A genetic-epidemiologic study of Alzheimer’s disease

textabstractAlzheimer's disease (AD) is the most frequent cause of dementia and thus is a major public-health problem. Age and genetic predisposition to the disease are the most important risk factors. In 2001 more than 24 million people in the western world had dementia. This number is expected to double every 20 years up to 81 million in 2040 because of the anticipated increase in life expectancy. Genetically, AD is a heterogeneous disorder with both familial and sporadic forms. Chapter 1 is a general introduction on epidemiological and genetic factors of AD and also describes the different studies performed in this thesis. In chapter 2 the linkage studies are presented. Chapter 2.1 describes a whole genome screen on 112 AD patients and their first-degree relatives from the Genetics Research in Isolated Populations (GRIP) study. Of the 112 patients, 103 could be connected into an extremely large and complex pedigree. This pedigree cannot be analyzed with available linkage software. In this study, we developed an algorithm for splitting complex pedigrees to allow us to conduct linkage analysis. Then we determined genome wide significance thresholds for linkage analysis using the sub pedigrees obtained by our pedigree cutting algorithm and finally, we performed linkage analysis sing these sub pedigrees. The strongest evidence of linkage was for chromosome 1q21 (HLOD = 5.20, at marker D1S498), which is a locus identified earlier. We confirmed the AD locus at 10q24 (HLOD = 4.15, at marker D10S185). There is also significant evidence of linkage at chromosomes 3q23 (HLOD = 4.44 at marker D3S1569) and! 18q12 (HLOD = 3.68, at marker D18S1152). Several single families contributed strong linkage signals on chromosomes 1, 3 and 18. Multiple families contributed moderate linkage signal to chromosome 10q24 locus. Finally, there is also suggestive evidence of linkage at chromosome 11q24 (HLOD = 3.29, at marker D11S1320). We sequenced the obvious candidate genes on chromosome 1, nicastrin (NCST) and C-reactive protein (CRP) but no mutations on these genes were found. We also sequenced 21 genes on the locus on chromosome 3 including the transferrin gene, the gene encoding for butyrilcholinesterase, the neprilysin gene and the somatostatin gene. We screened these genes for mutations but no variants were found. In chapter 2.2 we present a combined linkage and association study between the microtubule associated tau protein (MAPT) and granulin (GRN) genes and AD. In this study we investigated the association between the region including MAPT plus other eight confirmed genes (including the GRN gene) and five putative genes and AD in patients from the GRIP study. We studied 122 AD patients and 85 control subjects using an extensive Single Nucleotide Polymorphism (SNP) panel. No association was found for any individual SNP of the MAPT gene and linkage analysis including 7 tagging SNPs in MAPT excluded linkage. Nominally significant association was found for two SNPs located 722 kb upstream the MAPT gene in the N-myristoyltransferase 1 (NMT1) gene. The GRN gene was sequenced in 17 patients but no mutations were found. We also performed a meta analysis of all studies conducted to date on the associations between the MAPT gene and AD stratified by APOE*4. We found a very modest non-signif! icant association for carriers of the H2 haplotype compared to H1/H1 carriers, only in those with the APOE*4 allele. These findings suggest that the MAPT and GRN genes do not play a role in AD in our population. Aiming to find genes involved in AD, in chapter 2.3 we performed a genome wide linkage analysis on an extended Dutch family with nine affected individuals. Two linkage peaks above 1.0 were found on chromosomes 1 and 7. A further two locus linkage analyses using only affected individuals yielded a LOD score of 2.92 for the combination of these 2 loci suggesting an epistatic effect between these two loci. We used a literature mining strategy to prioritize candidate gene selection from the genes present in the defined loci. Our literature mining approach suggested as potential candidate genes in these two regions the CASP9, TNFRSF1B and MTHFR genes on chromosome 1 and CDK5 and NOS3 on chromosome 7. These genes have been previously associated with AD and apoptosis. CASP9 on chromosome 1 and CDK5 on chromosome 7 were selected for sequencing. We found no variants in the coding exons or the splice sites of these genes but we found the rare allele of SNP rs9872 on the CDK5 gene pres! ent in all affected individuals suggesting that this gene maybe involved in AD in this family. Chapter 3 describes the genetic association studies performed in this thesis. Three studies were carried out using the Rotterdam Study (chapters 3.1-3.3) a large prospective study of determinants of diseases in the elderly and one in the GRIP study (chapter 3.4). In chapter 3.1 we tested the association between 5 SNPs on the CDK5 gene and AD. We found a significantly increased risk of AD for carriers of the GG genotype of SNP rs2069442 (OR=1.8, 95% CI 1.16-2.79, p=0.009) in those without APOE*4. Our haplotype analysis showed that haplotype C, tagging the G allele of SNP rs2069442, was significantly associated (p=0.05) to AD. These result suggest that alterations in the expression of the Cdk5 protein may be relevant in neurodegenerative processes Taken together, the results of chapters 2.3 and 3.1 underscore the relevance of this gene on AD. We also evaluated the relationship between the cholesteryl ester transfer protein (CETP) and AD in chapter 3.2. Here we show that there is a positive association between the VV carriers of the I405V polymorphism of the CETP and AD independently of the APOE gene and systemic HDL levels. Since the VV genotype is associated with lower levels of CETP and higher HDL, our study is compatible with the view that low CETP levels may increase the risk of AD through a reduction in neuronal repair capacity. In chapter 3.3 we investigated the relationship between the ubiquilin 1 (UBQLN1) gene and AD, Parkinsonâ? Ts disease (PD) and cognitive function. This gene is involved in the ubiquitination machinery, related with general protein degradation. A SNP located downstream of exon 8 (rs12344615) of the UBQLN1 gene has been associated with AD. We found no significant difference in risk of AD or PD for carriers of the CC genotype of SNP 12344615 in our overall and stratified analyses in the Rotterdam Study. We did not find an association of this SNP and AD in our family based study, we did not find linkage of AD to the region including the UBQLN1 gene, and we detected no effect of this polymorphism on cognitive function. Our data suggest that it is unlikely that the SNP 12344615 of the UBQLN1 gene is related to AD, PD or cognitive function. Chapter 3.4 shows the relationship between a 14-kb deletion extending into the DJ-1 gene and dementia using a case-control study of dementia patients and controls ascertained from GRIP study. We analyzed 191 demented patients and 129 non-demented controls. We found no association between this deletion and the presence of dementia. There were 3 heterozygous carriers of the DJ-1 gene deletion (two affected individuals and one control) that were related within 8 generations suggestion a common origin of the deletion. Form our results we cannot exclude that carriers of this deletion in the DJ-1 gene have a higher risk of developing AD. Variations in the DJ-1 gene different from the deletion may be associated with the risk of dementia. However, our results show that haploinsufficiency in the DJ-1 gene is not a major risk factor for dementia in this isolated population. Finally, chapter 4 provides a general discussion on the results obtained on the studies on AD presented in this thesis and all findings are put into perspective for future studies.