Page 1
Archives of Medical Research 34 (2003) 70–75
0188-4409/03 $–see front matter. Copyright © 2003 IMSS. Published by Elsevier Science Inc.
PII S0188-4409(02)00452-6
CASE REPORT
Familial Hypercholesterolemia Due to Ligand-Defective
Apolipoprotein B100.
First Case Report in a Mexican Family
Ludivina Robles-Osorio,
a
Ma. Luisa Ordoñez,
a
Carlos A. Aguilar-Salinas,
a
Moisés Aurón-Gómez,
a
Ma. Teresa Tusié-Luna,
b
Francisco J. Gómez-Pérez
a
and Juan A. Rull-Rodrigo
a
a
Departamento de Endocrinología y Metabolismo,
b
Unidad de Biología Molecular y Medicina Genómica,
Instituto Nacional de Ciencias Médicas y de Nutrición Salvador Zubirán, Mexico City, Mexico
Received for publication April 25, 2002; accepted August 19, 2002 (02/082).
Background.
Familial defective apolipoprotein B100 (FDB) is one of the known causes
of familial hypercholesterolemia (FH). Its frequency among subjects with FH varies among
ethnic groups; information on FH is insufficient for populations from Latin America. We pro-
posed to describe prevalence of FDB in a cohort of Mexican FH probands (
n
�
30).
Methods.
We searched for the known FDB mutations using polymerase chain reaction as-
says. In this set of patients, mean lipid values were representative of FH (cholesterol 351 mg/dL,
LDL cholesterol 274 mg/dL, HDL cholesterol 51 mg/dL, and triglycerides 132 mg/dL).
Results.
One subject with Arg3500Gln mutation was found: a 44-year-old male with a
history of coronary heart disease (CHD) among paternal relatives. His lipid profile was
cholesterol 370 mg/dL, LDL-cholesterol 300 mg/dL, HDL-cholesterol 32 mg/dL, and tri-
glycerides 189 mg/dL. Tendinous xanthomata were detected. Three of four siblings, one
of three sons, and one of nine nieces and nephews carried the mutation. The mutation was
confirmed by automated sequencing. Tendinous xanthomata were absent in affected subjects
younger than age 20 years; additionally, the subjects had borderline cholesterol levels.
Conclusions.
Our data suggest that FDB explains the small number of FH cases in Mex-
ico. Inclusion of molecular biology assays to the clinical laboratory makes it possible to
diagnose affected individuals with borderline cholesterol levels or without tendinous
xanthomata. © 2003 IMSS. Published by Elsevier Science Inc.
Key Words:
Apolipoprotein B, Familial hypercholesterolemia, Cholesterol, Atherosclerosis, Mexico.
Introduction
Familial defective apoB100 (FDB) is one known cause of fa-
milial hypercholesterolemia (FH) (1). It is inherited in autosomal
dominant form with nearly 100% penetrance. Clinical manifes-
tations are explained by plasma accumulation of low-density
lipoproteins (LDL) bearing defective apolipoprotein B100
(apoB100). This change lowers its affinity for LDL receptor
(LDLR) responsible for 80% of its clearance from plasma. Con-
sequently, hypercholesterolemia, tendinous xanthomata, and
premature atherosclerosis are consequences of this disorder (2).
Increased cardiovascular risk was recently confirmed in a pro-
spective study including 205 patients from Denmark, Germany,
and The Netherlands. By age 70,
�
60% had suffered a cardio-
vascular event (3).
The apoB100 gene is localized in chromosome 2p24–p23.
It is composed of 29 exons and 28 introns. Three mutations
in exon 26 are associated with FDB; the first described is
the most common (4,5). Genomic DNA from affected indi-
vidual contained a CGG-to-CAG change in nucleotide
10699 at the codon for amino acid 3500, which results in
substitution of Arg for Gln (R3500Q) (6). This residue is the
apoB region involved with binding to LDL receptor (amino
Address reprint requests to: Carlos Alberto Aguilar-Salinas, M.D.,
INCMNSZ, Vasco de Quiroga #15, Col. Sección XVI, Tlalpan, 14000
México, D.F., México. Phone: (
�
52) (55) 5513-3891; FAX: (
�
52) (55)
5513-0002; E-mail: caguilarsalinas@yahoo.com
Page 2
Familial Hypercholesterolemia in Mexico
71
acids 2980–3780) (2,6,7). Lipoproteins with this abnormal
form of apoB have only 10% normal affinity to LDL recep-
tor. Another mutation, i.e., change of arginine to cysteine in
position 3531, is also a cause of FDB (8). Only two families
have been diagnosed with this defect and were from differ-
ent ethnic origins. This mutation causes a defect in receptor
affinity; however, defect magnitude is 30% lower than ob-
served with mutation in position 3500. Finally, the third
mutation, substitution of tryptophan for arginine in position
3500, was reported in one Scottish family and 13 Asian kin-
dreds (9,10).
Prevalence of these mutations differs among popula-
tions. Due to methodological problems, estimates of the ma-
jority of populations are biased. Cases due to mutations in
apolipoprotein B have discretely lower cholesterol concen-
trations than those caused by defects in LDL receptor (11).
As a result, prevalence is lower in studies performed in so-
called lipid clinics and higher when analysis is performed in
populations selected for having hypercholesterolemia with
prevalences between 1 and 6% (12–15). The majority of cases
are found in Caucasian populations. In fact, highest prevalence
of this defect is located in Central Europe and Switzerland
(1:200 adults); its frequency decreases gradually in Mediterra-
nean or Northern European populations (4,5,16), whose char-
acteristics suggest a founder-effect gene origin (17). In Germany,
UK, and the U.S., prevalence ranges between 1:500 and
1:700 adults. This mutation additionally has been reported
in Israel, Russia, China, and Japan (15–19). To the best of
our knowledge, no case has been described in Latin Ameri-
can populations.
Molecular epidemiology of familial hypercholesterolemia
is unknown in Mexico and Latin America, including per-
centage of FH cases due to defects in either LDL receptor or
apoB genes. This information has scientific and practical
implications. The search for mutations on LDL receptor or
apoB genes is the best tool to identify affected relatives.
Diagnosis based solely on lipid profile may be difficult, es-
pecially in children and young adults. However, it is not
possible to seek out these defects without knowing which
mutations are the most common in studied population. The
main goal of this report was to describe prevalence of
apoB100 mutations in Mexican patients with familial hy-
percholesterolemia. Because there are in excess of 860
known mutations in LDL receptor gene associated with FH
(20) and only three in apoB gene, we began to look for ab-
normalities in apoB gene in this survey. The first Mexican
FDB family is reported on here.
Materials and Methods
Subjects.
Thirty FH cases identified at the Instituto Nacional
de Ciencias Médicas y de Nutrición Salvador Zubirán Lipid
Clinic were included. All fulfilled clinical criteria for consider-
ation as heterozygous FH (autosomal dominant inheritance,
tendinous xanthomata, corneal arc, family history of coronary
heart disease, and LDL cholesterol
�
160 mg/dL).
Methods.
Patients fasted overnight for a minimum of 9 h after
which time blood was drawn and collected in Vacutainer tubes.
A blood sample was taken for lipid profile and DNA extraction.
All analytic measurements were done at the Endocrinology and
Metabolism Department of the Salvador Zubirán National Insti-
tute of Medical Sciences and Nutrition in Mexico City. All sam-
ples were kept frozen at
�
80
�
C; maximum time of storage was
12 months. Glucose was analyzed by glucose-oxidase method
(Boehringer Mannheim, Mannheim, Germany). Serum con-
centrations of total cholesterol and triglycerides were deter-
mined by enzymatic methods (Boehringer Mannheim). HDL-
cholesterol was measured after precipitation of VLDL and
LDL by phosphotungstate method (Boehringer Mannheim),
LDL-cholesterol was measured by immunochemical direct
method. Intra-assay CV values for total cholesterol, triglycer-
ides, and HDL-cholesterol were 3, 5, and 5%, respectively. Our
laboratory followed standardization procedures according to
World Health Organization (WHO) recommendations, includ-
ing use of external control sera. This laboratory is certified for
test standardization by the External Comparative Evaluation of
Laboratories Program of the College of American Pathologists.
Identification of apoB mutations.
Peripheral lymphocyte
DNA was extracted using phenol/chloroform extraction and
salt precipitation method. Polymerase chain reaction (PCR)
and restriction enzyme assays were used for identification
of Arg3500Trp, Arg3500Gln, and Arg3531Cys mutations;
oligonucleotides and enzymes employed in these assays are
mentioned elsewhere (8–10,12). Briefly, PCR mixture was
carried out using dNTPs (10 mM), MgCl
2
(25 mM), forma-
mide, the oligonucleotides, Taq polymerase, and the ex-
tracted DNA. Samples were placed in the thermocycler for
35 cycles. Successful DNA amplification was verified by
agarose gel electrophoresis. PCR fragments were digested
using restriction enzymes and fragments were analyzed in
Table 1.
Characteristics of the cases with familial hypercholesterolemia
Variable
n
�
30
Age (years) 47
�
16.22
Gender (M/F) 4/26
Body mass index (kg/m
2
) 24
�
4.72
Age at diagnosis (years) 34.5
�
17.64
Tendinous xanthomata,
n
(%) 30 (100)
Coronary heart disease,
n
(%) 4 (13.3)
Cholesterol (mg/dL) 351
�
61
Triglycerides (mg/dL) 132
�
52
HDL-cholesterol (mg/dL) 51.3
�
14.7
LDL-cholesterol (mg/dL) 274.9
�
60.4
High blood pressure,
n
(%) 5 (16.6)
Diabetes mellitus,
n
(%) 3 (10)
Page 3
72
Robles-Osorio et al./ Archives of Medical Research 34 (2003) 70–75
acrylamide gels. Resulting bands were compared against
fragments described in previous reports (8–10,12). The mu-
tation was confirmed using automatic sequencing with an
Applied Biosystem 310 sequencer (Perkin-Elmer Corp.,
Foster City, CA, USA).
The Ethics Committee of the Instituto Nacional de Ciencias
Médicas y de Nutrición approved the study on January 12,
2001. All participants gave informed consent to participate
in the study. DNA material was used solely for purposes of
the study.
Statistical analysis.
The database was validated through
recognition of missing values, outliers, and inconsistencies
among variables. Descriptive analysis included estimation
of mean values and standard deviations (SDs) for continu-
ous variables. Prevalence and frequencies were expressed as
percentages. All statistical analyses were conducted using
the Statgraphics statistical package (Statistical Graphics
Co., Rockville, MD, USA).
Results
Thirty FH cases were included (26 females and 4 males),
whose clinical characteristics are shown in Table 1. All sub-
jects were born and raised in Mexico. Coronary heart dis-
ease, ischemia, or myocardial infarction was found in four
study participants (13.3%).
DNA samples were obtained in each case. Only one sample
was positive for mutation Arg3500Gln of apoB gene. All 30
samples were negative for Arg3500Trp and Arg3531Cys muta-
tions. In this population, apoB100 mutation prevalence was
3.3
�
0.18% (95% confidence interval [CI] 0.08–17.22%).
Description of the FDB case.
A 44-year-old male was re-
ferred for evaluation of severe hypercholesterolemia. He
had a history of coronary heart disease (CHD) among pater-
nal relatives (see pedigree in Figure 1). Patient’s medical
history was unremarkable with the exception of hypercho-
lesterolemia diagnosed at age 40 years. His lipid profile was
cholesterol 370 mg/dL, LDL-cholesterol 300 mg/dL,
Figure 1. Proband case pedigree.
Page 4
Familial Hypercholesterolemia in Mexico
73
HDL-cholesterol 32 mg/dL, and triglycerides, 189 mg/dL.
Tendinous xanthomata (at Achilles tendon) were detected
on physical examination. Atorvastatin 40 mg/day and life-
style modifications were started. The LDL-cholesterol was
lower than 100 mg/dL in the follow-up measurements. The
mutation was confirmed by automated sequencing. CGG-
to-CAG change was found in nucleotide 10699 (Figure 3).
Lipid profile of the patient’s relatives is shown in Table 2.
Thirteen relatives were sampled; kindred structure is de-
picted in Figure 1. The proband’s father was born in Cata-
luña, Spain. The patient reported that his father had the
same lesions as he had on Achilles tendon; his father died at
age 52 years of myocardial infarction. Three of four
proband siblings had hypercholesterolemia; Arg3500Gln
mutation was found in their samples. All three had previ-
ously undetected tendinous xanthomata. Nine of 14 mem-
bers of the subsequent generation were also sampled. As
shown in Figure 2, two were positive for Arg3500Gln muta-
tion; their cholesterol levels were normal or slightly in-
creased. However, these values were the highest among
members of the third generation. These two individuals had
no tendinous lesions. Lifestyle modifications and atorvasta-
tin 20 mg/day were recommended in both cases.
Discussion
Familial defective ApoB100 is a form of familial hypercho-
lesterolemia that could be diagnosed with certainty using
simple molecular biology procedures. This disorder is associ-
ated with a high risk of premature coronary events (1–3,21).
However, it could be diagnosed early in life and hypo-
lipemiant treatment is capable of preventing cardiovascular
morbidity. These are strong arguments for efficient diagnostic
and therapeutic approaches. Inclusion of molecular biology as-
says at the clinical laboratory makes possible diagnosis of
affected cases with borderline cholesterol levels or without
tendinous xanthomata. These procedures have become nec-
essary in lipid clinics or referral centers. Identification of apoB
mutations requires only a few PCR assays. In contrast,
�
860
mutations were reported in LDL receptor gene (20). The
search for LDL receptor gene abnormalities in individual
cases requires description of molecular epidemiology of
these mutations in every ethnic group. However, there is in-
sufficient information on Latin American countries (22,23).
Thus, identification of the apoB mutations was selected as
our initial step for description of molecular epidemiology of
Mexican FH cases. Low prevalence found in this cohort
(one of 30 FH subjects) and absence of additional published
FDB cases from Mexico or other Latin American countries
suggest that this mutation is not a common cause of FH in
the Mexican population. Taking together our data and re-
ports derived from Brazilian populations (
n
�
124), esti-
mated FDB prevalence among FH patients from Latin
Table 2.
Characteristics of the cases with familial defective apolipoprotein B
Number on
electrophoresis gel
Number on
pedigree Age Cholesterol
a
Triglycerides
a
HDL
a
LDL
a
Glucose
a
0 II-8 73 238 94 70 149 70
1 III-2 45 251 68 49 188 76
2 III-4 44 251 93 50 182 86
- III-6 42 263 61 67 184 84
3 III-10 37 247 78 45 186 82
4 III-8 40 206 191 56 112 78
5 IV-4 14 149 96 53 77 69
6 IV-3 16 159 154 43 85 71
7 IV-5 12 256 91 49 189 75
8 IV-7 14 158 62 33 113 90
9 IV-8 11 155 206 36 77.8 78
10 IV-9 14 150 78 44 90 83
11 IV-10 14 168 70 49 105 71
12 IV-11 8 146 70 54 78 78
13 IV-12 8 210 90 33 159 71
a
Values expressed as mg/dL.
Figure 2. Polymerase chain reactions for detection of Arg 3500Gln muta-
tions. Subject characteristics are shown in Table 2. Patients 1, 2, 3, 7, and
13 are affected.
Page 5
74
Robles-Osorio et al./ Archives of Medical Research 34 (2003) 70–75
America is 0.8
�
0.8% (95% confidence interval [CI] 0.02–
5.5%). These data reinforced the importance of describing
molecular epidemiology of FH in Latin America because
the simplest procedures will be useful in a very small per-
centage of FH cases.
The case reported here was representative of the FDB clini-
cal picture. Increased atherogenicity of FDB was in accordance
with premature cardiovascular death of the proband’s father
and grandfather. Hypercholesterolemia was severe but slightly
lower in magnitude than that observed in subjects with LDL re-
ceptor defects. The difference may be explained because a por-
tion of LDLs contains apolipoprotein E on the surface that is a
ligand of LDL receptor. This feature resulted in alternative
clearance pathway for LDL particles. However, such differ-
ences are not sufficiently significant to distinguish clinically
the two known causes of hypercholesterolemia. However, re-
sponse to hypolipemiant treatment is better among FDB cases;
accordingly, the probands’s LDL cholesterol remained below
100 mg/dL during follow-up. Finally, molecular biology assay
usefulness was well exemplified in the kindred reported on
here. Two cases from the third generation had the mutation;
however, they could be considered incorrectly as normal based
solely on lipid levels and using biochemical criteria for fa-
milial hypercholesterolemia proposed in the Make Early
Diagnoses
�
Prevent Early Deaths (MEDPED) report (24,25).
The need to use these tests may be even greater in FDB cases
compared to other forms of FH because FDB individuals may
have lower prevalence of tendinous xanthomata (pathogno-
monic sign of FH) (26). Also, lower cholesterol concentrations
found in FDB cases compared to cases with LDL receptor
defects may limit applicability of cut-points utilized in the
MEDPED report. In addition to precise diagnosis, impact of
genetic testing is usually positive among patients and their rela-
tives. Andersen (2) studied attitudes found for family screening
in FDB. In 57 index patients and 93 hypercholesterolemic rela-
tives, only 6% regretted awareness of having FH. The majority
(84%) was in favor of screening for affected individuals in
their family. Increasing knowledge of the disease resulted in
better adherence to treatment, positive attitude, and effective
counseling.
These mutations modify LDL secondary structure, de-
creasing protein affinity for LDL receptor. When first de-
scribed, LDL accumulation was explained by interference
of abnormal amino acid with receptor binding site. However,
recent studies demonstrated that the effect is performed at a
distance, changing secondary structure; additionally, this de-
fect does not interfere directly with lipoprotein binding. The
carboxyterminal portion of apoB usually inhibits lipopro-
tein interaction with LDL receptor. The initial 89% of apoB
includes LDL particle as a belt; 11% of carboxyterminal
folds over apoB near residue 3500. The region that binds
with receptor (residues 3359–3369) is near the site of car-
boxyterminal region folding. Change in position 3500 allows
fold interference with the region of apoB that binds with
LDL receptor (27). The main difference among mutations
that cause FH is found in homozygotes (28,29). Four sub-
jects have been described to date as homozygous for the
mutation in position 3500, their ages range from 31 to 69
years, and it is uncommon in cases due to LDL receptor de-
fects. Only one subject showed ischemic heart disease when
reported. Homozygous subjects with defects in apolipoprotein
B have cholesterol concentrations similar to heterozygotes.
Statin treatment is able to normalize cholesterol concentra-
tions in these cases.
In conclusion, further studies are required to describe the
molecular epidemiology of this disorder in Latin American
Figure 3. Confirmation of Arg 3500Gln mutation. CGG-to-CAG change was found on position 10699.
Page 6
Familial Hypercholesterolemia in Mexico
75
groups. Molecular biology techniques allow easy detection
of cases explained by abnormalities of apolipoprotein B
gene. However, our data suggested that FDB explained only
a small number of FH cases in Mexico. Despite this, imple-
mentation of these tests is crucial for correct diagnosis of
FDB kindred members.
Acknowledgments
LR-O received a grant from Consejo Nacional de Ciencia y Tecnología
(CONACYT, México) as a postgraduate program student.
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