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Background The frequency of the RhD negative (D-) phenotype among the population of Taiwan is only 0·34% and so anti-D is a relatively rare antibody. Routine pretransfusion D typing of patients at Mackay Memorial Hospital (MMH) was discontinued in 1988, and this report is a look back and retrospective evaluation over 30-years (1988-2017). Study design and methods The incidence of anti-D among patients at MMH during the periods 1984-1988 (when D typing was performed) and 1988-2017 (when D typing was not performed) was reviewed. Also, the incidence of anti-D among both MMH patients and voluntary blood donors at the Taiwan Blood Foundation was compared. The importance of anti-'Mia ' in Taiwan is also discussed. Results The incidence of anti-D relative to other Rh antibodies among MMH patients when D typing was performed and D typing not performed has remained relatively unchanged (5%). The frequencies of anti-D and anti-'Mia ' among 38 537 patients who were transfused at MMH during 2008-2017 were found to be 0·06% and 2·6%, respectively. During the same period, among 3 510 131 blood donors at Taiwan Blood Foundation, the frequencies of anti-D and anti-'Mia ' were 0·004% and 0·2%, respectively. Conclusion The elimination of D typing of patients at MMH has proven to have been a correct and logical decision. D- patients, if they do not carry anti-D, can thus be safely transfused with D+ red cells.

Human CO2 respiration requires rapid conversion between CO2 and HCO3- Carbonic anhydrase II facilitates this reversible reaction inside red blood cells, and band 3 [anion exchanger 1 (AE1)] provides a passage for HCO3- flux across the cell membrane. These 2 proteins are core components of the CO2 transport metabolon. Intracellular H2O is necessary for CO2/HCO3- conversion. However, abundantly expressed aquaporin 1 (AQP1) in erythrocytes is thought not to be part of band 3 complexes or the CO2 transport metabolon. To solve this conundrum, we used Forster resonance energy transfer (FRET) measured by fluorescence lifetime imaging (FLIM-FRET) and identified interaction between aquaporin-1 and band 3 at a distance of 8 nm, within the range of dipole-dipole interaction. Notably, their interaction was adaptable to membrane tonicity changes. This suggests that the function of AQP1 in tonicity response could be coupled or correlated to its function in band 3-mediated CO2/HCO3- exchange. By demonstrating AQP1 as a mobile component of the CO2 transport metabolon, our results uncover a potential role of water channel in blood CO2 transport and respiration.-Hsu, K., Lee, T.-Y., Periasamy, A., Kao, F.-J., Li, L.-T., Lin, C.-Y., Lin, H.-J., Lin, M. Adaptable interaction between aquaporin-1 and band 3 reveals a potential role of water channel in blood CO2 transport.

Background and Aim Symptomatic gallstone disease (SGSD) induced several inflammatory responses and affected extrahepatic bile ducts. Although the pathology and environmental risk factors of gallstone disease are well documented, immune or inflammatory responses in SGSD development are still inconclusive. Interleukin 18 (IL18) is a pro-inflammatory cytokine that plays an important role in immune, infectious, and inflammatory diseases because of the induction of interferon-γ. In this study, we investigated whether polymorphisms of the IL18 gene were associated with SGSD susceptibility. Methods Genomic DNA was isolated from the whole blood samples of 445 patients with SGSD and 1121 gallstone-free controls. The IL18 rs549908T>G, rs5744247C>G, rs187238G>C, rs1946518T>G, and rs360719A>G polymorphisms were genotyped using predeveloped TaqMan allelic discrimination assay. Results We found IL18 rs5744247G allele conferred protection against SGSD in female patients (odds ratio = 0.75, corrected P-value = 0.015). Haplotype analysis revealed that TGGTA protected females from SGSD development (odds ratio = 0.75, corrected P-value = 0.02). Conclusions Based on our findings, IL18 rs5744247C>G polymorphism could be a potential genetic marker to predict SGSD susceptibility in Han Chinese women.

BACKGROUND: GP.Mur (Mi.III) is a glycophorin B-A-B hybrid sialoglycoprotein expressing several potent immunogens, including Mia, Mur, and Hil. GP.Mur is considered one of the most important red blood cell (RBC) phenotypes in blood banking in Southeast Asia. However, there are no antibodies commercially available for the screening of GP.Mur RBCs. STUDY DESIGN AND METHODS: To develop a direct blood polymerase chain reaction (PCR) approach for the screening of GP.Mur cells, we first confirmed the genomic sequence differences among four GP.Mur and three Mi(a–) samples by sequencing their GYP.Mur and GYPB genes. With these data, we designed PCR primers that best discriminate GYPB and GYP.Mur. Our primer design also allows the detection of other Hil+ glycophorin variants. We also constructed two plasmids—pGBi2i3 and pMiIIIi2i3—which serve as the negative and positive control DNA, respectively, for the PCR procedure. Additionally, we designed a control PCR to be run side by side with the typing PCR. RESULTS: Because of the high specificity of our primers, we found it unnecessary to extract DNA from blood samples for PCR. We have tested this PCR method on 379 fresh and frozen blood samples. The results were further validated by serology and DNA sequencing and were shown to be completely accurate in our hand. We also found that the rapid genotyping method—high-resolution melting—can be a timesaving alternative for DNA sequencing. CONCLUSION: This direct blood PCR approach for determination of GP.Mur and related Hil+ phenotypes is reliable and economical and is expected to be useful for blood banking in Southeast Asia.

Graves disease (GD) is an autoimmune thyroid disease with a female preponderance and a wide range of ages at onset, and human leukocyte antigen (HLA) gene plays a primary role in the susceptibility to GD. We aim to investigate the associations between HLA-DRB1 alleles and Taiwanese children with GD by both case-control and family-based studies. A total of 241 unrelated children with GD, 539 healthy controls, 115 trios of affected patients and their parents, and 121 trios of unaffected siblings and their parents were recruited. HLA-DRB1 genotyping was performed by polymerase chain reaction and sequence-based typing assays. We found that DRB1*09:01 (OR = 2.60, 95% CI 2.02–3.35, Pc = 6.55 × 10−13) was associated with GD risk, while DRB1*12:02 (OR = 0.32, 95% CI 0.20–0.53, Pc = 4.55 × 10−5) was protective against GD. Transmission/disequilibrium test further confirmed an overtransmission of the DRB1*09:01 (OR 3.37, 95% CI 2.13–6.22, Pc = 1.0 × 10−5) and an undertransmission of the DRB1*12:02 (OR 0.21, 95% CI 0.05–0.42, Pc = 1.7 × 10−3). The findings were similar in females when stratified by gender. In conclusion, our results clearly identify that HLA-DRB1*09:01 confers susceptibility to GD and DRB1*12:02 exerts protection against GD development in Taiwanese children.

Background and Objectives Miltenberger blood group antigen subtype III (Mi.III) is characterized by expression of a glycophorin B-A-B hybrid (Gp.Mur) on the erythrocyte surface. The two alleles of glycophorin B are substituted with the B–A–B hybrid alleles in homozygous Mi.III (Mi.III+/+), and thus, Mi.III+/+ erythrocytes lack glycophorin B (GPB) and express Gp.Mur only. Because GPB is a major component of the Rh complex on RBCs, in this study, we explored how the absence of GPB might affect Rh expression in Mi.III RBCs. Materials and Methods (1) Mi.III+ RBCs were serologically identified and further differentiated their homozygosity or heterozygosity by immunoblot or direct sequencing. (2) RhD and RhCcEe mRNA was cloned, and their sequences analysed. (3) The expression levels of Rh antigen, Rh-associated glycoprotein (RhAG) and the U antigen in MI.III vs. non-Mi.III RBCs were assessed by flow cytometry and Western blot. Results Compared with the non-Mi.III samples, the surface expression of the Rh antigen was reduced to 76·4% in Mi.III+/+ RBCs and 93·6% in Mi.III+/−. RhAG expression was also significantly reduced in Mi.III+/+, but not in Mi.III+/−. The U antigen expression in Mi.III+/− was only 14·9% relative to the control RBCs, while GPB was half the level of the controls. The mRNA sequences of Rh polypeptides from Mi.III+ samples were identical to the NCBI reference sequences. Conclusion Substitution of GPB with Gp.Mur significantly reduced the expression of Rh antigen and RhAG on the Mi.III+/+ erythrocyte membrane. The Mi.III phenotype is predicted to induce considerable structural variations within the band 3/Rh-associated macrocomplexes.

Background and Objective: Biliary atresia (BA) is a destructive inflammatory obliterative cholangiopathy of neonates that affects both intrahepatic and extrahepatic bile ducts. Although the etiology is unknown, immunologically mediated injury of the bile ducts triggered by as yet unidentified infectious agents is likely to play a critical role. Interleukin-18 (IL-18) is a proinflammatory cytokine that plays an important role in immune, infectious, and inflammatory diseases because of its induction of interferon-gamma. In this study, we investigated whether polymorphisms of the IL18 gene were associated with susceptibility to BA. Patients and Methods: Genomic DNA was extracted from whole-blood samples of 50 Taiwanese children with BA and 1117 ethnically matched healthy controls. The IL18 ―1297 T/C, ―607 C/A, ―137 G/C, and +105 A/C polymorphisms were genotyped using the TaqMan assay. Results: No statistically significant differences of genotype, allele, carrier, and haplotype frequencies of these IL18 gene variants were found between children with BA and healthy controls. Conclusions: Our data suggest that the IL18 gene does not play a major role in BA predisposition in Taiwanese children.

BACKGROUND: Thomsen-Friedenreich (T) activation in infants with neonatal necrotizing enterocolitis (NEC) has in some cases led to severe hemolysis after transfusions of plasma-containing blood components, causing some authors to advise routine screening for T activation in all infants with NEC. However, no data are available on the frequency of T activation in infants with NEC in Taiwan. STUDY DESIGN AND METHODS: We retrospectively reviewed the medical records of 43 infants with NEC managed in our hospital from 2000 to 2007. In all cases, Arachis hypogaea lectin was used to test for T activation. RESULTS: Of the 43 infants, 16 infants (37%) had Stage II and 27 (63%) had Stage III NEC. Four infants had trace T activation, two of whom received transfusions with washed red blood cells (RBCs) and two with unwashed RBCs. None had evidence of hemolysis. The overall mortality in this series was 16% (7/43), but none of the four babies with T activation died. CONCLUSION: In this series of Taiwanese infants with NEC, weak T activation was present in only 9% (4/43) of infants, and RBC transfusion did not result in hemolysis, regardless of whether washed or unwashed cells were administered. We considered routine screening for T activation and provision of prepared blood components in infants with NEC in Taiwan might be unnecessary.

Background and Objectives Miltenberger blood group antigens belong to the complex MNS system. Miltenberger antigen subtype III (Mi.III) was previously found to promote the expression of band 3. Here, we investigated whether the direct interaction between band 3 and Mi.III-specific Gp.Mur (a glycophorin B-A-B hybrid) might affect the expression of related blood group antigens such as the Wright b (Wrb) antigen. Materials and Methods (1) Band 3 genes of Mi.III+ and non-Mi.III (control) donors were sequenced to determine the genotypes of the Wright antigens. (2) The expression levels of Wrb, glycophorin A (GPA) and band 3 in Mi.III and the control erythrocytes were quantitatively assessed by flow cytometry. Results Mi.III erythrocytes expressed 22·5 ± 6·6% more Wrb antigen than the control cells. The increase in Wrb in Mi.III cells was independent of their GPA levels. Conclusion The elevated Wrb levels in Mi.III RBCs were likely linked to their higher band 3 levels. Higher band 3 densities on the Mi.III+ cell surface conceivably could drive complex formation between band 3 and GPA/Gp.Mur, thereby increasing the expression of Wrb.

Host factors play an important role in determining the immune response and development of leprosy. The human leukocyte antigen system (HLA) has repeatedly been found to be associated with the pathogenesis of leprosy. This study analyzes the polymorphism of the HLA class I and II antigens in multibacillary leprosy patients and a healthy control group to provide predictable prognostic indicators and/or a differential diagnostic for the disease. Sixty-5 multibacillary leprosy patients from Lo-Sheng Leprosarium and 190 healthy Taiwanese were used as cases and controls, respectively. A serologic method was initially used for HLA-A and HLA-B antigen determination, and sequence-based typing was later applied for HLA-DRB1 allele typing. Although no significant associations were found with HLA-A or HLA-B antigens, this study shows a strong HLA-DRB1*0405 association with resistance to multibacillary leprosy, supporting results previously reported in the literatures.

Biliary atresia (BA) is a devastating neonatal hepatobiliary disease characterized by bile duct inflammation and fibrosis. The pathogenesis remains unclear, but immunologically mediated injury to bile ducts following an infectious insult is likely to play a critical role. Interferon-gamma (IFN-gamma) is a key cytokine that affects immune-mediated inflammatory responses.This study aims to investigate whether polymorphisms of the IFN-gamma (IFNG) gene were associated with susceptibility to BA.The IFNG -1615 C/T, -183 G/T, +874 A/T, and +2197 A/G polymorphisms were genotyped using the TaqMan assay, and CA repeat microsatellite was analyzed using capillary electrophoresis in 50 children with BA and 788 ethnically matched healthy controls.The distribution of genotype, allele, and haplotype frequencies of these IFNG gene variants did not differ significantly between children with BA and controls.Polymorphisms of the IFNG gene do not appear to play a major role in the genetic predisposition to BA in Taiwanese children.

BACKGROUND: Anti-“Mia” is one of the most important irregular red blood cell antibodies found in Taiwan. The aim of this study was to investigate whether specific HLA-DRB1 alleles are associated with anti-“Mia” production. STUDY DESIGN AND METHODS: A case-control retrospective study was performed on 68 patients showing presence of anti-“Mia” and 219 unrelated control subjects from the Mackay Memorial Hospital. HLA-DRB1 genotyping was carried out using sequence-based typing method. Fisher's exact test using 2 × 2 contingency tables was used to analyze significance of the association between DRB1 polymorphisms and presence of anti-“Mia” in patients. RESULTS: HLA-DRB1*0901 allele frequency in the anti-“Mia” group (30%) was significantly higher than in the control group (16%) with an odds ratio of 2.27 (95% confidence interval, 1.44-3.55; p = 0.0005; pc = 0.016). CONCLUSION: HLA-DRB1*0901 is significantly more prevalent in the anti-“Mia” patients group than in the control group. It is suggested that cells from DR9 individuals might present processed “Mia” antigen-allospecific peptides more effectively than cells from individuals carrying other DR phenotypes. Finally, it was predicted that two epitopes, derived from the MiIII glycophorin amino acid sequence, were likely to bind preferentially with the DR9 molecule. Further work will be necessary to determine if these epitopes are responsible for anti-“Mia” alloimmunization.

Human papillomavirus (HPV) is considered to be a necessary but not sufficient cause for cervical cancer. The host immunogenetic background plays an important role in the persistence of HPV infection and subsequent development of cervical cancer. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a molecule expressed mainly on activated T cells and is important in the down-regulation of T-cell activation. The aim of this study was to determine if polymorphisms of the CTLA-4 gene are associated with HPV-induced cervical cancer in Taiwanese women. Polymerase chain reaction-restriction fragment length polymorphism was used to genotype -318 C/T, +49 A/G and CT60 A/G polymorphisms in 144 women with cervical squamous cell carcinoma (CSCC) and 378 ethnicity-matched healthy control women. The presence and genotypes of HPV in CSCC were determined by E6-, E7-based nested polymerase chain reaction. The frequency of C/T genotype of -318 C/T polymorphism was significantly higher in patients with HPV-16-positive CSCC compared with controls (odds ratio = 1.99, 95% confidence interval = 1.16-3.42, P(c) = 0.03). No significant associations were found for +49 A/G and CT60 A/G polymorphisms. Analysis of haplotypes, computationally inferred from genotype data, also revealed no significant differences in distribution among all subjects with CSCC, those with HPV-16-positive CSCC and controls. Our results suggest that the -318 C/T variant in the promoter region of the CTLA-4 gene is associated with HPV-16-associated CSCC in Taiwanese women.

Background Aneurysmal subarachnoid hemorrhage (SAH) is a highly morbid and fatal condition with high rate of cognitive impairment and negative impact in quality of life among survivors. Delayed cerebral infarction (DCI) is one the major factors for these negative outcomes. In this study we compared the circulating microRNA profiles of SAH patients and healthy individuals, and the circulating microRNA profiles of SAH patients with and without DCI. Methods Peripheral blood samples on Day 7 after the onset of SAH were subjected to microarray analysis with Affymetrix miRNA 3.0 array and quantitative PCR analysis. SAH patients with (N = 20) and without DCI (N = 20) and Healthy controls (N = 20) were included for analyses. Results We demonstrated that 99 miRNAs were found to be dysregulated in the SAH patient group with DCI. 81 miRNAs were upregulated and 18 were downregulated. Findings from KEGG pathway analysis showed that miRNAs and target genes for axon guidance and TGF-beta signaling were involved, implying that the resulted differential miRNA expression pattern reflect the results of SAH instead of etiology of the disease. miR-132-3p and miR-324-3p showed distinctive upregulations in qPCR [miR-132: 9.5 fold (95%CI: 2.3 to 16.7) in DCI group and 3.4 fold (95%CI: 1.0 to 5.8) in Non-DCI group; miR-324: 4924 fold (95%CI: 2620 to 7228) in DCI group and 4545 fold (95%CI: 2408 to 6683) in non-DCI group]. However, there were no significant differences in fold changes between SAH patients with and without DCI [fold change ratios (mean+/-SD): 2.7+/-4.2 and 1.1+/-1.1 for miRNA-132 and miRNA-324]. Conclusion Our study demonstrated that as compared to healthy control, miR-132 and miR-324 showed a upregulation in both SAH DCI and Non-DCI groups. However, the differences between the SAH DCI and non-DCI groups were not statistically significant.

Polymorphisms in human leukocyte antigen (HLA) genes have been implicated in the risk for cervical cancer. Association of certain class II HLA alleles with cervical cancer has been documented in various ethnic populations. The implications of such an association, however, are controversial. We analyzed 126 Chinese women with cervical squamous cell carcinoma (CSCC) and 289 healthy controls to test associations of certain HLA-DRB1 alleles. We then performed meta-analyses combining our own experimental data and data from nine other published studies. We found no significant differences in HLA-DRB1 allele frequencies in both CSCC and HPV-16-positive CSCC patients and control subjects. Meta-analysis provided evidence that four allele families (HLA-DRB1*04, *07, *11, and *15) and seven alleles (HLA-DRB1*0403, *0405, *0407, *0701, *1501, *1502, and *1503) were positively associated and two allele families (HLA-DRB1*09 and *13) and four alleles (HLA-DRB1*0901, *1301, *1302, and *1602) were negatively associated with CSCC in all studies or in Caucasian subgroups. In conclusion, our meta-analysis confirms the apparent association between certain HLA-DRB1 allele families and alleles and CSCC, suggesting that oncogenesis in this disease may be related to defects in immunoregulation. Larger studies may be needed, particularly in various ethnic groups, to further substantiate these associations.

BACKGROUND: The high frequency of RhD (D) antigen among Taiwanese persons (99.67%) often imposes unnecessary risks of undertransfusion on D– patients awaiting D– blood. Also because of the rare occurrence of anti-D among Taiwanese persons, routine pretransfusion D typing has been discontinued in the Mackay Memorial Hospital since 1988. This report is the retrospective evaluation of the outcome of abolishing RhD typing for Taiwanese. STUDY DESIGN AND METHODS: More than 10 years of alloantibody data at Mackay Memorial Hospital Blood Bank were reviewed. The cases with anti-D were further used to analyze the potency of D antigen and to observe whether there were differences in the incidence of anti-D before and after discontinuation of routine D typing among Taiwanese individuals. RESULTS: The incidence of anti-D before and after discontinuation of routine pretransfusion D typing has remained unchanged. The immunogenicity of D and “Mia” in Taiwanese persons is found to be similar. In terms of opportunity for immunization, however, the “Mia” antigen (phenotype frequency 7.3% in Taiwanese persons) has become the most important blood group antigen in Taiwan. CONCLUSION: The results strongly support the exclusion of D typing from routine compatibility testing for individuals of Taiwanese origin. Because the low incidence of D– and relatively high incidence of “Mia”+ phenotypes are common findings throughout southeast Asia, and because a population genetic study revealed that the Taiwanese people are genetically related to southern Asian populations, it is suggested that RhD typing for blood transfusion is unnecessary among southeast Asian populations.

Allopurinol, a commonly prescribed medication for gout and hyperuricemia, is a frequent cause of severe cutaneous adverse reactions (SCAR), which include the drug hypersensitivity syndrome, Stevens–Johnson syndrome, and toxic epidermal necrolysis. The adverse events are unpredictable and carry significant morbidity and mortality. To identify genetic markers for allopurinol–SCAR, we carried out a case-control association study. We enrolled 51 patients with allopurinol–SCAR and 228 control individuals (135 allopurinol-tolerant subjects and 93 healthy subjects from the general population), and genotyped for 823 SNPs in genes related to drug metabolism and immune response. The initial screen revealed strong association between allopurinol–SCAR and SNPs in the MHC region, including BAT3 (encoding HLA-B associated transcript 3), MSH5 (mutS homolog 5), and MICB (MHC class I polypeptide-related sequence B) ( P < 10 –7 ). We then determined the alleles of HLA loci A, B, C, and DRB1. The HLA-B*5801 allele was present in all (100%) 51 patients with allopurinol–SCAR, but only in 20 (15%) of 135 tolerant patients [odds ratio 580.3 (95% confidence interval, 34.4–9780.9); corrected P value = 4.7 × 10 –24 ] and in 19 (20%) of 93 of healthy subjects [393.51 (23.23–6665.26); corrected P value = 8.1 × 10 –18 ]. HLA alleles A*3303, Cw*0302, and DRB1*0301 were in linkage disequilibrium and formed an extended haplotype with HLA-B*5801. Our results indicated that allopurinol–SCAR is strongly associated with a genetic predisposition in Han Chinese. In particular, HLA-B*5801 allele is an important genetic risk factor for this life-threatening condition.

Cytokine gene polymorphisms may affect their transcription, influence their level of production, and may be implicated in inducing susceptibility or resistance to diseases. Cytokine single-nucleotide polymorphisms (SNPs) were used to determine allelic and genotypic frequencies in the Minnan, the Hakka, and in four indigenous tribes: the Ami, the Tsou, the Atayal, and the Tao (or Yami). The following cytokine gene polymorphisms were analyzed: interleukin-1alpha (IL-1alpha) (T/C -889), IL-1beta (C/T -511, T/C +3962), IL-1R (C/T Pst-I 1970), IL-1Ralpha (T/C Mspa1-I 1100), IL-2 (T/G -330, G/T +166), IL-4 (T/G -1098, T/C -590, T/C -33), IL-4Ralpha (G/A +1902), IL-6 (G/C -174, G/A nt565), IL-10 (G/A -1082, C/T -819, C/A -592), IL-12 (C/A -1188), interferon-gamma (A/T UTR 5644), transforming growth factor-beta (C/T codon 10, G/C codon 25), and TNF-alpha (G/A -308, G/A -238). Little differences were observed between the Minnan and the Hakka. On the other hand, the Minnan and Hakka showed significant differences with the indigenous people.

The human blood group i and I antigens are characterized as linear and branched repeats of N-acetyllactosamine, respectively. Conversion of the i to the I structure requires the activity of I-branching β-1,6-N-acetylglucosaminyltransferase (IGnT). Thus the blood group I gene is assigned to encode a β-1,6-N-acetylglucosaminyltransferase; however, its identity has not been confirmed. The null phenotype of I, the adult i phenotype, provides a means to identify the I gene. Interestingly, the adult i phenotype has been noted to be associated with congenital cataracts in Asians. Molecular genetic studies of 3 adult i pedigrees are reported here. The results obtained on mutation detection within the 2 I-branching enzyme encoding genes, segregation analyses, and enzyme function assays identify molecular changes associated with the adult i phenotype. The adult i phenotype in 2 of the pedigrees studied resulted from 1043G→A and 1148G→A mutations, which predict Gly348Glu and Arg383His alterations, respectively, in theIGnT gene. These amino acid changes abolished the original GlcNAc-transferase activity. Deletion of the IGnT gene was observed in the person with adult i phenotype in the third pedigree. These findings suggest that the IGnT gene, first reported in 1993, is the candidate for the blood group I gene. Confirmation of the blood group I gene will further assist in the investigations of the molecular genetics that control I antigen expression in secretions and the molecular basis for the association of the adult i phenotype with congenital cataracts in Asians.

G. L. Daniels (Chair), D. J. Anstee, J. P. Cartron, W. Dahr, A. Fletcher, G. Garratty, S. Henry, J. Jorgensen, W. J. Judd, L. K ornstad, C. Levene, M. Lin, C. Lomas-Francis, A. Lubenko, J. J. Moulds, J. M. Moulds, M. Moulds, M. Overbeeke, M. E. Reid, P. Rouger, M. Scott, P. Sistonen, E. Smart, Y. Tani, S. Wendel & T. Zelinski*

There are major differences in the distribution of blood group antigens and antibodies between the different population groups of Taiwan and whites. As a result, standard Western pretransfusion testing procedures have been modified for use in Taiwan, resulting in great reductions in cost and labor. These differences, in addition to their influence on the clinical practise of transfusion medicine in Taiwan, are also important anthropologically, and it is hoped that more population groups in Asia can be investigated in the near future. Further DNA studies on the B3 phenotype, the MiIII phenotype, and the Lewis phenotypes among our different population groups are in progress and further interesting findings are awaited.

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) near the human leukocyte antigen (HLA)-DP loci that were significantly correlated with outcomes of hepatitis B virus (HBV) infection. We performed a case–control study nested in a well-characterized cohort of booster recipients to assess whether genetic variants of HLA-DPB1 are also associated with response to hepatitis B (HB) vaccination. The cases and controls were 171 and 510 booster recipients whose post-booster titers of antibodies against HBV surface antigen (anti-HBs) were undetectable and detectable, respectively. The HLA-DPB1 genotype was determined using sequence-based techniques. The frequencies of HLA-DPB1 alleles were significantly different between cases and controls (p = 1.7 × 10−8). The HLA-DPB1 05:01 and 09:01 alleles were significantly more frequent in the cases, and 02:01:02, 02:02, 03:01:01, 04:01:01, and 14:01, were significantly more frequent in the controls. The adjusted odds ratio (OR) of undetectable post-booster anti-HBs titers was significantly correlated with the number of risk alleles (p for trend = 3.8 × 10−5). For the number of protective alleles, the trend was significantly inversed (p for trend = 1.3 × 10−5). As compared with subjects with two risk alleles, adjusted OR were 0.34 (95 % confidence interval [CI] 0.21–0.55) and 0.20 (95 % CI 0.08–0.48) for subjects with 1 and 2 protective alleles, respectively. The HLA-DPB1 02:02, 04:01:01, 05:01 and 09:01 alleles were also significantly correlated with the likelihoods of undetectable pre-booster anti-HBs titers. Our results indicated that HLA-DPB1 is significantly correlated with response to booster HB vaccination in adolescent who had received postnatal active HB vaccination. HLA-DBP1 may also determine the long-term persistence of response to HB vaccination.

Neonatal jaundice is known to be more severe in Taiwanese infants than in Caucasian infants. Although ABO fetomaternal incompatibility and glucose-6-phosphate dehydrogenase deficiency have been shown to play a role in the etiology of neonatal jaundice in some Taiwanese infants, the etiology in the majority ofcases is unknown. In this study we found that in Taiwanese newborn infants, the red cell Le a antigen appeared later in infants who were jaundiced (peak serum bilirubin levels of >12 mg/dl during the first week of life) than in infants who were not. However, the Le b antigen, and hence the transferases encoded by the Se and Se w genes, did not appear to be similarly involved in the etiology of physiological jaundice. Thus it would appear that the Le gene-specified transferase is less active or has a delayed function, in jaundiced infants. The relationship between the Le gene-specified transferase and bilirubin has yet to be established.

A fatal transfusion reaction due to anti-Ku in a Knull (Ko) patient is reported. The patient was transfused with 34 units of incompatible RBCs during 44 days of hospitalization. Apart from the first transfusion, all subsequent transfusions failed to raise the patient’s Hb.No serum antibody was identified until he was transferred to another hospital for dialysis. A compatibility test demonstrated a weak antibody and autocontrol reacting at room temperature by a manual polybrene method. The antibody was considered to be a “cold agglutinin.” A blood sample was sent to a reference laboratory where the patient was found to be Knull and the antibody was identified as anti-Ku. Immunohematology 2003;19:19–21.

Transfusion-associated respiratory distress occurred more often in the past in Taiwan but recently only very rarely. From the results of our studies it would appear that the former cases did not represent immune reactions involving red blood cells, serum proteins (especially IgA), HLA or granulocyte-specific antigens. Other causes, such as improper handling of the blood units may have been involved, but further study is also needed to investigate the possibility of the role of cytokine release from leukocytes or other factors. In addition, it appears that both plasma and serum obtained by recalcification of plasma may cause false-positive reactions in the granulocyte microagglutination test.

Lewis phenotyping of 487 blood samples from Chinese newborn infants and young children, revealed that 50% of cord cells were Le(a-b+) and 50% Le(a-b-). The weak Leb antigen of Le(a-b+) cord cells is most likely produced by the newborn infant rather than of maternal origin and it appears that these infants eventually develop by way of an intermediate Le(a+b+) stage into the adult Le(a-b+) phenotype. Most infants with Le(a-b-) cord cells, but not all, appear to develop through a transitional Le(a+b-) stage, into Le (a+b+) by about 1 month of age, most likely continuing as such into adulthood. This development of Le(a-b-) cord cells into the adult Le(a+b+) phenotype is postulated to be the result of the weak secretor gene Se omega. Those infants with Le(a-b-) cord cells that do not convert to Le(a+b+) during the first month of life, most likely remain as such into adulthood. The blood of 120 adult voluntary blood donors, used as controls, reconfirmed adult Chinese phenotypic frequencies of approximately 70% Le(a-b+), 22% Le(a+b+) and 8% Le(a-b-).

Biliary atresia (BA) is a neonatal cholangiopathy of unknown etiology that leads to biliary cirrhosis and is the most common cause of liver transplantation in children. A still undetermined hepatobiliary viral infection may elicit an uncontrollable autoimmune response against the biliary epithelial cells in genetically predisposed children and culminates in atresia of the biliary trees. Interleukin 4 (IL4) is crucial for the differentiation of naive T helper cells into the T helper 2 effector cells that promote humoral immunity. This study aims to investigate whether polymorphisms of the IL4 gene are associated with susceptibility to BA. Genomic DNA was extracted from whole blood samples of 53 Taiwanese children with BA and 904 ethnically-matched healthy controls. The IL4 –590 C/T, –33 C/T, and 8375 A/G polymorphisms were genotyped using the Pre-Developed TaqMan Allelic Discrimination Assay in a real-time polymerase chain reaction system. No significant difference between children with BA and healthy controls were found when comparing genotype, allele, carrier, and haplotype frequencies of these IL4 gene variants. These results suggest that the tested polymorphisms of IL4 gene are unlikely to contribute significantly to BA susceptibility in Taiwanese children.

BACKGROUND: Graves' disease (GD) is the leading cause of hyperthyroidism and thyroid eye disease inherited as a complex trait. Although geoepidemiology studies showed relatively higher prevalence of GD in Asians than in Caucasians, previous genetic studies were contradictory concerning whether and/or which human leukocyte antigen (HLA) alleles are associated with GD in Asians. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a case-control association study (499 unrelated GD cases and 504 controls) and a replication in an independent family sample (419 GD individuals and their 282 relatives in 165 families). To minimize genetic and phenotypic heterogeneity, we included only ethnic Chinese Han population in Taiwan and excluded subjects with hypothyroidism. We performed direct and comprehensive genotyping of six classical HLA loci (HLA-A, -B, -C, -DPB1, -DQB1 and -DRB1) to 4-digit resolution. Combining the data of two sample populations, we found that B*46:01 (odds ratio under dominant model [OR] = 1.33, Bonferroni corrected combined P [P(Bc)] = 1.17 x 10⁻²), DPB1*05:01 (OR = 2.34, P(Bc) = 2.58 x 10⁻¹⁰), DQB1*03:02 (OR = 0.62, P(Bc) = 1.97 x 10⁻²), DRB1*15:01 (OR = 1.68, P(Bc) = 1.22 x 10⁻²) and DRB1*16:02 (OR = 2.63, P(Bc) = 1.46 x 10⁻⁵) were associated with GD. HLA-DPB1*05:01 is the major gene of GD in our population and singly accounts for 48.4% of population-attributable risk. CONCLUSIONS/SIGNIFICANCE: These GD-associated alleles we identified in ethnic Chinese Hans, and those identified in other Asian studies, are totally distinct from the known associated alleles in Caucasians. Identification of population-specific association alleles is the critical first step for individualized medicine. Furthermore, comparison between different susceptibility/protective alleles across populations could facilitate generation of novel hypothesis about GD pathophysiology and indicate a new direction for future investigation.

A new human leukocyte antigen-B allele, B*51:84 (B*5184), has been found in Taiwan. It differs from B*51:01:01(B*510101) by one nucleotide substitution at codon 145 (CGC →GGC), resulting to one amino acid change (Arginine to Glycine).

Poster Session: Biochemistry and Molecular Biology: no. 674. Membrane Proteins as Drug Targets: B121 abstract no. 674.2

Biliary atresia (BA) is a chronic inflammatory disease of the bile ducts resulting in biliary cirrhosis. Vascular endothelial growth factor (VEGF) has been implicated in cell-mediated inflammatory reactions. We aimed to study the relationship between genetic variations of the VEGF gene and susceptibility to BA using both case-control and family-based methodologies.A total of 45 Taiwanese children with BA, 160 ethnically matched healthy controls, and 40 families (consisting of parents, affected children, and unaffected siblings) were studied. Three functional VEGF polymorphisms (-2578 A/C, -634 G/C, and +936 C/T) were assessed by using TaqMan assay.The +936 CC genotype [odds ratio (OR) 3.51, 95% confidence interval 1.54-8.01, P(c)=0.006] and C allele (OR 3.19, 95% confidence interval 1.48-6.90, P(c)=0.004) were significantly associated with increased risk of BA. The association of the +936 C allele with BA was also confirmed in a family-based association study (OR 5.7, chi2=9.8, P(c)=0.005). None of the haplotypes studied significantly influenced the risk to BA in either the case-control or family data sets.The VEGF +936 C/T polymorphism and particularly the C allele are associated with BA, possibly conferring increased susceptibility to the disease.

The special blood group antigen Mi.III exhibits a characteristic hybrid structure of glycophorin A (GPA) and glycophorin B, termed Gp.Mur. This phenotype has exceptionally high occurrence rates in several indigenous tribes in Taiwan ( approximately 21.2%-88.4%). Because glycophorin/Miltenberger begins interaction with anion exchanger-1 (AE1) in the endoplasmic reticulum, we hypothesized that the AE1-based macrocomplexes on erythrocyte membranes obtained from Mi.III(+) people could be differentiated from those obtained from non-Miltenberger people. Quantitative mass spectrometric comparison of the AE1-based complexes by iTRAQ (Applied Biosystems) revealed 25% to 67% higher expression of AE1 in Mi.III(+) erythrocytes. In accordance with the higher AE1 level, the Mi.III(+) erythrocytes exhibited superior HCO(3)(-) capacities, pH homeostasis, and osmotic resistance. Cotransfection experiments in HEK293 cells showed that Gp.Mur, like GPA, enhanced trafficking of AE1 to the plasma membrane. In summary, the increased surface expression of AE1 in Mi.III(+) erythrocytes could be attributed to the additive effect of GPA and Gp.Mur coexpression.

To evaluate the association of human leukocyte antigen (HLA)–DRB1 alleles with systemic lupus erythematosus (SLE) and lupus nephritis (LN) in the Taiwanese population, and to investigate the possible association of HLA-DRB1 alleles with disease severity in LN. HLA-DRB1 alleles were studied in 105 SLE patients (82 patients with LN, 23 patients without LN) and 855 healthy controls by polymerase chain reaction and sequence-based typing assays. The frequency of the HLA class II alleles DRB1*0301 (Odds ratio [OR] = 2.01, 95% confidence interval [CI] = 1.31–3.10, Pc = 0.02) and DRB1*1501 (OR = 2.06, 95% CI = 1.36–3.13, Pc = 0.01) were both increased in SLE patients, compared to healthy controls. The frequency of DRB1*1202 was significantly lower in LN patients than in SLE patients without nephritis (OR = 0.23, 95% CI = 0.09–0.57, Pc = 0.01). No specific allele was significantly associated with an increased or decreased risk for severity of LN in this sample. In Taiwanese people, the DRB1*0301 and DRB1*1501 alleles are significant risk factors for SLE, while the DRB1*1202 allele is protective for LN.

An alpha-D-galactosidase (EC 3.2.1.21), capable of converting group B into group O red cells, was isolated from the stem portion of taro. It was purified about 3000 fold by gel filtration and ion-exchange chromatography. The blood group-converting activity was demonstrated by hemolysis and hemagglutination studies. This activity is comparable to that of alpha-D-galactosidase isolated from coffee beans. Taro alpha-D-galactosidase also hydrolyzes (1----4)- and (1----6)-linked alpha-D-galactopyranosyl groups from D-galactose-containing glycoconjugates. Taro alpha-D-galactosidase has a low Km value (0.28mM), a low molecular weight (40,000), and a neutral optimal pH (6.0). At a final enzyme concentration of 30 units/mL in the incubation mixture, the conversion of group B into group O activity was completed within two hours, without apparent changes in the shape of the red cells.

Although some previous studies have reported that genetic and immunological factors play important roles in the pathogenesis of Kawasaki disease (KD), the etiological factors of this enigmatical pediatric disease are still poorly understood.This study aims to investigate whether polymorphisms of the CD40 ligand (CD40L) gene are associated with KD and the development of coronary artery lesions (CAL) in the Taiwanese children.The CD40L -3459 A/G and IVS4+121 A/G single nucleotide polymorphisms (SNPs) were genotyped in 167 children with KD and 1,010 ethnically matched healthy controls by TaqMan assay.None of the CD40L polymorphisms was associated with susceptibility or CAL development of KD, and this finding was supported by the haplotype analysis.In summary, these results provide little support for specific CD40L SNPs in the susceptibility or CAL development of KD in Taiwanese children. However, it will be necessary to validate or replicate this association in other independent large-size ethnic groups.

Background The CTLA4 gene is involved in the activity of T cells. Aim To determine the association between Graves' disease (GD) susceptibility and CT60 polymorphism of the CTLA4 gene. Patients 189 children with GD and 620 healthy controls. Methods We determined the genotype with restriction fragment length polymorphism and compared results. Results Genotype G/G was significantly associated with GD (odds ratio [OR] = 1.71, 95% confidence interval [CI] 1.20-2.44, Pc = 0.006); however, allele A could reverse its effect. Allele G was significantly more frequent (OR = 1.61, 95% CI 1.18-2.19, Pc = 0.0049) but allele A (OR = 0.62, 95% CI 0.46-0.85, Pc = 0.0049) and phenotype A (OR = 0.58, 95% CI 0.41-0.83, Pc = 0.006) were less frequent in patients with GD than in controls. Conclusion The CT60 SNP was associated with susceptibility to GD. The G allele increased the risk of GD.

Kawasaki disease (KD) is an acute febrile vasculitis of unknown etiology that mainly occurs in infants and children. Clinical and histopathologic findings suggest that vascular endothelial growth factor (VEGF) is involved in the coronary artery lesions (CALs) development in KD. This study hypothesized that specific VEGF gene polymorphisms and their haplotypes are associated with KD susceptibility and CAL development in Taiwanese children.The VEGF -2578 A/C, -634 G/C, and +936 C/T single-nucleotide polymorphisms (SNPs) were genotyped in 156 children with KD and 672 ethnically matched healthy controls using the Pre-Developed TaqMan Allelic Discrimination Assay.No significant differences in genotype, allele, carrier, and haplotype frequencies of the three SNPs were found between healthy controls and children with KD or between patients with and without CAL.Our data suggest that VEGF -2578 A/C, -634 G/C, and +936 C/T SNPs do not confer increased susceptibility to KD or to CAL development.

Although some previous studies have reported that genetic and immunologic factors play important roles in the pathogenesis of Kawasaki disease (KD), the etiologic factors of this enigmatic pediatric disease are still poorly understood. The purpose of this study was to investigate whether polymorphisms of the human leukocyte antigen DRB1 (HLA-DRB1) gene are associated with KD and the development of coronary artery lesions (CAL) in Taiwanese children. Genomic DNA was extracted from whole blood samples from 145 children with KD and 331 healthy controls. The HLA-DRB1 gene was genotyped by polymerase chain reaction (PCR) and sequence-based typing assays. We found that the distribution of HLA-DRB1 allele families and alleles in children with KD did not differ from that in healthy controls. Stratified analysis did not demonstrate any association between particular HLA-DRB1 allele families or alleles and the development of CAL in children with KD. These findings suggest that susceptibility to KD and CAL is not associated with the HLA-DRB1 gene in a Taiwanese population. If immunogenetic determinants are involved in this disease and its complications in Taiwanese children, they must involve genes other than HLA-DRB1.

The CBLB gene functions as a negative regulator of autoimmunity. Impairment of the Cbl-b signaling pathway may contribute to human autoimmune disease. dbSNP rs2305035 is a C/T polymorphism located in exon 10 of the CBLB gene. We report an association study of this polymorphism in children with Graves' disease. The patients were 158 unrelated children (125 girls) with Graves' disease, aged 9.8 +/- 3.3 years. The controls consisted of 237 adults without a history of autoimmune disease. The C allele and phenotype frequencies of patients and controls were 247 (78.2%) vs 356 (75.1%) (OR = 1.19, p >0.05) and 151 (95.6%) vs 221 (93.2%) (OR = 1.56, p >0.05), respectively. The allelic polymorphism in patients and controls with and without DRB1*09012 were also not significantly different. This study demonstrates that the C/T polymorphism in exon 10 of the CBLB gene is not associated with Graves' disease in children.

BACKGROUND: The A m phenotype has been characterized as a weak expression of the A antigen on red blood cells but the presence of a normal quantity of the A antigen in saliva. This study describes a molecular genetic analysis of members of an A m family. STUDY DESIGN AND METHODS: The eight exon regions of the ABO genes of the A m proposita were amplified by polymerase chain reaction and cloned, and their sequences were analyzed. The α-1,3-N-acetylgalactosaminyltransferase (A-transferase) activities of the A m serum and the expressed A m transferase were analyzed. RESULTS: An A gene with a 664G>A mutation, which predicts an amino acid alteration of Val222Met, was identified in the A m proposita. This A m 664A allele was demonstrated in other three family members with the A m phenotype. The A-transferase activity was virtually undetectable in the A m sera, and the expressed A m transferase showed weak A-transferase activity, when compared with the expressed A 1 transferase, in assays that use acceptor substrates mimicking the Type 2 H structure and Type 1 H structure. CONCLUSION: A novel A allele with 664G>A mutation was demonstrated in a pedigree with the A m phenotype. The mechanism leading to the formation of the A m phenotype still awaits elucidation.

Background The human leukocyte antigen (HLA) system is widely used as a strategy in the search for the etiology of infectious diseases and autoimmune disorders. During the Taiwan epidemic of severe acute respiratory syndrome (SARS), many health care workers were infected. In an effort to establish a screening program for high risk personal, the distribution of HLA class I and II alleles in case and control groups was examined for the presence of an association to a genetic susceptibly or resistance to SARS coronavirus infection. Methods HLA-class I and II allele typing by PCR-SSOP was performed on 37 cases of probable SARS, 28 fever patients excluded later as probable SARS, and 101 non-infected health care workers who were exposed or possibly exposed to SARS coronavirus. An additional control set of 190 normal healthy unrelated Taiwanese was also used in the analysis. Results Woolf and Haldane Odds ratio (OR) and corrected P-value (Pc) obtained from two tails Fisher exact test were used to show susceptibility of HLA class I or class II alleles with coronavirus infection. At first, when analyzing infected SARS patients and high risk health care workers groups, HLA-B*4601 (OR = 2.08, P = 0.04, Pc = n.s.) and HLA-B*5401 (OR = 5.44, P = 0.02, Pc = n.s.) appeared as the most probable elements that may be favoring SARS coronavirus infection. After selecting only a "severe cases" patient group from the infected "probable SARS" patient group and comparing them with the high risk health care workers group, the severity of SARS was shown to be significantly associated with HLA-B*4601 (P = 0.0008 or Pc = 0.0279). Conclusions Densely populated regions with genetically related southern Asian populations appear to be more affected by the spreading of SARS infection. Up until recently, no probable SARS patients were reported among Taiwan indigenous peoples who are genetically distinct from the Taiwanese general population, have no HLA-B* 4601 and have high frequency of HLA-B* 1301. While increase of HLA-B* 4601 allele frequency was observed in the "Probable SARS infected" patient group, a further significant increase of the allele was seen in the "Severe cases" patient group. These results appeared to indicate association of HLA-B* 4601 with the severity of SARS infection in Asian populations. Independent studies are needed to test these results.