Dombrock Genotyping In Brazilian Blood Donors Reveals Different Regional Frequencies Of The Hy Allele.

Dombrock blood group system genotyping has revealed various rearrangements of the Dombrock gene and identified new variant alleles in Brazil (i.e., DO*A-SH, DO*A-WL and DO*B-WL). Because of the high heterogeneity of the Brazilian population, interregional differences are expected during the investigation of Dombrock genotypes. The present study aims to determine the frequencies of Dombrock genotypes in blood donors from Minas Gerais and compare the frequencies of the HY and JO alleles to those of another population in Brazil. The frequencies of the DO alleles in Minas Gerais, a southeastern state of Brazil, were determined from the genotyping of 270 blood donors. Genotyping involved polymerase chain reaction and restriction fragment length polymorphism analysis to identify the 323G>T, 350C>T, 793A>G, and 898C>G mutations, which are related to the HY, JO, DO*A/DO*B, and DO*A-WL/DO*B-WL alleles, respectively. Moreover, the frequencies of rare HY and JO alleles were statistically compared using the chi-square test with data from another Brazilian region. The HY allele frequency in Minas Gerais (2.4%) was almost twice that of the JO allele (1.5%). The frequency of the HY allele was significantly higher (p-value = 0.001) than that in another Brazilian population and includes a rare homozygous donor with the Hy- phenotype. In addition, the DO*A-WL and DO*B-WL alleles, which were first identified in Brazil, were found in the state of Minas Gerais. The data confirm that the frequencies of DO alleles differ between regions in Brazil. The population of Minas Gerais could be targeted in a screening strategy to identify the Hy- phenotype in order to develop a rare blood bank.35400-

A New Strategy To Identify Rare Blood Donors: Single Polymerase Chain Reaction Multiplex Snapshot Reaction For Detection Of 16 Blood Group Alleles.

As an alternative to phenotyping, large-scale DNA-based assays, which are feasible for high-throughput donor red blood cell typing, were developed for determination of blood group polymorphisms. However, high-throughput genotyping platforms based on these technologies are still expensive and the inclusion of single nucleotide polymorphisms and analysis of the alleles depend on the manufacturer's determination. To overcome this limitation and in order to develop an assay to enable the screening of rare donors, we developed a SNaPshot assay for analysis of nine single nucleotide polymorphisms related to antigens that are difficult to assess using conventional serology. The single polymerase chain reaction multiplex SNaPshot reaction was optimized to identify nine single nucleotide polymorphisms determining 16 alleles: KEL*3/KEL*4, KEL*6/KEL*7, DI*1/DI*2, DI*3/DI*4, YT*1/YT*2, CO*1/CO*2, DO*1/DO*2, DO*4, DO*5. We designed a single multiplex PCR with primers encompassing the blood group single nucleotide polymorphisms and performed an internal reaction with probe primers able to discriminate the alleles after fragment analysis. The SNaPshot assay was validated with 140 known alleles previously determined by PCR restriction fragment length polymorphism. We were able to simultaneous detect nine single nucleotide polymorphisms defining 16 blood group alleles on an assay based on a multiplex PCR combined with a single base extension using genomic DNA. This study demonstrates a robust genotyping strategy for conducting rare donor screening which can be applied in blood centers and could be an important tool for identifying antigen-negative donors and, therefore, for providing rare blood.12 Suppl 1s256-6

An easy and efficient strategy for KEL genotyping in a multiethnic population

BACKGROUND: The Kell blood group system expresses high and low frequency antigens with the most important in relation to transfusion including the antithetic KEL1 and KEL2; KEL3 and KEL4; KEL6 and KEL7 antigens. Kell is a clinically relevant system, as it is highly immunogenic and anti-KEL antibodies are associated with hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Although required in some situations, Kell antigen phenotyping is restricted due to technical limitations. In these cases, molecular approaches maybe a solution. This study proposes three polymerase chain reaction genotyping protocols to analyze the single nucleotide polymorphisms responsible for six Kell antithetic antigens expressed in a Brazilian population. METHODS: DNA was extracted from 800 blood donor samples and three polymerase chain reaction-restriction fragment length polymorphism protocols were used to genotype the KEL*1/KEL*2, KEL*3/KEL*4 and KEL*6/KEL*7 alleles. KEL*3/KEL*4 and KEL*6/KEL*7 genotyping was standardized using the NlaIII and MnlI restriction enzymes and validated using sequencing. KEL*1/KEL*2 genotyping was performed using a previously reported assay. RESULTS: KEL genotyping was successfully implemented in the service; the following distribution of KEL alleles was obtained for a population from southeastern Brazil: KEL*1 (2.2%), KEL*2 (97.8%), KEL*3 (0.69%), KEL*4 (99.31%), KEL*6 (2.69%) and KEL*7 (97.31%). Additionally, two individuals with rare genotypes, KEL*1/KEL*1 and KEL*3/KEL*3, were identified. CONCLUSION: KEL allele genotyping using these methods proved to be reliable and applicable to predict Kell antigen expressions in a Brazilian cohort. This easy and efficient strategy can be employed to provide safer transfusions and to help in rare donor screening.9910

Estudo molecular dos alelos DI A/DI B e da banda 3-memphis na população brasileira

Orientador : Lilian Maria de CastilhoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: O sistema de grupo sanguíneo Diego é constituído por 21 antígenos localizados na glicoproteína banda 3 da membrana eritrocitária. Compreende 4 antígenos antitéticos Dia/Dib e Wra/Wrb e 17 antígenos de baixa incidência populacional. O fenótipo Dia resulta de uma mutação de ponto no nucleotídeo 2561 (T>C) que leva à substituição do aminoácid6 Prolina (Dib) para Leucina (Dia) na posição 854. O antígeno Dib é um antígeno de alta incidência na população geral, enquanto que o antígeno Dia é de baixa incidência em caucasianos, mas pode ser comum entre índios e japoneses. A mutação 166A>G no gene SLC4A1 (AE 1) que codifica a banda 3 dá origem a uma proteína variante, chamada banda 3-Memphis. Podem ser distinguidos dois tipos de banda 3 Memphis: variantes 1 e 11. A banda 3 Memphis II está associada a presença do antígeno Dia e apresenta maior afinidade de reação covalente com o H2DIDS do que a Memphis 1 e a banda 3 normal. De acordo com a literatura, o antígeno Dia está sempre associado a presença da banda 3-Memphis, caracterizando a variante Memphis 11. Entretanto, nenhum estudo de freqüência dos alelos DI A e 166G foi realizado até o momento. Com a finalidade de determinar a freqüência dos alelos DI A/DI B e da mutação 166A>G na população brasileira por métodos moleculares, estudamos 318 amostras de DNA de 4 diferentes populações (93 doadores voluntários de sangue, 71 descendentes de japoneses, 84 descendentes de africanos e 70 índios da tribo dos Parakanãs).Nossos resultados demonstraram que existe forte associação entre os alelos DI A e 166G e que o alelo DI A pode também ocorrer sem o alelo 166G. Em nosso trabalho foram identificadas 4 formas alélicas da associação DI / Memphis na população brasileira: alelo DI A 1 66A, DI A 1 66G, DI B166A e DI B166G . Os resultados da genotipagem 166A/G demonstraram que a banda 3-Memphis é um polimorfismo comum entre as populações estudadas. Devido a alta freqüência da mutação 166A>G encontrada, o alelo 166G pode ser considerado como um bom marcador genético de populações não miscigenadas e que o alelo 166G pode também ser considerado um protótipo do gene SLC4A1. Além disso, considerando a importância clínica dos antígenos Dia e Dib, a possibilidade em determinar os genótipos DI A/DI B, poderá contribuir substancialmente na segurança transfusional e materno-fetalAbstract: The Diego blood group system consists of21 antigens located on band 3 of the red blood cell membrane. It includes the 4 antithetical antigens Dia / Dib and Wra / Wrb, and 17 low incidence antigens. The Dia phenotype results from a point mutation at nucleotide 2561 (T>C) leading to a single amino acid substitution from Leu (Dia) to Pro (Dib) in position 854. Dib is a high incidence antigen while Dia has low incidence among Caucasians but is particularly common among American Indians and Japanese. The Memphis variants of band 3 result from a point mutation 166 A>G in SLC4Al leading to an amino acid substitution (56 Lys>Glu). There appears to be a strong association of Memphis (56Glu) with Dia (854Leu) among Native Americans. The initial aim of this study was to determine the frequency of Diego (DI A/DI B) and Memphis (166A>G) mutations in the Brazilian population. We studied samples from 70 Amazonian Indians, 71 individuals of Japanese descent, 93 regular blood donors and 84 Sickle Cell Disease patients by PCR-RFLP. PCR assays were designed to amplify a sequence of 149 bp from exon 18 (DI A/DI B alleles), and of 84 bp from exon 3 SLC4Algene that determine band 3-Memphis. Al1eles were identified by digestion of amplified products with the restriction enzymes Msp I to discriminate DI A from DI B in exon 18, and Mnl I to identify band 3-Memphis in exon 3. Both the DI A allele and band 3-Memphis had high gene frequency among Amazonian Indians (0.571 and 0,543, respectively). Unexpectedly, four Amazonian Indians had the DI A allele without the band-3 Memphis mutationMestradoClinica MedicaMestre em Clinica Medic

Molecular analysis of Dombrock blood grou'p system in brazilian people

Orientador : Lilian Maria de CastilhoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: O sistema de grupo sangüíneo Dombrock é constituído por um par de antígenos antitéticos, Doa e Dob, e três antígenos de alta freqüência populacional, Gregory (Gya), Holley (Hy) e Joseph (Joa). O gene DO, que codifica os antígenos do sistema Dombrock, foi recentemente clonado e suas bases moleculares foram elucidadas. Os antígenos Doa e Dob. são controlados pelos alelos DOA e DOB que estão associados à troca de três nucíeotídeos no exon 2 do gene DO: 378C/T, 624T/C e 793A/G, respectivamente. O fenótipo Jo(a-) está associado com a substituição 350C/T que caracteriza o alelo JO. O fenótipo Hy-negativo está associado à presença de um dos alelos: HY1 ou HY2. O alelo HY2 é caracterizado pela substituição 323G/T. Uma substituição adicional 898C/G, no exon 3, caracteriza o gene HY/1. Recentemente dois novos alelos foram descritos: DOB- SH (378C, 624C, 793G) e DOA-HA (378T, 624T, 793A). Considerando que: as bases moleculares dos antígenos Dombrock foram elucidadas; que novos alelos têm sido descritos; a miscigenação da população brasileira e que até o momento nenhum estudo foi realizado sobre a freqüência dos genes Dombrock na nossa população, estudamos 450 amostras de DNA, através das técnicas de PCR-RFLP e Microarrays® - HEA Beadchip para determinar a freqüência dos aletos DO {DOA, DOB, HY1, HY2, JO). Dois novos alelos foram identificados baseados em novas combinações de SNPs: alelo DOB- WL (793G, 898G, 323C) e alelo DOA-SH (378C, 624C, 793A). Nossos dados demonstram uma grande complexidade e heterogeneidade dos alelos Dombrock na população brasileira e confirmam a necessidade do estudo molecular em diferentes populaçõesAbstract: Background: The molecular basis of the Doa and Dob polymorphism, are associated with three single-nucleotide polymorphisms (SNPs) on exon 2 of DO gene: 378C>T, 624T>C and 793A>G, DOA and DOB alleles, respectively. The SNPs 350OT (JO allele) and 323G>T (HY allele) are associated with: Jo(a-) and Hy-negative phenotype. Recently, two new DO alleles were identified using the microarray technology, DOB-SH (378C, 624C, 793G) and DOA-HA (378T, 624T, 793A). Although the molecular background of Dombrock system is well defined, no studies were carried out in the Brazilian population. Methods: We employed PCR-RFLP based assays and a microarray assay to determine the frequency of the DO alleles {DOA, DOB, HY1, HY2 and JO) in Brazilians. We tested DNA of 288 Brazilians people by PCR-RFLP to determine the 793A>G (DOA/DOB), 323G>T {HY), 350OT (JO) and 8980G {HY1IHY2) SNPs. We also tested DNA from 162 blood donors by the HEA Beadchip¿ (BioArray Solutions, USA) to determine the 3780T, 624T>C, 793A>G (DOAIDOB), 350OT (JO allele) and 323G>T (HY) SNPs. Results: Two novel alleles combinations were found in our samples: the 793G SNP (DOB allele) associated with 898G and 323G (DOB-WL) and the SNPs 378C, 624C, 793A and 323G (DOA-SH). We also found the DOB-SH and DOA-HA alleles recently reported. Conclusion: Our data demonstrate high heterogeneity of DO alleles in the Brazilian population and highlight the importance of testing a cohort of different populations to determine the DO allele combinations and establish reliable genotyping to predict the Doa/Dob antigen statusDoutoradoCiencias BasicasDoutor em Clínica Médic