Inherited retinal degenerations are a group of disorders characterised by great genetic heterogeneity. Clinically, they can be divided into two large groups of diseases, those associated with night blindness, e.g. retinitis pigmentosa (RP), and those with macular malfunction, e.g. cone/cone-rod dystrophy (COD/CORD). This thesis is focused on finding the genetic basis of disease in families with autosomal dominant COD, autosomal dominant RP, and Bothnia dystrophy (BD), a regional variant of RP.   A variant of COD was previously mapped to 17p12-p13 in a family from northern Sweden. One additional family originating from the same geographical area was included in fine mapping of this chromosome region. Using 12 microsatellite markers in linkage and haplotype analysis, the region was refined from 26.9 to 14.3 cM. A missense mutation, Q626H, in an evolutionarily conserved region of PITPNM3, phosphatidylinositol transfer membrane-associated protein, was identified. The mutation segregated with the disease in both families and was absent from normal control chromosomes. PITPNM3 is a human homologue of the Drosophila retinal degeneration (rdgB) protein, which is highly expressed in the retina and has been proposed to be required for membrane turnover of photoreceptor cells. With the intention of establishing the global impact that PITPNM3 has on retinal degenerations 165 DNA samples from COD and CORD patients were obtained from Denmark, Germany, the UK, and USA and screened for mutations. The Q626H mutation found in the Swedish families was also found in one British family and a novel Q342P variant was detected in a German patient. In addition, two intronic variants were identified: c.900+60C>T and c.901-45G>A. Thus, we concluded that mutations in PITPNM3 represent a rare cause of COD worldwide. In two large families from northern Sweden showing autosomal dominant RP with reduced penetrance, the disease locus was mapped using genome-wide linkage analysis to 19q13.42 (RP11). Since mutation screening of eight genes on 19q13.42 revealed no mutations, multiplex ligation-dependent probe amplification (MLPA) was used to screen for large genomic abnormalities in PRPF31, RHO, RP1, RPE65, and IMPDH1. A large deletion spanning 11 exons of PRPF31 and three genes upstream was identified. Using long-range PCR, the breakpoints of the deletion were identified and the size of the deletion was determined to encompass almost 59 kb. BD is an autosomal recessive type of RP with high prevalence in northern Sweden. The disease is associated with a c.700C>T mutation in RLBP1. In a screening of recessive RP in northern Sweden, 67 patients were found to be homozygous for c.700C>T and 10 patients were heterozygous. An evaluation with arrayed primer extension (APEX) technology revealed a second mutation, c.677T>A, in RLBP1 giving rise to compound heterozygosity in these patients. In addition, a c.40C>T exchange in CAIV was detected in a patient with BD and in 143 healthy blood donors. The c.40C>T substitution in CAIV has been reported to cause autosomal dominant RP in South African families with European ancestry. However, in the population of northern Sweden it appears to be a benign polymorphism. In summary, a first mutation in PITPNM3, encoding a human homologue of the Drosophila retinal degeneration protein, was detected in two large families with COD. A large deletion in PRPF31 was discovered in two families with autosomal dominant RP showing reduced penetrance and in 10 patients BD was shown to be caused by two allelic mutations in RLBP1.

Prostate cancer is a major health burden throughout the world, yet the etiology of prostate cancer is poorly understood. Evidence has accumulated supporting the existence of a hereditary form of this disease. Improved understanding of the genetic mechanisms underlying the development and progression of prostate cancer would be a major advance for improved prevention, detection and treatment strategies. This thesis evaluates different aspects of the genetic epidemiology of prostate cancer. In a genomic scan two chromosomal regions with evidence for linkage was observed. The strongest support was found on chromosome 19p with an allele sharing LOD score of 2.91 (genome-wide P = 0.032). The second region, showing suggestive evidence of linkage, was observed in the centromeric region of chromosome 5. Linkage analyses of densely spaced markers on chromosome 8p22-23 confirmed (P = 0.03) previously reported linkage to this region. A systematic evaluation of the possible impact that the RNASEL gene have on prostate cancer was performed. Overall, limited evidence for association with prostate cancer risk was found. The results provide strong evidence against a role of RNASEL in prostate cancer etiology in Sweden. In a comprehensive evaluation of occurrence of other malignancies in HPC families, previously reported association between gastric and prostate carcinoma was confirmed. The increased risk was of the same magnitude in early and late onset HPC families and confined to only male relatives. A genome-wide linkage analysis, stratified by occurrence of gastric carcinoma, identified a novel susceptibility locus on chromosome Xp21. In summary, chromosome 5q and 19p represents the regions most likely to harbor susceptibility genes predisposing to prostate cancer in the Swedish population. A common genetic basis for both gastric and prostate cancer has been confirmed and a novel susceptibility locus on chromosome Xp21 has been identified.

The aim with this thesis has been to identify genes involved in the pathogenesis of Systemic Lupus Erythematosus (SLE). SLE is a systemic autoimmune disorder, most likely caused by both several genetic and environmental factors. In order to identify susceptibility loci for the disease we performed linkage analyses on data from 70 families of various ethnic origins. Significant linkage was found in two regions. One region (chromosome 17p12-q11) was linked to SLE in a set of Argentine families. Since the same region had been previously identified in several linkage studies on Multiple Sclerosis patients, we propose that this locus may contain a genetic variant that affects not only SLE, but also autoimmunity in general. The second locus is located on chromosome 4p14-13 and has only been identified in a set of Icelandic families. We suggest that this locus contains a mutation that has been enriched in the Icelandic population due to its population history. The BCL2 gene has been suggested as a candidate gene for SLE. Three markers in this gene were investigated for association with the disease in two different populations. However, no association could be found with any of the markers or when these markers were analysed together as a haplotype. We conclude that the BCL2 gene is not associated with SLE in our material. This result contradicts previously published results of an association between BCL2 and SLE. We suggest that the PD-1 pathway (involved in inhibition of T- and B-cell responses) is an important component in SLE pathogenesis. A regulatory variant in the PD-1 gene had previously been associated with SLE and here we show strong association (p<0.0001) to a haplotype containing SNPs in both PD-L1 and PD-L2. Our results indicate that SLE is a disease caused by several genetic variations that differ between families and populations.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-18T13:21:11Z No. of bitstreams: 2 Dissertação - Ana Paula Simplício Mota - 2015.pdf: 2622408 bytes, checksum: 964fc3216d1a8c8e5e27bc1ef2a5d5be (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-18T13:21:34Z (GMT) No. of bitstreams: 2 Dissertação - Ana Paula Simplício Mota - 2015.pdf: 2622408 bytes, checksum: 964fc3216d1a8c8e5e27bc1ef2a5d5be (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-18T13:21:34Z (GMT). No. of bitstreams: 2 Dissertação - Ana Paula Simplício Mota - 2015.pdf: 2622408 bytes, checksum: 964fc3216d1a8c8e5e27bc1ef2a5d5be (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-10-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The aim of this study was to validate molecular markers specifically associated with alleles of the Co-4 gene that controls the reaction to anthracnose of common bean, in particular the Co- 42 allele, and foment the integration of these markers in breeding programs aimed at developing resistant cultivars. For this, we evaluated 261 individuals F2 and 197 F2:3 progenies coming from crosses between SEL 1308 (carrier of Co-42 allele) and BRS Cometa. The studies confirm that the inheritance of reaction to anthracnose in SEL1308, using race 73 of Colletotrichum lindemuthianum, it is conditioned by an allele with complete dominance. In all, 15 markers were analyzed, ten STS (Sequence-Tagged Sites) and two SSR (Simple Sequence Repeat), recently identified by the Embrapa research group, and three SCAR (Sequence Characterized Amplified Regions) previously reported in the literature. Of these, 13 were polymorphic between the parents and segregated in the expected proportions to generations analyzed (3:1 or 1:2:1). Among the markers linked in repulsion phase, six STS (P8283-V1, P8284 -V1, P8285-V2, P8286-V1, P8286-V2 and P8286-V3) co-segregated at a distance of 2,64 cM from the allele Co-42 and 0,0 cM apart. As regards the SCAR markers, it was found that all are linked in coupling phase of the Co-42 allele at distances ranging from 2,93 cM (SAS13) at 6,42 cM (SH18). Of codominant markers, the STS P8286-V6 was mapped closest to Co-42 allele at a distance of 2,58 cM. Thus, the STS tested are excellent tools markers for assisted selection of genotypes resistant to anthracnose, since that present the highest selection efficiency estimates (84 - 99%) and the detection of plants carriers of different alleles of the Co-4 gene. However, only the SCAR SH18 possible the specific breakdown of Co- 42 allele, despite having less selection efficiency (85%) compared to the other analyzed markers. Therefore, it is recommend the combined or sequential use of P8286-V6 and SH18 markers for Co-42 selection at the expense of other alleles of the Co-4 gene. As for the selection of resistant genotypes in populations in which only the Co-42 allele is present, it is suggested to use only the P8286-V6 marker, which stands out for being codominant and strongly associated with the resistance allele. From a practical standpoint, the molecular markers validated in this study have shown great potential for use in the development of common bean cultivars resistant to anthracnose, being affordable to laboratories with different levels of infrastructure and highly efficient in monitoring genotypes carriers of resistance alleles of the loco Co-4. / O objetivo desse estudo foi validar marcadores moleculares especificamente associados aos alelos do gene Co-4 que controla a reação à antracnose do feijoeiro-comum, em particular ao alelo Co-42, e fomentar a integração destes marcadores a programas de melhoramento visando ao desenvolvimento de cultivares resistentes. Para isso, foram avaliados 261 indivíduos F2 e 197 progênies F2:3, provenientes de cruzamentos entre SEL 1308 (portadora do alelo Co-42) e BRS Cometa. Os estudos confirmaram que a herança da reação à antracnose em SEL 1308, usando a raça 73 de Colletotrichum lindemuthianum, é condicionada por um alelo com dominância completa. Ao todo, foram analisados 15 marcadores, sendo dez Sequence-Tagged Sites (Sítios Marcados por Sequências) e duas Simple Sequence Repeat (Sequências simples repetidas), identificadas recentemente pelo grupo de pesquisa da Embrapa, e três Sequence Characterized Amplified Regions (Regiões Amplificadas Caracterizadas por Sequências) previamente relatados na literatura. Destes, 13 foram polimórficos entre os genitores e segregaram nas proporções esperadas para as gerações analisadas (3:1 ou 1:2:1). Dentre os marcadores ligados em fase de repulsão, seis STS (P8283-V1, P8284-V1, P8285-V2, P8286-V1, P8286-V2 e P8286-V3) co-segregaram a uma distância de 2,64 cM do alelo Co-42 e a 0,0 cM entre si. No que se refere aos marcadores SCAR, constatou-se que todos estão ligados em fase de acoplamento ao alelo Co-42, a distâncias que variaram de 2,93 cM (SAS13) a 6,42 cM (SH18). Dos marcadores codominantes, o STS P8286-V6 foi mapeado como mais próximo do alelo Co-42, a uma distância de 2,58 cM. Dessa forma, os marcadores STS testados constituem excelentes ferramentas para a seleção assistida de genótipos resistentes à antracnose, visto que apresentaram as maiores estimativas de eficiência de seleção (84 a 99%) e a detecção de plantas portadoras dos diferentes alelos do gene Co-4. Contudo, somente o SCAR SH18 possibilitou a discriminação específica do alelo Co-42, apesar de apresentar menor eficiência de seleção (85%) em comparação aos demais marcadores analisados. Diante disso, recomenda-se a utilização combinada ou sequencial dos marcadores P8286-V6 e SH18 para seleção do Co-42 em detrimento dos demais alelos do gene Co-4. Já para a seleção de genótipos resistentes em populações nas quais somente o alelo Co-42 está presente, sugerese a utilização apenas do marcador P8286-V6, que se destaca por ser codominante e fortemente associado ao alelo de resistência. Do ponto de vista prático, os marcadores moleculares validados neste estudo demonstraram grande potencial para utilização no desenvolvimento de cultivares feijoeiro-comum resistentes à antracnose, por serem acessíveis a laboratórios com diferentes níveis de infraestrutura e altamente eficientes no monitoramento de genótipos portadores de alelos de resistência para o loco Co-4.

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