cDNA sequencing reveals an unexpected splice variation leading to weak KEL2 expression

BACKGROUND: KEL1 antigen expression is routinely tested in blood donors in Switzerland. A donor sample with an apparent rare KEL:1,-2 phenotype was genotyped KEL*01.01/KEL*02. A series of detailed molecular analyses were performed to solve this discrepancy, and a novel variant KEL*02 allele was identified. MATERIALS AND METHODS: Standard serological column agglutination and in-house adsorption-elution testing were used for the detection of KEL antigens. Genomic DNA was isolated and analyzed by commercial sequence-specific primer (SSP)-PCR, in-house multiplex SSP-PCR, and KEL exon sequencing. Total RNA was isolated from blood samples; polyadenylated RNA was reverse-transcribed, and cDNA was amplified with allele-specific primers and sequenced. SpliceAI and PolyPhen-2 were used to evaluate the impact of nucleotide variations and amino acid changes on splice effects and protein function, respectively. RESULTS: The donor sample was initially typed KEL:1,-2. However, SSP-PCR revealed the genotype KEL*01.01/KEL*02 and subsequent adsorption-elution testing indicated very weak KEL2 expression. Exon sequencing showed the heterozygous missense mutation c.139C>T leading to the amino acid substitution p.(Arg47Trp). PolyPhen-2 predicted this change to be benign, whereas SpliceAI analysis indicated a putative change in splice sites. Allele-specific amplification and sequencing revealed that the KEL*02 derived transcript lacks a significant portion of exon 3, causing a frameshift. CONCLUSION: We identified a novel missense mutation c.139C>T in KEL*02. Although the variant nucleotide locates in the center of exon 3, far away from the exon/intron boundary, it leads to variant splicing of the transcript, resulting in very weak expression of a truncated protein only detectable by adsorption-elution testing.