Locus on chromosome 6p linked to elevated HDL cholesterol serum levels and to protection against premature atherosclerosis in a kindred with familial hypercholesterolemia.

Locus on Chromosome 6p Linked to Elevated HDL
Cholesterol Serum Levels and to Protection Against
Premature Atherosclerosis in a Kindred With
Familial Hypercholesterolemia
Samuel Canizales-Quinteros, Carlos A. Aguilar-Salinas, Eduardo Reyes-Rodríguez, Laura Riba,
Maribel Rodríguez-Torres, Salvador Ramírez-Jiménez, Adriana Huertas-Vázquez,
Verónica Fragoso-Ontiveros, Alejandro Zentella-Dehesa, José L. Ventura-Gallegos,
Gerardo Vega-Hernández, Angelina López-Estrada, Moise´s Aurón-Gómez,
Francisco Gómez-Pérez, Juan Rull, Nancy J. Cox, Graeme I. Bell, Maria Teresa Tusié-Luna
Abstract—Heterozygous familial hypercholesterolemia (FH) is a highly atherogenic genetic disorder leading to premature
coronary heart disease (CHD), usually before 60 years of age. We studied an extended multigenerational kindred with
FH linked to chromosome 1p32 in which atherosclerotic complications were either delayed or prevented in individuals
with elevated HDL cholesterol (HDL-C) levels or hyperalphalipoproteinemia (HA). Premature CHD was observed in
FH individuals without HA. The study of this family established that the HA trait in the family also followed an
autosomal dominant mode of inheritance with a pattern of segregation independent from FH. We identified a locus on
chromosome 6 linked to elevated HDL-C levels (HA) in this family. Haplotype analysis refined the localization to a
7.32-cM interval (73 to 80 cM from pter) flanked by markers D6S1280 and D6S1275. Parametric 2-point and multipoint
analyses yielded maximum LOD scores of 3.05 and 3.17, respectively. This finding was confirmed with a nonparametric
multipoint score of 3.78 (P�0.0009). We propose that this locus, linked to elevated HDL-C levels, confers protection
against premature CHD within an FH context. (Circ Res. 2003;92:569-576.)
Key Words: LDL cholesterol
�
HDL cholesterol
�
genetics
�
familial hypercholesterolemia
�
hyperalphalipoproteinemia
Epidemiological studies have associated elevated LDLcholesterol (LDL-C) levels with an increased risk for
coronary heart disease (CHD).1 Familial hypercholesterol-
emia (FH) was the first entity directly associated with the
development of premature atherosclerosis and CHD.2
FH (see Mendelian Inheritance in Man [MIM]-143890, which
can be accessed online [OMIM] at http://www.ncbi.
nlm.nih.gov/omim), also referred to as autosomal dominant
hypercholesterolemia or ADH, is an inherited metabolic disorder
characterized by an autosomal dominant pattern of transmission,
high LDL-C levels, which give rise to tendon xanthomata and
arcus corneae, and premature atherosclerosis and early death
from cardiovascular complications. It is also often referred to as
familial heterozygous hypercholesterolemia because of its
dosage-dependent effect; ie, homozygous individuals exhibit a
more severe clinical phenotype than do heterozygotes. The
prevalence of the latter in the majority of populations is 1/500,
making this disorder one of the most prevalent dyslipidemias
associated with CHD. Several studies in FH individuals support
the high atherogenicity associated with the disease. In England,
the prevalence of CHD in men is 85% by age 60 years and 75%
in women by age 70 years. Another study in Norway showed
that the mean ages of death for male and female FH heterozy-
gotes were 55 and 64 years, respectively.2
In addition to high cholesterol and LDL-C levels, several
other factors have been associated with an elevated risk for
cardiovascular disease, including hypertension, apoE 3/4 or
4/4 genotypes, and high levels of lipoprotein(a).3–5
Original received August 21, 2002; resubmission received December 17, 2002; revised resubmission received February 14, 2003; accepted February
14, 2003.
From Unidad de Biología Molecular y Medicina Genómica (S.C.-Q., L.R., M.R.-T., S.R.-J., A.H.-V., V.F.-O., M.T.T.-L.), Instituto de Investigaciones
Biomédicas de la Universidad Nacional Autónoma de México e Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; Departamento de
Sistemas Biológicos (S.C.-Q.), Universidad Autónoma Metropolitana-Xochimilco; Departamento de Endocrinología y Metabolismo de Lípidos
(C.A.A.-S., E.R.-R., A.L.-E., M.A.-G., F.G.-P., J.R.), Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; and Departamento de
Biología Celular, Instituto de Fisiología Celular (A.Z.-D., J.L.V.-G.), and Dirección General de Servicios de Cómputo Académico (G.V.-H.), Universidad
Nacional Autónoma de México, México City, Mexico; and the Howard Hughes Medical Institute (G.I.B.) and Departments of Biochemistry and
Molecular Biology (G.I.B.) and Medicine and Human Genetics (N.J.C., G.I.B.), the University of Chicago, Chicago, Ill.
Correspondence to Maria Teresa Tusié-Luna, MD, PhD, Unidad de Biología Molecular y Medicina Genómica, Instituto Nacional de Ciencias
Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15 Colonia Sección 16, Tlalpan 14000, México DF. E-mail tusie@servidor.unam.mx
© 2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000064174.69165.66
569
Clinical Research

FH is genetically heterogeneous with mutations in different
genes affecting different families. The most common cause is
mutations in the LDL receptor (LDLR)6 on chromosome 19
(19p13), with �800 mutations reported to date (LDLR
Mutation Database; http://www.ucl.ac.uk/fh/ and http://
www.umd.necker.fr are databases exclusive for LDLR muta-
tions). A less common cause is mutations in the apoB gene on
chromosome 2p23-p24 (FDB, MIM 144010), with only 3
reported mutations.7,8 Finally, genetic studies have identified
in third locus on chromosome 1p34.1-32.9,10
As opposed to hypercholesterolemia, increased levels of
HDL cholesterol (HDL-C) or hyperalphalipoproteinemia
(HA, MIM 143470) (�60 mg/dL) are inversely correlated
with CHD.11,12 High levels of HDL-C are usually associated
with late onset of atherosclerosis and longevity.13 Specifi-
cally, HDL2 cholesterol levels are inversely correlated with
the incidence of CHD and are considered the HDL-C particle
subclass, with the most prominent effect against the develop-
ment of atherosclerosis.14
Up to 70% of the variation in HDL-C levels in humans is
genetically determined. Segregation analyses have found
evidence of a major gene-type influence affecting high HDL
levels under both mendelian and nonmendelian models.15,16
So far, there are 10 published reports of genome-wide scans
for the identification of quantitative trait loci that determine
HDL levels in humans. These studies have shown evidence of
linkage with 22 different loci on 14 chromosomes.17
Genes that influence the concentration and nature of lipid
content in HDL-C particles have an important effect on its
role on overall lipid metabolism.18 In humans, a mutation in
the gene encoding apoA-I, the major component of the
HDL-C particle, results in increased stability (Milano muta-
tion) and confers protection against cardiovascular disease.19
The mechanisms by which elevated HDL-C levels reduce
cardiovascular risk are not completely understood. This effect
is probably mediated mostly through its induction of cellular
cholesterol efflux and reverse cholesterol transport from
peripheral tissue to the liver.20–22 HDL-C has additional
beneficial antiatherosclerotic effects, such as inhibition of
LDL particle oxidation,23 inhibition of cytokine-induced ex-
pression of adhesion molecules by endothelial cells,24 and the
activation of endothelial NO synthase.25
We identified a kindred (CGZ) with FH and HA, in which
both traits exhibit an autosomal dominant pattern of inheri-
tance and segregate independently. Atherosclerotic compli-
cations were either delayed or prevented in members of the
family affected with FH who also exhibited elevated HDL-C
levels, whereas premature coronary disease was observed in 2
individuals with FH but no evidence of HA. We have mapped
the FH trait in this family to markers on chromosome 1p32,
a region identified as the third locus for FH.9,10 Through a
whole genome scan, we identified a novel locus for the HA
trait on chromosome 6p.
Materials and Methods
Subjects and Clinical Features
The proband is a 94-year-old woman first evaluated at 88 years. She
was admitted to the Instituto Nacional de Ciencias Médicas y
Nutrición, where she was diagnosed with gastritis and arterial
hypertension. The patient smoked from age 15 to 30 years. At age 91
years, she suffered transient ischemic episodes. A Doppler ultra-
sound of the carotid arteries showed multiple plaques on both sides,
especially on the left carotid artery. The largest lesion caused a 50%
obstruction of the vessel lumen. The symptoms disappeared after
dipyridamole treatment. An ECG showed a left bundle branch
blockade. Hypercholesterolemia was diagnosed (425 mg/dL), and
the patient was referred to the Lipid Clinic. On physical examination,
she had a body mass index of 24.6 and blood pressure of 140/
80 mm Hg. Pulses at the lower limbs were normal. Symmetrical
arcus corneae and bilateral xanthomata at the Achilles tendons were
detected and confirmed by ultrasound. Blood chemistry and thyroid-
stimulating hormone concentrations were normal. A complete lipid
profile revealed the following: total cholesterol level 395 mg/dL,
LDL-C 220 mg/dL, HDL-C 98 mg/dL, apoA-I 190 mg/dL, and
triglycerides (TGs) 110 mg/dL. The LDL-C accumulation was
mainly explained by increased concentrations of the smaller and
denser LDL-C particles. The HDL-C elevation was mainly due to the
HDL2 subclass (which represented 52% of the total). Increased
lipoprotein(a) concentrations were also detected (136 mg/dL).
A low-fat low-cholesterol diet and lovastatin (20 to 40 mg/d)
treatment were prescribed but were followed irregularly. A lipid
profile during the follow-up was as follows: cholesterol 281�21
mg/dL, LDL-C 179�18 mg/dL, HDL-C 90�8 mg/dL, and TGs
131�29 mg/dL.
The family history is consistent with FH. Her brother (II3) had a
similar lipid profile and tendinous xanthomata (see online Table,
available at http://www.circresaha.org). The proband (II2) had 2 sons
and 1 daughter. Her oldest son (III1) died at 52 years of age of a
myocardial infarction, and his lipid and clinical profiles were
consistent with FH. Her second son (III3) and her daughter (III4) are
also affected, according to their lipid profiles and the presence of
tendinous xanthomata.
Forty members of the family were studied (all available subjects
gave informed consent). Of these, 12 were diagnosed as affected
according to the criteria outlined by Kwiterovich26; 7 of these
exhibited tendinous xanthomata. All affected individuals showed
LDL-C levels above the 90th percentile according to age and sex in
the Mexican population.27 As shown in Figure 1, the FH and HA
phenotypes are consistent with an autosomal dominant pattern of
transmission and independent segregation.
Besides the proband, 3 other individuals (II3, aged 84 years; III4,
aged 57 years; and III18, aged 49 years) exhibited elevated total
cholesterol and LDL-C levels but no evidence of premature CHD.
Individual II5, (proband’s brother) died of natural causes at 75 years
of age. Of his offspring, 1 of his daughters (III16) had the HA
phenotype, another (III20) had the HF phenotype, and his son had
both phenotypes, which would support the assumption that this
individual also carried both traits. A closer examination of the lipid
profiles of the entire family led us to the observation that several
members of the family had high levels of serum HDL-C. These data
are available in the online data supplement, which can be found at
http://www.circresaha.org. The criteria for HA was considered to be
HDL-C �60 mg/dL because that this level has been established to
confer protection against CHD.28–30 This value corresponds to the
90th percentile (according to age and sex) in the Mexican
population.27
Biochemical Determinations
Plasma lipids and blood chemistry were performed in 33 available
individuals through fully automated tests with commercially avail-
able reagents. Serum concentrations of total cholesterol and TGs
were determined by enzymatic methods (Boehringer-Mannheim).
The LDL-C and HDL-C particle distributions were characterized
through density gradient ultracentrifugation as described.31 The
double precipitation method was used to measure the HDL-C
subclasses as described.32 ApoA-I was measured by immunophelom-
etry.33 Sitosterol levels were determined by capillary-gas-liquid
chromatography.34
The diagnosis or status assignment for all family members was
carefully established. Members with a single measurement of lipid
570 Circulation Research March 21, 2003

levels or borderline lipid values were considered as “unknown” for
linkage analysis.
Analysis of Candidate Genes
DNA was extracted from whole blood using a phenol-free extraction
protocol adapted from Buffone and Darlington.35 Analysis of mic-
rosatellite markers was performed by polymerase chain reaction
amplification using end-labeled ([�-32P]dCTP) primers. The role of
different candidate genes in the expression of FH and HA was
evaluated using genetic markers closely linked to these genes
selected from various databases (Center for Medical Genetics,
available at http://research.marshfieldclinic.org/genetics, and Ge-
nome Database, available at http://www.gdb.org). Candidate genes
analyzed were as follows: LDLR and apoB for HF and apoA-I,
apoA-II/apoC-III/apoA-IV cluster, apoC-II, apoE, ATP-binding cas-
sette A type 1, peroxisome proliferator–activated receptor-�, scav-
enger receptor class B type I, hepatocyte nuclear factor-4�, cho-
lesteryl ester transfer protein, lecithin-cholesterol acyltransferase,
hepatic lipase, lipoprotein lipase, and paraoxonase 1 for HA. In
addition, apoE haplotyping was performed as described.36
Genomewide Scan
Thirty-four family members were genotyped with a set of 238
autosomal markers (average spacing of 15 to 20 cM). Most markers
used were trinucleotide or tetranucleotide repeats. Polymerase chain
reaction products were analyzed on an ABI 377 sequencer through
standard methods. Allele numbers were internally assigned and do
not refer to allele numbers in public databases.
Linkage Analysis
A model assuming an autosomal dominant pattern of inheritance was
used with a gene frequency of 0.001 and 90% penetrance. The
maximum expected LOD score for this family was estimated through
SIMULATE (LINKAGE software) using 1000 replicas.37 Two-point
LOD scores were calculated with the use of FASTLINK.38 Allele
frequencies for all markers were calculated from unrelated individ-
uals from the Mexican population. Parametric multipoint analysis
was performed with the use of GENEHUNTER-PLUS.39 To avoid
any potential uncertainty in the exact mode of inheritance of HA, the
data were analyzed with the model-free multipoint allele-sharing
method implemented in this same program. Haplotypes were ob-
tained with this same program and adjusted manually.
Results
Mapping of the FH Locus
We identified a kindred (CGZ) displaying all clinical char-
acteristic of FH, including high total cholesterol and LDL-C,
tendon xanthomata, arcus corneae, and premature CHD. We
estimated the power of the family for linkage analysis
through simulation. With the use of 1000 replicates, a
maximum expected Zmax of 5.9 was obtained (the average
expected Zmax was 2.1, with 57% �3.00).
Through linkage analysis, both LDLR and the apoB genes
were ruled out as the underlying cause of this condition. For
the apoB gene, screening for the 2 most frequent mutations
(R3500Q and R3531C) was also performed for the proband
and an affected brother. The possibility of sitosterolemia was
excluded by direct sitosterol measurement in the proband.
A genome-wide scan (chromosomes 1 to 22) was performed
with 238 markers. Positive pairwise LOD scores were obtained for
adjacent markers on chromosome 1. The maximum pairwise LOD
score obtained was 2.94 at ��0.05 for marker D1S197. Further
analysis with a higher density of markers in this region showed
positive LOD scores at ��0.0 for 2 additional markers closely
linked to D1S197 (D1S386 and D1S1661) (Table 1). We also
analyzed the data set under an “affecteds-only” model (Table 1).
Multipoint analysis gave a maximum LOD score of 3.29 between
markers D1S2134 and D1S200 (Figure 2). Haplotype reconstruc-
tion showed that all 12 affected individuals for FH share a common
haplotype encompassing markers D1S197 through D1S417. The
centromeric boundary of this interval was defined by a recombina-
tion event between markers D1S417 and D1S200 in individual III4,
and the telomeric boundary was defined by a recombination event
between markers D1S197 and D1S2134 in individual II3 (Figure
3). This interval corresponds to a 6.75-cM region located 75.6 to
82.4 cM from pter. Five asymptomatic individuals and 2 with
unknown affection status also share this same haplotype, implying
incomplete penetrance.
Figure 1. Family drawing of FH and HA kin-
dred CGZ. Open symbols indicate unaffected
individuals; half-filled left symbols,
FH-affected individuals; half-filled right sym-
bols, HA-affected individuals; and filled sym-
bols, FH- and HA-affected individuals. Gray
symbols represent unknown status.
TABLE 1. Pairwise LOD Scores for Chromosome 1p32 Markers
and HF
Whole Family Affecteds Only
Locus Distance* ��0.00 Zmax �max ��0.00 Zmax �max
D1S3721 72.59 �5.27 0.00 0.49 �1.56 0.21 0.26
D1S2134 75.66 �1.21 1.00 0.18 0.25 1.50 0.10
D1S197 76.27 �1.59 2.94 0.05 1.68 1.68 0.00
D1S386 77.18 0.80 1.18 0.10 2.41 2.41 0.00
D1S1661 78.25 1.82 1.82 0.00 1.60 1.60 0.00
D1S417 79.80 �1.41 0.14 0.30 1.90 1.90 0.00
D1S200 82.41 �2.99 0.60 0.22 �0.23 1.28 0.10
D1S2867 85.68 �7.91 0.12 0.30 �1.00 0.24 0.15
*Distance in Kosambi centimorgans (cM) from pter.
Canizales-Quinteros et al Locus on Chromosome 6p Linked to Elevated HDL-C 571

Mapping of the HA Locus
On further clinical evaluation of the proband and other family
members, we identified 10 individuals with elevated levels of
HDL-C. Examination of the extended family led to the
observation that HA segregated as an independent trait with
an autosomal dominant pattern of inheritance and indepen-
dent of FH (Figure 1). Thus, we tested the hypothesis that a
locus associated with elevated HDL-C levels is present in this
family.
Thirteen candidate genes were examined through linkage
analysis to evaluate their possible contribution to the HA
phenotype, including genes for several apolipoproteins, cho-
lesteryl ester transfer protein, the scavenger receptor class B
type I, and transcription factors, such as hepatocyte nuclear
factor-4� and peroxisome proliferator–activated receptor-�
(see Materials and Methods). No evidence of linkage was
found to any of the analyzed genes.
Genome-wide multipoint analysis showed a single statisti-
cally significant locus, with a maximum LOD score of 3.17
and a maximum nonparametric (NPL) score of 3.78
(P�0.0009) between markers D6S1280 and D6S1275. The
maximum 2-point LOD score and NPL values in this region
were 3.05 and 3.08 (P�0.004), respectively, for the marker
D6S1662 (Figure 4 and Table 2).
All 10 individuals with HA shared a common haplotype
encompassing markers D6S2410 and D6S1053 (Figure 5).
The centromeric boundary of this interval is defined by a
recombination event between markers D6S1053 and
D6S1275 in individual II2, and the telomeric boundary is
defined by a recombination event between markers D6S1280
and D6S2410 in individual II3, defining a 7.32-cM interval
on chromosome 6p located 73.1 to 80.4 cM from pter. There
are no obvious candidate genes that directly regulate HDL
metabolism in this region.
Figure 2. Multipoint LOD scores for
chromosome 1p32 and HF. Markers are
indicated below the x-axis.
Figure 3. Suggested haplotypes of chromosome 1p32 linked to FH in the CGZ family. Open and filled symbols represent unaffected
and affected individuals, respectively; gray symbols represent unknown status. The haplotypes in brackets were deduced. The common
region shared in affected individuals in the family is indicated by gray bars.
572 Circulation Research March 21, 2003

However, there are two proinflammatory cytokine genes in
this interval: interleukin-17 (IL-17) and interleukin-17F (IL-
17F).40,41 Several proinflammatory cytokines have been im-
plicated in the overall regulation of plasma HDL-C concen-
trations.42– 44 Sequence analysis identified two
polymorphisms in the promoter region of the IL-17 gene and
one polymorphism (E126G) in exon 3 of IL-17F. None of
these nucleotide changes cosegregates with elevated HDL
levels in this family.
Discussion
Atherosclerosis is a multifactorial entity in which genetics
and environmental factors play a role in the pathophysiology
of the disease.45 Mutations in genes associated with FH, as
well as mutations in genes that lower HDL-C levels, such as
ABC1, are known to cause accelerated atherogenesis.2,8,46,47
On the other hand, factors such as cigarette smoking, arterial
hypertension and dietary cholesterol consumption are associ-
ated with an increased risk of CHD.3,48,49
Several epidemiological and genetic studies confirm the
association between elevated HDL-C levels and protection
against atherogenesis.15,50,51 In the Framingham Heart Study,
individuals with HDL-C concentrations of 1.5 mmol/L (60
mg/dL) or higher are protected against the development of
CHD even in the presence of elevated LDL-C serum lev-
els.28–30 In a different study, an association was found
between an increase of HDL2 �45% and a decreased fre-
quency of atherosclerosis.52
In addition, animal models have demonstrated the role of
HDL-C as cardioprotective particles. For instance, hypercho-
lesterolemic Watanabe heritable hyperlipidemic rabbits over-
expressing the apoA-I protein showed a reduction in the
formation of atherosclerotic plaques.53 Furthermore, overex-
pression of human apoA-I in transgenic mice led to an
increase in HDL-C levels �150 mg/dL and a 95% reduction
of the atherosclerotic plaques in C57BL/6 mice on a high-fat
diet.54
We identified an FH kindred in which elevated HDL-C
levels (HA) are displayed as an independent trait within the
family. Finding concurrent expression of FH and HA within
a family is highly unusual because elevated LDL-C levels are
generally inversely correlated with HDL-C levels.55,56 There-
fore, this kindred provides a unique opportunity to dissect the
genetic component underlying the HA trait in a background
of atherogenicity.
A striking feature found in this family is that 2 affected
individuals aged �80 years (proband II2, aged 94 years, and
her brother II3, aged 84 years) do not display the expected
TABLE 2. Pairwise LOD Scores for Chromosome 6p Markers and HAL
LOD Score at �
Locus Distance* 0.00 0.01 0.05 0.10 0.20 0.30 0.40 Zmax �max
D6S1280 73.13 �0.55 0.68 1.63 1.92 1.83 1.36 0.65 1.95 0.13
D6S2410 73.13 1.53 1.52 1.45 1.32 0.97 0.54 0.14 1.53 0.00
D6S1956 75.45 1.58 1.54 1.40 1.19 0.86 0.49 0.17 1.58 0.00
D6S1960 76.62 1.50 1.78 2.15 2.21 1.92 1.34 0.60 2.21 0.10
D6S1662 77.78 3.05 3.03 2.89 2.67 2.09 1.39 0.60 3.05 0.00
D6S1276 78.85 2.20 2.21 2.16 2.02 1.60 1.05 0.43 2.21 0.01
D6S1952 79.92 0.39 0.38 0.34 0.30 0.22 0.14 0.07 0.39 0.00
D6S1053 80.45 1.92 1.88 1.71 1.50 1.06 0.61 0.21 1.92 0.00
D6S1275 80.45 �1.86 �0.74 0.12 0.47 0.60 0.45 0.17 0.61 0.18
D6S1031 88.63 �7.35 �3.11 �1.25 �0.43 0.14 0.23 0.12 0.23 0.30
*Distance in Kosambi centimorgans (cM) from pter.
Figure 4. Multipoint linkage analysis of
HA to markers on chromosome 6p12.13-
q13. The x-axis represents the relative
distance in centimorgans, and the y-axis
represents the multipoint LOD (solid line)
and NPL (broken line) scores from para-
metric and nonparametric linkage analy-
ses, respectively. Markers are indicated
below the x-axis.
Canizales-Quinteros et al Locus on Chromosome 6p Linked to Elevated HDL-C 573

atherosclerotic manifestations associated with this condition
in spite of their elevated total cholesterol and LDL-C levels
and additional risk factors [hypertension, elevated Lp(a), or
hypertriglyceridemia]. The absence of symptomatic CHD in
these individuals is in stark contrast to the observed incidence
of CHD in FH heterozygotes aged �60 years.1
The observed protection against premature CHD in these 2
individuals is correlated with a concurrent elevation of their
HDL-C levels. Conversely, there are 2 documented affected
FH individuals with premature CHD: (1) individual III3, who
does not have the HA trait or the haplotype associated with it
and has presented with several ischemic episodes since 52
years of age, and (2) individual III1, who died at 52 years of
age as a result of myocardial infarction. Even though HDL-C
levels were not available for the latter individual, he did not
have the haplotype linked to HA (his haplotype was unequiv-
ocally inferred). Also, none of his 3 daughters has either
elevated HDL-C levels or the chromosome 6 haplotype,
which would support the assumption that his HDL-C levels
were below what is regarded as protective. In light of these
observations, we propose that in this family, the HDL-C
elevation explains the antiatherogenic effect seen in those FH
individuals also carrying the HA trait.
Through a genome-wide scan, we mapped the FH trait to a
region on chromosome 1p32, a region previously linked to
FH in 2 separate studies.9,10,57 Based on recombination events
in 2 affected individuals, our results localize the responsible
gene to an interval of 6.75 cM flanked by markers D1S2134
and D1S200. The integration of the mapping data from all 3
studies yields a common interval of �0.61 cM flanked by
markers D1S2134 to D1S197. Some likely candidate genes in
the vicinity of this region are EPS15 (an epidermal growth
factor receptor pathway substrate),58 APOER2 (an apoE
receptor),59 and SCP2 (a sterol carrier protein).60
In the family we studied, this locus displays incomplete
penetrance, because in addition to all 12 affected individuals
sharing a common 4 marker haplotype, several other mem-
bers share the same region but appear to be asymptomatic. Of
these, 5 are individuals with total cholesterol and LDL-C
levels within the normal range, and 2 are individuals who
showed either borderline lipid values according to age and
sex or are individuals with unknown status (see Materials and
Methods). The affecteds-only analysis showed 5 consecutive
markers (D1S2134 through D1S417) with positive LOD
scores (all at ��0). A comparison of the results obtained
under the 2 models used (whole family versus affecteds only)
is consistent with the observed incomplete penetrance in this
kindred. Although incomplete penetrance for this FH locus
has been reported for both French and Spanish families,9 the
penetrance found in our kindred may be lower. This suggests
the possible involvement of additional loci influencing the
phenotype.
For the HA trait, a genome-wide scan identified a locus on
chromosome 6p. Haplotype analysis defined a region span-
ning 7.32 cM between markers D6S1280 and D6S1275. This
haplotype is shared by all individuals with HA and 1
individual with levels within the normal range, implying a
penetrance of 90% in this family. Our region on chromosome
6p (73.13 to 80.45 cM from pter) overlaps the region reported
by Knoblauch et al61 influencing LDL levels in an FH Arab
family and an independent sample of healthy white monozy-
gotic and dizygotic twins from Germany. This same region
has also been linked with TG/HDL levels in a study in a
population from Minnesota,62 and it is close to a peak for TG
levels (LOD score 1.24 at 71.3 cM) reported in African
American families from the HyperGEN Study.63 Therefore, it
is possible that the locus mapped in chromosome 6p12.3-q13
in our family for high HDL levels may also influence lipid
concentrations in other populations. The critical interval
defined in our family has �30 known genes, with
interleukin-17 and interleukin-17F as the only candidate
genes showing a possible biologically related function. Se-
quence analysis of these genes showed no evidence that they
were responsible for the HA phenotype. We have not ana-
Figure 5. Suggested haplotypes of chromosome 6p12.13-q13 linked to HA in the CGZ family. Open and filled symbols represent unaf-
fected and affected individuals, respectively; gray symbols represent unknown status. The haplotypes in brackets were deduced. The
common region shared in affected individuals in the family is indicated by gray bars.
574 Circulation Research March 21, 2003

lyzed other positional candidate genes within this chromo-
somal region.
Although the 6p12.3-q13 locus has already been associated
with the modulation of cholesterol, TG, and TG/HDL lev-
els,61–63 it has not been previously linked to high HDL levels
or to an antiatherogenic effect in humans. As to the extent of
the observed protective effect conferred by this locus in the
family we studied, it is noteworthy that there are independent
cardiovascular risk factors besides FH in affected individuals
with no manifestations of CHD. This suggests that the
antiatherogenic effect conferred by this locus may also
provide protection against atherogenesis-promoting condi-
tions other than FH. In this regard, it would be of interest to
determine the possible influence of this locus on lipid
concentrations and its associated cardiovascular risk in Mex-
ican population.
The identification of the responsible gene within the
6p12.3-q13 locus will provide new information regarding the
role of the HDL-C particles as antiatherogenic agents and
may result in the identification of new therapeutic targets for
the prevention and treatment of atherosclerotic disease.
Acknowledgments
This work was supported by grants IN217898 and IN217501 from
DGAPA, Universidad Nacional Autónoma de México, and from
Fundación Miguel Alemán, 2000. S. Canizales-Quinteros was sup-
ported by a PhD fellowship from Consejo Nacional de Ciencia y
Tecnología, México. We are very grateful to the participating family
for their kindness and enthusiastic collaboration in this study. We
thank S. Patel, who kindly performed the sitosterol measurements at
Southwestern Medical Center, Dallas, Tex. We also thank S. Romero
for providing technical informatics assistance and S. Curiel for
access to a Workstation and informatics support during the early part
of this project.
References
1. Wilson PW, D’Agostino RB, Levy D, Belanger AM, Silbershattz H,
Kannel WB. Prediction of coronary heart disease using risk factor cate-
gories. Circulation. 1998;97:1837–1847.
2. Goldstein J, Hobbs HH, Brown MS. Familial hypercholesterolemia. In:
Scriver CR et al, eds. The Metabolic and Molecular Bases of Inherited
Disease. New York, NY: McGraw-Hill; 2000:2863–2913.
3. Kannel WB. Coronary heart disease risk factors in the elderly. Am J
Geriatr Cardiol. 2002;11:101–107.
4. Davignon J, Cohn JS, Mabile L, Bernier L. Apolipoprotein E and ath-
erosclerosis: insight from animal and human studies. Clin Chim Acta.
1999;286:115–143.
5. Kraft HG, Lingenhel A, Raal FJ, Hohenegger M, Utermann G. Lipopro-
tein(a) in homozygous familial hypercholesterolemia. Arterioscler
Thromb Vasc Biol. 2000;20:522–528.
6. Goldstein JL, Brown MS. Binding and degradation of low density
lipoproteins by cultured human fibroblasts: comparison of cells from a
normal subject and from a patient with homozygous familial hypercho-
lesterolemia. J Biol Chem. 1974;249:5153–5162.
7. Innerarity TL, Weisgraber KH, Arnold KS, Mahley RW, Krauss RM,
Vega GL, Grundy SM. Familial defective apolipoprotein B-100: low
density lipoproteins with abnormal receptor binding. Proc Natl Acad Sci
U S A. 1987;84:6919–6923.
8. Rabès JP, Varret M, Saint-Jore B, Erlich D, Jondeau G, Krempf M,
Giraudet P, Junien C, Boileau C. Familial ligand-defective apolipoprotein
B-100: simultaneous detection of the ARG35003GLN and
ARG35313CYS mutations in a French population. Hum Mutat. 1997;
10:160–163.
9. Varret M, Rabès JP, Saint-Jore B, Cenarro A, Marinoni JC, Civeira F,
Devillers M, Krempf M, Coulon M, Thiart R, Kotze MJ, Schmidt H,
Buzzi JC, Kostner GM, Bertolini S, Pocovi M, Rosa A, Farnier M,
Martinez M, Junien C, Boileau C. A third major locus for autosomal
dominant hypercholesterolemia maps to 1p34.1-p32. Am J Hum Genet.
1999;64:1378–1387.
10. Hunt SC, Hopkins PN, Bulka K, McDermott MT, Thorne TL, Wardell
BB, Bowen BR, Ballinger DG, Skolnick MH, Samuels ME. Genetic
localization to chromosome 1p32 of the third locus for familial hyper-
cholesterolemia in a Utah kindred. Arterioscler Thromb Vasc Biol. 2000;
20:1089–1093.
11. Miller GJ, Miller NE. Plasma high density lipoprotein concentration and
development of ischemic heart disease. Lancet. 1975;1:16–20.
12. Reichl D, Miller NE. Pathophysiology of reverse cholesterol transport:
insights from inherited disorders of lipoprotein metabolism. Arterioscle-
rosis. 1989;9:785–797.
13. Glueck CJ, Garstside P, Fallat RW, Sielski J, Steiner PM. Longevity
syndromes: familial hypobeta and familial hyperalpha lipoproteinemia.
J Lab Clin Med. 1976;88:941–957.
14. Cheung MC, Brown GB, Wolf AC, Albers JJ. Altered particle size
distribution of apolipoprotein A-I-containing lipoproteins in subjects with
coronary artery disease. J Lipid Res. 1991;32:383–394.
15. Mahaney MC, Blangero J, Rainwater DL, Comuzzie AG, VandeBerg JL,
Stern MP, MacCluer JW, Hixson JE. A major locus influencing plasma
high-density lipoprotein cholesterol levels in the San Antonio Family
Heart Study: segregation and linkage analyses. Arterioscler Thromb Vasc
Biol. 1995;15:1730–1739.
16. Cupples LA, Myers RH. Segregation analysis for high density lipoprotein
in the Berkeley data. Genet Epidemiol. 1993;10:629–634.
17. Wang X, Paigen B. Quantitative trait loci and candidate genes regulating
HDL cholesterol: a murine chromosome map. Arterioscler Thromb Vasc
Biol. 2002;22:1390–1401.
18. Rader DJ, Ikewaki K. Unravelling high density lipoprotein-
apolipoprotein metabolism in human mutants and animal models. Curr
Opin Lipidol. 1996;7:117–123.
19. Franceschini G, Calabresi L, Tosi C, Gianfranceschi G, Sirtori CR,
Nichols AV. Apolipoprotein AIMilano: disulfide-linked dimers increase
high density lipoprotein stability and hinder particle interconversion in
carrier plasma. J Biol Chem. 1990;265:12224–12231.
20. Glomset JA. High-density lipoproteins in human health and disease. Adv
Intern Med. 1980;25:91–116.
21. Ho YK, Brow MS, Goldstein JL. Hydrolysis and excretion of cytoplasmic
cholesteryl esters by macrophages: stimulation by high density
lipoprotein and others agents. J Lipid Res. 1980;21:391–398.
22. Hara H, and Yokoyama S. Interaction of free apolipoproteins with mac-
rophages: formation of high density protein-like lipoproteins and
reduction of cellular cholesterol. J Biol Chem. 1991;266:3080–3086.
23. Mackness MI, Mackness B, Durrington PN, Fogelman AM, Berliner J,
Lusis AJ, Navab M, Shih D, Fonarow GC. Paraoxonase and coronary
heart disease. Curr Opin Lipidol. 1998;9:319–324.
24. Cockerill GW, Rye KA, Gamble JR, Vadas MA, Barter PJ. High-density
lipoproteins inhibit cytokine-induced expression of endothelial cell
adhesion molecules. Arterioscler Thromb Vasc Biol. 1995;15:1987–1994.
25. Yuhanna IS, Zhu Y, Cox BE, Hahner LD, Osborne-Lawrence S, Lu P,
Marcel YL, Anderson RG, Mendelsohn ME, Hobbs HH, Shaul PW.
High-density lipoprotein binding to scavenger receptor-BI activates en-
dothelial nitric oxide synthase. Nat Med. 2001;7:853–857.
26. Kwiterovich PO Jr. Identification and treatment of heterozygous familial
hypercholesterolemia in children and adolescents. Am J Cardiol. 1993;
72:30–37.
27. Aguilar-Salinas CA, Olaiz G, Valles V, Torres JM, Perez FJ, Rull JA,
Rojas R, Franco A, Sepulveda J. High prevalence of low HDL cholesterol
concentrations and mixed hyperlipidemia in a Mexican nationwide
survey. J Lipid Res. 2001;42:1298–1307.
28. National Cholesterol Education Program. Report of the expert panel on
detection, evaluation, and treatment of high blood cholesterol in adults.
Arch Intern Med. 1988;148:36–39.
29. Summary of the Second Report of the National Cholesterol Education
Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment
of High Blood Cholesterol in Adults (Adult Treatment Panel II). JAMA.
1993;269:3015–3023.
30. Wilson PW, Abbott RD, Castelli WP. High density lipoprotein choles-
terol and mortality: the Framingham Heart Study. Arteriosclerosis. 1988;
8:737–741.
31. Lossow WJ, Shah SN, Chaikoff IL. Isolation of cholesterol-esterifying
activity in rat serum by ultracentrifugal flotation. Biochim Biophys Acta.
1966;116:172–174.
32. Martini S, Baggio G, Baroni L, Enzi GB, Fellin R, Baiocchi MR, Crepaldi
G. Evaluation of HDL2 and HDL3 cholesterol by a precipitation pro-
Canizales-Quinteros et al Locus on Chromosome 6p Linked to Elevated HDL-C 575

cedure in a normal population and in different hyperlipidemic pheno-
types. Clin Chim Acta. 1984;137:291–298.
33. Anderson RJ, Stember JC. A rate nephelometer for immunoprecipitin
measurement of specific serum proteins. In: Ritchie RF, ed. Automated
Immunoanalysis. New York, NY: Marcel Dekker; 1987:410–469.
34. Beaty TH, Kwiterovich PO Jr, Khoury MJ, White S, Bachorik PS, Smith
HH, Teng B, Sniderman A. Genetic analysis of plasma sitosterol, apo-
protein B, and lipoproteins in a large Amish pedigree with sitosterolemia.
Am J Hum Genet. 1986;38:492–504.
35. Buffone GJ, Darlington GJ. Isolation of DNA from biological specimens
without extraction with phenol. Clin Chem. 1985;31:164–165.
36. Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E
by gene amplification and cleavage with HhaI. J Lipid Res. 1990;31:
545–548.
37. Ott J. Analysis of Human Genetic Linkage. Rev ed. Baltimore,
Md/London, UK: The Johns Hopkins University Press; 1991.
38. Cottingham RW Jr, Idury RM, Schaffer AA. Faster sequential genetic
linkage computations. Am J Hum Genet. 1993;53:252–263.
39. Kong A, Cox NJ. Allele-sharing models: LOD scores and accurate
linkage tests. Am J Hum Genet. 1997;61:1179–1188.
40. Yao Z, Painter SL, Fanslow WC, Ulrich D, Macduff BM, Spriggs MK,
Armitage RJ. Human IL-17: a novel cytokine derived from T cells.
J Immunol. 1995;155:5483–5486.
41. Starnes T, Robertson MJ, Sledge G, Kelich S, Nakshatri H, Broxmeyer
HE, Hromas R. Cutting edge: IL-17F, a novel cytokine selectively
expressed in activated T cells and monocytes, regulates angiogenesis and
endothelial cell cytokine production. J Immunol. 2001;167:4137–4140.
42. Khovidhunkit W, Memon RA, Feingold KR, Grunfeld C. Infection and
inflammation-induced proatherogenic changes of lipoproteins. J Infect
Dis. 2000;181(suppl 3):S462–S472.
43. Hardardottir I, Moser AH, Fuller J, Fielding C, Feingold K, Grunfeld C.
Endotoxin and cytokines decrease serum levels and extra hepatic protein
and mRNA levels of cholesteryl ester transfer protein in Syrian hamsters.
J Clin Invest. 1996;97:2585–2592.
44. Khovidhunkit W, Moser AH, Shigenaga JK, Grunfeld C, Feingold KR.
Regulation of scavenger receptor class B type I in hamster liver and
Hep3B cells by endotoxin and cytokines. J Lipid Res. 2001;42:
1636–1644.
45. Lusis AJ. Atherosclerosis. Nature. 2000;407:233–241.
46. Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van Dam
M, Yu L, Brewer C, Collins JA, Molhuizen HO, Loubser O, Ouelette BF,
Fichter K, Ashbourne-Excoffon KJ, Sensen CW, Scherer S, Mott S, Denis
M, Martindale D, Frohlich J, Morgan K, Koop B, Pimstone S, Kastelein
JJ, Hayden MR, et al. Mutations in ABC1 in Tangier disease and familial
high-density lipoprotein deficiency. Nat Genet. 1999;22:336–345.
47. Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, Diederich W,
Drobnik W, Barlage S, Buchler C, Porsch-Ozcurumez M, Kaminski WE,
Hahmann HW, Oette K, Rothe G, Aslanidis C, Lackner KJ, Schmitz G.
The gene encoding ATP-binding cassette transporter 1 is mutated in
Tangier disease. Nat Genet. 1999;22:347–351.
48. Willett WC, Green A, Stampfer MJ, Speizer FE, Colditz GA, Rosner B,
Monson RR, Stason W, Hennekens CH. Relative and absolute excess
risks of coronary heart disease among women who smoke cigarettes.
N Engl J Med. 1987;317:1303–1309.
49. Renaud S, de Lorgeril M. Dietary lipids and their relation to ischaemic
heart disease: from epidemiology to prevention. J Intern Med Suppl.
1989;225:39–46.
50. Gordon T, Castelli WP, Hjortland MC, Kannel WB, Dawber TR. High
density lipoprotein as a protective factor against coronary heart disease:
the Framingham Study. Am J Med. 1977;62:707–714.
51. Gordon DJ, Rifkind BM. High-density lipoprotein: the clinical impli-
cations of recent studies. N Engl J Med. 1989;32:1311–1316.
52. Assmann G, Schulte H. Relation of high-density lipoprotein cholesterol
and triglycerides to incidence of atherosclerotic coronary artery disease
(the PROCAM experience): Prospective Cardiovascular Munster study.
Am J Cardiol. 1992;70:733–737.
53. Emmanuel F, Caillaud JM, Hennuyer N, Fievet C, Viry I, Houdebine JC,
Fruchart P, Denèfle P, Duverger N. Overexpression of human apoli-
poprotein A-I inhibits atherosclerosis development in Watanabe rabbits.
Circulation. 1996;94(suppl I):I-632. Abstract.
54. Rubin EM, Krauss RM, Spangler EA, Verstuyft JG, Clift SM. Inhibition
of early atherogenesis in transgenic mice by human apolipoprotein AI.
Nature. 1991;353:265–267.
55. Schmidt HH, Gregg RE, Tietge UJ, Beisiegel U, Zech LA, Brewer HB Jr,
Manns MP, Bojanovski D. Upregulated synthesis of both apolipoprotein
A-I and apolipoprotein B in familial hyperalphalipoproteinemia and
hyperbetalipoproteinemia. Metabolism. 1998;47:1160–1166.
56. Frenais R, Ouguerram K, Maugeais C, Marchini JS, Benlian P, Bard JM,
Magot T, Krempf M. Apolipoprotein A-I kinetics in heterozygous
familial hypercholesterolemia: a stable isotope study. J Lipid Res. 1999;
40:1506–1511.
57. Eden ER, Naoumova RP, Burden JJ, McCarthy MI, Soutar AK. Use of
homozygosity mapping to identify a region on chromosome 1 bearing a
defective gene that causes autosomal recessive homozygous hypercho-
lesterolemia in two unrelated families. Am J Hum Genet. 2001;268:
653–660.
58. Wong WT, Kraus MH, Carlomagno F, Zelano A, Druck T, Croce CM,
Huebner K, Di Fiore PP. The human eps15 gene, encoding a tyrosine
kinase substrate, is conserved in evolution and maps to 1p31-p32.
Oncogene. 1994;9:1591–1597.
59. Kim DH, Magoori K, Inoue TR, Mao CC, Kim HJ, Suzuki H, Fujita T,
Endo Y, Saeki S, Yamamoto TT. Exon/intron organization, chromosome
localization, alternative splicing, and transcription units of the human
apolipoprotein E receptor 2 gene. J Biol Chem. 1997;272:8498–8504.
60. Ohba T, Rennert H, Pfeifer SM, He Z, Yamamoto R, Holt JA, Billheimer
JT, Strauss JF III. The structure of the human sterol carrier protein
X/sterol carrier protein 2 gene (SCP2). Genomics. 1994;24:370–374.
61. Knoblauch H, Muller-Myhsok B, Busjahn A, Ben Avi L, Bahring S,
Baron H, Heath SC, Uhlmann R, Faulhaber HD, Shpitzen S, Aydin A,
Reshef A, Rosenthal M, Eliav O, Muhl A, Lowe A, Schurr D, Harats D,
Jeschke E, Friedlander Y, Schuster H, Luft FC, Leitersdorf E. A choles-
terol-lowering gene maps to chromosome 13q. Am J Hum Genet. 2000;
66:157–166.
62. Klos KL, Kardia SL, Ferrell RE, Turner ST, Boerwinkle E, Sing CF.
Genome-wide linkage analysis reveals evidence of multiple regions that
influence variation in plasma lipid and apolipoprotein levels associated
with risk of coronary heart disease. Arterioscler Thromb Vasc Biol.
2001;21:971–978.
63. Coon H, Leppert MF, Eckfeldt JH, Oberman A, Myers RH, Peacock JM,
Province MA, Hopkins PN, Heiss G. Genome-wide linkage analysis of
lipids in the Hypertension Genetic Epidemiology Network (HyperGEN)
Blood Pressure Study. Arterioscler Thromb Vasc Biol. 2001;21:
1969–1976.
576 Circulation Research March 21, 2003