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 A2 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-Ds produces no D

and is polymorphic in Africans

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 , 71G > A, 72T > G S1L‡, G5E‡ 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 Lub/Lua 230G > A R77H Aua/Aub 1615A > G T539A KEL KEL k/K 578C > T T193M Kpb/Kpa 841C > T R281W Jsb/Jsa 1790T > C L597P FY FY Fya/Fyb 125G > A G42D Fyb/Fy –67T > C Not coding JK SLC14A1 Jka/Jkb 838G > A D280N

Blood group polymorphisms arising from SNPs (Geoff Daniels; Transplant Immunology,2005)

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

Seltsam A et al (2003). Blood 102 (8): 3035

 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)

 O1 & O1v allele has single-base deletion  O2 allele : no deletion but nt substitutions,

:abolish the activity of the transferase

differ in 8 positions of nt with 4 aa substitutions

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

Genotype Chinese Malay Japan O1 O1 O1 O1v O2 O2 18 96.67 43 53 3.33 53 22

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 (DII to

DVII, DI being obsolete)

 Characterization of partial D is performed by

differential reactivity with monoclonal anti-D antibodies

DVI :most

important partial D.

 HDN occurred in

RhD +ve babies born to DVI mothers with anti-D

DVI occurs

 due to RHD-RHCE

hybrid

Adapted Geoff Daniels; Transplant Immunology,2005

Population data for the Rh D factor and the RhD neg allele Population Rh(D) Neg Rh(D) Pos European Basque approx 35% 65%

• ther Europeans

16% 84% African American approx 7% 93% Native Americans approx 1% 99% African descent less 1%

• ver 99%

Asian less 1%

• ver 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)

Rh genotype Percentage cde/cde 55.9 Cde/cde 32.4 Cde/Cde 8.8 cdE/cde cdE/cdE CdE/cde CdE/cdE

*0.44% of blood donor were Rh-neg in Transfusion Medicine Unit (TMU), Kelantan

*Rapiaah M, Rosline H ; Transfusion Alternatives in Transfusion med.

2006;7(2) supplement:42

RHD exons polymorphi sm

Rhesus Phenotype

Total ccee Ccee ccEe CcEe CCee All absent 14 14 Partial absent 4 4 One present 1 1 2 total 14 5 1 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

Del units able to induce anti-D in RhD-neg

recipients

Serology Del RBCs are detectable only by

adsorption and elution tests.

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

. . Shao, N Engl J Med 362(5):472-473 February 4, 2010

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

 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 inactive RHD gene require a monitoring of the pregnancy

neonatal alloimmune thrombocytopenia

 Fetal status is determined by testing

fetal DNA for HPA-1a/1b from cells

• btained 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.

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 typing

inaccurate

DNA-based methods overcome these

limitations

 regions of genes common to all alleles

are targeted

 minor amounts of donor DNA

• utcompeted by patient DNA

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

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

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

• ur population