Figure 2. Results of the discovery phase analysis. Manhattan plot presenting the 2log P-values from the discovery phase analysis of cases aged 85 years (A) and 90 years (B). The loci that showed a genome-wide significant association after the joint analysis of the discovery and replication phase (chromosome 19q13.32 and 5q33.3) are shown in red.

Despite the highly interesting association of the minor allele of rs2149954 with low blood pressure and a decreased risk for CAD, stroke, and mortality, its association with decreased all-cause mortality was not influenced by blood pressure in two studies of participants aged 75 years (PROSPER and Leiden 85-plus study Cohort II; Supplementary Material, Table S10). This may indicate that at higher ages, this locus influences longevity via pathways additional to those involved in blood pressure regulation.

DISCUSSION: 

We have performed the largest genome-wide association meta-analysis for human longevity, in which a novel locus on chromosome 5q33.3 associating with survival beyond 90 years was identified. The minor allele of rs2149954 (T) promotes human longevity by reducing the risk of mortality owing to stroke and non-cardiovascular causes. In addition, this allele has previously been associated with low blood pressure, which may explain the protection from CVD mortality risk in middle age. At ages above 80 years, however, low SBP associates with increased mortality (37, 38). Hence, the observed blood pressure-independent association of the minor allele with mortality at 75 years may be due to pleiotropic effects on other mortality-related clinical parameters. Examination of publicly available data of several large GWAS consortia for association of the locus with parameters related to glucose and fat metabolism provided as yet no clues for other potentially involved mechanisms.

Phenotypic characterization and pathway analysis: 

In an attempt to identify the underlying mechanism by which this novel longevity locus at chromosome 5q33.3 could influence human longevity, we examined rs2149954 in the published data of several large GWAS consortia for association with metabolic traits in generally middle-aged individuals. None of the investigated traits, i.e., 2 h glucose (OGTT), Hb Ac, fasting glucose, fasting insulin, insulin resistance (HOMA-IR), b-cell activity (HOMA-B), total/HDL/LDL cholesterol, triglycerides and20q13.2 rs8126377 20 51 590 254 TSHZ2, ZNF217 G Discovery 5209 14 893 0.057 0.068 0.75 0.66 0.85 3.38 1025 Replication 7278 60 647 0.063 0.054 1.04 0.95 1.13 0.309 Joint 12,487,755,540 0.94 0.87-1.00, with a heterogeneity of only 0.117 and an EAF of the analyzed individuals pooled for the effect allele; OR represents the odds ratio for the effect allele; and the abbreviation CI refers to the confidence interval of the analysis, which is set at a confidence level of 95%. It should be noted that genotyping of this SNP with the Sequenom MassARRAY system for the replication phase failed, and that SNPs in bold overlap with Table 1.

The logio(p-value) and position on chr19 (Mb) are shown in the table above along with other relevant data on this topic. The figure below shows regional association plots for chromosome19q13.32 and chromosome5q33.3 loci based on data from cases aged over ninety years.

Figure3: Regional association plots for chromosome19q13.32 and chromosome5q33.3 loci

In Figure4, forest plots for rs4420638 and rs2149954 are represented in A and B, respectively, showing odds ratios with a confidence interval of about ninety-five percent for cohorts analyzed in both discovery and replication phases.

Figure4: Forest plots for rs4420638 and rs2149954

The newly annotated lincRNA is not yet available in the large eQTL databases, and further functional studies are required to illuminate the mechanism by which this locus influences human longevity. Rs2149954 is located in an intergenic region on chromosome 5q33.3 between CLINT1 and EBF1. The presence of several regulatory elements in this region implies that transcription factor binding and/or expression of (nearby) genes could be influenced. The currently available eQTL databases did not provide evidence for such effects, which might be due to the limited tissue diversity of the databases. The effects of the chromosome 5q33.3 locus on human longevity might be exerted through the lincRNA, which has recently been annotated.

Figure5 shows the chromosomal region around rs2149954, which contains a lincRNA (RP11-524N5.1) for which the poly(A) features are supported by PolyA-seq reads from liver, muscle, and testis.

Figure5: Chromosomal region around rs2149954

The low contrast between cases and controls likely has reduced our probability of identifying longevity loci. There were no genome-wide significant hits for human longevity or mortality besides the well-known TOMM40/APOE/APOC1 locus. The FOXO3A locus showed only moderate evidence for association with survival at ninety years in the discovery phase of our GWAS.