ABCMdb

ABCA1 p.Arg1068His

Predicted by SNAP2:	A: D (85%), C: D (91%), D: D (95%), E: D (91%), F: D (95%), G: D (91%), H: D (85%), I: D (91%), K: D (85%), L: D (91%), M: D (80%), N: D (91%), P: D (95%), Q: D (85%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Homology modeling and functional testing of an ABC... Atherosclerosis. 2011 Oct;218(2):404-10. Epub 2011 Jun 29. Suetani RJ, Sorrenson B, Tyndall JD, Williams MJ, McCormick SP
Homology modeling and functional testing of an ABCA1 mutation causing Tangier disease.
Atherosclerosis. 2011 Oct;218(2):404-10. Epub 2011 Jun 29., [PMID:21763656]

Abstract [show]
OBJECTIVE: To investigate the impact of the p.R1068H mutation on the structure and function of the ATP-binding cassette A1 (ABCA1) protein. METHODS: A homology model of the nucleotide binding domains of ABCA1 was constructed to identify the three-dimensional orientation of R1068. Cholesterol efflux assays were performed on fibroblasts obtained from members of a Tangier disease (TD) family carrying the p.R1068H mutation and in HEK293 cells transfected with a p.R1068H mutant cDNA vector. Confocal microscopy was used to investigate the localisation of the wildtype and mutant p.R1068H protein in HEK293 cells. RESULTS: Sequence alignments and modeling indicated residue R1068 to be located in an alpha-helix downstream of the Walker B motif in the first nucleotide binding domain (NBD-1), in a position to form ionic interactions with D1092 and E1093. Cholesterol efflux studies showed the efflux from TD fibroblasts and HEK293 cells expressing the mutant p.R1068H protein to be markedly reduced compared to wildtype. Localisation of the mutant p.R1068H protein in HEK293 cells showed intracellular retention of the protein indicating a defect in trafficking to the plasma membrane. CONCLUSION: Homology modeling of the ABCA1 protein showed that the p.R1068H mutation would likely disrupt the conformation of NBD-1. Functional studies of p.R1068H showed a lack of cholesterol efflux function due to defective trafficking to the plasma membrane, most likely caused by impaired oligomerisation.

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0 Atherosclerosis 218 (2011) 404-410 Contents lists available at ScienceDirect Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis Homology modeling and functional testing of an ABCA1 mutation causing Tangier disease Rachel J. Suetania , Brie Sorrensona , Joel D.A. Tyndallb , Michael J.A. Williamsc , Sally P.A. McCormicka,* a Department of Biochemistry, University of Otago, Dunedin, New Zealand b School of Pharmacy, University of Otago, Dunedin, New Zealand c Department of Medical and Surgical Sciences, University of Otago, Dunedin, New Zealand a r t i c l e i n f o Article history: Received 27 January 2011 Received in revised form 8 June 2011 Accepted 8 June 2011 Available online 29 June 2011 Keywords: ABCA1 HDL Cholesterol efflux Homology modeling Tangier disease a b s t r a c t Objective: To investigate the impact of the p.R1068H mutation on the structure and function of the ATP-binding cassette A1 (ABCA1) protein. Login to comment
2 Cholesterol efflux assays were performed on fibroblasts obtained from members of a Tangier disease (TD) family carrying the p.R1068H mutation and in HEK293 cells transfected with a p.R1068H mutant cDNA vector. Login to comment
3 Confocal microscopy was used to investigate the localisation of the wildtype and mutant p.R1068H protein in HEK293 cells. Login to comment
5 Cholesterol efflux studies showed the efflux from TD fibroblasts and HEK293 cells expressing the mutant p.R1068H protein to be markedly reduced compared to wildtype. Login to comment
6 Localisation of the mutant p.R1068H protein in HEK293 cells showed intracellular retention of the protein indicating a defect in trafficking to the plasma membrane. Login to comment
7 Conclusion: Homology modeling of the ABCA1 protein showed that the p.R1068H mutation would likely disrupt the conformation of NBD-1. Login to comment
8 Functional studies of p.R1068H showed a lack of cholesterol efflux function due to defective trafficking to the plasma membrane, most likely caused by impaired oligomerisation. Login to comment
26 We previously reported a missense mutation (c.3516G>A) in exon 22 of the ABCA1 gene, causing a p.R1068H amino acid substitution in a highly conserved region of the ABCA1 protein [11]. Login to comment
28 Heterozygotes with the p.R1068H mutation displayed a wide range of HDL levels (0.48-1.22 mmol L-1) [11]. Login to comment
31 doi:10.1016/j.atherosclerosis.2011.06.019 model for the human ABCA1 protein in order to better predict the effect of the p.R1068H mutation. Login to comment
32 This was followed with direct testing of the functional impact of the p.R1068H mutation on the ABCA1 protein via cholesterol efflux assays and analysis of sub-cellular localisation. Login to comment
34 Materials and methods 2.1. Computational modeling The functional impact of the p.R1068H substitution was predicted with the PANTHER [12] and SIFT [13] programmes, using the NCBI reference sequence NP005493.2. Login to comment
38 Study subjects We have previously described the p.R1068H proband and her extended pedigree [11]. Login to comment
50 Mutagenic primers were used to introduce the c.3516G>A nucleotide change in exon 22 of the ABCA1 sequence causing the p.R1068H mutation. Login to comment
78 Results 3.1. Computational analysis of p.R1068H Previous computational analysis of the p.R1068H mutation using PolyPhen suggested that the mutation was 'probably damaging` [11], however, further analysis of the mutation here using PANTHER and SIFT predicted the mutation to be 'damaging` and 'tolerated`, respectively. Login to comment
109 Functional activity of the p.R1068H protein Cholesterol efflux assays in primary fibroblast cultures established from the TD subject showed a markedly reduced efflux compared to wildtype family members (0.9% versus 7.3%; Fig. 2A). Login to comment
110 Heterozygous carriers of the p.R1068H mutation displayed an intermediate level of cholesterol efflux (2.9%) which was significantly lower than wildtype. Login to comment
113 ABCA1 protein levels in both unstimulated and stimulated cells appeared unaffected by the presence of the p.R1068H mutation (Fig. 2C and D). Login to comment
114 This was confirmed by quantification of triplicate Western blots that showed no significant difference in ABCA1 protein levels in p.R1068H heterozygotes versus wildtype fibroblasts under unstimulated (Fig. 2E) or stimulated (Fig. 2F) conditions. Login to comment
116 The presence of the p.R1068H mutation did not affect ABCA1 expression, as the levels of wildtype R1068 and the mutant H1068 protein were comparable (Fig. 3A). Login to comment
117 Cholesterol efflux assays showed that HEK293 cells expressing the p.R1068H vector had markedly reduced cholesterol efflux compared to cells Fig. 3. Login to comment
119 Cultured HEK293 cells were transiently transfected with a GFP-tagged ABCA1 cDNA expression vector for either wildtype or p.R1068H. Login to comment
127 Indeed, there was no statistical difference in cholesterol efflux between nontransfected HEK293 cells and cells expressing the mutant p.R1068H protein (p = 0.163), again confirming that the p.R1068H mutation renders the ABCA1 protein completely dysfunctional. Login to comment
128 3.3. Sub-cellular localisation of the ABCA1 protein in vitro Confocal microscopy was used to investigate the effect of the p.R1068H mutation on localisation of the GFP-tagged ABCA1 protein. Login to comment
130 Wildtype ABCA1 protein primarily localised to the extracellular membrane as expected while the p.R1068H ABCA1 protein localised to intracellular regions of the cells indicative of a defect in trafficking of the mutant protein (Fig. 4). Login to comment
136 The p.R1068H mutation studied here was deemed to be damaging by PolyPhen and PANTHER, yet tolerated by SIFT. Login to comment
137 Indeed, SIFT predicts any residue substitution at position 1068 to be tolerated except R1068W which does not align with the observation that p.R1068H and the p.R1068C mutation are associated with extremely reduced HDL levels [11,23]. Login to comment
139 For these reasons, we constructed a model of the dimeric NBDs of ABCA1 based on the crystal structure of the E. coli HlyB ABC transporter in order to visualise the p.R1068H mutation in a three-dimensional context. Login to comment
141 The three dimensional model indicates potential ionic interactions between R1068 and two adjacent carboxylic acid containing residues, D1092 and E1093, which would be disrupted by the p.R1068H mutation. Login to comment
143 The p.R1068H mutation does not appear to affect expression or stability of the ABCA1 protein as shown by Western blot analysis of both primary human fibroblasts and transfected HEK293 cells. Login to comment
148 Preliminary studies investigating the oligomerisation state of the p.R1068H mutant showed only limited formation of a tetramer band (>800 kDa) and no dimer (Supplementary Fig. 1). Login to comment
150 Furthermore, higher molecular weight species were apparent with p.R1068H suggesting a tendency to aggregate. Login to comment
151 This could potentially explain why a much lower amount of the p.R1068H protein was detected on native gels compared to wildtype, despite similar amounts of ABCA1 being detected under denaturing conditions (Supplementary Fig. 1). Login to comment
152 Based on our model (Fig. 1B), we propose that the R1068H mutation disrupts the structure of the ABCA1 monomer around the ATP-binding site altering the conformation of NBD-1 to disrupt dimerisation and subsequent tetramer formation. Login to comment
155 The virtual absence of HDL in the TD proband would indicate that the p.R1068H mutation was highly detrimental to ABCA1 function. Login to comment
156 Functional testing of the p.R1068H mutation in primary fibroblast cell lines established from p.R1068H family members confirmed this, with the mutant protein failing to mediate efflux to apoA-I even after stimulation. Login to comment
157 Cholesterol efflux assays were also performed in HEK293 cells with cDNA expression vectors for either wildtype or p.R1068H ABCA1. Login to comment
158 These assays also confirmed that the p.R1068H protein was completely dysfunctional. Login to comment
160 HEK293 cells were transiently transfected with GFP-tagged ABCA1 cDNA expression vectors for either wildtype or p.R1068H. Login to comment
163 To conclude, functional studies confirm that the p.R1068H mutation produces a dysfunctional ABCA1 protein due to defective trafficking. Login to comment
166 Acknowledgements We wish to thank the p.R1068H family members for their participation in this study. Login to comment
51 Mutagenic primers were used to introduce the c.3516G>A nucleotide change in exon 22 of the ABCA1 sequence causing the p.R1068H mutation. Login to comment
79 Results 3.1. Computational analysis of p.R1068H Previous computational analysis of the p.R1068H mutation using PolyPhen suggested that the mutation was 'probably damaging` [11], however, further analysis of the mutation here using PANTHER and SIFT predicted the mutation to be 'damaging` and 'tolerated`, respectively. Login to comment
111 Heterozygous carriers of the p.R1068H mutation displayed an intermediate level of cholesterol efflux (2.9%) which was significantly lower than wildtype. Login to comment
115 This was confirmed by quantification of triplicate Western blots that showed no significant difference in ABCA1 protein levels in p.R1068H heterozygotes versus wildtype fibroblasts under unstimulated (Fig. 2E) or stimulated (Fig. 2F) conditions. Login to comment
118 Cholesterol efflux assays showed that HEK293 cells expressing the p.R1068H vector had markedly reduced cholesterol efflux compared to cells Fig. 3. Login to comment
120 Cultured HEK293 cells were transiently transfected with a GFP-tagged ABCA1 cDNA expression vector for either wildtype or p.R1068H. Login to comment

[hide] High-density lipoprotein reduces the human monocyt... Arterioscler Thromb Vasc Biol. 2008 Nov;28(11):2071-7. Epub 2008 Jul 10. Murphy AJ, Woollard KJ, Hoang A, Mukhamedova N, Stirzaker RA, McCormick SP, Remaley AT, Sviridov D, Chin-Dusting J
High-density lipoprotein reduces the human monocyte inflammatory response.
Arterioscler Thromb Vasc Biol. 2008 Nov;28(11):2071-7. Epub 2008 Jul 10., [PMID:18617650]

Abstract [show]
OBJECTIVE: Whereas the anti-inflammatory effects of high-density lipoprotein (HDL) on endothelial cells are well described, such effects on monocytes are less studied. METHODS AND RESULTS: Human monocytes were isolated from whole blood followed by assessment of CD11b activation/expression and cell adhesion under shear-flow. HDL caused a dose-dependent reduction in the activation of CD11b induced by PMA or receptor-dependent agonists. The constituent of HDL responsible for the antiinflammatory effects on CD11b activation was found to be apolipoprotein A-I (apoA-I). Cyclodextrin, but not cyclodextrin/cholesterol complex, also inhibited PMA-induced CD11b activation implicating cholesterol efflux as the main mechanism. This was further confirmed with the demonstration that cholesterol content of lipid rafts diminished after treatment with the cholesterol acceptors. Blocking ABCA1 with an anti-ABCA1 antibody abolished the effect of apoA-I. Furthermore, monocytes derived from a Tangier disease patient definitively confirmed the requirement of ABCA1 in apoA-I-mediated CD11b inhibition. The antiinflammatory effects of apoA-I were also observed in functional models including cell adhesion to an endothelial cell monolayer, monocytic spreading under shear flow, and transmigration. CONCLUSIONS: HDL and apoA-I exhibit an antiinflammatory effect on human monocytes by inhibiting activation of CD11b. ApoA-I acts through ABCA1, whereas HDL may act through several receptors.

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26 Study Subjects: Healthy and R1068H Tangier Family The study was approved by the Human Ethics committees of the Alfred Hospital and the University of Otago; informed consent was obtained from all donors. Login to comment
104 Tangier Patient Derived Monocytes For simplification of comparison between members of the R1068H Tangier family, results are expressed as percentage of CD11b expression after activation with PMA. Login to comment
138 Finally, we explored the antiinflammatory role of HDL and apoA-I in a Tangier disease subject along with a heterozygote subject and an unaffected member from the R1068H family.48 Tangier disease patients have a dysfunctional ABCA1 unable to support cholesterol efflux to apoA-I.49 The response of monocytes from the unaffected family member was similar to that of healthy subjects, monocytes from the heterozygote subject responded to both HDL and apoA-I albeit less than compared to monocytes of unaffected family member. Login to comment
145 Acknowledgments We acknowledge Rachel Brace for her efforts in helping with the blood collection and providing medical care for the R1068H family. Login to comment
19 Study Subjects: Healthy and R1068H Tangier Family The study was approved by the Human Ethics committees of the Alfred Hospital and the University of Otago; informed consent was obtained from all donors. Login to comment
97 Tangier Patient Derived Monocytes For simplification of comparison between members of the R1068H Tangier family, results are expressed as percentage of CD11b expression after activation with PMA. Login to comment
131 Finally, we explored the antiinflammatory role of HDL and apoA-I in a Tangier disease subject along with a heterozygote subject and an unaffected member from the R1068H family.48 Tangier disease patients have a dysfunctional ABCA1 unable to support cholesterol efflux to apoA-I.49 The response of monocytes from the unaffected family member was similar to that of healthy subjects, monocytes from the heterozygote subject responded to both HDL and apoA-I albeit less than compared to monocytes of unaffected family member. Login to comment

[hide] Novel rare mutations and promoter haplotypes in AB... Clin Genet. 2008 Feb;73(2):179-84. Slatter TL, Jones GT, Williams MJ, van Rij AM, McCormick SP
Novel rare mutations and promoter haplotypes in ABCA1 contribute to low-HDL-C levels.
Clin Genet. 2008 Feb;73(2):179-84., [PMID:18199144]

Abstract [show]
The ATP-binding cassette A1 (ABCA1) protein regulates plasma high-density lipoprotein (HDL) levels. Mutations in ABCA1 can cause HDL deficiency and increase the risk of premature coronary artery disease. Single nucleotide polymorphisms (SNPs) in ABCA1 are associated with variation in plasma HDL levels. We investigated the prevalence of mutations and common SNPs in ABCA1 in 154 low-HDL individuals and 102 high-HDL individuals. Mutations were identified in five of the low-HDL subjects, three having novel variants (I659V, R2004K, and A2028V) and two with a previously identified variant (R1068H). Analysis of four SNPs in the ABCA1 gene promoter (C-564T, G-407C, G-278C, and C-14T) identified the C-14T SNP and the TCCT haplotype to be over-represented in low-HDL individuals. The R1587K SNP was over-represented in low-HDL individuals, and the V825I and I883M SNPs over-represented in high-HDL individuals. We conclude that sequence variation in ABCA1 contributes significantly to variation in HDL levels.

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7 Mutations were identified in five of the low-HDL subjects, three having novel variants (I659V, R2004K, and A2028V) and two with a previously identified variant (R1068H). Login to comment
57 One mutation (R1068H), detected in two low-HDL subjects, had previously been identified in an Otago family with Tangier disease (25). Login to comment
59 The R1068H mutation was predicted to be Ôprobably damaging`, the R2004K mutation Ôpossibly damaging` and the I659V and A2028V mutations Ôbenign`. Login to comment
88 Of those subjects carrying ABCA1 mutations, two were heterozygous for the R1068H mutation that was previously identified in an Otago family with Tangier disease (25). Login to comment

[hide] Promoter haplotype of a new ABCA1 mutant influence... Atherosclerosis. 2006 Aug;187(2):393-400. Epub 2005 Oct 12. Slatter TL, Williams MJ, Frikke-Schmidt R, Tybjaerg-Hansen A, Morison IM, McCormick SP
Promoter haplotype of a new ABCA1 mutant influences expression of familial hypoalphalipoproteinemia.
Atherosclerosis. 2006 Aug;187(2):393-400. Epub 2005 Oct 12., [PMID:16225879]

Abstract [show]
Mutations in the ATP-binding cassette A1 (ABCA1) transporter cause the high-density lipoprotein (HDL) deficiency syndromes of Tangier disease and familial hypoalphalipoproteinemia (FHA). Between individuals carrying ABCA1 mutations, the expression of FHA can be highly variable. Using denaturing HPLC (dHPLC) and direct promoter sequencing we screened the ABCA1 gene of a family with Tangier disease and variable expression of FHA. A new mutation (R1068H) within the first ATP-binding domain was identified in homozygous form in the Tangier disease individual and was present in several family members. Haplotyping of both 1068H alleles in the proband showed homozygosity in the coding region, however, the maternal 1068H allele had three single nucleotide polymorphisms (SNPs) in the promoter previously reported to be associated with reduced ABCA1 expression and HDL levels. An analysis of HDL levels based on 1068H allele haplotype showed the paternal 1068H heterozygotes to have the expected low HDL levels (0.67+/-0.16mmol/L), while maternal 1068H heterozygotes showed normal HDL levels (1.18+/-0.14mmol/L). Haplotype analysis of the wildtype allele amongst heterozygotes showed no haplotype that was common to the paternal or maternal side. We propose that the paternal 1068H ABCA1 allele causes a negative effect on the function of the wildtype allele and is associated with low HDL levels. In contrast, the maternal 1068H allele has less effect and is associated with a relatively normal HDL level. We conclude that haplotypes of mutant ABCA1 alleles may contribute to the phenotypic variance shown between FHA individuals.

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3 A new mutation (R1068H) within the first ATP-binding domain was identified in homozygous form in the Tangier disease individual and was present in several family members. Login to comment
35 (A) Pedigree and genotype of the R1068H family. Login to comment
36 The proband with Tangier disease (represented by an arrow and filled circle) is homozygous for the R1068H mutation. Login to comment
40 (B) Detection of the R1068H mutation by dHPLC. Login to comment
41 The dHPLC profiles of the exon 22 amplicon of three members of the R1068H family are shown. Login to comment
44 (C) Detection of the R1068H mutation by DNA sequencing. Login to comment
47 The mutation predicts an arginine to histidine substitution at amino acid 1068 in the ABCA1 protein. Login to comment
83 Statistical analysis The Mann-Whitney test was used to compare the mean differences in lipid levels in the presence and absence of the R1068H mutation and in the presence of the maternal versus paternal 1068H allele in heterozygotes. Login to comment
89 An HDL-C measurement was not available Table 1 Plasma lipid levels and genotypes in the R1068H family Subject Sex Age TC TG LDL-C HDL-C R1068H II:2 M 76 5.40 1.20 3.50 1.37 -/- II:3 M 66 5.70 1.60 3.30 1.63 -/- II:4 F 67 6.07 2.62 3.75 1.12 +/- II:5a M 58 3.90 0.63 NT II:7 F 60 7.10 2.10 4.60 1.53 -/- II:8 M 66 5.10 2.60 3.30 0.60 +/- III:1 F 46 5.40 1.00 3.60 1.39 -/- III:3 F 42 3.50 1.50 2.80 0.04 +/+ III:4 M 38 4.00 0.60 2.50 1.22 +/- III:5 F 49 5.40 0.90 2.70 2.26 -/- III:6 M 48 6.50 2.02 4.60 1.00 -/- III:7 F 44 3.98 1.31 2.90 0.48 +/- III:8 F 40 7.50 1.42 5.85 1.08 -/- III:9 F 41 2.60 0.70 1.70 0.60 +/- III:10 F 37 4.50 1.10 3.10 0.90 +/- III:11 M 35 5.53 1.20 4.02 1.10 -/- IV:1 F 23 3.60 0.40 2.40 1.03 +/- IV:2 F 21 4.00 0.80 2.30 1.35 +/- IV:3 F 24 3.50 0.50 2.50 0.76 +/- TC indicates total cholesterol; TG, triglycerides; HDL-C, high-density cholesterol; LDL-C low-density cholesterol. All measurements are expressed in mmol/L. Login to comment
90 (-/-) wildtype; (+/-) heterozygous for the R1068H mutation; (+/+) homozygote for the R1068H mutation. Login to comment
104 (A) Schematic diagram showing the position of the R1068H mutation within the ATP-binding region of ABCA1. Login to comment
106 (B) Alignment of the ABCA protein sequences from different species around the R1068H region. Login to comment
111 A computer analysis of the effect of the R1068H mutation on ABCA1 function using PolyPhen (www.bork.embl-heidelberg.de/PolyPhen) predicts that the mutation is "possibly damaging". Login to comment
114 Sequencing of exon 22 from the nine family members containing the exon 22 heteroduplex revealed them all to be heterozygous for the R1068H mutation (Fig. 1A). Login to comment
115 The R1068H mutation was absent in all other family members. Login to comment
136 Table 2 Mean plasma lipid and lipoprotein levels in the R1068H family based on genotype R1068H Genotype Sex Age TC TG LDL-C HDL-C M F -/- (n = 8) 4 4 53 ± 14 6.07 ± 0.85 1.43 ± 0.45 4.02 ± 0.98 1.42 ± 0.40 -/+ (n = 9) 7 2 40 ± 17 4.15 ± 0.99* 1.18 ± 0.86 2.72 ± 0.61** 0.90 ± 0.31*** -/+m (n = 4) 1 4 37 ± 21 4.42 ± 1.12 1.10 ± 1.02 2.74 ± 0.68 1.18 ± 0.14 -/+p (n = 5) 1 3 42 ± 15 3.94 ± 0.96**** 1.24 ± 0.82 2.70 ± 0.63***** 0.67 ± 0.16******,******* TC indicates total cholesterol; TG, triglycerides; HDL-C, high-density cholesterol; LDL-C low-density cholesterol. All measurements are expressed in mmol/L. Login to comment
137 (-/-), wildtype; (+/-), heterozygous for the R1068H mutation; (-/+m), heterozygous for the maternal R1068H allele; (-/+p), heterozygous for the paternal R1068H allele. Login to comment
151 A further analysis of lipid levels based on genotype (Table 2) revealed a significantly lower mean total cholesterol, LDL-C and HDL-C in R1068H heterozygotes compared to unaffected family members (4.15 ± 0.99 mmol/L versus 6.07 ± 0.85 mmol/L, p = 0.003; 2.72 ± 0.61 mmol/L versus 4.02 ± 0.98 mmol/L, p = 0.006 and 0.90 ± 0.31 mmol/L versus 1.42 ± 0.40 mmol/L, p = 0.012, respectively). Login to comment
159 In the current study, we report a new mutation (R1068H) within the first ATP-binding cassette of ABCA1. Login to comment
165 Since the R1068H mutation is likely to produce a dysfunctional protein, one would expect it to be associated with FHA in the heterozygous state. Login to comment
187 Furthermore, a recent sequence analysis of the 3 untranslated region of both R1068H alleles has revealed a SNP(NCBISNPIDNumberrs4149341)inthepaternalallele (T Slatter, unpublished data), which strengthens the case for non-consanguinity. Login to comment

[hide] Variations on a gene: rare and common variants in ... Annu Rev Nutr. 2006;26:105-29. Brunham LR, Singaraja RR, Hayden MR
Variations on a gene: rare and common variants in ABCA1 and their impact on HDL cholesterol levels and atherosclerosis.
Annu Rev Nutr. 2006;26:105-29., [PMID:16704350]

Abstract [show]
Cholesterol and its metabolites play a variety of essential roles in living systems. Virtually all animal cells require cholesterol, which they acquire through synthesis or uptake, but only the liver can degrade cholesterol. The ABCA1 gene product regulates the rate-controlling step in the removal of cellular cholesterol: the efflux of cellular cholesterol and phospholipids to an apolipoprotein acceptor. Mutations in ABCA1, as seen in Tangier disease, result in accumulation of cellular cholesterol, reduced plasma high-density lipoprotein cholesterol, and increased risk for coronary artery disease. To date, more than 100 coding variants have been identified in ABCA1, and these variants result in a broad spectrum of biochemical and clinical phenotypes. Here we review genetic variation in ABCA1 and its critical role in cholesterol metabolism and atherosclerosis in the general population.

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555 Since a complete loss of function allele would be expected to result in a 50% reduction in HDL levels, a greater than 50% reduction in HDL is most likely explained by a dominant negative allele, in which TABLE 3 Patient phenotypes associated with heterozygous ABCA1 mutations Mutation HDL (mmol/L) HDL (% of control) Number of patients M1091T 0.48 ± 0.5 30 ± 30 4 G1216V 0.50 40 1 R2144X 0.56 ± 0.2 41 ± 18 12 R282X 0.52 41 1 R909X 0.59 ± 0.3 42 ± 19 5 K776N 0.55 ± 0.1 47 ± 5 2 R587W 0.61 ± 0.1 47 ± 8 7 S364C 0.60 48 1 P1065S 0.80 51 1 c-ter deletion 0.75 53 1 N1800H - 56.5 33 P85L 0.72 ± 0.4 57 ± 33 5 Del693L 0.79 ± 0.2 57 ± 15 8 D1289N 0.80 ± 0.1 59 ± 12 4 R2081W 0.80 ± 0.1 59 ± 12 4 2203X 0.80 ± 0.2 59 ± 20 4 DelED1893,4 0.77 ± 0.2 59 ± 18 8 2145X 0.82 ± 0.1 59 ± 9 4 A1046D 0.70 ± 0.1 60 ± 8 2 Q597R 0.82 ± 0.1 60 ± 5 5 C1477R 0.82 ± 0.2 61 ± 15 9 IVS25 + 1G > C 0.78 ± 0.1 62 ± 12 4 D1099Y 0.83 ± 0.3 63 ± 21 5 1552X 1.00 64 1 F2009S 0.82 ± 0.2 64 ± 19 6 R587W 0.86 ± 0.1 65 ± 17 2 R1068H 0.90 ± 0.3 67 ± 26 9 N935S 1.00 ± 0.3 74 ± 16 7 T929I 1.01 ± 0.2 76 ± 7 8 1284X 1.11 ± 0.2 83 ± 14 5 A937V 1.15 ± 0.6 85 ± 28 2 R1680W 1.22 ± 0.2 87 ± 17 3 635X 1.24 ± 0.5 90 ± 32 7 W590S 1.32 ± 0.6 103 ± 46 15 the mutant protein actually interferes with the activity of the remaining wild-type protein. Login to comment
562 Support for this concept comes from a recent study showing that HDL cholesterol levels in carriers of the R1068H mutation are significantly influenced by the ABCA1 promoter haplotype associated with the mutation (100). Login to comment

[hide] Functional rescue of mutant ABCA1 proteins by sodi... J Lipid Res. 2013 Jan;54(1):55-62. doi: 10.1194/jlr.M027193. Epub 2012 Oct 20. Sorrenson B, Suetani RJ, Williams MJ, Bickley VM, George PM, Jones GT, McCormick SP
Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.
J Lipid Res. 2013 Jan;54(1):55-62. doi: 10.1194/jlr.M027193. Epub 2012 Oct 20., [PMID:23087442]

Abstract [show]
Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

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16 Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Login to comment
18 In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Login to comment
53 The Pearson`s correlation coefficient between the GFP and AlexaFluor594 of the ABCA1 mutations were previously identified in low HDL-C subjects and included three uncharacterized mutations, p.I659V, p.R2004K, and p.A2028V (18) and three variants, p.R1068H, p.T1512M, and p.N1800H, known to have reduced localization and cholesterol efflux (19, 20). Login to comment
85 4-PBA rescues mutant ABCA1 localization and improves cholesterol efflux function in transfected HEK293 cells 4-PBA treatment was applied to the six uncharacterized ABCA1 mutants as well as to three mutants that we have previously shown to have reduced cholesterol efflux function, p.R1068H (19), p.T1512M (20), and p.N1800H (20, Fig. 1). Login to comment
109 Upon 4-PBA treatment, efflux function was significantly increased relative to the untreated level for the p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, and p.A2028V mutants (Fig. 3B). Login to comment
112 Treatment with 4-PBA induced a significant increase in colocalization for the p.A594T, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, and p.R2004K mutants. Treatment with 4-PBA did not affect the colocalization of the wild-type ABCA1-GFP protein (supplementary Fig. II). Login to comment
122 Fibroblasts were obtained from two p.R1068H heterozygote carriers (RH1 and RH2), two p.N1800H heterozygote carriers (NH1 and NH2), and two ABCA1 wild-type subjects (WT1 and WT2). Login to comment
126 However, the cholesterol efflux of the p.R1068H mutant was unable to be restored to wild-type levels. Login to comment
141 In contrast, there was a clear trend for increased efflux in the mutants fibroblast cells with statistically significant increases for the p.R1068H heterozygote RH1 (p < 0.05) and the p.N1800H heterozygote NH2 (p < 0.001). Login to comment
156 Fibroblast cultures established from wild-type (WT1, WT2), p.R1068H carriers (RH1, RH2) and p.N1800H carriers (NH1, NH2) were treated with 10 mM 4-PBA for 24 h. Login to comment
164 For example, the p.Y1767D mutant showed a much enhanced localization and dramatic increase in cholesterol efflux after 4-PBA treatment whereas the efflux function for the p.R1068H mutant remained low despite showing a similar enhancement in localization. Login to comment
165 The p.R1068H mutant, located in the first ATP binding domain, has been shown to be defective in oligomerisation (19) and it is likely that although localization was improved for this mutant with 4-PBA treatment, the oligomerisation and therefore efflux function remained defective. Login to comment
185 The magnitude by which 4-PBA rescued function was mutant-dependent as restored localization did not always result in a restoration of function, as was the case for the p.R1068H mutant. Login to comment
188 For two of the most dramatically affected mutants, p.R1068H and p.N1800H, a functional improvement with 4-PBA treatment was also confirmed ex vivo using primary fibroblast cells. Login to comment
201 The authors thank the p.R1068H and p.N1800H family members for their participation in this study. We are grateful to Professor Christiane Albrecht for kindly providing the pCIneo-ABCA1-GFP expression vector. Login to comment

[hide] Subfraction analysis of circulating lipoproteins i... Clin Chim Acta. 2016 Jan 15;452:167-72. doi: 10.1016/j.cca.2015.11.021. Epub 2015 Nov 23. Murano T, Yamaguchi T, Tatsuno I, Suzuki M, Noike H, Takanami T, Yoshida T, Suzuki M, Hashimoto R, Maeno T, Terai K, Tokuyama W, Hiruta N, Schneider WJ, Bujo H
Subfraction analysis of circulating lipoproteins in a patient with Tangier disease due to a novel ABCA1 mutation.
Clin Chim Acta. 2016 Jan 15;452:167-72. doi: 10.1016/j.cca.2015.11.021. Epub 2015 Nov 23., [PMID:26616730]

Abstract [show]
Tangier disease, characterized by low or absent high-density lipoprotein (HDL), is a rare hereditary lipid storage disorder associated with frequent, but not obligatory, severe premature atherosclerosis due to disturbed reverse cholesterol transport from tissues. The reasons for the heterogeneity in atherogenicity in certain dyslipidemias have not been fully elucidated. Here, using high-performance liquid chromatography with a gel filtration column (HPLC-GFC), we have studied the lipoprotein profile of a 17-year old male patient with Tangier disease who to date has not developed manifest coronary atherosclerosis. The patient was shown to be homozygous for a novel mutation (Leu1097Pro) in the central cytoplasmic region of ATP-binding cassette transporter A1 (ABCA1). Serum total and HDL-cholesterol levels were 59mg/dl and 2mg/dl, respectively. Lipoprotein electrophoretic analyses on agarose and polyacrylamide gels showed the presence of massively abnormal lipoproteins. Further analysis by HPLC-GFC identified significant amounts of lipoproteins in low-density lipoprotein (LDL) subfractions. The lipoprotein particles found in the peak subfraction were smaller than normal LDL, were rich in triglycerides, but poor in cholesterol and phospholipids. These findings in an adolescent Tangier patient suggest that patients in whom these triglyceride-rich, cholesterol- and phospholipid-poor LDL-type particles accumulate over time, would experience an increased propensity for developing atherosclerosis.

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123 Arg1068His has been identified to cause Tangier disease [24, 25]. Login to comment