1. Molecular Characterization Of Rare D--/D--variants In Individuals Of Indian Origin
Dr. Swati Kulkarni
Transfusion Medicine Department
ICMR-National Institute of Immunohaematology

2. RH gene organization on chromosome 1
INTRODUCTION
The Rh system is the most polymorphic and immunogenic protein based blood group system.
The two RH genes, RHD and RHCE are about 30,000 bp apart, have opposite orientation and are homologous, retaining more than 96% identity.
RH gene organization on chromosome 1
Alloantibodies produced against five Rh antigens D, C, c, E and e (encoded by RHD and RHCE) are known to be major cause of HDFN and HTR

3. Unusual Rh phenotypes such as Rh null and D- - are rarely encountered in routine blood bank testing.
Rh null phenotype shows complete absence of D, C, c, E, and e antigens.
However D-- phenotype lack expression of C, c, E and e on the red cells because of mutations in both alleles of the RHCE gene but shows presence of D antigen.
Frequencies have been estimated at 0·0005 (in Sweden), 0·0047 (in Iceland) and 0·0032 (in Japan) (Daniels, 1995).
In absence of RHCE antigens, the D antigen expression is exalted (approximately
– per D-- red cell compared with – in normal individuals) to the extent that IgG anti-D can agglutinate the RBCs in saline phase (IgM phase).
Such individuals show presence of anti-Rh17 or anti-Hro.
This phenotype is strongly linked to consanguinity and has been found in disperse populations including white Americans and Europeans, American indigenous populations, Black Americans, Japanese, Chinese, Indians and mixed race South Africans.

4. Molecular mechanisms of RHCE null phenotype…
Gene rearrangements( RHCE-D-CE hybrid)- Most common mechanism
-large segments encompassing one or more exons of the RHCE gene
are missing eg. RHCE-D(3-9)-CE
2) Frameshifts - Due to a deletion of one of more nucleotides, which introduces a
premature STOP codon .eg. RHCE*cE907delC
3)Single-nucleotide polymorphisms(SNPs). eg. (RHCE*Ce659A)
4) Splice site mutations - A single-nucleotide polymorphism at a splice site can
prevent mRNA splicing eg. RHCE*ceIVS4+1t
RHD gene
RHCE-D(3-9)-CE

5. MATERIALS AND METHODS
Five RhD positive postnatal women who had produced antibodies against all Rh antigens except D, leading to HDFN and fetal loss were referred to ICMR -NIIH for further evaluation. 
Extensive serological and molecular analysis was carried out.
Serological Typing:
1. Tested with anti- C,c,E,e.
2. RhD typing by routine commercial blend(IgM+IgG) and IgG anti- Ds
3. Adsorption-Elution of D-- samples to detect presence of D, C, c, E, and e antigens.
4. Flow cytometric analysis to detect presence of D, C, c, E, and e antigens
5.Antibody detection and titration.
Molecular Genotyping:
DNA extraction from whole blood (Phenol- Chloroform method)
Determination of copy number of specific RHD exons by QMPSF.
QMPSF (Quantitative Multiplex Polymerase Chain reaction of Short Fluorescent
fragment) is an invaluable tool to detect quantitative variants such
as deletion, duplication, hybrids etc. .

6. RESULTS
Serological testing with anti-C, anti-c, anti-E, and anti-e showed absence of C, c, E and e antigen, thus identifying the rare Rh variant as D--/D--
Sample No.
Anti-C
Anti-c
Anti-E
Anti-e
Anti-D
Rh status
Patient 1 - +
D--
Patient 2
Patient 3
Patient 4
Patient 5
Control 1
R1R1
Control 2
R1R2
Control 3
R1r
Control 4
R2r
Control 5 rr
Ether-elution of D-- samples treated with Anti-C, c, E and e showed negative reaction with antigen positive cells thus confirming the total absence of RhCE antigens.
Patient’s serum showed presence of anti-Rh17 in high titer (> 1:128).

7. C, c, E and e antigens along with exalted expression of D antigen.
Flow Cytometry analysis of RHCE antigens
Flow cytometry analysis carried out on FACSCalibur confirmed absence of
C, c, E and e antigens along with exalted expression of D antigen.
Flow cytometry image of an individual with D - - phenotype showed absence of C,c,E and e antigens and presence of the D antigen
Positive control having ryr/r’r’’ phenotype showed presence of C,c,E and e antigens and absence of D antigen

8. Comparison of MFI of D antigen between D-- individuals and RIRI individual
Homozygous D--/D-- individual showed elevated expression of D antigen when compared with R1R1 individual and heterozygous D-- individuals

9. Molecular genotyping: By QMPSF of RHD and RHCE genes
RHD QMPSF analysis of RhD positive control with R1R2 phenotype
RHCE QMPSF analysis of RhD positive control with R1R2 phenotype
exon3
exon7
exon5
exon9
exon1C
exon 6
exon4 F9
exon2c
HFE
exon8
exon10
RHD QMPSF analysis of RhD negative control with rr phenotype
RHCE QMPSF of rr phenotype will have all the RHCE exons

10. RHCE QMPSF of D - - /D - - samples
Wild-type
Cc control
RHCE-D(3-9)-CE
RHCE-D(3-8)-CE
RHCE-D(2-6)-CE e3 e7 e5 e9
e1C e6 e4 F9
e2c
HFE e8
e10 *
RHD QMPSF of D - - /D - - samples showed presence of Entire RHD gene
*All RHD exons have been sequenced and showed wild-type profiles.

11. Most common hybrid was found to be RHCE-D(3-9)-CE (n=3)
Molecular analysis by QMPSF showed gene conversion event between RHCE and RHD causing D- -/D - - phenotype.
In this hybrid genes, recombination between RHD and RHCE results in replacement of regions of RHCE by regions of RHD gene of different length.
Most common hybrid was found to be RHCE-D(3-9)-CE (n=3)
followed by RHCE-D(3-8)-CE(n=1) and
RHCE-D(2-6)-CE(n=1).
Sample No.
RH Genotype
Rh Phenotype
RHCE
RHD 1
RHCE-D(3-9)-CE
Intact RHD
D-- 2 3 4
RHCE-D(3-8)-CE 5
RHCE-D(2-6)-CE

12. Molecular mechanisms of D--/D-- phenotype reported in various populations
Origin
Mutations
reference
RHCE GENE
RHD GENE 1
Italian, Icelandic, UK
RHCE-D(3-9)-CE
Intact RHD
Kemp et al (1996)
Italian, UK
RHCE-D(3-8)-CE 2
Caucasian ,Cajun
Intact RHCE
(RHCE*cE221A)
Ochoa-Garay et al(2013)
Turkish
(RHCE*CeIVS1+5a) 3
White
RHCE-D(3-7)-CE
Cheng et al (2000) 4
Japanese
Okuda et al (2000) 5
Malaysian
Flatt et al(2012)
RHCE-D(4-9)-CE 6
Italian
RHCE-D(3-8) –CE(9)-D(10)
RHD-CE(10)
Cherif-Zahar et al (1996) 7
Hispanic
Deletion of ‘c907′ (Frameshift)
Westhoff et al (2011)

13. CONCLUSION
This is the first study reporting molecular mechanism of D-- phenotype in Indian population.
Formation of RHCE-D-CE hybrids responsible for D-- phenotype in our population
Lack of RhCE protein results in exalted expression of D antigen.
Identification of RHCE ‐ null variants facilitates confirmation of D-- phenotypes in patients and donors, helping improve transfusion safety.
Identification of D-- phenotype helps detect other family members harboring the same mutation and helps council females of child bearing age in the family about the complications involved in such pregnancies.
Rare donor registry
Thanks