Wei-Ting Wang

Identification of a novel A1v-O1v hybrid allele with G829A mutation in a chimeric individual of AelBel phenotype.

BACKGROUND:u2002 Many A and B suballeles responsible for ABO subgroup formation have been identified. Some of these minor alleles have mutations in the ABO gene coding sequence. Most of these mutations are due to single‐nucleotide substitution and lead to amino acid alteration. Several alleles at the ABO locus appear to be caused by crossing over between dissimilar alleles.

Real-time biallelic polymorphism-polymerase chain reaction for chimerism monitoring of hematopoietic stem cell transplantation relapsed patients.

BACKGROUNDnAn accurate analysis of chimerism kinetics permits early detection of hematopoietic stem cell transplantation (HSCT) in patients with high risks of graft-versus-host disease or those liable to relapse. Although short tandem repeats-PCR (STR-PCR) is the golden standard for quantitative chimerism analysis in most of the clinical laboratories, it has a relatively low sensitivity of 5% and the detection of low percentage in mixed chimerism is usually delayed. In this study, we developed a real-time PCR for chimerism analysis based on the informative biallelic polymorphisms (BP).nnnMETHODSnThe allele frequencies of 19 selective biallelic polymorphic markers were analyzed using the genomic DNA from 100 healthy Taiwanese volunteers. The informative biallelic polymorphic markers with high discrimination power in the Taiwanese population were identified. The TaqMan probe-based real-time BP-PCR for amplification of the informative loci was designed and the detection sensitivity was determined. Clinical application of real-time BP-PCR in chimerism monitoring was evaluated and was compared with the conventional STR-PCR by analyzing the DNA samples obtained at different time points post-HSCT from 4 relapsed and 10 non-relapsed patients.nnnRESULTSnAllele distribution analysis revealed that the loci of S01a, S03, S04a, S05b, S06, S07b, S08b, S09b, S10b and S11a had a relatively high discrimination power and were the informative BP for chimerism monitoring in the Taiwanese population. Real-time BP-PCRs for these 10 BP loci were set up with the detection sensitivity equivalent to 0.003-0.006%. Real-time BP-PCR of the 4 HSCT patients revealed the presence of recipient-specific DNA at early time point than STR-PCR for 3 of the patients, whereas real-time BP-PCR was as effective as STR-PCR in uncovering the sign of relapse for one of the patients. In addition, the baseline value for the patients with no sign of relapse was 0.127 ± 0.193% of recipient DNA.nnnCONCLUSIONnWe conclude that real-time BP-PCR is a sensitive and reliable method for chimerism monitoring and is superior to the STR-PCR in identifying patients who are at high risk for relapse after transplantation.

MNSs Blood Group Glycophorin Variants in Taiwan: A Genotype-Serotype Correlation Study of ‘Mia’ and Sta with Report of Two New Alleles for Sta

Background Glycophorin variants of the MNSs blood group are important in Taiwan. For more than 20 years, screening for the most frequent irregular antibody, anti-‘Mia’, has been conducted by using ‘Mia’(+) RBCs, with a significant success. However, the sensitivity and the specificity of this screening strategy have never been validated, and the true incidences of different glycophorin variants in Taiwan have been in controversy. Also, the significance of another less frequent and usually separately reported variant, Sta, has never been evaluated. Methodology/Principal Findings We ran a population-based screening (from unselected patients in our hospital) for MNSs blood group glycophorin variants by PCR-sequencing method. GP.Mur (Mil.III) was confirmed by sequence from 57 out of 1027 samples (5.6%), and there was no other Miltenberger subtype glycophorin variant found. Glycophorin variant Sta was found from 35 out of 1027 samples (3.4%). In contrast to anti-‘Mia’, which is the most frequently identified irregular antibody in Taiwan, the prevalence of anti-Sta was only 0.13% as determined by serologic method. In addition, two new alleles for Sta were found and reported. Conclusion/Significance We confirm the long-standing assumption that GP.Mur is the only prevalent Miltenberger subtype in Taiwan. The current anti-‘Mia’ screening method used in Taiwan, although neither sensitive nor specific, is still a suitable practice. Although Sta antigen has a high prevalence in Taiwan, routine screening for anti-Sta is not warranted based on current evidence.

Effect of HLA mismatching at HLA-A, -B, and -DRB1 for umbilical-cord blood transplantation in Taiwan

Unrelated cord blood transplantation has become a reliable alternative therapy for children and adults owing to that one or two antigen/allele mismatches between a patient and the cord blood donor are acceptable without occurrence of graft-versus-host disease. To investigate the relationship between the number and types of mismatches and relapse, we compared the number of mismatched and non-mismatched donor-recipient pairs, number of mismatched alleles, and number of mismatched antigens at each of the Human Leukocyte Antigen (HLA) -A, -B, and -DR loci, respectively. The result indicates that the number of mismatched antigens at the HLA-A locus was significantly associated with occurrence of relapse (X2P-value=0.0243; RR=1.49, 95% CI: 1.04-2.13). Additionally, the number of mismatched donor-recipient pairs and the number of mismatched alleles at the HLA-DR locus was negatively associated with risks of relapse (X2P-value=0.0028; RR=0.52, 95% CI: 0.31-0.89). In this study, we found that the mismatch at the HLA-A locus is associated with increased risk of relapse; while the mismatch at the HLA-DR locus is innocuous. Hence, we suggest that the well-matched HLA-A alleles were most critical for matching HLA alleles between umbilical-cord blood transplantation donors and recipients. In other words, cord blood transplantation requires less stringent HLA matching, if there are two 5/6 or 4/6 HLA matched donors, its better to choose HLA-A matched donor at least.

Single nucleotide polymorphisms within HLA region are associated with disease relapse for patients with unrelated cord blood transplantation

Disease relapse occurs in unrelated cord blood transplantation (CBT) even when the alleles of human leukocyte antigen (HLA) are fully matched between donor and recipient. This is similar to that observed in other types of hematopoietic stem cell transplantation. Fourteen single nucleotide polymorphisms (SNPs) within the HLA region have been reported previously by Petersdorf et al. and Piras et al. as transplantation determinants in unrelated hematopoietic cell transplantation. In this study, the genomic sequences within 500 base pairs upstream and downstream of the fourteen transplantation-related SNPs from 53 patients and their HLA-matched unrelated donors were analyzed for determining whether or not genetic variants, conferred by either recipient or donor SNP genotype or by recipient-donor SNP mismatching, were associated with the risk of relapse. Seven SNPs were associated with the risk of relapse in unrelated CBT. These included the donor genotype with the SNPs of rs2523675 and rs2518028 at the telomeric end of HCP5 gene, rs2071479 in the intron of the HLA-DOB gene, and rs2523958 in the MICD gene; and the recipient genotype with SNPs of rs9276982 in the HLA-DOA gene, and rs435766 and rs380924 in the MICD gene. As measured by pair-wise linkage disequilibrium (LD) with D′ as the parameter for normalized standard measurement of LD which compares the observed and expected frequencies of one haplotype comprised by alleles at different loci, rs2523675 had high LD with rs4713466 (D′ = 0.86) and rs2523676 (D′ = 0.91) in the HCP5 gene. The rs2518028 had no LD with all other SNPs except rs2523675 (D′ = 0.76). This study provides the basis for developing a method or algorithm for selecting better unrelated CBT candidate donors.

Rapid rare ABO blood typing using a single PCR based on a multiplex SNaPshot reaction

BACKGROUNDnABO subgroups would be considered when discrepancies in ABO grouping occur. Serological methods including adsorption-elution test, salivary ABH inhibition test, and anti-A1 (lectin) saline method could be used. However, these serological methods are laboring and obscure. Therefore, reliable and affordable method to assess the ABO subgroups is of particular interest.nnnMETHODSnTo solve this problem, the multiplex SNaPshot-based assays were designed to determine rare A and B subgroups. Primers used as probes for determination of rare ABO blood groups known in Taiwanese population were designed. Many ABO subtype samples were used to validate the accuracy and reproducibility of our SNaPshot panel.nnnRESULTSnA panel of primer probes were successfully designed in determining 8 SNP sites (261, 539, 838, 820, 745, 664, IVS6 +5, and 829 in exon 6 and 7) for A phenotype and 6 SNP sites (261, 796, IVS3 +5, 247, 523, and 502 in exon 2, 6 and 7 and intron 3) for B phenotype. SNaPshot analysis for defining blood group A alleles (A1, A2, A3, Am and Ael) and blood group B alleles (B1, B3, Bw and Bel) was therefore available.nnnCONCLUSIONnSNaPshot analysis could be used in reference laboratories for typing known rare subgroups of A and B without DNA cloning and traditional sequencing. Moreover, this method would help to construct databases of genotyped blood donors, and it potentially plays a role in determining fetal-maternal ABO incompatibility.

Real-time amplification of glyceraldehyde-3-phosphate dehydrogenase gene for quality control of leukopoor platelets.

Leukoreduction of blood products is crucial to prevent white blood cell (WBC)‐associated complications during transfusion. Of the widely accepted methods for quantifying WBCs in blood components, Nageotte hemocytometry is time‐consuming and laborious whereas a specialized instrument is required for flow cytometry. A reliable and affordable method to assess WBC count in blood products is of particular interest.

Hematopoietic Stem Cell Transplant Relapse with Loss of an Allele and Insertion of One Repeat at D13S317 Locus

Post-transplantation chimerism testing is important to monitor the engraftment of donor stem cells and the diagnosis of relapse. Because short tandem repeats (STRs) are interspersed throughout the genome and the commercial applications are available, the analysis of STRs has become a frequently used method for the study of engraftment. The case reported here was diagnosed as multiple myeloma, and showed an almost complete engraftment after allogeneic hematopoietic stem cell transplantation. However, after 4.5 months, the result of chimerism test indicated an acute relapse. Interestingly, the data of D13S317 locus showed beside two donor alleles (8 and 12) and one shift allele (13). Meanwhile, the recipient-specific allele (9) was missing. The possible reason for the corresponding loss of an STR allele is chromosome loss, and the insertion of one repeat is through the slippage mechanism during DNA replication. These circumstances are potential sources of error in the interpretation of disease relapse.