A large deletion spanning XG and GYG2 constitutes a genetic basis of the Xg_null phenotype, underlying anti‐Xg^a production

Abstract

BACKGROUND The PBDX/XG gene encoding the Xga blood group antigen was described in 1994, but the genetic determinant of XG expression on RBCs was reported only in 2018. However, the frequencies of Xg(a−) individuals could not explain the rarity of anti‐Xga makers. We therefore sought to elucidate the molecular basis of the Xg(a−) phenotype in people producing anti‐Xga. STUDY DESIGN AND METHODS Two genomic DNA (gDNA) and 13 plasma‐derived cell‐free DNA (cfDNA) samples from anti‐Xga makers were investigated (14 males and one female). PBDX/XG exon sequencing was attempted on one gDNA sample. Polymerase chain reaction assays were developed and bioinformatics used to define a suspected deletion in all samples. RESULTS Investigation of one gDNA sample revealed a 114‐kb deletion (esv2662319) on the X chromosome that spans XG exons 4 through 10 and the downstream GYG2 gene. A 3555‐bp fragment bridging this deletion was amplified to confirm its presence. Another deletion‐specific polymerase chain reaction of 714 bp enabled identification of esv2662319 in both gDNA samples and eight cfDNA samples while ruling it out in one cfDNA. Males were hemizygous for esv2662319 and the female likely homozygous. Four cfDNA sample results were inconclusive, probably due to poor sample quality. Sanger sequencing recognized the recombination junctions as a heterogeneous LTR6B sequence. CONCLUSION We identified a large deletion on the X chromosome, resulting in a true, tissue‐wide Xgnull phenotype. This deletion was found in 10 of 11 anti‐Xga makers from which DNA could be amplified. One sample remained unexplained, indicating further heterogeneity to be explored.

Genetic background of anti‐Xga producers in Japanese blood donors

“…Genomic DNA was extracted from peripheral blood leucocytes using a QIAamp DNA Blood Mini Kit (Qiagen, Tokyo, Japan). To detect the XGdel , we performed PCR using SVD‐F primer specific for XG intron 3 and SVD‐R primer specific for the downstream region of the breakpoint of the 114‐kb deletion (Table S2) [11]. Genomic DNA (20–40 ng) was amplified by 35 cycles of PCR in a 10 μl volume containing PCR buffer with 0.2 units of Phusion DNA polymerase (New England BioLabs, Tokyo, Japan), 0.2 μl of 10 mM dNTPs, 0.3 μl of 99.5% dimethyl sulphoxide (Wako, Osaka, Japan), 0.5 μl of 20 nM SVD‐F and 0.5 μl of 20 nM SVD‐R under the following conditions: one cycle of 30 s at 98°C and 35 cycles of 10 s at 98°C, 30 s at 64°C and 30 s at 72°C (Veriti, Applied Biosystems, Waltham, MA).…”

“…Another mechanism of the Xg(aÀ) phenotype is the lack of the XG gene on the X chromosome. Lee et al reported that individuals producing anti-Xg a had a rare allele of 114 kb deletion (esv2662319), including exon 4 through exon 10 of the XG (Figure 1a) [11]. The Xg null individuals have no Xg a antigen not only in erythrocytes but also in tissues.…”

The Xg blood group system: no longer forgotten

“…1). 11 These individuals therefore are truly devoid of the Xg glycoprotein in all tissues and are thus Xg null . Interestingly, this large deletion was reported elsewhere but was not associated with Xg status, and its relatively high frequency is attributed to non-allelic homologous recombination between two LTR6B endogenous retroviral sequences on the X and Y chromosomes.…”

“…[12][13][14] The genetic basis of the sole outlying anti-Xg a maker has yet to be elucidated. 11 Less is known about anti-CD99 producers. An abstract in 2015, however, reported that the CD99− phenotype and associated anti-CD99 can be attributed to various large deletions spanning exons 2-8 of the CD99 gene that retain the correct reading frame.…”

“…The authors of this review would like to acknowledge the co-authors who helped with the discoveries reported by our group. 6,11…”