Tier 3 — preclinical

A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A

Matthew Simon, Jiping Yang, Jonathan Gigas, Eric J Earley, Eric Hillpot, Lei Zhang, Maria Zagorulya, Greg Tombline, Michael Gilbert, Samantha L Yuen, Alexis Pope, Michael Van Meter, Stephan Emmrich, Denis Firsanov, Advait Athreya, Seyed Ali Biashad, Jeehae Han, Seungjin Ryu, Archana Tare, Yizhou Zhu, Adam Hudgins, Gil Atzmon, Nir Barzilai, Aaron Wolfe, Kelsey Moody, Benjamin A Garcia, David D Thomas, Paul D Robbins, Jan Vijg, Andrei Seluanov, Yousin Suh, Vera Gorbunova

The EMBO Journal 2022 Volume 41, Issue 21, Article e110393

Bibliography

DOI: 10.15252/embj.2021110393
PubMed: PMID 36215696
PubMed Central: PMC9627671
Funding: US National Institutes of Health grants AG056278 (VG), AG027237 (VG), AG064706 (VG), AG064704 (VG), AG046320 (AS), AG047200 (VG and AS), AG051449 (VG and AS), AG056278 (YS), AG076040 (YS), AG057433 (YS), AG061521 (YS), AG055501 (YS), AG057341 (YS), AG057706 (YS), AG057909 (YS), and AG17242 (YS); grant GCRLE-1320 (YS) from the Global Consortium for Reproductive Longevity and Equality at the Buck Institute, made possible by the Bia-Echo Foundation; a grant from the Michael Antonov Foundation; and a grant from the Simons Foundation (YS).
Competing interests: VG serves on the SAB of Genflow and DoNotAge. Other authors declare no competing interests or other interests that might be perceived to influence the results and/or discussion reported in this paper.

Study snapshot

Design	Human targeted capture sequencing of the SIRT6 locus in a centenarian case-control cohort, followed by biochemical and cell-based functional characterisation of the identified centSIRT6 allele in vitro and in human cell lines.
Model	Ashkenazi Jewish centenarian cohort (450 individuals ≥95 years) versus AJ controls (550 individuals without family history of exceptional longevity); GnomAD 141,456-individual database for validation. Functional characterisation in recombinant purified SIRT6 protein, telomerase-immortalised HCA2 human fibroblasts with cumate-inducible SIRT6 expression on a SIRT6-KO background, HT1080 and HeLa cancer cell lines, and human ESC-derived mesenchymal stem cells carrying CRISPR knock-in of the centenarian allele.
Sample	Human genetics: 450 centenarians vs 550 controls, plus 141,456 GnomAD individuals. In vitro assays: 3 technical replicates. Cell-based assays: 3 biological replicates; ≥80 cells scored per condition for foci quantification.
Intervention	Not an interventional trial. Comparison of wild-type SIRT6, single-mutant N308K, single-mutant A313S, and the linked double-mutant centSIRT6 (N308K/A313S) across enzymatic, DNA repair, LINE1 suppression, oxidative-stress resistance, cancer-cell killing, and interactome assays.
Duration	Not applicable (in vitro and cell-based assays; hours to 72 hours per assay). Human cohort ages captured at time of sequencing (centenarians mean 100.4 ± 3.3 years; controls 78.3 ± 8.1 years).
Endpoints	Allele frequency of SIRT6 variants in centenarians vs controls; SIRT6 deacetylase activity (Vmax, Km for NAD⁺ and peptide substrates) on H3K9ac and H3K18ac; SIRT6 mono-ADP-ribosylation activity (auto-ribosylation, PARP1 ribosylation, KAP1 ribosylation); LINE1 retrotransposon expression (qRT-PCR ORF1 and ORF2) and retrotransposition efficiency; Non-homologous end joining and homologous recombination DSB repair efficiency; γH2AX foci resolution kinetics after 2 Gy gamma irradiation; Oxidative stress resistance after paraquat exposure; Cancer cell viability and apoptosis in HT1080 and HeLa lines; SIRT6 and Lamin A/C interactome (TMT mass spectrometry); LMNA ribosylation state

What the study showed, in plain terms

Some people live past 100 in good health. Their genes offer clues to what protects a body from ageing. This study looked at the SIRT6 gene in 450 Ashkenazi Jewish centenarians and compared them with 550 people from the same population who did not reach exceptional old age. The researchers found a rare version of SIRT6 — carrying two linked changes called N308K and A313S — that appeared roughly twice as often in centenarians. The cohort was too small for this difference to reach statistical significance, but a follow-up look at a much larger public genetic database showed the same variant was enriched in people over 75.

The researchers then rebuilt this centenarian version of SIRT6 in the lab and tested what it does differently. SIRT6 has two enzymatic jobs: removing acetyl groups from histones (the proteins DNA winds around), and adding a small chemical tag called mono-ADP-ribose to other proteins. The centenarian version was slightly worse at the first job — but roughly twice as good at the second. This shift in activity mattered: cells carrying the centenarian variant repaired DNA breaks more efficiently, kept a class of "jumping gene" called LINE1 more tightly silenced, resisted oxidative stress better, and killed cancer cells more aggressively than the standard version.

A striking finding was that the centenarian SIRT6 also bound more strongly to Lamin A — a scaffolding protein at the edge of the nucleus that is itself linked to ageing. The two proteins appear to work together to keep DNA repair, chromatin organisation, and genome stability functioning well. The study does not test any supplement or intervention. It provides mechanistic and human-genetic evidence that enhanced SIRT6 mono-ADP-ribosylation activity — not deacetylation — is what matters for the aspects of SIRT6 function most tied to longevity.

Key findings

The centSIRT6 allele (linked N308K + A313S substitutions) appeared at 1.0% frequency in AJ centenarians versus 0.55% in AJ controls and 0.60% in the AJ GnomAD cohort. The nominal centenarian enrichment did not reach statistical significance (P=0.3 vs study controls; P=0.27 vs GnomAD AJ) due to limited cohort size.
In the full GnomAD database (141,456 individuals of diverse ancestry), the centSIRT6 allele ranked in the top 5th percentile of chromosome 19 SNPs enriched in the 75+ age group and the 9th percentile of missense mutations.
An intronic SIRT6 SNP linked to higher SIRT6 expression across 18 tissues (rs350845) was associated with living beyond 100 years (P=0.009).
Recombinant centSIRT6 showed reduced deacetylase Vmax on myristoylated peptide substrates (24.3 ± 2.9 RFU/s versus 37.2 ± 4.1 for wild type, P=0.02) and slower deacetylation of H3K9ac and H3K18ac on designer nucleosomes.
centSIRT6 auto-ribosylation was approximately 2-fold higher than wild type across a range of NAD⁺ concentrations (max signal ~5.2×10⁶ vs 1.85×10⁶). PARP1 ribosylation and KAP1 ribosylation by centSIRT6 were also significantly enhanced.
Non-homologous end joining DSB repair was stimulated 2.5-fold and homologous recombination 2-fold more by centSIRT6 than by wild-type SIRT6. Basal γH2AX foci in centSIRT6-expressing cells were reduced by roughly 50%.
LINE1 retrotransposon expression (ORF1 and ORF2) was more strongly suppressed by centSIRT6 than by wild-type SIRT6, and LINE1 retrotransposition efficiency was reduced.
centSIRT6 killed HT1080 and HeLa cancer cells at roughly 2-fold higher rates than wild-type SIRT6 in transient overexpression assays; apoptosis induction was similarly increased.
centSIRT6 co-immunoprecipitated with Lamin A/C more strongly than wild-type SIRT6 (reciprocal IPs confirmed the interaction), and LMNA showed increased mono-ADP-ribosylation in cells expressing centSIRT6.
CRISPR knock-in of the centenarian mutations into human ESCs increased SIRT6 protein levels, and ESC-derived mesenchymal stem cells showed improved DSB repair, better MMS survival, and fewer 53BP1 foci compared with wild-type controls.

What this study can and cannot tell us

The centenarian enrichment of centSIRT6 in the primary Ashkenazi Jewish cohort was not statistically significant. The 1.0% versus 0.55% allele frequency difference is directionally supportive but underpowered — the authors state explicitly they lacked the power to detect statistically significant differences. The supporting GnomAD analysis is a secondary observational signal, not a replication cohort.

The human genetic component is confined to an Ashkenazi Jewish founder population. Genetic homogeneity aided variant identification but limits generalisability to other ancestries. Whether centSIRT6 is enriched in centenarians of other populations remains untested.

Functional characterisation is entirely in vitro or in human cell lines with induced SIRT6 expression. There are no lifespan, healthspan, or clinical outcome data in humans carrying the allele. The authors could not model centSIRT6 in mice because the C-terminal region of SIRT6 that harbours the centenarian mutations is not conserved in mouse.

The study does not test any dietary intervention, supplement, or SIRT6-activating compound. Its relevance to SIRT6 activator supplementation is indirect — it strengthens the mechanistic rationale that enhanced SIRT6 mono-ADP-ribosylation activity is beneficial, but it does not demonstrate that increasing SIRT6 activity pharmacologically produces the same outcomes as inheriting the centenarian allele.

Commercial disclosure relevant to Biohack Blueprint readers. Senior author Vera Gorbunova serves on the Scientific Advisory Board of DoNotAge — the manufacturer of the SIRT6 Activator product Biohack Blueprint reviews. This is disclosed in the paper's competing-interests statement. The finding that enhanced SIRT6 activity may promote longevity aligns with the commercial interest of a company selling a SIRT6 activator. This does not invalidate the biochemical or genetic data, but readers should be aware.