Estudis d'associació i funcionals en gens candidats per a l'osteoporosi

L'osteoporosi, una malaltia complexa determinada tant per factors genetics com ambientals, esta caracteritzada per una reduida densitat mineral ossia (DMO), un deteriorament de la microarquitectura ossia i un augment del risc de patir fractures osteoporotiques. En el present treball ens vam plantejar estudiar les causes genetiques que determinen l'aparicio de l'osteoporosi mitjancant la realitzacio d'estudis d'associacio i estudis funcionals. Els gens candidats classics per a l'osteoporosi que hem analitzat han estat el gen "COL1A1", el gen "ESR1", el gen "VDR" i el gen "TGFB1". Els estudis d'associacio s'han dut a terme en la cohort BARCOS formada per dones postmenopausiques de l'area de Barcelona. Els resultats obtinguts en la cohort BARCOS han estat incorporats en el projecte GENOMOS (metaanalisi prospectiva amb 20.000 mostres). En la cohort BARCOS vam observar que polimorfismes de canvi puntual de nucleotid (SNP) del gen "COL1A1" aixi com tambe determinats haplotips i la interaccio entre ells o be amb SNPs del gen "VDR" es trobaven associats a la DMO. Pel que fa al gen "ESR1", vam trobar que l'haplotip LPX es trobava associat a la DMO femoral. Els resultats del projecte GENOMOS, van mostrar que nomes el polimorfisme Sp1 del gen "COL1A1" es trobava associat a la DMO. En el projecte GENOMOS, els polimorfismes "XbaI (ERSR1)", Cdx2 "(VDR)" i Sp1 ("COL1A1") es van trobar associats al risc de patir fractures. En la cohort BARCOS tambe vam analitzar l'efecte de SNPs situats en gens candidats no classics ("RUNX2" i "IL6R"). Pel que fa a "RUNX2" vam trobar que un SNP del promotor 2, pero no un SNP del promotor 1 es trobava associat a la DMO femoral. Aquesta ha estat la primera vegada que SNPs del gen "IL6R" s'han analitzat en relacio a fenotips osteoporotics. Vam observar que SNPs i haplotips en aquest gen es trobaven associats a la DMO femoral i a l'index de massa corporal (IMC). Com a segon objectiu ens vam plantejar completar l'estudi funcional dels SNPs del promotor del gen COL1A1 (-1997 G/T i -1663 indelT) en relacio a les proteines CIZ/NMP4 i BMP2. Previament s'havia descrit que la proteina CIZ/NMP4, un factor de transcripcio arquitectonic que inhibeix la via osteoblastogenica de la BMP2, s'unia a la regio nucleotidica on es troba el SNP -1663 indelT. En les cellules Saos-2 vam observar que el patro de transcripcio de diferents construccions del promotor del gen "COL1A1" fusionat al gen de la luciferasa era similar tant si aquestes eren tranfectades de manera estable o be de forma transitoria. En estudiar l'efecte de la BMP2 sobre l'activitat transcripcional del gen "COL1A1", vam observar que mentre el promotor basal disminuia la seva activitat en presencia de BMP2, les altres construccions analitzades l'augmentaven. Les construccions que eren estimulades en major grau presentaven la regio del promotor on es troben els polimorfismes. Tot i aixo, analitzant els diferents haplotips, vam observar que aquests no participaven en l'estimulacio induida per BMP2. "In silico" la regio estimulada per BMP2, presenta dues possibles caixes d'unio de factors de transcripcio de la via de la BMP2. En mutagenitzar-les per separat, pero, aquestes no van semblar participar en l'estimulacio induida per la BMP2. Seguidament vam analizar el patro d'expressio de CIZ/NMP4 en diversos teixits humans i vam observar que diverses isoformes s'expressaven simultaniament en tots ells i tambe en les cellules d'osteosarcoma humanes Saos-2 i MG-63. Es van identificar les isoformes 11H, CIZ6.1, 21H i 21H-I1. Aquestes dues ultimes van ser clonades i sobrexpressades de manera estable en les cellules Saos-2, les quals es van transfectar transitoriament amb el promotor del gen "COL1A1" i es van tractar amb BMP2. Els resultats van indicar que CIZ/NMP4 augmentava lleugerament la transcripcio del gen "COL1A1" en absencia de BMP2. En presencia de BMP2, i contrariament al que s'esperava, vam observar que la sobrexpressio de CIZ/NMP4 no tenia cap efecte. "SUMMARY: "ASSOCIATION AND FUNCTIONAL STUDIES WITH CANDIDATE GENES FOR OSTEOPOROSIS" TEXT: Osteoporosis, a complex disease determined by genetic and environmental factors, is characterized by a reduced bone mineral density (BMD) and an increased risk of fracture. In this thesis we studied the genetic component of osteoporosis through association and functional studies. The classical candidate genes for osteoporosis we analyzed were COL1A1, ESR1, VDR and TGFB1. The association studies were performed with the BARCOS cohort, a group of postmenopausal women from Barcelona. The results obtained in this cohort were included in a prospective metaanalysis called GENOMOS (20,000 samples). In the BARCOS cohort we observed that SNPs in the COL1A1 gene, their haplotypes and interactions, and interactions with SNPs in the VDR gene were associated with BMD. The haplotype LPX (ESR1) was also associated with BMD. In GENOMOS, although several SNPs were found to be associated with fracture risk, the Sp1 polymorphism (COL1A1) was the only one associated with BMD. In the BARCOS cohort we also found that one SNP situated in promoter 2 of RUNX2 gene, but not one situated in promoter 1, was associated with BMD. Finally, this was the first time that SNPs in IL6R gene were analyzed in relation to osteoporosis and we found that they were associated with BMD and also with body mass index (BMI). The second aim of this thesis was the functional study of two SNPs situated in the promoter of COL1A1 gene in relation to CIZ/NMP4 and BMP2 proteins. It was known that CIZ/NMP4, an architectural transcription factor that inhibits the BMP2 osteoblastogenic pathway, binded to the COL1A1 promoter. We observed that the transcription pattern of differnt constructs of COL1A1 promoter was similar between transient and stable transfections. BMP2 stimulated all these constructs, except for the basal promoter. The constructs that were stimulated more, contained the region where the SNPs lie, but when the haplotypes were analyzed no differences were observed between them. In this region there are two putative transcription factor sites for the BMP2 pathway, but they seemed not to participate in the BMP2 stimulation. Several isoforms of CIZ/NMP4 were simultaneously expressed in different tissues. We identified 11H, CIZ6.1, 21H and 21H-I1 isoforms. These two last were stably overexpressed in Saos-2 cells, which were then transiently tranfected with COL1A1 promoter and treated with BMP2. In absence of BMP2, overexpression of CIZ/NMP4 isoforms slightly increased COL1A1 transcription. "