Familial hypercholesterolemia (FH) is characterized
by increased levels of plasma LDL cholesterol that
leads to the formation of tendon xanthomas, acceler-
ated atherosclerosis, and premature coronary heart
disease. In most cases, FH is an autosomal dominant
disorder caused by mutations in the LDL receptor
gene that lead to defective clearance of plasma LDL.
There is a strong gene-dosage effect, and homozygous
FH patients exhibit a severe and highly characteristic
clinical phenotype (1).
We described previously two kindreds with a clinical
diagnosis of homozygous FH whose Epstein-Barr
virus–transformed lymphocytes (EBV-lymphocytes) in
culture showed defective LDL receptor–dependent
internalization of LDL, despite normal expression of
LDL receptor mRNA and protein (2). The disorder was
clearly inherited, but as an autosomal recessive, rather
than dominant, trait, suggesting that the probands
were homozygous for a defective gene whose product
is involved in internalization or trafficking of the LDL.
We mapped the defect in the two families to chromo-
some 1p36 (3), a region that has since been shown to
harbor a novel gene (ARH1), mutations in which coseg-
regate with autosomal recessive hypercholesterolemia
(ARH) in several families of different ethnic origin (4).
The gene encodes a putative adaptor protein that was
reported to bind to the cytoplasmic tail of the LDL
receptor (4). However, although earlier studies in vivo
showed that some of the patients with mutations in
this gene exhibit defective clearance of LDL from the
circulation, no defect in LDL receptor function has
been observed in their cultured skin fibroblasts (5).
We now show that our original patients, as well as
patients in a third kindred of English origin, are
homozygous for mutations in ARH1 that are all pre-
dicted to result in synthesis of truncated forms of the
protein. We have confirmed, by repairing the cellular
defect by retroviral expression of normal ARH1 cDNA,
that defective LDL receptor–dependent internalization
and degradation of LDL by EBV-lymphocytes from the
patients are caused by defects in ARH1.
The Journal of Clinical Investigation|December 2002|Volume 110|Number 11
Restoration of LDL receptor function in cells from patients
with autosomal recessive hypercholesterolemia
by retroviral expression of ARH1
Emily R. Eden,1Dilipkumar D. Patel,1Xi-Ming Sun,1Jemima J. Burden,1
Michael Themis,2Matthew Edwards,3Philip Lee,4Clare Neuwirth,1
Rossitza P. Naoumova,1and Anne K. Soutar1
1Medical Research Council, Clinical Sciences Centre, and
2Biomedical Sciences Division, Faculty of Medicine, Imperial College, London, United Kingdom
3Cytogenetics Unit, St. Mary’s Hospital, London, United Kingdom
4Great Ormond Street Hospital for Sick Children, London, United Kingdom
Familial hypercholesterolemia is an autosomal dominant disorder with a gene-dosage effect that is
usually caused by mutations in the LDL receptor gene that disrupt normal clearance of LDL. In the
homozygous form, it results in a distinctive clinical phenotype, characterized by inherited hyper-
cholesterolemia, cholesterol deposition in tendons, and severe premature coronary disease. We
described previously two families with autosomal recessive hypercholesterolemia that is not due to
mutations in the LDL receptor gene but is characterized by defective LDL receptor–dependent inter-
nalization and degradation of LDL by transformed lymphocytes from the patients. We mapped the
defective gene to chromosome 1p36 and now show that the disorder in these and a third English fam-
ily is due to novel mutations in ARH1, a newly identified gene encoding an adaptor-like protein. Cul-
tured skin fibroblasts from affected individuals exhibit normal LDL receptor activity, but their mono-
cyte-derived macrophages are similar to transformed lymphocytes, being unable to internalize and
degrade LDL. Retroviral expression of normal human ARH1restores LDL receptor internalization in
transformed lymphocytes from an affected individual, as demonstrated by uptake and degradation
of 125I-labeled LDL and confocal microscopy of cells labeled with anti–LDL-receptor Ab.
J. Clin. Invest. 110:1695–1702 (2002). doi:10.1172/JCI200216445.
Received for publication July 18, 2002, and accepted in revised form
October 22, 2002.
Address correspondence to: Anne K. Soutar, Hammersmith
Hospital, Ducane Road, London W12 ONN, United Kingdom.
Phone: 44-208-383-2324; Fax: 44-208-383-2077;
Conflict of interest: The authors have declared that no conflict of
Nonstandard abbreviations used: familial hypercholesterolemia
(FH); Epstein-Barr virus (EBV); autosomal recessive
hypercholesterolemia (ARH); lipoprotein-deficient serum
(LPDS); 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI);
1,1-dioctylacyl 3,3,3′,3′-tetramethylindocyanine perchlorate (DiI).
Subjects. The patients in families 1 and 2 have been
described in detail previously (2, 3). Two further
patients, brothers of English origin, first presented at
Great Ormond Street Hospital for Sick Children at
the ages of 9 and 7 years with a clinical diagnosis of
“pseudo” homozygous FH. DNA samples and cul-
tured skin fibroblasts were sent to us by J.V. Leonard
(Great Ormond Street Hospital for Sick Children,
London, United Kingdom) for investigation of possi-
ble mutations in the LDL receptor gene. All individu-
als gave informed consent.
Nucleotide sequencing of ARH1. Total RNA was isolated
from cultured EBV-lymphocytes (6). Overlapping frag-
ments of ARH1 cDNA were amplified by RT-PCR, and
their sequences (ABI3700) were compared with ARH1
cDNA (GenBank accession no. AL117654). Genomic
fragments comprising exons or pairs of exons were
amplified from genomic DNA isolated from whole
blood or cells (7), with primers located in the introns at
least 50 bp from the intron/exon junction. Primers for
exons 5–9 were designed from the sequence of ARH1 in
BAC clone AL606491; others were as published (8, 9) (for
all primer sequences and PCR conditions see supple-
mentary data at http://www.jci.org/cgi/content/full/
Genotyping. Polymorphic markers flanking ARH1
were selected from the Ensembl database and geno-
typed as described previously (3).
Fluorescent in situ hybridization. Metaphase spreads (10)
from EBV-lymphocytes of affected individual FH3.1
were hybridized with DIG-labeled probes to ARH (DIG-
Nick Translation Mix; Roche Diagnostics Ltd., Lewes,
United Kingdom) and a biotin-labeled probe to chro-
mosome 1 α-satellite (Qbiogene-Alexis Ltd. Notting-
ham, United Kingdom) (10). ARH probe 1 was an 11-kb
Eag1-Pac1 fragment of BAC clone AL031280 (Sanger
Centre, Hinxton, Cambridge, United Kingdom), and
ARH probe 2 comprised three PCR products amplified
from BAC clone 121-03 (ResGen Invitrogen Corp., Pais-
ley, United Kingdom), resulting in a 9.5-kb probe
encompassing exons 2–7 of ARH1 (see supplementary
data). ARH probes were detected with FITC anti-DIG
and α-satellite with Cy3 anti-biotin Ab (Sigma-Aldrich,
Poole, Dorset, United Kingdom) and viewed under an
Olympus BX40 microscope with the CytoVision system
(Applied Imaging International Ltd., Newcastle Upon
Tyne, United Kingdom).
Cell culture. Skin fibroblasts and EBV-lymphocytes
were maintained as described (6, 11). Mononuclear
cells were isolated from 20–30 ml of blood, seeded at
2.5 ×106cells per 4.5-cm-diameter well in 12-place mul-
tiwell dishes (Linbro; ICN Pharmaceuticals Ltd., Bas-
ingstoke, United Kingdom), incubated for 1.5 hours,
and washed to remove nonadherent lymphocytes (11).
Adherent monocytes were incubated for 7 days in
RPMI-1640 medium (GIBCO BRL; Life Technologies,
Paisley, United Kingdom) containing autologous
serum (20% vol/vol) or in serum-free medium
(Macrophage-SFM; GIBCO BRL; Life Technologies)
containing human recombinant GM-CSF (0.1 µg/ml;
Sigma-Aldrich). PA317 amphotropic retroviral pack-
aging cells (ECACC/89032007) were grown in DMEM
supplemented with GlutaMAX (GIBCO BRL; Life
Technologies), 4.5 g/l D-glucose, and 10% FCS.
For measurement of uptake or degradation of
labeled LDL, cells were preincubated for 16 hours in
medium containing 10% (vol/vol) lipoprotein-defi-
cient serum (LPDS). Degradation of 125I-labeled LDL
was determined as described (6, 11). Western blotting
of cell extracts to detect c-myc-ARH1 was as described
previously for the LDL receptor, with the exception
that the primary Ab was mouse monoclonal anti–
c-myc (Santa Cruz Biotechnology Inc., Santa Cruz,
California, USA) diluted 1:3,000 (2).
Measurement of ARH1 mRNA by real-time PCR. Cells
were preincubated in medium containing LPDS (10%
vol/vol) and compactin (0.1 µg/ml; Sigma-Aldrich) for
16 hours before isolation of total RNA with RNA-Bee
(Biogenesis Ltd., Poole, Dorset, United Kingdom).
ARH1 mRNA was assayed by real-time PCR using an
ABI PRISM Sequence Detection System (Applied
Biosystems, Warrington, United Kingdom) (for probes
and primers see supplementary data). Primers and
probes for GAPDH mRNA were included in each assay
as an internal standard. All assays were carried out in
triplicate, and all values were related to a standard
curve generated from control mRNA, combined from
two normal cell lines.
Retroviral expression of c-myc-ARH in EBV-transformed
B cells. ARH1 cDNA was amplified from plasmid
The Journal of Clinical Investigation| December 2002| Volume 110| Number 11
Pedigrees of family 1 (a) and family 2 (b). The plasma cholesterol con-
centration (mmol/l) is shown below each symbol. Filled symbols,
homozygous for the Q136X (a, family 1) or the delGG86,87(b, family
2) mutations in ARH1; half-filled symbols, heterozygous carriers of
the mutation, confirmed by sequencing amplified fragments of
genomic DNA. Individuals whose cells were analyzed for LDL recep-
tor activity or ARH1 mRNA levels are identified by a number in italics.
DKFZp586D0624 (Deutsches Ressourcenzentrum für
Genomforschung GmbH, Berlin, Germany) with
primers that introduced a c-myc tag at the amino-ter-
minus, and cloned into the HindIII/ClaI sites of the
LNCX retroviral vector (12). PA317 cells (5 × 104
cells/ml) were transfected with LNCX/c-myc-ARH1
using LipofectAMINE PLUS reagent (GIBCO BRL; Life
Technologies). Virus-containing medium was harvest-
ed 48 hours later, filtered (0.45 µm), and incubated
with 5 µg/ml DEAE dextran (Sigma-Aldrich) for 20
minutes and then with an equal volume of EBV-lym-
phocytes (1 ×106cells/ml) for 48 hours before selection
for neomycin resistance (0.6 mg/ml G418; GIBCO BRL;
Life Technologies). The cells were maintained in 40%
conditioned medium containing 15% FBS and 0.6
mg/ml G418. After 1 month, G418-resistant cells start-
ed to divide, while uninfected cells failed to survive.
Microscopy. EBV-lymphocytes were preincubated for
16 hours in medium containing 10% (vol/vol) LPDS
and compactin (0.1 µg/ml), and allowed to adhere to
poly-L-lysine–coated (Sigma-Aldrich) coverslips (Scien-
tific Laboratory Supplies Ltd., Nottingham, United
Kingdom). Cells were blocked with 1% (wt/vol) BSA in
PBS (wash buffer) and incubated at 4°C for 1 hour
with rabbit anti–LDL receptor Ab (diluted 1:200 in
PBS; Progen Biotechnik GmbH, Heidelberg, Germany).
To examine internalization of the LDL receptor, the
washed cells were incubated at 37°C for 10 minutes.
Cells were fixed in 4% (wt/vol) paraformaldehyde, per-
meabilized in PBS containing 0.1% Triton X-100 and
10 mM glycine, incubated sequentially for 1 hour at
ambient temperature with mouse monoclonal anti–α-
adaptin Ab (Santa Cruz Biotechnology Inc.; diluted
1/100), Alexa 568–conjugated goat anti-rabbit IgG
(highly cross-absorbed; Molecular Probes Europe BV,
Leiden, The Netherlands; diluted 1/100), and Alexa
488–conjugated goat anti-mouse IgG (highly cross-
absorbed; Molecular Probes, diluted 1/100), and then
mounted on slides with VECTASHIELD plus 4′,6-
diamidine-2′-phenylindole dihydrochloride (DAPI)
(Vector Laboratories, Peterborough, United Kingdom).
Cells were viewed on a Leica confocal microscope using
the ×100 oil objective, and the images were analyzed
using Leica confocal software (Leica Microsystems Ltd.,
Milton Keynes, United Kingdom).
Preparation of fluorescently-labeled LDL. Human LDL
was labeled with the fluorescent probe 1,1-dioctylacyl
3,3,3′,3′-tetramethylindocyanine perchlorate (DiI) as
described (13). Monocyte-derived macrophages on
coverslips were preincubated for 16 hours with medi-
um containing 10% LPDS and then for 3 hours at
37°C with DiI-LDL (7.5 µg/ml) in the presence or
absence of an excess of unlabeled LDL (1.0 mg/ml).
Cells were fixed and mounted as described above.
Results and Discussion
Clinical characteristics of the patients. Two of the families,
one of Turkish and the other of Asian Indian origin,
have been described in detail previously (2, 3); their
pedigrees are shown in Figure 1. Two further patients,
brothers of English origin, presented at the ages of 9
and 7 years with a clinical phenotype of “pseudo”
homozygous FH. There was no evidence of consan-
guinity (family 3, Figure 2). The elder brother (3.1) pre-
sented with xanthomata on his elbows and wrists.
These were confirmed by biopsy to contain cholesterol,
and at that time his total serum cholesterol was 14.2
mmol/l. The younger brother (3.2) was found to have
total cholesterol of 13.4 mmol/l but did not have any
xanthomata or other cutaneous lesions. The past med-
ical history of patient 3.1 is unremarkable. In 1999 he
had an exercise electrocardiogram, which was normal.
The echocardiography performed at that time showed
that, apart from having a tricuspid aortic valve and a
mild central jet of aortic regurgitation, he had no evi-
dence of aortic stenosis. Both brothers are completely
asymptomatic from the cardiovascular point of view.
The Journal of Clinical Investigation| December 2002|Volume 110|Number 11
Pedigree and haplotype analysis of family 3. The plasma cholesterol
(chol.) concentration (mmol/l) is shown below each symbol. Filled
symbols, homozygous for the insC mutation in ARH1; half-filled sym-
bols, heterozygous carriers of the mutation, confirmed by sequenc-
ing amplified fragments of genomic DNA. Haplotypes for the ARH1
locus are shown for polymorphic markers flanking the gene, known
polymorphisms (8), and the two novel base substitutions within the
coding region of ARH1; the position of each marker on Ensembl is
shown (Mb). A recombination in the paternal uncle of the proband
is indicated (X). The maternal allele carrying the insC620mutation is
boxed; the paternal allele carrying the putative deletion is boxed and
shaded. The haplotype in the father, shown in italics, was deduced.
Individuals whose cells were analyzed for LDL receptor activity or
ARH1 mRNA levels are identified by a number in italics.
They respond well to treatment with statins. We had
failed previously to detect any mutations in the LDL
receptor or apoB genes of these two brothers (unpub-
lished data), but this family was not included in our
earlier gene-mapping study (3), because it was unlikely
that they were homozygous for any mutant allele.
Characterization of point mutations in ARH1. The
sequence of ARH1 cDNA, amplified from EBV-lym-
phocyte mRNA, was determined. The proband of Turk-
ish origin (individual 1.1, Figure 1a), her affected sib-
ling (1.2), and her affected cousin (1.3) were found to
be homozygous for a single base substitution of C427to
T (nucleotide numbering based on GenBank accession
no. AL117645). This mutation has previously been
described in a Lebanese proband and is predicted to
introduce a premature termination codon (Q136X) in
exon 4 (4). Further investigation revealed that
antecedents in the Turkish family were Lebanese, and
thus it is likely that this allele was inherited from a
common ancestor. Sequencing of genomic DNA con-
firmed that the three affected individuals were
homozygous for the mutation and revealed that both
parents and several other apparently unaffected sib-
lings were heterozygous carriers (Figure 1a), as expect-
ed from their previously determined haplotypes (3).
The two affected siblings of Asian Indian origin (2.1
and 2.2, Figure 1b) were found to be homozygous for a
2-bp deletion of GG in a run of seven consecutive G
nucleotides (bp 86–92) in exon 1. The resultant
frameshift is predicted to introduce a premature ter-
mination codon eight residues after Gly23. This muta-
tion has not been described previously, but two others
have been identified within this short section (4). Both
parents were heterozygous for the mutation, as were
two apparently unaffected siblings in the family (Fig-
ure 1b), in agreement with previous genotyping (3).
The two affected English siblings (3.1 and 3.2, Figure
2) were both found to be homozygous in ARH1 mRNA
for two single base substitutions. The first was an inser-
tion of C in a run of eight consecutive C residues (bp
620–627) in exon 6 that results in a frameshift and is
predicted to give rise to a premature termination codon
17 residues after Pro202. The second was substitution
of C733 with T in exon 7 and is predicted to change
residue Arg238 to Trp. When genomic DNA was
sequenced, both affected siblings were homozygous for
both variants. This was somewhat surprising, because
there was no suggestion of consanguinity in the fami-
ly. Their unaffected sister carried neither mutation and
their mother was heterozygous for both, confirming
that both mutations lay on the same allele. The father
of the affected boys was deceased, but neither of the
paternal grandparents carried the mutant allele (Figure
2) and thus the origin of the second mutant allele in
the probands was not apparent.
One possible explanation was that the second allele
carried a deletion of the ARH1 gene encompassing at
least exons 6 and 7. However, Southern blotting of
genomic DNA digested with several enzymes and
hybridized with cDNA probes representing the entire
transcript did not reveal any additional bands in the
probands (data not shown). This suggested that any
deletion must encompass the majority of the ARH1
gene, a view supported by haplotype analysis (Figure 2).
The pattern of inheritance of polymorphic markers
flanking and within ARH1 implied that the probands
inherited an allele with a deletion at this locus between
D1S2674 and D1S2639 from their paternal grand-
mother (Figure 2), and this was demonstrated by fluo-
rescent in situ hybridization (FISH). When metaphase
chromosomes from affected individual 3.1 were
hybridized with a probe extending from exon 5 to 7 kb
beyond the 3′ end of ARH1 (Figure 3a, probe 1), both
copies of chromosome 1, as visualized with a chromo-
some 1 α-satellite probe, were labeled (Figure 3b, probe
1). However, when hybridized with a probe comprising
The Journal of Clinical Investigation| December 2002| Volume 110| Number 11
Determination of ARH1 mRNA levels by quantitative PCR
Cells LDLR mRNA
1.00 ± 0.102
0.99 ± 0.101
0.90 ± 0.123
0.79 ± 0.098
1.02 ± 0.194
1.05 ± 0.161
1.15 ± 0.099
0.48 ± 0.043B
0.85 ± 0.100
0.93 ± 0.122
0.05 ± 0.009B
0.13 ± 0.004B
Unaffected sibling 1.5
AARH1 and LDLR mRNA levels were expressed relative to GAPDH mRNA
assayed in the same tube; threshold cycle values were related to a standard
curve derived from assays of pooled mRNA from normal cells. Values shown
(± SE) are the mean of triplicate assays of three different preparations of RNA
from each cell line. Significance of differences between samples and control
was determined by unpaired Student’s t test. BP < 0.005. LDLR, LDL receptor.
Identification of a partial deletion of ARH1 in family 3. (a) Diagram
showing the approximate size of the ARH1 DNA probes used for
fluorescent in situ hybridization (FISH). The vertical black bars rep-
resent the nine exons of ARH1. (b) Metaphase chromosomes from
individual FH3.1 were hybridized with either DIG-labeled ARH1
DNA probe 1 or probe 2 as indicated (detected with FITC-labeled
anti-DIG Ab; small filled arrows) and biotin-labeled chromosome
1–specific α-satellite probe DNA (detected with Cy3-labeled anti-
biotin Ab; large open arrows).
exons 2–7 of ARH1 (Figure 3a, probe 2), only one copy
of chromosome 1 was labeled (Figure 3b, probe 2).
Thus, as implied by the sequence data (Figure 2), the
deletion includes at least exons 2–7 but does not extend
far downstream beyond exon 7.
Expression of ARH1. The amount of ARH1 mRNA in
EBV-lymphocytes was measured by real-time RT-PCR
(Table 1). In EBV-lymphocytes from individual 2.1
(ARH1 delGG86,87), ARH1 mRNA was not significant-
ly reduced compared with normal cells, but in cells
from individual 1.1 (ARH1 Q136X), there was an
approximately 50% reduction. In cells from individu-
als 3.1 and 3.2 (ARH1 insC620), there was a 90% reduc-
tion in ARH1 mRNA. Even when the absence of
mRNA due to the deletion of one allele is taken into
account, the insC620mutation has the most marked
effect on cellular ARH1 mRNA, reducing it to one-
fifth of the level expected from that allele. The differ-
ent effects of the three mutations in ARH1 on mRNA
levels is a little surprising; the mutations might be
expected to induce nonsense-mediated decay to a sim-
ilar extent, since all three introduce a premature ter-
mination codon in the mRNA 5′to the 50 nucleotides
that precede the final exon/intron boundary (14).
Despite near-normal levels of mRNA in cells from the
probands in family 2, there is no doubt that ARH1
protein is nonfunctional in all three families, because
the mutations are predicted to result in the produc-
tion of a severely truncated protein. ARH1 mRNA lev-
els, expressed relative to GAPDH mRNA, were identi-
cal in EBV-lymphocytes and skin fibroblasts from
subject 3.1 (data not shown).
LDL receptor function in ARH1-negative cells. We have
previously shown that EBV-lymphocytes from the
probands in families 1 and 2 are unable to take up and
degrade LDL (2). We have recently obtained an explant
from the proband in family 1 (1.1) and now show that
cultured skin fibroblasts from this subject exhibit the
same saturable, high-affinity uptake and degradation
of LDL as do fibroblasts from a normolipemic individ-
ual (Figure 4a). Others have also reported the absence
of any defect in LDL receptor function in skin fibrob-
lasts from patients homozygous for mutations in
ARH1(4, 8). EBV-lymphocytes from a heterozygous sib-
ling of the proband (individual 1.4 in Figure 1) and
from another sibling who does not carry the mutant
allele (1.5) degrade LDL normally (Figure 4b). This
finding is supported by the observation that the het-
erozygous carriers of a mutant allele of ARH1 in the
three families have no obvious clinical phenotype. The
same pattern was observed with cells from individual
3.1 in family 3, in that cultured skin fibroblasts were
able to degrade LDL normally (Figure 5b), while no
degradation occurred in EBV-lymphocytes from this
proband or his affected brother (Figure 5a).
To determine whether the cellular phenotype in EBV-
lymphocytes was a consequence of their transforma-
tion with EBV, we also determined the ability of mono-
cyte-derived macrophages from ARH1-defective
individuals to degrade LDL. Blood monocytes were iso-
lated and maintained in culture for 7 days, during
which time they developed into macrophages (15).
Even after upregulation of LDL receptor expression,
monocyte-derived macrophages from the proband in
family 3 (3.1) were totally unable to degrade 125I-labeled
The Journal of Clinical Investigation| December 2002| Volume 110| Number 11
Degradation of 125I-labeled LDL by cultured skin fibroblasts and
EBV- lymphocytes from individuals in family 1. Cells were preincu-
bated for 16 hours in medium containing LPDS and then for 4
hours with 125I-labeled LDL. Saturable degradation of LDL was
determined as the difference in the amount of trichloroacetic
acid–soluble (TCA-soluble), non-iodide radioactivity in the medi-
um of cells incubated in the presence and absence of an excess of
unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes.
Nonsaturable degradation of LDL by normal cells was always less
than 5% of the total. Data shown are representative of at least two
separate experiments. (a) Cultured skin fibroblasts from the
proband in family 1 (ARH–/–), and from a normolipemic control
(ARH+/+). (b) EBV-lymphocytes from the proband in family 1
(ARH–/–), her heterozygous sibling (ARH+/–), and a sibling who does
not carry the mutant allele (ARH+/+).
Degradation of 125I-labeled LDL by skin fibroblasts, EBV-lympho-
cytes, and monocyte-derived macrophages from individuals in fam-
ily 3. Cells were preincubated for 16 hours in medium containing
LPDS and then for 4 hours with 125I-labeled LDL. Saturable degra-
dation of LDL was determined as the difference in the amount of
TCA-soluble, non-iodide radioactivity in the medium of cells incu-
bated in the presence and absence of an excess of unlabeled LDL (1
mg/ml); values are the mean of duplicate dishes. Nonsaturable
degradation of LDL by normal cells was always less than 5% of the
total. Data shown are representative of at least two separate exper-
iments. (a) EBV-lymphocytes, (b) skin fibroblasts, and (c) mono-
cyte-derived macrophages, from probands 3.1 (filled triangles) and
3.2 (open triangles) in family 3 and from unrelated normolipemic
controls (filled circles).
LDL (Figure 5c), confirming that internalization of the
LDL receptor was defective in these cells. Somewhat
surprisingly, monocyte-derived macrophages from his
affected brother, 3.2, who was homozygous for the
same mutation(s) in ARH1, showed saturable, high-
affinity degradation of LDL that was approximately
20% of that exhibited by normal cells. Identical results
were obtained with two different preparations of cells
from both individuals.
In preliminary investigations, microscopy of mono-
cyte-macrophages incubated with fluorescently-
labeled LDL suggested that the low level of degrada-
tion of 125I-labeled LDL observed in individual 3.2 was
due to normal uptake by a few of the cells in the cul-
ture, and not to a low level of uptake by all cells (data
not shown). There were no obvious morphological dif-
ferences between cells that were able to take up LDL
and those that were not, but further studies to investi-
gate different cell types in the culture were hampered
by strictly limited availability of the cells. We conclude
that a proportion of the cells in the monocyte-
macrophage cultures from individual 3.2 shared a phe-
notype with fibroblasts, in that LDL receptor function
was independent of ARH1 function. The underlying
reason for this is currently unknown.
Expression of exogenous ARH1 in mutant EBV-lympho-
cytes. A retroviral vector containing a cDNA for
human ARH1 tagged with c-myc at its amino-termi-
nus was transfected into PA317 cells. The transfected
viral-packaging cells produced a myc-tagged protein
of approximately 37 kDa (Figure 6, lane 2), the expect-
ed size for c-myc-ARH1 (lane 1), that was absent in
nontransfected cells (lane 3). EBV-lymphocytes from
proband 1.1 (ARH1 hmz Q136X) were infected with
medium from the PA317 producer cells, and cells
expressing viral genes were selected for neomycin
resistance. These cells also expressed a myc-tagged
protein of the expected size (lane 4) that was absent
from nontransfected cells (lane 5), although the level
of expression declined with prolonged time in culture
(lane 7). Maximum expression was restored by incu-
bation of the infected cells for 16 hours with 0.3 µM
trichostatin A, a histone deacetylase inhibitor (16)
(lane 8), showing that the viral construct had not been
lost, but unfortunately this treatment reduced LDL
receptor protein levels (lanes 9 and 10) and was not
useful for future experiments.
Unlike the mutant cells, infected cells were able to
take up and degrade 125I-labeled LDL (Figure 6c). This
showed that LDL receptor activity in the internaliza-
tion-defective cells could be restored in the mutant cells
by expression of c-myc-ARH1 and demonstrated that a
defect in this gene is responsible for the phenotype in
the patients. The apparent affinity of the LDL receptor
for labeled LDL was lower in the mutant cells express-
ing c-myc-ARH1 than that in control cells, which may
be due to a rate-limiting level of expression of exoge-
nous ARH1 or to the c-myc tag interfering with normal
function to some extent. We are unable to distinguish
between these possibilities at present.
The subcellular localization of the LDL receptor in
EBV-lymphocytes in which LDL receptor expression
was upregulated was examined by confocal micro-
scopy. No LDL receptor staining was observed with
cells that had not been preincubated with LPDS or
with cells incubated with second Ab alone (data not
The Journal of Clinical Investigation| December 2002| Volume 110| Number 11
Effect of expression of c-myc-ARH1 on LDL receptor activity in mutant
EBV-lymphocytes. (aand b) Cells were preincubated for 16 hours with
LPDS before preparation of cell extracts. Proteins were fractionated
on nonreduced SDS-polyacrylamide gels (13%), transferred to nylon
membranes, and immunoblotted with anti–c-myc (lanes 1–8) or
anti–LDL receptor Ab (lanes 9 and 10). Bound Ab was detected with
peroxidase-conjugated anti-mouse IgG and chemiluminescence. (a)
Whole-cell extracts (approximately 50 µg of protein per lane) of Chi-
nese hamster ovary (CHO) cells transiently transfected with pcDNA3-
c-myc-ARH1PA317 (lane 1), PA317 cells transfected with ARH1 retro-
viral construct (lane 2), PA317 cells (lane 3), EBV-lymphocytes from
affected individual 1.1 (ARH1.1 cells) 1 month after infection with
c-myc-ARH1 retrovirus (lane 4), and uninfected EBV-lymphocytes
from affected individual 1.1 (lane 5). (b) Whole-cell extracts (approx-
imately 50 µg of protein per lane) of EBV-lymphocytes from affected
individual 1.1 (lane 6), the same cells 3 months after stable infection
with c-myc-ARH1 retrovirus (lanes 7 and 9), and the same infected
cells after preincubation for 16 hours with 0.3 µM trichostatin A (lane
8 and 10). (c) Virus-infected (ARH–/c-myc-ARH) and uninfected
(ARH–) EBV-lymphocytes from individual 1.1 were preincubated for
16 hours in medium containing LPDS and then for 4 hours with 125I-
labeled LDL. Saturable degradation of LDL was determined as the dif-
ference in the amount of TCA-soluble, non-iodide radioactivity in the
medium of cells incubated in the presence and absence of an excess
of unlabeled LDL (1 mg/ml); values are the mean of duplicate dishes.
Data shown are representative of two separate experiments.
shown). In normal and mutant EBV-lymphocytes
incubated with anti–LDL receptor Ab at 4°C, LDL
receptor protein (red) was only visible on the cell sur-
face (Figure 7, a and b). In LDL receptor Ab–labeled
cells incubated at 37°C, LDL receptor protein was vis-
ible as a punctate pattern inside control cells (Figure
7d) but remained on the surface of ARH1-negative
cells, presumably because LDL receptor internaliza-
tion is defective (Figure 7e). In ARH1-negative cells
expressing c-myc-ARH1, LDL receptor protein was vis-
ible on the surface of cells incubated at 4°C (Figure
7c), and as a punctate pattern similar to that in normal
cells after incubation at 37°C (Figure 7f), showing that
the LDL receptor had been internalized.
LDL receptor–mediated endocytosis is thought to
occur via clathrin-coated pits, of which the adaptor
complex AP2 is a component (17). However, in normal
or ARH1-negative EBV-lymphocytes, α-adaptin–
stained AP2 was visible as punctate staining on the
inner cell membrane (green, Figure 7, a–f) but did not
colocalize with the LDL receptor (no yellow visible).
Similar results were obtained with cells from three dif-
ferent individuals (Figure 7, g–i); very little of the LDL
receptor colocalized with AP2 at the cell membrane
when viewed either in cross section (Figure 7, g and h)
or at the surface (Figure 7i). In marked contrast, in
three normal skin fibroblast cell lines, the majority of
the LDL receptor colocalized with AP2 (Figure 7, j–l).
The surprisingly small degree of colocalization between
the LDL receptor and AP2 observed in lymphocytes is
consistent with previous observations in hepatocytes
from transgenic mice expressing the human LDL
receptor (18), suggesting that EBV-lymphocytes might
present a better model for hepatic LDL receptor–medi-
ated endocytosis than do skin fibroblasts.
ARH resembles a putative adaptor protein, since it
contains a phosphotyrosine-binding (PTB) domain
similar to that found, for example, in the Drosophila
numb protein (4). In many adaptor proteins, the PTB
domain binds to a phosphorylated NPXpY motif
(where X is any amino acid and pY is phosphotyro-
sine), but in some, such as Disabled-1 and Disabled-2,
it binds with high affinity to a nonphosphorylated
NPXY sequence (19). The PTB domain of ARH1 has
recently been shown to interact in vitro with the NPVY
internalization sequence in the cytoplasmic tail of the
LDL receptor (20). It was not possible to examine
interaction of ARH1 with the LDL receptor in EBV-
lymphocytes expressing c-myc-ARH1, because retrovi-
ral expression of c-myc ARH1 declined to almost unde-
tectable levels in the cells. Although preincubation of
the cells with trichostatin A increased expression of
c-myc-ARH1, this resulted in very low levels of LDL
receptor protein. As a result, the two proteins could
not both be detected in any one cell by confocal
microscopy (data not shown).
In this study we have shown that the defect in LDL
receptor internalization that we have observed previ-
ously in EBV-lymphocytes from patients with autoso-
mal recessive hypercholesterolemia is due to defects in
ARH1 and can be corrected in these cells by retroviral
expression of normal ARH1. We have further shown
that ARH1 is required for LDL receptor function in
normal macrophages, but not in skin fibroblasts from
the same individuals. Our observation that the LDL
receptor colocalizes with AP2 at 4°C in fibroblasts, but
not in EBV-lymphocytes, suggests a possible mecha-
nism for the difference in LDL receptor phenotype
between EBV-lymphocytes and fibroblasts lacking
ARH1 and clearly warrants further investigation.
The Journal of Clinical Investigation| December 2002| Volume 110| Number 11
Confocal microscopy of cells labeled with anti–LDL receptor Ab.
(a–f) EBV-lymphocytes were incubated with rabbit anti–LDL recep-
tor (red) at 4°C, washed at 4°C, and either directly permeabilized
(a–c) or incubated for 10 minutes at 37°C before permeabiliza-
tion to allow internalization of LDL receptor/Ab complexes (d–f).
Permeabilized cells were then incubated with mouse anti–α-
adaptin (AP2), washed, and incubated with Alexa 568–conjugat-
ed goat anti-rabbit IgG (LDL receptors, red) and Alexa 488–con-
jugated goat anti-mouse IgG (AP2, green). Nuclei were stained
with DAPI. The plates shown are an overlay of red and green
images. The bar represents 5.0 µm. (a and d) Control cells; (b and
e) cells from proband 1.1; (c and f) cells from proband 1.1 express-
ing viral c-myc-ARH. (g–l) EBV-lymphocytes (g–i) or cultured skin
fibroblasts (j–l) from three different control subjects were incu-
bated with anti–LDL receptor Ab at 4°C, permeabilized, and then
incubated with anti–α-adaptin Ab (AP2) as described for a–f
above. The bars represent 5.0 µm (in g for g–i, and in j for j–l).
Acknowledgments Download full-text
We are very grateful to the members of the families for
their willing cooperation in these studies and to J.V.
Leonard for providing samples from family 3. We are
indebted to Nina Krausewicz (Medical Research Coun-
cil, Clinical Sciences Centre) for suggesting the use of tri-
chostatin A. Bruce Pottinger provided excellent help
with tissue culture. Simon Gregory (Sanger Centre,
Hinxton, Cambridge) kindly provided BAC clone
AL031280. This work was supported in part by a project
grant from the British Heart Foundation (PG 98062).
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