Assessment of Low-density Lipoprotein Cholesterol Levels and Non-invasive Vascular Health in School-aged Children: A Study in Ogasa District, Shizuoka Prefecture

Abstract

Aim: The present study assessed low-density lipoprotein cholesterol (LDL-C) levels in school-aged children from the Ogasa District of Shizuoka Prefecture and evaluated the utility of non-invasive vascular tests, namely flow-mediated dilation (FMD) and intima-media thickness (IMT), in pediatric patients with familial hypercholesterolemia (FH).

Method: We analyzed the lipid test results of 8,568 students screened for prevention of lifestyle-related diseases and 78 children under 15 years old with cholesterol levels exceeding 220 mg/dL who visited Chutoen General Medical Center. We examined the LDL-C distribution from school-age screenings and conducted FMD and IMT assessments on those meeting the 2022 Pediatric FH Guidelines criteria.

Results: Among the screened students, 186 (2.2%) exhibited LDL-C levels above 140 mg/dL, including 123 fourth-graders (2.8%) and 63 first-year junior high students (1.5%). The mean LDL-C level across all students was 90.0 mg/dL (standard deviation: 21.3 mg/dL), with the 95th percentile at approximately 125.0 mg/dL. Of the 78 children who visited the hospital, 65 met the FH diagnostic criteria. In children ≥ 10 years old, no significant IMT differences were observed between the Definitive and Probable FH groups and the Possible FH group; however, a significant difference in the FMD percentage was noted between these groups (9.9% [8.1%-11.9%] vs. 14.2% [11.6%-16.3%], P=0.003).

Conclusions: Our findings highlight the LDL-C distribution in FH screening and suggest a potential reduction in FMD in pediatric FH patients ≥ 10 years old. These results emphasize the importance of initiating pharmacological interventions in school-aged children to maintain optimal LDL-C levels for lifelong cardiovascular health.

See editorial vol. 32: 306-307

Introduction

Atherosclerotic pathological changes begin to develop during childhood. This is evidenced by autopsy findings of the Bogalusa Heart Study¹⁾ and Pathological Determinants of Atherosclerosis in Youth (PDAY)²⁾. Familial hypercholesterolemia (FH) is an autosomal dominant genetic disorder that affects approximately 1 in every 200–500 individuals in the general population^{3, 4)}. Approximately 80% of individuals diagnosed with FH present with mutations in one of the three genes: low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin/kexin type 9 (PCSK9)⁵⁾. The early detection of FH in children can enable the establishment of lifestyle interventions before the onset of puberty³⁾. Numerous randomized controlled trials (RCTs) on statin treatment in children have been conducted since 1997, with some spanning up to two years^6-9). These trials have demonstrated that statin treatment is well tolerated, with no adverse effects on growth or maturation, thereby providing a clinical foundation for recommending the initiation of statin therapy starting at eight years old^{10, 11)}. However, there is a paucity of long-term data on individuals who begin statin therapy during childhood^{12, 13)}.

In the 2022 Japanese FH Guidelines¹⁴⁾, the established cutoff value of 180 mg/dL for low-density lipoprotein cholesterol (LDL-C) screening was confirmed as an appropriate threshold for identifying atherosclerotic lesions in pediatric populations. In Kagawa Prefecture, Japan, the attendance rate for FH universal screening among school-age children was high (90%-95%), but the lack of an effective follow-up system led to difficulties in conducting secondary checkups, with only 50%-70% participation⁴⁾.

In recent years, various non-invasive methods have been developed for detecting atherosclerotic lesions, which are widely applied across diseases, including FH^15-18). These methods facilitate the evaluation of the key characteristics of atherosclerosis, such as endothelial dysfunction and vascular wall thickening, through subclinical measures such as flow-mediated dilation (FMD)¹⁹⁾ and intima-media thickness (IMT)²⁰⁾. FMD, a non-invasive assessment of the endothelial function, is recognized for its clinical and research utility, especially in assessing the health of the vasculature by measuring blood vessel dilation capacity in response to increased blood flow²¹⁾. Ultrasound-based measurement of brachial reactivity has become the most established technique in adults for FMD¹⁵⁾. This technique has been utilized to evaluate endothelial health, with its significance highlighted in studies on Kawasaki disease¹⁸⁾, low-birth-weight children²²⁾, and FH²¹⁾. Ultrasound-based measurement of carotid IMT is also recognized as an established technique in adults¹⁵⁾, with its utility in childhood assessments demonstrated in studies of abdominal obesity¹⁶⁾, low-birth-weight infants¹⁷⁾, and FH²¹⁾.

A meta-analysis revealed that children with FH exhibit a significantly greater IMT than controls²³⁾. In addition, the cardio-ankle vascular index (CAVI) has been used as a crucial atherosclerosis indicator to non-invasively assess arterial stiffness, measuring stiffness between the heart and ankle while accounting for blood pressure during the test. FMD, the IMT, and the CAVI are safe and typically well tolerated in the pediatric population. They provide valuable insights into the vascular health of children and can assist in the identification of early disease markers.

This study evaluated the levels of LDL-C in school-aged children during lifestyle checkups within the Ogasa District medical area of Shizuoka Prefecture and assessed the efficacy of noninvasive vascular testing in pediatric patients with FH.

Materials and Methods

Study Population

This study was conducted at Chutoen General Medical Center, a tertiary referral hospital located in Shizuoka, Japan. The hospital serves a catchment area comprising approximately 465,000 individuals, of which approximately 63,000 are children ≤ 15 years old. Within our medical catchment region, three municipalities have consistently carried out health checkups aimed at preventing lifestyle-related diseases among fourth-graders in elementary school and first-year students in junior high school. Annually, in our local region, around 2,800 students, encompassing fourth-graders in elementary school and first-year students in junior high school, participate in blood testing as part of a screening program for preventing lifestyle-related diseases. Although participation in these screenings was voluntary, the rate consistently exceeded 90%. The study population included children ≤ 15 years old who attended the Department of Pediatrics at Chutoen Medical Center between June 2016 and March 2023 and were found to have serum total cholesterol (TC) levels exceeding 220 mg/dL. We primarily identified these children through two screening protocols: a routine school-based health checkup aimed at preventing lifestyle-related diseases for fourth-grade elementary school students and a similar checkup for first-year junior high school students. In addition, opportunistic blood testing was conducted when children visited pediatricians for unrelated reasons, such as the evaluation of short stature or treatment for infectious diseases, and were incidentally found to have TC levels ＞220 mg/dL.

Data Collection

The FH Diagnosis

This was a single-center, retrospective cohort study. The diagnosis of pediatric FH in this study was determined based on the three criteria outlined in the guidelines¹⁴⁾, as shown in Table 1. FH is diagnosed when (1) criteria 1 (LDL-C ≥ 140 mg/dL) and 2 are met, (2) the individual’s LDL-C level is ≥ 180 mg/dL along with the presence of criterion 3, or (3) the LDL-C level is ≥ 250 mg/dL (indicated in red in Table 1). Upon a diagnosis of FH, patients were classified into either heterozygous or homozygous forms, as appropriate. Furthermore, the diagnosis of FH can be confirmed through the identification of a pathogenic mutation via genetic testing. A diagnosis of “probable FH” is considered if (1) criteria 1 and 3 are met, or (2) the individual’s LDL-C level is ≥ 180 mg/dL without additional criteria being met (indicated as orange in Table 1). A diagnosis of “possible FH” is considered if either criterion 2 or 3 is met with an LDL-C level of 100–139 mg/dL, or if LDL-C levels are between 140 and 179 mg/dL without a known FH family history. In such cases, annual follow-up assessments were recommended (indicated in yellow in Table 1). Conversely, if there is a family history of FH including criterion 3 but the LDL-C level is ＜100 mg/dL, or if there is no family history and the LDL-C level is ＜140 mg/dL, FH is excluded “unlikely” (not inherited), and no follow-up is deemed necessary (indicated as white in Table 1). In cases where the diagnosis is challenging, consultation with a specialist and consideration of genetic testing are advised. If there is an increase in LDL-C levels during follow-up or if an FH family history becomes evident, a re-evaluation of the diagnosis is recommended.

Table 1.Criteria for Pediatrics FH diagnosis

Criteria    1.LDL-C level more than 140mg/dL    2.Family history of FH (parents or siblings)    3.Parental LDL-C more than 180mg/dL or family history of premature coronary artery disease (Grandparent of the parent)
	LDL-C (mg/dL)
	＜100	100-139	140-179	180-249	≥ 250
Family history of FH	Unlikely	Possible	FH	FH	FH
Parent’s LDL ≥ 180mg/dL or Family history of premature coronary artery disease	Unlikely	Possible	Probable	FH	FH
No family history of FH	Unlikely	Unlikely	Possible	Probable	FH

Guidelines for the Diagnosis and Treatment of Pediatric Familial Hypercholesterolemia 2022 14). Abbreviations: FH; Familiar Hypercholesterolemia, LDL-C; low-density lipoprotein-cholesterol,

In a screening program aimed at preventing lifestyle-related diseases, serum lipid levels, including TC, LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG), were measured. These measurements were obtained from fasting samples using an enzymatic method. Specifically, TC levels were measured using the Determiner-L TC II system, LDL-C with the MetaboLead LDL-C system, HDL-C with the MetaboLead HDL-C system, and TG with the Determiner-L TG II system, all of which were supplied by Minaris Medical Co., Ltd. (Tokyo, Japan).

At our institution, TC, LDL-C, HDL-C, and TG levels were measured in fasting samples using a direct method. Specifically, TC levels were determined using the Cholestest TC system, LDL-C using the Cholestest LDL-C system, HDL-C using the Cholestest HDL-C system, and TG using the Cholestest TG system, all supplied by Sekisui Medical Co., Ltd. (Tokyo, Japan).

In this study, a definitive diagnosis of FH was established at our institution in accordance with 2022 guidelines¹⁴⁾. The classification of FH, as outlined in the FH guidelines, which includes three distinct groups – definitive FH, probable FH, and possible FH – was restructured into two consolidated categories for the purposes of this analysis: Definitive and Probable FH and Possible FH. A comparative study was conducted to analyze clinical data across these newly defined groups.

Non-Invasive Vascular Assessments

We have incorporated non-invasive vascular function assessments involving FMD, the IMT, and the CAVI, the accuracy of which have also been established in childhood²⁴⁾, into our follow-up evaluations. FMD evaluations were performed using a high-resolution ultrasonography system (UNEXEF18G; UNEX Co., Nagoya, Japan). The measurement protocol was consistent with previously described methods²⁵⁾. For the FMD assessment, participants were instructed to fast and abstain from consuming any food or beverage for 6 h prior to the test. Specifically, on the day of the test, they were advised to avoid fatty foods, caffeine, and beverages containing vitamin C. Participants were also asked to rest in a supine position for approximately 10 minutes before the commencement of the FMD procedure. During the test, the patient remained at rest, and the brachial artery was meticulously monitored. Subsequently, a cuff placed on the upper arm was inflated for 5 min to induce arterial occlusion and facilitate the assessment. While nitroglycerine-induced dilation is generally employed in FMD measurements, in this study, we utilized a method that does not involve the use of nitroglycerine.

For IMT measurements, the common carotid arteries 1-2 cm proximal to the bulb region were scanned, focusing on the posterior (far) arterial wall. These measurements were conducted using a LOGIQ S8 ultrasound system (GE Healthcare Japan, Tokyo, Japan) equipped with an 11.0-MHz linear transducer. All assessments followed a standardized scanning protocol for the right and left common carotid arteries. The IMT value was determined as the average of 50–100 points spanning a width of 1–2 cm. The CAVI was evaluated using a Vasera VS-1000 automatic system (Fukuda Denshi, Tokyo Japan), as previously described^{26, 27)}, following a rest period in the supine position. Electrocardiogram electrodes were applied to both the wrists and sternum for heart-sound detection. Cuffs were secured around both the upper arms and ankles. The cardio-ankle pulse wave velocity (PWV) was gauged by dividing the distance from the aortic valve to the ankle artery by the time sum of the pulse wave propagation from the brachium to the ankle and the time difference between the second heart sound on the phonocardiogram and that on the notch of the brachial pulse wave. The CAVI is articulated as the stiffness parameter β according to the following equation: CAVI=a×[2ρ/PP×[ln Ps/Pd] PWV2]＋b, where a and b are constants, ρ represents the blood density, PP is the pulse pressure, Ps is the systolic pressure, and Pd is the diastolic pressure. Theoretically, CAVI measurement is independent of blood pressure, and the mean CAVI from the left and right sides of the body was utilized for analyses.

Ethical Considerations

The study protocol adhered to the principles outlined in the Declaration of Helsinki and was approved by the Ethics Committee of Chutoen Medical Center (approval date, January 25, 2022; approval number, 177). Consent for participation was obtained from the guardians of all subjects using an opt-out approach in alignment with the Japanese Ethical Guidelines for Medical and Health Research Involving Human Subjects. The details of this study are publicly available on our website. Data from subjects whose guardians opted out were excluded from the analysis.

Outcome Measures

The primary outcome of this study was to elucidate the distribution patterns of TC, LDL-C, and other lipids in school-aged children and to elucidate the serial changes in noninvasive vascular assessments in patients diagnosed with FH.

Data Analyses

Normally distributed data were expressed as the mean±standard deviation (SD), while data that were not normally distributed were expressed as median (interquartile range). The results were expressed as the median and interquartile range (IQRs). Two-sided comparisons between groups were performed using the Mann–Whitney U-test, chi-square test, or Fisher’s exact test, as appropriate.

All statistical analyses were performed using the IBM SPSS Statistics software program, version 28 (IBM Corp., Armonk, NY, USA). The threshold for statistical significance was set at P＜0.05.

Results

Distribution of TC, LDL-C, and other Lipids in a Routine School-Based Health Checkup Aimed at Preventing Lifestyle-Related Diseases

The data from Kakegawa City, representing one of the three municipalities involved, related to the screening for lifestyle-related diseases among schoolchildren from 2018 to 2022, are illustrated in Fig.1 and Table 2. Over 5 years, among fourth-graders and first-year junior high school students in Kakegawa City, 186 students (2.2%) exhibited LDL-C levels exceeding 140 mg/dL. Of these, 123 were fourth-grade elementary school students (2.8%), and 63 were first-year junior high school students (1.5%). Table 2 presents the sex-specific distributions of TC, LDL-C, HDL-C, and TG for fourth-graders in elementary school and first-year students in junior high school. This study established cutoff values for these lipid levels as follows: TC, ≥ 220 mg/dL; LDL-C, ≥ 140 mg/dL; HDL-C, ＜40 mg/dL; and TG, ≥ 140 mg/dL. The table details the number and percentage of patients whose lipid levels exceeded the reference values.

Fig.1. Distribution of low-density lipoprotein cholesterol (LDL-C) levels observed in lifestyle-related disease screenings conducted for school-aged children in Kakegawa City, one of the three municipalities in the Ogasa region, from 2018 to 2022

(A) LDL-C distribution for 4,321 fourth-grade elementary school students, and (B) LDL-C distribution for 4,247 first-year junior high school students.

Table 2.The results of 5-year screening for lifestyle-related diseases among school students

	Fourth-grade elementary school			First-grade junior high school
n	2180	2141	p-value	2185	2062	p-value
Sex	Boys	Girls		Boys	Girls
Height (cm)	133.5 (5.6)	133.56 (6.2)	0.894	152.6 (8.0)	151.7 (5.8)	＜0.001
Weight (kg)	30.9 (6.4)	30.1 (5.8)	＜0.001	43.8 (9.5)	43.1 (7.6)	0.014
sysBP(mmHg)	105.5 (11.9)	105.5 (12.0)	0.835	112.5 (11.9)	112.9 (12.1)	0.226
diaBP(mmHg)	59.5 (10.0)	59.1 (10.0)	0.240	62.8 (10.0)	63.9 (10.3)	＜0.001
TC (mg/dL)	172.9 (24.7)	176.0 (26.3)	＜0.001	161.4 (25.3)	168.6 (24.9)	＜0.001
95%CI	171.9-173.9	174.9-177.1		160.3-162.5	167.5-169.7
＊≥ 220mg/dL_n (%)	90 (4.1)	124 (5.8)		110 (5.0)	90 (4.4)
LDL-C (mg/dL)	91.1 (20.2)	95.6 (22.3)	＜0.001	83.9 (20.6)	89.4 (20.4)	＜0.001
95%CI	90.3-91.9	94.7-96.5		83.0-84.8	88.5-90.3
＊≥ 140mg/dL_n (%)	37 (1.7)	86 (4.0)		25 (1.1)	38 (1.8)
HDL-C (mg/dL)	66.1 (13.6)	64.3 (12.8)	＜0.001	62.2 (13.4)	63.9 (12.5)	＜0.001
95%CI	65.5-66.7	63.8-64.8		61.6-62.8	63.4-64.4
＊＜40mg/dL_n (%)	28 (1.3)	27 (1.3)		42 (1.9)	18 (0.9)
TG (mg/dL)	81.0 (54.3)	82.2 (50.3)	0.432	80.2 (48.0)	83.3 (48.4)	0.034
95%CI	78.7-83.3	80.1-84.3		78.2-82.2	81.2-85.4
＊≥ 140mg/dL_n (%)	232 (10.6)	235 (11.0)		227 (10.4)	195 (9.5)

Result, mean (standard deviation), ^＊Each lipid cutoff value_n (%), Abbreviations: sysBP; systolic blood pressure, diaBP; diastolic blood pressure, CI; Confidence interval, TC; Total cholesterol, LDL-C; low-density lipoprotein-cholesterol, HDL-C; high-density lipoprotein, TG; triglycerides. TC, LDL-C, HDL-C, and TG levels were measured using fasting samples using the enzymatic method, Specifically, TC was measured with the Determiner -L TC II system, LDL-C with the MetaboLead LDL-C system, HDL-C with the MetaboLead HDL-C system, and TG with the Determiner -L TG II system, all provided by Minaris Medical Co., Ltd., Tokyo, Japan.

In the overall analysis, the mean LDL-C level was 90.0 mg/dL with an SD of 21.3 mg/dL, and the 95th percentile was approximately 125.0 mg/dL. Levels of ≥ 140 mg/dL correspond to approximately the 99.1th percentile. For 4th-grade boys, the mean LDL-C level was 91.1 mg/dL (SD: 20.2 mg/dL), with the 95th percentile at about 124.3 mg/dL, and levels of ≥ 140 mg/dL corresponding to approximately 99.2nd percentile. For 4th-grade girls, the mean LDL-C level was 95.6 mg/dL (SD: 22.3 mg/dL), with the 95th percentile at about 132.3 mg/dL, and levels of ≥ 140 mg/dL corresponding to the 97.7th percentile. For first-year junior high school boys, the mean LDL-C level was 83.9 mg/dL (SD: 20.6 mg/dL), and the 95th percentile was approximately 117.8 mg/dL, with ≥ 140 mg/dL corresponding to the 97.7th percentile. For first-year junior high school girls, the mean LDL-C level was 89.4 mg/dL (SD: 20.4 mg/dL), the 95th percentile was 123.0 mg/dL, and ≥ 140 mg/dL corresponded to approximately the 99.3rd percentile. For 4th-grade students, the mean TC level was 174.5 mg/dL (SD:25.6 mg/dL) with the 95th percentile was approximately 175.3 mg/dL, and the mean HDL-C level was 65.2 mg/dL (SD: 13.2 mg/dL), with the lower 95th percentile approximately 43.5 mg/dL, and the mean TG level was 81.6 mg/dL (SD: 52.4 mg/dL), with the 95th percentile approximately 167.8 mg/dL. For first-year junior high school students, the mean TC level was 164.9 mg/dL (SD: 25.4 mg/dL), with the 95th percentile approximately 165.7 mg/dL, and the mean HDL-C level was 63.0 mg/dL (SD: 13.0 mg/dL), with the lower 95th percentile approximately 41.6 mg/dL, and the mean TG level was 81.7 mg/dL (SD: 48.1 mg/dL), with the 95th percentile approximately 160.8 mg/dL. In both the fourth-grade elementary school and first-year junior high school students, TC and LDL-C levels were significantly higher in girls than in boys, while HDL-C levels were significantly lower in girls than in boys (Table 2).

Characteristics of FH and Non-Invasive Vascular Assessments

Fig.2 illustrates a flowchart of the study. From June 2016 to March 2023, 78 children under 15 years old presented to our hospital with serum TC levels exceeding 220 mg/dL. Of these 78 students, 48 were screened in the fourth grade of elementary school and 22 in the first year of junior high school during the lifestyle-related disease prevention checkup for children; 8 were incidentally included through blood sampling. Within the cohort of 78 children, 14 were excluded because they were unlikely to be diagnosed with FH. Among the 14 children excluded from the study, 4 presented with LDL-C levels below 100 mg/dL at the time of blood collection at our hospital. In addition, 8 patients had LDL-C levels ranging from 100 to 139 mg/dL, with an ambiguous FH family history. Furthermore, one case was excluded due to a parental LDL-C level ＜180 mg/dL and another case was excluded due to the presence of obesity.

Fig.2. Flowchart of the study

Of the 78 subjects examined, 64 (2.1%) were categorized as having FH, probable FH, or Possible FH.

Of the 64 subjects enrolled, 40 (62.5%) were categorized as FH probable and 24 as FH possible. None of the families underwent cascade screening during the study period. A clear family history of hyperlipidemia was documented in 57 patients (89.1%). Possible FH was identified in 19 patients (79.2%), and Definitive and Probable FH was confirmed in 38 patients (95.0%). Table 3 presents a detailed overview of the demographic and clinical characteristics of the study population at the initial visit. Of the 64 patients, 57 (89.1%) were identified through a screening program to prevent lifestyle-related diseases. Notably, 38 patients (59.4%) were diagnosed during the examination conducted in the fourth grade. The z-scores for TC, LDL-C, HDL-C, and TG levels were computed using the values presented in Table 1. The median LDL-C z-score in the Definitive and Probable FH group was 5.05.

Table 3.Characteristics in the present study

	Definitive and Probable FH	Possible FH	p-value
n	40	24
Category
Sex (%)
male	18 (45.0)	9 (37.5)	0.609
female	22 (55.0)	15 (62.5)
Screening_Opportunity
＊Chance (%)	6 (15.0)	1 (4.2)	0.451
Forth-grade elementary (%)	23 (57.5)	15 (62.5)
First-grade Junior high (%)	11 (27.5)	8 ( 33.3)
Continuous variable
	Median [IQR]	Median [IQR]
Age (y)	10.9 [9.9-13.0]	10.8 [9.7-12.7]	0.454
Height (cm)	144.4 [132.5-152.1]	143.6 [138.7- 150.8]	0.635
Weight (kg)	36.0 [28.9-42.9]	39.8 [33.5-43.5]	0.341
TC (mg/dL)	275.5 [245.0-301.0]	218.0 [202.8- 237.0]	＜0.001
TC_z score	3.98 [2.94-5.12]	1.97 [1.24-2.42]	＜0.001
LDL-C (mg/dL)	196.5 [159.5-232.3]	126.5 [111.0- 137.3]	＜0.001
LDL_z score	5.05 [3.36-6.75]	1.71 [1.09-2.41]	＜0.001
HDL-C (mg/dL)	56.5 [49.0-65.3]	62.5 [49.5-72.0]	0.206
HDL-C_z score	-0.54 [-1.08- 0.14]	-0.06 [-1.07- 0.63]	0.197
TG (mg/dL)	74.0 [60.5-112.5]	94.0 [59.3- 190.3]	0.361
TG_z score	-0.14 [-0.44-0.61]	0.25 [-0.47-2.17]	0.273
CRP (mg/dL)	0.01 [0.00-0.01]	0.01 [0.00-0.02]	0.589
Glucose (mg/dL)	92.5 [88.3-98.0]	94.0 [89.5-97.0]	0.882
Vascular health
n	34	15
sysBP (mmHg)	114.0 [106.0-121.0]	108.5 [104.3- 115.8]	0.243
diaBP (mmHg)	63.0 [59.0-68.0]	64.0 [61.8-67.0]	0.856
FMD (%)	12.3 [10.0-13.5]	13.2 [10.7-14.4]	0.372
IMT (mm)	0.41 [0.38-0.49]	0.43 [0.40-0.47]	0.777
CAVI	5.05 [4.50-5.45]	4.75 [4.35-5.19]	0.414

Abbreviations: Table 1. IQR; Interquartile Range, ^＊Chance; A case was identified by chance with a TC level exceeding 220 mg/dL at the hospital, CRP; C-reactive-protein. Glucose; Fasting plasma glucose. FMD; Flow-mediated dilation, IMT; Intima-media thickness, CAVI; Cardio-ankle vascular index.

TC, LDL-C, HDL-C, and TG levels were measured from fasting samples using the direct method. Specifically, TC levels were determined with the Cholestest TC system, LDL-C with the Cholestest LDL-C system, HDL-C with the Cholestest HDL-C system, and TG with the Cholestest TG system, all supplied by Sekisui Medical Co., Ltd.

The z-score was computed based on the mean and standard deviation for each grade and gender within the screening for lifestyle-related diseases among school students.

Noninvasive vascular assessments were conducted on 34 patients categorized as having Definitive and Probable FH and 14 patients with Possible FH. The results of the secondary outcomes are presented in Tables 3 and 4 and Fig.3. Post-initial visits and non-invasive vascular assessments were conducted annually. Throughout the study period, these assessments were performed at a median frequency of 2 times (IQR: 1–4 times) per patient. The patients were divided into 2 age categories for the analysis of vascular assessment outcomes: those ≥ 10 years old and those ＜10 years old. A comparative analysis of these vascular assessment results was then conducted between Definitive and Probable FH and Possible FH. Upon analyzing the vascular assessment results in patients ≥ 10 years old, it was found that there was no significant difference in the IMT between the Definitive and Probable FH and Possible FH groups. However, a significant difference was noted in %FMD between the two groups (Fig.3). No significant sex-based disparities were observed in the %FMD, IMT, and CAVI measurements between participants ≥ 10 years old and those ＜10 years old. In patients ＜10 years old, no significant correlation was observed between the %FMD and IMT.

Table 4.Follow-up observation of the assessment of vascular health between the two groups.

	Definitive and Probable FH	Possible FH	p-value
By age group
Less than 10y
FMD (%) (n = 23)	13.2 [11.2-15.4]	14.3 [12.4-15.3]	0.561
IMT (mm) (n = 25)	0.44 [0.41-0.50]	0.43 [0.41-0.47]	0.484
CAVI (n = 25)	4.75 [4.50-5.25]	4.85 [4.54-5.09]	1.000
More than 10y
FMD (%) (n = 27)	9.9 [8.1-11.9]	14.20 [11.6-16.3]	0.003
IMT (mm) (n = 38)	0.46 [0.43-0.54]	0.45 [0.40-0.50]	0.417
CAVI (n = 38)	5.35 [4.82-5.75]	4.75 [3.70-5.25]	0.146

Abbreviations: Table 1, and Table 2.

Fig.3. A comparative analysis of non-invasive vascular assessments, specifically flow-mediated dilation (FMD) and the intima-media thickness (IMT), between the Definitive and Probable FH and Possible FH groups

Notably, FMD values in the Definitive and Probable FH groups, comprising individuals ≥ 10 years old, were significantly lower than those in the Possible FH group.

Other Findings

Table 5 shows the maximum adverse values of TC, LDL-C, HDL-C, and TG, the rate of loss to follow-up (LTFU) during the study period, and the progression of statin therapy initiation in the study population. LTFU was defined as instances where participants, despite their initial engagement in the study, became uncontactable or failed to attend subsequent scheduled visits. This could be attributed to a multitude of reasons. For instance, a participant may have canceled a one-year follow-up appointment without providing a reason and then ceased all further communication. In this study, LTFU was observed in 18 of 64 patients, representing 28.1% of the total patients. Notably, the LTFU rate was significantly higher in the Definitive and Probable FH groups, with 15 of the 40 patients (37.5%) compared to 3 of the 24 patients (17.5%) with Possible FH.

Table 5. Follow-up observation results between the two groups

	Definitive and Probable FH	Possible FH	p.value
n	40	24
Category
Lost to follow-up (%)	15 (37.5)	3 (12.5)	0.044
Statin-treated cases (%)	10 (25.0)	0 (0.0)	0.010
Continuous variable
	Median [IQR]	Median [IQR]
Follow_up (m)	25.1 [12.3-41.3]	11.1 [0.6-21.7]	0.008
The number of blood tests.	3.0 [1.5-4.5]	2.0 [1.0-2.5]	0.074
TC (mg/dL)	292.0 [252.0-327.0]	222.5 [201.3-252.5]	＜0.001
TC_z score	4.82 [3.24-6.47]	2.29 [1.34-3.38]	＜0.001
LDL-C (mg/dL)	205.0 [166.5-249.5]	133.0 [118.0-151.5]	＜0.001
LDL-C_z score	5.58 [3.72-7.87]	2.04 [1.11-2.94]	＜0.001
HDL-C (mg/dL)	55.0 [45.5-63.5]	62.00 [47.8-72.0]	0.197
HDL-C_z score	-0.73 [-1.38-0.03]	-0.10 [-1.29-0.63]	0.200
TG (mg/dL)	106.0 [74.3-144.3]	111.0 [62.0-219.0]	0.618
TG_z score	0.46 [-0.15-1.21]	0.57 [-0.44-2.67]	0.553
CRP (mg/dL)	0.01 [0.01-0.04]	0.01 [0.00-0.03]	0.083

Abbreviations: Table 1, Lost to follow-up (LTFU)

TC, LDL-C, HDL-C, and TG levels were measured from fasting samples using the direct method. Specifically, TC levels were determined with the Cholestest TC system, LDL-C with the Cholestest LDL-C system, HDL-C with the Cholestest HDL-C system, and TG with the Cholestest TG system, all supplied by Sekisui Medical Co., Ltd., Tokyo, Japan.

The z-score was computed based on the mean and standard deviation for each grade and gender within the screening for lifestyle-related diseases among school students.

In the present study, comprehensive information regarding the normal values of IMT¹⁶⁾ and FMD³⁰⁾ was provided to patients and their families. It has been emphasized that a decline in these vascular assessment parameters can necessitate the initiation of statin therapy, as recommended in the guidelines. Furthermore, guidelines¹⁴⁾ also indicates that statin therapy might be appropriate even when these indices are within normal limits. In alignment with these guidelines, detailed explanations of starting statin treatment were provided to the families and children participating in the study. Consequently, during the study period, statin therapy was initiated in 10 of the 40 patients with Definitive and Probable FH, representing 25% of this group.

Discussion

In this study, 2.2% of school-age children had LDL-C levels of ≥ 140 mg/dL, with the 95th percentile at approximately at 125.0 mg/dL. A previous study⁴⁾ reported that approximately 4% of children in Kagawa Prefecture had LDL-C levels ≥ 140 mg/dL. In addition, a national survey of Japanese children²⁸⁾ indicated that the 95th percentile for LDL-C was approximately 140 mg/dL. In comparison, the corresponding values in our catchment area were lower than those previously reported. Considering that LDL-C levels are influenced by various factors, such as genetic predispositions and regional dietary habits, variations in the cutoff values for screening tests across different regions may occur. Nonetheless, levels exceeding 140 mg/dL are markedly abnormal and deemed an appropriate threshold for screening purposes. Future studies are needed to further explore and refine the cutoff values for LDL-C screening.

In clinical evaluations involving adult patients, the use of a combination of multiple noninvasive assessments for detecting atherosclerotic lesions reportedly offers enhanced predictive accuracy for future vascular events, as opposed to employing each test individually²⁹⁾. Similarly, we considered the application of this integrative approach, incorporating various noninvasive vascular assessments, in the management of FH in school-aged children. A comprehensive medical care framework integrating non-invasive vascular assessments, such as FMD, which assesses the vascular endothelial function; CAVI, which evaluates vascular stiffness; and IMT, which provides a morphological assessment of arteriosclerosis, for FH in Japanese pediatric populations remains unreported. Early detection of FH during school age, effective management of incipient arteriosclerotic lesions, and the development of a healthcare system that incorporates statin therapy are essential for the prevention of future cardiovascular events and strokes.

In the present study, we observed pronounced differences in LDL-C levels between the Definitive and Probable FH groups and the Possible FH group at their initial consultation. Despite this, the results from the three distinct noninvasive vascular assessments showed no significant disparities between the groups. In addition, these groups, stratified by age categories of ＜10 and ≥ 10 years old, showed no significant age-related variations in IMT. Nevertheless, in the Definitive and Probable FH groups, FMD exhibited a significant decrease when comparing children ＜10 and ≥ 10 years old. Concerning non-invasive vascular assessments of children with FH around 10 years old, previous studies have reported that while no significant difference was observed in IMT values in FH patients ≥ 10 years old compared to a cohort of healthy children, a significant divergence was noted in FMD measurements³⁰⁾. They also reported a correlation between IMT and FMD. However, our study did not include a cohort of healthy children, thus limiting our ability to clarify the relationship between IMT and FMD in childhood. In pediatric patients with FH, IMT increases by 0.0056 mm per annually³¹⁾. On average, carotid atherosclerosis may develop at 17 and 26 years old in male and female patients with heterozygous FH, respectively³²⁾. IMT is acknowledged to be a more effective predictor of future cardiovascular events in the elderly population than FMD²⁹⁾. However, owing to its gradual progression rate, FMD can be considered a more sensitive non-invasive vascular indicator for detecting atherosclerotic lesions in children with FH than IMT. In addition, in children with FH ≥ 10 years old, a reduction in FMD is regarded as a valuable indicator for initiating statin therapy around 10 years old, in alignment with current guideline recommendations¹⁴⁾.

Within our region, approximately 90% of school-age children participate in health checkups aimed at preventing lifestyle-related diseases. Nonetheless, following secondary screening, a concerning pattern emerged with a total of 18 cases (28.1%) of LFTU, predominantly within the Definitive and Probable FH group, and statin treatment goals were achieved in only 9% of these patients. These findings highlight the importance of enhancing education for providers and offering readily accessible information for the screening and management of pediatric FH. Receiving an FH diagnosis at a young age can have psychological implications for children and their families, including increased stress and worry about future health outcomes³³⁾. The observed reduction in LFTU from primary screening to detailed secondary examination in FH can likely be attributed to insufficient education and understanding of the disease among primary care physicians and medical staff, as previously reported³⁴⁾. The societal repercussions of the COVID-19 pandemic³⁵⁾ during the study period may have markedly affected the incidence of LTFU, an issue that necessitates mitigation strategies.

In real-world clinical settings, the introduction and maintenance of drug therapy in asymptomatic children with FH during school years is challenging. Pediatric participants in drug intervention trials for FH have been reported to struggle with appropriate long-term medication management³⁶⁾. There is an ongoing debate suggesting that the decision to initiate lipid-lowering therapy in adult patients with hypercholesterolemia should be based on vascular imaging results rather than solely on circulatory biomarkers³⁷⁾. We conducted non-invasive vascular assessments in pediatric patients diagnosed with FH, provided imaging data concurrently with blood test results, and emphasized the importance of regular follow-up and statin treatment. However, the efficacy of this comprehensive approach is yet to be validated.

Presumably, to reduce LTFU and promote the initiation of drug therapy, it is essential for primary care physicians in the community and patients’ families to recognize the importance of understanding the future cardiovascular risks associated with FH and its prevention. Moving forward, there appears to be a need for further research on the establishment of comprehensive medical care provision for patients with FH.

In the global diagnostic criteria for FH, the LDL-C value (140-180 mg/dL)^{4, 14)} in Japan’s FH guidelines is different from that in other countries and studies (approximately 160-190 mg/dL)^{3, 6, 8-10)}. Reviews of noninvasive vascular assessments for pediatric FH indicate that deviations in IMT, FMD, and CAVI manifest from childhood to adolescence²³⁾. However, much of the existing research aligns with guidelines from outside of Japan. In particular, the prevalence of FH is known to vary across different regions of the world^{38, 39)}, emphasizing the need for individual countries to formulate their own diagnostic criteria and treatment approaches. These approaches are crucial for the effective identification and management of FH in pediatric populations, highlighting its significance^{6, 31)}.

In the present study, our objective was to risk-stratify asymptomatic pediatric patients with FH by integrating noninvasive vascular assessments into the follow-up protocol for individuals diagnosed with FH during childhood, in accordance with the Japanese FH guidelines. This was achieved through universal screening of school-aged children. Our findings demonstrated the feasibility of this approach. Future research should aim to expand the sample size and further investigate the effects of statin therapy.

However, several limitations associated with the present study warrant mention. First, it involved a relatively small number of subjects and had a retrospective design. Second, we incorporated the evaluation of Achilles tendon thickening, as recommended by the guidelines, in some patients. However, because most patients did not exhibit abnormal findings, they were often omitted during follow-up. Moving forward, we plan to include the routine assessment of Achilles tendon thickening in our examinations. Third, while we acquired data pertaining to lifestyle-related disease prevention screenings among children in Kakegawa City, official records detailing the precise number of children who visited local medical facilities were not accessible. Fourth, in this study, we were unable to perform evaluations for apolipoprotein tests, including lipoprotein (a). However, in FH, the assessment of apolipoproteins, such as lipoprotein (a), APOA-1, APOA-2, APOB, APOC-2, APOC-3, and APOE, plays a crucial role in disease detection. These tests are essential for an accurate diagnosis, risk stratification, and the formulation of effective treatment strategies. Moving forward, we intend to develop a comprehensive risk assessment and treatment framework for FH that includes critical apolipoprotein evaluations. The current investigation did not include an adequate evaluation of non-invasive vascular assessments, particularly FMD and post-statin therapy initiation. We did not have the opportunity to assess FMD, the IMT, or the CAVI in pediatric patients with FH who commenced statin therapy. In pediatric patients with FH exhibiting abnormal FMD and IMT values, a limited number of reports have suggested potential normalization of FMD⁷⁾ and IMT^{6, 31)} following the initiation of statin therapy. However, the scarcity of cases and insufficient follow-up duration contribute to the overall lack of conclusive evidence.

Future studies should focus on identifying the LDL-C cutoff value essential for maintaining normal FMD during childhood and enhancing FMD longitudinally following the commencement of statin therapy. In this study, in accordance with the Japanese FH guidelines¹⁴⁾, we incorporated non-invasive vascular assessments, including FMD, into the follow-up protocol for pediatric FH patients. Our investigation highlighted FMD’s utility as an effective surrogate marker for early identification and risk stratification of atherosclerotic lesions in asymptomatic pediatric patients with FH. Future research should aim to expand the study population and evaluate the impact of statin therapy in these patients.

Our findings provide novel insights into the management of pediatric FH. By synthesizing data from lifestyle-related disease prevention screenings and various noninvasive vascular assessments, we demonstrate the feasibility of a more holistic assessment of potential cardiovascular risks in pediatric patients with FH. This comprehensive approach not only enhances our understanding of FH in children but also facilitates improved communication with patients and their families regarding cardiovascular risk management.

Conclusions

We elucidated the distribution of LDL-C during screening for FH within the catchment area. In addition, we integrated various noninvasive vascular assessments in the evaluation of pediatric FH patients and identified a potential reduction in FMD, particularly in those over 10 years old. Moving forward, we need to create a program that can initiate pharmacological interventions during school age and maintain appropriately low LDL-C levels.

Acknowledgements

The authors are grateful for the valuable assistance in treating patients admitted to the Department of Pediatrics at the Chutoen General Medical Center and the Ogasa Medical Association for their invaluable support. This research received no specific grants from any funding agency in the public, commercial, or not-for-profit sectors. Equipment from Chutoen General Medical Center was used.

Disclosures

The authors declare no conflicts of interest. This research received no specific grants from any funding agency in the public, commercial, or not-for-profit sectors. Equipment from Chutoen General Medical Center was used.

References

• 1)  Li S, Chen W, Srinivasan SR, Bond MG, Tang R, Urbina EM, Berenson GS. Childhood cardiovascular risk factors and carotid vascular changes in adulthood: the Bogalusa Heart Study. JAMA, 2003; 290: 2271-2276
• 2)  Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group. Natural history of aortic and coronary atherosclerotic lesions in youth. Findings from the PDAY Study. Arterioscler Thromb, 1993; 13: 1291-1298
• 3)  Wiegman A, Gidding SS, Watts GF, Chapman MJ, Ginsberg HN, Cuchel M, Ose L, Averna M, Boileau C, Borén J, Bruckert E, Catapano AL, Defesche JC, Descamps OS, Hegele RA, Hovingh GK, Humphries SE, Kovanen PT, Kuivenhoven JA, Masana L, Nordestgaard BG, Pajukanta P, Parhofer KG, Raal FJ, Ray KK, Santos RD, Stalenhoef AFH, Steinhagen-Thiessen E, Stroes ES, Taskinen MR, Tybjærg-Hansen A, Wiklund O; European Atherosclerosis Society Consensus Panel. Familial hypercholesterolaemia in children and adolescents: gaining decades of life by optimizing detection and treatment. Eur Heart J, 2015; 36: 2425-2437
• 4)  Matsunaga K, Mizobuchi A, Ying Fu H, Ishikawa S, Tada H, Kawashiri MA, Yokota I, Sasaki T, Ito S, Kunikata J, Iwase T, Hirao T, Yokoyama K, Hoshikawa Y, Fujisawa T, Dobashi K, Kusaka T, Minamino T. Universal Screening for Familial Hypercholesterolemia in Children in Kagawa, Japan. J Atheroscler Thromb, 2022; 29: 839-849
• 5)  Talmud PJ, Futema M, Humphries SE. The genetic architecture of the familial hyperlipidaemia syndromes: rare mutations and common mutations in multiple genes. Curr Opin Lipidol, 2014; 25: 274-281
• 6)  Wiegman A, Hutten BA, de Groot E, Rodenburg J, Bakker HD, Büller HR, Sijbrands EJG, Kastelein JJP. Efficacy and safety of statin therapy in children with familial hypercholesterolemia: a randomized controlled trial. JAMA, 2004; 292: 331-337
• 7)  de Jongh S, Ose L, Szamosi T, Gagné C, Lambert M, Scott R, Perron P, Dobbelaere D, Saborio M, Tuohy MB, Stepanavage M, Sapre A, Gumbiner B, Mercuri M, van Trotsenburg ASP, Bakker HD, Kastelein JJP. Efficacy and safety of statin therapy in children with familial hypercholesterolemia: a randomized, double-blind, placebo-controlled trial with simvastatin. Circulation, 2002; 106: 2231-2237
• 8)  McCrindle BW, Ose L, Marais AD. Efficacy and safety of atorvastatin in children and adolescents with familial hypercholesterolemia or severe hyperlipidemia: a multicenter, randomized, placebo-controlled trial. J Pediatr, 2003; 143: 74-80
• 9)  Avis HJ, Hutten BA, Gagné C, Langslet G, McCrindle BW, Wiegman A, Hsia J, Kastelein JJP, Steinet EA. Efficacy and safety of rosuvastatin therapy for children with familial hypercholesterolemia. J Am Coll Cardiol, 2010; 55: 1121-1126
• 10)  Nordestgaard BG, Chapman MJ, Humphries SE, Ginsberg HN, Masana L, Descamps O, Wiklund O, Hegele RA, Raal FJ, Defesche JC, Wiegman A, Santos RD, Watts GF, Parhofer KG, Hovingh GK, Kovanen PT, Boileau C, Averna M, Borén J, Bruckert E, Catapano AL, Kuivenhoven JA, Pajukanta P, Ray K, Stalenhoef AFH, Stroes E, Taskinen MR, Tybjærg-Hansen, and for the European Atherosclerosis Society Consensus Panel. Familial hypercholesterolaemia is underdiagnosed and undertreated in the general population: guidance for clinicians to prevent coronary heart disease: consensus statement of the European Atherosclerosis Society. Eur Heart J, 2013; 34: 3478-3490a
• 11)  Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents, National Heart, Lung, and Blood Institute. Expert panel on integrated guidelines for cardiovascular health and risk reduction in children and adolescents: summary report. Pediatrics, 2011; 128 Suppl 5: S213-256
• 12)  Elis A, Zhou R, Stein EA. Treatment of familial hypercholesterolaemia in children and adolescents in the last three decades. Cardiol Young, 2014; 24: 437-441
• 13)  Kusters DM, Avis HJ, de Groot E, Wijburg FA, Kastelein JJP, Wiegman A, Huttenet BA. Ten-year follow-up after initiation of statin therapy in children with familial hypercholesterolemia. JAMA, 2014; 312: 1055-1057
• 14)  Harada-Shiba M, Ohtake A, Sugiyama D, Tada H, Dobashi K, Matsuki K, Minamino T, Yamashita S, Yamamoto Y. Guidelines for the Diagnosis and Treatment of Pediatric Familial Hypercholesterolemia 2022. J Atheroscler Thromb, 2023; 30: 531-557
• 15)  Tomiyama H, Yamashina A. Non-invasive vascular function tests: their pathophysiological background and clinical application. Circ J, 2010; 74: 24-33
• 16)  Iwashima S, Nakagawa Y, Ishikawa T, Satake SSE, Nagata E, Ohzeki T. Abdominal obesity is associated with cardiovascular risk in Japanese children and adolescents. J Pediatr Endocrinol Metab, 2011; 24: 51-54
• 17)  Iwashima S, Ishikawa T, Ohira A, Itou H. Association of abdominal aortic wall thickness in the newborn with maternal factors. Am J Perinatol, 2012; 29: 441-448
• 18)  Ishikawa T, Iwashima S. Endothelial dysfunction in children within 5 years after onset of Kawasaki disease. J Pediatr, 2013; 163: 1117-1121
• 19)  Anderson TJ, Uehata A, Gerhard MD, Meredith IT, Knab S, Delagrange D, Lieberman EH, Ganz P, Creager MA, Yeung AC, Selwyn AP. Close relation of endothelial function in the human coronary and peripheral circulations. J Am Coll Cardiol, 1995; 26: 1235-1241
• 20)  Salonen JT, Salonen R. Ultrasound B-mode imaging in observational studies of atherosclerotic progression. Circulation, 1993; 87(3 Suppl): II56-65
• 21)  Thijssen DHJ, Black MA, Pyke KE, Padilla J, Atkinson G, Harris RA, Parker B, Widlansky ME, Tschakovsky ME, Green DJ. Assessment of flow-mediated dilation in humans: a methodological and physiological guideline. Am J Physiol Heart Circ Physiol, 2011; 300: H2-12
• 22)  Engan B, Engan M, Greve G, Vollsæter M, Hufthammer KO, Leirgul E. Vascular Endothelial Function Assessed by Flow-Mediated Vasodilatation in Young Adults Born Very Preterm or With Extremely Low Birthweight: A Regional Cohort Study. Front Pediatr, 2021; 9: 734082
• 23)  Narverud I, Retterstøl K, Iversen PO, Halvorsen B, Ueland T, Ulven SM, Ose L, Aukrust P, Veierød MB, Holven KB. Markers of atherosclerotic development in children with familial hypercholesterolemia: a literature review. Atherosclerosis, 2014; 235: 299-309
• 24)  Urbina EM, Williams RV, Alpert BS, Collins RT, Daniels SR, Hayman L, Jacobson M, Mahoney L, Mietus-Snyder M, Rocchini A, Steinberger J, McCrindle B. Noninvasive assessment of subclinical atherosclerosis in children and adolescents: recommendations for standard assessment for clinical research: a scientific statement from the American Heart Association. Hypertension, 2009; 54: 919-950
• 25)  Maruhashi T, Soga J, Fujimura N, Idei N, Mikami S, Iwamoto Y, Kajikawa M, Matsumoto T, Hidaka T, Kihara Y, Chayama K, Noma K, Nakashima A, Goto C, Higashi Y. Nitroglycerine-induced vasodilation for assessment of vascular function: a comparison with flow-mediated vasodilation. Arterioscler Thromb Vasc Biol, 2013; 33: 1401-1408
• 26)  Saiki A, Ohira M, Yamaguchi T, Nagayama D, Shimizu N, Shirai K, Tatsuno I. New Horizons of Arterial Stiffness Developed Using Cardio-Ankle Vascular Index (CAVI). J Atheroscler Thromb, 2020; 27: 732-748
• 27)  Kinoshita M, Yokote K, Arai H, Iida M, Ishigaki Y, Ishibashi S, Umemoto S, Egusa G, Ohmura H, Okamura T, Kihara S, Koba S, Saito I, Shoji T, Daida H, Tsukamoto K, Deguchi J, Dohi S, Dobashi K, Hamaguchi H, Hara M, Hiro T, Biro S, Fujioka Y, Maruyama C, Miyamoto Y, Murakami Y, Yokode M, Yoshida H, Rakugi H, Wakatsuki A, Shizuya Yamashita S; Committee for Epidemiology and Clinical Management of Atherosclerosis. Japan Atherosclerosis Society (JAS) Guidelines for Prevention of Atherosclerotic Cardiovascular Diseases 2017. J Atheroscler Thromb, 2018; 25: 846-984
• 28)  Okada T, Murata M, Yamauchi K, Harada K. New criteria of normal serum lipid levels in Japanese children: the nationwide study. Pediatr Int, 2002; 44: 596-601
• 29)  Nagai K, Shibata S, Akishita M, Sudoh N, Obara T, Toba K, Kozaki K. Efficacy of combined use of three non-invasive atherosclerosis tests to predict vascular events in the elderly; carotid intima-media thickness, flow-mediated dilation of brachial artery and pulse wave velocity. Atherosclerosis, 2013; 231: 365-370
• 30)  Vlahos AP, Naka KK, Bechlioulis A, Theoharis P, Vakalis K, Moutzouri E, Miltiadous G, Michalis LK, Siamopoulou-Mavridou A, Elisaf M, Milionis HJ. Endothelial dysfunction, but not structural atherosclerosis, is evident early in children with heterozygous familial hypercholesterolemia. Pediatr Cardiol, 2014; 35: 63-70
• 31)  Luirink IK, Wiegman A, Kusters DM, Hof MH, Groothoff JW, de Groot E, Kastelein JJP, Hutten BA. 20-Year Follow-up of Statins in Children with Familial Hypercholesterolemia. N Engl J Med, 2019; 381: 1547-1556
• 32)  Tada H, Kawashiri MA, Okada H, Nakahashi T, Sakata K, Nohara A, Inazu A, Mabuchi H, Yamagishi M, Hayashi K. Assessments of Carotid Artery Plaque Burden in Patients With Familial Hypercholesterolemia. Am J Cardiol, 2017; 120: 1955-1960
• 33)  Lozano P, Henrikson NB, Dunn J, Morrison CC, Nguyen M, Blasi PR, Anderson ML, Whitlock EP. Lipid Screening in Childhood and Adolescence for Detection of Familial Hypercholesterolemia: Evidence Report and Systematic Review for the US Preventive Services Task Force. JAMA, 2016; 316: 645-655
• 34)  Mikat-Stevens NA, Larson IA, Tarini BA. Primary-care providers- perceived barriers to integration of genetics services: a systematic review of the literature. Genet Med, 2015; 17: 169-176
• 35)  Liu YC, Kuo RL, Shih SR. COVID-19: The first documented coronavirus pandemic in history. Biomed J, 2020; 43: 328-333
• 36)  Langslet G, Bogsrud MP, Halvorsen I, Fjeldstad H, Retterstøl K, Veierød MB, Ose L. Long-term follow-up of young adults with familial hypercholesterolemia after participation in clinical trials during childhood. J Clin Lipidol, 2015; 9(6): 778-785
• 37)  Tokgozoglu L, Morrow DA, Nicholls SJ. Great debate: lipid-lowering therapies should be guided by vascular imaging rather than by circulating biomarkers. Eur Heart J, 2023; 44: 2292-2304
• 38)  Hu P, Dharmayat KI, Stevens CAT, Sharabiani MTA, Jones RS, Watts GF, Genest J, Ray KK, Vallejo-Vaz AJ. Prevalence of Familial Hypercholesterolemia Among the General Population and Patients With Atherosclerotic Cardiovascular Disease: A Systematic Review and Meta-Analysis. Circulation, 2020; 141: 1742-1759
• 39)  Beheshti SO, Madsen CM, Varbo A, Nordestgaard BG. Worldwide Prevalence of Familial Hypercholesterolemia: Meta-Analyses of 11 Million Subjects. J Am Coll Cardiol, 2020; 75: 2553-2566