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
); their pedigrees are shown in Figure . 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 consanguinity (family 3, Figure ). The elder brother (3.1) presented 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 medical 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 evidence of aortic stenosis. Both brothers are completely asymptomatic from the cardiovascular point of view. 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 We had failed previously to detect any mutations in the LDL receptor or apoB genes of these two brothers (unpublished data), but this family was not included in our earlier gene-mapping study (3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.
Figure 1 Pedigrees of family 1 (a) and family 2 (b). The plasma cholesterol concentration (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 (more ...)
Figure 2 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 symbols, heterozygous carriers of the mutation, confirmed (more ...) Characterization of point mutations in ARH1.
The sequence of ARH1 cDNA, amplified from EBV-lymphocyte mRNA, was determined. The proband of Turkish origin (individual 1.1, Figure a), her affected sibling (1.2), and her affected cousin (1.3) were found to be homozygous for a single base substitution of C427
to 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 confirmed that the three affected individuals were homozygous for the mutation and revealed that both parents and several other apparently unaffected siblings were heterozygous carriers (Figure a), as expected from their previously determined haplotypes (3
The two affected siblings of Asian Indian origin (2.1 and 2.2, Figure b) 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 termination codon eight residues after Gly23. This mutation 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 (Figure b), in agreement with previous genotyping (3
The two affected English siblings (3.1 and 3.2, Figure ) were both found to be homozygous in ARH1 mRNA for two single base substitutions. The first was an insertion 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 family. 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 ) 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 ). 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 grandmother (Figure ), and this was demonstrated by fluorescent 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 a, probe 1), both copies of chromosome 1, as visualized with a chromosome 1 α-satellite probe, were labeled (Figure b, probe 1). However, when hybridized with a probe comprising exons 2–7 of ARH1 (Figure a, probe 2), only one copy of chromosome 1 was labeled (Figure b, probe 2). Thus, as implied by the sequence data (Figure ), the deletion includes at least exons 2–7 but does not extend far downstream beyond exon 7.
Figure 3 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 represent the nine exons of ARH1. (b) Metaphase chromosomes (more ...) Expression of ARH1.
The amount of ARH1 mRNA in EBV-lymphocytes was measured by real-time RT-PCR (Table ). In EBV-lymphocytes from individual 2.1 (ARH1
), ARH1 mRNA was not significantly reduced compared with normal cells, but in cells from individual 1.1 (ARH1
Q136X), there was an approximately 50% reduction. In cells from individuals 3.1 and 3.2 (ARH1
), there was a 90% reduction in ARH1 mRNA. Even when the absence of mRNA due to the deletion of one allele is taken into account, the insC620
mutation has the most marked effect on cellular ARH1 mRNA, reducing it to one-fifth of the level expected from that allele. The different 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 similar extent, since all three introduce a premature termination 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 production of a severely truncated protein. ARH1 mRNA levels, expressed relative to GAPDH mRNA, were identical in EBV-lymphocytes and skin fibroblasts from subject 3.1 (data not shown).
Determination of ARH1 mRNA levels by quantitative PCR
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 individual (Figure a). Others have also reported the absence of any defect in LDL receptor function in skin fibroblasts from patients homozygous for mutations in ARH1
). EBV-lymphocytes from a heterozygous sibling of the proband (individual 1.4 in Figure ) and from another sibling who does not carry the mutant allele (1.5) degrade LDL normally (Figure b). This finding is supported by the observation that the heterozygous 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 b), while no degradation occurred in EBV-lymphocytes from this proband or his affected brother (Figure a).
Figure 4 Degradation of 125I-labeled LDL by cultured skin fibroblasts and EBV- lymphocytes from individuals in family 1. Cells were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125I-labeled LDL. Saturable degradation of LDL was (more ...)
Figure 5 Degradation of 125I-labeled LDL by skin fibroblasts, EBV-lymphocytes, and monocyte-derived macrophages from individuals in family 3. Cells were preincubated for 16 hours in medium containing LPDS and then for 4 hours with 125I-labeled LDL. Saturable degradation (more ...)
To determine whether the cellular phenotype in EBV-lymphocytes was a consequence of their transformation with EBV, we also determined the ability of monocyte-derived macrophages from ARH1
-defective individuals to degrade LDL. Blood monocytes were isolated 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 125
I-labeled LDL (Figure c), 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 monocyte-macrophages incubated with fluorescently-labeled LDL suggested that the low level of degradation of 125I-labeled LDL observed in individual 3.2 was due to normal uptake by a few of the cells in the culture, and not to a low level of uptake by all cells (data not shown). There were no obvious morphological differences between cells that were able to take up LDL and those that were not, but further studies to investigate 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 phenotype 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-lymphocytes.
A retroviral vector containing a cDNA for human ARH1 tagged with c-myc at its amino-terminus was transfected into PA317 cells. The transfected viral-packaging cells produced a myc-tagged protein of approximately 37 kDa (Figure , lane 2), the expected 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 incubation 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.
Figure 6 Effect of expression of c-myc-ARH1 on LDL receptor activity in mutant EBV-lymphocytes. (a and b) Cells were preincubated for 16 hours with LPDS before preparation of cell extracts. Proteins were fractionated on nonreduced SDS-polyacrylamide gels (13%), (more ...)
Unlike the mutant cells, infected cells were able to take up and degrade 125I-labeled LDL (Figure c). This showed that LDL receptor activity in the internalization-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 expressing c-myc-ARH1 than that in control cells, which may be due to a rate-limiting level of expression of exogenous 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 microscopy. 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 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 surface (Figure , a and b). In LDL receptor Ab–labeled cells incubated at 37°C, LDL receptor protein was visible as a punctate pattern inside control cells (Figure d) but remained on the surface of ARH1-negative cells, presumably because LDL receptor internalization is defective (Figure e). In ARH1-negative cells expressing c-myc-ARH1, LDL receptor protein was visible on the surface of cells incubated at 4°C (Figure c), and as a punctate pattern similar to that in normal cells after incubation at 37°C (Figure f), showing that the LDL receptor had been internalized.
Figure 7 Confocal microscopy of cells labeled with anti–LDL receptor Ab. (a–f) EBV-lymphocytes were incubated with rabbit anti–LDL receptor (red) at 4°C, washed at 4°C, and either directly permeabilized (a–c) or (more ...)
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 , a–f) but did not colocalize with the LDL receptor (no yellow visible). Similar results were obtained with cells from three different individuals (Figure , g–i); very little of the LDL receptor colocalized with AP2 at the cell membrane when viewed either in cross section (Figure , g and h) or at the surface (Figure i). In marked contrast, in three normal skin fibroblast cell lines, the majority of the LDL receptor colocalized with AP2 (Figure , 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–mediated 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 phosphotyrosine), 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 retroviral expression of c-myc ARH1 declined to almost undetectable 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 previously in EBV-lymphocytes from patients with autosomal 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 mechanism for the difference in LDL receptor phenotype between EBV-lymphocytes and fibroblasts lacking ARH1 and clearly warrants further investigation.