Discussion
In this study, we identified rare PCSK9 and APOB variants that are associated with lower serum LDL-C levels among a population of healthy older individuals without a history of CHD events. Most of the variants detected were enriched in frequency among the healthy older CHD-free ASPREE population, compared with a large reference population of similar genetic ancestry (gnomAD-NFE). Serum LDL-C and TC concentrations for rare variant carriers were consistently lower than non-carriers in the study, and rare variant carrier status was associated with 19.4 mg/dL (14.6%) lower serum LDL-C and TC concentrations, after adjusting for statin use. The prevalence of statin prescriptions in variant carriers were less than half that observed in non-carriers. Together, these results indicate that lipid-lowering genetic variants are enriched in healthy older individuals without CHD, and play a role in coronary disease-free survival throughout the human lifespan.
Consistent with other studies,1–9 we observed that LoF variants in the PCSK9 and APOB gene are associated with lower serum LDL-C concentrations. However, to our knowledge, the prevalence shown in ASPREE during ageing free of atherosclerotic cardiovascular disease manifestations has not previously been demonstrated. Variants analysed were found to lower serum LDL-C and TC levels among the ASPREE population, with the difference in LDL-C concentrations between ASPREE rare variant carrier and non-carriers being ~20 mg/dL after adjusting for statin use. At an average participant age of 75 years, this represents the effect of potentially a lifetime of exposure to genetically determined lower LDL-C.
Meta-analysis of statin trials suggests for a 38 mg/dL reduction in LDL-C, there is a 20%–22% reduction in CHD risk, in the setting of relatively short clinical trials.27 Mendelian randomisation studies, however, demonstrate the importance of lifetime exposure to low LDL-C, suggesting that genetically determined low LDL-C is associated with a greater magnitude of CHD risk-reduction, compared with equivalent reduction through statin use.28 It is therefore likely that ASPREE rare variant carriers detected in this study, who have experienced a lifetime of exposure to genetically lower LDL-C, have benefited substantially from lower CHD risk. However, it is noteworthy that the rare variants detected likely account for only a fraction of the reduced CHD risk in the ASPREE population (n=13 131), with a range of other genetic and lifestyle factors contributing.
Strengths of the study include the sample size and unique ascertainment of the ASPREE population. The sequenced cohort comprised 13 131 individuals with an average age of 75 years, with no previous diagnosis of CHD or other cardiovascular events. It is rare for a population ascertained with these characteristics to be made available for genetic analysis. The sequenced cohort was the result of a unique set of circumstances made possible by the strict ASPREE inclusion criteria and age cut-off, and associated research biobank.14 29
Another strength of the study is that ASPREE participants were well characterised, each receiving a medical assessment by a general practitioner at enrolment, to confirm eligibility for the trial, and to rule out previous diagnoses of CHD.14 This provided confidence that detected variant carriers were CHD event-free at enrolment. Other strengths of the study include the depth of sequencing, focus on canonical lipid metabolism genes with established biological effect and stringency of variant curation used, to ensure only high-confidence variants were included in analyses.
Limitations of the study include our results not necessarily being generalisable to populations of non-European ancestry. Furthermore, we caution the comparison of rare variant prevalence between ASPREE and reference populations such as gnomAD, due to the potential for technical artefacts introduced by differences in sequencing technologies and variant curation, and population stratification related to differences in genetic ancestry. These well-known sources of variability are compounded when attempting to compare rare variant frequencies between studies.30 Nonetheless, our results suggest that rare PSCK9/APOB variants are enriched in healthy older CHD-free individuals (table 2), consistent with previous studies showing that LDL-C-lowering variants are associated with reduced risk for CHD and longevity.1–9
Regarding the genes analysed in our study, we focused on only the most established two canonical lipid metabolism genes where rare LoF variants have been demonstrated to have a high effect in reducing LDL-C levels (PCSK9 and APOB).1 3 8 9 We did not examine gain-of-function variants in the LDLR gene, or LoF variants in other genes that have been associated with LDL-C reduction (eg, NPC1L1, LPA, APOC3, ANGPTL3/4 and ASGR1) as these genes were not included on the sequencing panel used. We also could not calculate polygenic scores due to the targeted nature of the sequencing assay, and could not assess subclinical atherosclerosis due to absence of data on coronary artery calcium and carotid intima-media thickness. We did not functionally validate the detected rare variants.
Our study demonstrates the unique contribution of healthy older populations to exploring genetic determinants of health and lifespan. Historically, healthy older populations have not been the focus of large human genetic studies, mainly due to the difficulties in ascertaining large numbers of samples in this age group. However, studies focused on well-characterised populations of healthy older individuals represent an underexplored opportunity for the detection and improved understanding of protective genetic variants, especially in the context of lipid regulation and CHD.29 An improved understanding of the role played by protective lipid-lowering variants may help inform novel future approaches to CHD risk-reduction and lipid management.