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Isolated Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by cold-induced slow ‘passive leak’ 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 unaccompanied by clinical symptoms or signs except for 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).

 

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 in pcDNA3.1 vector. The novel point mutations found in our patients: c.1361T>C, p.V454A; c.826C>T, p. R276W; c.2168G>A, p.R723Q were introduced into pcDNA3.1-ABCB6 by site-directed mutagenesis. WT and mutants constructs were transfected into HEK-293 cells for 72h. The cells were maintained at 30°C to evaluate the effects of reduced temperature. After transfection, the cells were incubated in a medium containing ⁸⁶rubidium (⁸⁶Rb⁺) as a tracer for K⁺. ⁸⁶Rb was determined in cell lysate, and K was determined in the supernatant by atomic absorption spectrometry.

In one Bolivian patient we found the homozygous mutation c.1361T>C, p.V454A. In one patient from Cardiff we found two heterozygous mutations in trans: c.826G>T, p. R276W and c.2168G>A, p.R723Q.  In Ireland family we found the heterozygous mutation c.826G>T, p. R276W. All these mutations are annotated in public databases as single nucleotide variants (SNVs), suggesting that many patients with FP could be present in donor blood population and are predicted to be damaging by in silico analysis by PolyPhen2 and SIFT tools. V454A, R276W, and R723Q as well as the previously identified, R375Q and R375W, were expressed into HEK-293 cells. Expression analysis of all mutants showed no alterations in levels of expression of mutant RNA or polypeptide, and molecular modeling predicted minimal structural changes resulting from the novel missense mutations. However, measurement of ouabain- and bumetanide-resistant net cation flux demonstrated a greater loss of cell K from ABCB6 mutant-expressing cells compared to ABCB6 WT-expressing cells. The coexpressed R276W/R723Q mutations, in particular, elicited greater efflux of cellular K⁺ into extracellular medium than did the other mutants studied.

Our findings demonstrate that missense mutations in ABCB6 lead to increased cellular K⁺ efflux as exhibited in RBCs of FP patients. Storage of FP blood can cause a significant increase in blood K⁺ levels, with serious clinical implications mostly in 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. In the future, genetic tests for FP could be added to blood donor prescreening.