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Clinical Features and Genetic Analysis of Autosomal Recessive Hypercholesterolemia

Departments of Bioscience (M.H.-S.), Pharmacology (A.T.), and Etiology and Pathophysiology (Y.I., A.Y.), National Cardiovascular Center Research Institute, and Department of Medicine (Y.M., M.T.), National Cardiovascular Center, Osaka 565-8565; and Department of Biochemistry, Cell Biology, and Metabolism (S.Y.), Nagoya City University Graduate School of Medical Sciences, Aichi 467-8601, Japan

Address all correspondence and requests for reprints to: Dr. Mariko Harada-Shiba, Department of Bioscience, National Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Osaka 565-8565, Japan. E-mail: mshiba{at}ri.ncvc.go.jp.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Previously we have reported on siblings with severe hypercholesterolemia, xanthomas, and premature atherosclerosis without any impairment of low-density lipoprotein receptor in their fibroblasts as a first characterization of autosomal recessive hypercholesterolemia (ARH). Recently, mutations were identified for this disease in a gene encoding a putative adaptor protein. The purpose of this study was to examine the molecular pathogenesis of ARH in Japanese siblings. A novel insertion mutation was discovered in the ARH gene of the siblings. An insertion of an extra cytosine residue was identified in a locus comprising eight consecutive cytosines at positions 599 through 606 in exon 6, resulting in a sequence of nine cytosines and generating an early stop codon at 657–659. The mother was heterozygous for this mutation. Neither transcription product nor protein of ARH was detected in the fibroblasts of the homozygous patients. A single nucleotide polymorphism was discovered among the normal control subjects at position 604 (cytosine to thymine: ARH-604C to ARH-604T), which changes the proline residue at 202 to serine. Interestingly, ARH is caused by a mutation of cytosine to adenine at this same position. Both siblings exhibited fatty liver, which may also be related to this mutation.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
IN 1973 KHACHADURIAN and Kuthman (1) first described two categories of hereditary hypercholesterolemia, autosomal dominant and recessive. A few years later, extensive studies on hypercholesterolemia with autosomal dominant inheritance led to the discovery of the low-density lipoprotein (LDL) receptor. A genetic defect in this receptor protein characterizes familial hypercholesterolemia (FH). Patients with homozygous FH show severe elevation of plasma LDL, cutaneous and tendon xanthomas, and atherosclerotic vascular lesions during the first decade of life (2). On the other hand, severe hypercholesterolemia with autosomal recessive inheritance exhibits almost the same clinical features as those of FH homozygotes but is apparently rare and had never been characterized until our first reports of this disease (3, 4). We described the siblings as having severe elevation of plasma LDL levels despite normal LDL receptor activity in their fibroblasts as the first characterization of severe hypercholesterolemia with autosomal recessive inheritance. The binding, incorporation, and degradation of ¹²⁵I-LDL by their fibroblasts were normal (3). Their LDL also bound, internalized, and degraded normally in their fibroblasts (3). The pulse-chase pattern for the LDL receptor protein in their fibroblasts was normal, suggesting that their LDL receptor protein was synthesized and processed normally (3). Their LDL receptor genes were shown to have different haplotypes, excluding the possibility that the disorder is caused by homozygosity of this gene. The fractional catabolic rates of plasma cholesterol were calculated from the rebound increase curve after LDL apheresis and were found to be severely impaired in both patients. This indicates a similarity between homozygous FH disorder and autosomal recessive hypercholesterolemia (ARH) with respect to the LDL metabolism in plasma (4).

Several authors subsequently reported severe hypercholesterolemia with autosomal recessive inheritance, and this disease was named autosomal recessive hypercholesterolemia (5, 6, 7). Using isotope-labeled LDL in vivo, ARH was shown to have selective reduction in hepatic LDL uptake (6, 7).

Recently, Garcia et al. (8) mapped the ARH locus to chromosome 1p35 and identified six mutations in a gene encoding a putative LDL receptor adaptor protein in the families with ARH. ARH protein contains a phosphotyrosine-binding domain, which in other cells binds NPXY motifs in the cytoplasmic tails of cell-surface receptors. This suggests that ARH has a tissue-specific role in LDL receptor function because it is required in liver but not in fibroblasts. However, the underlying mechanism linking gene mutations and impairment of LDL metabolism still remains unclear. Thus, it is very important to identify the nature of the mutations for the patients in whom the clinical characterization has been established to investigate a role of this new gene product in LDL metabolism. For this reason we undertook the analysis of ARH for the Japanese family with ARH. We report a novel insertion mutation in exon 6 of the LDL receptor adaptor protein gene in these homozygous siblings with ARH. We describe the detail clinical features of these patients as well as a new single nucleotide polymorphism occurring at the same locus in healthy volunteers.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Characteristics of the patients

The proband is a 48-yr-old Japanese male who first noticed xanthomas in his elbows and knees at age 9 or 10 yr. He visited the National Cardiovascular Center Hospital for the first time when he was 30 yr old in 1982. Large cutaneous and tendinous xanthomas were identified in his fingers, elbows, and knees (Fig. 1, A and B). The thickness of his Achilles tendons was 23 mm in the right and 28 mm in the left. Before he began medical treatment, he was diagnosed as having fatty liver by abdominal computed tomography and ultrasonogram. Alcohol consumption was examined by a questionnaire. The kind of alcohol beverage, quantity per day, and frequency per week were asked. He was a light drinker (22 g alcohol/d). He has been treated with LDL-apheresis once every other week since 1983. In 1987 at the age of 35 yr, the patient developed coronary symptoms, and significant stenoses were found in the coronary artery lesions by angiography. Percutaneous transluminal coronary angioplasty was performed five times, and he has not experienced any cardiac symptom since then.

View larger version (89K): [in this window] [in a new window]   Figure 1. Multiple extensive xanthomas in the proband’s hand (A) and foot (B). C, Microscopic findings of the patient’s (sister of the proband) liver. The specimen was stained by hematoxylin and eosin. D, The specimen was stained by Sudan III.

The siblings were born to consanguineous parents of an uncle and a niece (4). Their father died of stroke at the age of 61 yr, never having any signs of hypercholesterolemia or xanthomas. Their mother is healthy with normal plasma cholesterol level (200 mg/dl) and has no xanthomas. No known relative has a record of coronary artery disease. The major clinical parameters are listed in Table 1.

Fibroblasts were established as cell lines from skin biopsy specimens of the proband, the sister of the proband, their mother, and a normal control subject. Stock cultures were maintained as monolayers in a humidified incubator (5% CO₂) at 37 C in minimum essential culture medium (S-MEM) (Life Technologies, Inc., Rockville, MD) containing penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% (vol/vol) fetal calf serum (FCS) (Hyclone Laboratories, Inc. AB, Lund, Sweden).

Northern blot analysis

Total cellular RNA was isolated from fibroblasts using the acid guanidium thiocyanate-phenol-chloroform method, described by Chomczynski and Sacchi (9). Total RNA (10 µg) was electrophoresed in 0.9% agarose gels containing 2.2 M formaldehyde and then transferred onto nylon membranes (Biodyne Nylon Membranes, Pall BioSupport, East Hills, NY). The membrane was hybridized at 68 C for 3 h, with the following probes labeled with [³²P]dCTP (Amersham Biosciences, Buckinghamshire, UK) using the multipriming method (10). The human ARH cDNA was purchased from RZPD (Berlin, Germany) as DKFZp586D0624 (GenBank accession no. AL117654), and the human LDL receptor cDNA was a kind gift from Dr. D. W. Russell (University of Texas). Full-length cDNAs were used as probes. The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). The membrane was washed and exposed to an imaging plate (Fuji Photo Film Co., Ltd., Tokyo, Japan). The radioactive bands were detected and quantitative analysis was done by a BAS 2500 image analyzer (Fuji Photo Film Co., Ltd.).

DNA sequence

Genomic DNA was isolated from fibroblasts (the proband, the sister of the proband, and their mother) or peripheral mononuclear cells of normal control human subjects and used as a template for PCR. The sequence of the exon and intron boundaries of the ARH gene was obtained from Celera human sequence data except for exon 1 (11). DNA fragments containing each exon and exon-intron boundaries of the ARH gene were amplified from the genomic DNA by Gene Amp, PCR System 9700 (Applied BioSystem, Foster City, CA). PCR was performed using the Advantage 2 protocol (CLONTECH) with 25 pmol of each pair of primers listed in Table 2. The exon 1-intron 1 boundary information did not exist in the Celera human sequence data or the database of the Human Genome Project Consortium. To obtain the sequence of the proband’s information, mRNA was reverse transcribed into cDNA using Superscript II reverse transcriptase (Life Technologies, Inc.). The primer used in reverse transcription is shown as exon 1-RT in Table 2. This primer is an antisense sequence located in exon 2. The cDNA was amplified by PCR using exon 1-F sense primer and a nested antisense primer exon 1-R of primer exon 1-RT. PCR products were purified by Microcon (Millipore Corp., Bedford, MA) and used as templates for direct sequencing. DNA sequencing was carried out according to the manufacturer’s instructions using a dye terminator method (BigDye Terminator Cycles Sequencing Ready Reaction, Applied BioSystem).

Western blot analysis

The fibroblasts from the proband, the sister of the proband, their mother, and a normal control subject were seeded into a 100-mm dish. On d 5 of cell growth, cells received S-MEM containing 10% of lipoprotein-deficient serum prepared from FCS. After incubation for 48 h, the cells were suspended in PBS and centrifuged at 5000 rpm for 5 min. The cell pellets were washed in buffer containing 100 mM NaCl, 50 mM HEPES, 10 mM EDTA, 10 mM EGTA, 2.2% dimethyl sulfoxide, and protease inhibitor mixture (Sigma, St. Louis, MO) and lysed in the same buffer containing 1% (vol/vol) triton X-100 (Sigma). Lysates were centrifuged in a microfuge for 5 min at 15,000 rpm, and the supernatant was collected. Ten micrograms protein were separated by electrophoresis (25 V, 3 h) on a 10% polyacrylamide-sodium dodecyl sulfate gel. Proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA). The membrane was incubated with serum (1:100) from a rabbit immunized with a peptide containing the C-terminal 18 amino acids of human ARH. Horseradish peroxidase-conjugated goat antibody against rabbit IgG and the enhanced chemiluminescence Western blotting detection kit (Amersham Biosciences) were used for the signal detection.

Lipid analysis of liver specimens

An autopsy liver specimen was obtained from the sister of the proband. The tissue was homogenized and the lipid fraction was extracted with chloroform:methanol, 2:1 (vol/vol). After the sample was filtered and washed using Folch’s procedure three times, the extract was dried with N₂ (12). The lipid fraction was measured for triglyceride, total and free cholesterol, and phospholipid using enzymatic assay kits (13, 14, 15).

All procedures for the analysis of patients have been approved by the institutional ethics committee, and written informed consent was obtained from each subject or their legal representatives.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Mutation in the ARH gene

For each DNA segment of exon 2–9, an exon and exon-intron boundary was amplified with the primers described in Patients and Methods. Because the information for intron 1 was not found in the available data banks, a different strategy had to be used. The information for exon 1 was found by using RT-PCR, with primers of 5'-flanking sequence and from the sequence in exon 2. The PCR products of each exon of the siblings and mother of the patients were sequenced and compared with their counterparts in control subjects. There are eight sequential cytosines between the nucleotide positions 599 and 606 (nucleotides are numbered from the first nucleotide that encodes the starting methyonine codon) in exon 6 of the normal ARH gene. An extra cytosine is inserted into this region of the ARH gene in the affected siblings. This insertion mutation causes a frame shift and results in a change of amino acid residue sequence at position 204 and generating a stop codon at position 220 (Fig. 2, A and B). For the mother’s sample, the amplified DNAs were cloned and sequenced. From among 10 independent clones, four showed eight cytosines and six showed nine cytosines, suggesting that the mother is heterozygous for the mutation (Fig. 2A).

View larger version (44K): [in this window] [in a new window]   Figure 2. A, DNA sequence data of the affected siblings, their mother, and control subjects. The mother of the patients has two alleles detected by subcloning. B, The predicted amino acid sequence in the patient and control subjects. The asterisks show the mutation locus of the DNA. C, Northern blot analysis of the ARH gene. On d 0, fibroblasts from each subject were seeded into 10-cm plastic dishes and cultivated in S-MEM containing 10% FCS. On d 7, cells were collected. Total RNA (10 µg) was electrophoresed in 0.9% agarose in the presence of 2.2 M formaldehyde, transferred onto nylon membrane, and hybridized with 32P-labeled probes. The membrane was rehybridized with human GAPDH as an internal standard. Quantitative analysis was done and is shown in the right panel. Lane 1, The proband; lane 2, the sister of the proband; lane 3, their mother; lane 4, normal control subject. D, Northern blot analysis of LDL receptor gene. On d 0, fibroblasts from each subject were seeded into 10-cm plastic dishes and cultivated in S-MEM containing 10% FCS. On d 5, the medium was changed to S-MEM containing 10% lipoprotein-deficient serum. On d 7, the cells were collected. Total RNA (10 µg) was electrophoresed in 0.9% agarose in the presence of 2.2 M formaldehyde, transferred onto nylon membrane, and hybridized with 32P-labeled probes. The membrane was rehybridized with human GAPDH as an internal standard. Quantitative analysis was carried out and is shown in the right panel. Lane 1, The proband; lane 2, the sister of the proband; lane 3, their mother; lane 4, normal control subject. E, Western blot analysis of ARH protein in cultured skin fibroblasts from each subject. Lane 1, The proband; lane 2, the sister of the proband; lane 3, their mother; lane 4, normal control subject.

Total RNA was isolated from fibroblasts of the affected siblings, their mother, and a control subject and was analyzed by Northern blot. When hybridizing with the full-length ARH cDNA as a probe, no band was detected for either patient (the proband or sister of the proband) (Fig. 2C). Quantitative analysis of the blotting bands is also shown in Fig. 2C. The ARH RNA band of the mother appeared a little fainter than that of the control subject. The RNA of the LDL receptor of the patients appeared normal both in size and expression level (Fig. 2D).

Western blot analysis

No immunodetectable ARH protein could be demonstrated for either patient (the proband or sister of the proband), but the ARH band was visualized significantly in the heterozygous patient (Fig. 2E).

Findings in the liver

Both homozygous patients were diagnosed with fatty liver by means of liver biopsy or abdominal computed tomography around age 30 yr. After the sister of the proband died, an autopsy sample of the liver was microscopically examined and analyzed for a lipid profile. The hepatocytes were swollen (Fig. 1C), and large lipid droplets were identified with Sudan III staining (Fig. 1D). Lipid was extracted from the liver. Triglyceride, cholesterol, and phospholipid levels were determined. An increase of triglyceride was prominent among other lipids, being consistent with the diagnosis of fatty liver (Table 1).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
Here, we described clinical features and genetic analysis of an autosomal recessive hypercholesterolemia family. When this family had previously been described as the first clinical characterization of this disease (3, 4), other factors known at that time to be potentially involved in hypercholesterolemia were excluded. From the rebound curve after the LDL-apheresis treatment, the fractional catabolic rate of plasma cholesterol was calculated and found to have decreased (4). We, therefore, surmised that the patients had a catabolic disorder in the plasma cholesterol caused by a factor(s) other than defective LDL receptors.

Zuliani et al. (7) described Sardinian families in which the probands had a marked reduction in the catabolic rate of ¹²⁵I-LDL and a reduction in the rate of hepatic uptake of LDL, demonstrated using the ⁹⁹technetium-LDL biodistribution study. Schmidt et al. (6) reported a Turkish subject with clinical homozygous FH phenotype whose parents, siblings, and children all had normal cholesterol levels. An LDL turnover study of the patient showed a delayed catabolism despite a functionally intact LDL receptor and apolipoprotein B, suggesting that the genetic impairment involves plasma LDL metabolism. Thus, these patients, classified as ARH, have been categorized in the clinical entity of the genetic disorder of LDL catabolism by reason(s) other than dysfunction of the LDL receptor gene.

Recently, Garcia et al. (8) mapped the ARH locus to chromosome 1p35. After screening 13 genes within this locus, they identified six mutations in a gene encoding a putative adaptor protein in ARH families including Sardinian ones who had been reported to have retardation in LDL turnover (7). Notably, an LDL receptor adaptor protein contains a phosphotyrosine-binding (PTB) domain that interacts with the NPXY motif in the cytoplasmic tails of cell surface receptors including the LDL receptor. On the basis of this information, we analyzed this gene in the family we previously reported as ARH and identified a novel insertion mutation in the ARH gene in exon 6. There are eight sequential cytosines between nucleotide positions 599 and 606 in exon 6 of the normal ARH gene, and an extra cytosine is inserted into this region of the ARH gene in the affected siblings to cause a frame shift, generating a stop codon after amino acid position 219. A predicted, truncated ARH protein contains an intact PTB domain. In preparation of this manuscript, two reports of genetic analysis of ARH have been published (17, 18). Arca et al. (17) reported that two ARH mutations were present in all the unrelated families with ARH in Sardinia and Italy. Al-Kateb et al. (18) reported an intron 1 acceptor splice-site mutation in the ARH gene in a Syrian family. They also found the 13q22-q32 locus as a gene strongly influencing LDL levels in another family with ARH and normal dizygotic twins.

In addition, we identified one single nucleotide polymorphism in the region in which an insertion mutation was found. This nucleotide substitution of cytosine by thymine at position 604 changes the coded amino acid from a proline to a serine at position 202 (ARH-604C and ARH-604T). Among 20 healthy normolipidemic volunteers, four are homozygous and 10 are heterozygous for this variation. According to a report by Garcia et al. (8), a mutation of cytosine to adenine at position 605 changes the coded amino acid residue from a proline to a histidine at position 202 and causes ARH. Thus, this position with eight sequential cytosines appears to be a mutation hot spot. It is noteworthy that the amino acid change from a proline to a serine at position 202 does not cause any apparent disorder, but the substitution by histidine at the same position produces severe clinical symptoms of ARH. Additional large-scale screening studies are required to identify the clinical significance of this nucleotide polymorphism.

Neither ARH mRNA nor protein was detected in the fibroblasts of homozygous patients. It is plausible that extreme instability of mRNA coding for ARH protein could be responsible for all clinical manifestations of the affected siblings rather than expression of the truncated protein. The phenomenon that early termination of translation caused by nonsense mutation or frame shift is called nonsense-mediated mRNA decay (19). Although the mechanism of nonsense-mediated mRNA decay has not been fully explained, this phenomenon is universally observed in yeast, plants, and mammals (19). We have previously reported such a mutation in LPL deficiency family who had a one-base deletion in exon 5 of the LPL gene resulting in premature termination by frame shift (20).

The function of the protein encoded by ARH needs to be clarified. The amount of LDL receptor mRNA is normal, and the receptor protein functions normally in the fibroblasts of homozygote patients in a cell culture system. This includes binding, internalization, and degradation of LDL (3). However, homozygous mutations of ARH result in the symptoms apparently similar to homozygous FH. The retarded turnover of LDL in the patients’ plasma strongly indicates that the product of ARH modulates clearance of plasma LDL. Thus, ARH putatively alters the function of the LDL receptor in vivo, especially in the liver because 80% of human plasma LDL is cleared in the liver (21). Garcia et al. (8) suggested that ARH encodes a putative LDL receptor adaptor protein and may participate in the regulation of localization of the LDL receptor in polarized cells. The PTB domain of ARH is considered to interact with the cytoplasmic tail of the LDL receptor, which is required for trafficking of the LDL receptor to the basolateral surface (22, 23). In fact, mutation of the cytoplasmic domain of the LDL receptor results in its misdirected targeting to the apical surface in hepatic epithelial cells (24). Recently, He et al. (25) reported that ARH interacts with LDL receptor, clathrin, and adaptor protein complex 2 and may play a role in endocytosis. The precise roles of ARH on LDL receptor protein remain to be elucidated.

In addition, both patients were found to have fatty livers, which has never been described either for FH or ARH. They were diagnosed with the fatty liver at around the age of 30 yr despite not having any of the common causes of this disorder such as obesity, hyperinsulinemia, alcoholism, or the use of specific drugs. Therefore, this finding may also be associated with the mutation of ARH and the function of the product of this gene may be related to regulation of intracellular lipid metabolism, although we cannot exclude the possibility of mere coincidence.

Here, we report homozygous patients with ARH, who have a novel insertion mutation in the ARH gene. A putative major single nucleotide polymorphism has also been identified at this region of ARH.

	Acknowledgments

 
We thank Drs. Kenji Kangawa, Hisayuki Matsuo, and Yasunao Yoshimasa for helpful discussion and advice; Dr. Yoshitane Tsukamoto for taking the histological pictures; and Dr. Patrick Leahy for proofreading this manuscript. We also thank Keiko Jinno, Eri Abe, and Moto Ohira for excellent technical assistance.

	Footnotes

 
This work was supported in part by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR); Setsuro Fujii Memorial, the Osaka Foundation for Promotion of Fundamental Medical Research; and a Grant-in-Aid for Scientific Research (C) (no. 12670384) from the Ministry of Education, Science, Sports, and Culture, Japan.

Abbreviations: ARH, Autosomal recessive hypercholesterolemia; FCS, fetal calf serum; FH, familial hypercholesterolemia; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDL, low-density lipoprotein; PTB, phosphotyrosine binding; S-MEM, minimum essential culture medium.

Received September 23, 2002.

Accepted November 20, 2002.

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