Analysis of biomarkers for complex human diseases

The aims of this study were to analyse known and potential biomarkers of common and genetically complex human disorders and to identify genetic and environmental variation associated with plasma biomarker concentrations. Two groups of protein biomarkers were analysed. First, plasma complement factor H (CFH) was selected as a potential biomarker for age-related macular degeneration (AMD), since common variants in the CFH gene show strong association with this disorder. Secondly, two isoforms of amyloid-β (Aβ40 and Aβ42) were selected as biomarkers for Alzheimer disease (AD) since Aβ deposits are major constituents of the amyloid plaques characteristic of this disorder. Physiological and anthropometric measurements and samples of human and genomic DNA were collected from a population sample of 1,021 individuals from the Croatian island of Vis. Quantitative determination of plasma Aβ40 and Aβ42 concentrations was performed using enzyme-linked immunosorbent assays. Heritabilities and significant covariate effects were estimated for each trait in the Croatian data set. Genome-wide linkage and association analyses were conducted for the biomarker traits. A novel finding was the genome-wide significant association between a CFH and several polymorphisms close to and within the CFH gene. The strongest association was with an intronic SNP within CFH, which explained 28% of the total trait variance (P < 10-50). The association was also replicated in a Dutch sample set. A SNP haplotype was identified which accounted for a higher proportion of the phenotypic variance. Conditional haplotype analysis showed that the effect of this haplotype on plasma CFH concentration was independent of the CFH Y402H variant, and significantly stronger than a deletion of the adjacent CFHR3/CFHR1 which was already known to affect AMD susceptibility. Genetic analysis of 382 AMD cases and 201 controls was consistent with the CFH Y402H variant being the strongest AMD susceptibility locus. Variation in plasma CFH concentration was found to explain up to 1.8% of the variation in susceptibility to AMD with an odds 2.1 (95% C.I. 1.3-3.4, P = 0.003). SNPs that were strongly associated with a CFH concentration also influenced AMD susceptibility (P < 0.05) independently of the CFH Y402H polymorphism. Functional analysis of genomic regions associated with plasma CFH is needed to identify the causal variants. Associations were observed between plasma Aβ40 concentration and several novel candidate loci, spanning regions of approximately 0.2 Mb, on chromosomes 9 and X. Similarly, novel associations with plasma Aβ42 were found in several regions, each spanning 0.2-0.4 Mb, on chromosomes 2, 5, 9, 15 and 20. The proportion of the phenotypic variance in plasma Aβ42 explained by these putative associations ranged between 1.8 and 2.8%. However, none of the associated SNPs was significant after correction for multiple testing, therefore replication is required. Finally, attempts were made to identify and quantitate new protein biomarkers of disease in human plasma using mass spectrometry. Development and optimisation of techniques was initially undertaken to deplete high-abundance plasma proteins and improve signal:noise ratio. This allowed the assessment of downstream proteomic approaches including MALDI-TOF mass spectrometry (MS), capillary electrophoresis (CE) and ion exchange chromatography (IEC), each with the potential for large-scale quantitation of plasma proteins. Although the analysis of single protein analytes, using CE and IEC proved promising, the results highlighted the difficulty associated with MALDI-TOF and protein ionisation techniques in analysing complex mixtures such as plasma