ABCMdb

PMID: 19019193

Pisciotta L, Bocchi L, Candini C, Sallo R, Zanotti I, Fasano T, Chakrapani A, Bates T, Bonardi R, Cantafora A, Ball S, Watts G, Bernini F, Calandra S, Bertolini S
Severe HDL deficiency due to novel defects in the ABCA1 transporter.
J Intern Med. 2009 Mar;265(3):359-72. Epub 2008 Oct 25., [PubMed]

Sentences

No. Mutations Sentence Comment

13 ABCA1 p.Ala1046Asp Two patients were homozygous for mutations in the coding region of the ABCA1 gene, resulting in an amino acid substitution (p.A1046D) and a truncated protein (p.I74YFsX76). Login to comment 16 ABCA1 p.Arg587Trp ABCA1 p.Trp1699Cys The fourth patient, with preclinical atherosclerosis, was a compound heterozygote for two missense mutations (p.R587W/p.W1699C). Login to comment 17 ABCA1 p.Arg587Trp ABCA1 p.Ala1046Asp ABCA1-mediated cholesterol efflux was abolished in fibroblasts from patients with p.A1046D and del p.D1567_K1591 mutants and in fibroblasts homozygous for p.R587W. Login to comment 19 ABCA1 p.Trp1699Cys The mutant p.W1699C was largely retained in the endoplasmic reticulum, when expressed in HEK293 cells. Login to comment 75 ABCA1 p.Trp1699Cys Mutant ABCA1-GFP cDNA (corresponding to the naturally occurring mutant p.W1699C) was obtained by site-directed mutagenesis (Stratagene, La Jolla, CA, USA). Login to comment 93 ABCA1 p.Ala1046Asp The proband was found to be homozygous for a C>A transversion in exon 22 (c.3137 C>A) of the ABCA1 gene, causing a nonconservative amino acid substitution (p.A1046D). Login to comment 97 ABCA1 p.Ala1046Asp He had a mild dysmorphism (coarse facial features), developmental and speech delay, abnormal gait, congenital cataracts (removed at 2 weeks of life), hypertrophied tonsils and adenoids (tonsillo-adenoidectomy at 2 years of age), yellowish pharyngeal deposits, no Table 1 ABCA1 alleles, demographics and plasma lipid, lipoprotein and apolipoprotein concentrations in members of the four families Subject ABCA1 gene alleles Age (years) Sex BMI (kg m)2 ) TC (mmol L)1 ) LDL-C (mmol L)1 ) HDL-C (mmol L)1 ) TG (mmol L)1 ) Apo A-I (mg dL)1 ) Apo B (mg dL)1 ) APOE genotype CAD Family 1 I.1 W / W 74 M 24.0 5.33 3.38 1.47 1.02 - - - I.2 M1 / M1 72 M 23.0 1.24 0.75 0.08 1.29 <5 59 33 + Family 2 I.2 M2 / W 34 F 34.0 2.51 1.40 0.70 0.91 - - - II.1 M2 / M2 7 M 14.6 1.89 0.90 0.10 1.93 <5 - 34 Family 3 I.1 M3 / W 33 M - 5.61 ND 0.77 ND 88 141 33 I.2 M3 / W 27 F - 3.26 ND 1.08 ND 126 60 33 II.1 M3 / M3 4 M - 1.47 0.95 0.02 1.07 <5 82 33 Family 4 I.1 M4 / W 70 M - 8.01 6.30 1.19 1.13 145 160 33 +++ I.2 M5 / W 68 F - 6.69 4.76 1.24 1.50 153 145 33 I.4 M5 / W 67 F - 5.48 3.82 1.24 0.90 151 89 33 II.2 M4 / W 47 F - 6.59 4.34 1.19 2.29 148 129 33 II.3 M4 / M5 42 M - 4.70 3.98 0.10 1.35 <5 114 33 II.4 M5 / W 48 F - 3.64 2.76 0.59 0.60 92 73 33 + II.5 W / W 47 F - 4.70 3.05 1.24 0.90 150 80 23 II.6 M5 / W 42 M - 4.83 3.64 0.54 1.40 78 85 23 + II.7 M5 / W 41 F - 3.89 2.02 1.08 0.60 138 65 23 III.1 M4 / W 24 F - 3.70 1.65 1.73 0.70 170 71 34 W, ABCA1 wild type allele; M, ABCA1 mutant allele: M1 (p.A1046D); M2 (del. Login to comment 98 ABCA1 p.Arg587Trp ABCA1 p.Trp1699Cys p.D1567_K1591); M3 (p.I74YFsX76); M4 (p.W1699C); M5 (p.R587W); ND, not determined. Login to comment 147 ABCA1 p.Arg587Trp ABCA1 p.Trp1699Cys The proband was found to be a compound heterozygote for the following mutations of the ABCA1 gene: (i) a C>T transition in exon 14 (c.1759 C>T, p.R587W), inherited from the mother; (ii) a G>T transversion in exon 37 (c.5097 G>T, p.W1699C), inherited from the father (Fig. S3). Login to comment 149 ABCA1 p.Trp1699Cys The sister (subject II.2) and the niece (subject III.1) of the proband were heterozygous for the p.W1699C mutation. Login to comment 150 ABCA1 p.Arg587Trp The sister of the proband`s mother (subject I.4) and her children (subjects II.4, II.6 and II.7) were carriers of the p.R587W mutation. Login to comment 155 ABCA1 p.Ala1046Asp Cholesterol efflux from cultured fibroblasts ABCA1-mediated cholesterol efflux was evaluated in fibroblasts from the probands of families 1 and 2, homozygous for the p.A1046D substitution and for the splice site mutation in intron 35 (c.4773 + 1g>a, del p.D1567_K1591) respectively. Login to comment 157 ABCA1 p.Arg587Trp The cholesterol efflux in fibroblasts of these probands was similar to that observed in fibroblasts of another patient with severe HDL deficiency (homozygous for the p.R587W mutation) we had previously identified [24] (Fig. 3). Login to comment 158 ABCA1 p.Arg587Trp Enrichment of cholesterol in fibroblast plasma membrane We also measured the increase in cholesterol content in the fibroblast plasma membrane from the probands of families 1 and 2 and from the patient homozygous for p.R587W mutation, induced by the stimulation of ABCA1 expression with 9-cis-retinoic acid and 22-hydroxycholesterol. Login to comment 161 ABCA1 p.Arg587Trp In the probands` fibroblasts and in the fibroblasts of the patient homozygous for the ABCA1 p.R587W mutation, the increase in membrane cholesterol varied considerably. Login to comment 162 ABCA1 p.Arg587Trp ABCA1 p.Ala1046Asp In the p.A1046D (family 1) and homozygous p.R587W cells this increase was significant, although much lower than that of control cells, whilst in c.4773 + 1g>a mutant cells (family 2) no increase in membrane cholesterol was observed. Login to comment 168 ABCA1 p.Arg587Trp Figure 5 also shows (lane 3) that the ABCA1 protein in fibroblasts from the previously reported patient [24] homozygous for p.R587W (the missense mutation found in the compound heterozygote of family 4) is clearly detect- Fig. 3 3 H-cholesterol efflux to Apo A-I in skin fibroblasts under basal conditions and following stimulation of ABCA1 gene expression with 22-hydroxycholesterol and 9-cis-retinoic acid (22OH / cRA). Login to comment 169 ABCA1 p.Arg587Trp ABCA1 p.Ala1046Asp Ctrl, control cells; p.R587W, fibroblasts from a homozygous patient reported previously [24]; p.A1046D, fibroblasts from the proband of family 1; del p.D1567_K1591, fibroblasts of the proband of family 2. able, even though its content appears to be lower than that seen in control fibroblasts. Login to comment 181 ABCA1 p.Arg587Trp ABCA1 p.Ala1046Asp Ctrl, control cells; p.R587W, fibroblasts from a homozygous patient reported previously [24]; p.A1046D, fibroblasts from the proband of family 1; del p.D1567_K1591, fibroblasts of the proband of family 2. Login to comment 184 ABCA1 p.Ala1046Asp Lanes 1 and 2: control fibroblasts before (lane 1) and after (lane 2) stimulation of ABCA1 gene expression with 22OH / cRA; lane 3: fibroblasts from a homozygous patient for p.R578W mutation reported previously [24]; lane 4: fibroblasts of a compound heterozygote for ABCA1 null alleles reported previously [21]; lane 5: p.A1046D fibroblasts from the proband of family 1; lane 6: del p.D1567_K1591 fibroblasts from the proband of family 2. Login to comment 187 ABCA1 p.Trp1699Cys Finally the mutant ABCA1-GFP p.W1699C (found in the compound heterozygote of family 4) was largely retained in the cytoplasm (Fig. 6c), mainly in the endoplasmic reticulum as demonstrated by the co-localization with calnexin (Fig. 6c, right panel). Login to comment 197 ABCA1 p.Cys1477Arg ABCA1 p.Ser1506Leu Previous studies have shown that two missense ABCA1 mutants (p.C1477R and p.S1506L) located in the second extracellular loop, when expressed in transfected cells, exhibited a dramatic reduction in the interaction capacity with Apo A-I [27]. Login to comment 200 ABCA1 p.Arg1901Ser We previously reported a 16-year-old girl with severe HDL deficiency due to ABCA1 mutations (p.H160FsX173/p.R1901S) who had a mild mental retardation and radial-ulnar synostosis [18]. Login to comment 203 ABCA1 p.Trp1699Cys HEK293 cells were transiently transfected with pcDNA3.1 ABCA1-GFP wild type (a) ABCA1-GFP del p.K422_K1524 (an artificial mutant containing a large deletion spanning from exon 11 to exon 33) (b) and ABCA1-GFP p.W1699C (c) mutant vectors. Login to comment 210 ABCA1 p.Ala1046Asp The proband of family 1 was homozygous for a missense mutation (p.A1046D) previously reported by Wang et al. [31] in a kindred with HDL deficiency, which included three compound heterozygotes (whose second mutant allele was a nucleotide insertion in exon 34 - c.4630 ins A - causing a frameshift) and two heterozygotes for this mutation. Login to comment 211 ABCA1 p.Ala1046Asp Previous in vitro experiments have shown that the p.A1046D mutation, which occurs in the intracellular domain between the first Walker A and B motifs, severely impairs cholesterol efflux from transfected cells, even if it does not abolish the localization of ABCA1 in the plasma membrane, where it retains a residual capacity to bind Apo A-I [32]. Login to comment 215 ABCA1 p.Arg587Trp ABCA1 p.Trp1699Cys The proband of family 4 was a compound heterozygote carrying two missense mutations (p.R587W and p.W1699C). Login to comment 216 ABCA1 p.Arg587Trp The p.R587W mutation was first reported by Lawn et al. [33] in a compound heterozygote with HDL deficiency, in whom the second allele failed to produce detectable mRNA. Login to comment 218 ABCA1 p.Arg587Trp Previous in vitro experiments in transfected cells expressing the p.R587W mutant (designed to define the alteration in the ABCA1 pathway) were somewhat inconsistent. Login to comment 219 ABCA1 p.Arg587Trp Fitzgerald et al. [27] showed that the p.R587W mutant reached the cell surface just like its wild type counterpart, but showed a 50% reduction in the cross-linking efficiency to Apo A-I. Login to comment 220 ABCA1 p.Arg587Trp Singaraja et al. [32] showed that the p.R587W mutation prevents the migration of the protein to the plasma membrane, causing a 75% decrease in ABCA1-mediated cholesterol efflux. Login to comment 221 ABCA1 p.Arg587Trp In our patient homozygous for the p.R587W mutation [24] the ABCA1 protein content was lower than that observed in control fibroblasts (Fig. 5, lane 3), possibly suggesting an increased rate of intracellular degradation (an event expected if the mutant protein is retained, at least in part, in the endoplasmic reticulum) [32]. Login to comment 223 ABCA1 p.Trp1699Cys The second missense mutation (p.W1699C) found in the proband of family 4 is a novel mutation, expected to have a deleterious effect on ABCA1 function, as indicated by computational analysis with PolyPhen, PANTHER and SIFT algorithms (Table S2). Login to comment 227 ABCA1 p.Trp1699Cys ABCA1 p.Trp1699Cys As an alternative, we expressed the p.W1699C mutant (ABCA1-GFP-W1699C) in HEK293 cells and examined its intracellular localization (Fig. 6c). Login to comment 229 ABCA1 p.Trp1699Cys It is possible that the p.W1699C mutant retains some residual function, as the plasma HDL-C levels found in the three family members carrying this mutation were not as low as might be expected in carriers of null ABCA1 alleles. Login to comment