Homozygosity, Allele Distribution, and Genotype Distribution in the Two Age Groups

The frequency of homozygous individuals was analyzed in the two age groups composed of 137 centenarians and 275 control participants, and resulted to be significantly higher in the long-lived group with respect to the control group (Table 2). Within each age group, homozygosity and heterozygosity were not significantly different from those expected under Hardy–Weinberg equilibrium (Table 2).

To better understand the meaning of the homozygosity increase observed in centenarians (see Discussion), the distributions of the most common alleles and genotypes (i.e., alleles and genotypes whose frequencies were higher than 10% in either centenarians or young participants) were also compared in the two age groups; all other alleles and genotypes were respectively grouped together. A statisti- cally significant difference (p ¼ .014) emerged when the

Table 2. Observed and Expected (Under Hardy–Weinberg [HW] Equilibrium) Values of Homozygosity and Heterozygosity at YTHDF2 (TG)12-27 Locus (rs28969505) in Young Control Participants and Centenarians

Young Participants (N ¼ 275) Centenarians (N ¼ 137) Homozygosity 0.182 (expected according to HW: 0.180) 0.277 (expected according to HW: 0.231) Heterozygosity 0.818 (expected according to HW: 0.820) 0.723 (expected according to HW: 0.769)

Note: Fisher’s Exact Test for observed homozygosity and heterozygosity in young control participants versus centenarians, p ¼ .030; Fisher’s Exact Test for observed versus expected homozygosity and heterozygosity in young participants, p ¼ 1.00; Fisher’s Exact Test for observed versus expected homozygosity and heterozygosity in centenarians, p ¼ .49.

frequency of the most common alleles were compared between young participants and centenarians (Table 3). In particular, an increase of the ‘‘15’’ allele was evident in centenarians, balanced by a decreased frequency of the cate- gory composed of the less common alleles. The frequency of the most common genotypes (Table 4) was also different between the two age classes, albeit with a marginal statis- tical significance ( p ¼ .047). In particular, the frequency of the most common genotype (‘‘15-15’’) was nearly doubled in the group of centenarians compared to young participants. Also, the odds ratio of the ‘‘15-15’’ genotype resulted to be significant (odds ratio ¼ 2.186, Table 4), when the most abundant category (the class of less frequent genotypes) was used as the reference category.

evident between YTHDF2 mRNA relative expression and YTHDF2 genotype. In particular, lymphocytes with the ‘‘15-15’’ genotype showed a more-than-doubled average relative expression of YTHDF2 mRNA with respect to that observed in lymphocytes carrying other genotypes (Figure 3). The expression level in lymphocytes with the ‘‘15- 15’’genotype was also compared with the expression levels in lymphocytes with every single other genotype. The results showed that ‘‘15-15’’ cells were still those with the highest mean expression level, and the result was statisti- cally significant when cell lines with the ‘‘15-15’’ genotype were compared with cell lines with the ‘‘16-23’’ (Student t test, p ¼ .042), ‘‘16-16’’ (Student t test, p ¼ .039), or ‘‘16-21’’ genotypes (Student t test, p ¼ .015). Finally, lymphocytes derived from individuals of different age and gender did not show any significant variations in YTHDF2 mRNA levels (data not shown).

An analysis performed to test for possible gender differences regarding the frequency of the ‘‘15’’ allele and the ‘‘15-15’’ genotype in centenarians gave negative results. Taking into account that the number of male centenarians was relatively low (n ¼ 26), this analysis was repeated using the results from the genotyping of an additional 17 samples of male centenarians recently recruited following identical inclusion criteria, thus reaching a total number of 43 male centenarians. Even after such an increase, we did not observe any significant difference between male and female centenarians as far as the frequency of the ‘‘15’’ allele and of the ‘‘15-15’’ genotype is concerned (data not shown). Such a result, even if does not rule out the possibility that a gender difference might emerge in a much larger number of centenarians and controls, suggests that it is unlikely.

The mRNA expression level of the YTHDF2 gene, normalized against two housekeeping genes (ACTB and GAPD), was determined in 16 different human tissues. YTHDF2 resulted to be expressed in all tissues examined, but its mRNA level showed important variations. In particular, the highest relative expression was observed in testis, followed by placenta and pancreas; the relative YTHDF2 mRNA expression in testis was 7-fold to 8-fold higher than in small intestine, skeletal muscle, and heart, and 3-fold to 5- fold higher than in the majority of other tissues (Figure 4).

Additional data about the expression profile of the YTHDF2 gene have been obtained by checking the ESTs

YTHDF2 mRNA Abundance in Immortalized Lymphocytes

The relative mRNA expression levels between the YTHDF2 gene and the housekeeping gene beta-actin (ACTB) were assayed in immortalized lymphocytes by

Table 4. Frequencies (%) of Most Common Genotypes at YTHDF2 (TG)12-27 Locus (rs28969505) in Young Control Participants and Centenarians

Young Participants Centenarians Odds Ratio (95% Genotypes* (N ¼ 275) (N ¼ 137) Confidence Interval) p** 15-15 8.0 15.3 2.186 (1.146–4.170) p ¼ .023 15-16 14.5 16.8 1.317 (0.746–2.324) p ¼ .372 Other genotypes 77.5 67.9 1

Table 3. Frequency (%) of Most Frequent Alleles at YTHDF2 (TG)12-27 Locus (rs28969505) in Young Control Participants and Centenarians

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