Heterozygous familial hypercholesterolemia (FH) is an autosomal co-dominant genetic condition characterized by elevated low-density lipoprotein cholesterol (LDL-C) and increased risk of coronary heart disease (CHD). Genetic testing facilitates diagnosis of FH through detection of pathogenic variants in canonical genes (LDLR, APOB, PCSK9). Pathogenic FH variants, especially in patients with elevated LDL-C and/or with family history of coronary disease, increases risk of premature CHD1. However, heterozygous FH is not fully phenotypically penetrant, implying that a subset of people who are carriers of a gene variant, and hence are at high genetic risk, may survive to advanced ages without developing CHD.
The prevalence of pathogenic FH variants in the general US population is estimated at 1 in 2562. Prevalence is higher in patients with dyslipidaemia and premature CHD. However, the prevalence of pathogenic FH variants in older individuals who remain CHD-free is unknown. Measuring FH variant prevalence in CHD-free healthy older populations may help overcome historic clinical ascertainment bias and help understand the survival risk conferred by FH variants during middle years of life. Healthy elderly populations may also provide new insights into the role polygenic risk and protective alleles play in modifying FH penetrance.
To measure the prevalence of pathogenic FH variants in healthy older individuals without CHD, we sequenced 13,131 participants from the ASPirin in Reducing Events in the Elderly (ASPREE) study3. Consistent with the exclusion criteria of the ASPREE trial, participants had no current symptoms or prior history of cardiovascular events to age 70 years and older (mean age 75 years) including: myocardial infarction, heart failure, stroke, transient ischemic attack, atrial fibrillation or high blood pressure. Participants also had no dementia diagnosis, physical disability, or illness likely to cause death within 5 years at enrolment4.
We sequenced the DNA of 13,131 participants using the Thermo Fisher Scientific S5TM XL system following standard protocols (average 200X depth, alignment to GRCh37). Single nucleotide variants and small insertions/deletions with ‘pathogenic’ or ‘likely pathogenic’ annotation and/or high-confidence predicted loss-of-function were curated following ACMG/AMP guidelines5. Variants of uncertain significance or conflicting interpretations of pathogenicity were excluded. FH variant prevalence was compared with 50,726 unselected individuals from the Geisinger Health System for LDLR, APOB and PCSK9 genes combined2. We assessed blood lipid levels and statin medication use at enrolment, and cardiovascular endpoints during mean 4.5 years of follow-up3. We generated polygenic risk scores (PRS) for coronary artery disease using 50 SNPs6 measured on the Axiom Precision Medicine Diversity Array (Thermo Fisher). The study was approved by the Alfred Hospital Human Research Ethics Committee and data are available from the corresponding author upon request.
Sequenced participants from the ASPREE trial were mostly white/Caucasian (99% self-reported), female (54%), with low rates of obesity (28%, BMI≥30 kg/m2) and current smoking (4%). Among 13,131 participants, we detected 13 individuals harbouring pathogenic FH variants. We detected 11 different pathogenic FH variants passing clinical variant curation (Table).
Table 1:
Characteristics of carriers of a pathogenic gene variant causative of familial hypercholesterolemia in ASPREE
| Gene | Variant | rsID | Consequence | Sex | Age (years) | BMI | LDL | HDL | Tg | TC | Statin use* | CVD endpoint† | CAD PRS Quintile† | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Start* | End† | (mg/DL)* | (mg/DL)* | (mg/DL)* | (mg/DL)* | |||||||||
| LDLR | 11213381C>T (p.Arg78Cys) | rs370860696 | Missense | F | 82 | 87 | 26.1 | 259.1 | 65.7 | 203.7 | 367.4 | - | - | Q4-Mid |
| LDLR | 11216083C>A (p.Cys167Ter) | rs752596535 | Stop gained | M | 79 | 84 | 40.9 | 112.1 | 54.1 | 203.7 | 208.8 | Atorvastatin | - | Q5-High |
| LDLR | 11216084G>A (p.Asp168Asn) | rs200727689 | Missense | M | 71 | 77 | 44.0 | 92.8 | 34.8 | 70.9 | 143.1 | Atorvastatin | - | Q4-Mid |
| LDLR | 11218138C>A (p.Cys296Ter) | rs879254708 | Missense | F | 70 | 77 | 30.1 | 162.4 | 46.4 | 168.3 | 243.6 | Atorvastatin | - | Q1-Low |
| LDLR | 11221336G>A (p.Glu317Lys) | rs746834464 | Stop gained | F | 72 | 76 | 30.1 | 150.8 | 58.0 | 115.1 | 232 | - | - | Q2-Mid |
| LDLR | 11221336G>A (p.Glu317Lys) | rs746834464 | Stop gained | F | 78 | 81 | 27.5 | 154.7 | 58.0 | 168.3 | 247.5 | Atorvastatin | Non-fatal MI | n/a |
| LDLR | 11221414G>A (p.Gly343Ser) | rs730882096 | Missense | M | 71 | 76 | 23.8 | 135.3 | 104.4 | 97.4 | 259.1 | Simvastatin | - | Q4-Mid |
| LDLR | 11221435C>T (p.Arg350Ter) | rs769737896 | Stop gained | M | 71 | 74 | 25.4 | 100.5 | 42.5 | 62.0 | 154.7 | Simvastatin | - | Q1-Low |
| LDLR | 11224005C>T (p.Thr413Met) | rs368562025 | Missense | F | 78 | 81 | 30.2 | 139.2 | 73.5 | 159.4 | 243.6 | Atorvastatin | - | n/a |
| LDLR | 11227685C>G | rs370245937 | Intron variant | M | 71 | 75 | 26.7 | 150.8 | 50.3 | 79.7 | 216.6 | Atorvastatin | - | Q5-High |
| LDLR | 11240330G>A (p.Gly844Asp) | rs121908037 | Missense | F | 71 | 74 | 33.2 | 119.9 | 38.7 | 230.3 | 204.9 | - | - | n/a |
| APOB | 21229160C>T (p.Arg3527Gln) | rs5742904 | Missense | F | 73 | 78 | 23.1 | 143.1 | 54.1 | 97.4 | 212.7 | Atorvastatin | - | Q4-Mid |
| APOB | 21229160C>T (p.Arg3527Gln) | rs5742904 | Missense | M | 80 | 85 | 26.2 | 112.1 | 50.3 | 44.3 | 174 | Atorvastatin | - | n/a |
| Mean | 74 | 79 | 29.8 | 141 | 56.2 | 130.8 | 223.7 | 10 of 13 | - | - | ||||
At ASPREE study enrolment (time of randomization to aspirin).
At end of ASPREE trial (time of last secondary end point screen).
CAD PRS = Coronary Artery Disease Polygenic Risk Score (Khera et al 20166), Q1=Low, Q2/3/4=Mid, Q5=High
n/a = not available. MI = myocardial infarction. BMI = Body mass index (weight in kilograms / height in m2)
The carrier rate for heterozygous FH in the ASPREE population was 1 in 1010, markedly lower than the 1 in 256 reported in the general US population (OR=0.25 [0.14 to 0.47], P<0.001). Among the 13 heterozygous FH variant carriers detected, mean LDL-C at enrolment was 141 mg/DL (min 92.8, max 259), with 10 of 13 taking statin medication (Table). All 13 FH variant carriers survived a mean 4.5 years of follow-up, with only one developing a cardiovascular event (non-fatal myocardial infarction). The remaining 12 FH variant carriers (6 male, 6 female) survived CHD event-free to mean age 79 years (min 74, max 87) at completion of the ASPREE trial.
The low prevalence of FH observed in ASPREE suggests a selective depletion of FH carriers at enrolment, due to the strict cardiovascular exclusion criteria4. FH carriers, being more prone to premature CHD, were less likely to be enrolled, consistent with their survival disadvantage to age 70. Despite this, we identified 12 surviving FH variant carriers who met the study criteria and completed the trial to mean age 79 years (some to over 85 years) without developing CHD.
We hypothesised that these CHD-free FH carriers may be enriched with protective genetic modifiers and/or low polygenic risk, modifying FH penetrance. Among the 12 CHD-free FH carriers detected, PRS data passing quality control was available on 9 individuals. A low PRS (in the bottom quintile or lowest 20% of the ASPREE population) was only observed for 2 of 9 (Table). This suggests PRS alone did not explain reduced penetrance. Another 2 of 9 had high PRS (in the top quintile or highest 20%), and the remaining 4 had average PRS (in quintiles 2/3/4). The fact that PRS alone did not explain reduced penetrance suggests that environmental factors, adherence to a healthy lifestyle, long-term statin use, or yet undiscovered rare protective alleles may also have played a role.
Older FH variant carriers who survive CHD-free are typically not the focus of large genetic research studies. We suggest that with larger sample sizes and whole-genome analysis, elderly unaffected FH carriers may provide important clues towards understanding polygenic risk and the role protective variants may play. Further genomic evaluation of FH carriers that remain unaffected by CHD to advances ages may also provide a new opportunity for novel target discover for preventive therapies.
Acknowledgments -
We thank the trial staff in Australia and the United States, the participants who volunteered for this trial, and the general practitioners and staff of the medical clinics who cared for the participants.
Sources of Funding: The ASPREE Healthy Ageing Biobank is supported by a Flagship cluster grant (including the Commonwealth Scientific and Industrial Research Organisation, Monash University, Menzies Research Institute, Australian National University, University of Melbourne); and grants (U01AG029824) from the National Institute on Aging and the National Cancer Institute at the National Institutes of Health, by grants (334047 and 1127060) from the National Health and Medical Research Council of Australia, and by Monash University and the Victorian Cancer Agency.
Disclosures: Watts has received honoraria and/or research grants from Arrowhead, AstraZeneca, Kowa, Regeneron, Sanofi, Amgen, and Novartis. Nicholls has received research support and/or honoraria for Amgen, AstraZeneca, Eli Lilly, Esperion, Novartis, Merck, Pfizer, Iowa and Sanofi-Regeneron. Sebra serves as Vice-President of Technology Development at Sema4. Schadt serves as Chief Executive Officer at Sema4. No other conflicts were reported.
Nonstandard Abbreviations and Acronyms
- FH
Familial hypercholesterolemia
- LDL-C
Low-density lipoprotein cholesterol
- CHD
Coronary heart disease
- ASPREE
ASPirin in Reducing Events in the Elderly
- ACMG/AMP
American College of Medical Genetics and Genomics/Association for Molecular Pathology
- PRS
Polygenic risk score
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