ABCMdb

PMID: 11785958

Nishida Y, Hirano K, Tsukamoto K, Nagano M, Ikegami C, Roomp K, Ishihara M, Sakane N, Zhang Z, Tsujii Ki K, Matsuyama A, Ohama T, Matsuura F, Ishigami M, Sakai N, Hiraoka H, Hattori H, Wellington C, Yoshida Y, Misugi S, Hayden MR, Egashira T, Yamashita S, Matsuzawa Y
Expression and functional analyses of novel mutations of ATP-binding cassette transporter-1 in Japanese patients with high-density lipoprotein deficiency.
Biochem Biophys Res Commun. 2002 Jan 18;290(2):713-21., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCA1 p.Arg1851* ABCA1 p.Ala255Thr ABCA1 p.Asn1611Asp In order to know the molecular basis for FHD, we characterized three different ABCA1 mutations associated with FHD (G1158A/ A255T, C5946T/R1851X, and A5226G/N1611D) with respect to their expression in the passaged fibroblasts from the patients and in the cells transfected with the mutated cDNAs. Login to comment 2 ABCA1 p.Ala255Thr Fibroblasts from the all patients showed markedly decreased cholesterol efflux to apolipoprotein (apo)-Al. In the fibroblasts homozygous for G1158A/A255T, the immunoreactive mass of ABCA1 could not be detected, even when stimulated by 9-cis- retinoic acid and 22-R-hydroxycholesterol. Login to comment 3 ABCA1 p.Arg1851* In the fibroblasts homozygous for C5946T/R1851X, ABCA1 mRNA was comparable. Login to comment 4 ABCA1 p.Arg1851* ABCA1 p.Arg1851* Because the mutant ABCA1 protein (R1851X) was predicted to lack the epitope for the antibody used, we transfected FLAG-tagged truncated mutant (R1851X/ABCA1-FLAG) cDNA into Cos-7 cells, showing that the mutant protein expression was markedly reduced. Login to comment 5 ABCA1 p.Asn1611Asp The expression of N1611D ABCA1 protein was comparable in both fibroblasts and overexpressing cells, although cholesterol efflux from the cells was markedly reduced. Login to comment 58 ABCA1 p.Arg1851* ABCA1 p.Ala255Thr ABCA1 p.Asn1611Asp TABLE 1 Clinical Profiles of Patients with Familial HDL Deficiency Case 1 Case 2 Case 3 ABCA1 substitutions found (nt/aa) G1158A/A255T C5946T/R1851X A5226G/N1611D Age (years)/sex (M, F) 56/M 71/F 53/F Total cholesterol (mmol/L) 0.72 1.47 2.7 HDL-cholesterol (mmol/L) 0.16 0.05 0.11 Triglyceride (mmol/L) 2.6 3.27 1.75 Apo-Al (mg/dL) 3.9 5.0 11.0 Atherosclerosis af9; af9; af9; Typical TD phenotype af9; af9; afa; Cholesterol efflux (% of control) 5.0 2.0 7.0 Note. Login to comment 59 ABCA1 p.Arg1851* ABCA1 p.Ala255Thr ABCA1 p.Asn1611Asp TABLE 1 Clinical Profiles of Patients with Familial HDL Deficiency Case 1 Case 2 Case 3 ABCA1 substitutions found (nt/aa) G1158A/A255T C5946T/R1851X A5226G/N1611D Age (years)/sex (M, F) 56/M 71/F 53/F Total cholesterol (mmol/L) 0.72 1.47 2.7 HDL-cholesterol (mmol/L) 0.16 0.05 0.11 Triglyceride (mmol/L) 2.6 3.27 1.75 Apo-Al (mg/dL) 3.9 5.0 11.0 Atherosclerosis ϩ ϩ ϩ Typical TD phenotype ϩ ϩ - Cholesterol efflux (% of control) 5.0 2.0 7.0 Note. Login to comment 102 ABCA1 p.Ala255Thr Recently, the existence of extracellular loop at the amino-terminus, where the A255T may be located, was proposed (Refs. Login to comment 103 ABCA1 p.Ala255Thr Recently, the existence of extracellular loop at the amino-terminus, where the A255T may be located, was proposed (Refs. Login to comment 104 ABCA1 p.Arg1851* R1851X appeared to lack the second nucleotide binding domain. Login to comment 105 ABCA1 p.Arg1851* ABCA1 p.Asn1611Asp R1851X appeared to lack the second nucleotide binding domain. Login to comment 106 ABCA1 p.Asn1611Asp N1611D appeared to be located in the middle of extracellular loop from codons 1369 to 1654 (Ref. Login to comment 107 ABCA1 p.Ala255Thr We could not find the G1158A/A255T substitution in 48 unrelated Americans or 176 Japanese control subjects (data not shown). Login to comment 109 ABCA1 p.Arg1851* ABCA1 p.Ala255Thr We could not find the G1158A/A255T substitution in 48 unrelated Americans or 176 Japanese control subjects (data not shown). Login to comment 110 ABCA1 p.Asn1611Asp Case 3 was homozygous for a A to G substitution at nucleotide position 5226, causing a change of amino acid from asparagine (N) to aspartic acid (D) at codon 1611 (N1611D), as shown in Fig. 1C. The codon 1611 appeared to be located around the proximal part of the second transmembrane domain (Fig. 2). Login to comment 111 ABCA1 p.Arg1851* This mutation caused a premature stop at the codon 1851 (R1851X), resulting that the predicted ABCA1 protein was truncated from lacking the second nucleotide binding domain (NBD) (Figs. 1B and 2). Login to comment 112 ABCA1 p.Asn1611Asp Case 3 was homozygous for a A to G substitution at nucleotide position 5226, causing a change of amino acid from asparagine (N) to aspartic acid (D) at codon 1611 (N1611D), as shown in Fig. 1C. The codon 1611 appeared to be located around the proximal part of the second transmembrane domain (Fig. 2). Login to comment 113 ABCA1 p.Ala255Thr Fibroblasts from Case 1 (Ho/A255T) had markedly low levels of ABCA1 mRNA in the condition without the stimulation, whereas these levels became comparable after the stimulation. Login to comment 114 ABCA1 p.Arg1851* ABCA1 p.Asn1611Asp In Cases 2 (Ho/ R1851X) and 3 (Ho/N1611D), the expression of mRNA did not appear to be altered with and without the stimulation (Figs. 3A and 3B). Login to comment 115 ABCA1 p.Ala255Thr Fibroblasts from Case 1 (Ho/A255T) had markedly low levels of ABCA1 mRNA in the condition without the stimulation, whereas these levels became comparable after the stimulation. Login to comment 116 ABCA1 p.Arg1851* ABCA1 p.Asn1611Asp In Cases 2 (Ho/ R1851X) and 3 (Ho/N1611D), the expression of mRNA did not appear to be altered with and without the stimulation (Figs. 3A and 3B). Login to comment 117 ABCA1 p.Ala255Thr It was likely that no-trace amount of ABCA1 protein contributed to the FHD phenotype in Case 1 (Ho/A255T). Login to comment 118 ABCA1 p.Arg1851* ABCA1 p.Arg1851* As it was noted that the predicted mutant ABCA1 (R1851X) lacked the epitope for the antibody we used, we could not see any immunoreactive mass in fibroblasts from Case 2 (Ho/ R1851X). Login to comment 119 ABCA1 p.Ala255Thr ABCA1 p.Asn1611Asp It was likely that no-trace amount of ABCA1 protein contributed to the FHD phenotype in Case 1 (Ho/A255T). Login to comment 120 ABCA1 p.Arg1851* ABCA1 p.Arg1851* As it was noted that the predicted mutant ABCA1 (R1851X) lacked the epitope for the antibody we used, we could not see any immunoreactive mass in fibroblasts from Case 2 (Ho/ R1851X). Login to comment 121 ABCA1 p.Asn1611Asp We could observe the comparable expression of ABCA1 protein in fibroblasts from Case 3 (Ho/ N1611D). Login to comment 122 ABCA1 p.Ala255Thr ABCA1 p.Ala255Thr It was noted that we obtained the comparable protein expression of A255T/ABCA1-FLAG cDNA construct, though we could observe no ABCA1 protein in the fibroblasts from Case 1 (Ho/A255T). Login to comment 123 ABCA1 p.Arg1851* It was striking that the protein expression of cDNA construct carrying the truncated mutation (R1851X) was extremely low, though we could see the expected band with smaller size. Login to comment 124 ABCA1 p.Ala255Thr ABCA1 p.Ala255Thr It was noted that we obtained the comparable protein expression of A255T/ABCA1-FLAG cDNA construct, though we could observe no ABCA1 protein in the fibroblasts from Case 1 (Ho/A255T). Login to comment 125 ABCA1 p.Arg1851* It was striking that the protein expression of cDNA construct carrying the truncated mutation (R1851X) was extremely low, though we could see the expected band with smaller size. Login to comment 127 ABCA1 p.Ala255Thr We could detect a significant amount of cholesterol efflux from cells expressing A255T/ABCA1-FLAG. Login to comment 128 ABCA1 p.Arg1851* As expected, no significant cholesterol efflux could be detected from cells transfected with R1851X/ ABCA1-FLAG cDNA. Login to comment 129 ABCA1 p.Ala255Thr ABCA1 p.Asn1611Asp We could detect a significant amount of cholesterol efflux from cells expressing A255T/ABCA1-FLAG. Login to comment 130 ABCA1 p.Arg1851* As expected, no significant cholesterol efflux could be detected from cells transfected with R1851X/ ABCA1-FLAG cDNA. Login to comment 131 ABCA1 p.Asn1611Asp From cells expressing N1611D/ ABCA1-FLAG, no significant cholesterol efflux could be observed, although we obtained the comparable expression of the mutant protein (Figs. 4A and 4B). Login to comment 133 ABCA1 p.Ala255Thr In Case 1, we found a novel substitution (G1158A/ A255T). Login to comment 135 ABCA1 p.Ala255Thr ABCA1 p.Ala255Thr In Case 1, we found a novel substitution (G1158A/ A255T). Login to comment 137 ABCA1 p.Ala255Thr Because the recent clinical mutational analyses in patients with TD appeared to show that many potential loss-of-function mutations are located around this lesion (8-11), we had initially speculated that this predicted mutant ABCA1 protein (A255T) could have any dysfunction of this loop. Login to comment 139 ABCA1 p.Ala255Thr The ABCA1 cDNA construct carrying the G1158A/A255T substitution appeared to be functionally normal in the overexpressing cells (Figs. 5A and 5B). Login to comment 141 ABCA1 p.Ala255Thr The ABCA1 cDNA construct carrying the G1158A/A255T substitution appeared to be functionally normal in the overexpressing cells (Figs. 5A and 5B). Login to comment 143 ABCA1 p.Arg1851* In Case 2, we found another novel mutation resulting in the presence of truncated ABCA1 which lacks the second NBD (R1851X). Login to comment 144 ABCA1 p.Arg1851* Our transfection study demonstrated an interesting data to show that the expression level of truncated mutant (R1851X) was much lower than those of other mutants and wild type tested. Login to comment 145 ABCA1 p.Arg1851* In Case 2, we found another novel mutation resulting in the presence of truncated ABCA1 which lacks the second NBD (R1851X). Login to comment 146 ABCA1 p.Arg1851* Our transfection study demonstrated an interesting data to show that the expression level of truncated mutant (R1851X) was much lower than those of other mutants and wild type tested. Login to comment 149 ABCA1 p.Arg1851* Based upon the results of our transfection experiment (Fig. 3), we speculated that ABCA1 expression was severely impaired at the protein level in Case 2 (Ho/R1851X). Login to comment 151 ABCA1 p.Arg1851* Based upon the results of our transfection experiment (Fig. 3), we speculated that ABCA1 expression was severely impaired at the protein level in Case 2 (Ho/R1851X). Login to comment 165 ABCA1 p.Asn1611Asp In Case 3, who did not have typical TD phenotype such as orange tonsils and hepatosplenomegaly, we found the other novel substitution (A5226G/N1611D). Login to comment 167 ABCA1 p.Asn1611Asp In Case 3, who did not have typical TD phenotype such as orange tonsils and hepatosplenomegaly, we found the other novel substitution (A5226G/N1611D). Login to comment