Screening the Expression of ABCB6 in Erythrocytes Reveals an Unexpectedly High Frequency of Lan Mutations in Healthy Individuals

Lan is a high-incidence blood group antigen expressed in more than 99.9% of the population. Identification of the human ABC transporter ABCB6 as the molecular basis of Lan has opened the way for studies assessing the relation of ABCB6 function and expression to health and disease. To date, 34 ABCB6 sequence variants have been described in association with reduced ABCB6 expression based on the genotyping of stored blood showing weak or no reactivity with anti-Lan antibodies. In the present study we examined the red blood cell (RBC) surface expression of ABCB6 by quantitative flow cytometry in a cohort of 47 healthy individuals. Sequencing of the entire coding region of the ABCB6 gene in low RBC ABCB6 expressors identified a new allele (IVS9+1G>A, affecting a putative splice site at the boundary of exon 9) and two nonsynonymous SNPs listed in the SNP database (R192Q (rs150221689) and G588 S (rs145526996)). The R192Q mutation showed co-segregation with reduced RBC ABCB6 expression in a family, and we found the G588 S mutation in a compound heterozygous individual with undetectable ABCB6 expression, suggesting that both mutations result in weak or no expression of ABCB6 on RBCs. Analysis of the intracellular expression pattern in HeLa cells by confocal microscopy indicated that these mutations do not compromise overall expression or the endolysosomal localization of ABCB6. Genotyping of two large cohorts, containing 235 and 1039 unrelated volunteers, confirmed the high allele frequency of Lan-mutations. Our results suggest that genetic variants linked to lower or absent cell surface expression of ABCB6/Langereis may be more common than previously thought.This work was supported by the Lendulet Program of the Hungarian Academy of Sciences (GS), OTKA 83533 and by the Polish POIG grant 01.01.02-10-005/08 TESTOPLEK, supported by the EU through the European Regional Development Fund. Hajnalka Andrikovics is a recipient of the Janos Bolyai Research Scholarship from the Hungarian Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Dr. Camilo Toro and Dr. William Gahl of the NIH Undiagnosed Diseases Program for an affected patient specimen; that work was supported by the Intramural Research Program of the National Human Genome Research Institute and the Office of the Director of the NIH. We thank Lionel Arnaud (National Institute of Blood Transfusion (INTS), Paris, France) for helpful discussions

Characterization of ABCB6 mutations responsible for the Lan- blood type.

<p>RBC membrane lysates (80 ug/lane) were prepared from a compound heterozygote individual carrying the R276W and the G588S mutations (lane 1); homozygotes carrying the R192W mutation (c.574C>T, p.Arg192Trp); lanes 2, 3); or individuals carrying two wild-type alleles (lanes 4, 5). ABCB6 expression is revealed by the ABCB6-567 monoclonal antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111590#pone.0111590-Paterson1" target="_blank">[9]</a>, Band III is shown as loading control.</p

Molecular genetics of the <i>ABCB6</i> gene.

<p>Abbreviations: DUH: dyschromatosis universalis hereditaria; del: deletion; dup: duplication; fs: frameshift; ins: insertion; N.D.:not determined; rs: RefSNP.</p><p>Molecular genetics of the <i>ABCB6</i> gene.</p

Erythrocytic ABCB6 expression in 47 healthy individuals.

<p>Histogram illustrating the distribution of erythrocytic ABCB6 expression in 47 unrelated healthy individuals, as measured by a quantitative FACS assay using the OSK43 monoclonal antibody specifically recognizing a surface epitope of ABCB6 (see Methods). The four individuals exhibiting significantly lower ABCB6 levels (below the 10^th percentile) are labeled by asterisks.</p

Pedigree of a family carrying the ABCB6 R192Q mutation– co-segregation of the heterozygous mutation with reduced erythrocytic ABCB6 expression levels.

<p>Blood samples obtained from the family members of the healthy volunteer proband carrying the R192Q ABCB6 mutation (indicated with black arrow) were analyzed for ABCB6 expression and the presence of the mutation. The upper panel represents a heterozygous R192Q (c.575G>A) sequence. The lower panel shows the segregation of R192Q heterozygosity (marked with green half circles and squares for females and males, respectively) with reduced ABCB6 expression in erythrocytes (values in parentheses).</p

Determination of the subcellular localization of ABCB6 variants.

<p>A. Double immunofluorescence labeling and laser-scanning confocal microscopy images of HeLa cells overexpressing ABCB6-HA, ABCB6-HA-G588S, ABCB6-HA-R192Q or ABCB6-HA-R192W, probed with the ABCB6-567 antibody (green) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111590#pone.0111590-Paterson1" target="_blank">[9]</a> in the context of LysoTracker (red) or Calnexin (red). B. Colocalization of ABCB6-variants with various intracellular markers. Bars represesent averaged Pearson’s coefficients +/− standard deviation for each subcellular marker from 5 random pictures. Blue: ABCB6-HA; red: ABCB6-HA-G588S; green: ABCB6-HA-R192Q; purple: ABCB6-HA-R192W.</p

Detection of relative plasma membrane ABCB6 levels by flow cytometry.

<p>K562 cells were stably transduced with retroviral constructs encoding wild-type (WT), or mutated ABCB6 variants (R192W, R192Q, G588S). Cell surface (A, C) and total (B, D) ABCB6 expression was analyzed by flow cytometry using the monoclonal anti-Lan OSK43 antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111590#pone.0111590-Helias1" target="_blank">[16]</a> in intact and permeabilized cells, respectively. The effect of pharmacological chaperoning and the inhibition of the proteasome (C, D) was studied by culturing the cells in the presence of 2 mM 4-PBA or 0.5 uM MG132, respectively. Bars represent average ABCB6 expression +/− standard deviation of at least 3 independent experiments.</p