Novel Mutations in ABCA1 Gene in Japanese Patients with Tangier Disease and Familial High Density Lipoprotein De¢Ciency with Coronary Heart Disease

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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

Tangier disease (TD) is an inherited condition * Corresponding author. International University of Health and Welfare, 1-3-1, Ngahama Chuo-ku, Fukuoka 810-0072, Ja- characterized by almost complete absence of high pan. Fax: +81-92-739-4320. density lipoprotein (HDL) cholesterol and by the ac- E-mail address: [email protected] (J. Sasaki). cumulation of cholesteryl ester in various tissues, in-

BBADIS 62046 19-7-01 72 W. Huang et al. / Biochimica et Biophysica Acta 1537 (2001) 71^78 cluding tonsils, liver, spleen, intestines, and Schwann mones [13]. The full-size ABC proteins are character- cells [1]. The reduced HDL is inherited in an auto- ized by two nucleotide binding folds (NBF) with somal co-dominant mode, with heterozygotes show- conserved Walker A and B motifs and two trans- ing approximately half normal HDL undiscernible membrane domains, each consisting of six mem- from other cases of familial HDL de¢ciency (FHA) brane-spanning helicals [10]. [1]. On the other hand, FHA has been described in Mutations in speci¢c ABC transporters have been patients with a markedly decreased plasma HDL reported as the underlying defects of several human cholesterol (less than ¢fth percentile), and reduced diseases. For example, cystic ¢brosis is caused by apolipoprotein (apo) A-I level, but absence of mutations of the cystic ¢brosis transmembrane con- LCAT or apoA-I gene defect or clinical manifesta- ductance regulator gene [14]. Recently, the human tion of TD [2,3]. Patients with TD show a moderate ABCA1 gene has been cloned and shown to play reduction in low density lipoprotein (LDL) and high an important role in lipid transport of macrophages levels of triglycerides. Fibroblasts from patients with [15]. In the present study, we isolated and cultivated TD are defective in apolipoprotein-mediated e¥ux of monocyte-derived macrophages from Japanese pa- both cholesterol and phospholipid, suggesting that tients with TD and FHA, and identi¢ed three muta- the markedly decreased HDL level in TD may result tions in the ABCA1 gene in these patients. 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 2. Materials and methods patients with FHA [8]. The genetic defect in TD has recently been con- 2.1. Description of patients ¢ned to chromosome 9q31 using a graphical linkage analysis by Rust et al. [9]. One of the genes that had Two siblings with marked reduction of plasma been previously broadly mapped to the respective HDL were previously identi¢ed in a Japanese family region was ABC1 [10]. Further narrowing and anal- (Fig. 1A, II-3 and II-4) [16]. The serum concentra- ysis of the human candidate region demonstrated tion of HDL cholesterol in the proband (Fig. 1A, II- human ABC1 within the narrowed candidate region 4) was 2.7 mg/dl, and the concentrations of apoA-I [11]. Subsequent studies have demonstrated that mu- and apoA-II were almost undetectable in the serum. tations in the ABCA1 gene are the underlying defect In addition, the concentrations of LDL cholesterol in TD [3,11,12], and are also found in patients with and triglycerides in plasma were 30 mg/dl and 83 mg/ FHA, the genetic mutation of which was also local- dl, respectively. Hepatosplenomegaly was noted in ized in the same region (9q31) as TD [3]. The both siblings; lipid deposition was noted in reticu- ABCA1 is a member of the superfamily of ATP loendothelial cells on liver biopsy of the proband. binding cassette (ABC) transporters involved in The proband was free from ischemic heart disease membrane transport of diverse substrates including at the age of 29 years when she was diagnosed amino acids, peptides, vitamins, and steroid hor- 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.

BBADIS 62046 19-7-01 W. Huang et al. / Biochimica et Biophysica Acta 1537 (2001) 71^78 73 gina at the age of 37 years and was diagnosed with a terol and apoA-I levels of his mother, three daugh- single vessel disease at the age of 44 years. She also ters and son were 27 and 30% lower than those of had mitral regurgitation (grade III), ventricular pre- control subjects. All patients gave informed consent mature contraction, impaired glucose tolerance and prior to participation in this study. manic depression illness. Her elder sister (Fig. 1A, II- 3) also had manic depression illness but no signs of 2.2. Cell culture ischemic heart disease. Further details of the clinical features and biochemical data of these two patients Peripheral blood monocytes were isolated using have been described previously [16]. Lymphoprep Separation Solution (Nycomed Phar- The proband with HDL de¢ciency (Fig. 1B, II-2) ma, Norway), then suspended in RPMI 1640 me- was a 62 year old Japanese man who presented at dium supplemented with 10% human type AB serum age 45 years at the Amami Central Hospital with (Sigma, St. Louis, MO, USA), penicillin and strepto- bronchial asthma. He began to notice chest oppres- mycin. Monocytes were plated in 10 cm culture sion at age 50 and entered the hospital for treatment dishes (Falcon Labware) and incubated for 1^3 h at age 53. He was 154 cm tall and weighed 55 kg. at 37³C. Non-adherent cells were subsequently re- Physical examination on admission revealed hyper- moved by washing the dishes with PBS, and the re- tension (168/80 mmHg) and anemia. There was no maining adherent cells were grown in culture me- tonsillar abnormality, lymphadenopathy, hepatosple- dium for 2 weeks, with replacement of the medium nomegaly, or xanthomas, and no evidence of neuro- every 3 days. pathy. Laboratory tests showed normocytic anemia (hemoglobin: 8.9 g/dl) and hypolipoproteinemia (to- 2.3. Reverse transcription (RT)-PCR ampli¢cation tal cholesterol 55 mg/dl, triglyceride 42 mg/dl, HDL and sequence analysis cholesterol 7 mg/dl; these values were measured at age 45 years, see [16] for details). The electrophoretic Total cellular RNA was isolated from monocyte- separation of serum lipoproteins on agarose gel re- derived macrophages using a commercially available vealed no de¢nite K band. There were no sings of kit (Isogen, Wako Pure Chemical Industries, Japan). diabetes mellitus, thyroid dysfunction, or renal dis- In the next step, 1 Wg RNA was reverse transcribed ease. Coronary angiography revealed 99% stenosis using the Ready-To-Go T-primed ¢rst-strand kit on the left coronary artery and required percutane- (Amersham Pharmacia Biotech). An aliquot of the ous transcutaneous coronary angioplasty. Plasma cDNA was ampli¢ed by AmpliTaq DNA polymerase and lipoprotein lipid levels of the proband and his (Perkin-Elmer, Norwalk, CT, USA). The primers for family members are shown in Table 1. HDL choles- 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 Age Sex ABCA1 4 bp Cholesterol (mg/dl) Triglyceride Apolipoprotein (mg/dl) (years) deletion (mg/dl) Total Total HDL A-I A-II B E I 2 82 F +/3 122 37 78 101 22 65 3 II 1a 62 M +/+ 66 3.5 69 6 2 27 1.5 III 1 36 F +/3 150 47 58 112 21 66 4.2 234M+/3 184 39 100 89 27 86 6.2 324F+/3 116 40 62 11 20 53 3.9 421F+/3 149 51 139 117 25 78 2.9 Normal controlsb Males (n = 15) 192 þ 27 52 þ 18 118 þ 32 146 þ 20 32 þ 5 102 þ 24 4.2 þ 1.1 Females(n = 16) 195 þ 26 58 þ 20 108 þ 40 154 þ 22 34 þ 6 98 þ 25 4.1 þ 1.0 aProband. bMean þ S.D.

BBADIS 62046 19-7-01 74 W. Huang et al. / Biochimica et Biophysica Acta 1537 (2001) 71^78 to those described previously [15]. All PCR products tide sequence for the human ABCA1 gene and the were subcloned into the pT7Blue-T vector (Novagen) intron^exon boundaries of the ABCA1 gene are after puri¢cation with a GeneClean kit (Bio101). based on published data [3,15,21]. A total of 141 Twelve clones of both strands in opposite directions healthy Japanese individuals were recruited as con- of each subcloned fragment were sequenced using a trol subjects. Control subjects gave informed consent DNA sequencing kit (Dye terminator cycle sequenc- prior to participation in this study. ing ready reaction, Perkin-Elmer) with an ABI 373 DNA sequencer according to the protocol recom- 2.5. Plasma lipoprotein characterization mended by the manufacturer. In addition, exons 27 and 47 of ABCA1 from genomic DNA of two sib- Blood samples were collected in Na2 EDTA after a lings were also sequenced to con¢rm the identi¢ed 12 h fast. Lipoproteins were isolated by sequential mutations. The ABCA1 gene sequence is based on ultracentrifugation. The following density fractions a published sequence in GenBank, accession No. were obtained: very low density lipoprotein AJ012376. The numbering of the exon is based on (VLDL) (d 6 1.019 g/ml), LDL (1.019^1.063 g/ml) newly published data [17]. Sequence analyses of and HDL (1.063^1.21 g/ml) [18]. Plasma cholesterol apoA-I and LCAT genes of the proband of FHA and triglyceride values were determined by enzymatic were performed as described previously [18^20]. methods [18]. HDL cholesterol was quantitated by the heparin-manganese precipitation method [18]. 2.4. Restriction fragment length polymorphism Apolipoproteins were measured by the single radial analysis (RFLP) immunodi¡usion method [18].

Genomic DNA was isolated from peripheral blood as described previously [18]. Genotyping for the TD 3. Results and discussion patient was performed by PCR ampli¢cation of exon 27 followed by restriction digestion with TspEI for In the present study, we isolated and cultivated G3805A, and ampli¢cation of exon 47 followed by monocyte-derived macrophages from one of two pre- digestion with AgeI for C6181T, respectively. The viously described siblings with Tangier disease [16]. primers for exon 27 were: (forward) 5P-TTGT- Nine overlapping PCR fragments spanning the whole GCCTTCAGATGGTACCTTGC-3P, (reverse) 5P- ABCA1 cDNA coding the mature product were am- TCAGAATCATTTGGATCAGCAGAAT-3P; the pli¢ed by RT-PCR, as described in Section 2. Both primers for exon 47 were: (forward) 5P-CACAGG- strands were sequenced throughout. It should be CATGGATCCCAAAGACCG-3P, (reverse) 5P-CTA- noted that we used the primers by Langmann et al. CGGACCTATGAGATGTAAGCAC-3P. Genotyp- [15] to sequence cDNA of the ABC1 gene that en- ing was performed by PCR ampli¢cation of exon 7 code the mature product of the ABC1 gene, and followed by restriction digestion with Eco81I for cover exon 2 to exon 50 based on updated sequence G596A, and ampli¢cation of exon 27 followed by information [17]; thus our sequencing did not in- digestion with NspV for the CGCC3787-3790 dele- clude the updated sequence (exon 1) coding 60 amino tion, respectively. The primers for exon 7 were: (for- acids. Two novel homozygous missense mutations ward) 5P-GTTTCTGAGCTTTGTGGCCTACCTA- were identi¢ed, at nucleotide 3805 with a G to A 3P, (reverse) 5P-CTACTCACCAGGATTGGCTTC- transition, and at nucleotide 6181 with a C to T AGG-3P; the primers for exon 27 were: (forward) transition, in cDNA from the sister of the proband 5P-TTGTGCCTTCAGATGGTACCTTGC-3P, (re- (Fig. 2). The two mutations resulted in the substitu- verse) 5P-TCAGCAGCATCATCTTCAGTGTTCG- tion of Asp 1229 with Asn in exon 27, and substitu- 3P. The underlined bases indicate mutagenized nu- tion of Arg 2021 with Trp in exon 47, respectively. cleotides so that the restriction cutting sites for Both mutations were also con¢rmed by sequencing TspEI, AgeI, Eco81I, and NspV are generated in exons 27 and 47 of ABCA1 from genomic DNA of the case of G3805A, C6181T, G596A, and the the two siblings (data not shown). RFLP analysis of CGCC3787-3790 deletion, respectively. The nucleo- genomic DNA from the two siblings further con-

BBADIS 62046 19-7-01 W. Huang et al. / Biochimica et Biophysica Acta 1537 (2001) 71^78 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 Fig. 2. Sequence analysis of the ABCA1 gene in a sister of the so far been reported in 19 families with TD. Muta- proband with Tangier disease. (A) cDNA sequences of a tions in TD are distributed throughout the ABCA1 healthy control subject and a sister of the proband (Fig. 1, II- encoded protein, and many of them are clustered 3) are shown for part of exon 27 of the ABCA1 gene. Arrow close to the ATP-binding cassette domain. The mu- indicates the G to A transition at nucleotide 3805. The patient is homozygous for the mutation. (Bottom) Restriction digestion tation at amino acid 1229 is close to the ¢rst ATP- polymorphism analysis of patients with Tangier disease. The binding cassette domain, and the mutation at amino presence of the G3805A mutation was detected by restriction acid 2021 is located within the second ATP-binding digestion with TspEI, which cuts the mutant allele only. Lanes: cassette domain. Furthermore, amino acids 1229 and M, molecular weight marker; 1, proband; 2, sister of the pro- 2021 are conserved between human and mouse band; 3, normal control. (B) Sequence analysis of the ABCA1 ABCA1 and in a Caenorhabditis elegans homologue, cDNA region containing the C to T transition at nucleotide 6181. Sequences of a healthy control subject and a sister of the indicating that these two amino acids are probably of proband (Fig. 1, II-3). The patient is homozygous for the muta- functional importance. At present, however, which of tion. Arrow indicates the C to T transition at nucleotide 6181. the two mutations is mainly associated with the TD (Bottom) Restriction digestion polymorphism analysis of pa- phenotype could not be determined in our case. Ini- tients with Tangier disease. The presence of the C6181T muta- tially, we thought that one of these mutations would tion was detected by restriction digestion with AgeI, which cuts the mutant allele only. Lanes: M, molecular weight marker; be a polymorphism, but we screened 141 normal 1, proband; 2, sister of the proband; 3, control subject. 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 ¢rmed the mutations (Fig. 2). Sequence analysis of Brousseau et al. [23], and one of these, from Japa- the ABCA1 gene from the HDL de¢ciency patient nese TD kindreds (TD 18-1, TD 18-2), was identical revealed a homozygous 4 bp deletion at nucleotides to ours. We also con¢rmed that the TD siblings re- 3787^3790 of the cDNA in exon 27, resulting in pre- ported by Brousseau et al. were identical to ours. mature termination by frameshift at codon 1224 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 by the family members, we could not determine mistakenly reported the substitution of Asp 1229 apoA-I-mediated cellular cholesterol e¥ux. The pro- with Leu instead of Asn in exon 27. They showed band had severe coronary heart disease but did not that apoA-I-mediated cholesterol e¥ux from the ¢- show the typical TD phenotype, and he had no ton- broblasts of these subjects was 10% of that of control sillar abnormality, lymphadenopathy, hepatospleno- subjects. megaly, or evidence of neuropathy. Mutations were Mutations in the ABC1 gene have been also re- not detected in apoA-I and LCAT genes of the pro- ported in some patients with FHA [10,11]. In the band. present study we checked three unrelated families The ABCA1 transporter plays a crucial role in the with FHA, and found the mutation in one family apolipoprotein-mediated lipid removal pathway in with a 4 bp deletion, resulting in a premature stop monocytes and ¢broblasts [15,22]. Both probands at codon 1224 by a frameshift (Fig. 4). Loo and of TD and FHA in our study developed coronary 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]. Mis- processing 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^ Fig. 4. Diagram of the portion of the ABCA1 gene showing 3790 of the ABCA1 molecule might result in mispro- the 4 nucleotide deletion, leading to a frameshift mutation and cessing and degradation intracellularly and is prob- premature termination in exon 27. The amino acid sequences of a section of exon 27 from a normal subject and the FHA pro- ably non-functional. The truncation could occur band are illustrated, and the underlined are the deleted nucleo- between the predicted seventh and eight trans- tides. Numbers at the top of the sequence indicate the amino membrane domains. Since skin biopsy was refused acid residues of native ABCA1.

BBADIS 62046 19-7-01 W. Huang et al. / Biochimica et Biophysica Acta 1537 (2001) 71^78 77 heart disease. This may imply the important role of Canadian kindreds. Clinical, biochemical, and molecular this pathway in intracellular cholesterol transport as characterization, Arterioscler. Thromb. Vasc. Biol. 15 a key mechanism in atherogenesis. During the isola- (1995) 1015^1024. [3] A. Brooks-Wilson, M. Marcil, S.M. Clee, L.-H. Zhang, K. tion of monocytes, we observed that the number of Roomp, M. van Dam, L. Yu, C. Brewer, J.A. Colins, monocytes from the patient was clearly lower than H.O.F. Molhuizen, O. Loubser, F.F.B. Ouelette, K. Fichter, that of control subjects (unpublished observation), K.J.D. Ashbourne-Exco¡on, C.W. Sensen, S. Scherer, M. implying that monocytes may be target cells in TD. Denis, S. Mott, D. Martindale, J. Frohlich, K. Morgan, B. This is consistent with previously published data Koop, S. Pimstone, J.J.P. Kastelein, J. Genest Jr., M.R. Hayden, Mutations in ABC1 in Tangier disease and familial [15,22]. However, in our case with TD, tonsillar en- high-density lipoprotein de¢ciency, Nat. Genet. 22 (1999) largement was not observed in either sibling [16]. 336^345. Although mutations in ABCA1 are associated with [4] M. Walter, U. Gerdes, U. Seedorf, G. Assmann, The high TD and FHA [3,11,12], the molecular mechanism density lipoprotein- and apolipoprotein A-I-induced mobili- related to phenotypic heterogeneity of the disease zation of cellular cholesterol is impaired in ¢broblasts from and FHA remains to be de¢ned. Tangier disease subjects, Biochem. Biophys. Res. Commun. 205 (1994) 850^856. In conclusion, we isolated RNA from monocyte- [5] G. Rogler, B. Trumbach, B. Klima, K.J. Lackner, G. derived macrophages from one of two previously de- Schmitz, HDL-mediated e¥ux of intracellular cholesterol is scribed Japanese patients with TD and FHA to gen- impaired in ¢broblasts from Tangier disease patients, Arte- erate ABCA1 cDNA by RT-PCR. We identi¢ed rioscler. Thromb. Vasc. Biol. 15 (1995) 683^690. three novel mutations at Asp 1229 with Asn substi- [6] A.T. Remaley, U.K. Schumacher, J.A. Stonik, B.D. Farsi, H. Nazih, H.B. Brewer, Decreased reverse cholesterol trans- tution in exon 27, Arg 2021 with Trp in exon 47 and port from Tangier disease ¢broblasts: acceptor speci¢city a 4 bp deletion at nucleotides 3787^3790 of the and e¡ect of brefeldin on lipid e¥ux, Arterioscler. Thromb. cDNA in exon 27. The three mutations were not Vasc. Biol. 17 (1997) 1813^1821. found in 141 Japanese control subjects. Further stud- [7] G.A. Francis, R.H. Knopp, J.F. Oram, Defective removal of ies are necessary to elucidate which of the two muta- cellular cholesterol and phospholipids by apolipoprotein A-I tions is mainly related to the phenotype of TD in our in Tangier disease, J. Clin. Invest. 96 (1995) 78^87. [8] M. Marcil, L. Yu, L. Krimbou, B. Boucher, J.F. Oram, J. case. Genest Jr., Cellular cholesterol transport and e¥ux in ¢broblasts are abnormal in subjects with familial HDL de¢ciency, Arterioscler. Thromb. Vasc. Biol. 29 (1999) 159^169. Acknowledgements [9] S. Rust, M. Walter, H. Funke, A. von Eckardstein, P. Cull- en, H.Y. Kroes, R. Hordijk, J. Geisel, J. Kastelein, H.O.F. Molhuizen, M. Schreiner, A. Mischke, H.W. Hahmann, G. This work was partly supported by grants from the Assmann, Assignment of Tangier disease to chromosome Japanese Ministry of Education, Science, Sports, and 9q31 by a graphical linkage exclusion strategy, 20 (1998) Culture (No. 08671195 to J.S.), and the Science Re- 96^98. search Promotion Fund from the Japan Private [10] M.F. Luciani, F. Denizot, S. Savary, M.G. Mattei, G. Chi- School Promotion Foundation. We wish to thank mini, Cloning of 2 novel ABC transporters mapping on Dr. Ken-ichi Hirano from the Department of Inter- chromosome 9, Genomics 21 (1994) 150^159. [11] S. Rust, M. Rosier, H. Funke, J. Real, Z. Amoura, J.-C. nal Medicine and Molecular Science, Osaka Univer- Piette, J.-F. Deleuze, H.B. Brewer, N. Duverger, P. Dene'£e, sity, for technical advise on cell culture studies. G. Assmann, Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1, Nat. Genet. 22 (1999) 352^355. References [12] M. Marcil, A. Brooks-Wilson, S.M. Clee, K. 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