Genetics in Medicine (2022) 24, 2103–2111

www.journals.elsevier.com/genetics-in-medicine

ARTICLE
Universal screening for familial hypercholesterolemia
in 2 populations
Ursa Sustar1,2, Olga Kordonouri3, Matej Mlinaric1, Jernej Kovac2,4, Stefan Arens3,
Katarina Sedej5, Barbara Jenko Bizjan4, Katarina Trebusak Podkrajsek2,4, Thomas Danne3,
Tadej Battelino1,2, Urh Groselj1,2,6,*
1
Department of Endocrinology, Diabetes and Metabolism, University Children's Hospital, University Medical Centre
Ljubljana, Ljubljana, Slovenia; 2Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; 3Children’s Hospital Auf
der Bult, Janusz-Korczak-Allee, Hanover, Germany; 4Clinical Institute of Special Laboratory Diagnostics, University
Children's Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia; 5Unit Siska, Community Health Centre
Ljubljana, Ljubljana, Slovenia; 6Division of Cardiovascular Medicine, Department of Medicine, Stanford University,
Stanford, CA

ARTICLE INFO ABSTRACT

Article history: Purpose: In Europe, >2 million individuals with familial hypercholesterolemia (FH) are
Received 6 April 2022 currently undiagnosed. Effective screening strategies for FH diagnosis in childhood are urgently
Received in revised form needed. We assessed the overall performances of 2 different FH screening programs in children:
24 June 2022 universal screening program with opt-out and opt-in type participation.
Accepted 24 June 2022 Methods: We analyzed the data from 2 independent populations based on >166,000 individuals
Available online 1 August 2022 screened for hypercholesterolemia. Genetic analyses of FH-related genes were finalized in 945
children and 99 parents.
Keywords: Results: A total of 305 (32.3%) children were genotyped as positive or with a variant of un-
Children certain significance in FH-related genes. For low-density lipoprotein cholesterol levels of
Cholesterol 3.5 mmol L (135.3 mg/dL), the overall sensitivity and specificity for confirming FH were
Familial hypercholesterolemia 90.5% and 55.3%, respectively. As part of child–parent screening, in >90% of the families,
Genetic testing the parent with reported higher cholesterol levels was positive for the familial genetic variant.
Universal screening The cohort-based prevalence of FH from the opt-out universal screening program was
estimated to be 1 in 431 individuals (95% CI = 1/391-1/472).
Conclusion: Universal 3-step FH screening approach in children enabled detection of most
children and their parents in every generation screened at reasonable costs. Opt-out screening
strategy might be preferable over opt-in screening strategy.
© 2022 American College of Medical Genetics and Genomics.
Published by Elsevier Inc. All rights reserved.

*
Correspondence and requests for materials should be addressed to Urh Groselj, University Children's Hospital, University Medical Centre Ljubljana,
Bohoriceva 20, 1000 Ljubljana, Slovenia. E-mail address: urh.groselj@kclj.si

doi: https://doi.org/10.1016/j.gim.2022.06.010
1098-3600/© 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.
2104
Introduction
Familial hypercholesterolemia (FH) is an autosomal domi-
nant disorder characterized by elevated serum total choles-
terol (TC) and/or low-density lipoprotein cholesterol (LDL-
C) and/or pathogenic variant in one of the FH-related
genes.1 Owing to the cumulative exposure to elevated
cholesterol levels from birth, individuals with FH are more
likely to develop cardiovascular disease.2 Half of the
affected men and 30% of affected women will have a heart
attack by age 60 years if not adequately treated.3 The fre-
quency of FH is estimated to be 1 in 250 to 1 in 500, making
it possibly the most common life-threatening monogenic
dominant disorder in humans.4
Whereas in adults, the low-density lipoprotein serum
cholesterol levels of patients with FH and other individuals
frequently overlap, in children, the discrepancy of the LDL-
C levels between both groups is more noticeable. Early FH
detection and treatment in childhood also leads to preventive
gain during the latency period before the manifestation of
atherosclerotic and cardiovascular sequelae and reduces the
later risk of cardiovascular disease.5-8 Currently, diagnosis
in adults occurs mostly after the occurrence of a cardio-
vascular event. Furthermore, globally about 90% of adults
and around 95% of children remain undiagnosed.1,9,10 Its
frequency and the rate of undertreatment make FH a public
health concern.1,11,12 Universal child–parent screening at
immunizations is shown to be effective.13 Moreover,
cascade FH screening was successfully implemented in the
Netherlands.14,15 We assessed the overall performance of
the universal FH screening program in Slovenia (opt-
out),16,17 compared it with a pilot universal screening pro-
gram in Lower Saxony, Germany (opt-in),18 and evaluated
the genotype–phenotype correlations in children referred
through both programs.
Materials and Methods
Study design and cohort description
Two different screening approaches of FH screening are
presented in our study (Supplemental Figure 1). In total,
154,658 children were screened as part of the opt-out pro-
gram and 12,140 children were screened as part of the opt-in
program (Supplemental Figure 2). The National Medical
Ethics Committee approved the study in Slovenia (#25/12/
10, #63/07/13, 0120-14/2017/5, and 0120-273/2019/9) and
the Ethics Committee of Hannover Medical School (Medi-
zinische Hochschule Hannover) approved the study in
Lower Saxony (approval number 7089/20.01.2016). The
principles of the Declaration of Helsinki were followed.
Written consent of at least 1 parent or other primary
caregiver was obtained before inclusion.
The opt-out universal screening program in Slovenia con-
sisted of a TC screening at the primary pediatrician level that is
U. Sustar et al.
offered to all 5-year old children as part of the mandatory
check-up in each generation and was gradually implemented
since 1995 (approximately 20,000 children live-born per each
generation)16,17 with significant improvement in the last few
years owing to upgraded guidelines for primary care pediatri-
cians. Genetic testing for FH-related genes was introduced in
2011. As of 2017, the first 2 steps of the 3-step algorithm for
early and efficient FH detection were implemented: TC mea-
surement in the general pediatric population and LDL-C
measurement in children with elevated TC levels with ge-
netic testing of the children with elevated LDL-C levels. In
2019, the third step of the screening program was systemati-
cally included—the child–parent cascade screening of a parent
with a higher LDL-C level (Supplemental Figure 1A). Of
169,767 live-born children of 8 generations (born 2006-2013),
154,658 are estimated to have participated in the study
(Supplemental Figure 2). Genetic analyses were finalized in
813 children with elevated LDL-C levels. So far, 99 parents
from 83 families of genetically positive children were geno-
typed as part of the child–parent cascade screening. A total of
41 siblings were screened as part of the sibling-cascade
screening program. The detailed screening algorithm is pre-
sented in the Supplemental Methods.
The opt-in universal screening program in Lower Saxony
started in November 2016 as part of the Fr1dolin-Trial.18 The
flowchart of the participants is presented in Supplemental
Figure 2B. LDL-C measurement was offered to approxi-
mately 150,000 children aged between 2 to 6 years during the
programed checkups and at any voluntary visits to the pedi-
atrician's office. Altogether, 12,140 children from the Lower
Saxony cohort participated in the study. Following the
screening algorithm (Figure 1B), 132 children positive in the
first step of the screening had finalized genetic analyses.
Genetic analyses
All the genetic analyses for both cohorts were performed
centrally, at the University Children’s Hospital Ljubljana in
Slovenia. Genomic DNA was isolated, and genetic analyses
were finalized in 945 children (813 children from the opt-out
cohort, 132 children from the opt-in cohort) and 99 parents of
genetically positive children. DNA sequencing and variant
classification methods are presented in the Supplemental
Methods.
Statistical analyses
Descriptive statistics (median, interquartile range (IQR))
were used to analyze the demographic and clinical data of
the study participants. Differences in disease-associated
variant accumulation between positive/variant of uncertain
significance (VUS) and negative groups and among partic-
ipants (sex and cardiovascular-positive family history),
and their corresponding odds ratio (OR) and/or relative risk
were evaluated using Fisher exact test for 2 × 2 contingency
tables.
U. Sustar et al. 2105

Figure 1 Distribution of the LDL-C/TC in general pediatric and familial hypercholesterolemia screening positive populations.
A. Distribution of the TC levels of 5-year old children measured at the primary care pediatrician as the first step of the Slovenian opt-out
universal screening program (n = 3782). B. Distribution of the LDL-C levels for FH screening positive children according to the genetic
results for the Slovenian opt-out cohort (n = 813). C. Distribution of the LDL-C levels of the preschool children measured as part of Fr1dolin
study from the Lower Saxony opt-in cohort (n = 12,140). D. Distribution of the LDL-C levels for FH screening positive children according to
the genetic results of the Lower Saxony opt-in cohort (n = 132). Vertical lines represent medians of the samples. LDL-C, low-density
lipoprotein cholesterol; TC, total cholesterol; VUS, variant of uncertain significance.

The prevalence of possible FH in the opt-out universal
screening program in the children born between 2006 and
2013 (defined as positive in the first step of opt-out uni-
versal screening and LDL-C measurement of >3.5 mmol/L
(135.3 mg/dL)), and FH (defined as (1) phenotypic—
positive in the first step of the opt-out universal screening
and at least 1 additional LDL-C measurement of >4 mmol/
L (154.7 mg/dL), and/or (2) monogenic—with confirmed
pathogenic genetic variant) were estimated according to the
number of live-born children in each generation (all gen-
erations together: N = 169,767; data available at https://
pxweb.stat.si/SiStatData/pxweb/sl/Data/-/05J1002S.px).
The costs per new genetically confirmed case were esti-
mated considering the direct costs for all the steps of the
screening algorithm for the opt-out universal screening and
the first 2 steps for the opt-in universal screening program.
Additional information is presented in the Supplemental
Methods.
Genotype–phenotype correlations
Subpopulations of a minimum of 10 children were generated to
visualize the genotype–phenotype correlations -between the
most common pathogenic and VUS genetic variants in the
APOB and LDLR genes by variant types and protein domains.
Differences between the subpopulated groups were calculated
by the Wilcoxon signed-rank test. P values were adjusted using
the Benjamini-Hochberg correction. All statistical calculations
were performed in R 4.0.4 (R Core Team, Austria) and
GraphPad Prism 8.4.2 (GraphPad Software, USA) software.
2106 U. Sustar et al.

Results positive, 42 (5.2%) as VUS heterozygotes, and 552 (67.9%)

The first step of the universal FH screening
program—TC/LDL-C measurement
In Slovenia, a cohort of 154,658 from 169,767 live-born
children was estimated to participate in the universal FH
screening program (8.9% opted out from the study). Com-
plete screening data from the primary care pediatricians
were collected for 3782 children (1821 female and 1961
male). The median age at the first TC measurement was 5.1
(IQR = 5.0-5.2) years. The median TC level of the cohort
was 4.1 (IQR = 3.7-4.6) mmol/L (158.5 (IQR = 143.1-
177.9 mg/dL)) (Figure 1A).
In total, 12,140 individuals from the Lower Saxony had
opted in in the study. Complete screening data were avail-
able for 12,133 children (5789 female and 6344 male). The
median age of the children at the first LDL-C measurement
was 4.0 (IQR = 3.0-5.1) years. The median LDL-C level of
the screened cohort was 2.4 (IQR = 2.0-2.8) mmol/L (93.0
(IQR = 79.0-108.0 mg/dL)) (Figure 1C).
The second step of the universal FH screening
program—genetic characterization
The demographic and clinical characteristics of the 813
children from the Slovenian cohort and 132 from the Lower
Saxony cohort with finalized genetic analyses are repre-
sented in Table 1 and Table 2, respectively. Of 813 children
from the Slovenian cohort, 219 (26.9%) were genotyped as
as negative. The distribution of the LDL-cholesterol ac-
cording to the genetic result is presented in Figure 1B.
Furthermore, of genetically positive children, 67 (30.6%),
151 (68.9%), and 1 (0.5%) had pathogenic variants in the
APOB, LDLR, and PCSK9 gene, respectively. Of the chil-
dren genotyped as VUS heterozygotes, 27 (64.3%) had a
variant in the APOB, 13 (30.9%) in the LDLR, and 2 (4.8%)
in the PCSK9 gene. One child (0.5%) was homozygous for
variant in the LDLR gene, 1 (0.5%) was double heterozy-
gous for variants in APOB and LDLR gene, and other
children were heterozygous. Furthermore, LDL-C levels for
the LDLR positive group were significantly higher in com-
parison with the APOB positive group (P < .01). Moreover,
a positive family history, according to the Simon Broome
Register criteria, was observed in 117 (21.2%), 99 (45.2%),
and 6 (14.3%) children in negative, positive, and VUS
groups, respectively.
Of 132 children from the Lower Saxony cohort, 26
(19.7%) were classified as positive, 18 (13.6%) as VUS
heterozygotes, and 88 (66.7%) as negative. The distribution
of the LDL-cholesterol according to the genetic result is
presented in Figure 1D. Furthermore, of genetically positive
children, 6 (23.1%) and 20 (76.9%) had a pathogenic variant
in the APOB and LDLR gene, respectively. Of the children
genotyped as VUS heterozygotes, 10 (55.5%) had a variant
in the APOB gene, 3 (16.7%) in the LDLR gene, and 5
(27.8%) in the PCSK9 gene. Moreover, positive family
history was observed in 48 (54.5%), 18 (69.2%), and 11
(61.1%) children in negative, positive, and VUS groups,
respectively (Table 1).

Table 1 Demographic and clinical characteristics of the Slovenian (opt-out) cohort with regard to genetic results
Demographic and Clinical Characteristics Negative, n = 552 (%) Positive, n = 219 (%) VUS, n = 42 (%) Total, N = 813 (%)
Sex
Male 210 (38.0) 109 (49.8) 19 (45.2) 338 (41.6)
Female 342 (62.0) 110 (50.2) 23 (54.8) 475 (58.4)
Family history
Positive 117 (21.2) 99 (45.2) 6 (14.3) 222 (27.3)
Negative 435 (78.8) 120 (54.8) 36 (85.7) 591 (72.7)
At first evaluation at the tertiary outpatient clinic
Age (y) 6.4 (5.8-7.2) 6.0 (5.6-6.6) 6.4 (5.9-7.3) 6.2 (5.7-7.1)
BMI SDS 0.1 (–0.5 to 0.8) –0.05 (–0.5 to 0.6) 0.4 (–0.3 to 1.1) 0.06 (–0.5 to 0.8)
TC (mmol/L) 5.4 (5.0-5.9) 6.5 (5.9-7.1) 5.4 (5.1-5.7) 5.6 (5.1-6.2)
TC (mg/dL) 208.9 (193.4-228.2) 251.4 (228.2-274.6) 208.8 (198.2-220.4) 216.6 (197.2-239.8)
LDL-C (mmol/L) 3.4 (2.9-3.8) 4.7 (4.0-5.4) 3.4 (3.0-3.7) 3.6 (3.1-4.3)
LDL-C (mg/dL) 131.5 (112.1-146.9) 181.7 (154.7-208.8) 129.5 (117.0-142.1) 139.2 (119.9-166.3)
HDL-C (mmol/L) 1.5 (1.3-1.8) 1.4 (1.2-1.6) 1.7 (1.4-1.9) 1.5 (1.3-1.7)
HDL-C (mg/dL) 58.0 (50.3-69.6) 54.1 (46.4-61.9) 65.7 (54.1-72.5) 58.0 (50.3-65.7)
TG (mmol/L) 0.9 (0.6-1.3) 0.8 (0.7-1.2) 0.9 (0.6-1.1) 0.9 (0.6-1.2)
TG (mg/dL) 79.7 (53.1-115.1) 70.9 (53.1-104.0) 79.7 (53.1-97.4) 79.7 (53.1-106.3)
Therapy 19 (3.4) 56 (25.6) 1 (2.4) 76 (9.3)
Demographic and clinical characteristics of the genotyped Slovenian (opt-out) cohort for genetically negative, positive, and VUS subgroups. Data were
collected at the first evaluation at the Lipid Clinic. Data are presented as median (IQR).
BMI SDS, body mass index SD score; HDL-C, high-density lipoprotein cholesterol; IQR, inter quartile range; LDL-C, low-density lipoprotein cholesterol;
TC, total cholesterol; TG, triglyceride; VUS, variant of uncertain significance.
U. Sustar et al. 2107

Table 2 Demographic and clinical characteristics of the Lower Saxony (opt-in) cohort with regard to genetic results
Demographic and Clinical Characteristics Negative, n = 88 (%) Positive, n = 26 (%) VUS, n = 18 (%) Total, N = 132 (%)
Sex
Male 36 (40.9) 18 (69.2) 6 (33.3) 60 (45.5)
Female 52 (59.1) 8 (30.8) 12 (66.7) 72 (54.5)
Family history
Positive 48 (54.5) 18 (69.2) 11 (61.1) 77 (58.3)
Negative 38 (43.2) 6 (23.1) 7 (38.9) 51 (38.6)
Data NA 2 (2.3) 2 (7.7) 0 (0.0) 4 (3.0)
At first evaluation at the Lipid Clinic
Age (y) 4.5 (3.6-6.2) 4.7 (3.4-6.6) 3.6 (3.2-4.5) 4.5 (3.5-6.2)
BMI SDS 0.2 (–0.4 to 0.8) 0.3 (–0.4 to 0.7) 0.5 (–0.9 to 1.1) 0.3 (–0.4 to 0.8)
TC (mmol/L) 5.3 (4.8-5.7) 5.8 (5.4-7.0) 5.1 (4.8-5.8) 5.3 (4.8-5.9)
TC (mg/dL) 204.0 (184.0-220.0) 226.0. (210.5-270.5) 196.0 (186.3-225.5) 206.0 (188.0-228.0)
LDL-C (mmol/L) 3.6 (3.2-4.0) 4.3 (3.8-5.4) 3.3 (3.2-3.8) 3.7 (3.2-4.2)
LDL-C (mg/dL) 140.0 (124.0-155.0) 166.0 (146.3-209.5) 126.5 (123.5-146.5) 143.0 (124.0-163.0)
HDL-C (mmol/L) 1.4 (1.1-1.6) 1.5 (1.2-1.7) 1.3 (1.1-1.6) 1.4 (1.1-1.6)
HDL-C (mg/dL) 54.0 (43.5-62.0) 58.0 (48.0-64.0) 50.0 (43.0-62.0) 55.0 (44.0-63.0)
TG (mmol/L) 0.7 (0.5-0.9) 0.6 (0.5-0.8) 0.6 (0.5-0.9) 0.7 (0.5-0.9)
TG (mg/dL) 58.0 (47.0-80.0) 55.5 (46.5-68.0) 53.0 (44.5-80.5) 58.0 (47.0-78.0)
Therapy 0 (0) 0 (0) 0 (0) 0 (0)
Demographic and clinical characteristics of the genotyped Lower Saxony (opt-in) cohort for genetically negative, positive, and VUS subgroups. Data were
collected at the first evaluation at the Lipid Clinic. Data are presented as median (IQR).
BMI SDS, body mass index SD score; HDL-C, high-density lipoprotein cholesterol; IQR, interquartile range; LDL-C, low-density lipoprotein cholesterol;
NA: data not applicable; TC, total cholesterol; TG, triglyceride; VUS, variant of uncertain significance.

The third step of the universal FH screening
program—child–parent FH screening
As part of the FH child–parent cascade screening, 99 parents
(50 female and 49 male) from 83 families from the Slove-
nian cohort were included in the study. Their median age at
the time of genetic testing was 40.8 years (IQR = 37.5-
45.3). In 9.6% of the families, the parent with reported
higher cholesterol level was negative for the tested variant.
All positive parents identified at the third step of the uni-
versal FH screening program were recommended to start
LDL-C lowering therapy and referred to the adult lipidology
clinic for further investigation and treatment.
[NM_000384.3:c.5066G>A] in APOB and p.Gly20Arg
[NM_000527.5:c.58G>A] in LDLR genes). Children with
frameshift deletions had more severe phenotypes than the
children carrying missense variants (p.Arg3527Gln variant
[NM_000384.3:c.10580G>A] in APOB and p.Cys27Trp
NM_000527.5:c.81C>G], p.Ser286Arg [NM_000527.5:c.858
C>G], and p.Gly478Arg [NM_000527.5:c.1432G>C] vari-
ants in LDLR gene). This is consistent with Figure 3B, where
we stratified genetic variants of the LDLR positive children

Genotype–phenotype correlations

The sensitivity and specificity for the cutoff value of

3.5 mmol/L (135.3 mg/dL) were 90.5% and 55.3%,
respectively (Figure 2). For the cutoff value of 3.8 mmol/L
(146.9 mg/dL), the sensitivity was 83.0% and the specificity
72.5%. Furthermore, for the cutoff value 4.0 mmol/L
(154.7 mg/dL), the sensitivity was 78.0% and the specificity
80.1%. For reaching the 90.0% specificity, the cutoff value
was 4.3 mmol/L (166.3 mg/dL) with 67.0% sensitivity.
The genotype–phenotype correlations were assessed for Figure 2 Sensitivity vs specificity for different LDL-C levels
the most common pathogenic and VUS genetic variants in acquired from children with completed genetic analysis.
the APOB and LDLR genes and were compared with the A. Sensitivity vs specificity of genetic analysis for different LDL-C
genetically negative group (Figure 3A). Statistically signifi- levels for children with completed genetic analysis. Vertical line
cant differences were observed between all variants in represents sensitivity (90.5%) and specificity (55.3%) at LDL-C
comparison with the negative group (P < .001), except level of 3.5 mmol/L (135.3 mg/dL). LDL-C, low-density lipopro-
for 2 variants previously classified as VUS (p.Arg1689His tein cholesterol.
2108 U. Sustar et al.

Figure 3 LDL-C levels for the most frequent genetic variants. A. LDL-C levels for the most frequent genetic variants in APOB and
LDLR genes compared with the genetically negative group. B. LDL-C levels for the variants in the LDLR gene are grouped by the protein
domain on which the pathogenic variant affects compared with the genetically negative group. APOB, apolipoprotein B; LDL-C, low-density
lipoprotein cholesterol; LDLR, low-density lipoprotein receptor; TC, total cholesterol.

into groups by protein domains: all groups with the patho-
genic variants had significantly more severe phenotypes then
the genetically negative children, except for the variant
p.Gly20Arg (NM_000527.5:c.58G>A) that encodes for the
signal sequence domain and was previously classified as
VUS.19 In Supplemental Table 1, we present 26 novel variants
from both cohorts. Following the American College of
Medical Genetics and Genomics and the Association for
Molecular Pathology guidelines, 18 of the novel variants were
classified as a VUS and 8 as (likely) pathogenic.
The relative risk of having confirmed a pathogenic genetic
variant for a child with a positive family history was 1.9 (95%
CI = 1.5-2.4; P < .0001). The OR for having a pathogenic
genetic variant in children with LDL-C levels of ≥4.0 mmol/
L (154.7 mmol/L) was 6.3 (95% CI = 4.1-9.5, P < .0001)
compared with the children with LDL-C levels between 3.5
and 4.0 mmol/L (135.3 and 154.7 mg/dL, respectively). The
OR for having a pathogenic genetic variant in children with
an LDL-C level of ≥3.5 mmol/L (135.3 mmol/L) was 6.6
(95% CI: 4.6-9.7, P < .0001) compared with the children
with LDL-C level of <3.5 mmol/L (135.3 mg/dL).
Prevalence and cost estimation
The prevalence for all children referred through the first step
of the opt-out universal screening program was 1 in 164
(95% CI = 1/117-1/211). The prevalence of positive result at
the first step of the universal screening program and another
LDL-C measurement of >3.5 mmol/L (135.3 mg/dL) as part
of the second step of the screening program (possible FH)
was 1 in 320 individuals (95% CI = 1/273-1/368).
The prevalence of FH in children (monogenic and extreme
phenotypic) was 1 in 431 individuals (95% CI = 1/391-1/
472). For the opt-in cohort, the prevalence could not be
determined.
The direct costs per new genetically confirmed FH case
were 938$ and 905$, respectively, including the costs for all
3 steps of the screening algorithm of the opt-out screening
and 2 steps of the screening algorithm of the opt-in screening
program.
Discussion
As recently re-emphasized by the worldwide FH community
globally, around 30 million people with undiagnosed FH are
at a high but preventable cardiovascular disease risk.1,9,11
The initiation of statin therapy in children with FH reduces
the risk of cardiovascular disease in adulthood.5 However,
FH is diagnosed at a median age of 46.2 years, and among
those, only 2.1% represent children and adolescents.7
Various FH screening strategies have been reported,15 with
most evidence on cascade FH screening, successfully
implemented in several countries.20 We assessed the perfor-
mance of the opt-out universal FH screening program and
compared it with a pilot opt-in FH screening program. Re-
sults indicated that both approaches are feasible for early and
cost-effective FH detection, and by child–parent FH
screening simultaneously, they also target the undiagnosed
adult population.
Traditionally, the FH prevalence was estimated to be 1 in
500, but current research suggests it to be as high as 1 in 250
U. Sustar et al.
or higher in some populations.4 The prevalence of possible
FH in the Slovenian national opt-out screening program was
1 in 320 individuals (including all children positive at the
universal screening independent of genetic result) and 1 in
431 individuals for FH. Our results are consistent with FH
prevalence in previous studies and recent meta-analyses.4,21
The prevalence of the opt-in program was not representative
because the study participation was voluntary with possible
selection bias toward the engagement of families with a
predisposition to FH, bringing the results from this opt-in
screening closer to the results of selective screening pro-
grams.21 Approximately 3.4% of the children opted in in the
opt-in universal screening program and 8.9% of the children
opted out of the universal screening program. Considering
the percentage of the children who opted in in the opt-in
universal screening program, opt-out screening strategy
was overall more effective than the opt-in strategy.
Because routine FH genetic testing is not widely imple-
mented worldwide, the diagnosis of FH still primarily relies
on the established clinical criteria.11 However, clinical
criteria might be less suitable for the pediatric population or
are even not directly applicable.5 In this study, >100
different FH pathogenic variants contributed to a heteroge-
neous pool in both cohorts. Most different pathogenic var-
iants were discovered in the LDLR gene (Supplemental
Table 2). Of all detected variants, 27 (24.7%) were novel.
In total, 7 of the most frequent pathogenic variants
explained 50.5% of all positive cases. Variant p.Arg3527
Gln (NM_000384.3:c.10580G>A) in the APOB gene
explained 20.6% of positive cases, probably owing to its
known Central European presence.22 A large proportion of
children with clinically diagnosed FH have not been
confirmed with a pathogenic genetic variant in APOB,
LDLR, or PCSK9.23,24 Moreover, systematically testing for
copy number variants in genes other than LDLR gene and
polygenic risk score calculation may be reasonable to be
implemented in near future as part of routine genetic testing.
Genetic testing is not just diagnostically conclusive, but
also prognostically important,25 because one-third of the
children with FH pathogenic variant has cholesterol levels
below the 95th percentile.21 Owing to some phenotypic
overlap between the FH-negative children and FH-positive
children, mainly in children with APOB variants and in
those with LDLR variants associated with a milder FH
phenotype, an optimal relationship between sensitivity and
specificity should be rationally determined. For elevated
LDL-C levels (3.5 mmol/L [135.3 mg/dL]), the overall
sensitivity was >90%, with a specificity of 55%. As a trade-
off for lower specificity, children negative for FH-related
genes should be tested for polygenic hypercholesterolemia
and may also benefit from an early diagnosis.26 In case of
extreme cholesterol levels, other dyslipidemias should be
considered.
A positive family history, according to Simon Broome
Register criteria, was observed in 45.2% of genetically
positive children. Therefore, family history may not allow
reliable identification of children through selective and
2109
cascade screening owing to the relatively young age of the
parents. Child–parent FH screening by testing the parent
with the reported higher cholesterol levels enabled diag-
nosing a parent of an FH-positive child in >90% of the
screened families at a modest additional cost.13 Besides,
healthy parents are in the direct interest of the affected child,
which is another important argument for introducing
child–parent FH screening.15,21,27 This study’s one of
the drawbacks is the limited cohort of parents participating
in the child–parent cascade screening owing to the
delayed systematic implementation of the third phase of the
screening program, resulting in a lower response rate to
involvement in the study.
The average cost of 1277$ per new FH case identified by
cascade screening,27 already proven to be highly cost-
effective,28 is comparable to our cost calculations per newly
detected case (938$ for the opt-out universal and 905$ for
the opt-in universal screening program). As genetic testing
costs are rapidly declining, the costs of our screening pro-
grams are expected to decline several folds in the near
future.25 However, the costs for TC/LDL-C level measure-
ment as a part of the first step of the FH screening program
remain unchanged. Moreover, after 1 full generation of
testing, it can be assumed that most of those diagnosed with
FH would have children and that all at-risk patients would
be detected by cascade screening.
In conclusion, early FH detection provides continuous
screening of the trailing population of preschool children
and a preventive improvement in cardiovascular health
within the overall population. A universal 3-step approach:
TC or LDL-C measurement in the general pediatric popu-
lation, fasting LDL-C measurement in those with elevated
initial cholesterol levels, followed by genetic testing of the
children with elevated levels, and child–parent cascade
screening of a parent with higher LDL-C level, and cascade
screening of older siblings if untested, as an applicable
strategy for early detection of the patients with FH. Both the
opt-out universal and the opt-in universal FH screenings
were feasible and effective for the early detection of undi-
agnosed FH cases. Compared with cascade screening
programs, both versions of the universal screening programs
were more economic and tend to diagnose presymptomatic
patients of 2 generations (proband children and their par-
ents). With the opt-out screening program, every child has
an opportunity to participate in the screening, whereas the
opt-in screening acts as a more selective approach, targeting
a large part (but not all) of the population. Furthermore,
>90% of the included population had their cholesterol levels
measured as a part of the opt-out screening program.
Therefore, universal participation in FH screening programs
(with opting-out possibility) might be preferred and more
reliable over opt-in participation.
Data Availability
Data is available upon request.
2110
Acknowledgments
We thank Gasper Klancar for his past work in the program
of familial hypercholesterolemia screening. We are also
thankful to all the primary care pediatricians, fellow clini-
cians, laboratory personnel, and nursing team for their vast
efforts with the successful implementation of universal fa-
milial hypercholesterolemia screening, particularly to Mar-
usa Debeljak, Natasa Bratina, Nevenka Bratanic, and Neza
Molk. We would also like to acknowledge all the children
and their families for participating in the study.
We would like to further acknowledge persons involved
in the Fr1dolin project from Hanover, particularly Erika
Marquardt, Kerstin Semler, Frank Roloff, Thekla von dem
Berge, Claire Tombois, Alisa Arens, Isa Boettcher, Karin
Lange, and Bärbel Aschemeier-Fuchs, and of course pedi-
atricians and families.
This work was supported by the Slovenian Research
Agency (grants P3-0343, J3-2536, J3-4116, J3-6800,
J3-6798). The Fr1dolin study has been supported by a
grant from the Juvenile Diabetes Research Foundation
(2-SRA-2016-243-Q-R), the Liona M. and Harry B.
Helmsley Charitable Trust, and Sanofi-Aventis Deutschland
GmbH. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation
of the manuscript.
Author Information
Conceptualization: O.K., K.T.P., T.B., U.G.; Data Curation:
U.S., O.K., M.M.; Formal Analysis: U.S.; Funding Acqui-
sition: T.B., O.K.; Investigation: O.K., S.A., K.S.; Meth-
odology: J.K., U.S.; Visualization: U.S.; Writing-original
draft: U.S., U.G.; Supervision: U.G.; Writing-review and
editing: O.K., M.M., J.K., S.A., K.S., B.J.B., K.T.P., T.D.,
T.B.
Ethics Declaration
The National Medical Ethics Committee approved the study
in Slovenia (#25/12/10, #63/07/13, 0120-14/2017/5, and
0120-273/2019/9) and the Ethics Committee of Hannover
Medical School (Medizinische Hochschule Hannover)
approved the study in Lower Saxony (approval number
7089/20.01.2016). The principles of the Declaration of
Helsinki were followed. Written consent of at least 1 parent
or other primary caregiver was obtained before inclusion.
Conflict of Interest
The authors declare no conflicts of interest.
U. Sustar et al.
Additional Information
The online version of this article (https://doi.org/10.1016/j.
gim.2022.06.010) contains supplementary material, which
is available to authorized users.
References
1. Nordestgaard BG, Chapman MJ, Humphries SE, et al. Familial
hypercholesterolaemia is underdiagnosed and undertreated in the gen-
eral population: guidance for clinicians to prevent coronary heart dis-
ease: consensus statement of the European Atherosclerosis Society. Eur
Heart J. 2013;34(45):3478-90a. Published correction appears in Eur
Heart J. 2020;41(47):4517. https://doi.org/10.1093/eurheartj/eht273.
2. Ference BA, Ginsberg HN, Graham I, et al. Low-density lipoproteins
cause atherosclerotic cardiovascular disease. 1. Evidence from genetic,
epidemiologic, and clinical studies. A consensus statement from the
European Atherosclerosis Society Consensus Panel. Eur Heart J.
2017;38(32):2459–2472. http://doi.org/10.1093/eurheartj/ehx144.
3. Marks D, Thorogood M, Neil HAW, Humphries SE. A review on the
diagnosis, natural history, and treatment of familial hyper-
cholesterolaemia. Atherosclerosis. 2003;168(1):1–14. http://doi.org/10.
1016/S0021-9150(02)00330-1.
4. Hu P, Dharmayat KI, Stevens CAT, et al. Prevalence of familial hy-
percholesterolemia among the general population and patients with
atherosclerotic cardiovascular disease: a systematic review and meta-
analysis. Circulation. 2020;141(22):1742–1759. http://doi.org/10.
1161/CIRCULATIONAHA.119.044795.
5. Luirink IK, Wiegman A, Kusters DM, et al. 20-year follow-up of
statins in children with familial hypercholesterolemia. N Engl J Med.
2019;381(16):1547–1556. http://doi.org/10.1056/NEJMoa1816454.
6. 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 cardio-
vascular health and risk reduction in children and adolescents: sum-
mary report. Pediatrics. 2011;128 Suppl 5(Suppl 5):S213–S256. http://
doi.org/10.1542/peds.2009-2107C.
7. EAS Familial Hypercholesterolaemia Studies Collaboration (FHSC).
Global perspective of familial hypercholesterolaemia: a cross-sectional
study from the EAS Familial Hypercholesterolaemia Studies Collabo-
ration (FHSC). Lancet. 2021;398(10312):1713–1725. http://doi.org/10.
1016/S0140-6736(21)01122-3.
8. Tromp TR, Hartgers ML, Hovingh GK, et al. Worldwide experience of
homozygous familial hypercholesterolaemia: retrospective cohort
study. Lancet. 2022;399(10326):719–728. http://doi.org/10.1016/
S0140-6736(21)02001-8.
9. Gidding SS, Champagne MA, de Ferranti SD, et al. The agenda for
familial hypercholesterolemia: a scientific statement from the American
Heart Association. Circulation. 2015;132(22):2167-2192. Published
correction appears in Circulation. 2015;132(25):e397. https://doi.org/1
0.1161/CIR.0000000000000297.
10. EAS Familial Hypercholesterolaemia Studies Collaboration, Vallejo-
Vaz AJ, De Marco M, et al. Overview of the current status of familial
hypercholesterolaemia care in over 60 countries – the EAS Familial
Hypercholesterolaemia Studies Collaboration (FHSC). Atheroscle-
rosis. 2018;277:234–255. http://doi.org/10.1016/j.atherosclerosis.
2018.08.051.
11. Representatives of the Global Familial Hypercholesterolemia Com-
munity, Wilemon KA, Patel J, et al. Reducing the clinical and
public health burden of familial hypercholesterolemia: a global call
to action. JAMA Cardiol. 2020;5(2):217-229. Published correction
appears in JAMA Cardiol. 2020;5(5):613. https://doi.org/10.1001/
jamacardio.2019.5173.
U. Sustar et al.
12. World Heart Federation. Improving prevention and control of raised
cholesterol: A Call to Action. World Heart Federation. Published 2021.
Accessed September 20, 2021. https://world-heart-federation.org/
wp-content/uploads/2021/05/World-Heart-Federation-Cholesterol-White-
paper.pdf
13. Wald DS, Bestwick JP, Wald NJ. Child-parent screening for familial
hypercholesterolaemia: screening strategy based on a meta-analysis. BMJ.
2007;335(7620):599. http://doi.org/10.1136/bmj.39300.616076.55.
14. Wiegman A, Gidding SS, Watts GF, et al. Familial hypercholesterolaemia
in children and adolescents: gaining decades of life by optimizing
detection and treatment. Eur Heart J. 2015;36(36):2425–2437. http://doi.
org/10.1093/eurheartj/ehv157.
15. Groselj U, Wiegman A, Gidding SS. Screening in children for familial
hypercholesterolaemia: start now. Eur Heart J. 2022:ehac224. https://
doi.org/10.1093/eurheartj/ehac224.
16. Klančar G, Grošelj U, Kovač J, et al. Universal screening for familial
hypercholesterolemia in children. J Am Coll Cardiol.
2015;66(11):1250–1257. http://doi.org/10.1016/j.jacc.2015.07.017.
17. Groselj U, Kovac J, Sustar U, et al. Universal screening for familial
hypercholesterolemia in children: the Slovenian model and literature
review. Atherosclerosis. 2018;277:383–391. http://doi.org/10.1016/j.
atherosclerosis.2018.06.858.
18. Kordonouri O, Lange K, Boettcher I, et al. New approach for detection
of LDL-hypercholesterolemia in the pediatric population: the Fr1dolin-
Trial in Lower Saxony, Germany. Atherosclerosis. 2019;280:85–91.
http://doi.org/10.1016/j.atherosclerosis.2018.11.011.
19. Thormaehlen AS, Schuberth C, Won HH, et al. Systematic cell-based
phenotyping of missense alleles empowers rare variant association
studies: A case for LDLR and myocardial infarction. PLoS Genet.
2015;11(2):e1004855. Published correction appears in PLoS Genet.
2015;11(3):e1005060. https://doi.org/10.1371/journal.pgen.1004855.
20. Umans-Eckenhausen MA, Defesche JC, Sijbrands EJ, Scheerder RL,
Kastelein JJ. Review of first 5 years of screening for familial
2111
hypercholesterolaemia in the Netherlands. Lancet. 2001;357(9251):
165–168. http://doi.org/10.1016/S0140-6736(00)03587-X.
21. Wald DS, Bestwick JP, Morris JK, Whyte K, Jenkins L, Wald NJ.
Child-parent familial hypercholesterolemia screening in primary care.
N Engl J Med. 2016;375(17):1628–1637. http://doi.org/10.1056/
NEJMoa1602777.
22. Liyanage KE, Burnett JR, Hooper AJ, van Bockxmeer FM. Familial
hypercholesterolemia: epidemiology, Neolithic origins and modern
geographic distribution. Crit Rev Clin Lab Sci. 2011;48(1):1–18. http://
doi.org/10.3109/10408363.2011.565585.
23. Civeira F, International Panel on Management of Familial Hyper-
cholesterolemia. Guidelines for the diagnosis and management of het-
erozygous familial hypercholesterolemia. Atherosclerosis. 2004;173(1):
55–68. http://doi.org/10.1016/j.atherosclerosis.2003.11.010.
24. Palacios L, Grandoso L, Cuevas N, et al. Molecular characterization of
familial hypercholesterolemia in Spain. Atherosclerosis. 2012;221(1):
137–142. http://doi.org/10.1016/j.atherosclerosis.2011.12.021.
25. Sturm AC, Knowles JW, Gidding SS, et al. Clinical genetic testing for
familial hypercholesterolemia: JACC Scientific Expert Panel. J Am
Coll Cardiol. 2018;72(6):662–680. http://doi.org/10.1016/j.jacc.2018.
05.044.
26. Zhang Y, Vittinghoff E, Pletcher MJ, et al. Associations of blood
pressure and cholesterol levels during young adulthood with later
cardiovascular events. J Am Coll Cardiol. 2019;74(3):330–341. http://
doi.org/10.1016/j.jacc.2019.03.529.
27. Wald DS, Bestwick JP. Reaching detection targets in familial hyper-
cholesterolaemia: comparison of identification strategies. Atheroscle-
rosis. 2020;293:57–61. http://doi.org/10.1016/j.atherosclerosis.2019.
11.028.
28. Ademi Z, Watts GF, Juniper A, Liew D. A systematic review of eco-
nomic evaluations of the detection and treatment of familial hyper-
cholesterolemia. Int J Cardiol. 2013;167(6):2391–2396. http://doi.org/
10.1016/j.ijcard.2013.01.280.