Dr Rosline Hassan Haematology Department, School of Medical Sciences, Universiti Sains Malaysia, Kelantan
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1 Dr Rosline Hassan Haematology Department, School of Medical Sciences, Universiti Sains Malaysia, Kelantan THE FIRST ASEAN FEDERATION OF HAEMATOLOGY AND THE VIIITH MALAYSIAN NATIONAL HAEMATOLOGY SCIENTIFIC MEETING
2 ABO blood group was discovered by Karl Landsteiner in s : Biochemical basis was elucidated carbohydrate structure of glycoproteins was worked out 1990 : ABO gene was determined
3 Existence of a character in two or more variant forms in a population and the least common form present is more than 1% of individuals[1]. Eg: blood group has a frequency of more than 1% and less than 99%, it is polymorphic. [1] Kendrew J. (Ed.) The encyclopedia of molecular biology. Oxford1994. BlackweI1 Science.
4 1- Insight about RBC antigens and antibodies 2-Implication in management of transfusion medicine
5 To date nearly 300 blood groups phenotypes identify from an almost 30 blood group system The most common cause of blood group polymorphism missense mutation nucleotide change encoding substitution of one amino acid for another.
6
7 Gene deletion. Deletion of a whole gene only applies to the D polymorphism of the Rh system Homozygosity : deletion of the whole region of GYPB accounts for : S-s-Uphenotype Single nucleotide deletion. Deletion of single nucleotide : shift in reading-frame for the common O alleles and A 2 allele of the ABO system
8 Sequence duplication plus nonsense mutation : inactive RHD gene (RHDΨ), Intergenic recombination between closelylinked genes, : hybrid genes MNS system :GYP(B-A-B) gene responsible for the GP.Mur phenotype in the Far East. Rh systems include RHD-CE-D s produces no D and is polymorphic in Africans
9 Blood group polymorphisms arising from SNPs (Geoff Daniels; Transplant Immunology,2005) System Gene Polymorphism SNP Amino acid change ABO ABO A/B 526C > G, 703G > A, 796C > A, 803G > C R176G, G235S, L266M, G268A MNS GYPA M/N 59C > T, S1L, G5E 71G > A, 72T > G GYPB s/s 143C > T T29M RH RHCE C/c 48C > G, 178A > C, 203G > A, 307T > C C16W, I60L, S68N, S103P e/e 676G > C A226P LU LU Lu b /Lu a 230G > A R77H Au a /Au b 1615A > G T539A KEL KEL k/k 578C > T T193M Kp b /Kp a 841C > T R281W Js b /Js a 1790T > C L597P FY FY Fy a /Fy b 125G > A G42D Fy b /Fy 67T > C Not coding JK SLC14A1 Jk a /Jk b 838G > A D280N
10 H antigen is an essential precursor to the ABO blood group antigens. H locus located on chromosome 19. contains 3 exons and encodes a fucosyltransferase that produces the H Ag. ABO locus is located on chromosome 9 7 exons & encodes glycosyltransferase three alleleic forms: A, B, and O.
11 A allele encodes A transferase :transfer GlcNAc- > fucosylated galactosyl B allele encodes transferase: transfer gal -> fucosylated galactose O allele :deletion of single nt guanine at position 261 in exon 6 results in a loss of enzymatic activity.
12 A and B Ag differ by 4 aa substitutions Arg176Gly Gly235Ser Leu266Met Gly268Ala Aa at 266 & 268 : most important to determine A-transferase or B- transferase
13 Six common alleles in white individuals of the ABO gene A A101 (A1); A201 (A2); B B101 (B1) ; O O01 (O1); O02 (O1v) :O03 (O2) O 1 & O 1v allele has single-base deletion differ in 8 positions of nt with 4 aa substitutions O 2 allele : no deletion but nt substitutions, :abolish the activity of the transferase Seltsam A et al (2003). Blood 102 (8): 3035
14 *Rapiaah M, et al; Transfusion Bulletin, 2005 O:40% A: 35% B: 15% AB: 5%
15 Genotype Chinese Malay Japan O 1 O 1 O 1 O 1v O 2 O Ogasawara et al, Hum Genet Jun;97(6): * Study performed using BAGene ABO-Type; 2010
16 P. Han et at showed incidence of HDN due to ABO incompatibility In Singapore was 3.7% of all group O mothers Correlate with low distribution of grp 0 among Asian pop Homogenous grp 0 alelle P. Han et al: J.of Paed and Child Health; 2008
17 Great importance for transfusion medicine High immunogenicity Rh system are encoded by two genes, RHD and RHCE. These genes located on chromosome 1 Both have high level of homology with 93.8% identity
18 Adapted Geoff Daniels; Transplant Immunology,2005
19 D antigen comprises several different antigenic epitopes. It is classified into 6 distinct categories (D II to D VII, D I being obsolete) Characterization of partial D is performed by differential reactivity with monoclonal anti-d antibodies
20 D VI :most important partial D. HDN occurred in RhD +ve babies born to D VI mothers with anti-d D VI occurs due to RHD-RHCE hybrid Adapted Geoff Daniels; Transplant Immunology,2005
21 Population data for the Rh D factor and the RhD neg allele Population European Basque Rh(D) Neg approx 35% Rh(D) Pos 65% other Europeans 16% 84% African American Native Americans approx 7% approx 1% 93% 99% African descent less 1% over 99% Asian less 1% over 99% Mack, Steve (March 21, 2001). MadSci Network. ml.
22 Rh neg haplotypes in Africans & Asian : 1. RHD deletion & normal RHCE 2. RHD pseudogene, RHDΨ. RHD gene duplication: premature stop codon 3. RHD-CE-D, a hybrid gene Exons from RHD, plus exons from RHCE, followed by exons from RHD. hybrid gene produces no D Ag, but prob produce abnormal C Ag. Geoff Daniels; Transplant Immunology,2005)
23 *0.44% of blood donor were Rh-neg in Transfusion Medicine Unit (TMU), Kelantan Rh genotype cde/cde 55.9 Cde/cde 32.4 Cde/Cde 8.8 cde/cde 0 cde/cde 0 CdE/cde 0 CdE/cdE 0 Percentage *Rapiaah M, Rosline H ; Transfusion Alternatives in Transfusion med. 2006;7(2) supplement:42
24 RHD exons polymorphi sm Rhesus Phenotype ccee Ccee ccee CcEe CCee Total All absent Partial absent One present total
25
26 Allelic frequency of RhDel phenotype among Rh neg donor : 4/14 or 1 in 3.5 All 4 donors with RhDel assoc with Ce phenotype D el units able to induce anti-d in RhD-neg recipients Serology D el RBCs are detectable only by adsorption and elution tests.
27 .. Shao, N Engl J Med 362(5): February 4, 2010 Transfusion of RhD-Positive Blood in Asia Type DEL Recipients The RhD status of transfusion recipients and donors is routinely matched for red-cell transfusion. This worldwide practice is due to the potent immunogenicity of RhD. In East Asians, the frequency of RhDnegative status is only about 0.3%, which sharply limits the supply of RhD-negative blood. However, approximately 30% of RhD-negative persons carry an RhD variant, termed "Asia type" DEL. 1 Beginning in 2008, my colleagues and I organized a collaborative group of 10 laboratories, located in 10 cities in northern, central, and southern China
28 Antibody-based technology has been the basis for blood group typing Current expansion in molecular knowledge of RBC and platelet has made a progression in the laboratory aspect of Transfusion Medicine
29 Polymorphism of blood group in a population Patient with AIHA or positive DAT Recently transfused patient Rare blood group phenotypes or discrepancies in blood group testings Prenatal testing Investigate ABO and Rhesus HDN Determine fetal bld grp & rhesus Determine RHD zygosity for fathers
30 Rhesus antigen is highly polymorphic eg Asian Required further type Rhesus negative donors and recipients Safe transfusion can be assured To identify RHDel Shao et al,2010 found RHD gene intact but antigen D alleles in the Ce haplotype and highly associated with the RHD 1227A allele.
31 Determine paternal zygosity & gene expression HDN : homozygous for the gene, all children Rh +ve father with deletion in the RHD gene or has inactive RHD gene require a monitoring of the pregnancy
32 neonatal alloimmune thrombocytopenia Fetal status is determined by testing fetal DNA for HPA-1a/1b from cells obtained by amniocentesis or Testing fetal-derived DNA present in maternal plasma at > 5 weeks gestation If fetus antigen is negative, mother and fetus need not undergo invasive, costly monitoring or receive immune-modulating agents.
33 Not indicated for routine use of DNAbased to determine variants of D especially in area with low prevalence Extensive pretransfusion matching of donor blood for patients with diseases that have a high risk of alloimmunization sickle cell anemia thalassemia
34 Presence of donor RBCs makes typing inaccurate DNA-based methods overcome these limitations regions of genes common to all alleles are targeted minor amounts of donor DNA outcompeted by patient DNA
35 Accurate typing in massive transfusions with non leukocyte-reduced blood DNA isolated from a buccal swab Another indication of DNA arrays genetic screening to establish susceptibility to common diseases
36 DNA-based blood group typing is to complement conventional ABO typing and Ab screening but not as independent test Replacement of Ag & Ab by conventional methods for pretransfusion testing with molecular methods is not straightforward. Majority of transfusion does not require cross matching beyond ABO and D type
37 Identifying blood group polymorphism in a population is the basis for future planning in the application DNA technology in transfusion medicine It is highly recommended to do further typing for Rhesus negative donor to detect RHDel which is highly prevalent in our population
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