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

Abstract

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.

Decreased calcium pump expression in human erythrocytes is connected to a minor haplotype in the ATP2B4 gene

“…According to our previous experience, strongly reduced RBC expression levels of the ABCG2 [7] and the ABCB6 [8] membrane proteins were connected to specific mutations and/or polymorphisms in the coding genes. Therefore, we analyzed the DNA extracts of the blood samples of the volunteers with low RBCPMCA4b expression by Sanger sequencing of the ATP2B4 gene, coding for this protein.…”

“…RBC membrane protein determinations were carried out according to our recently developed method [6][7][8][9]. In brief, we fixed and permeabilized the RBC membranes by using 1% formaldehyde solution, resulting in RBC "ghosts", and then equal number of cells were incubated with the PMCA-specific primary antibody (JA3, mouse monoclonal antibody, RRID: AB 628160) followed by a secondary, Alexa Fluor 488 labeled goat anti-mouse (H + L) antibody (Thermo Fisher/Life Technologies), in 96 well plates.…”

Functional characterization of novel ABCB6 mutations and their clinical implications in familial pseudohyperkalemia

“…Through screening erythroid ABCB6 expression, Koszarska et al found an unexpectedly high frequency of Lan mutations in healthy individuals. 21 Indeed, in public databases (1000 Genomes, NHLBI Exome Sequencing Project, Exome Aggregation Consortium) the allele frequencies of V454A, R723Q and R276W are 0.43%, 0.08% and 1.5%, respectively. Moreover, the high frequency of ABCB6 variations in FP, including two FP patients found in a Cardiff blood donor cohort recently described by Bawazir et al, 22 has clinical implications for blood transfusion screening and practice.…”

Association of five SNPs with human hair colour in the Polish population

“…All subjects were adult volunteers at the age of over 18 years living in Poland, who donated saliva samples to the Biobank Lab at the Department of Molecular Biophysics of the University of Lodz. Sample collection and anthropological data acquisition through a questionnaire were performed as previously described (Koszarska et al, 2014; Strapagiel et al, 2016). For this study, 186 subjects were randomly chosen and categorised into three distinct groups according to their declared, self-assessed natural hair colour.…”

“…The isolation of the genomic DNA from saliva was performed as previously reported (Koszarska et al, 2014). Genotyping of the variants within the studied genes (Table 2) was performed with the HRM (High Resolution Melt) method accompanied by direct sequencing.…”