Young Participants Centenarians Alleles (Alleles ¼ 550) (Alleles ¼ 274) 15 27.5 36.1 16 25.6 26.6 22 13.6 13.9 Other alleles 33.2 23.4

Notes: *Genotypes with frequencies higher than 10% in either centenarians or young participants are listed, whereas all other genotypes are grouped together and used as the reference category for odds ratio calculations.

**p values refer to the Pearson chi-square tests comparing the reference category ‘‘Other genotypes’’ with ‘‘15-15’’ or with ‘‘15-16’’ genotypes, respectively. For the global Pearson chi-square test comparing the three genotype categories in young subjects vs centenarians, p ¼ .047.

Note: Pearson chi-square, p ¼ .014. Alleles were analyzed if their frequencies were higher than 10% in either centenarians or young participants, and all other alleles were grouped together.

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  “15-15”fs4,92“other genotypes”        e©rtaowmortno(yun Aregique)uopsseidxo @AREIAI WNW ZACHLA 

Figure 3. Relative YTHDF2 messenger RNA (mRNA) expression determined by real-time polymerase chain reaction in two groups of cell lines carrying, respectively, the ‘‘15-15’’ genotype or other genotypes. Graph shows the geometric mean of the relative expression values obtained in the samples of the two groups; bars represent 95% confidence intervals. Relative expression was calculated as the ratio of YTHDF2 mRNA of each sample versus a control sam- ple (calibrator), normalized against the ACTB housekeeping gene. Cell lines (immortalized lymphocytes) with ‘‘15-15’’ genotype, n ¼ 10; cell lines with ‘‘othergenotypes,’’n¼41(‘‘16-16,’’n¼3;‘‘22-22,’’n¼2;‘‘20-20,’’n¼1;‘‘15- 21,’’n¼5;‘‘15-20,’’n¼1; ‘‘15-22,’’n¼6;‘‘15-23,’’n¼1;‘‘16-20,’’n¼3;‘‘16- 21,’’n¼4;‘‘16-22,’’n¼7;‘‘16-23,’’n¼6;‘‘17-22,’’n¼2).One-wayanalysisof variance to test differences of the YTHDF2 mRNA expression in ‘‘15-15’’ vs ‘‘other genotypes,’’ df ¼ 1; F ¼ 10.35; p ¼ .002 (executed on log-transformed data).

Figure 4. Relative messenger RNA (mRNA) expression levels of the YTHDF2 gene determined in 16 different human tissues by real-time polymerase chain reaction. Height of the histograms represents the relative abundance of YTHDF2 mRNA of each sample vs the sample with the highest expression level (testis), normalized against two housekeeping reference genes (ACTB and GAPD).

obtained on a total sample of 581 Italian individuals, ranging from 17 to 109 years of age, including 206 centenarians. It is interesting to note that, in the present study, the level of homozygosity did not show deviations from the expected (under Hardy–Weinberg equilibrium) homozygosity in both age groups. This observation suggests that it is unlikely that the increased homozygosity in centenarians results from a selection in favor of homozy- gotes, which would have presumably caused a deviation from the expected homozygosity, according to Hardy– Weinberg equilibrium. An alternative explanation is that the increased homozygosity at YTHDF2 intron 4 polymorphic locus might result as a consequence of a shift towards the most abundant alleles. Indeed, for any multiallelic system,

of this gene present in the public databases. In particular, Table 5 refers to data accessible through the UniGene section of GenBank (http://www.ncbi.nlm.nih.gov), and lists the number of transcripts associated with the YTHDF2 gene, derived from different organs. The organ with the highest relative number of YTHDF2 transcripts present in the database is testis (179 YTHDF2 transcripts per million ESTs from this organ), but a relatively high number of YTHDF2 ESTs is also associated with skin (165 transcripts per million), whereas the other organs are associated with a lower number of ESTs (from 135 to 34 per million). Additionally (not reported in Table 5), the relative number of YTHDF2 ESTs is higher in embryo (112 transcripts per million, i.e., 69/615,270) than in adult (61 transcripts per million, i.e., 62/1,001,530).

Table 5. Number of YTHDF2 Transcripts in Different Organs

YTHDF2 YTHDF2 Transcripts Per ESTs/Total Organ Million ESTs ESTs in Pool Testis 179 25/139,594 Skin 165 28/168,972 Eye 135 24/176,958 Prostrate 115 16/138,018 Liver 111 15/134,735 Uterus 106 20/187,614 Kidney 96 14/144,709 Placenta 93 23/245,330 Brain 87 42/481,991 Mammary gland 83 12/143,252 Lymph node 82 11/132,558 Lung 70 21/298,117 Colon 65 12/183,722 Muscle 58 6/103,284 Stomach 53 6/111,340 Pancreas 34 7/203,564

D ISCUSSION

The results of this study indicate that a (TG)n micro- satellite polymorphism in the fourth intron of the YTHDF2 gene is associated with human longevity. This polymorphic microsatellite was identified through the characterization of the ‘‘anonymous’’ inter-Alu marker ‘‘QM376-400,’’ which in our previous study (16) showed an increased homozy- gosity in centenarians. In the present study, a PCR assay specific for the YTHDF2 intron 4 (TG)12-27 locus (GenBank rs28969505) was applied on a novel and independent set of 412 Italian individuals, and the results confirmed, at this locus, a significantly increased homozy- gosity in centenarians compared to younger controls. Considering the whole set of data from the two studies, the association of longevity and homozygosity at YTHDF2 (TG)12-27 locus is currently sustained by the results

Notes: Only tissues for which the total number of Expressed Sequence Tags (ESTs) in pool is higher than 100,000 have been reported. Data obtained from the EST database (http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi).