1. Molecular Basis of Weak D in Indians
Dr Swati Kulkarni
Head, Dept. of Transfusion Medicine
ICMR- National Institute of Immunohaematology,
Mumbai

2. Introduction The Rh blood group system - most polymorphic system
High immunogenicity - correct determination of the RhD status important as implicated in HTR and HDFN.
Serologically, the task of determining an individual’s RhD status seems simple enough unfortunately, it is not always that simple.
The Rh antigen complexities have long been recognized
initially in serologic testing,
more recently as a result of more number of antigens recognized,
the variable expression of antigens,
availability of monoclonal anti-D
Although most people are either D pos or D neg, there is a plethora of D variants.
Most of these D variants are clinically important as they can lead to production of anti-D when their blood is transfused to RhD negative individuals or they are transfused with normal D pos blood.
Therefore identification of these individuals important.

3. RhD variants
Schematic representation of antigens and epitopes on weak D, partial D, and normal D positive red cells.
1: Normal D antigen with all epitopes,
2: Weak D with reduced D antigenic sites and all epitopes present,
3: Partial D with normal D antigenic sites but lacking one D epitope,
4: Partial D with normal D antigenic sites but lacking two D epitope,
5 : Partial weak D with reduced D antigenic sites and lacking one D epitope.

4. Clinical implications of the D antigen
D phenotypes
Changes in amino acid in RhD
D antigen expression
Test used to detect D antigen
In patient
In donor
Can make anti-D through transfusion or pregnancy
Type of RBC components for transfusion
Rh IgG prophylaxis recommended
Can cause immunisation if transfused to D neg recipient
D positive
None
Normal
Direct agglutination No
D positive or
D negative
Yes
Partial D
Extracellular
Altered (some D epitopes present, some absent)
Direct agglutination & IAT (depending on reagents used
D negative or matched partial D
Partial weak D
Extracellular (sometimes also transmembrane or intracellular
Altered (some D epitopes present, some absent) and weak or variable
D negative or matched partial weak D
Unlikely but possible
Weak D
transmembrane or intracellular
Normal but weak
IAT
No ?
D negative ?
RhD absent
Absent

5. Confusions Over Weak Expression of D Individual’s Rh status
Variables Affecting D Typing Results
Multiple methods:
test tube, gel, solid phase,
automation, molecular
may or may not proceed for Du testing
Different for Donors/patients
Variability in expression of D - same D variant type reacts differently with one reagent and different methods
Reagents
Methods
Strength of reaction
Interpretation of results
Donor / Recipient / Cord blood / ANC
Confusions Over Weak Expression of D Individual’s Rh status
Donor
Recipient
Prenatal RhIg?
Newborn Postpartum RhIg?
Autologous
A donor can be a patient tomorrow – correct RhD status important

6. As various commercial anti-D reagents directed against the different epitopes and the techniques used in determining the RhD status vary from one laboratory to another, a RhD variant can be mistyped as D-negative in routine testing.
DEL variants only detected by adsorption elution techniques, are not performed routinely thus not identified
Limitations of serology warrant - molecular genotyping - to predict RhD phenotype
This phenotypic variability - genetic polymorphisms, involves several mutational mechanisms, ranging from single nucleotide variations to whole RHD gene deletion

7. Promotes genetic exchange Produces clinically relevant Rh variants.
Genetics of Rh Blood Group System
Two closely linked and highly homologous Rh genes (RHD and RHCE)
chromosome location 1p36.1.
Both RHD and RHCE genes have 10 exons each spanning over 75 Kb of DNA and face each other with their 3’-end.
They encode for 417 AA which form transmembrane protein with 6 extracellular loops.
The RHD gene -D antigen and RHCE -C/c and E/e antigen
Close proximity
Opposite orientation
96% homology
Promotes genetic exchange
Produces clinically relevant Rh variants.

8. RhD Positive
RhD Negative
(Deletion)
RhD Negative
(Pseudogene)
RhD Negative
( Hybrid allele)
Representative diagram of RH genes in RhD positive and RhD negative phenotypes.
Coding regions consist of 10 exons, depicted as black and grey boxes for RHD and RHCE respectively.

9. Representative examples
RhD variant phenotypes and their molecular basis
Classification
Molecular basis
Representative examples
Protein alteration
Molecular Mechanism
Partial D (Qualitative)
Extracellular surface aminoacid substituition
Missense
(Point mutations)
Hybrid protein
Gene
Conversion (Rearrangement)
Weak D
(Quantitative)
Intracellular
Missense (Point mutation)
Del (Quantitative)
Strongly reduced protein expression
Missense mutation
RHD exon RHCE exon

10. Several phenotype studies have reported the incidence of RhD-negative individuals, weak D and partial D antigen carriers in India.
Phenotypic data in terms of D antigen density and their distribution in different castes and communities of the Indian population have also been provided, but extensive genetic studies on Rh antigens have not been carried out in Indians.

11. Weak D –Indian studies Weak D prevalence 0.01% in 1, 84,072 donors
The incidence of weak D Rh antigen was 0.189% in donors
The prevalence of weak D (Du) in Western Indian population is % in 38,962 donors

12. DEL variants in Indians
The frequency of DEL phenotype detected by adsorption and elution in Rh negative donor population of North India is 1.5%. (3 variants in 200 D-negative donors)
Serologically DEL phenotype found to be absent in 900 RhD negative individuals by adsorption elution. PCR-SSP for RHD(K409K) common in Asians and RHD(M295I) common in Caucasians also screened.

13. Aim was to characterize the molecular bases of weak expression of D antigen in Indians,
with an objective of creating a database of RHD variant alleles prevalent in Indians
Samples obtained by screening RhD negative individuals -3019
ambiguous RhD typing (n = 156) due to
discrepancy in strength of agglutination reaction between two anti-D reagents,
weak strength of agglutination,
D+ samples producing anti-D and
samples showing discrepancy in present and historical test results
Serological screening:
5 different commercial monoclonal anti-Ds reagents
Advanced Partial RhD typing Kit
DEL variants, by ether elution method
Rh phenotyping
D antigenic sites flow cytometric method

14. Molecular analysis
Genomic DNA was extracted from whole blood using Qiagen FlexiGene DNA kit
Initially, the strategy was to screen for presence of the weak D type 1, 2 and 3 alleles by Real Time PCR as these are the most common mutations in the Caucasian population.
For the uncharacterized samples, the ten RHD exons and their flanking intronic sequences were amplified in a single tube by a multiplex PCR
Alternatively copy number variations (CNVs) of RHD exons were analyzed by RHD Quantitative Multiplex PCR of Short Fragments (QMPSF) for exon quantitation.
Briefly, the ten RHD and RHCE exons were amplified in two separate single tube reactions by a multiplex PCR with HFE and F9 as control genes.
The reaction ends within the exponential quantitative phase. Two control HFE and F9 gene-specific sequences.
The fragments were then analysed on the automated capillary DNA sequencer in presence of a size standard using a dedicated software.
RHCE QMPSF was carried out when a hybrid RHD-CE-D gene was suspected

15. Results Serology 2.2% of serological D negative – D variants
No DEL variants
Total – 223 weak D variants
220 (+2-+w) at IgM phase
Advanced Partial D kit- varied reactivity pattern, mostly Ep 1.2 absent
Molecular
Screen for presence of the weak D type 1, 2 and 3
No variant allele was identified by this method, suggesting that the molecular bases of weak D phenotype are different between Indians and Caucasians.
Example of melting curves after specific amplifications for the genotyping of the weak D
All the ten RHD exons were then directly sequenced in these samples.
Single nucleotide variations were found in only in few samples, suggesting that this mutational mechanism is not so common in the Indian population

16. QPMSF analysis RHD gene
Presence of entire RHD gene in D positive
Deletion of entire RHD gene in D negative

17. Screening of D variants samples by RHD QMPSF
a wild-type pattern with all RHD exons present
two copies of RHD exon 3 in many samples
Tested for all RHCE exons as suspecting hybrids with RHCE, but all RHCE exons present
A novel duplication in exon 3 and flanking regions identified in (~58%) weak D allele tested A B C
HFE F9 e3 e8 e7
e10 e1 e4 e6 e2 e5 e9
Hemizygous, wild-type RHD
Hemizygous exon 3 duplication
Homozygous exon 3 duplication
Exon 3 dup

18. RHD alleles found in the Indian weak D samples screened by serological analyses
Genotype
Occurrence %
Exon 3 duplication
130
58.30
Wild-type 37
16.59
Whole gene deletion 26
11.66
RHD(G63C)* 3
1.35
RHD(A244V)
RHD(L216F) 2
0.90
Weak D type 8
Weak D type 25
DFW
Others 16
7.17
Total
223
100.00

19. Other novel variant D alleles
3 missense variations - RHD(A59T), RHD(G63C), RHD(A237V)
3 complex hybrid alleles - RHD-CE(5:E233Q,V238M,V245L)-D
RHD(T201R)-CE(5)-D(I342T)
RHD(L214F)-CE(7)-D)
Sequencing patterns of novel variant RHD alleles
Control
Sample
Exon 5
c.710C>T
p.Ala327Val
c.697G>C
p.Glu233Gln
c.712G>A
p.Val238Met
c.733G>C
p.Val245Leu
c.744C>T
Synonymous
RHD(A327V)
RHD-CE(5:E233Q,V238M,V245L)-D B C D A
Exon 2
c.187G>T
p.Gly63Cys
RHD(G63C)
c.175G>A
p.Ala59Thr
RHD(A59T)
Single nucleotide variations identified by direct sequencing
Arrowheads indicate nucleotide variations in samples compared with wild-type sequences.

20. Serological characteristics of the novel mutations
Exon 3 duplication
(+2-+w) at IgM phase barring three samples which could only be identified in the IgG phase.
Rh phenotyping
124/130 samples positive for the C antigen
6/130 individuals (ccee: n = 4; ccEe: n=2)
Three samples homozygous for the exon 3 duplication presented with a C+c–E–e+ phenotype.

21. On comparison with reaction profile results
Extended phenotyping: D epitope profile
Common agglutination patterns
Testing with Advanced Partial RhD Typing Kit (set of 12 monoclonal anti-D reagents) - varying patterns of agglutination and reaction strength
absence of epitope 1.2 (clones LHM174/102 and LHM70/45) - 86%
absence of epitope 8.1 (LHM76/58 and LHM59/19) - 79%
absence of epitope 6.3 (LHM50/2B, LHM169/80, and LHM57/17) - 61%
Sample ID
Pattern 1
Pattern 2
Pattern 3
Clone
Epitope
LHM76/58
8.1 +
LHM76/59
5.1
LHM174/102
1.2
[-]
LHM50/2B
6.3
LHM169/81
1.1
ESD1
4.1
LHM76/55
3.1
LHM77/64
9.1
LHM70/45
LHM59/19
LHM169/80
LHM57/17
On comparison with reaction profile results
DFR
DOL
No matching pattern

22. Flow cytometric analysis for exon 3 duplication
Exon 3 positive sample with different monoclonal anti-D reagents.
Flow cytometry analysis in exon 3 duplication positive samples (n=41) showed a varying weak D antigen density
average mean density  SD: 2753 ± 1351 D antigens/cell; (range: 1276–6050)

23. Serology of other novel alleles
Sample ID
RHD-008
RHD-064
RHD-105
RHD-188
RHD-257
RHD-091
RHD-239
RHD-249
Clone
Epitope
LHM76/58
8.1 + NT
[-]
LHM76/59
5.1
LHM174/102
1.2
LHM50/2B
6.3
LHM169/81
1.1
ESD1
4.1
LHM76/55
3.1
LHM77/64
9.1
LHM70/45
LHM59/19
LHM169/80
LHM57/17
Rh phenotype
CcDee
ccDee
Novel allele
RHD
(G63C)
RHD (A59T)
RHD (A237V)
Hybrid 1*
Hybrid 2*
Hybrid 3*
Anti-D production No
Yes
 * Epitope in accordance with Scott M. Section 1A: Rh serology. Coordinator’s report. Transfus Clin Biol. 2002;9(1):23-29.
† Hybrid 1: RHD-CE(E233Q,V238M,V245L)-D; Hybrid 2: RHD(T201R)-CE(5)-D(I342T); Hybrid 3: RHD(L214F)-CE(7)-D.

24. Minimal duplicated region (>10 kb)
RHD genotyping and mapping of duplicated region: To delineate the duplicated region another QMPSF assay based on universal fluorescent labeling was designed.
This customized QMPSF identified a >10-kb duplicated region in weak D individuals e2 e3 e4
i2a
i3c
i3b
i3a
i2b *
Intron 2
(5.9 kb)
Intron 3
(10.2 kb)
Breakpoint regions
Markers
Minimal duplicated region (>10 kb) 1 2 10 9 8 7 6 5 4 3 A
(A)- A novel QMPSF assay was designed by positioning additional markers (*) in introns 2 (i2a, and i2b) and 3 (i3a, i3b, and i3c).
i3b e7
HFE F9 e4 e2
i2b
i2a
i3c
i3a
Calibrator
Sample B
(B)- Typical QMPSF profiles obtained with a hemizygous, wild-type RHD calibrator (Rh C/c, E/e phenotype: Ccee) (top panel); and a hemizygous (exon 3 duplication) sample (Ccee). Calibrator: hemizygous, wild-type RHD control sample; sample: weak D, hemizygous exon 3 duplication sample.

25. Functional consequences of exon 3 duplication
The novel, Indian weak D allele involves duplication of a 12-kb fragment inserted within RHD intron 3.
Breakpoint was PCR-amplified and sequenced with primers RHD_i3ex3dup_F and RHD_i2ex3dup_R in standard conditions
Functional consequences of exon 3 duplication
To get insights into the mechanism involved in the expression of a weak D phenotype due to exon 3 duplication we sought to characterize the functional consequences of the allele as follows:-
Total RNA extracted of wild-type, RHD-negative and exon 3 duplication samples- Trizol based method,
Reverse transcribed with cDNA synthesis kit
cDNA amplified by PCR (RHD exon 2-4, exon 6-8, and ACTB). PCR products further subcloned into commercial vector and directly sequenced.

26. (A) RT-PCR products on gel.
The novel variant RHD allele including duplication of exon 3 impairs cellular splicing.
(A) RT-PCR products on gel.
(B) RT-PCR products from RHD(2-4) amplification were extracted from the gel, subcloned into a commercial vector and sequenced. Four products were found from an exon 3 duplication sample:
(1) exon 2 - exon 3 - exon 3 - exon 4;
(2) exon 2 - exon 3 - exon 3 - exon 4 deleted from the first four base pairs;
(3) exon 2 - exon 3 - exon 4, identical to what observed in a wild-type sample; and
(4) exon 2 - exon 3 - exon 4 deleted from the first four base pairs.
Sample product 1
Exon 2
Exon 3
Exon 4
Sample product 2
Wild-type
Sample
product 3
product 4
ACTB
RHD(2-4) D+ S D-
NTC
D+: wild-type, RHD-positive sample
S: exon 3 duplication sample
D-: RHD-negative sample
NTC: no template control
RHD(6-8) A B

27. Important observations from functional studies
This result provides important information about both the genomic rearrangement and the functional mechanism involved in the expression of the weak D phenotype and suggests that
an additional exon 3 is located between exons 2 and 4 in the same orientation; and
although splicing is severely altered and production of wild-type transcript decreases, a wild-type RhD protein may be generated at a low level in agreement with the expression of a weak D phenotype.
Two additional subproducts with deletion of 4 base pairs in either aforementioned amplicons were found, suggesting that processing of the transcript is severely altered by the variation.

28. Indian RhD specific genotyping assay primers designed
Design of an “Indian-specific, RHD genotyping assay”
Next we thought to design a molecular test that may be easily implemented at the laboratory level for genotyping RHD in Indians.
A standard, multiplex PCR assay was designed, including:
GAPDH (internal amplification control),
RHD exon 10 (present in the vast majority of variant RHD alleles),
RHD exon 5 (absent in several partial/null RHD alleles), and
RHD exon 3 duplication-specific marker
Indian RhD specific genotyping assay primers designed
Primers
RHD exon bp
RHD_e5seqF ATACCTTTGAATTAAGCACTTCACAGAG
RHD_e5seqR ACTGTGACCACCCAGCATTCTA
Ex3dup breakpoint 407 bp
RHD_i3ex3dupF ACGTGTTGAGGGCATGACCTC
RHD_i2ex3dupR GCCTGGATTCCTTGTGATACACG
RHD exon bp
RHD_e10seqF AGGCTGTTTCAAGAGATCAAGCCA
RHD_e10seqR GATGTTGTTATGTGGTACATGGCTG
GAPDH intron bp
GAPDH_i2F CCCCACACACATGCACTTACC
GAPDH_i2R CCTAGTCCCAGGGCTTTGATT

29. Indian-specific, multiplex PCR RHD genotyping
RHD-004 Exon 3 duplication
RHD-009 Exon 3 duplication
RHD-030 Exon 3 duplication
RHD-048 D cat VI, type 1 (D-CE(4-5)-D)
RHD-049 D cat VI, type 3 (D-CE(3-6)-D)
RHD-221 Weak D type 4.2.2
Interpretation guide for genotyping assay
GAPDH
RHD exon 3 dup
RHD exon 5
RHD exon 10
Results -
Failed* +
D negative
Partial/D negative †
D positive†
Weak D
+: positive PCR amplification
- : no PCR amplification
* Test again
† Other genotyping methods may be considered (sequencing, microarray, QMPSF…).

30. Novel, predominant RHD variant allele specific to the Indian population.
Novel RhD variants gave weaker reaction at IgM phase of testing and did not require IAT for detection.
Majority of the variants show ‘C’ antigen positivity
Discovery of new molecular mechanism involved in the production of novel weak RhD variant.
Development of an Indian specific genotyping assay for the RHD gene.
This study extends the current knowledge of RH molecular genetics but also extends the spectrum of mutational mechanisms involved in the variability of Rh expression.
The study contributes to improve Rh blood group diagnostics significantly in more than one billion Indians.

31. Rh variants around the world
Rh phenotype variability studied in Caucasian, African and East Asian origins and hundreds of variations have been documented and registered in the RhesusBase (
Caucasians
Africans
East Asians
Indians
Weak D type 1-3
> 90%
rare
Not reported
Weak D type 4.0, 4.1, 4.3
> 2%
common
Weak D type 4.2
Reported (Weak D type 4.2.2)
Weak D type 15
<1%
Partial D
Reported common (DVI)
Reported common (DAU, DAR)
Reported common (DFR)
DEL
Apart from the less common (RHDM295I) mostly single reports
Very Common (RHD1227G<A)
Phenotypically reported in North India.
RHD1227G>A & RHDM295I mutations absent in western India
Exon 3 Duplication
Common ≈ 60%
(Sandler et al 2017, Daniels 2013)

32. THANKS