Risk Assessment for Cardiovascular Events using Achilles Tendon Thickness and Softness and Intima-Media Thickness in Familial Hypercholesterolemia

Abstract

Aims: This was a retrospective cohort study that aimed to determine cutoff values for major adverse cardiovascular events (MACEs) in patients with heterozygous FH (HeFH) for Achilles tendon (AT) thickness (ATT) measured by ultrasonography (US-ATT) and radiography (Xp-ATT), AT softness, and intima-media thickness of carotid artery (C-IMT), and to examine the effectiveness of these values as well as AT calcification as indexes in assessing risk for MACEs.

Methods: The subjects were 391 clinically diagnosed HeFH patients. Kaplan-Meier curves were drawn based on the threshold values for the individual indexes calculated from ROC curves, and multivariate analysis was used to examine whether they were predictors of the development of MACEs.

Results: The median observation period was 1,239 days (700-1,827 days). Twenty-one subjects (5%) had MACEs during the observation period. The cutoff values for MACEs for US-ATT were 9.9 mm in males and 7.1 mm in females, and those for C-IMT were 1.6 mm in males and 1.5 mm in females. Subjects were classified into two groups according to whether they were above or below the cutoff values and presence of calcification, and we compared MACE rates between them. MACE rates were significantly increased in groups with AT thickening determined by ultrasonography (P＜0.001), AT softening (P＜0.001), presence of calcification in AT (P=0.016) and greater C-IMT (P＜0.001). However, classification according to Xp-ATT revealed no significant difference in MACE rate (P=0.112).

Conclusions: These thresholds and examination for AT calcification will help in risk assessment for patients in Japanese FH practice and encourage stricter and more comprehensive management for patients who exceed the thresholds.

Introduction

Familial hypercholesterolemia (FH) is a genetic disorder characterized by marked high low-density lipoprotein (LDL)-cholesterol (LDL-C) levels, premature atherosclerotic cardiovascular disease (ASCVD), and tendon xanthomas^{1, 2)}. Since FH is reported to account for 5-10% of patients with acute coronary syndrome in Japan^3-5), its prevention is an important public health issue. Xanthomas are a physical finding specific to FH, and the Dutch Lipid Clinic Network FH criteria⁶⁾, Simon-Broome diagnostic criteria⁷⁾ and Japan Atherosclerosis Society (JAS) FH criteria^{5, 8)} include tendon xanthomas for diagnosis of FH.

In Japan, Achilles tendon thickness (ATT) is measured quantitatively and cutoff values are set for FH diagnosis. The original Japanese diagnostic criteria for FH had used a cutoff value for ATT measured by an X-ray method (Xp-ATT); however, based on our research results⁹⁾, the new diagnostic criteria published in 2022 ⁵⁾ also include cutoff values for ATT measured by an ultrasound method (US-ATT). As an important finding, we have shown that ATT is associated with degree of carotid intima-media thickness (C-IMT) and history of ASCVD, suggesting that ATT is useful not only as a diagnostic criterion for FH, but also as a risk assessment parameter for atherosclerosis⁶⁾. The cutoff values for ATT in the latest FH diagnosis criteria⁵⁾ are 8.0 mm for men and 7.5 mm for women by the X-ray method, in consideration of the results of our study¹⁰⁾, and 6.0 mm for men and 5.5 mm for women by the ultrasound method.

In addition, in our investigation using ultrasound elastography, we reported that the Achilles tendon (AT) in FH is softer than in non-FH patients due to lipid deposition¹¹⁾. AT softness can be quantified using the Elasticity Index (EI); the lower the value of EI, the softer the AT. We have also reported that combined use of ATT and EI (AT softness) cutoff values improves FH diagnostic accuracy¹¹⁾ and that both EI (AT softness) and ATT predict severity of C-IMT and history of ASCVD among heterozygous FH patients¹²⁾. On the other hand, there have been no studies on whether respective cutoff values for AT thickness or softness are associated with a higher risk of ASCVD in patients with FH.

In both FH¹³⁾ and non-FH^{14, 15)}, C-IMT has been reported to be a predictive indicator of ASCVD. Although several C-IMT cutoff values for ASCVD risk in non-FH have been reported to date^{16, 17)}, none have been reported for FH.

In FH practice, we considered that if we knew thresholds of ATT, EI (AT softness), and C-IMT that predict future ASCVD development, we would be able to take actions, such as more aggressive LDL-C lowering, for high-risk FH patients who exceed these thresholds. We also thought that this might improve the prognosis of patients with FH. Therefore, we conducted a retrospective observational study to determine thresholds of Xp-ATT, US-ATT, EI (AT softness), and C-IMT for risk of major adverse cardiovascular events (MACEs) in patients with heterozygous FH.

We also investigated the impact of presence or absence of AT calcification on MACE prevalence. We often see patients with calcification of the Achilles tendon in FH practice and it seems to be especially frequent in patients with marked AT thickening. Since atherosclerosis and AT thickening are pathologically similar, we thought that calcification in the AT could be a means of evaluating MACE severity.

2.Patients and Methods

2.1. Subjects

This study initially included a total of 582 patients visiting the National Cardiovascular Center Hospital (NCVC) between September 2013 and May 2021, who had a clinical diagnosis of FH based on the 2017 JAS FH criteria¹⁸⁾ and whose carotid IMT and Achilles tendon thickness were measured by echocardiography. Sixteen patients with homozygous FH, 16 patients younger than 15 years (Japanese adult FH diagnostic criteria are for those 15 years of age and older), 122 untreated patients, and 37 patients who could not be followed up after the first visit were excluded. Thus, the final number of patients included in the analysis for this study was 391. It was performed in conformity with the Helsinki Declaration and approved by the ethics committee of NCVC (Approval No. M25-112). Written informed consent was obtained from all subjects.

2.2. Measurement of Achilles Tendon Thickness and Softness

Measurement of US-ATT was performed according to the standardized method recommended by the Japanese Society of Ultrasound Medicine and the Japanese Society of Arteriosclerosis. Radiography was performed using the conventional method¹⁹⁾. The cutoff values of US-ATT for FH diagnosis were 6.0 mm for males and 5.5 mm for females in accordance with the 2022 JAS FH criteria⁵⁾ and those of Xp-ATT for FH diagnosis were 8.0 mm for males and 7.5 mm for females, also in accordance with the 2022 JAS FH criteria⁵⁾. However, it should be noted US-ATT data are the latest obtained during the observation period, while Xp-ATT data are those obtained at the time of the initial examination. EI (AT softness) was measured using ultrasonic elastography and softening was defined as a value less than 4.7 ¹²⁾. The Aplio 500 (Canon Medical Systems, Tochigi, Japan) system was used for ATT measurement with a 9 MHz high-frequency linear ultrasonic probe, and the LOGIQ S8 system (GE Healthcare Japan, Tokyo, Japan) was used for AT softness measurement with a 9 MHz high-frequency linear ultrasonic probe. ATT and EI (AT softness) were averaged for the left and right sides for the analysis.

We defined calcification in the AT as “a structure with an X-ray signal intensity comparable to that of the calcaneus” for X-ray and “a structure with an acoustic shadow of similar brightness to that of the calcaneus” for ultrasound. A patient was determined to have calcification if it was present on either the left or right side. However, it should be noted that in patients with AT calcification, EI (AT softness) was not measured in principle. This is because calcification of an area to be measured presents a challenge as it interferes with accurate determination of softening associated with lipid deposition^{11, 12)}. Therefore, in the case of patients in whom EI could not be measured due to AT calcification, EI (AT softness) data were handled as missing values in the analysis.

2.2. Ultrasonography of Carotid Artery

C-IMT was measured using the same equipment as for the ATT measurements. The greater of the maximal IMT of the right and left common carotid arteries was used for analysis. Greater IMT was defined as an increase of 1.1 mm or more according to the “Standardized Method for Evaluation of Carotid Artery Lesions by Ultrasound” of the Japanese Society of Ultrasound Medicine¹⁹⁾. We chose maximal IMT as our index rather than mean IMT because it has been reported that maximal IMT is a better predictor of ASCVD development in Japanese patients^20-22).

2.3. Clinical and Laboratory Characteristics

Since the subjects of this study were outpatients, blood data are non-fasting. Total cholesterol (TC), triglycerides, HDL cholesterol (HDL-C), LDL-C, and lipoprotein (a) (Lp(a)) were measured using a LABOSPECT 008 instrument (Hitachi High-Technologies Co, Tokyo, Japan) and Qualigent reagents (Sekisui Medical Co, Tokyo, Japan). HbA1c was measured using an HLC 723 G9 system (A&T Co, Kanagawa, Japan). These items were measured in the NCVC clinical laboratory. Body mass index (BMI) was calculated by dividing weight (kg) by the square of height (m), using the weight and height at the ultrasound examination. Smoking history was assessed by interview at the ultrasound examination, and those with a current smoking or past smoking were defined as subjects with smoking history. MACEs were defined as cardiovascular event death, myocardial infarction, coronary angioplasty or coronary artery bypass graft surgery, or stenosis of 75% or greater on coronary angiography, or aortic aneurysm exacerbation requiring treatment.

The observation period started on the date we measured US-ATT and ended on the date of the last visit to the hospital or the date of a cardiovascular event occurring up to August 31, 2021.

2.4. Statistical Analysis

Tests for comparisons between two groups used T-tests or Mann-Whitney’s U test for continuous variables. The chi-square test was used for categorical variables, and ANOVA (post hoc test) or the Kruskal-Wallis test was used for multiple group comparisons. Continuous variables are presented as mean±SD or median (interquartile range, IQR) and categorical variables as percentages. In developing the ROC curves for predicting MACEs, we chose cutoff values where both sensitivity and specificity were greater than or equal to 0.5 and well balanced The cardiovascular event rates among groups were estimated by the Kaplan-Meier method and compared by the log-rank test. Logistic regression analysis was performed for MACEs with US-ATT, XP-ATT, C-IMT and presence of calcification in AT as adjustment factors, with p＜0.05 considered statistically significant. Statistical analyses were performed using JMP14 (SAS Institute Inc., Cary, NC, USA).

3.Results

3.1. Clinical Background

Table 1 summarizes the clinical characteristics of the study subjects. The average age was 53±16 years, and 42% were male. Seventy-five patients (19%) had a history of ASCVD, and greater C-IMT (1.1 mm or more increase) was determined in 172 patients (44%). The mean US-ATT was 7.8±3.6 mm and 65% had thickening (≥ 5.5 mm in women, ≥ 6.0 mm in men). The mean Xp-ATT was 11.0±4.4 mm and 75% had thickening (≥ 7.5 mm in women, ≥ 8.0 mm in men). BMI, TG and C-IMT as well as prevalences of smoking history, hypertension and previous ASCVD were higher in male subjects. Conversely, age, TC, HDL-C and Lp(a) levels and EI measurements were lower in male subjects. There were no differences between male and female subjects regarding presence of diabetes, apheresis treatment, LDL-C and HbA1c levels, or presence of calcification in the AT. US-ATT and Xp-ATT were significantly greater in male subjects, whereas there was no difference in the proportion of patients with AT thickening between men and women. The AT was softer (EI values were lower) in male subjects, while there was no difference in the proportion of those with softening between male and female subjects.

Table 1.Baseline characteristics

	Total (n= 391)	Male (n= 163)	Female (n= 228)	P value
Age (years)	53±16	50±17	55±15	0.005
Body mass index	22.6±3.3	23.6±2.9	21.9±3.4	＜0.001
Smoking, n (%)	142 (36)	99 (61)	43 (19)	＜0.001
Diabetes mellitus, n (%)	52 (13)	27 (17)	25 (11)	0.108
Hypertension, n (%)	94 (24)	55 (34)	39 (17)	＜0.001
Cardiovascular disease history, n (%)	75 (19)	47 (29)	28 (12)	＜0.001
Lipoprotein apheresis, n (%)	7 (2)	5 (3)	2 (1)	0.107
Total cholesterol (mg/dl)	208±50	196±43	216±53	＜0.001
Triglycerides (mg/dl)	87 (63 – 132)	94 (64 – 139)	84 (61 – 116)	0.034
HDL-cholesterol (mg/dl)	56±15	49±11	61±15	＜0.001
LDL-cholesterol (mg/dl)	129±44	126±40	132±47	0.152
Lipoprotein (a) (mg/dl)	17.2 (7.4 – 38.0)	15.4 (5.9 – 31.9)	19.0 (8.6 – 42.7)	0.036
HbA1c (%)	5.8±0.7	5.9±0.8	5.8±0.6	0.094
US-AT thickness (mm)	7.8±3.6	8.5±4.4	7.3±2.9	0.011
US-AT thickening, n (%)	253 (65)	99 (61)	154 (68)	0.165
Xp-AT thickness (mm)	11.0±4.4	11.8±5.4	10.4±3.4	0.010
Xp-AT thickening, n (%)	230 (75)	92 (72)	138 (79)	0.160
Elastic index (AT softness)	4.4±0.7	4.4±0.7	4.5±0.7	0.035
AT softening, n (%)	178 (55)	83 (60)	95 (52)	0.180
Calcification in the AT	34 (9)	12 (7)	22 (10)	0.439
C-IMT (mm)	0.9 (0.7 -1.4)	1.2 (0.7 – 1.6)	0.8 (0.7- 1.3)	＜0.001
Greater C-IMT, n (%)	172 (44)	93 (57)	79 (35)	＜0.001

Values are presented as mean±standard deviation or median (interquartile range 25% - 75%) or number. HDL-C: high density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol, US-AT: Achilles tendon using ultrasonography, Xp-AT: Achilles tendon using radiography, C-IMT: maximum intima-media thickness of common carotid artery, AT: Achilles tendon

Supplemental Table 1 shows the results of classification by whether LDL-C control target levels (primary prevention: ＜100 mg/dl, secondary prevention: ＜70 mg/dl) according to JAS Guidelines⁸⁾ were achieved or not. The frequency for unachieved LDL-C target levels was 81% (319/391, 135 males and 184 females). In the evaluation using ultrasound, AT was thicker (achieved group: 6.5±2.7 mm, unachieved group: 8.1±3.7 mm, P＜0.001) and softer (lower EI values; achieved group: EI=4.7±0.5, unachieved group: 4.4±0.7, P＜0.001) in the unachieved group than in the achieved group. In the evaluation by radiography, the frequency of AT thickening in the unachieved group was higher than that in the achieved group (achieved group: 62%, unachieved group: 79%, P=0.0070), but the difference in ATT was not significant (achieved group: 10.0±3.7 mm, unachieved group: 11.3±4.5 mm, P=0.064). There were no significant differences in C-IMT, presence of calcification or incidence of cardiac events during the observation period.

Supplemental Table 1.Comparison according to achievement of target LDL cholesterol levels

	Achieved group (n= 72)	Unachieved group (n= 319)	P value
Age (years)	55±15	52±17	0.254
Male, n (%)	28 (39)	135 (42)	0.594
Body mass index	21.7±2.7	22.8±3.4	0.017
Smoking, n (%)	21 (29)	121 (38)	0.163
Diabetes mellitus, n (%)	10 (14)	42 (13)	0.870
Hypertension, n (%)	15 (21)	79 (25)	0.480
MACE, n (%)	3 (4)	18 (6)	0.616
Lipoprotein apheresis, n (%)	0 (0)	7 (2)	0.205
Total cholesterol (mg/dl)	165±26	218±48	＜0.001
Triglycerides (mg/dl)	77 (54 – 121)	89 (64 – 133)	0.129
HDL-cholesterol (mg/dl)	60±16	55±15	0.015
LDL-cholesterol (mg/dl)	82±16	140±41	＜0.001
Lipoprotein (a) (mg/dl)	14.9 (5.4– 30.9)	17.8 (7.8 – 39.0)	0.141
HbA1c (%)	5.9±1.0	5.8±0.6	0.245
US-AT (mm)	6.5±2.7	8.1±3.7	＜0.001
US-AT thickening, n (%)	29 (40)	224 (70)	＜0.001
Xp-AT thickness (mm)	10.0±3.7	11.3±4.5	0.064
Xp-AT thickening, n (%)	34 (62)	196 (79)	0.007
Elastic index	4.7±0.5	4.4±0.7	＜0.001
AT softening, n (%)	22 (39)	156 (59)	0.005
Calcification in AT	4 (6)	30 (9)	0.308
C-IMT (mm)	0.9 (0.7 – 1.4)	0.9 (0.7- 1.4)	0.796
Greater C-IMT, n (%)	30 (42)	142 (45)	0.630

Values are presented as mean±standard deviation or median (interquartile range 25% - 75%) or number. MACE: major adverse cardiovascular events, HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol, US-AT: Achilles tendon using ultrasonography, Xp-AT: Achilles tendon using radiography, C-IMT: maximum intima-media thickness of common carotid artery, AT: Achilles tendon

3.2. Comparison by Presence or Absence of MACEs during Observation Period

MACEs occurred in 21 patients (5%). Supplemental Table 2 gives the details of MACEs in individual patients. Table 2 summarizes the characteristics of patients who had MACEs during the observation period (MACE group) and those who did not (non-MACE group). In the MACE group, age and serum Lp(a) levels were higher and HDL-C levels lower than in the non-MACE group, whereas there were no differences in LDL-C, TG and HbA1c levels. Prevalences of patients with a history of smoking, hypertension, history of ASCVD, and those receiving apheresis therapy were higher in the MACE group. The AT of patients in the MACE group was thicker (both ultrasonically and radiographically) and softer (lower EI values) than in the non-MACE group. However, the percentage of patients with Achilles tendon thickening as assessed by radiography did not differ between the two groups. The proportion of patients with calcification of the AT was higher in the MACE group. C-IMT was greater in the MACE group, as was the proportion of patients with thickening (＞1.1 mm).

Supplemental Table 2.MACEs that occurred and their numbers

Specific details of MACEs in individual patients	N
CAG was performed due to chest discomfort, 75% stenosis in LAD	1
Myocardial scintigraphy showed ischemia, MSCT angiography showed 75% stenosis in RCA	1
CABG was performed due to ischemia on myocardial scintigraphy and 3-vessel disease on CAG	1
PCI was performed due to ischemia on RI and severe stenosis in LAD on CAG	2
MSCT angiography was performed for suspected silent myocardial infarction, with severe stenosis in LAD	1
CABG was performed due to severe stenosis in LAD and the LCX on CAG	2
PCI was performed due to suspected silent myocardial infarction and 75% stenosis in LAD on CAG	1
Reoperation for exacerbation of thoracoabdominal aortic aneurysm	1
PCI was performed due to suspected unstable angina pectoris and severe stenosis in LAD on CAG	1
MSCT angiography performed for screening purposes showed stenosis of more than 75% stenosis in LAD	2
PCI was performed due to chest discomfort and 75% stenosis in LAD	2
CAG was performed due to chest discomfort and ischemia on myocardial scintigraphy, 75% stenosis in LAD and LCX on CAG	1
Cardiovascular death	5
Total	21

CAG, coronary angiography; LAD, left anterior descending artery; RCA, right coronary artery; LCX, left circumflex artery; MSCT, multi-slice computed tomography; CABG, coronary artery bypass graft surgery; PCI, percutaneous coronary intervention

Table 2.Comparison between patients with and without MACE

	Patients with MACE (n= 21)	Patients without MACE (n= 370)	P value
Age (years)	69±13	52±16	＜0.001
Male, n (%)	12 (57)	151 (41)	0.140
Body mass index	22.4±2.7	22.6±3.3	0.809
Smoking, n (%)	13 (62)	129 (35)	0.012
Diabetes mellitus, n (%)	1 (5)	51 (14)	0.236
Hypertension, n (%)	10 (48)	84 (23)	0.009
Cardiovascular disease history, n (%)	10 (48)	65 (18)	＜0.001
Lipoprotein apheresis, n (%)	3 (14)	4 (1)	＜0.001
Total cholesterol (mg/dl)	199±50	208±50	0.407
Triglycerides (mg/dl)	81 (56 – 120)	87 (63 – 134)	0.315
HDL-cholesterol (mg/dl)	47±16	57±15	0.003
LDL-cholesterol (mg/dl)	133±42	129±44	0.734
Lipoprotein (a) (mg/dl)	33.4 (9.3 – 70.2)	17.2 (7.4 – 35.7)	0.010
HbA1c (%)	5.6±0.4	5.8±0.7	0.252
US-AT thickness (mm)	11.9±6.9	7.6±3.2	＜0.001
US-AT thickening, n (%)	20 (95)	233 (63)	0.003
Xp-AT thickness (mm)	14.7±8.5	10.8±4.0	＜0.001
Xp-AT thickening, n (%)	15 (88)	215 (75)	0.221
Elasticity index	4.0±0.7	4.5±0.7	0.001
AT softening, n (%)	18 (86)	160 (53)	0.004
Calcification in AT	5 (24)	29 (8)	0.012
C-IMT (mm)	1.9 (1.4 – 2.5)	0.9 (0.7 – 1.4)	＜0.001
Greater C-IMT, n (%)	20 (95)	152 (41)	＜0.001

Values are presented as mean±standard deviation or median (interquartile range 25% - 75%) or number. HDL-C: high density lipoprotein cholesterol, MACE: major adverse cardiovascular events, LDL-C: low-density lipoprotein cholesterol, US-AT: Achilles tendon using ultrasonography, Xp-AT: Achilles tendon using radiography, AT: Achilles tendon, C-IMT: maximum intima-media thickness of common carotid artery

3.3. Cutoff Values of ATT, AT Softness and C-IMT for Prediction of MACE

Fig.1 shows the receiver-operating characteristic (ROC) curves by sex for determining the respective thresholds of US-ATT, Xp-ATT, EI (AT softness), and C-IMT for predicting the onset of MACEs. The cutoff values for US-ATT were 9.9 mm (area under the ROC curve (AUC) 0.83, sensitivity 0.83, specificity 0.75) in male subjects and 7.1 mm (AUC 0.66, sensitivity 0.67, specificity 0.62) in female subjects (Fig.1-a) and the cutoff value for Xp-ATT was 13.0 mm (AUC 0.75, sensitivity 0.67, specificity 0.75) in male subjects and 10.0 mm (AUC 0.56, sensitivity 0.50, specificity 0.54) in female subjects (Fig.1-b). The cutoff values for EI (AT softness) were 3.9 (AUC 0.75, sensitivity 0.75, specificity 0.72) in male subjects and 4.4 (AUC 0.63, sensitivity 0.67, specificity 0.62) in female subjects (Fig.1-c). The cutoff values for C-IMT were 1.6 mm (AUC 0.78, sensitivity 0.67, specificity 0.76) in male subjects and 1.5 mm (AUC 0.84, sensitivity 0.78, specificity 0.85) in female subjects (Fig.1-d).

Fig.1. Receiver-operating curves for ATT measured by ultrasonography and radiography, Elastic index (AT softness), and C-IMT for prediction of cardiovascular disease

(1-a) ATT measured by ultrasonography, (1-b) ATT measured by radiography, (1-c) Elastic Index (AT softness), (1-d) maximum IMT of common carotid artery. AUC: area under the curve

3.4. Comparison of MACEs according to Calculated Cutoff Values

Fig.2 show the results of Kaplan-Meier analysis and the log-rank test regarding occurrence of MACEs during the observation period when patients were divided into two groups according to the calculated cutoff values of each index shown in Fig.1 and presence or absence of AT calcification. The median observation period was 1,239 days (700-1,827 days). Cardiovascular event rates were significantly increased in the groups with AT thickening evaluated by ultrasonography, AT softening (EI lowering), greater C-IMT (P＜0.001, Fig.2-a, c, d) and presence of calcification (P=0.0157, Fig.2-e). However, for classification by Xp-ATT, there was no significant difference in the cardiovascular event rate (P=0.112, Fig.2-b).

Fig.2. Kaplan-Meier curve for MACE in classification according to calculated cutoff values

(2-a) ATT measured by ultrasonography, (2-b) ATT measured by radiography, (2-c) Elastic Index (AT softness), (2-d) maximum IMT of common carotid artery, (2-e) Presence of calcification in AT.

3.5. Impact of AT Thickening and Softening, C-IMT and Calcification of AT on MACEs during Observation Period

The results of logistic regression analysis of the impact of AT thickening and softening, C-IMT and AT calcification on MACEs during the observation period are shown in Table 3. The odds of cardiovascular events increased 6.7-fold for AT thickening evaluated by ultrasonography, according to the calculated cutoff values (odds ratio (OR): 6.67, 95% confidence interval (CI): 2.39 - 18.63, P＜0.001), 4.9-fold for AT softening according to the calculated EI cutoff values (OR: 4.93, 95% CI: 1.86 - 13.08, P＜0.001), and 11.0-fold for C-IMT thickening according to the calculated cutoff values (OR: 11.03, 95% CI: 4.13 - 29.47, P＜0.001). However, for AT thickening evaluated by radiography, according to the calculated cutoff values, there was no significant association with cardiovascular events (OR: 2.43, 95% CI: 0.90 - 6.56, P=0.078).

Table 3.Logistic regression analysis for presence of AT thickening and softening, C-IMT, calcification in AT and MACE

	Cut off values	Reference	Odds ratio	95% CI	P value
US-AT thickness	Male: 9.9 mm Female: 7.1 mm	Calculated using ROC curve	6.67	2.39 – 18.63	＜0.001
	Male: 6.0 mm Female: 5.5 mm	2022 JAS FH criteria＊1	11.76	1.57 – 88.56	0.017
Xp-AT thickness	Male: 13.0 mm Female: 10.0 mm	Calculated using ROC curve	2.43	0.90 – 6.56	0.078
	Both sexes: 9.0 mm	2017 JAS FH criteria＊2	3.14	0.88 – 11.17	0.077
	Male: 8.0 mm Female:7.5 mm	2022 JAS FH criteria	2.48	0.55 – 11.10	0.236
Elastic index (AT softness)	Male: 3.9 Female: 4.4	Calculated using ROC curve	4.93	1.86 – 13.08	＜0.001
	Both genders: 4.7	From previous study＊3	5.24	1.51 – 18.20	0.002
C-IMT	Male: 1.6 mm Female: 1.5 mm	Calculated using ROC curve	11.03	4.13 – 29.47	＜0.001
	Both genders: 1.1 mm	2017 JSUM guideline＊4	28.42	3.77 – 214.05	0.001
Calcification in the AT	Presence		3.66	1.25 – 10.72	0.018

^＊1: 2022 Japan Atherosclerosis Society FH criteria, ^＊2: 2017 Japan Atherosclerosis Society FH criteria, ^＊3: Michikura M. et al. Achilles Tendon Softness as a New Tool for Diagnosing Familial Hypercholesterolemia. J Am Coll Cardiol Img. 2021 Jul, 14 (7) 1483–1485. ^＊4: 2017 Standard method for ultrasound evaluation of carotid artery lesions - The Japan Society of Ultrasonics in Medicine. MACE: major adverse cardiovascular events, CI: confidence interval, US-AT: Achilles tendon using ultrasonography, Xp-AT: Achilles tendon using radiography, C-IMT: maximum intima-media thickness of common carotid artery, AT: Achilles tendon, ROC: receiver-operating characteristic

We obtained the following results when calculating the odds ratios for MACEs using the cutoff values in existing diagnostic criteria. The odds of cardiovascular events increased 11.8-fold for AT thickening evaluated by ultrasonography according to the 2022 JAS FH criteria⁵⁾ (OR: 11.76, 95% CI: 1.57 - 88.56, P=0.017), 5.2-fold for AT softening according to criteria in a previous study⁹⁾ (OR: 5.24, 95% CI: 1.51 - 18.20, P=0.002), 28.4-fold for C-IMT thickening according to the guidelines established in a previous study²²⁾ (OR: 28.42, 95% CI: 3.77 - 214.05, P=0.001) and 3.7-fold for presence of calcification in AT (OR: 3.66, 95% CI: 1.25 - 10.72, P=0.018). However, when evaluated by radiography according to the cutoff values in both the 2017 JAS FH criteria (OR: 3.14, 95% CI: 0.88 - 11.17, P=0.077) and 2022 JAS FH criteria (OR: 2.48, 95% CI: 0.55 - 11.10, P=0.236) AT thickening did not have an impact on cardiovascular events.

4.Discussion

In this retrospective cohort study of clinically diagnosed FH patients, we established gender-specific thresholds for ATT, EI (AT softness), and C-IMT to predict development of MACEs and found that calcification in the Achilles tendon is also a risk factor for developing MACEs in patients with FH. We believe that these thresholds and identification of AT calcification will help in risk assessment for patients in Japanese FH practice and encourage stricter and more comprehensive management for patients who exceed the thresholds.

Both AT thickening and AT softening thresholds calculated using ROC curves for ATT and EI measured by ultrasonography were predictive of MACE development (Fig.1, Fig.2). These findings support the results of the cross-sectional studies we previously reported^{9, 12)} and further strengthen the usefulness of ATT and AT softness for risk assessment. Although our study was retrospective, we believe that it is clinically significant in that the longitudinal analysis showed that AT status at the time of the initial ATT and EI (AT softness) measurements (including at diagnosis) can predict future MACE development.

The AUC for US-ATT and Xp-ATT in women was smaller than that in men (Fig.1-a, b). We consider the following to explain why the AUC for ATT was smaller in women. According to the diagnostic criteria, the gender difference in ATT is 0.5 mm for both Xp (8.0 mm versus 7.5 mm) and ultrasound (6.0 mm versus 5.5 mm). On the other hand, for Xp, the cutoff values of ATT for MACEs in this study differed by 3 mm between men and women - 13.0 mm and 10.0 mm, respectively, and, for ultrasound by 2.8 mm - 9.9 mm for men and 7.1 mm for women. In other words, the difference between the cutoff values in the diagnostic criteria and the cutoff values for MACEs in the present study was much smaller in women than in men. We believe that this resulted in the lower sensitivity and specificity of the cutoff values to predict MACEs and a smaller overall AUC in women. It is difficult to determine from the data in this study why both sensitivity and specificity were lower in women, although we speculate that it is because the Achilles tendon is less likely to thicken due to body size and foot size, and the presence of other risk factors.

Interestingly, even though this cohort population included only those with clinical FH, the ATT thresholds in the 2022 version of the JAS diagnostic criteria (≥ 6.0 mm for men and ≥ 5.5 mm for women)⁸⁾ also predicted future MACE development (Table 3). The EI (AT softness) threshold for FH diagnosis in our previous results (＜4.7 for both sexes)⁷⁾ similarly predicted the development of MACE in an FH-only population (Table 3). These findings may imply that FH patients with ATT measurements that meet the thresholds of the diagnostic criteria and those that meet the above diagnosis threshold for EI that we previously established are at higher risk of developing future MACE and require more aggressive lipid-lowering treatment.

In contrast, for measurements made using radiography, neither the thresholds for ATT calculated from the ROC curves of the study subjects (≥ 13 mm for men and ≥ 10 mm for women) nor those in the JAS diagnostic criteria (≥ 8.0 mm for men and ≥ 7.5 mm for women in 2022 version) could predict future MACE development. We speculate that this is because in many patients, the attachment of the Achilles tendon to the heel bone is twisted, and the angle of twist varies from patient to patient, making the ATT measurements by radiography inaccurate. However, the failure of the Xp-ATT (JAS 2022) criteria⁸⁾ to predict the development of MACEs is of limited significance because our study population consisted of FH patients clinically diagnosed by the JAS 2017 diagnostic criteria¹⁸⁾ and it is unreasonable to evaluate them by 2 different sets of criteria. Also, the reason for the downward revision for Xp-ATT in the JAS 2022 FH guidelines is to increase sensitivity in diagnosis¹⁰⁾, so care should be taken in interpretation. Nevertheless, the results of this study suggest that ultrasound measurements may be more useful in determining AT thickening than radiographic measurements in predicting the development of MACEs.

The risk of MACEs was increased for patients with calcification in the AT, with an odds ratio of 4.12. The mechanisms of the process of inflammation, lipid deposition, and calcification are similar for AT thickening and atherosclerosis^{23, 24)}. Thus, lipid deposition in the AT is related to inflammation, and calcification of the AT is assumed to be similar to the end-stage degeneration in atherosclerosis. However, in this study, calcification of the AT was not seen in patients without thickening, and the ultrasound image of calcification approximated that of AT rupture so we speculate that the calcification in the AT was not due to atherosclerotic degeneration but was a scar of rupture. We believe that we were looking at calcification that had occurred during the repair process of an area where the fibrous component had been stretched and broken due to lipid deposition within the Achilles tendon²⁵⁾.

The odds of a cardiovascular event increased 11.0-fold for greater C-IMT according to the calculated cutoff values (≥ 1.6 mm for men and ≥ 1.5 mm for women). Several studies have reported that C-IMT is associated with LDL-C and history of cardiovascular disease in HeFH patients^26-28), so FH patients with greater C-IMT may already have advanced coronary atherosclerosis. We therefore consider that aggressive screening for coronary lesions (e.g., coronary CT) may be warranted for patients with FH and greater C-IMT, even if they are asymptomatic. HeFH patients with an increased C-IMT should also undergo more detailed examinations for atherosclerosis and aggressive lipid-lowering treatment.

Only 19% of the FH patients in this study had achieved LDL-C control targets. In addition, the AT of patients who had not achieved LDL-C control targets was thicker and softer than that of those who had. However, it is difficult to achieve target LDL-C levels for FH management with existing oral medications alone²⁹⁾, and the patient’s financial situation is often a hindrance to aggressive treatment³⁰⁾. Moreover, physicians’ clinical inertia¹²⁾ may be an obstacle to achieving management targets.

On the other hand, aggressive lipid-lowering therapy has been reported to reduce ATT and C-IMT^{31, 32)}. Therefore, a study is needed for determining whether reductions in ATT and C-IMT with aggressive lipid-lowering therapy in patients with FH can prevent future MACEs, and if the results are positive, it will make stricter treatment more acceptable to patients and physicians. In addition, we believe that it is debatable whether a uniform LDL-C control target of less than 100 mg/dL is sufficient for both primary prevention patients who already have markedly increased AT thickness or IMT and those who do not.

In the MACE group, higher proportions of patients had hypertension and a history of smoking, and lower HDL-C and higher Lp(a) levels than those in the non-MACE group. These MACE-related factors are consistent with our previous findings^33-35). Therefore, for the MACE group patients, although some risks remain unmanageable (HDL-C, Lp(a), etc.), it would be important to conduct comprehensive management covering those that are readily manageable (smoking and blood pressure) as well as those that are manageable with aggressive treatment, including LDL-C.

This study has several limitations. First, since the FH subjects had been clinically diagnosed, non-FH patients may also have been included, so there was the possibility of cutoff values for each indicator being underestimated. However, given that the number of institutions in Japan that can perform genetic testing is limited and many general clinicians use diagnostic criteria to diagnose FH, the cutoff values we determined should be useful in clinical practice. Second, the subjects of this study were only Japanese patients at a single institution, and it was a retrospective study. However, we consider that the adoption of ultrasonography for the 2022 diagnostic criteria will enable a multicenter prospective study to be conducted. Third, PCSK9 inhibitors were launched in 2016, and the FH guidelines in 2017 included LDL-C target values and recommended strict control for secondary prevention. As the present study enrolled patients from as far back as 2013, it is possible that the medications and intensity of treatment at the time were different from that at present. Fourth, the 95% CIs for US-AT thickness (JAS 2022 Guidelines) and C-IMT (The Japan Society of Ultrasonics in Medicine 2017 Guidelines) were very wide: 1.57-88.56 (US-ATT) and 3.77-214.05 (C-IMT) (Table 3). This was as a result of classification by the diagnostic thresholds in the guidelines and may have been due to the majority of those who developed MACEs being above the diagnostic thresholds. Therefore, it should be noted that classification by diagnostic thresholds was statistically significant in predicting MACE, but not necessarily very precise. Finally, the times of measuring Xp-ATT and US-ATT differed even for the same patient. In some cases, ATT may have been smaller than at the initial visit because it was measured post-treatment. On the other hand, Xp-ATT was measured at the time of the first visit, so some of the data are for pre-treatment. This could have affected the results.

In conclusion, the present retrospective cohort study determined cutoff values of ATT and EI (AT softness) and C-IMT for MACE in clinically diagnosed HeFH patients on statins, and confirmed their usefulness. We consider that ATT and C-IMT measured by noninvasive ultrasonography would be useful for assessing the prognostic risk in FH at the time of diagnosis or relatively early in treatment. However, we need to address the question of whether aggressive LDL-C lowering therapy in HeFH patients with AT thickening, softening and calcification deposits and increased C-IMT would improve the prognosis.

Financial Support

This work is supported by a Labor and Welfare Sciences Research Grant for Research on Rare and Intractable Diseases (21FC1009), and JSPS KAKENHI (JP 20H01112 and 21K08143).

Credit Authorship Contribution Statement

Masahito Michikura: Conceptualization, Formal analysis, Data curation, Writing – original draft. Masatsune Ogura: Data curation, Writing – original draft, review & editing. Kota Matsuki: Conceptualization, Data curation, Writing – original draft. Makoto Yamaoka: Data curation, Hisashi Makino: Data curation. Mariko Harada-Shiba: Data curation, Writing – review & editing.

Declaration of Competing Interest

Masatsune Ogura has received lecture fees from Amgen and Kowa. Mariko Harada-Shiba is holding stocks: Liid Pharmaceuticals and received payments or honoraria from Amgen, MEDPACE, Novartis, Protosera, BML, and Kowa. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We sincerely thank Ms. Chizuru Fuke for excellent assistance with the sonographic studies and Mr. Yoshiyuki Sumita for expert technical advice.

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