Detection of Familial Pseudohyperkalemia Among Italian Blood Donors By Genetic Screening for the R276W Mutation in ABCB6

Introduction Isolated Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by cold-induced slow 9passive leak9 of red cell K+ into plasma, first described in a large Scottish family from Edinburgh (Stewart GW, et al., 1979). Although in freshly obtained blood samples plasma [K+] was normal, it was increased when measured in blood stored at or below room temperature. This trait was accompanied by mild abnormalities of red cell shape. Functional gene mapping and sequencing analysis of the candidate genes within the 2q35-q36 critical interval in three multigenerational FP families with 20 affected individuals identified two novel heterozygous missense mutations in the ABCB6 gene that cosegregated with disease phenotype (Andolfo I. et al., 2013). The two genomic substitutions altered two adjacent nucleotides within codon 375 of ABCB6, a porphyrin transporter that in erythrocyte membranes bears the Langereis blood group antigen system (Krishnamurthy PC, et al., 2006; Helias V, et al., 2012). Recently, the ABCB6 mutation R723Q was found in two patients with FP (Bawazir W, et al.,2014). Of note, both patients presented as blood donors, and increased cold-induced potassium leak was demonstrated. The transfusion of pseudohyperkalemic blood has clinical implications especially for neonates and infants receiving large-volume RBC transfusions. In this study we analyzed one additional family and report the first functional characterization of an ABCB6 mutation, towards understanding the pathogenic mechanism of FP. Moreover, we screened an Italian blood donor population for the R276W variant of the ABCB6 gene. Methods DNA was obtained for genetic analysis from affected and unaffected family members, after signed informed consent, according to the Declaration of Helsinki. The search for mutations was performed by direct sequencing of the ABCB6 gene. cDNAs encoding full-length wildtype ABCB6 were cloned into pcDNA3.1. Our patients9 novel point mutation (c.826C>T, p.R276W) was introduced into pcDNA3.1-ABCB6 by site-directed mutagenesis. WT and mutant constructs were transfected into HEK-293 cells and the cells were maintained at 30°C for 72 hrs to evaluate the effects of reduced temperature. After transfection, the cells were incubated in a medium containing 86 rubidium ( 86 Rb + ) as a surrogate for K + . 86 Rb + was determined in cell lysate, and K content of extracellular medium was determined by atomic absorption spectrometry. Results We found the heterozygous mutation c.826G>T, p. R276W in an Irish family. This mutation is annotated in public databases as single nucleotide variants (SNVs), and is predicted by PolyPhen2 and SIFT to be damaging. Variant R276W showed a minor allele frequency (MAF) of 1.3:100 confirming that many patients with FP could be present in the blood donor population. R276W and previously identified ABCB6 variants R375Q and R375Wwere overexpressed in HEK-293 cells to characterize the functional properties of these variants. Expression of ABCB6 mutants showed no change in RNA or polypeptide levels. However, measurement of ouabain- and bumetanide-resistant net cation flux demonstrated a greater loss of cell K from mutant ABCB6-expressing cells than from WT ABCB6-expressing cells. The high allele frequency of ABCB6 variant R276W prompted a genetic screen of 327 blood donors. The variant was present in 0.3% (1/327) of the donor cohort, and this single subject with ABCB6 R276W exhibited slightly increased MCV and variably increased plasma K + concentrations. Conclusions: Our findings demonstrate that missense mutations in ABCB6 lead to increased K + efflux from RBCs in FP patients. Storage of FP blood can cause a significant increase in blood K+ levels, with especially serious clinical implications for neonates and infants receiving large-volume transfusions of whole blood. Furthermore, the prevalence of FP might be underestimated, since patients with FP can be asymptomatic and thus undetected in the donor population. Screening for the most frequent ABCB6 variation, R276W, confirmed that FP patients are present in the Italian blood donor population. In the future, genetic tests for FP could be added to blood donor prescreening to improve the quality and safety of donor units. Disclosures No relevant conflicts of interest to declare.