School of Medical Sciences, Universiti Sains Malaysia, Kelantan - PowerPoint PPT Presentation

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

 ABO blood group was discovered by Karl Landsteiner in 1900  1970’s : Biochemical basis was elucidated  carbohydrate structure of glycoproteins was worked out  1990 : ABO gene was determined

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.

 1- Insight about RBC antigens and antibodies  2-Implication in management of transfusion medicine

 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 .

 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-U- phenotype  Single nucleotide deletion.  Deletion of single nucleotide : shift in reading-frame for the common O alleles and A 2 allele of the ABO system

 Sequence duplication plus nonsense mutation :  inactive RHD gene ( RHDΨ ),  Intergenic recombination between closely- linked 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

Blood group polymorphisms arising from SNPs ( Geoff Daniels; Transplant Immunology,2005 ) System Gene Polymorphism SNP Amino acid change † ABO ABO A/B 526C > G, R176G, G235S, 703G > A, L266M, G268A 796C > A, 803G > C MNS GYPA M/N 59C > T , S1L ‡ , G5E ‡ 71G > A, 72T > G GYPB s/S 143C > T T29M ‡ RH RHCE C/c 48C > G, C16W, I60L, 178A > C, S68N, S103P 203G > A, 307T > C 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

 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.

 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.

 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

 Six common alleles in white individuals of the ABO gene  A A101 (A1); A201 (A2); differ in 8 positions of  B B101 (B1) ; nt with 4 aa substitutions  O O01 (O1); O02 (O1v) : O03 (O2)  O 1 & O 1v allele has single-base deletion  O 2 allele : no deletion but nt substitutions, :abolish the activity of the transferase Seltsam A et al (2003). Blood 102 (8): 3035

O:40% A: 35% B: 15% AB: 5% *Rapiaah M, et al; Transfusion Bulletin, 2005

Genotype Chinese Malay Japan O 1 O 1 18 96.67 43 53 3.33 53 O 1 O 1v 22 O 2 O 2 Ogasawara et al, Hum Genet. 1996 Jun;97(6):777-83. * Study performed using BAGene ABO-Type; 2010

 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

 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

Adapted Geoff Daniels; Transplant Immunology,2005

 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

 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

Population data for the Rh D factor and the RhD neg allele Rh(D) Population Rh(D) Pos Neg approx European Basque 65% 35% other Europeans 16% 84% approx African American 93% 7% approx Native Americans 99% 1% African descent less 1% over 99% Asian less 1% over 99% Mack, Steve (March 21, 2001). MadSci Network. http://www.madsci.org/posts/archives/mar2001/985200157.Ge.r.ht ml.

 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 )

*0.44% of blood donor were Rh-neg in Transfusion Medicine Unit (TMU), Kelantan Rh genotype Percentage cde/cde 55.9 Cde/cde 32.4 Cde/Cde 8.8 cdE/cde 0 cdE/cdE 0 CdE/cde 0 CdE/cdE 0 * Rapiaah M, Rosline H ; Transfusion Alternatives in Transfusion med. 2006;7(2) supplement:42

RHD exons Rhesus Phenotype Total polymorphi sm ccee Ccee ccEe CcEe CCee All absent 14 0 0 0 0 14 Partial 0 4 0 0 0 4 absent One 0 1 1 0 0 2 present total 14 5 1 0 0 20

 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.

. . Shao, N Engl J Med 362(5):472-473 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 EastAsians, the frequency of RhD- negative 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

 Antibody-based technology has been the basis for blood group typ in g  Current expansion in molecular knowledge of RBC and platelet has made a progression in the laboratory aspect of Transfusion Medicine

 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

 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.

 Determine paternal zygosity & gene expression  HDN :  homozygous for the gene, all children Rh +ve  father with deletion in the RHD gene or has in active RHD gene require a monitor in g of the pregnancy

 neonatal alloimmune thrombocytopenia  Fetal status is determ in ed by test in g fetal DNA for HPA-1a/1b from cells obta in ed by amniocentesis or  Test in g fetal-derived DNA present in maternal plasma at > 5 weeks gestation  If fetus antigen is negative,  mother and fetus need not undergo in vasive, costly monitor in g or receive immune-modulat in g agents.

 Not indicated for routine use of DNA- based 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

 Presence of donor RBCs makes typ in g in accurate  DNA-based methods overcome these limitations  regions of genes common to all alleles are targeted  m in or amounts of donor DNA outcompeted by patient DNA

 Accurate typ in g 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