Maurizio Cardelli, Francesca Marchegiani, Luca Cavallone, Fabiola Olivieri, Simona Giovagnetti, Elena Mugianesi, Raffaella Moresi, Rosamaria Lisa, and Claudio Franceschi conducted a study to identify a locus associated with human longevity in one of the chromosomal regions with the highest density of Alu elements, in 1p35. They identified a (TG)n microsatellite in the YTHDF2 gene by characterizing an anonymous marker detectable through inter-Alu fingerprinting. This marker previously evidenced an increased homozygosity in centenarians. After genotyping 412 participants of different ages, including 137 centenarians, they confirmed the increased homozygosity in centenarians at this locus and observed a concomitant increase in the frequency of the most frequent allele and corresponding homozygous genotype. The same genotype was associated with increased YTHDF2 messenger RNA levels in immortalized lymphocytes. Finally, YTHDF2 messenger RNA was mainly expressed in testis and placenta. The data suggest a possible role of this locus in human longevity.

Alu repetitive elements are present on all human chromosomes and have more than one million copies. They are the most abundant human SINEs (Short Interspersed Nuclear Elements) and transcribed by RNA polymerase III. Alu-rich genomic regions are considered potentially unstable due to unequal recombination events. Alu density positively correlates with gene density and is highly clustered near genes involved in metabolism, transport, and signaling processes.

The authors used an Alu-based DNA fingerprinting method to screen multiple loci in peculiar genomic regions particularly rich in Alu sequences. The method amplifies DNA fragments located between two closely spaced (usually no more than 1 kb) Alu sequences with opposite orientation, which occurs most frequently in Alu-rich genomic regions.

The aims of the present work were to identify and characterize the genomic locus corresponding to the anonymous QM-376-400 inter-Alu marker, previously reported to show an increased homozygosity in centenarians. They wanted to assess the existence of a genotype longevity association at this locus in a new, independent set of very old participants (centenarians) and younger controls. They also collected preliminary information about the functional role of the genetic variants of this locus eventually associated with longevity.

The PCR assay used primers R12A/267 labeled with tetrachlorofluorescein (TET), and R14B/264 labeled with 6-carboxyfluorescein (6-FAM); total reaction volume 50 ll, genomic DNA template 100 ng; 27 PCR cycles of 948C denaturation, 508C annealing, 728C extension. Capillary electrophoresis evidenced, in the resulting inter-Alu pattern, the presence of a 384 bp fragment corresponding to an allele of the QM 376-400 polymorphism. An aliquot of the PCR product was subjected to electrophoresis on a 1.5% agarose gel. The gel slice corresponding to an estimated length of 350-400 bp was cut, and the DNA was eluted and analyzed through capillary electrophoresis to confirm the presence and purity of the 384 bp fragment.

DNA was extracted from blood samples following standard procedures. Peripheral blood mononuclear cells were obtained from some samples. Peripheral blood mononuclear cell isolation, culture, and B-lymphocyte immortalization were performed as previously described. After immortalization, cells were harvested and frozen in liquid nitrogen two months later.