Effect of Cumulative Exposure to Low-Density Lipoprotein-Cholesterol on Cardiovascular Events in Patients With Familial Hypercholesterolemia

Abstract

Background: Recent studies suggest that cumulative exposure to low-density lipoprotein-cholesterol (LDL-C) leads to the development of atherosclerotic cardiovascular disease (ASCVD). However, few studies have investigated whether this link extends to individuals with familial hypercholesterolemia (FH), a relevant patient population.

Methods and Results: We retrospectively investigated the health records of 1,050 patients with clinical FH diagnosis between April 1990 and March 2019. We used Cox proportional hazards models adjusted for established ASCVD risk factors to assess the association between cholesterol-year-score and major adverse cardiovascular events (MACEs), including death from any cause or hospitalization due to ASCVD events. Cholesterol-year-score was calculated as LDL-C max × [age at diagnosis/statin initiation] + LDL-C at inclusion × [age at inclusion − age at diagnosis/statin initiation]. The median follow-up period for MACE evaluation was 12.3 (interquartile range, 9.1–17.5) years, and 177 patients experienced MACEs during the observation period. Cholesterol-year-score was significantly associated with MACEs (hazard ratio, 1.35; 95% confidence interval, 1.07–1.53; P=0.0034, per 1,000 mg-year/dL), independent of other traditional risk factors including age and LDL-C, based on cross-sectional assessment. Cholesterol-year-score improved the discrimination ability of other traditional risk factors for ASCVD events (C-index, 0.901 vs. 0.889; P=0.00473).

Conclusions: Cumulative LDL-C exposure was strongly associated with MACEs in Japanese patients with FH, warranting early diagnosis and treatment initiation in these patients.

Familial hypercholesterolemia (FH), caused primarily by mutations in the low-density lipoprotein (LDL) receptor (LDLR), proprotein convertase subtilisin/kexin type 9 (PCSK9), or apolipoprotein B (APOB) genes, is characterized by the clinical triad of primary hyper-LDL cholesterolemia, tendon xanthomas, and premature atherosclerotic cardiovascular disease (ASCVD).^1,2 The prevalence of FH is ≈1 in 300 in general populations, 1 in 31 among individuals with ischemic heart disease, and 1 in 15 among those with premature ischemic heart disease.³ Cumulative exposure to LDL-cholesterol (LDL-C) since birth is suggested to be detrimental in individuals with FH, leading to ASCVD beyond a certain threshold.⁴ This cumulative exposure hypothesis has been widely accepted, based on the results of Mendelian randomization studies showing that particular genetic variations associated with elevated LDL-C levels throughout an individual’s lifetime increase the risk;^5–8 however, validation studies using real-world data, especially those including patients with FH, are lacking. Conversely, we have recently reported that patients with FH identified through cascade screening at a younger age exhibit significantly better prognosis of ASCVD than their first relatives who seek medical attention at an older age.⁹

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Therefore, we aimed to determine whether cholesterol-year-score, an indicator of cumulative exposure to LDL-C, was associated with ASCVD in Japanese patients with FH referred to Kanazawa University Hospital in Japan.

Methods

Study Population

This retrospective study included the health records of 1,980 patients who met the Japanese clinical diagnostic criteria for FH and were diagnosed at Kanazawa University Hospital between April 1, 1990 and March 31, 2019. After the exclusion of 442 patients lacking clinical data and/or genetic analysis, 6 patients with homozygous or compound heterozygous FH, and 482 individuals who were lost to follow-up, a total of 1,050 patients were included in the final analyses (Supplementary Figure 1). The mean age was 48 years, 47% of the cohort was male, and 295 patients (28%) had a history of ASCVD. The median follow-up period was 12.3 (interquartile range (IQR), 9.1–17.5) years.

Clinical Diagnosis of FH

All individuals included in the study fulfilled at least 2 of the 3 clinical criteria for FH specified by the Japan Atherosclerosis Society: (1) LDL-C ≥180 mg/dL, (2) tendon xanthoma (tendon xanthoma on the dorsal side of hands, elbows, knees, etc.; Achilles tendon hypertrophy, or X-ray assessed Achilles tendon thickness ≥9 mm) or xanthoma tuberosum, and (3) family history of FH or premature coronary artery disease among 2nd-degree relatives of the patient (Supplementary Table 1).¹⁰

Genetic Analysis

The exome regions of 21 dyslipidemia-related Mendelian genes, including 3 FH genes (LDLR, APOB, and PCSK9) and LDLR adaptor protein-1 (LDLRAP1), were sequenced in all study participants. The pathogenicity of variants was based on allele frequency information obtained from the ExAC Asian population database, in silico annotation tools, and the ClinVar database. An allele frequency <5% was defined as a rare mutation among Asian populations. Finally, variants were classified as pathogenic in the presence of supporting evidence based on the standard American College of Medical Genetics and Genomics criteria. The details are described elsewhere.¹¹

Ethical Considerations

This study was approved by the Ethics Committee of Kanazawa University (2015-219). Written informed consent was not mandatory for this observational and retrospective study. Instead, we provided the chance to opt out of this study. However, informed consent for genetic analyses was obtained from all patients with FH included in the present study. All procedures were conducted in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and the Helsinki Declaration of 1975, revised in 2008.

Biochemical Analysis

Blood samples were drawn after overnight fasting. Serum levels of total cholesterol, triglycerides, and high-density lipoprotein-cholesterol were determined by enzymatic assays (Qualigent, Sekisui Medical, Tokyo, Japan). LDL-C levels were calculated using the Friedewald formula in patients with triglyceride levels <400 mg/dL; otherwise, LDL-C levels were determined enzymatically. Serum lipid levels were determined in overnight fasting state. Cholesterol-year-score was calculated as LDL-C max × [age at diagnosis/statin initiation] + LDL-C at inclusion × [age at inclusion − age at diagnosis/statin initiation]. We adopted the threshold of cholesterol-year-score 6,000 mg-year/dL based on the proposal by Professor Nordestgaard.⁴ In addition, we adopted another threshold of cholesterol-year-score 16,000 mg-year, assuming that LDL-C of 200 mg/dL for 80 years corresponded to the situation of whole-life accumulation of LDL-C among patients with untreated FH.

Clinical Evaluation

Major adverse cardiovascular events (MACEs) were defined as death associated with ASCVD, acute coronary syndromes including myocardial infarction (MI) and unstable angina, staged percutaneous coronary intervention, and coronary artery bypass grafting. Most MACEs were evaluated with reference to patient health records; some patients were assessed by telephone interview.

Smoking was defined as current smoking habit assessed by medical interview. Hypertension was defined as systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg, or the use of antihypertensive medication. Diabetes was defined based on the criteria of the Japan Diabetes Society or the use of diabetes medication. ASCVD was defined as the presence of angina pectoris, MI, or severe stenotic region(s) in coronary arteries, identified either by angiogram or computed tomography;¹² ischemic stroke; or severe cerebrovascular stenotic region(s).

Statistical Analysis

Categorical variables are expressed as percentages and were evaluated by Fisher’s exact test or the chi-square test, as appropriate. Continuous variables with a normal distribution are presented as mean±standard deviation, whereas medians with IQRs are reported for non-normally distributed variables. Mean values for continuous variables were compared using Student’s t-test for independent data, and median values were compared using the nonparametric Wilcoxon Mann-Whitney rank-sum test or the chi-square test for categorical variables with Fisher’s post hoc test. Differences in cumulative MACE rate were assessed by the log-rank test. Multivariate Cox regression analysis was performed using all variables, including age, male sex, hypertension, diabetes, smoking, LDL-C, Achilles tendon thickness, prior ASCVD, FH mutations, and cholesterol-year-score. Cumulative Kaplan-Meier survival curves starting at birth were constructed to compare time to first MACE among patients with FH categorized according to cholesterol-year-scores (<6,000, 6,000–16,000, and >16,000 mg-year/dL).

All statistical analyses were conducted using the R statistical software version 3.6.4 (The R Project for Statistical Computing, Vienna, Austria), and P values <0.05 were considered to indicate statistical significance.

Results

Characteristics of the Study Population

The clinical characteristics of the study participants with FH are shown in Table 1. The mean age was 48 years, the mean LDL-C level was 250 mg/dL, and 295 patients (28%) already had a history of prior ASCVD events at the time of diagnosis. Compared with the patients without MACEs, the mean age of those with MACEs was significantly higher (59 vs. 46 years, P<0.001). Additionally, there were significant differences in other clinical parameters associated with ASCVD, including hypertension, diabetes, smoking, and Achilles tendon thickness ≥9.0 mm (Table 1). Lipid-lowering therapies given to the patients are listed in Table 2. We found that the proportion of patients under intensive therapies was much higher in patients with MACE compared with those without MACE. The pathological genetic mutations associated with FH that were identified in the study cohort are presented in Supplementary Table 2. In the study cohort, 91 mutations in LDLR and 2 mutations in PCSK9 were identified and a nonsense mutation in LDLR, c.2431A>T/p.Lys811Ter, was the most commonly detected mutation (23.6%).

Table 1. Baseline Characteristics of FH Patients Included in the Study

	All (n=1,050)	MACE (n=177)	No MACE (n=873)	P value
Age (years)	48±18	59±14	46±18	<0.001
Male (%)	489 (46.6)	125 (70.6)	364 (41.7)	<0.001
Hypertension (%)	276 (26.3)	130 (73.4)	146 (16.7)	<0.001
Diabetes (%)	83 (7.9)	50 (28.2)	33 (3.8)	<0.001
Smoking (%)	301 (28.7)	127 (71.8)	174 (19.9)	<0.001
Total cholesterol (mg/dL)	327±59	335±59	324±60	<0.001
Triglyceride (mg/dL)	124 (81–168)	136 (95–178)	123 (80–166)	0.0039
HDL-cholesterol (mg/dL)	50±14	45±14	51±15	<0.001
LDL-cholesterol (mg/dL)	250±60	258±59	248±60	<0.001
Family history of FH and/or premature ASCVD (%)	839 (79.9)	153 (86.4)	686 (78.6)	0.023
Achilles tendon thickness (%)	533 (50.8)	148 (83.6)	385 (44.1)	<0.001
FH mutations (%)	777 (74.0)	133 (75.1)	644 (73.7)	0.78
History of prior ASCVD (%)	295 (28.1)	138 (78.0)	147 (13.8)	<0.001

ASCVD, atherosclerotic cardiovascular disease; HDL, high-density lipoprotein; FH, familial hypercholesterolemia; LDL, low-density lipoprotein; MACE; major adverse cardiac event.

Table 2. Lipid-Lowering Therapies Given to FH Patients During Study Period

	All (n=1,050)	MACE (n=177)	No MACE (n=873)	P value
Statins/maximum dose* (%)	1,031 (98.2)/790 (75.2)	175 (98.9)/134 (75.7)	856 (98.0)/656 (75.1)	0.66/0.95
Ezetimibe (%)	627 (59.7)	165 (93.2)	462 (52.9)	<0.001
Colestimide (%)	415 (39.5)	86 (31.6)	329 (22.3)	0.0088
Probcol (%)	2 (0.2)	2 (1.1)	0 (0.0)	0.028
PCSK9 inhibitor (%)	22 (2.1)	12 (6.8)	10 (1.1)	<0.001
LDL apheresis (%)	2 (0.2)	2 (1.1)	0 (0.0)	0.028
Fibrates (%)	3 (0.3)	1 (0.6)	2 (0.2)	1.00
n-3 PUFA (%)	16 (1.5)	8 (4.5)	8 (0.9)	0.0012

*Atorvastatin 40 mg/day, pitavastatin 4 mg/day, rosuvastatin 20 mg/day, fluvastatin 60 mg/day, pravastatin 20 mg/day. MACE, major adverse cardiac event; PCSK9, proprotein convertase subtilisin/kexin type 9; PUFA, polyunsaturated fatty acid.

Cholesterol-Year-Scores

The median cholesterol-year-score was 11,805 mg-year/dL. Additionally, the median cholesterol-year-scores of patients with and without MACEs were 15,076 and 11,188 mg-year/dL, respectively (Figure 1).

Figure 1.

Histogram of cholesterol-year-scores. MACE, major adverse cardiac event.

Type of MACEs

During the observation period, 177 patients experienced MACEs (Supplementary Table 3), including 66 deaths (6.3%) associated with ASCVD, 19 cases (1.8%) of MI, 28 cases (2.7%) of unstable angina, 56 cases (5.3%) of staged percutaneous coronary intervention, and 8 cases (0.8%) of coronary artery bypass grafting.

Factors Associated With MACEs

Multivariate analysis showed that several factors were independently associated with incident MACEs, including age (hazard ratio [HR], 1.06; 95% confidence interval [CI], 1.04–1.08; P<0.001), male sex (HR, 1.92; 95% CI, 1.21–2.82; P=0.0022), hypertension (HR, 2.59; 95% CI, 1.88–3.46; P<0.001), diabetes (HR, 1.81; 95% CI, 1.12–2.25; P=0.047), smoking (HR, 2.44; 95% CI, 1.51–3.11; P<0.001), LDL-C (per 10 mg/dL, HR, 1.03; 95% CI, 1.01–1.05; P=0.0018), Achilles tendon thickness ≥9.0 mm (HR, 2.01; 95% CI, 1.29–3.06; P=0.0058), prior ASCVD (HR, 3.49; 95% CI, 2.10–4.88; P<0.001), and FH genetic mutation status (HR, 1.71; 95% CI, 1.21–2.34; P=0.0078) (Table 3). Under these conditions, cholesterol-year-score was significantly associated with an increased risk of MACEs independent of other risk factors (HR, 1.35; 95% CI, 1.07–1.53; P=0.0034, per 1,000 mg-year/dL). In Supplementary Figure 2, associations between each component of MACE and cholesterol-year-score are shown. We found that death associated with ASCVD and staged PCI/CABG were major drivers of association.

Table 3. Factors Associated With MACE in FH Patients Included in the Study

Variable	HR	95% CI	P value
Age (per year)	1.06	1.04–1.08	<0.001
Male (yes vs. no)	1.92	1.21–2.82	0.0022
Hypertension (yes vs. no)	2.59	1.88–3.46	<0.001
Diabetes (yes vs. no)	1.81	1.12–2.25	0.0047
Smoking (yes vs. no)	2.44	1.51–3.11	<0.001
LDL-cholesterol (per 10 mg/dL)	1.03	1.01–1.05	0.0018
Achilles tendon thickness (≥9.0 mm)	2.01	1.29–3.06	0.0058
Prior ASCVD (yes vs. no)	3.21	2.00–4.78	<0.001
FH mutation (yes vs. no)	1.71	1.21–2.34	0.0078
Cholesterol-year-score (per 1,000 mg-year/dL)	1.35	1.07–1.53	0.0034

CI, confidence interval; HR, hazard ratio. Other abbreviations as in Table 1.

Additive Value of Cumulative Exposure to LDL-C for Risk Prediction

We investigated whether the discrimination ability of a model based on established traditional risk factors, including age, sex, hypertension, diabetes, smoking, and LDL-C, differed from that of a model that also included cholesterol-year-score. The C-statistic for the traditional risk factor model was 0.889, which increased to 0.901 (Figure 2, P=0.00473) after the addition of cholesterol-year-score to the model.

Figure 2.

Receiver-operating characteristic (ROC) curves predicting MACE. The blue line indicates ROC curve using traditional risk factors and cholesterol-year-score, and the black line indicates ROC curve using traditional risk factors only. The traditional risk factors included in the analyses were age, sex, hypertension, diabetes, smoking, and low-density lipoprotein-cholesterol. MACE, major adverse cardiac event.

Effect of Cumulative LDL-C Exposure on the Occurrence of ASCVD Events

When the patients were divided into 3 groups based on cholesterol-year score (<6, 000, 6,000–16,000, and >16,000 mg-year/dL), we found that ASCVD events occurred earlier among those with higher cholesterol-year-scores (P<0.001, Figure 3).

Figure 3.

Kaplan-Meier survival curves of MACE incidence. The x-axis represents age (years). The y-axis represents proportion of patients without MACE. Red: cholesterol-year-score, >16,000 mg-year/dL; Green: cholesterol-year-score, 6,000–16,000 mg-year/dL; Blue: cholesterol-year-score, <6,000 mg-year/dL. MACE, major adverse cardiac event.

Discussion

In the present study, we aimed to determine whether the cholesterol-year-score, an indicator of cumulative exposure to LDL-C, was associated with ASCVD events among patients with FH. Our analyses revealed that cholesterol-year-score was significantly associated with ASCVD events independent of other traditional risk factors, including age and LDL-C, based on cross-sectional assessment. These results have several clinical implications. First, our findings demonstrated that LDL-C is a causal factor for the development of ASCVD in patients with FH. Additionally, cumulative exposure, rather than temporary or transient elevation of LDL-C, appears to be a driving factor in the development of ASCVD in these patients. Finally, other modifiable risk factors, including hypertension and smoking, were also associated with ASCVD.

There is no doubt that LDL-C should be lowered in patients with FH. However, the timing for initiation of LDL-lowering therapy remains controversial. Mabuchi et al reported that patients with FH started to develop MI in their 30s.¹³ We recently reported that carotid and coronary atherosclerosis might start to develop in the 20 s.^14,15 Accordingly, initiation of LDL-lowering therapies might be necessary in patients as young as 20 years of age. In fact, we and others have shown that earlier interventions for patients with FH have resulted in better prognoses.^9,16 Various guidelines worldwide recommend statins as a safe therapeutic approach in pediatric patients with FH (≥8–10 years),^10,17,18 and state that earlier interventions are associated with significantly improved prognosis.¹⁹ Conversely, Mendelian randomization studies report that genetic variations that increase or decrease LDL-C levels through a patient’s lifetime are associated with increased or decreased ASCVD risk, respectively. Interestingly, the extent of LDL-C’s effect on ASCVD is far greater in the Mendelian randomization studies than in the clinical trials; the primary underlying cause of this difference is considered to be life-long exposure to LDL-C.²⁰ Several studies have demonstrated that this hypothesis may be applicable to the general American population,^21,22 as well as to French patients with FH.²³ The present study lends strong support to this hypothesis in Japanese patients with FH, by additional consideration of the mutation status of FH genes and Achilles tendon thickness, clearly demonstrating that LDL-C is the causal factor for the development of ASCVD.

Study Limitations

First, this was a retrospective, observational analysis performed in a single center. However, the study included one of the largest sample sizes related to patients with FH in Japan and the study findings can potentially further our understanding of the contribution of LDL-C exposure to ASCVD across different ethnic groups. Second, we could not perform full assessments of all patients with FH, which might have introduced some biases. Third, cholesterol-year-score in this study cannot reflect all of the LDL-C-lowering therapies during the patients’ clinical courses. Ideally, the cholesterol-year-score should account for changes in LDL-C levels at every point of blood sampling; however, we could not perform such assessments because of missing values. However, cholesterol-year-scores in the current study were significantly associated with MACEs, independent of the LDL-C value assessed cross-sectionally, suggesting that the cholesterol-year-scores calculated in this study can add useful information in terms of cumulative exposure to LDL-C among patients at this moment in time. Finally, some of the MACEs were evaluated via telephone interview and not by review of the patient’s health records.

Conclusions

Cumulative exposure to LDL-C was strongly associated with MACEs in Japanese patients with FH. Early diagnosis and treatment initiation for this common disease are warranted.

Acknowledgments

We express our special thanks to Kazuko Honda, Yoko Iwauchi, and Sachio Yamamoto (staff at Kanazawa University) for their outstanding technical assistance.

Author Contributions

All named authors meet the International Committee of Medical Journal Editors criteria for authorship of this manuscript and take responsibility for the integrity of the work as a whole.

Data Availability Statement

All data used in this study are provided upon official request to Dr. Hayato Tada (E-mail: [e-mail]).

Funding

This work was supported by scientific research grants from the Ministry of Education, Science, and Culture of Japan (19K08575); Astellas Foundation for Research on Metabolic Disorders; ONO Medical Research Foundation; Ministry of Health, Labor, and Welfare Sciences Research Grant for Research on Rare and Intractable Diseases; and Japanese Circulation Society (Project for Genome Analysis in Cardiovascular Diseases).

Declaration of Competing Iinterest

None.

IRB Information

Ethics Committee of Kanazawa University (2015-219).

Supplementary Files

Please find supplementary file(s);

http://dx.doi.org/10.1253/circj.CJ-21-0193

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