Yanli Ji

Comprehensive genotyping for 18 blood group systems using a multiplex ligation-dependent probe amplification assay shows a high degree of accuracy

In recent years genotyping methods have been implemented in blood banks as alternative to comprehensive serologic typing. We evaluated a newly developed assay for convenient and comprehensive genotyping of blood group alleles based on multiplex ligation‐dependent probe amplification (MLPA) technology.

The summary of FUT1 and FUT2 genotyping analysis in Chinese para‐Bombay individuals including additional nine probands from Guangzhou in China

The para‐Bombay phenotype is characterized by the absence or weak expression of ABH antigens on the surface of red blood cells, but normal expression in saliva.

The distribution of MNS hybrid glycophorins with Mur antigen expression in Chinese donors including identification of a novel GYP.Bun allele.

MNS hybrid glycophorins are identified by characteristic antigen profiles. One of these is the Mur antigen, which is expressed on red cell hybrid glycophorins of several phenotypes of the ‘Miltenberger’ series found predominantly in East Asian population. The aim of this study was to investigate the distribution of Mur‐positive hybrid glycophorins and clarify the genetic basis in the donors from southern China.

A novel 519_525dup mutation of KLF1 gene identified in a Chinese blood donor with Lu(a-b-) phenotype.

The Lutheran blood group system consists of 20 antigens encoded by LU gene and includes four pairs of antithetical antigens. The remainders are high-frequency antigens. Lu(a–b–)/Lunull is a rare phenotype first described by Crawford and colleagues in 1961 characterized by the absence of all Lutheran system antigens, which distributed with a frequency of 1:3000 in South East England and 1:5000 in South Wales blood donors. Three molecular backgrounds account for this rare phenotype. First, the rare recessive Lunull phenotype arises from homozygosity for inactivating mutations in the LU gene, which is generally ascertained through the presence of anti-Lu3. Second, a mutation of erythroid transcription factors GATA1 (GATA1-binding protein 1) has been shown to be responsible for X-linked Lu(a–b–) phenotype in one family. Third, the majority of probands with dominant inherited Lu(a–b–)/In(Lu) phenotype result from inactivating heterozygous mutations in KLF1. The red blood cells (RBCs) with this phenotype express traces of Lutheran antigen that could only be detected by absorption/ elution technique; therefore, their blood has been used to satisfy the requirement of patients with anti-Lu3 since they are relatively common. So far, four inactive mutations of LU gene accounting for Lunull phenotype and 11 KLF1 mutations accounting for In(lu) phenotype have been described (see Web Resource). In our study, one male donor with Lu(a–b–) phenotype (20 years old and without transfusion history) was identified accidentally while high-throughput genotyping and corresponding serologic typing were conducted in 90 Chinese donors. Anti-Lu and anti-Lu (Sanquin Reagents, Amsterdam, the Netherlands) were used to type Lu(a–b–) phenotype. Absorption/elution testing was conducted by using the anti-Lu serum. The donor’s RBCs were also tested with anti-AnWj and anti-INFI since the highincidence AnWj (901009) antigen and Indian blood group system antigens including INFI (IN3) are also known to be weakened in the In(Lu) phenotype. HbF level was also tested by using high-performance liquid chromatography (Variant II hemoglobin testing system, Bio-Rad, Hercules, CA). Unfortunately, the samples of the other family members were not available. Furthermore, both LU and KLF1 genes were analyzed by direct sequencing to clarify the molecular background. Genomic DNA was extracted from peripheral blood of Lu(a–b–) proband and 90 random individuals as controls. Exons 1 to 15 of LU gene, the promoter and Exons 1 to 3 of KLF1 gene, and the exon– intron boundaries were amplified by polymerase chain reaction (PCR). Amplification primers, annealing temperature, and PCR condition have been described previously, except that GC Buffer II (Takara, Dalian, China) was used to amplify the Exon_2 of KLF1. PCR products were sequenced using a sequencing kit on a genetic analyzer (ABI BigDye Terminator, Version 3.1, and ABI 3130XL, respectively, Applied Biosystems, Foster City, CA). Heterozygous mutations identified were further examined by TOPO TA cloning (Invitrogen, Carlsbad, CA). The novel mutation identified was tested in 90 random blood donors by direct sequencing. Negative agglutination with anti-Anwj and anti-INFI serum and negative antibody screening results (absence of anti-Lu3) were obtained in this donor. Absorption/ elution testing showed very weak expression of lu antigen compared with normal controls. No mutations were identified in all 15 exons of LU gene. However, a novel heterozygous seven-base duplicated insertion in Exon_2 of KLF1 gene (c.519_525dupCGGCGCC) was detected (Fig. 1A), which resulted in frameshift mutations from Amino Acid 176 (p.Gly176Argfs*179) and created a truncated protein with a premature stop codon (Fig. 1B). This heterozygous mutation was further confirmed by cloning analysis (registered in GenBank as Number JX877554) and was not found in 90 random donors. EKLF encoded by KLF1 gene is an essential transcription factor in the process of erythroid differentiation. The duplicated insertion will disrupt the EKLF transactivation domain in the proline-rich N-terminal region (Amino Acids 1-275) and three zinc finger domain in the C-terminal region (Amino Acids 276-358) that maybe influence the erythroid-specific genes expression resulting in In(lu) phenotype. Based on the weak expression of lu antigen, the absent expression of highfrequency Anwj and INFI antigens, and the novel mutation identified in KLF1 gene, In(lu) phenotype is confirmed in this Chinese donor. Besides, KLF1 mutations are also responsible for persistent hyper– hemoglobin F syndrome. In this donor, mean 0.8% HbF level was obtained compared with 0.7% in controls that indicate the effects of this mutation may be additive for this syndrome. Recently, the heterozygous mutation and compound heterozygous with L51R missense mutation were found in one Korean patient with 1.7% HbF level and one Vietnamese patient with 9.5% HbF level respectively, but the In(lu) phenotype was not tested.

GYP*Kip, a novel GYP(B‐A‐B) hybrid allele, encoding the MNS48 (KIPP) antigen

G P(B-A-B) hybrid glycophorins, designated GP.HF, GP.Mur, GP.Bun, and GP.Hop, arise from insertion of a homologous segment from the Exon 3 region of the GYPA gene into the GYPB gene, including the 50 end of Intron 3. This insertion repairs the inactive splice site (that makes the Exon 3 region of the GYPB gene a pseudoexon) and leads to the generation and expression of characteristic profiles of novel antigens. A novel red blood cell serologic profile for a GP(B-AB) hybrid (positive with anti-Mur, -Hil, -MINY, -MUT, and -Hop1Nob) was reported by the International Blood Group Reference Laboratory (IBGRL). It was postulated that this was a new GP(B-A-B) hybrid with a proposed designation GP.Kip after the German propositus. In 1992, the IBGRL reported detection of an identical serologic pattern for an Australian blood donor. The genetic bases of four GP(B-A-B) MNS system hybrid glycophorins (GP.HF, GP.Mur, GP.Hop, and GP.Bun) that result from rearrangement between the homologous GYPA and GYPB genes are well characterized. This report describes a new GYP(B-A-B) hybrid allele from the Australian blood donor reported with the GP.Kip antigen profile.

Genotyping for Glycophorin GYP(B - A - B) Hybrid Genes Using a Single Nucleotide Polymorphism-Based Algorithm by Matrix-Assisted Laser Desorption/Ionisation, Time-of-Flight Mass Spectrometry

The genetic basis for five GP(B-A-B) MNS system hybrid glycophorin blood group antigens results from rearrangement between the homologous GYPA and GYPB genes. Each hybrid glycophorin displays a characteristic profile of antigens. Currently, no commercial serological reagents are currently available to serologically type for these antigens. The aim of this study was to develop a single nucleotide polymorphism (SNP) mapping genotyping technique to allow characterisation of various GYP(B-A-B) hybrid alleles. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) assays were designed to genotype five GYP(B-A-B) hybrid alleles. Eight nucleotide positions were targeted and incorporated into the SNP mapping protocol. The allelic frequencies were calculated using peak areas. Sanger sequencing was performed to resolve a GYP*Hop 3′ breakpoint. Observed allelic peak area ratios either coincided with the expected ratio or were skewed (above or below) from the expected ratio with switching occurring at and after the expected break point to generate characteristic mass spectral plots for each hybrid. Sequencing showed that the GYP*Hop crossover in the intron 3 region, for this example, was identical to that for GYP*Bun reference sequence. An analytical algorithm using MALDI-TOF MS genotyping platform defined GYPA inserts for five GYP(B-A-B) hybrids. The SNP mapping technique described here demonstrates proof of concept that this technology is viable for genotyping hybrid glycophorins, GYP(A-B-A), GYP(A-B) and GYP(B-A), and addresses the gap in current typing technologies.

RHD genotype and zygosity analysis in the Chinese Southern Han D+, D− and D variant donors using the multiplex ligation‐dependent probe amplification assay

Several comprehensive genotyping platforms for determining red blood cell (RBC) antigens have been established and validated for use in the Caucasian and Black populations, but not for the Chinese. The multiplex ligation‐dependent probe amplification (MLPA) assay was validated for RHD genotyping in the Chinese.

Identification of a novel frequent RHCE*ce308T variant allele in Chinese D- individuals, resulting in a C+c- phenotype

The RHCE allele is highly polymorphic; more than 60 variants have been described leading to diminished expression of C, c, E, and e antigens. Not much is known about the prevalence of RHCE variants in the Chinese population. Individuals carrying a variant are at risk to develop alloantibodies in response to mismatched pregnancy or transfusion. In this study, phenotyping and genotyping of the RHCE allele in Chinese donors revealed a new clinically relevant mutation.

A variant RhAG protein encoded by the RHAG*572A allele causes serological weak D expression while maintaining normal RhCE phenotypes: RHAG*572A causing a serological weak D phenotype

The molecular events resulting in a weak D phenotype include missense mutations, in‐frame insertion, or deletion mutations of the RHD gene and hybrid RHD‐CE‐D hybrid alleles. Mutations in genes encoding the proteins that are required for proper membrane expression of Rh proteins, such as RhAG and ankyrin 1, can lead to absent or weakened expression of Rh antigens.

Genotyping analysis of MNS blood group GP(B-A-B) hybrid glycophorins in the Chinese Southern Han population using a high-resolution melting assay: GENOTYPING OF GP(B-A-B) HYBRIDS BY HRM

MNS hybrid GP(B‐A‐B) glycophorins are more commonly found in Southeast Asians and alloantibodies to antigens they carry are clinically significant. Detection of hybrid glycophorins by serologic techniques is limited due to lack of commercial reagents. In this study, a genotyping method for GP(B‐A‐B) hybrid glycophorins based on high‐resolution melting (HRM) analysis was applied for genotyping analysis in the Chinese Southern Han population.