Transfusion · 2023
Two new JK silencing alleles identified by single molecule sequencing with 20-Kb long-reads.
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Isa Kazumi, Takada Shinnosuke, Takeda Hiromi, Tsuneyama Hatsue, Ogasawara Kenichi, Takahashi Daisuke, Miyazaki Toru, Miyata Shigeki, Satake Masahiro
Аннотация
BACKGROUND: The Kidd blood group gene SLC14A1 (JK) accounts for approximately 20 Kb from initiation codon to stop codon in the genome. In genomic DNA analysis using Sanger sequencing or short-read-based next generation sequencing, it is difficult to determine the cis or trans positions of single nucleotide variations (SNVs), which are occasionally more than 1 Kb away from each other. We aimed to determine the complete nucleotide sequence of a 20-Kb genomic DNA amplicon to characterize the JK allelic variants associated with Kidd antigen silencing in a blood donor.
STUDY DESIGN AND METHODS: The Jk(a-b-) phenotype was identified in this donor by standard serological typing. A DNA sample obtained from whole blood was amplified by long-range PCR to obtain a 20-Kb fragment of the SLC14A1 gene, including the initiation and stop codons. The fragment was then analyzed by Sanger sequencing and single-molecule sequencing. Transfection and expression studies were performed in CHO cells using the expression vector construct of JK alleles.
RESULTS: Sanger sequencing and single-molecule sequencing revealed that the donor was heterozygous with JK*01 having c.276G>A (rs763262711, p.Trp92Ter) and JK*02 having c.499A>G (rs2298719, p.Met167Val), c.588A>G (rs2298718, p.Pro196Pro), and c.743C>A (p.Ala248Asp). The two JK alleles identified have not been previously described. Transfection and expression studies indicated that the CHO cells transfected with JK*02 having c.743C>A did not express the Jkb and Jk3 antigens.
CONCLUSIONS: We identified new JK silencing alleles and their critical SNVs by single-molecule sequencing and the findings were confirmed by transfection and expression studies.
STUDY DESIGN AND METHODS: The Jk(a-b-) phenotype was identified in this donor by standard serological typing. A DNA sample obtained from whole blood was amplified by long-range PCR to obtain a 20-Kb fragment of the SLC14A1 gene, including the initiation and stop codons. The fragment was then analyzed by Sanger sequencing and single-molecule sequencing. Transfection and expression studies were performed in CHO cells using the expression vector construct of JK alleles.
RESULTS: Sanger sequencing and single-molecule sequencing revealed that the donor was heterozygous with JK*01 having c.276G>A (rs763262711, p.Trp92Ter) and JK*02 having c.499A>G (rs2298719, p.Met167Val), c.588A>G (rs2298718, p.Pro196Pro), and c.743C>A (p.Ala248Asp). The two JK alleles identified have not been previously described. Transfection and expression studies indicated that the CHO cells transfected with JK*02 having c.743C>A did not express the Jkb and Jk3 antigens.
CONCLUSIONS: We identified new JK silencing alleles and their critical SNVs by single-molecule sequencing and the findings were confirmed by transfection and expression studies.