ABO Genotyping Research Articles

Highlights of This Issue

Although mammographic density is a strong marker of breast cancer risk, it is unclear whether tumors arise specifically within dense breast tissue. Pinto Pereira and colleagues studied 231 British women diagnosed with breast cancer and assessed whether tumor location was related to localized mammographic density 5 years prior to diagnosis. Tumor locations, identified on digitized diagnostic films, were aligned with serial images obtained from the same woman before diagnosis. The authors report that prediagnostic mammographic density was higher in areas that subsequently contained tumors. This work supports a model in which tumors predominantly arise within radiodense breast tissue and suggests that localized mammographic density may be a predictor of subsequent tumor location.To uncover new biomarkers for bladder cancer, Eissa and colleagues retrospectively analyzed the methylation status of the RARβ2 and APC genes in urine samples from bladder cancer patients. The authors report that the methylation of these two genes was higher in cancer patients compared with healthy controls. Methylated RARβ2 and APC were found in all bladder cancer grades and stages. These studies suggest that methylated RARβ2 and APC might represent valuable urinary molecular markers for the early detection of bladder cancer.Previous studies, using serotype-derived blood typing, have shown an association between ABO blood type and gastric cancer. In this study, Nakao and colleagues further explored this association using single-nucleotide polypeptide analysis to evaluate ABO blood genotype and the risks of gastric cancer, atrophic gastritis and Helicobacter pylori infection. They report a significant association between ABO genotype and gastric cancer risk. In addition, the study revealed that ABO genotype may also influence atrophic gastritis prevalence and H. pylori infection.The microsomal epoxide hydrolase enzyme (mEH), encoded by EPHX1, has an established role in the detoxification of smoking-induced oxidative substances. Lee and colleagues explored the association between two EPHX1 polymorphisms and cancer risk in a large Danish population. In these studies, 47,000 individuals were genotyped and divided into groups with predicted fast, intermediate, and slow mEH phenotypes. The authors then evaluated the risks of several cancer diagnoses and found that individuals with predicted intermediate and slow mEH activities were at higher risk of tobacco-related cancers, compared with subjects with fast mEH phenotypes. This work represents one of the largest studies testing the hypothesis that genetically lowered mEH activities are associated with cancer risk.

Cloning and sequence analysis of 5'untranslated region of human ABO gene

To clone and investigate the polymorphism of the 5'-untranslated region (5'-UTR) of the human ABO gene, in order to provide the basis for exploring the transcriptional regulation of the human ABO histo-blood group genes. ABO phenotypes of 30 unrelated healthy blood donors were determined by serological technique, their genotypes were analyzed by sequencing the exons 6 and 7 of the ABO gene. Nearly 5 kb of the 5'-UTR of ABO gene was obtained by PCR amplification and sequencing was performed bidirectionally. Haplotypes of samples with heterozygous sites in the 5'-UTR of ABO gene were separated and analyzed after cloning. Twenty polymorphic sites were identified in these samples where ABO genotypes were consistent with serological phenotypes. It included sixteen nucleotide sequence variations, one 8 bp deletion, one 6 bp deletion/insertion, one 36 bp insertion and one 43 bp repeats. Among them, 11 were novel polymorphic sites. Seven different haplotypes of 5'-UTR of ABO gene were defined to the cis/trans linkage of those mutations. There were polymorphisms in the 5'-UTR of ABO gene and the nucleotide sequences near the proximal promoter were conservative.

A Novel Method for ABO Genotyping Using a DNA Chip

ABO genotyping is often performed to identify the blood type of decomposed samples, which is difficult to be determined by a serological test. In this study, we developed a simple method for ABO genotyping using a DNA chip. In this method, polymerase chain reaction-amplified and fluorescent-labeled fragments in the ABO gene and primate-specific D17Z1 were hybridized with DNA probes on a chip designed to detect single nucleotide polymorphisms (SNPs) in the ABO gene and part of the D17Z1 sequence. Using blood samples from 42 volunteers and 10 animal species, we investigated whether the chip could be used to detect SNPs in the ABO gene and the D17Z1 sequence. This method was then applied to various forensic samples, and it was confirmed that this method was suitable for the simultaneous analyses of ABO genotyping and species identification. This method fulfills the recent need for the development of rapid and convenient methods for criminal investigations.

Rapid One-Tube Long PCR-Sequencing for ABO Genotyping From Exons 2 to 7.

AbstractAbstract 1116 Background:ABO is the most clinically important blood group system in transfusion and transplantation medicine. DNA-based methods for determining the ABO genotype have markedly advanced in recent years and have been increasingly introduced in clinical laboratories. Most studies using sequencing methods for ABO genotyping have focused on exons 6 and 7, ignoring the other exon and intron sequences. Although these approaches usually can distinguish among common ABO alleles, erroneous or ambiguous typing results might be obtained in cases of hybrid and subgroup alleles with various missense or nonsense mutations. For more accurate ABO genotype results, the entire exon sequence including the introns and regulatory regions should be analyzed, but such sequencing methods are time-consuming, labor intensive, and expensive. Methods:To investigate the entire sequence of ABO exons 2–7 including introns 2–6 and excluding non-coding region of exon 7, long shuttle one-tube PCR-sequencing method was designed. We used a rapid DNA polymerase with high fidelity (Phusion® Flash II DNA Polymerase; Finnzyme OY, Espoo, Finland). ABO genotyping was performed using this technique in peripheral blood of two unrelated families. Results:Time requirement of the PCR amplification and purification processes were about 1.8 h and 20 min, respectively. Also, this technique could read the entirety of exons 2–7 including introns, required one PCR tube, one purification step, and 27 sequencing reactions for the entire sequencing. A total of seven different ABO alleles from two families were analyzed. All genotyping results agreed with serologic findings and results expected by Mendelian inheritance. The ABO phenotypes (genotypes) of family A members were A1 (A105/A105), O (O01/O02), A1 (A105/O01), A1 (A105/O02), and A1 (A105/O02) in the husband, wife, and three sons, respectively. The ABO genotype of husband in family B was cis-ABO1/O01 and his serologic ABO typing result was ABweak. His wife had the common AB phenotype (A1B); however, her genotype revealed a hybrid of A and B alleles and the B101 allele. The hybrid allele comprised the consensus ABO allele in exons 2–5 (including introns 2–4), the B101 allele in exon 6 (with introns 5 and 6), and the Al01 allele in exon 7. Conclusion:This method is suitable for the fast analysis of ABO genotypes and may be valuable in clinical transfusion or forensic applications. Disclosures:No relevant conflicts of interest to declare.

Quantitative Trait Locus (QTL) Linkage Analysis of a Large Pedigree with Von Willebrand Disease Identifies Novel Modifier Loci of Von Willebrand Factor Levels and Confirms Some Previously Reported Modifier Loci

AbstractAbstract 240Von Willebrand disease (VWD) is the most common inherited bleeding disorder with a prevalence ranging from 0.8 to 1.3%. VWD is characterized by incomplete penetrance and variable expressivity, even in families with a unifying VWF mutation, suggesting the presence of modifier genes. We diagnosed and characterized a 1500-member multigenerational Amish family in which 132 individuals exhibit a single autosomal-dominant C4120T mutation (R1374C) in the A1 domain of the mature von Willebrand Factor (VWF) molecule. When accounting for the C4120T mutation, analyses of coagulation factor VIII levels (FVIII:C) and log transformed, sex- and age-adjusted VWF levels in SOLAR estimated a heritability (h2) of 0.301 for VWF antigen levels (VWF:Ag; p=2.7×10−6), 0.386 for VWF ristocetin cofactor activity (VWF:RCo; p=1.16×10−8), and 0.280 for FVIII:C (p=1.52×10−5). These results demonstrate strong evidence of residual heritability of the traits despite the obvious effect of the 4120 mutation. Among individuals with the wild-type VWF CC4120 genotype, ABO genotype, the only known major modifier of VWF levels, explained 6.5, 10.0 and 4.2% of the variability in VWF:Ag, VWF:RCo and FVIII:C levels, respectively, suggesting that a significant genetic component of the variability of these traits remains unaccounted for.In order to search for modifier loci that contribute to the observed variability we then performed a primary genetic screen at an average 10 cM interval using a standard short tandem repeat polymorphism mapping set in a 384-member subset of the pedigree. Quantitative trait locus (QTL) linkage analysis was performed using SOLAR for VWF:Ag, VWF:RCo and FVIII:C. The highest LOD score generated demonstrated linkage of VWF:RCo to region 12p13 where VWF has been physically mapped (LOD=64.73). When accounting for the C4120T mutation, five other chromosomal regions were identified with LOD scores above 2.0 with linkage to FVIII:C, VWF:Ag, and/or VWF:RCo: 2q14 (LOD=2.48), 6p12 (LOD=2.22), 9q33 (LOD=2.84), 10q26 (LOD=2.29), and 16q11 (LOD=2.66). Notably, the ABO locus maps to chromosome 9q33, demonstrating by linkage the known effect of ABO on VWF levels.Five chromosomal regions demonstrated LOD scores between 1.5 and 2.0 for the above phenotypes: 3p14 (LOD=1.56), 4q34 (LOD=1.64), 5q33 (LOD=1.8373), 15q14 (LOD=1.78), and 21q21 (LOD=1.85). Comparison to other linkage and association studies such as the Genetic Analysis of Inherited Thrombophilia (GAIT) and the Framingham Study identified several concurrent peaks in our analysis. Specifically, 22q11and 9q34 were reported in the GAIT study as chromosomal regions linked to VWF:Ag and 6p22 was reported in the Framingham study as well as in the GAIT study as linked to the same phenotype. Furthermore the 22q11 and 21q21 signals display orthology to Mvwf6, a murine modifier region of VWF levels recently reported. Two other signals, 5q33 and 6p12 also display orthology with regions closely syntenic to Mvwf4, another murine modifier. These comparisons strengthen the validity of this multigenerational pedigree as a tool to identify genetic modifiers of the phenotypic variability of VWF levels and provide the basis to which chromosomal regions will be followed-up to identify causative alleles. While genome-wide association studies (GWAS) are characterized by the identification of common variants of relatively modest effect, linkage studies such as the one we present here may have the ability to identify rare variants as well as allelic heterogeneity at a given modifier locus, which may explain a significant portion of the observed variability and likely account for a portion of the genetic component not identified by GWAS analyses. Disclosures:No relevant conflicts of interest to declare.

Evaluation of 28 Human Embryonic Stem Cell Lines for Use as Unrelated Donors in Stem Cell Therapy: Implications of HLA and ABO Genotypes

For human embryonic stem cells (hESCs) to be used clinically, it is imperative that immune responses evoked by hESCs and their derivates after transplantation should be prevented. Human leukocyte antigens (HLA) and ABO blood group antigens are important histocompatibility factors in graft rejection. HLA matching between recipient and unrelated donors, in particular, is important in improving outcomes in hematopoietic cell transplantation (HCT). We have established and successfully maintained 29 hESC lines and analyzed the HLA and ABO genotypes of these lines. HLA-A, -B, -C and -DR (DRB1) genotyping was performed by polymerase chain reaction (PCR) sequence-based typing and ABO genotyping was carried out by PCR restriction fragment length polymorphism methods. To determine what proportion of the Korean population would be covered by these cell lines in organ transplantation, 27 cell lines with HLA-A, -B, and -DR data were evaluated for HCT (cord blood) donors and 28 cell lines with HLA-DR and ABO data were evaluated for solid organ (kidney) transplantation donors, and then compared the data with those from 6,740 donated cord bloods. When 2 HLA mismatches are allowed for HCT, as currently accepted for cord blood transplantation, it was estimated that about 16% and 25% of the possible recipients can find one or more donor cell lines with ≤2 mismatches at A, B, DRB1 allele level and at A, B antigen/DRB1 allele level, respectively. When HLA-DR antigen level matching and ABO compatibility was considered for solid organ (kidney) transplantation, it was estimated that about 29% and 96% of the possible recipients can find one or more ABO-compatible donor cell lines with 0 and 1 DR mismatches, respectively. We provided the first report on the HLA and ABO genotypes of hESC lines, and estimated the degree of HLA and ABO matching in organ transplantation for the Korean population.

A New Method for Human ABO Genotyping Using a Universal Reporter Primer System

We developed a new method for forensic ABO genotyping based on a universal reporter primer (URP) system. This allows for the simultaneous detection of six single nucleotide polymorphism (SNP) sites in the ABO gene (nucleotide positions 261, 297, 526, 703, 796, and 803). This URP system provides obvious peaks, ranging from 82 to 151 bp in length. ABO genotypes were classified and successfully genotyped by our method, including minor alleles that may cause a discrepancy between the genetic data and serological phenotypes. Full profiles were identified using as little as 0.1 ng (0.05 ng ⁄ reaction) of standard K562 and 9947A DNA. Moreover, the success rate of genotyping from a URP system was much higher than that from a conventional primer extension method in degraded DNA. This method enables simple and rapid detection of multiple SNP sites on human ABO genes and is highly specific and sensitive when using limited and degraded DNA.

Rapid Direct PCR for ABO Blood Typing*

Many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. However, these ABO genotyping methods are often time-consuming and call for an initial DNA isolation step that requires the use of expensive kits or reagents. We report here a rapid direct ABO genotyping method that eliminates the need for DNA extraction from fresh blood, hair, and body fluid stains before PCR. Using a fast PCR instrument and an optimized polymerase, the genotyping method-which employs a multiplex allele-specific primer set for the simultaneous detection of three single-nucleotide polymorphism (SNP) sites (nucleotides 261, 526, and 803)-identifies A, B, O01/O02, O03, and cis-AB01 alleles in around 70 min from sample collection to electropherogram. Not only will this ABO genotyping method be efficiently used in forensic practice for rapid screening of samples before full-blown multilocus short tandem repeat profiling, but it will also demonstrate an example of rapid direct genotyping of SNPs that offers the advantages of time- and cost-efficiency, convenience, and reduced contamination during DNA analysis.

Polymorphisms of Lewis and Secretor genes are related to breast cancer and metastasis in axillary lymph nodes

ABH and Lewis antigen expression has been associated with cancer development and prognosis, tumor differentiation, and metastasis. Considering that invasive ductal breast carcinoma (IDC) presents multiple molecular alterations, the aim of the present study was to determine whether the polymorphism of ABO, Lewis, and Secretor genes, as well as ABO phenotyping, could be associated with tumor differentiation and lymph nodes metastasis. Seventy-six women with IDC and 78 healthy female blood donors were submitted to ABO phenotyping/genotyping and Lewis and Secretor genotyping. Phenotyping was performed by hemagglutination and genotyping by the polymerase chain reaction with sequence-specific primers. ABO, Lewis, and Secretor genes were classified by individual single nucleotide polymorphism at sites 59, 1067, 202, and 314 of the Lewis gene, 428 of the Secretor gene, and 261 (O1 allele), 526 (O2 and B allele), and 703 (B allele). No association was found between breast cancer and ABO antigen expression (P = 0.9323) or genotype (P = 0.9356). Lewis-negative genotype was associated with IDC (P = 0.0126) but not with anatomoclinical parameters. Nonsecretor genotype was associated with axillary lymph node metastasis (P = 0.0149). In conclusion, Lewis and Secretor genotyping could be useful to predict respectively breast cancer susceptibility and axillary lymph nodes metastasis.

Comprehensive polymorphism analysis of ABO using allele‐specific separation by bead technology and subsequent sequencing

The ABO system is the most important blood group system in transfusion and transplantation medicine. Over the last decade ABO genotyping was introduced to complement serological analysis but because of the enormous diversity at the ABO locus, DNA-based typing is highly sophisticated. In some genotype combinations and especially when dealing with hybrid genes, cis/trans linkage analysis is necessary. Therefore, we developed a simple and reliable approach to isolate ABO haplotypes. The described method physically separates diploid genomic DNA using probe-dependent allele hybridization followed by magnetic bead separation. After this haplotype-specific extraction (HSE), sequencing based typing (SBT) was performed to analyze coding and non-coding respectively regulatory regions of ABO.

Many genetically defined ABO subgroups exhibit characteristic flow cytometric patterns

A flow cytometric method for detection of low levels of A/B antigen had been developed previously in our laboratory. The aim of this study was to investigate if this approach could be utilized to characterize different ABO subgroups and constitute a useful tool in a reference laboratory. Blood samples causing ABO discrepancies (n = 94) by routine serology were further analyzed by ABO genotyping and flow cytometry. To verify the specificity of the monoclonal anti-A and -B reagents and to establish normal flow cytometric patterns, samples from 80 blood donors with common phenotypes were also assessed. Distinguishable flow cytometric patterns were detected for several subgroups but were more apparent for A(weak) (n = 80) samples than B(weak) (n = 14). Two subgroups, A(finn) (n = 11) and A(3) (n = 10) displayed diagnostic features and were used to establish reproduciblity over time and between donors. In general, the consistency within subgroups was remarkable. The allelic enhancement phenomenon was clearly visualized among A(x) samples (n = 10) where different alleles in trans resulted in high, low, or no A antigen expression. Nonsubgroup samples including O/A and O/B chimeras or A(h) and B(h) para-Bombay phenotypes displayed clearly distinguishable histograms. Samples from pregnant women (n = 10) displayed acquired A antigen loss, apparently accentuated during the third trimester. Genetically defined ABO subgroups and other anomalous phenotypes displayed flow cytometric profiles that may contribute valuable information to the investigation of ABO discrepancies. We conclude that the presented assay may complement traditional serology and genetic analysis in the reference laboratory setting.

Pancreatic cancer risk and ABO blood group alleles: results from the pancreatic cancer cohort consortium.

A recent genome-wide association study (PanScan) identified significant associations at the ABO gene locus with risk of pancreatic cancer, but the influence of specific ABO genotypes remains unknown. We determined ABO genotypes (OO, AO, AA, AB, BO, and BB) in 1,534 cases and 1,583 controls from 12 prospective cohorts in PanScan, grouping participants by genotype-derived serologic blood type (O, A, AB, and B). Adjusted odds ratios (ORs) for pancreatic cancer by ABO alleles were calculated using logistic regression. Compared with blood type O, the ORs for pancreatic cancer in subjects with types A, AB, and B were 1.38 [95% confidence interval (95% CI), 1.18-1.62], 1.47 (95% CI, 1.07-2.02), and 1.53 (95% CI, 1.21-1.92), respectively. The incidence rates for blood types O, A, AB, and B were 28.9, 39.9, 41.8, and 44.5 cases per 100,000 subjects per year. An increase in risk was noted with the addition of each non-O allele. Compared with OO genotype, subjects with AO and AA genotype had ORs of 1.33 (95% CI, 1.13-1.58) and 1.61 (95% CI, 1.22-2.18), whereas subjects with BO and BB genotypes had ORs of 1.45 (95% CI, 1.14-1.85) and 2.42 (1.28-4.57). The population attributable fraction for non-O blood type was 19.5%. In a joint model with smoking, current smokers with non-O blood type had an adjusted OR of 2.68 (95% CI, 2.03-3.54) compared with nonsmokers of blood type O. We concluded that ABO genotypes were significantly associated with pancreatic cancer risk.

Frequency of ABO blood group system polymorphisms in Plasmodium falciparum malaria patients and blood donors from the Brazilian Amazon region

We investigated the ABO genotypes and heterogeneity of the O alleles in Plasmodium falciparum-infected and non-infected individuals from the Brazilian Amazon region. Sample collection took place from May 2003 to August 2005, from P. falciparum malaria patients from four endemic regions of the Brazilian Amazon. The control group consisted of donors from four blood banks in the same areas. DNA was extracted using the Easy-DNA(TM) extraction kit. ABO genotyping was performed using PCR/RFLP. There was a high frequency of ABO*O01O01. ABO*AO01 was the second most frequent genotype, and the third most frequent genotype was ABO*BO01. There were low frequencies of the ABO*O01O02, ABO*AA, ABO*AB, ABO*BB, and ABO*O02O02 genotypes. We analyzed the alleles of the O phenotype; the O(1variant) allele was the most frequent, both in malaria and non-malaria groups; consequently, the homozygous genotype O(1)(v)O(1)(v) was the most frequently observed. There was no evidence of the homozygous O(2) allele. Significant differences were not detected in the frequency of individuals with the various alleles in the comparison of the malaria patients and the general population (blood donors).

Multicolor Real‐Time PCR Genotyping of ABO System Using Displacing Probes

Rapid and informative ABO genotyping has become increasingly popular in forensic use. We developed a multiplex real-time polymerase chain reaction (PCR) approach to genotype ABO major groups and subgroups. Seven differently fluorophor-labeled displacing probes for O(1)(261delG), A(261G), A(796C/803C), B(796A/803C), O(2) (802G>A), A(2) (1059delC), and A(2) (1009A>G) were combined in one or two PCRs to determine either ABO major groups or subgroups. The method correctly detected 13 reference DNA samples. A blind test of 237 samples resulted in complete agreement with their phenotypes, and 110 of these 237 samples as well as with PCR-SSP method. The whole analysis could be finished in less than 100 min at substantially low material cost and the template DNA ranging from 0.16 to 500 ng per reaction could be quantitatively detected. Despite the limited informativeness of ABO genotyping, the developed methods could find application in rapid and inexpensive screening of forensic settings.

Distribution of ABO blood group allele and identification of three novel alleles in the Chinese Han population

The ABO blood group system is clinically important in blood transfusion. The molecular characterization of ABO blood group has clinical and anthropological importance. Here, we determined the ABO alleles distribution and identified three novel alleles in the Chinese Han population. Four hundred and seventeen Chinese Han individuals were determined by standard serologic techniques for the ABO blood group phenotypes. The ABO genotypes and alleles were analysed by polymerase chain reaction sequence-based typing (PCR-SBT) for sequencing exon 6 to 7 of the ABO gene. The polymorphisms of intron 5 and 6 of the ABO gene were also analysed by PCR-SBT. The two haplotypes including new alleles were separated by a Dynabeads M-270 Streptavidin protocol. All ABO genotypes of the samples were consistent with the phenotypes. Fourteen alleles were identified based on the nucleotide sequences of exon 6 and 7, with five common alleles (A101, A102, B101, O01 and O02), six known rare alleles (A205, B110, O04, O05, O07 and O50) and three novel alleles (B112, CisAB06 and 061). The three new alleles appeared with the frequencies of 0.12%, 0.12% and 0.36%, respectively. The detailed frequencies distribution of ABO alleles was studied in the Chinese Han population. We identified 14 alleles, including 3 novel alleles.

Defining the Molecular Mechanisms Responsible for the ABO High Expresser Phenotype.

Abstract 2119Poster Board II-96ABO(H) blood group antigen expression on platelets varies widely among normal blood donors. An ABO ‚High Expresser' phenotype (HXP) that exhibits significantly increased A and/or B antigen expression on platelets has been identified in ∼7% of normal donors. HXP has been implicated in both platelet-refractoriness and neonatal alloimmune thrombocytopenic purpura, however, the underlying molecular and genetic elements that mediate this phenomenon are not well-defined.To investigate the mechanisms underlying HXP, blood samples were collected from 231 group A (180 A1and 51 A2) and 310 group O individual apheresis platelet donors. Quantitative expression of platelet A and H antigen were then assessed by flow cytometry of platelet-rich plasma. In total, 10 A1 donors (5.6%) exhibited HXP. Analysis of the platelet A antigen expression in these individuals identified 8 HXP donors who exhibited ‚type I' HXP (normal bimodal population of platelets, but with predominant A antigen expression) whereas 2 individuals exhibited ‚type II' HXP (a single uniform population of platelets, strongly positive for blood group A expression). Both types of HXP were found to be a stable donor characteristic.ABO(H) determinants have also been identified on the N-linked glycans of the plasma von Willebrand factor (VWF), and influence plasma VWF levels and susceptibility to proteolysis by ADAMTS13. To determine whether HXP was platelet-specific, blood group A antigen expression on plasma VWF from group A donors was determined. Interestingly, blood group A antigen expression on plasma VWF was concordantly increased in donors with type I and type II HXP, indicative of increased glycosyltransferase expression in HXP individuals.To ascertain whether increased glycosyltransferase expression contributes to HXP, ABO genotype was determined for all 231 group A donors by PCR-RFLP analysis. Genotype at the ABO locus on 9q34 exerts a dosage effect on glycosyltransferase expression. 80% HXP (all type I) donors were genotyped A1A1. suggesting increased A transferase activity contributes to type I HXP. Despite this, the majority of A1A1individuals (67%) did not exhibit HXP, and 2 HXP donors were found to possess the A2 allele, which expresses limited A transferase enzymatic activity. Collectively, this data clearly demonstrates the contribution of additional factors to ABO genotype that contribute to HXP.To identify additional HXP modifiers, potential enhancer repeat elements upstream of the ABO gene were examined in group A donors, including those with HXP. Typically, A1alleles contain a single 43-base pair repeat within a minisatellite positive regulatory region upstream of the ABO gene. In contrast, A2and O1alleles contain four 43bp repeats, which are associated with a 100-fold enhancement of transcriptional activity. Analysis of this enhancer region demonstrated two HXP donors with A1alleles containing four copies of the 43-base pair repeat. Consequently, this allele would be predicted to modulate A transferase expression via enhanced ABO gene transcription.In conclusion, we have demonstrated the multi-factorial nature of the regulatory elements mediating platelet type I and type II HXP. A1alleles containing novel upstream enhancer repeats identified in donor individuals may represent a novel genetic mediator of HXP, and contribute to the pathophysiology associated with this phenomenon independently of ABO genotype. Disclosures:No relevant conflicts of interest to declare.

Loss of red cell ABH Antigens and Hypermethylation of the ABO Gene Promoter region is frequent in Myeloid Malignancies.

Abstract 3151Poster Board III-88 BackgroundLoss of red blood cell (RBC) antigens may occur in solid tumors and in hematological diseases; however the mechanisms involved in these changes are not fully understood. Aberrant DNA hypermethylation is thought to be involved in acute myeloid leukemia (AML) as well as in myelodysplastic syndromes (MDS), and the methylation of cytosines residues in the dinucleotide CpG may account for the expression patterns of the ABO genes. In this study we investigated loss of RBC ABH antigens and the possible role of DNA methylation in the ABO promoter gene in patients with myeloid diseases. MethodsForty-three patients were included in our study (MDS=16, chronic myeloid leukemia (CML)=12, AML=4, polycythemia vera (PV)=3, essential thrombocythemia (ET)=3, chronic myelomonocytic leukemia (CMML)=3, myelofibrosis (MF)=1 and chronic neutrophilic leukemia (CNL)=1). Forty-one patients were evaluated according to their ABO group using serological immunohematological tests (Diamed Inc., Brazil). ABH secreted antigens were investigated in saliva and a PCR-based ABO genotyping using restriction enzyme digestion (Alu and Kpn) was performed to confirm the ABO blood type. In addition, the expression of the A, B and H antigens was analyzed by flow cytometry in 26 patients (median age=63 yrs; 6M/17F) and 81 healthy controls (median age=33 yrs; 38M/43F). Methylation of CpG islands was investigated in 45 bone marrow samples using methylation specific PCR (MSP) technique with methylated and unmethylated primer sets for region from -200 to +26 sequence of the ABO gene. ResultsThe investigation of the secreted antigens in saliva and the genotyping studies confirmed the results of the ABO serological tests in 40 patients, but we found that the RBCs of one patient were not agglutinated by anti-B while his genotype result was compatible with BO indicating loss of the B antigen in his RBCs. Overall, loss of A, B or H antigen was detected by flow cytometry in 11/26 (42%) patients. Hypermethylation of the ABO promoter gene was detected in 51% (23/45) of the analyzed bone marrow samples. Among patients with hypermethylation in the ABO promoter gene, 44% had loss of A or B antigens confirmed by serological or flow cytometric tests compared with 28% in the group of patients with unmethylated ABO promoter gene (p=0.632). ConclusionsEpigenetic changes including methylation of cytosine residues are recognized as major contributors to gene silencing, disease progression and worse outcome in several cancer patients. Our data showed that the hypermethylation of the ABO gene is frequent in patients with myeloid malignancies corresponding to the pathogenesis already described for AML and MDS. However, not all patients showing loss of RBCs antigens had ABO methylation evidence, suggesting that other mechanisms may take place. Patients with myeloid malignancies often need blood transfusion support and loss of RBCs antigens can lead to changing in ABO blood group increasing the risk of serious blood transfusion reactions. The relatively high rate (42%) of loss of ABH antigens found in this study demonstrated that these patients have to be carefully managed to avoid such severe transfusion reactions. (Grants supported by CNPq, 478814/2006-2). DisclosuresNo relevant conflicts of interest to declare.

Development of a laboratory project to determine human ABO genotypes—Limitations lead to further student explorations

A multiplex allele-specific PCR analysis was developed to identify six "common" genotypes: AA, AO, BB, BO, OO, and AB. This project included a pre-laboratory exercise that provided active learning experiences and developed critical thinking skills. This laboratory resulted in many successful analyses, which were verified by student knowledge of their phenotypes. However, the design was found to be deficient for the analysis of variants. The limitations in the original allied-health multiplex design were verified through a student designed problem-based laboratory project in an advanced level biochemistry class. Variants were further analyzed in an undergraduate research project using SSCP analyses. The topic of ABO genotyping provides several opportunities for student-centered explorations.

Comparative analysis of ABO genotyping and serological typing in northern Chinese Han population

In this paper, the comparative analysis of ABO genotyping and serological typing was conducted in 360 unrelated blood samples from northern Chinese Han population using genotyping method and serological typing method, respectively. The results of ABO genotyping were obtained by Goldeneye 16BT STR plus ABO kit. The ABO serological types were determined by the antigen–antibody agglutination test. The ABO types were confirmed by the two methods and no contradiction types were found; two more types were obtained using the ABO genotyping method and the discrimination power was further improved; the information of ABO genotyping and 15 STRs could be obtained at the same time using the Goldeneye 16BT STR plus ABO kit.

ABO genotyping by duplex amplification and oligonucleotide arrays assay

Objective Research on the application feasibility of ABO genotyping for forensic identification by oligonucleotide arrays assay. Methods Oligonucleotide microarrays which detect three different SNPs in exon 6 and exon 7 for ABO genotyping were used. After hybridization wash, the arrays were scanned and fluorescence intensities were analyzed using microarray population studies on ABO was carried out in a sample of 115 unrelated Chinese Han individuals oligonucleotide arrays for genotype detection. The method was also applied to cases. Results Technique could identify six genotypes of ABO system and the results of GeneChip analyses confirmed by PCR–RFLP. According to the results of population studies, no significant deviations Hardy–Weinberg equilibrium could be found. The observed heterozygosity (H-obs) was 0.591. Expected heterozygosity (H-exp) was 0.616. The polymorphic information content (PIC) was the average exclusion probability in paternity testing for duos (PE (1)) was 0.188. The average exclusion probability in paternity testing for trios (PE(2)) was 0.344. The discrimination power 0.777. Conclusion The data and case application demonstrated that ABO typing by oligonucleotide probe arrays was a useful technique for paternity testing and individual identification.