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 polymorphismmissense 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

PopulationRh(D)

NegRh(D) Pos

European Basque approx

35%65%

other Europeans 16% 84%

African Americanapprox

7%93%

Native Americansapprox

1%99%

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)

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

*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 0 0 0 0 14

Partial

absent

0 4 0 0 0 4

One

present

0 1 1 0 0 2

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

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 RecipientsThe 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 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 obtained by amniocentesis or

Testing fetal-derived DNA present inmaternal plasma at > 5 weeks gestation

If fetus antigen is negative,

mother and fetus need not undergo invasive, costly monitoring or receiveimmune-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

outcompeted 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

our population