Title: Implication of a Genetic Variant at PICALM in Alzheimer's Disease Patients and Centenarians

Authors: Irene Piaceri, Silvia Bagnoli, Ersilia Lucenteforte, Michelangelo Mancuso, Andrea Tedde, Gabriele Siciliano, Silvia Piacentini, Laura Bracco, Sandro Sorbi and Benedetta Nacmias.

Affiliations: 
a Department of Neurological and Psychiatric Sciences, University of Florence and Centro di Ricerca, Trasferimento e Alta Formazione DENOTHE, University of Florence, Viale Morgagni, Firenze, Italy 
b Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy 
c Department of Neuroscience, Neurological Clinic, University of Pisa, Pisa, Italy

Handling Associate Editor: Daniela Galimberti 

Abstract: 
A common polymorphism (rs3851179) in the PICALM (phosphatidylinositol-binding clathrin assembly protein) gene has been recently associated with reduced risk of developing late-onset Alzheimer's disease (LOAD). We analyzed the genotype and allele distributions of the PICALM polymorphism in 813 Italian subjects, including LOAD patients and centenarians. The segregation of the PICALM rs3851179 showed no statistically significant difference between LOAD cases and controls. The implication of a genetic variant at PICALM is confirmed for the first time in centenarians, thus suggesting a possible role in longevity.

Keywords: Alzheimer's disease, apolipoprotein E, centenarian, genetic variation, PICALM 

Introduction:
For almost twenty years, numerous molecular genetic studies in patients with the familial form of Alzheimer's disease (FAD) have revealed more than 220 pathogenetic mutations in the three candidate genes (APP, PS1, PS2) contributing to the pathogenesis of Alzheimer's disease (AD) [1]. Despite numerous years of research, only one common genetic variant 4 (the 4 allele) in the gene encoding apolipoprotein E (APOE) has been considered a risk factor for the disease. It has become clear that the majority of common genetic variants contribute only slightly to an increased risk for the disease, but in combination, they have an important impact on disease predisposition. Recently, three large Genome-wide association (GWA) studies, including more than 48,000 subjects, tested hundreds of single nucleotide polymorphisms (SNPs) and verified strong evidence of five new susceptibility loci for AD, CLU (clusterin, chr 8), PICALM (phosphatidylinositol-binding clathrin assembly protein, chr 11), CR1 (complement component [3b/4b] receptor 1, Chr 1), BIN1 (bridging integrator 1, chr 2) and EXOC3L2 (exocyst complex component 3-like 2, chr 19) [24]. Several large case-control studies have recently confirmed the role of PICALM in more than 23,000 subjects [58].

Methods:
All subjects were enrolled at the Neurology Unit of the Department of Neurological and Psychiatric Sciences of the University of Florence and Department of Neuroscience, Neurological Clinic, of the University of Pisa. The studied group consisted of 813 Italian subjects: 349 sporadic LOAD patients, 359 sex-and age-matched healthy individuals as a control group and 105 centenarians (54.3% aged >100 years). Presence of a family history of dementia (at least two first-degree relatives in two generations) was considered an exclusion criterion. All controls were carefully assessed using a rigorous clinical history evaluation and a general/neurological examination in order to exclude the presence of any neurological disorder. They were interviewed to exclude the presence of relatives with exceptional longevity (>100 years) in past generations.

Total DNA was isolated from peripheral blood using standard methods. The analysis of the rs3851179 was performed using High-Resolution Melting Analysis (HRMA), and the three genotypes were identified through sequencing (310 ABI PRISM Genetic Analyzer, Applied Biosystem). Moreover, we determined the APOE genotypes in all the studied subjects using polymerase chain reaction (PCR) and the restriction fragment length polymorphism (RFLP) method.