Novel mutations in ABCA1 gene in Japanese patients with Tangier disease and familial high density lipoprotein deficiency with coronary heart disease

Biochimica et Biophysica Acta 1537 (2001) 71^78 www.bba-direct.com

Novel mutations in ABCA1 gene in Japanese patients with Tangier disease and familial high density lipoprotein de¢ciency with coronary heart disease Wei Huang a , Kengo Moriyama b , Takafumi Koga a , Han Hua a , Masato Ageta c , Seiro Kawabata d , Koji Mawatari e , Takuro Imamura f , Tanenao Eto f , Mitsunobu Kawamura g , Tamio Teramoto h , Jun Sasaki a; * a

Department of Internal Medicine, Fukuoka University School of Medicine, Nanakuma, Jonan-ku, Fukuoka, Japan b Department of Biochemistry, Fukuoka University School of Medicine, Nanakuma, Jonan-ku, Fukuoka, Japan c Department of Internal Medicine, Miyazaki Prefectural Nichinan Hospital, Miyazaki, Japan d Amami Central Hospital, Kagoshima, Japan e Department of Internal Medicine, Kagoshima Seikyo Hospital, Kagoshima, Japan f Department of Internal Medicine, Miyazaki Medical College, Miyazaki, Japan g Department of Endocrinology and Metabolism, Tokyo Teishin Hospital, Tokyo, Japan h Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan Received 10 January 2001; received in revised form 1 May 2001; accepted 1 May 2001

Abstract Mutations in the ATP-binding cassette transporter 1 (ABCA1) gene have been recently identified as the molecular defect in Tangier disease (TD) and familial high density lipoprotein deficiency (FHA). We here report novel mutations in the ABCA1 gene in two sisters from a Japanese family with TD who have been described previously (S. Ohtaki, H. Nakagawa, N. Kida, H. Nakamura, K. Tsuda, S. Yokoyama, T. Yamamura, S. Tajima, A. Yamamoto, Atherosclerosis 49 (1983)) and a family with FHA. Both probands of TD and FHA developed coronary heart disease. Sequence analysis of the ABCA1 gene from the patients with TD revealed a homozygous G to A transition at nucleotide 3805 of the cDNA resulting in the substitution of Asp 1229 with Asn in exon 27, and a C to T at nucleotide 6181 resulting in the substitution of Arg 2021 with Trp in exon 47. Sequence analysis of the ABCA1 gene from the FHA patient revealed a homozygous 4 bp CGCC deletion from nucleotide 3787 to 3790 resulting in premature termination by frameshift at codon 1224. These mutations were confirmed by restriction digestion analysis, and were not found in 141 control subjects. Our findings indicate that mutations in the ABCA1 gene are associated with TD as well as FHA. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Tangier disease; ABCA1; High density lipoprotein; Coronary heart disease; ATP-binding cassette

1. Introduction * Corresponding author. International University of Health and Welfare, 1-3-1, Ngahama Chuo-ku, Fukuoka 810-0072, Japan. Fax: +81-92-739-4320. E-mail address: [email protected] (J. Sasaki).

Tangier disease (TD) is an inherited condition characterized by almost complete absence of high density lipoprotein (HDL) cholesterol and by the accumulation of cholesteryl ester in various tissues, in-

0925-4439 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 4 3 9 ( 0 1 ) 0 0 0 5 8 - 8

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cluding tonsils, liver, spleen, intestines, and Schwann cells [1]. The reduced HDL is inherited in an autosomal co-dominant mode, with heterozygotes showing approximately half normal HDL undiscernible from other cases of familial HDL de¢ciency (FHA) [1]. On the other hand, FHA has been described in patients with a markedly decreased plasma HDL cholesterol (less than ¢fth percentile), and reduced apolipoprotein (apo) A-I level, but absence of LCAT or apoA-I gene defect or clinical manifestation of TD [2,3]. Patients with TD show a moderate reduction in low density lipoprotein (LDL) and high levels of triglycerides. Fibroblasts from patients with TD are defective in apolipoprotein-mediated e¥ux of both cholesterol and phospholipid, suggesting that the markedly decreased HDL level in TD may result from failure of nascent apoA-I to acquire lipids from cells [4^7]. It has been recently demonstrated that cellular cholesterol e¥ux is also abnormal in some patients with FHA [8]. The genetic defect in TD has recently been con¢ned to chromosome 9q31 using a graphical linkage analysis by Rust et al. [9]. One of the genes that had been previously broadly mapped to the respective region was ABC1 [10]. Further narrowing and analysis of the human candidate region demonstrated human ABC1 within the narrowed candidate region [11]. Subsequent studies have demonstrated that mutations in the ABCA1 gene are the underlying defect in TD [3,11,12], and are also found in patients with FHA, the genetic mutation of which was also localized in the same region (9q31) as TD [3]. The ABCA1 is a member of the superfamily of ATP binding cassette (ABC) transporters involved in membrane transport of diverse substrates including amino acids, peptides, vitamins, and steroid hor-

mones [13]. The full-size ABC proteins are characterized by two nucleotide binding folds (NBF) with conserved Walker A and B motifs and two transmembrane domains, each consisting of six membrane-spanning helicals [10]. Mutations in speci¢c ABC transporters have been reported as the underlying defects of several human diseases. For example, cystic ¢brosis is caused by mutations of the cystic ¢brosis transmembrane conductance regulator gene [14]. Recently, the human ABCA1 gene has been cloned and shown to play an important role in lipid transport of macrophages [15]. In the present study, we isolated and cultivated monocyte-derived macrophages from Japanese patients with TD and FHA, and identi¢ed three mutations in the ABCA1 gene in these patients. 2. Materials and methods 2.1. Description of patients Two siblings with marked reduction of plasma HDL were previously identi¢ed in a Japanese family (Fig. 1A, II-3 and II-4) [16]. The serum concentration of HDL cholesterol in the proband (Fig. 1A, II4) was 2.7 mg/dl, and the concentrations of apoA-I and apoA-II were almost undetectable in the serum. In addition, the concentrations of LDL cholesterol and triglycerides in plasma were 30 mg/dl and 83 mg/ dl, respectively. Hepatosplenomegaly was noted in both siblings; lipid deposition was noted in reticuloendothelial cells on liver biopsy of the proband. The proband was free from ischemic heart disease at the age of 29 years when she was diagnosed with TD. However, she developed typical e¡ort an-

Fig. 1. (A) Pedigree of the TD family. (B) Pedigree of the FHD family. Squares, males; circles, females. A¡ected individuals are marked by solid symbols and heterozygous family members by half-solid symbols. Arrow indicates the proband.

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terol and apoA-I levels of his mother, three daughters and son were 27 and 30% lower than those of control subjects. All patients gave informed consent prior to participation in this study.

gina at the age of 37 years and was diagnosed with a single vessel disease at the age of 44 years. She also had mitral regurgitation (grade III), ventricular premature contraction, impaired glucose tolerance and manic depression illness. Her elder sister (Fig. 1A, II3) also had manic depression illness but no signs of ischemic heart disease. Further details of the clinical features and biochemical data of these two patients have been described previously [16]. The proband with HDL de¢ciency (Fig. 1B, II-2) was a 62 year old Japanese man who presented at age 45 years at the Amami Central Hospital with bronchial asthma. He began to notice chest oppression at age 50 and entered the hospital for treatment at age 53. He was 154 cm tall and weighed 55 kg. Physical examination on admission revealed hypertension (168/80 mmHg) and anemia. There was no tonsillar abnormality, lymphadenopathy, hepatosplenomegaly, or xanthomas, and no evidence of neuropathy. Laboratory tests showed normocytic anemia (hemoglobin: 8.9 g/dl) and hypolipoproteinemia (total cholesterol 55 mg/dl, triglyceride 42 mg/dl, HDL cholesterol 7 mg/dl; these values were measured at age 45 years, see [16] for details). The electrophoretic separation of serum lipoproteins on agarose gel revealed no de¢nite K band. There were no sings of diabetes mellitus, thyroid dysfunction, or renal disease. Coronary angiography revealed 99% stenosis on the left coronary artery and required percutaneous transcutaneous coronary angioplasty. Plasma and lipoprotein lipid levels of the proband and his family members are shown in Table 1. HDL choles-

2.2. Cell culture Peripheral blood monocytes were isolated using Lymphoprep Separation Solution (Nycomed Pharma, Norway), then suspended in RPMI 1640 medium supplemented with 10% human type AB serum (Sigma, St. Louis, MO, USA), penicillin and streptomycin. Monocytes were plated in 10 cm culture dishes (Falcon Labware) and incubated for 1^3 h at 37³C. Non-adherent cells were subsequently removed by washing the dishes with PBS, and the remaining adherent cells were grown in culture medium for 2 weeks, with replacement of the medium every 3 days. 2.3. Reverse transcription (RT)-PCR ampli¢cation and sequence analysis Total cellular RNA was isolated from monocytederived macrophages using a commercially available kit (Isogen, Wako Pure Chemical Industries, Japan). In the next step, 1 Wg RNA was reverse transcribed using the Ready-To-Go T-primed ¢rst-strand kit (Amersham Pharmacia Biotech). An aliquot of the cDNA was ampli¢ed by AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT, USA). The primers for ampli¢ed human ABCA1 cDNA were exactly similar

Table 1 Lipoprotein pro¢les of subjects from a Japanese family with familial HDL de¢ciency Subject

I II III

Age (years)

2 82 1a 62 1 36 2 34 3 24 4 21 Normal controlsb Males (n = 15) Females(n = 16) a b

Sex

ABCA1 4 bp deletion

Cholesterol (mg/dl)

Total

Total

HDL

F M F M F F

+/3 +/+ +/3 +/3 +/3 +/3

122 66 150 184 116 149

37 3.5 47 39 40 51

192 þ 27 195 þ 26

52 þ 18 58 þ 20

Triglyceride (mg/dl)

Apolipoprotein (mg/dl) A-I

78 69 58 100 62 139 118 þ 32 108 þ 40

Proband. Mean þ S.D.

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101 6 112 89 11 117 146 þ 20 154 þ 22

A-II

B

E

22 2 21 27 20 25

65 27 66 86 53 78

3 1.5 4.2 6.2 3.9 2.9

32 þ 5 34 þ 6

102 þ 24 4.2 þ 1.1 98 þ 25 4.1 þ 1.0

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to those described previously [15]. All PCR products were subcloned into the pT7Blue-T vector (Novagen) after puri¢cation with a GeneClean kit (Bio101). Twelve clones of both strands in opposite directions of each subcloned fragment were sequenced using a DNA sequencing kit (Dye terminator cycle sequencing ready reaction, Perkin-Elmer) with an ABI 373 DNA sequencer according to the protocol recommended by the manufacturer. In addition, exons 27 and 47 of ABCA1 from genomic DNA of two siblings were also sequenced to con¢rm the identi¢ed mutations. The ABCA1 gene sequence is based on a published sequence in GenBank, accession No. AJ012376. The numbering of the exon is based on newly published data [17]. Sequence analyses of apoA-I and LCAT genes of the proband of FHA were performed as described previously [18^20]. 2.4. Restriction fragment length polymorphism analysis (RFLP) Genomic DNA was isolated from peripheral blood as described previously [18]. Genotyping for the TD patient was performed by PCR ampli¢cation of exon 27 followed by restriction digestion with TspEI for G3805A, and ampli¢cation of exon 47 followed by digestion with AgeI for C6181T, respectively. The primers for exon 27 were: (forward) 5P-TTGTGCCTTCAGATGGTACCTTGC-3P, (reverse) 5PTCAGAATCATTTGGATCAGCAGAAT-3P; the primers for exon 47 were: (forward) 5P-CACAGGCATGGATCCCAAAGACCG-3P, (reverse) 5P-CTACGGACCTATGAGATGTAAGCAC-3P. Genotyping was performed by PCR ampli¢cation of exon 7 followed by restriction digestion with Eco81I for G596A, and ampli¢cation of exon 27 followed by digestion with NspV for the CGCC3787-3790 deletion, respectively. The primers for exon 7 were: (forward) 5P-GTTTCTGAGCTTTGTGGCCTACCTA3P, (reverse) 5P-CTACTCACCAGGATTGGCTTCAGG-3P; the primers for exon 27 were: (forward) 5P-TTGTGCCTTCAGATGGTACCTTGC-3P, (reverse) 5P-TCAGCAGCATCATCTTCAGTGTTCG3P. The underlined bases indicate mutagenized nucleotides so that the restriction cutting sites for TspEI, AgeI, Eco81I, and NspV are generated in the case of G3805A, C6181T, G596A, and the CGCC3787-3790 deletion, respectively. The nucleo-

tide sequence for the human ABCA1 gene and the intron^exon boundaries of the ABCA1 gene are based on published data [3,15,21]. A total of 141 healthy Japanese individuals were recruited as control subjects. Control subjects gave informed consent prior to participation in this study. 2.5. Plasma lipoprotein characterization Blood samples were collected in Na2 EDTA after a 12 h fast. Lipoproteins were isolated by sequential ultracentrifugation. The following density fractions were obtained: very low density lipoprotein (VLDL) (d 6 1.019 g/ml), LDL (1.019^1.063 g/ml) and HDL (1.063^1.21 g/ml) [18]. Plasma cholesterol and triglyceride values were determined by enzymatic methods [18]. HDL cholesterol was quantitated by the heparin-manganese precipitation method [18]. Apolipoproteins were measured by the single radial immunodi¡usion method [18]. 3. Results and discussion In the present study, we isolated and cultivated monocyte-derived macrophages from one of two previously described siblings with Tangier disease [16]. Nine overlapping PCR fragments spanning the whole ABCA1 cDNA coding the mature product were ampli¢ed by RT-PCR, as described in Section 2. Both strands were sequenced throughout. It should be noted that we used the primers by Langmann et al. [15] to sequence cDNA of the ABC1 gene that encode the mature product of the ABC1 gene, and cover exon 2 to exon 50 based on updated sequence information [17]; thus our sequencing did not include the updated sequence (exon 1) coding 60 amino acids. Two novel homozygous missense mutations were identi¢ed, at nucleotide 3805 with a G to A transition, and at nucleotide 6181 with a C to T transition, in cDNA from the sister of the proband (Fig. 2). The two mutations resulted in the substitution of Asp 1229 with Asn in exon 27, and substitution of Arg 2021 with Trp in exon 47, respectively. Both mutations were also con¢rmed by sequencing exons 27 and 47 of ABCA1 from genomic DNA of the two siblings (data not shown). RFLP analysis of genomic DNA from the two siblings further con-

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Fig. 2. Sequence analysis of the ABCA1 gene in a sister of the proband with Tangier disease. (A) cDNA sequences of a healthy control subject and a sister of the proband (Fig. 1, II3) are shown for part of exon 27 of the ABCA1 gene. Arrow indicates the G to A transition at nucleotide 3805. The patient is homozygous for the mutation. (Bottom) Restriction digestion polymorphism analysis of patients with Tangier disease. The presence of the G3805A mutation was detected by restriction digestion with TspEI, which cuts the mutant allele only. Lanes: M, molecular weight marker; 1, proband; 2, sister of the proband; 3, normal control. (B) Sequence analysis of the ABCA1 cDNA region containing the C to T transition at nucleotide 6181. Sequences of a healthy control subject and a sister of the proband (Fig. 1, II-3). The patient is homozygous for the mutation. Arrow indicates the C to T transition at nucleotide 6181. (Bottom) Restriction digestion polymorphism analysis of patients with Tangier disease. The presence of the C6181T mutation was detected by restriction digestion with AgeI, which cuts the mutant allele only. Lanes: M, molecular weight marker; 1, proband; 2, sister of the proband; 3, control subject.

¢rmed the mutations (Fig. 2). Sequence analysis of the ABCA1 gene from the HDL de¢ciency patient revealed a homozygous 4 bp deletion at nucleotides 3787^3790 of the cDNA in exon 27, resulting in premature termination by frameshift at codon 1224

75

(Fig. 3). RFLP analysis of genomic DNA from the proband further con¢rmed the mutation (Fig. 3). The mother, three daughters and a son of the proband of FHA were found to be heterozygous for the 4 bp mutation (data not shown). In addition, we found a G596A mutation in exon 7, but this mutation was also detected in some of the control subjects. These three mutations were not found in 141 normal subjects with RFLP, indicating that they are not polymorphisms. However, the G596A mutation was considered to be a polymorphism, since we found 29 homozygous and 21 heterozygous in 141 normal control subjects. In addition, the sequences determined in the patient and one control subject di¡ered in eight nucleotides in the 6603 bp open reading frame from that registered in GenBank (accession No. AJ012376) [15]. They were C for T at nucleotide 705, C for A at 1980, G for A at 3805, C for T at 4604, C for T 4883, G for T at 5861, C for T at 6181, and C for T at 6443. All these eight nucleotides were found in both the patient and the normal subject, and are consistent with data reported previously [22]. To our knowledge, 22 mutations in ABCA1 have so far been reported in 19 families with TD. Mutations in TD are distributed throughout the ABCA1 encoded protein, and many of them are clustered close to the ATP-binding cassette domain. The mutation at amino acid 1229 is close to the ¢rst ATPbinding cassette domain, and the mutation at amino acid 2021 is located within the second ATP-binding cassette domain. Furthermore, amino acids 1229 and 2021 are conserved between human and mouse ABCA1 and in a Caenorhabditis elegans homologue, indicating that these two amino acids are probably of functional importance. At present, however, which of the two mutations is mainly associated with the TD phenotype could not be determined in our case. Initially, we thought that one of these mutations would be a polymorphism, but we screened 141 normal subjects, and none of them was found to have such a mutation. Recently, four novel ABCA1 mutations in unrelated TD kindreds have been reported by Brousseau et al. [23], and one of these, from Japanese TD kindreds (TD 18-1, TD 18-2), was identical to ours. We also con¢rmed that the TD siblings reported by Brousseau et al. were identical to ours. However, these investigators did not mention the

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Fig. 3. Sequence analysis of the ABCA1 cDNA region in the patient with FHA containing the 4 bp deletion at nucleotides 3787^ 3790. Sequences of a normal control subject and the proband are shown. The patient is homozygous for the mutation. Arrow indicates the 4 bp deletion at nucleotides 3787^3790. (Bottom) Restriction digestion polymorphism analysis of the patient with FHA. The presence of the 4 bp deletion at nucleotides 3787^3790 was detected by restriction digestion with NspV, which cuts the mutant allele only. Lanes: M, molecular weight marker; 1, proband; 2, control subject.

substitution of Arg 2021 with Trp in exon 47, and mistakenly reported the substitution of Asp 1229 with Leu instead of Asn in exon 27. They showed that apoA-I-mediated cholesterol e¥ux from the ¢broblasts of these subjects was 10% of that of control subjects. Mutations in the ABC1 gene have been also reported in some patients with FHA [10,11]. In the present study we checked three unrelated families with FHA, and found the mutation in one family with a 4 bp deletion, resulting in a premature stop at codon 1224 by a frameshift (Fig. 4). Loo and Clarke intensively investigated the biosynthesis of P-glycoprotein which belongs to the ABC superfamily of transport proteins. They found that changes in 49 of 401 arti¢cially mutagenized residues (12.2%) of P-glycoprotein resulted in misprocessing [24]. Misprocessing mutations are located throughout the molecule; they include the transmembrane domains, intracellular and extracellular loops, the linker region, and both nucleotide-binding domains [24]. In this regard, the 4 bp deletion at nucleotides 3787^ 3790 of the ABCA1 molecule might result in misprocessing and degradation intracellularly and is probably non-functional. The truncation could occur between the predicted seventh and eight transmembrane domains. Since skin biopsy was refused

by the family members, we could not determine apoA-I-mediated cellular cholesterol e¥ux. The proband had severe coronary heart disease but did not show the typical TD phenotype, and he had no tonsillar abnormality, lymphadenopathy, hepatosplenomegaly, or evidence of neuropathy. Mutations were not detected in apoA-I and LCAT genes of the proband. The ABCA1 transporter plays a crucial role in the apolipoprotein-mediated lipid removal pathway in monocytes and ¢broblasts [15,22]. Both probands of TD and FHA in our study developed coronary

Fig. 4. Diagram of the portion of the ABCA1 gene showing the 4 nucleotide deletion, leading to a frameshift mutation and premature termination in exon 27. The amino acid sequences of a section of exon 27 from a normal subject and the FHA proband are illustrated, and the underlined are the deleted nucleotides. Numbers at the top of the sequence indicate the amino acid residues of native ABCA1.

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heart disease. This may imply the important role of this pathway in intracellular cholesterol transport as a key mechanism in atherogenesis. During the isolation of monocytes, we observed that the number of monocytes from the patient was clearly lower than that of control subjects (unpublished observation), implying that monocytes may be target cells in TD. This is consistent with previously published data [15,22]. However, in our case with TD, tonsillar enlargement was not observed in either sibling [16]. Although mutations in ABCA1 are associated with TD and FHA [3,11,12], the molecular mechanism related to phenotypic heterogeneity of the disease and FHA remains to be de¢ned. In conclusion, we isolated RNA from monocytederived macrophages from one of two previously described Japanese patients with TD and FHA to generate ABCA1 cDNA by RT-PCR. We identi¢ed three novel mutations at Asp 1229 with Asn substitution in exon 27, Arg 2021 with Trp in exon 47 and a 4 bp deletion at nucleotides 3787^3790 of the cDNA in exon 27. The three mutations were not found in 141 Japanese control subjects. Further studies are necessary to elucidate which of the two mutations is mainly related to the phenotype of TD in our case.

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Acknowledgements This work was partly supported by grants from the Japanese Ministry of Education, Science, Sports, and Culture (No. 08671195 to J.S.), and the Science Research Promotion Fund from the Japan Private School Promotion Foundation. We wish to thank Dr. Ken-ichi Hirano from the Department of Internal Medicine and Molecular Science, Osaka University, for technical advise on cell culture studies. References

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