Next Generation Sequencing-Based Genotyping of Human Blood Groups: FY, JK and ABO Genes
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Serological discrepancies in matching blood group antigens between donors and patients for blood transfusion may lead to alloimmunisation, especially in multiply transfused patients. Blood group genotyping (BGG) has contributed in reducing this issue. ABO, Fy and Jk antigens are among those to be causative for alloimmunisation through transfusion or pregnancy. The number of alleles of these clinically significant blood groups is ever increasing. Currently, all commercially available high-throughput BGG platforms are only based on pre-defined polymorphisms. Consequently, novel or rare alleles that might have clinical significance are not identified. Next generation sequencing (NGS) circumvents this issue by providing high-throughput comprehensive genotyping of blood group genes in discovery mode to find all existing and novel mutations. Accordingly, a large number of individuals can be genotyped in a single run. Here, we describe an NGS-based method coupled with long-range polymerase chain reaction (LR-PCR) for high-throughput, rapid and extensive genotyping of FY, JK and ABO blood group genes. The Ion Torrent Personal Genome Machine (PGMTM) was used for sequencing the entire FY, JK and ABO blood group genes including flanking regions. Accordingly, high resolution genotyping was obtained. 53 genomic DNA samples were sequenced and genotyped for FY, 67 for JK and 47 for ABO. Sequencing data were aligned to the gene reference sequence derived from the human genome (hg19) to analyse variants. Analysis was accomplished by software packages, such as Ion Torrent SuiteTM plugins. Sanger sequencing of cDNA and cDNA clones was used to confirm findings in the JK gene. The sequencing data had a coverage depth of more than 5000x for FY, 700x for JK and 600x for ABO. NGS data matched with the serological phenotypes of FY alleles FY*A, FY*B and FY*02 Null main polymorphisms, such as FY*A/FY*B (125G>A) in exon 2 and (-67 T>C) in the promotor region. JK variant analysis revealed that the JK*01W.01 allele (130G>A) is common (10/67 samples) with normal antigenicity. The previously described silencing polymorphism (810G>A), leading to a purported JK*B null allele, restores a splice site and does not correlate with loss of Jkb antigenicity (10/67 samples). JK intron analysis revealed several new JK alleles described in this thesis. All 7 exons, introns and the flanking regions of the ABO gene were covered by only four amplicons. Several rare O alleles were found, such as O73 and O75, while one suggested novel O allele was characterised by a missense SNP 482G>A (Arg161His) in exon 7. The ABO reference sequence from hg19 appeared to resemble (O01 and O02) alleles. The intronic SNPs might be used to distinguish between alleles more accurately as a correlation of the intronic SNPs with the alleles was noted for the homozygous O alleles. It is predicted that NGS-based genotyping will replace not only microarray-based genotyping but also serology in the blood group typing of individuals, with great advancements in technology and molecular knowledge being expected in the near future.
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