IMMUNOHAEMAIMMUNOHAEMATOLTOLOGYOGY BULLETINBULLETIN Vol. 48 : No 3 Sept - Dec 2017

ICMR - NATIONAL INSTITUTE OF IMMUNOHAEMATOLOGY Dr. Manisha Madkaikar, was appointed as Director of National Institute of Immunohaematology from 14 Dec, 2017

Dr. Babu Rao Vundinti, Scientist E, received BGRC Silver Jubilee Award for the year 2016 on 11th October 2017 from Indian Council of Medical Research, New Delhi for the research work on “Understanding the molecular basis of Hematological diseases”. The award was given for his signicant contribution to the eld of Human Genetics of Hematological diseases in the past 20 years. He carried out extensive research on diagnosis, molecular pathology of Fanconi anemia and identied several novel mutations and also established its prenatal diagnosis.

Dr. Prabhakar Kedar, Scientist D, received ICMR Prize for Biomedical Research for Scientists belonging to Underprivileged Communities for the year 2013 on 11th October 2017 from Indian Council of Medical Research, New Delhi for his research work on “Studies on hereditary hemolytic anemia due to red cell enzymopathies”. The award was given for his signicant contribution to the eld of Enzymopathies for the past 20 years. He has reported several novel mutations in the human PKLR gene in Indian patients and has developed a new test for diagnosis of red cell membrane protein defects. Genetic modulators of fetal Priya Hariharan & Anita Nadkarni Summary : was far more resistant to alkali denaturation 1 The clinical heterogeneity of than the normal adult hemoglobin (HbA, α2β2) . and β-thalassemia is so variable that it prompted After almost 60 years of this discovery, in 1934 the researchers to identify the genetic Brinkman et al., proved that the variation in the modulators of the disease. Though the primary alkali resistant characteristic of hemoglobin was modulator of the disease are the type of β- due to the globin portion of the molecule and mutations that affects the degree of β-globin thus alkali resistant hemoglobin was termed as chain synthesis, the co-inheritance of α- (HbF, µ2¡2) since it formed the thalassemia and the fetal hemoglobin levels also major hemoglobin fraction of the growing fetus2. act as a potent secondary genetic modier of A string of experiments showed that the fetal . During the course of hemoglobin had an altered physio-chemical development the human body sequentially properties as compared to the adult expresses three different forms of hemoglobin: hemoglobin, like having increased resistance to embryonic, fetal and adult hemoglobin in the elution from the RBCs under acid conditions, maturing erythroblasts. Since increased decreased solubility in phosphate buffer, production of the fetal hemoglobin ameliorates increased resistance to heat and higher oxygen the severity of hemoglobinopathies, the study of afnity3. In the same time period, Drescher - genetic factors that play a role in the regulatory Kiinzer and Huehns et al., identied that the network of fetal to adult hemoglobin switch will embryonic hemoglobin in small human embryos facilitate development of new therapeutic (gestational age:7-12 weeks) had an alkali strategies for hemoglobinopathies. In this review denaturation between HbA and HbF4. These we have tried to summarise various mechanisms series of discoveries left behind a perplexing of hemoglobin switching and the genetic question; does the hemoglobin in humans during modulators lying within and outside the β-globin the ontogeny undergo successive switches? and gene cluster with their plausible role in governing if yes, is there a perspective for molecular cure the fetal hemoglobin (HbF) levels. by reactivating fetal globin genes in Introduction: patients ? Way back in 1866, Korber et al., discovered that the hemoglobin obtained from umbilical cord Priya Hariharan & Anita Nadkarni Department of Hematogenetics National Institute of Immunohematology (ICMR), 13th Floor, New Multistoreyed Building, KEM Hospital Campus, Parel, Mumbai 400 012. Email: [email protected]

1 Association of elevated HbF and condition, which is retarded with increased HbF hemoglobinopathies : concentration. Both HbF and its mixed hybrid S The possibility that hemoglobinopathies can be tetramer (α2β γ) cannot enter the deoxy sickle cured by induction of fetal hemoglobin was hemoglobin polymer phase, thus circumventing 8 realized as the symptoms are seen in six months the cellular damage evoked by HbS polymers . In of age after the birth when there is gradual both the hemoglobinopathy conditions, reduction in the HbF levels. In late 1950s, it was increased g-globin gene expression acts as a well- observed that patients with Cooleys anemia known disease modier. Hence understanding survived beyond the period of the fetal to adult the molecular mechanism of hemoglobin hemoglobin switch because of the prolonged switching and identication of molecular targets, synthesis of fetal hemoglobin in the erythroid for reversing this switch, is a subject of intense 5 cells5. In β-thalassemia, the main cause of the research . disease is imbalance in the globin chain ratio, The hemoglobin switch : wherein the excess unbound α globin chain G A precipitates, inhibiting the maturation of the During the ontogeny, globin genes (ε, γ, γ, δ, β) erythroid precursors, ultimately leading to in the β-globin cluster are sequentially ineffective erythropoiesis and anemia5. The expressed, leading to production of different clinical pathogenicity of β-thalassemia develops hemoglobin molecules with distinct physiological properties. The site for primary hemoglobin during the rst year of life when the g-globin synthesis is the embryonic yolk sac and the rst genes are gradually silenced with the activation wave of hemoglobin switch (primitive to of δ and β- globin genes. Thus continued g-globin denitive) occurs in the fetal liver after 5 weeks gene expression in β-thalassemics, may lead to of gestation, wherein there is a switch from reduction in the globin chain imbalance as g- 2 2 9 embryonic (ζ2ε2) to fetal hemoglobin (µ ¡ ) . globin synthesized will combine with the excess rd Towards the end of 3 trimester, there is a 6 unbound α-globin chain . Similarly appreciating 2 2 gradual change from fetal (µ ¡ ) to adult the role of fetal hemoglobin (HbF;α2γ2) in sickle hemoglobin (α2β2). This second major hemoglobin cell disease started more than 60 years ago when switch occurs in the bone marrow and lasts until Janet Watson conrmed that infants with sickle 9 6 months of age after birth . Figure 1 shows a cell disease had few symptoms and that their schematic representation of different deoxygenated erythrocytes took longer time to hemoglobin progressively expressed during sickle and did not deform as extensively as did distinct stages of development. With the dawn of their sickle cell trait-carrying mother's cells7. molecular era, models for gene switching Sickle cell disease is caused by the mutation in suggested, that this process involved complex codon 6th position in the β-globin gene by interaction between the cis-acting elements that substitution of glutamic acid by valine, which includes the locus control region of the beta affects the biophysical properties of the adult globin gene cluster and trans acting transcription hemoglobin5. The pathophysiology of sickle cell factors which co-ordinately carry out chromatin disease is dependent on the polymerization of 10 remodelling activities . deoxy sickle hemoglobin under low oxygen

2 Figure 1: Expression of globin genes during the ontogeny. The figure describes the site of hemoglobin synthesis and 2 major hemoglobin switches with respect to the gestational age of the fetus. Keys: HBG1 and HBG2 : A g-globin and G g-globin gene . HBB : β globin gene . The cis- acting elements : concert to facilitate long range looping activity. The β- globin gene cluster contains a 5' distal The cis- acting promoter sequences in the regulatory element known as Locus Control individual globin genes and the LCR serve as a Region (LCR), which has ve active DNAase I template for binding of various transcription 11 hypersensitive sites (HS) 1-5, that is required for factors . It is observed that naturally occurring appropriate globin gene expression. This is mutations in g-globin promoter region are followed with the series of globin genes (ε, Gγ, associated with hereditary persistence of fetal Aγ, δ, β) arranged sequentially in their order of hemoglobin phenotype (HPFH).The mechanisms expression. To the downstream of the β globin underlying the continued expression of HbF with gene is another HS site 3' HS1 [Figure 2]. The these mutations are thought to involve β–globin cluster is anked with olfactory alterations in the protein binding motifs that receptors at both 5' and 3' ends11. Wijgerde et ultimately ablates the binding of repressors or 12 al., proposed a competitive model for globin enhancers affecting the gene expression . The gene regulation, where in the LCR participates most extensively studied variation in several in the long range looping interaction, that leads population groups is the XmnI polymorphism to only one productive LCR-globin gene (C®T) residing at -158 position (HBG2 c.-211 G interaction11. The potent transcriptional C®T) in the ¡globin promoter region. The T activation of the LCR is also due to the clustering allele of XmnI polymorphism is linked to the of various erythroid transcription factors that raised HbF levels and milder hemoglobinopathy 13 bind to the hypersensitivity sites which act in conditions . Though T allele in its homozygous state seems to have little effect on HbF levels in

3 Figure 2: Structure of β -globin gene cluster showing embryonic (ε), fetal ( G gamma , A gamma and adult (δ, β) globin genes arranged according to the order of their expression that is controlled by locus control region ( LCR ). normal individuals, it has been associated with v B- cell CLL / lymphoma 11 A [BCL 11 A]: increase of HbF especially during the B- cell CLL / lymphoma 11 A is a zinc nger erythropoietic stress, suggesting its role in HbF transcription protein which is encoded by induction 14. In clinically milder patients where BCL11A gene located in chromosome 2p16 . no mutation is detected in the g-globin This protein expressed in 4 different iso-forms ( promoter region, the other genetic modulators XS, S, L, XL) plays an important role in 15 [HBS1L-MYB intergenic region, BCL11A, KLF1] lymphopoiesis and neurogenesis . The leading to raised HbF phenotype are screened. pioneering role of BCL11A polymorphisms in intron 2 in association with fetal hemoglobin Role of factors involved in the levels was rst demonstrated by Uda et al., hemoglobin switch and new 2008 after conducting genome wide association studies in the Sardanian population, where in therapeutic targets: variants in BCL11A were found to be More recently recognised variability in HbF signicantly associated with increase in F-cell 16 expression both in non anemic individuals and numbers in hemoglobinopathy patients .This patients with β-hemoglobin disorders stimulated suggested that the 3kb second intronic region of efforts to detect the genetic factors that lead to BCL11A had regulatory elements or functional this deviation. Genome wide association motifs responsible for modulating the g-globin gene expression. Further Sankaran et al., mapping studies have identied quantitative proved that BCL11A may function as a potent trait loci in the nuclear factors that play a role in repressor of the g- globin gene as lower levels the globin gene regulation and switching. The of BCL11A m-RNA is expressed in variants role of major transcription factors that are 17 causing higher levels of HbF . This was involved in the process of hemoglobin switching validated by performing gene knockdown of are as follows: [Figure 3]. BCL11A using short-hairpin RNA (shRNA)

4 Figure 3: Schematic representation of hemoglobin switching in presence of various transcription factors. During the switch process from fetal to adult hemoglobin , the g- globin repressor complex occupies the g-globin promoter region, thus blocking the g- promoter interaction with LCR and enhancing its interaction with the β-globin gene in presence of KLF1, thus leading to increase in adult hemoglobin.

approaches in primary adult erythroid includes erythroid lineage commitment, the g progenitors17.Chromatin Immuno Precipitation to β globin gene switch and activation or (ChIP) technology demonstrated that BCL11 A, suppression of certain erythroid specic interacts with NuRD chromatin remodelling and genes ( red cell metabolism and structure repressor complex, erythroid transcription proteins) by formation of chromatin factors – GATA -1, FOG -1, SOX6 and Matrin-3 to remodelling complex19. KLF1 transcription silence the gamma globin gene, suggesting the factor was thought to take part in β-globin pivotal role of BCL11A in gamma globin gene activation by binding to the CACCC box silencing 17. of the beta-globin promoter, as ablating this v Kruppel like factor 1 [KLF 1]: region lead to β-thalassemia intermedia Erythroid Kruppel like factor (EKLF) or KLF1, a condition. This phenomenon was validated by erythroid specic transcription factor plays a knockdown of KLF1 gene in mouse embryo critical role in erythropoiesis18. This founding that succumbed to severe beta thalassemia19. member of Kruppel like factor family was rst Though Singleton et al., 2008 primarily identied by Ira Miller and James Bieker in reported KLF1 gene mutations leading to 1992 and named it as KLF1/ EKLF due to its clinically benign In (Lu) blood group, the rst zinc nger homology with that of Drosophila event of raised HbF phenotype was body pattern determining Kruppel–gap mentioned by Borg et al., in an extended gene18. This master erythroid gene regulator, Maltese family where in the proposita co-ordinates three important processes that inheriting two KLF1 gene variants p.K288X

5 and p.M39L showed a HPFH phenotype with effective of these, are the variants residing HbF levels of 19.5%20,21. The mechanism that within the core enhancer binding region in 23 KLF1 participates in repressing the gamma HMIP-2A and -2B . A three -bp deletion globin levels via BCL11A activation was (rs66650371) at HMIP-2A is suspected to proved by Zhou et al by conducting gene directly cause HbF variability. Recent reports knock down assay, in which reduction of have described a transcriptional network within erythroid cells whereby KLF1 and MYB KLF1 gene markedly decreases the BCL11A activate each other by formation of an Active levels and indirectly increasing the gamma β- Chromatin Hub(ACH) structure that brings globin mRNA levels22. Thus established the intergenic enhancer regions in proximity with bipartite role of KLF1 gene, in indirectly 23 the MYB promoter . This same study showed reducing the ¡ globin gene expression by that perturbations to the ACH structure, or to BCL11A activation and directly activating β- KLF1 expression, do not affect gene globin gene by favouring looping interactions expression of HBS1L, suggesting that the between the LCR and the β-globin gene primary role of the GWAS-identied HBS1L- against g-globin gene promoter 22. MYB intergenic region is to regulate the expression of MYB23. v HBS1L- MYB intergenic zone [HBS1L- MYB]: v GATA 1: With the aim of studying the genetic variants The GATA family of transcription factors associated with the HbF regulation, genome (GATA 1 to GATA 6) are a group to zinc mapping studies identied another loci containing transcription regulators that bind located in chromosome 6, residing within 126- to consensus (WGATAR) DNA motif present in kb intergenic region between the HBS1L and the regulatory region of various erythroid MYB genes. This region is distributed in 3 development genes 24, 25. The role of GATA 1 as disequilibrium blocks which are referred to as a lineage specic transcription factor and in HPIM block 1, 2, 3 amongst which block 2 that regulating the HbF levels was rst suggested spans 24 kb region is found to be associated by Philips et al., in a patient suffering from with reduced disease severity in patients with congenital erythropoietic porphyria with hemoglobinopathy by increasing the HbF elevated HbF levels thus suggesting its levels. Though the function of HBS1L in red paramount role in erythroid maturation and blood cell development is uncharacterized, hemoglobin switching26. GATA 1 has 2 regions, the MYB gene (encoding the c-Myb the N-terminal transactivation that stabilizes transcription factor [TF]) is a key regulator of DNA binding and a C terminal zinc nger hematopoiesis and erythropoiesis23. The domain that binds to the GATA 1 motifs26. functional importance of the intergenic region Bottardi et al., proved that GATA 1 works in was rst observed when transgene insertion concert with other co-factors like friend of within the murine HBS1L-MYB intergenic GATA 1 (FOG-1), histone acetyltransferase region completely disrupted the binding of CBP/P300 / Mediator complex subunit MED–I core complex proteins LDB1, GATA1, TAL1, and a chromatin modeller–BRG1 to bring and ETO2 and abolished MYB transcription about long range chromatin interactions which resulted in severe anemia23. The most between β-LCR and the globin genes to

6 regulate their gene expressions27. Further Targeted translational research for Chromatin Immuno Precipitation assays i n c r e a s i n g t h e H b F l e v e l s : showed that GATA 1 along with IKAROS : transcription factor, forms a repressome Opportunities and challenges! complex, that limits the recruitment of The common transcription factors that govern transcription activators to the g-globin the developmental switches execute their action promoters, thus by silencing the g-globin by complex inter-dependent networks. The gene and progressively increasing the mechanisms underlying the function of these transcription of β-globin gene27. regulators are so stringent, that any deviations in their expression or protein-protein interaction v SOX6 : may evoke disastrous phenotype leading to SOX6 a member of the SOX transcription anemia, leukemia or lymphomas 30. Hence it is factor family is characterised by high mobility pivotal to elucidate their mechanisms, with the group (HMG) DNA binding domain. This gene basic step of placing these factors in a hierarchy codes for a 79 amino acid DNA binding protein in which they function. Despite of vigorous that binds to the minor groove of the DNA, leading to conformational changes in the DNA research for the search of HbF inducers, only structure 2 8 . SOX 6 thus acts as an Hydroxyurea (HU) – a ribonucleotide reductase architectural protein by dynamically inhibitor, remains the sole FDA approved remodelling the chromatin structure and medication for sickle cell disease. However the assisting the binding of other transcription HU utility is limited by its unpredictable HbF factors28. Its role in silencing the embryonic induction that varies patient to patient and globin gene was rst shown in a murine myelo-suppression. Thus there is a need for model, as SOX6 decient mice persistently developing other potential strategies to elevate expressed the embryonic globin. This HbF levels in hemoglobinopathy patients. One hypothesis was further proved by Zanhua et such approach is to develop target based therapy al., that 36–base pair region of the embryonic which includes knocking down the target by RNA globin proximal promoter is a binding site for interference with high specicity and selectivity. SOX6 mediated repression that leads to the The other way it could be achieved is by 28 silencing of the ε-globin gene . Chromosome controlling small molecules by using therapeutic conformation capture (3C) assay, ChlP drugs. However in either technique, the greatest suggested that SOX6 co-occupies with BCL11 challenge is to selectively impair the activity of A and GATA1, in the β-globin gene cluster and universally expressed transcription factors conducts silencing of g-globin transcription in specically in the erythroid lineage without the adult erythroid proginetors29. These affecting its universal function in the other results were further validated by carrying out cells31. combined knockdown experiments of SOX6 and BCL11A in primary human erythroblast Indian scenario and NIIH experience: culture which greatly enhanced the HbF In India, the rst study by Dadheech S. et al., production thus suggesting that both the transcription factors played a combined role showed that the C allele of rs 11886868 (C®T) in in g-globin gene regulation 29. BCL11A gene is associated with amelioration of

7 the severity of β-thalassemia as well as sickle cell promoter which when co-inherited with the β- anemia (SCA)32. Roy P. et al., showed that,A®G thalassemia trait condition lead to an elevated allele on the rs4895441 in the HBS1L-MYB HbF levels [HbA2: 3.6%, HbF: 31.7% and HbA2 : intergenic region is associated with the raised 3.9%, HbF : 23.6%]36. HbF levels in the eastern population of India33. Conclusion : Previous study by Upadhye D et al., have tried to correlate the SNPs in the BCL11A and HBS1L-MYB Increase in HbF levels has potential therapeutic gene with the HbF levels and clinical severity in implications as it ameliorates the clinical severity paediatric sickle cell anemia patients. They of β-hemoglobinopathies. With this review we identied A allele of the SNP rs4671393 in BCL conclude that SNPs located at various quantitative trait loci, are associated with 11A gene and G allele of rs9389268 in the HBS1L- meagre elevation of HbF levels in the range of 3- MYB gene to be associated with raised HbF levels 5%, nevertheless they can increase the HbF and with milder clinical symptoms34. In our recent output to clinically benecial levels when co- study we have identied the C allele of inherited with hemoglobinopathies. Identication rs11886868 and rs66650371 (-TAC) (3 base of such variants are of importance as in future deletion) in the MYB gene to be associated with they can be used in genome editing to naturally raised HbF levels in adult SCA and thalassemia increase the HbF levels in patients with β- intermedia patients. Similarly, the rst event of hemoglobinopathies. The study of transcription KLF1 variations associated with raised HbF factors and their co-enzymes that play role in 22 phenotype was mentioned by Borg et al , gamma globin gene silencing may help in however no such studies have been carried out in determining new potential interacting surfaces India to understand the prevalence and effect of and enzymatic mechanisms. Curtailing the KLF1 variations in our population.We have activity of these proteins may thus lead to the identied 12 different KLF1 gene mutations after development of novel therapies for treating screening 325 hemoglobinopathy patients, most hemoglobinopathies. of the mutations associated with mild increase in References : HbF levels. We found a novel KLF1 gene variation 1. Lorkin PA. Fetal and embryonic . codon 211 A®G (c.632 A>G)to be associated with J Med Genet.1973;10:50-64. increased HbF levels in thalassemia trait [HbA2: 2. Brinkman R, Wildschut A, Wittermans A, et 4.1%, HbF: 8.6%, β-globin gene mutation: codon al. On the occurrence of two kinds of 15 G®A heterozygous] and speculate that co- hemoglobin in normal human blood. J. inheritance of this novel KLF1 variant might Physiol. 1934;80:377-87. have ameliorated the clinical manifestations of 3. Giardina B. Fetal hemoglobin: Structure and her six year old untransfused β-thalassemia f u n c t i o n . S c a n d J C l i n L a b homozygous child [HbA2 :1.6 %, HbF:97.9%, β- Invest.1982;42:32-37. globin gene mutation: codon 15 (GàA) 4. Huehns R., Dance N, Beaven H, et al. Human 35 homozygous] . Also we identied novel 5 bp embryonic hemoglobins. Nature.1964; deletion -ATAAG (-533 to -529) in the Gγ globin 201:1095-97.

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9 25. Phillips J, Steensma D, Pulsipher M, et al. HBG2 XmnI polymorphism On Hb F levels. Congenital erythropoietic porphyria due to a Hemoglobin. 2012;36:592-9. mutation in GATA1: the rst trans-acting 34. Upadhye D, Jain D, Trivedi Y, et al. mutation causative for a human porphyria. I n  u e n c e o f s i n g l e n u c l e o t i d e Blood. 2007;109:2618-21. polymorphisms in the BCL11A and HBS1L- 26. Ferreira R, Ohneda K, Yamamoto M, et al. MYB gene on the HbF levels and clinical GATA1 function, a paradigm for transcription severity of sickle cell anaemia patients. factors in hematopoiesis. Mol Cell Biol. Ann Hematol.2016;95:1201-03. 2005;25:1215-27. 35. Hariharan P, Gorivale M, Colah R, et al. Does 27. Bottardi S, Zmiri F, Bourgoin V, et al. Ikaros the Novel KLF1 Gene Mutation Lead to a interacts with P-TEFb and cooperates with Delay in Fetal hemoglobin Switch? Ann Hum GATA-1 to enhance transcription elongation. Genet. 2017;81:125-28. Nucleic Acids Res.2011;39:3505-19. 36. Hariharan P, Sawant M, Gorivale M, et al. 28. Yi Z, Cohen-Barak O, Hagiwara N, et al. Sox6 Synergistic effect of two β globin gene Directly Silences Epsilon Globin Expression in cluster mutations leading to hereditary Denitive Erythropoiesis. PLoS Genet. persistence of fetal hemoglobin (HPFH) 2006;2:14. phenotype. Mol Biol Rep. 2017;44:413-17. 29. XuJ, Sankaran V, Ni M, et al. Transcriptional silencing of {gamma}-globin by BCL11A 37. Wienert B, Funnell A, Norton L, et al.Editing involves long-range interactions and the genome to introduce a benecial naturally cooperation with SOX6. Genes Dev. occurring mutation associated with increased 2010;24:783-98. fetal globin. Nat Commun. 2015;14:6.

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Bcl-xL . Nat Cell Biol. 2001;3:173 - 82 32. Dadheech S, Madhulatha D, Jain S, et al. Association of BCL11A genetic variant (rs11886868) with severity in β-thalassaemia major & sickle cell anaemia. Indian J Med Res . 2016;143:449-54.

33. Roy P, Bhattacharya G, Mandal A, et al. Inuence of BCL11A, HBS1L-MYB, HBBP1 single nucleotide polymorphisms and the

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12 Award Winning Abstracts

1. Abstract awarded Harold Gunson fellowship variant samples identied were further at the 28th Regional conference of ISBT held at characterized by using the Advanced Partial RhD Guangzhou, China on 25-28 November, 2017 Typing Kit (Alba Bioscience, Edinburgh, UK).

PHENOTYPIC CHARACTERISTICS OF NOVEL RHD Serological data of D antigen epitope prole and VARIANTS IN INDIANS antigenic sites on these D variant samples was also evaluated. D S Parchure, S Kulkarni, H Gogri, V Gopalkrishnan, JM Chen, C Le Maréchal, L Raud, C Results: Out of the 223 RhD variants identied in Férec, M Madkaikar, K Ghosh, Y Fichou. National the study seven variants were found to be novel. Institute of Immunohaematology ICMR, Mumbai, They are as follows: Exon 3 duplication (n = 130), India and EFS - Inserm UMR1078, BREST, France. RHD(G63C) (3), RHD(A59T) (1), RHD(A237V) (1), RHD-CE(5:E223Q,V238M,V245L)-D (1), Background: The RhD antigen being unarguably RHD(T201R)- CE(5)-D(I342T) (1), RHD(L214F)- the most clinically signicant blood group antigen CE(7)-D (1). Majority of the exon 3 duplication has led to it being studied in several variants were detected in the IgM phase with comprehensive studies in Caucasians, Black weaker strength and showed presence of the 'C' Africans and East Asians. Several phenotypic antigen. They also showed a heterogeneous studies have been carried out in Indians but an pattern of agglutination with varying strengths on extensive study was lacking. India being a testing with panel of anti-D reagents from formidable heterogeneous population a study of ALBAclone Advanced Partial RhD Typing Kit (Alba the phenotypic variability would denitely add to Bioscience, Edinburgh, UK). Absence of epitopes the repertoire of RhD variants and a better 1.2, 8.1 and 6.3 in different combinations was understanding of the same. observed. Additionally, ow cytometry analysis in Aims: To phenotype and study novel RhD variants a subset of samples showed a range of D antigenic in Indians. sites (range: 1276-6050 D antigens/cell). Another Methods: Samples (n=223) presenting with a weak novel weak D mutation RHD(A237V) predicted to D phenotype by serological analysis using routine be present on the top of transmembrane domain commercial anti-D reagents were genotyped for eight which is close to the extracellular domain the RHD gene by conventional molecular showed absence of 1.1, 1.2, 6.3, 8.1 and 9.1 approaches, i.e by melting curve analysis and epitopes and was found to be responsible for the sequencing the RHD coding regions. Quantitative production of anti-D. For three samples showing multiplex PCR of short uorescent fragments weak D mutation RHD(G63C) predicted to be (QMPSF) assay was performed for assessment of located in the second transmembrane domain the RHD exon copy number variations. The novel D mean antigenic density was found to be in the range of 5088-7406 D antigens/cell. Though the variability of Rh expression has been

Summary / Conclusions: All the novel RhD documented in Indian population, extensive variants gave weaker reaction at IgM phase of genetic studies on Rh antigens have not been testing and did not require indirect antiglobulin carried out. In the present study we aim to test for detection. Overall we describe a major characterize the molecular bases of weak D novel weak D allele resulting due to exon 3 expression in Indians. duplication in Indians. The varying pattern of Material & Methods: Samples (n=223) presenting agglutination, strength and the antigenic density with a weak D phenotype by serological analysis observed for this novel variant can be attributed were genotyped in the RHD gene by conventional to the altered transcript biosynthesis. molecular approaches. Firstly, all samples were Preliminary data also suggest inter individual tested for the three common variant RHD alleles variation in the relative amount of transcripts (weak D, type 1, 2, and 3) in the Caucasian produced. The study contributes to improve Rh population by melting curve analysis. Samples blood group diagnostics signicantly in more than were then analyzed by sequencing the RHD one billion Indians. coding regions for identication of base substitutions/ insertions /deletions. 2. Best oral paper award rst prize at the 6th Quantitative multiplex PCR of short uorescent National Conference of Indian Society of fragments (QMPSF) assay was performed for Transfusion Medicine held at Lucknow, on 3-5th assessment of RHD exon copy number variations. November, 2017 This test aims to calculate the number of both NOVEL MOLECULAR MECHANISM PRODUCING the RHD and the RHCE genes/exons in two WEAK D PHENOTYPE IN INDIANS separate tests and is performed mainly to look

1 1 1 for partial D variants resulting by the formation Swati Kulkarni , Disha Parchure , Harita Gogri ,

1 1 of hybrid genes by rearrangement. Data of Vidhya G , Manisha Madkaikar , Kanjaksha

1 2 1 serological D epitope prole and antigenic site on Ghosh , Yann Fichou . National Institute of these D variant samples was also evaluated. Immunohaematology ICMR, Mumbai, India, 2EFS - Inserm UMR1078, BREST, France Results: Three common variant RHD alleles (weak D, type 1, 2, and 3) were absent in study The Rh blood group system is the most population and sequencing analysis identied polymorphic system and is implicated in only a limited number of variations, including hemolytic transfusion reaction and hemolytic novel single nucleotide substitutions. disease of the fetus and newborn. Molecular Conversely, QMPSF approach revealed a genetics of the RH genes has been extensively common duplication of exon 3 in a signicant studied in Caucasians, Africans and East Asians proportion of weak D samples (58.3%), suggesting and the variant alleles giving rise to weak and a novel, predominant rare allele specic of the partial RhD phenotypes have been reported. Indian population. Further functional analysis by cloning / samples showed a varying D antigen density in sequencing, minigene splicing assay showed that range of 1276–6050 D antigens/cell. Conclusion: this genetic variation results in the expression of Overall, we have identied a novel, predominant several transcripts, including a wild-type RHD variant allele specic to the Indian product. These results suggest that this allele population. As most of the weak D variants quantitatively affects the expression of the reported in literature are due to SNP and normal transcript, and then subsequently the formation of hybrid RHD-CE genes, our study of expression of the normal RhD protein, nally genotyping of the RhD variants led us to discover resulting in a weak RhD phenotype. Genomic a new molecular mechanism producing weak RhD characterization of the duplicated region nally variants in Indians. This discovery not only identied a ˜12 kilobase region encompassing extends the current knowledge of RH molecular the very 3′-end of exon 2, full intron 3, exon 3, genetics but also extends the spectrum of and a partial sequence of intron 3. Serological mutational mechanisms involved in the epitope prole in this novel variant samples variability of Rh expression. The study will also showed a variable pattern. Flow cytometry help us to develop Indian specic genotyping analysis in a subset of duplication positive assay.

Dr Ajit Gorakshakar, Scientist F, superannuated on 30th Nov 2017 after completing his service for 42 years at Institute. Hindi Pakhwada conducted at NIIH Seminar hall on 26- 27th Sept 2017. Essay writing and elocution competition were conducted. Chief guest for this occasion were Mr. Vipul Lakhnavi and Mr. Vinod Kumar Sharma.

NIIH staff and students conducted Sadbhavna Diwas Oath at Seminar Hall on 24th Nov 2017

Program on Swachh Bharat Abhiyaan was conducted in N.P. High School, Lalbaug Mumbai- 21st Nov 2017. Drawing & Essay writing competition was held in which 30 students participated and in elocution competition ve students took part. The topic for all the three competitions, were on Swachh Bharat Abhiyaan. Dr. Swati Kulkarni, Scientist D from the Department of Transfusion Medicine, was awarded Best oral paper award rst prize for presentation entitled "Novel molecular mechanism producing weak D phenotype in Indians" at the 6th National Conference of Indian Society of Transfusion Medicine held at Lucknow, on 3-5th November, 2017.

Miss. Disha Parchure, PhD student from Department of Transfusion Medicine, was awarded the prestigious Harold Gunson fellowship (for transfusion medicine students/researchers) at the 28 t h Regional conference of ISBT held at Guangzhou, China on 25- 28 November, 2017. This fellowship is a travel grant exclusively for young researchers. She was awarded the fellowship for her abstract entitled "Phenotypic characteristics of novel RHD variants in Indians"

“Award of Excellence” was given to Dr. Swati Kulkarni, Scientist D for extraordinary contribution in the eld of Transfusion Medicine by Indian Society of Blood Transfusion and Immunohaematology during 42nd Annual conference held at Kota from 8-10th Dec 2017. Flowcytometry workshop on Diagnosis of Primary Immunodeciency Disorders (PID) was organized by Pediatric Immunology and leukocyte biology department, on 7-8th Sept 2017 at NIIH.

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