• Alzheimer’s disease
• association study
• BDNF
• microarray
• neurotrop
• neurotrophic factor
• neurotrophic factor receptor
• NGF
• NGFR
• NTRK1
• NTRK2
• Single Nucleotide Polymorphism (SNP)

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the most widespread genetic variations and represent the main investigation target in genetic association studies, which aim at revealing the genetic contribution to complex disease vulnerability. In the last few years, efficient high-throughput methodologies for SNP genotyping have been developed, many of which rely on microarray technology. Genotypes are assessed by comparing the signal intensities of allele-specific probes, which are labeled either by using four fluorescent dyes (one for each base), and hybridized on a single array, or by using only two dyes, thus investigating the polymorphic alleles, two by two, on separate arrays. The Two-Array approach shows the advantage of being practicable in every microarray laboratory, since it requires a dual-laser scanner, rather than a more expensive tetra-laser scanner. As a drawback, inferring all the six possible genotypes is not always feasible, because a number of systematic errors in the experimental protocol may differently affect signal intensities on separate arrays, thus making their comparison difficult.
AIM OF THE STUDY: The first part of this work was aimed to develop and setup TAMGeS (Three-Array Method for Genotyping of SNPs), an exhaustive method for SNP genotyping through SBE (Single Base Extension) on dual-color microarrays, which allows for normalization of signals on distinct arrays by a bilinear regression theory-based approach.
TAMGeS was then employed to genotype 21 SNPs within genes coding for nerve growth factor beta (NGFB), brain-derived neurotrophic factor (BDNF), and their receptors, nerve growth factor receptor (NGFR), neurotrophin tyrosine kinase receptor 1 and 2 (NTRK1 and 2), in Italian sporadic (n = 151) and familial (n = 100) Alzheimer’s disease (AD) patients (SAD and FAD, respectively), and healthy subjects (CTRL, n = 97), since growing evidence indicates these genes as implicated in AD pathogenesis.
RESULTS: TAMGeS efficiency was evaluated, in comparison with the Two-Array method, on both experimental and synthetic data. With synthetic data, TAMGeS reduced the frequency of errors by an order of magnitude, when the incidence of systematic errors was not negligible. With the experimental data (87 samples), TAMGeS reduced the percentage of missing genotypes from 54% (Two-Array Method) to 16%. Global performance of TAMGeS resulted in 84.1% genotype and 84.4% allele calls on average. The association study between neurotrophin system genes and AD showed that in single SNP analysis, allele-wise association was detected for rs2072446 (Ser205Leu) on NGFR in FAD (P = 0.047), while the intronic SNP rs2289656 on NTRK2 showed genotype-wise association in SAD (P = 0.0036). After sample stratification according to age at onset (65 years as cutoff), rs6336 (Hys568Tyr) on NTRK1 resulted associated to early-onset SAD in both allele- and genotype-wise analysis (P = 0.028 and 0.014, respectively), while rs1048218 (Gln75His) on BDNF showed allele-wise association in late-onset SAD (P = 0.047). Other SNPs demonstrated a trend to association in SAD and/or in FAD. In multi-locus analysis, rs1048218 and rs6336 showed epistatic interaction in FAD (P < 0.001).
CONCLUSIONS: TAMGeS represents an innovative method, which proved to be very effective in producing reliable SNP genotyping data by dual-color microarrays. The requirement of a third array is well balanced by the strong enhancement in data quality and by the greater flexibility of the experimental protocol. The results of the association study suggest that the neurotrophin system might be implicated in conferring genetic vulnerability to AD in our Italian sample. However, further investigations are required to better elucidate the role of neurotrophins and their receptors in AD pathogenesis.