This document provides an overview of blood group genetics. It discusses basic genetics principles like chromosomes, genes, alleles, and genotypes and phenotypes. It covers inheritance patterns such as autosomal dominant, recessive, and sex-linked inheritance. It also discusses concepts like polymorphism, linkage, and gene interactions as they relate to blood group systems. The key blood group systems and their chromosomal locations are identified.

1. BlOOD GROUP GENETICS
Presented by: Dr Atif Irfan Khan

2. Outline
• INTRODUCTION
• BASIC PRINCIPLES OF GENETICS
• GENOTYPE AND PHENOTYPE
• POLYMORPHISM
• INHERITANCE OF GENETIC TRAIT
• POPULATION GENETICS
• BLOOD GROUP GENOMICS

3. Introduction
• Genetics is the study of heredity
• It describes the mechanism by which specific characteristics is passed from parents to
offspring
• Certain characteristics are also inherited on WBC, Platelet or RBC as antigens
• Blood groups are inherited was first shown by Van Dungen and Hiszfeld in 1910

4. Introduction
• Red cell antigen are like markers of RBC, the detection of these antigen and the different
types of antigens forms the basis of transfusion
• We require an understanding of the gene structure, function and expression
• An understanding of blood group antigen knowing the serologic and other possible
techniques to characterise their expression
• An ability to correlate the results to clinical problem being addressed

5. Basic principle of genetics
Chromosome
• This is the gene carrying structure visible at nuclear cell division
• 46 chromosomes present in humans in 23 pairs as homologous pair with one sex
chromosome
• Morphologically similar but differ in other characteristics as length position of centromere
• Each chromosome has two chromatids

6. Basic principle of genetics
Chromosome
• Internationally recognised terminology is used to describe chromosomes
• “p” is labelled for short arm
• “q” is labelled for long arm
• Numbers are given to various “bands” labelled outwards from the centromere
• Each bands have further sub-bands
• Example- a gene located at 1p11.2

7. Basic principle of genetics
Chromosome

8. Basic principle of genetics
Chromosomal gene locations of blood group systems
Gene Name- ISBT Chromosome Location
ABO 9q34.2
Rh 1p36.11
Kell 7q34
Duffy 1q23.2
Lewis 19p13.3
P 22q13.2
MNS 4q31.21
Lutheran 19q13.32
Xg Xp22.33

9. Basic principle of genetics
Gene
• Segment of DNA that encodes for a particular protein-
• Locus- location of a gene on the chromosome
• Gene at a given locus is called allele; there maybe more than one form of allele for that gene
• Each person has two alleles one derived from the mother and the other from the father
• Simplified example- ABO gene locus consist of three alleles: A,B and O
• Six possible variants can be inherited from 3 alleles- A/A, B/B, A/B, A/O, B/O and O/O

10. Genotype and phenotype
• Phenotype gives only observable expression of a gene
• Genotype is the set of alleles on a locus
• Presence or absence of antigen on RBC is detected by serologic method i.e phenotype,
this gives only partial information about the genotype
• Identical alleles for a given locus present on both chromosome is denoted homozygous
eg- A,A or B,B
• Presence of only one copy of allele is termed hemizygous eg AO or BO

11. Genotype and phenotype
• Presence of different (non identical) alleles at a locus is termed heterozygous eg KEL for
alleles of antigen K and k, when both are present it will be K+k+
• K and k alleles are said to be antithetical as both allele are found on the same locus
Antigen expression
• Is influenced by whether the individual is homozygous or heterozygous
• If homozygous for an antigen, the RBC is said to be double dose for the same
• if heterozygous for different antigen it is said to be single dose
• In some blood groups the antibody reacts more strongly to antigen with double dose than
in heterozygous expression

12. Genotype and phenotype
Antigen expression
• Example in the MNS blood group system
• M+N+(heterozygous) react more weakly with anti-M than phenotype of M+N-(homozygous for M)
• Sometime heterozygous expression of antigen may not react or will be weakly reactive with the
corresponding antibody
• This observable difference in strength of reaction based on homozygosity or
heterozygosity for an allele is termed “dosage effect”.
• Blood groups that show dosage effects- Rh, MNS, Kidd, duffy and lutheran

13. Polymorphism
• More than one allele occupying a gene locus producing different phenotypes in a
population each with appreciable frequency(>1%)
• A change in allele is due to permanent change in DNA sequence
• A change not present in parents is caused by mutation: this leads to expression of new
phenotype
• When the mutation occur in germ cells it becomes inheritable
• Mutation can be spontaneous or induced (radiation, UV rays X-rays)

14. Polymorphism
• Other genetic events leading to DNA change are chromosome deletion, rearrangement or
translocation which can also occur at nucleotide/gene level
• Most significant/predominant mechanism of change in DNA sequence leading to variation
in blood groups is the single nucleotide polymorphism (SNPs)

15. Inheritance of Genetic Traits
• Genetic trait is observed expression of one or more gene
• The inheritance of a trait is determined if it is on a autosome or on the X chromosome and
weather it is dominant or recessive
Autosomal dominant inheritance
• Regardless if homozygous or heterozygous, if the gene is present it will be expressed as
same phenotype. Example blood group A phenotype can be A/A or A/O genotypically
• Autosomal Co dominance can occur if both antigens are expressed; example is “AB” in
ABO system or S+s+ phenotype in MNS system

16. Autosomal dominant inheritance
B group shows A.D inheritance over O
group in the ABO system

17. Inheritance of Genetic Traits
Autosomal recessive inheritance
• This trait is expressed in individual who is homozygous for both allele inherited from both
parents or one gene is recessive and the other is silent/deleted(null)
• Occur more frequently with rare alleles in consanguineous mating
• When recessive gene is common in a population consanguinity is not a prerequisite.
Example is “O” group of the ABO group system is autosomal recessive

18. Inheritance of Genetic Traits-Autosomal recessive inheritance

19. Inheritance of Genetic Traits
Autosomal recessive inheritance
• In blood groups a recessive trait almost always implies RBC with null phenotype because
of homozygosity for a silent or amorphic gene that either result in no or defective product
• Example Lu(a-b-), Rhnull , or O phenotype

20. Inheritance of Genetic Traits
Sex linked inheritance
• Sex linked trait inherited from X or Y chromosome
• Can be dominant or recessive inheritance; more applicable to female with XX sex
chromosome
• X borne gene don’t have homolog (similar DNA sequence) on Y chromosome hence the
recessive trait is more prevalent in males due to XY chromosomes

21. Inheritance of Genetic Traits
Sex linked inheritance
• X-linked dominant inheritance
• Example Xg antigen has a frequency in males-66% and females 89%
• If present in father all female children will have the gene
• If present in mother on one chromosome; 50% all children will have the gene, if on both chromosomes
all children will have the gene
• X-linked recessive inheritance
• Recessive gene carried by all males is expressed, while only homozygous female will express the same

22. Inheritance of Genetic Traits
Sex linked inheritance
• X-linked recessive inheritance
• XK gene on X chromosome encodes for Kx protien and demonstrates X-borne
recessive inheritance
• Mutation in XK gene results in Mcleod phenotype- these RBC lacks Kx protien and have reduced expression of kell antigens-
• McLeod syndrome; associated with neurodegeneration and CNS symptoms and compensated haemolytic anemia, it is found
exclusively in male
• X chromosome inactivation(lyonization)
• One of the two chromosome in female becomes inactivated at a very early stage of embryonic
development

23. Inheritance of Genetic Traits
Sex linked inheritance
• X chromosome inactivation(lyonization)
• Recessive gene on the other chromosome becomes expressed
• Some gene escape lyonization such as XG gene for Xg blood group
• Another gene that escapes is the XK gene, when this happens the female expresses
dual phenotype of McLeod and non McLeod

24. Inheritance of Genetic Traits
Independent segregation
• This is the separation of homologous chromosomes and there random distribution in
gametes during meiosis
• Only one pair of allele is passed on to the next generation, each having equal probability
during fertilization
Independent assortment
• Alleles determining various traits are inherited independently of one another
• Eg B antigen is inherited independently of antigen M

25. Inheritance of Genetic Traits
Linkage and crossing over
• Linkage is the physical association between two genes located on the same chromosome
• RHD and RHCE encoding the RH system are found on chromosome 1 which have a linked locie and do
not assort independently
• Crossing over is the exchange of genetic material between homologous chromosome
pairs, also can be referred to as shuffle or recombination
• Likelihood of crossing over of two genes on same chromosome increases if they are distant away and
decreases if they are closer

26. Inheritance of Genetic Traits
Linkage disequilibrium
• Tendency of specific combination of alleles at two or more linked loci to be inherited
together more frequently than expected by chance
• Eg in MNS system if M+ and S+ were not linked there frequency of occurring together by
independent assortment is 17% but due to linkage of the alleles M and S they do not
assort independently and there prevalence in the population is 24%

27. Inheritance of Genetic Traits
Gene interaction and Position effect
• Alleles on same chromosome are said to be in cis position where as those in opposite
position on a chromosome are in trans position
• The Rh blood system best explains this example DCe/DcE
• Position effect of some of the alleles interferes with expression of the antigens
• Eg when Ce haplotype trans to a D antigen i.e dCe/DCe encoding haplotype, the D
expression is dramatically reduced

28. Inheritance of Genetic Traits
Gene interaction and Position effect
• In kell system Kpa supresses expression of other kell antigens depending on Cis modifier
effect, best observed with K0 gene in tran
• RHAG on Ch6 encodes for RhAG which is required on RBC membrane for Rh Ag expression
defect in this gene was shown to result in Rhnull phenotype with normal gene at RH
locus(Ch1)

29. Population Genetics
Distribution pattern of gene frequencies in a population
Phenotype prevalence
• The prevalence of blood group antigen or phenotype determined from large random
sample of people of the same race

30. Population Genetics
Blood Group antigen Frequency(%)
A 21.9
B 36.51
AB 09.19
O 32.37
D Positive 94.36
Agarwal N, Thapliyal RM, Chatterjee K. Blood group phenotype frequencies in blood donors from a tertiary care hospital in north
India. Blood research. 2013 Mar 1;48(1):51-4.
Blood group antigen Frequency(%)
Phenotype prevalence

31. Population Genetics
Phenotype prevalence
• Calculation of antigen negative phenotypes- example- a patient having multiple antibodies
AntiK, AntiS and AntiJKa needing RBC transfusion
• We check percentage of negative phenotype of the antibody in the donor population
• K- is 91%, S- is 48%, Jka - is 23%
• Multiply them ie 0.91x0.48x0.23 which gives 0.1
• Meaning 10% of donor pool will have negative phenotype for all three antigens together or 1 in 10 RBC
units checked will give the same

32. Population Genetics
The Hardy-Weinberg Equilibrium
• Is a principle proposed by Hardy and the German physician Weinberg, that gene frequencies in a
population reach equilibrium.
• Five conditions are required in order for a population to remain at Hardy-Weinberg Equilibrium
1. A large breeding population
2. Random mating
3. No change in allelic frequency due to mutation
4. No migration or immigration
5. No natural selection

33. Population Genetics
The Hardy-Weinberg Equilibrium
• . This equilibrium can be expressed by an equation
in a two allele system P+q=1
(p+q)2 = 1
p2 + 2pq + q2 = 1
Alleles are labelled A and a then
Homozygous for A allele, AA= p2
Homozygous for a allele, aa= q2
Heterozygous phenotype Aa=2pq
• knowing frequency of one allele means we can calculate the frequency of the other
• It permits us to estimate genotype frequency from phenotype prevalence and vice versa

34. Population Genetics
The Hardy-Weinberg Equilibrium
• Example if we use kell alleles Kk, using the equation we can say, p is K and q is k then
homozygous for KK=p2
Homozygous for kk=q2
Heterozygous for Kk=2pq
• If the antigen frequency of one allele is know, in this case K=9% in European population
p2 + 2pq + q2 = 1
P2+2pq=0.09 meaning
q2=1-(P2+2pq)
q2=1-0.09
q2=0.91
q= 0.91 =0.95

35. Population Genetics
The Hardy-Weinberg Equilibrium
• Then k frequency is 95%(0.95) which means frequency for K is 5%(0.05)
• From this info we can now estimate k+(K+k+ and K-k+) and K+(K+k+ and K+k-)
k+=2pq+q2
2(0.05 × 0.95) + (0.95)2 = 0.9975
prevalance of k+ is 99.75%
K+ = 2pq + p2
2(0.05 × 0.95) + (0.05)2 = 0.0975
prevalence of K+ is 9.75%

36. Population Genetics
The Hardy-Weinberg Equilibrium
• For two alleles (p+q)2 =p2 + 2pq + q2 = 1
• For three alleles like in ABO system we have 6 possible outcomes of the three alleles
(p+q+r)2 considering p=A,
q=B
r=O
(p+q+r)2 = p2 + 2pq + 2pr + q2 + 2qr + r2 = 1
AA AB AO BB BO OO

37. Blood Group Genomics
• 36 blood group system are known and sequenced genetically
• Antigen introduced into an individual lacking it can mount an immune response to develop
antibody
• Antibody from such response cause problem in clinical practice
• Hemaglutinition is the simple method to detect phenotype but has its limitations
• In such cases DNA based methods becomes more valid approach as it is simple and reliable
• However detailed genetic study showed far more alleles than phenotypes especially in ABO and
Rh

38. Blood Group Genomics
Limitations of Hemagglutination/serologic methods
• Is a subjective test
• Requires use of reliable antisera
• Difficult to phenotype a recently transfused patient
• Difficult to phenotype RBCs coated with IgG
• Difficult to distinguish an alloantibody from an autoantibody in antigen-positive people
• Restricted ability to determine zygosity, especially RHD zygosity in D-positive individuals
• Many antibodies are not commercially available

39. Blood Group Genomics
Molecular methods of blood group gene detection
• DNA is sourced from any nucleated cell, but most commonly WBC from whole blood
• principles used- sequence specific primer PCR(SSP-PCR), and allele specific PCR(AS-PCR)
• Target is DNA amplification of the gene sequence through PCR followed by
• Manual
• Gel electrophoresis to separate PCR products by fragment size
• May involve digestion of PCR product with a RFLP followed by electrophoresis and
visualisation of fragments

40. Blood Group Genomics
Molecular methods of blood group gene detection
• Semi automated
• Real time PCR using fluorescent probes with quantitative and qualitative read out
• Automated analysis
• Allows for high throughput with larger number of target alleles in the PCR reaction
which makes possible multiple gene assessment

41. Blood Group Genomics
Molecular methods of blood group gene detection
• Different methods of DNA detection in blood group genetics
• TaqMan genotyping on OpenArray- uses multiplex PCR to interrogate multiple genes simultaneously.
• Single nucleotide primer extension mini sequencing assay- extension fragment fluorescence detection
and electrophoresis
• Fluidic microarray using XMAP technology- uses on-bead extension followed by fluorescence detection
and electrophoresis
• Multiplex ligation-dependent probe amplification (MLPA)- multiplex ligation-dependent probe
amplification
• Elongation-mediated multiplexed analysis of polymorphisms (eMAP)- uses on-bead extension followed
by fluorescence detection

42. Blood Group Genomics
Molecular methods of blood group gene detection
Whole blood sample or buccal swab
DNA extraction
PCR amplification and processing
Hybridization on the BeadChip
Strand extension with incorporation of fluorescent nucleotides
Imaging
Data analysis
• Elongation-mediated multiplexed
analysis of polymorphisms (eMAP)
• Fluidic microarray using XMAP
technology
Paris S, Rigal D, Barlet V, Verdier M, Coudurier N, Bailly P, Brès JC. Flexible automated platform for blood group genotyping on DNA microarrays. The Journal of Molecular Diagnostics. 2014 May 1;16(3):335-42.

43. Blood Group Genomics
Transfusion and Apheresis Science 48 (2013) 257–261
39 multiply transfused patients were selected- thalassemia(35), sickle cell disease
patients(2), congenital dyserythropoietic anemia (n:1) and undefined chronic anemia
(n:1). They had their Rh, and kell phenotyped previously by serologic method, Only
3(7%) patients had formed alloantibodies
Blood group Genotyping by Sequence Specific Primers (SSPs)-PCR method was done for
each Rh, Kell, kid, and Duffy
19 of the 37 patients had discrepancies between
genotyping and phenotyping results in a total of 25
alleles. In 12 patients, the discrepancies had
clinical significance

44. Blood Group Genomics- DNA test uses
To predict red cell phenotype
• Useful in chronically transfused like thalassemia or in massive transfusion
• Patients with AIHA that is transfused before minor antigen phenotyping
• Usually differential allogenic adsorption to determine presence or absence of
alloantibody but DNA analysis can predict exactly the antigen profile of the RBC
• It allows phenotype matching of donor RBC to that of the patients for clinically
significant common antigens; this avoid the use of “least compatible” blood and rather
transfuse “antigen matched for clinically significant blood group antigens”

45. Blood Group Genomics- DNA test uses
To predict red cell phenotype
• When red cells are coated with IgG
• Treatment of RBC with substance like chloroquine to detach IgG is normally done
which may also destroy some antigen, DNA testing allows exact phenotyping
• To distinguish alloantibody from autoantibody
• Resolving blood group discrepancies eg weak blood group antigen or novel amino acid
change in protein carrying the blood group antigen, chimerism- natural or induced- HSC
transplant patients

46. Blood Group Genomics- DNA test uses
To predict red cell phenotype
• Phenotype of fetus at risk of anemia from hemolysis due to maternal alloantibody
• E.g RhD phenotype of RhD+ fetus to RhD- mother and Rh+ father
• DNA sample can be obtained from amnioscentesis or cordocentesis
• Non invasive method- from maternal plasma – cell free fetal DNA(cffDNA)
• Confirming RhD antigen expression in weak D donors
• Predicting silenced or non expressive gene

47. REFERENCES
• AABB 19th edition
• Molecular protocols in transfusion medicine -Gregory A Denomme, Maria Rios,
Marion E Reid
• The role of red cell genotyping in transfusion medicine. Keller MA.