herencia recessiva lligada al cromosoma X i la seva incidencia es de 1 de cada 10000-30000 homes. El DGP d'aquestes malalties es basa en l'identificacio d'embrions lliures de les mutacions que porten els seus progenitors i moltes vegades requereixen de la posada a punt de protocols especifics de pacient, molt costosos d'optimitzar. A mes, la PCR d'una unica cel.lula presenta nombroses dificultats tecniques degut a la quantitat minima de DNA que es parteix, sent la fallada d'amplificacio i la contaminacio de la mostra dos dels problemes mes frequents que dificulten el diagnostic. Per tant, es necessari emprar tecniques molt sensibles, que ens permetin confirmar el diagnostic i detectar els casos de contaminacio. En aquest treball s'ha posat a punt un metode d'amplificacio total del genoma de celules aillades per tal d'augmentar la quantitat de DNA en la que poder realitzar el diagnostic. Aquest DNA preamplificat ens permet analitzar i identificar mitjansant un unic protocol basat en tecniques moleculars amb marcatge fluorescent, la presencia o absencia de finjs a 31 mutacions diferents responsables de FQ. En el cas de les Hemofilies, la sequenciacio del DNA preamplificat ens permet identificar qualsevol mutacio de tipu substitucio, petita insercio o delecio del promotor, exons, o regions introniques flanquejants dels gens F8 i F9. La metodologia desenvolupada presenta gran robustesa i sensibilitat i pot ser aplicada a fins un 80% de les parelles portadores de FQ, el 75% de les Hemofilies A i practicament la totalitat dels casos de Hemofilia B, sense la necesitat de posar a punt protocols especifics de pacient. A mes, s'han optimitzat unes PCR multiplex fluorescents de marcadors lligats de tipu microsatelit (STR) per a confirmar el diagnostic mitjansant l'analisi indirecte addicional de les mutacions i simultaniament, detectar la posible contaminacio de la mostra en l'aplicacio clinica. Aquesta analisi de lligament permet tambe la deteccio indirecta de mutacions no analitzables per secuenciacio, com ara la frequent inversio de l'intro 22 responsable d'Hemofilia A severa Les taxes d'amplificacio de les celules aillades emprant els protocols generics optimitzats en el present treball son comparables als resultats obtinguts per altres autors, i per tant demostren la elevada eficiencia de la metodologia desenvolupada. Finalment, l'estrategia diagnostica utilitzada seria transferible a moltes altres malalties monogeniques, facilitant el desenvolupament de protocols versatils d'elevada fiabilitat per tal d'aconseguir descendencia sana en families portadores.

[en] During the last decades prenatal diagnosis has been applied to couples at risk of transmitting a genetic disorder to their offspring. This technique implies that in case of detection of an affected fetus, the parents have two alternatives: continue the pregnancy (despite the fetus being affected) or the therapeutic abortion. More recently, assisted reproduction techniques permit an earlier option, preimplantation genetic diagnosis (PGD), based on the analysis of embryo biopsies which allow the selection of only those healthy embryos for transfer to the uterus. PGD requires in vitro fertilization and can be applied in two different stages, in first polar bodies of oocytes, or in blastomeres from embryos. For the analysis of chromosomal abnormalities in couples carrying a chromosome rearrangement or in cases of advanced maternal age, FISH (fluorescence in situ hibridization) is used. In case of couples carrying a genetic disorder the analysis is performed basically by means of PCR (polymerase chain reaction). Monogenic disorders are caused by mutations in their responsible gene, such as CFTR gene responsible for cystic fibrosis (CF). CF is the most common recessive monogenic disorder which affects 1/2500 newborns. Its main features are an increase in chloride in sweat and lung and pancreatic obstruction. Another example would be hemophilia A and B, caused by mutations of the clotting factors FVIII and FIX. HA and B are the congenital bleeding disorders with most clinical and social impact and are due to the alteration or lack of activity of the responsible coagulation factor. They have an X-linked recessive inheritance and the incidence is 1/10000-30000 males. PGD for these disorders is based on the identification of embryos free of mutations and many times require the tedious work up of patient-specific protocols, very complicated to optimize. Moreover, single cell PCR has many technical difficulties, being the failure of amplification and contamination the two most common issues that compromise the diagnosis. Consequently, it is necessary to use very sensitive techniques that allow the back-up of the diagnosis and that permit the detection of contamination. In this study we have optimized a whole genome amplification method for single cells in order to increase the amount of DNA in which the diagnosis is performed. This preamplified DNA is used to analyze and identify the presence of up to 31 different mutations responsible for CF using a single protocol. In the case of Hemophilia, direct sequencing of the preamplified DNA permits the detection of any point mutation, small insertion or deletion in the promoter, exons and intronic flanking regions of F8 and F9 genes. The methods developed perform with great robustness and sensitivity and can be used in up to 80% of the couples carriers of CF, 75% of HA and almost all the cases of HB, without developing a patient-specific protocol. Additionally, several fluorescent multiplex PCRs of linked microsatellite markers (STR) have been optimized in order to confirm diagnosis through indirect analysis of the mutations and, simultaneously, being able to detect the possible contamination of the samples in clinical application. This linkage analysis also permits the indirect detection of mutations not analyzable by sequencing, such as intron 22 inversion responsible for severe HA. The amplification rates of single cells using the optimized generic protocols from the present study are comparable to other authors' results, consequently the high efficiency of the developed methodology is demonstrated. Finally, the diagnostic strategy applied would be transferable to many other single gene disorders, making easier the development of versatile and very reliable protocols in order to achieve healthy offspring in carrier families.