Diversitat Genòmica a Les Poblacions Del Nord D'àfrica

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Diversitat Genòmica a Les Poblacions Del Nord D'àfrica Diversitat genòmica a les poblacions del nord d’Àfrica Elena Bosch Fusté 2000 Dip òsit legal: B.24348-2003 ISBN: 84-688-2285-X Departament de Ciències Experimentals i de la Salut (en constitució) UNIVERSITAT POMPEU FABRA Diversitat genòmica a les poblacions del nord d’Àfrica Memòria presentada per Elena Bosch Fusté per optar al grau de Doctor per la Universitat Pompeu Fabra Jaume Bertranpetit i Busquets Elena Bosch Fusté Director de tesi Barcelona, febrer del 2000 Als meus pares, al meu germà i a en Francesc Agraïments Des de finals d’agost de l’any 1995 són moltes les persones que amb paciència i bon humor m’han animat, recolzat i ajudat a realitzar aquest treball. Vull expressar-los ara el meu reconeixement. En primer lloc, m’agradaria agrair a en Jaume Bertranpetit, director d’aquesta tesi, l’oportunitat que em donà per introduir-me a aquest món de la ciència. Puc dir ben alt i amb seguretat que m’honora haver-lo tingut com a mestre. He gaudit enormement amb el seu entusiasme i gran tafaneria científica. També un agraïment ben carinyós a en Francesc Calafell per la seva extraordinària paciència, tant dins com fora el laboratori, i per la seva inestimable ajuda en l’estadística, redacció i discussió de tot el que ha passat entre les nostres mans. Voldria agrair de manera molt especial l’ajuda desinteressada, actitud diplomàtica en tota mena de reunions o comitès d’ètica d’hospitals i el gran esperit aventurer de la Cristina Junyent; el bon rotllo i les explicacions per a realitzar les meves primeres tècniques de laboratori per part d’en David Comas, també company de viatge juntament amb la Cristina i en Jaume; les pacients classes de tot el que necessitava saber i més sobre els microsatèl·lits i el seu tipatge, així com el bon humor i exageracions catastròfiques de l’Anna Pérez; els petits consells i secrets sobre alguns protocols a la Blanca Gutiérrez i a la Neus Valveny; l’ajuda de la Rosa Martínez i l’Eva Mateu en l’expedició de València i sortides de matinada cap a Vic; el gran esforç realitzat per la mateixa Rosa, l’Araceli Rosa i en Jordi Clarimón per mimar al màxim els cultius cel·lulars, així com l’ajuda d’en Jordi en l’extracció de DNA de les darreres mostres que obtinguerem del Marroc. El meu sincer agraïment també a tota la resta de companys de laboratori, per la vostra companyia, acudits, i aventures en els moments de feina, així com en els àpats i sobretaules compartides. A la Unitat d’Antropologia (UB) són la Bàrbara Arias, la Mireia Esparza, l’Esther Esteban, l’Antonio González, en Carles Lalueza, en Toni López, i en Marc Via; i ara, a Biologia Evolutiva (UPF), l’Aida Andrés, en Xavier Domingo, en Josep Marmi, en Bernal Morera, l’Stephanie Plaza, i la Mònica Vallès. M’agradaria reconèixer també el suport rebut en dues estades a l’estranger. A Anglaterra, al Departament de Bioquímica de la Universitat d’Oxford on vaig trobar la càlida companyia d’en Fabrício R. Santos, de l’Arpita Pandya, de la Giovanna Arpidia, d’en Williams, d’en John, de la Milli, d’en Carlos, de l’Angela, d’en Leslie i del professor Chris Tyler-Smith. Als Estats Units, al Departament de Genètica de la Universitat d’Stanford on vaig conèixer en Peter A. Underhill, l’Anna Hurlbut, en Peidong Shen, l’Alice Lin, en Peter J. Oefner, la Phillys, la Silvia, en Titus, l’Adrianne i la Wie. Gràcies també a la Kay, la Cher, la Paula, la Marcie, i la Marga per la seva companyia a Montain View. També vull agrair el suport dels professors de la Unitat d’Antropologia de la Universitat de Barcelona, Lourdes Fañanás, Clara García Moro, Miquel Hernández, Pedro Moral, Pasqual Moreno, Txomin Toja i Daniel Turbón. I un record especialment carinyós al doctor Josep Pons, company d’aventures científiques del meu avi, temps enrere. Tenen el meu reconeixement també tots aquells donants de sang que acceptaren participar en aquest estudi. Sens dubte, gràcies a ells aquest treball ha estat possible. Igualment, voldria agrair la col·laboració inestimable d’investigadors de diverses universitats i de metges, infermeres i ajudants de diferents hospitals i dispensaris durant la recol·lecció de mostres. Són Omar Akhayat, Zahra Brakez, Hassan Izaabel, Omar Ouakrim (Agadir); Oriol Vall (Barcelona); Noufissa Benchemsi (Casablanca); Xavier Balanzó, Alba Bosch, Jordi Colomer, Josep Lluís Fernández Roure, Carme i Glòria (Mataró); Abdelaziz Sefiani (Rabat); Khadietu, Adda Brahim, Omar Mansur i Baha Mustafa (Sàhara Occidental); Elisabeth Pintado (Sevilla); Anne Cambon-Thomsen, Jean-Michel Dugoujon, Ghania Hariti (Toulouse); Enric Bufill, Assumpta i Montse (Vic); Enric Padrés, Felip Pi, doctora Prats i Pere Simonet (Viladecans), entre altres. Gràcies també a l’Amaia, l’Anna, la Carme i en Ramon de la Unitat de Seqüenciació del Servei Científico-Tècnic de la Universitat de Barcelona per la seva assistència, ajuda i consells, setmanana rera setmana. Un record per a les amigues Anna Campalans i Sílvia Busquets, antigues companyes de carrera, per tots els dinars dels dimecres on sovint rèiem de les nostres petites potineries al laboratori. Finalment, també vull agrair als meus pares i al meu germà el seu suport continu. Aquest treball l’he dut a terme mentre gaudia d’una beca de formació de personal investigador del Comissionat per a Universitats i Recerca, de gener de 1996 a desembre de 1999 (FI/96- 1.153). A més, el treball ha comptat amb les subvencions de la Dirección General de Investigación Científica y Técnica als projectes “Variación del genoma humano: análisis de diversas regiones genómicas de especial interés” (PB95-0267-CO2-01); i del Comissionat per a Universitats i Recerca com a Grup de Recerca Consolidat (1996SGR00141, 1998SGR00009). ÍNDEX PRESENTACIÓ 19 INTRODUCCIÓ 1. Nord d’Àfrica 1.1 Marc geogràfic 28 1.2 El poblament prehistòric 29 1.3 Història de les poblacions del nord d’Àfrica 30 1.4 El poble berber, àrab i sahrauí 32 1.5 Les llengües del nord d’Àfrica..... 33 2. Marcadors clàssics 2.1 Descripció i principals característiques 36 2.2 Aplicacions a la genètica 37 3. Els microsatèl·lits 3.1 Descripció i principals característiques 39 3.2 Mutació en microsatèl·lits 41 3.3 Aplicacions a la genètica 42 4. El cromosoma Y 4.1 Caracterització 44 4.2 Tipus de polimorfismes 46 4.3 Estructura de la diversitat genètica: haplogrups i haplotips 47 4.4 Aplicacions a la genètica 48 OBJECTIUS 55 MATERIALS I MÈTODES 1. Poblacions estudiades 61 2. Obtenció, purificació i quantificació de DNA 65 3. Anàlisi de microsatèl·lits 3.1 Caracterització dels microsatèl·lits autosòmics estudiats 66 3.2 Caracterització dels microsatèl·lits específics del cromosoma Y estudiats 69 3.3 Tipatge 69 4. Anàlisi de polimorfismes bial·lèlics 4.1 Polimorfismes analitzats per RFLP o Restriction Fragment Length Polymorphism 72 4.2 Polimorfismes analitzats mitjançant hibridació amb sondes específiques 74 4.3 DHPLC o Denaturing High Performance Liquid Chromatography 74 4.4 Filogènia de la variació en el cromosoma Y 78 5. Tractament estadístic 5.1 Elaboració d’una base de dades de freqüències al·lèliques recopilada de la literatura 82 5.2 Components principals: concepte estadístic i aplicació en genètica de poblacions 82 5.3 Distàncies genètiques 85 5.4 Representació gràfica en arbre de les distàncies genètiques 86 5.5 Validació de distàncies genètiques i arbres: bootstrap 87 5.6 Coordenades principals 87 5.7 Detecció de fronteres genètiques 88 5.8 Analysis of Molecular Variance o AMOVA 88 5.9 Paràmetres emprats per a la descripció de la variació en haplotips de microsatèl·lits 89 RESULTATS Capítol I: Population History of North Africa: Evidence from Classical Genetic Markers 95 Capítol II: Genetic structure of northwestern Africa revealed by STR analysis 115 Capítol III: Y chromosome STR haplotypes in four populations from northwestern Africa 153 Capítol IV: Variation in Short Tandem Repeats is deeply structured by genetic background on the human Y chromosome 171 Capítol V: Y chromosome lineages and northwestern African populations 193 DISCUSSIÓ 227 BIBLIOGRAFIA 253 PRESENTACIÓ El treball que presento a continuació pretén estudiar la variabilitat genètica de les poblacions del nord d’Àfrica a partir de l’anàlisi de diverses regions genòmiques per tal d’entendre les poblacions analitzades per una banda, i compendre la dinàmica del genoma per l’altra. Comença amb una Introducció on es caracteritzen les poblacions del nord d’Àfrica, així com les diferents eines genètiques emprades per al seu estudi. Segueixen els Objectius on presento les principals fites que es pretenien assolir en aquesta tesi. A continuació, a Materials i Mètodes s’introdueixen les poblacions estudiades, els marcadors tipificats, les tècniques emprades per a la seva anàlisi i els principals mètodes estadístics utilitzats al llarg del treball. Els Resultats obtinguts s’exposen en cinc capítols que corresponen a un article publicat a la revista Human Biology on es realitza una anàlisi preliminar de la variació genètica al nord d’Àfrica a partir de marcadors clàssics (Capítol I); a dos articles acceptats, respectivament, a les revistes European Journal of Human Genetics i International Journal of Legal Medicine en els quals es treballa amb la informació genètica aportada pels microsatèl·lits autosòmics (Capítol II) i pels microsatèl·lits específics del cromosoma Y (Capítol III); a un article acceptat a la revista American Journal of Human Genetics on plantegem com s’estructura la variació dels microsatèl·lits en el cromosoma Y (Capítol IV); i a un article en preparació basat en la informació aportada per marcadors bial·lèlics específics del cromosoma Y tipats mitjançant DHPLC (Capítol V).
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