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Hereditary persistence of fetal haemoglobin (HPFH) and (δβ)0-thalassaemia are conditions caused by large deletions of δ- and β-globin genes and are characterized by high fetal haemoglobin (HbF) in heterozygotes of these mutations. Complete characterization of these deletions is important for understanding the transcriptional and epigenetic mechanisms involved in increase in HbF levels by these mutations. Previous studies demonstrated that these large deletions remove the regulatory sequences which suppress γ-globin gene expression and increase the interaction of enhancers with γ-globin promoter. However, the exact mechanism for elevated HbF in these conditions and differences in HbF levels and phenotypes between (δβ)0-thalassaemia and HPFH still remains unclear. We screened 90 individuals from 51 families by gap-PCR for previously reported common deletions and by MLPA and quantitative fluorescent multiplex -PCR (QFM-PCR) for new deletions in the β-globin cluster. After identification of the approximate locations of the deletions by MLPA and QFM-PCR, we developed PCR strategies to amplify across the breakpoints and the products were sequenced to characterize the breakpoints. We identified six different deletions in Indian population. The frequencies of the previously reported common mutations, Asian inversion deletion and HPFH-3, were 46.66% and 24.44%, respectively. The rare mutations which were not found earlier in Indian population include 49.3kb deletion (12.22%) and 32.6kb deletion (10%). We found 2 novel deletions, 49.98kb deletion and 86.7kb deletion, which accounted 5.55% and 1.11%, respectively. The breakpoints of the 49.98kb deletion were found to be very close to those of HPFH-3 and this mutation was found in compound heterozygous with β-thalassaemia in a 8 year old boy with thalassaemia intermedia (Hb=8.8g/dL). Four heterozygotes in the family had mean MCV = 79.7fL (range: 76-85.6), mean HbF%= 26.9% (range: 23.3-33.0) and pancellular distribution of HbF suggesting that this mutation causes HPFH. The 86.7kb region encompassing Aγ-, δ-and β- globin genes was found in heterozygous state in one individual and it also had haematological parameters of HPFH (MCV=80.8fL and HbF=32.2%). To develop an experimental model to study the molecular mechanisms for elevated HbF in these conditions we established ex-vivo erythropoiesis system with the peripheral blood mononuclear cells obtained from patients with Asian inversion deletion and HPFH-3. Quantitative real time PCR (qPCR) showed increased expression of γ-globin transcripts in the cultured erythroid cells and chromatin immunoprecipitation (ChIP) of RNA Polymerase II (RNAPII) also showed increased transcriptional activation of γ-globin gene in the erythroid cells obtained from the patients with these mutations. To understand the roles of non-coding transcripts at the 5' and 3' breakpoints of the deletions in the transcriptional activation of γ-globin, we performed qPCR with the cDNA obtained from cultured erythroid cells using the primers that bind upstream and downstream of these breakpoints. The results showed that the level of BGL3, the noncoding transcript present near the 5'breakpoint, is enhanced by these deletions. We found activation of a novel non-coding transcript at the 3' breakpoints and they were absent in the erythroid cells of normal individuals. ChIP also showed significant binding of RNAPII at the genomic regions from where these non-coding transcripts are produced. The role of non-coding transcripts has not been reported earlier as a mechanism for increased γ-globin transcription in HPFH and (δβ)0-thalassaemia. It is possible that these deletions remove the genetic elements that cause transcription repression of γ-globin gene in normal adults and they also cause activation of non-coding transcripts which change the epigenetic status of γ-globin gene resulting in its active transcription. To conclude, using QFM-PCRs we could perform a comprehensive study of (δβ)0-thalassaemia and HPFH in Indian population and detected rare and novel deletions that cause these conditions. Using ex-vivo erythropoiesis system which mimicked the pattern of globin gene expression in normal individuals and patients with globin gene mutations, we could identify the role of noncoding transcripts in increased HbF expression in the patients with HPFH and (δβ)0-thalassaemia.

Ex-vivo differentiation of hematopoietic stem cells and progenitors to erythroid cells helped tremendously in our understanding of transcriptional and epigenetic regulation of normal erythroid differentiation as large number of nucleated cells can be generated by these protocols. However, to study the disease mechanisms in haematological diseases, there are practical difficulties in obtaining mobilized CD34+cells from the patients. Alternatively, peripheral blood mononuclear cells (PBMNCs), which have a small fraction of haematopoietic progenitors, have also been used to obtain cultured erythroid cells (cErCs). Though PBMNCs is an alternative source, the current protocols using these cells do not generate sufficient number of cErCs, which is a major hurdle for performing transcriptional and epigenetic studies in erythroid cells from patients with β thalassaemia and sickle cell disease. We established a modified two phase culture protocol using a serum free basal medium with IL-3, IL-6, Flt3, SCF in phase I and Epo, SCF, IL-3 and iron compounds in phase II and could generate up to 108cells from PBMNCs isolated from 5-10ml blood of patients with homozygous β thalassaemia and those who were compound heterozygous for β thalassaemia and HPFH or δβ thalassaemia. Real time quantitative PCR (qPCR) analysis showed steady increase in the expression of α and β globin genes in different stages of erythropoiesis. As we obtained a large number of nucleated cells using this protocol, we could perform chromatin immunoprecipitation (ChIP) to study the binding of RNA Polymerase II (RNAPII), transcription cofactors, CBP and p300, and histone modifications at the regulatory sequences of β globin cluster. High levels of RNAPII was found at the coding sequences of β globin gene in the cErCs from both normal individuals and patients with splice site mutations suggesting that these mutations do not cause significant reduction in the transcription rate. Both CBP and p300 were found to bind at the HS3 and HS2 regions of locus control region in both normal and β thalassaemia cultures though they were not found at the promoter sequences of actively transcribed genes in the β globin cluster. In the patients compound heterozygous for β thalassaemia and HPFH-3 or (Aγδβ)° deletion/Inversion, we found increased γ globin gene expression compared to the normal controls and ChIP also showed high levels of RNAPII occupancy at the coding sequences of γ globin gene. As it has been shown earlier that alternative splicing is a transcriptional regulatory mechanism for cell specific expression of genes, we performed experiments to identify the alternatively spliced transcripts of β globin gene and their co-ordinated expression in human erythropoiesis. RT-PCRs and sequencing of β globin cDNA using various sets of primers that bind at the upstream, downstream and coding sequences showed 3 aberrantly spliced transcripts formed by the cryptic splice sites in exon1 and 4 novel transcripts by the activation of splice sites at the 5'UTR. Interestingly, 5' splice site mutations in intron 1 did not activate cryptic splice sites in the coding sequences and instead, they activated specific cryptic splice sites upstream b globin gene. Q-PCR with chimeric primers for the exact quantitation of the splice variants showed that the splice site mutations also cause significant reduction (> 90%) in expression of normally spliced transcripts suggesting that nonsense mediated decay is not the major mechanism for the reduced β globin production by these mutations. We found high levels of one of the aberrantly spliced transcripts with 5' splice site in exon 1 (∼20%) in normal individuals. All the splice variants showed steady increase in their expression from early to late stages of erythropoiesis. Our study shows that ex-vivo erythropoiesis system is an invaluable tool for studying the mechanism of globin gene regulation and switching. Identification of specific splice variants in cErCs of normal individuals and in patients with β thalassaemia could define the molecular mechanisms by which splice site mutations cause β-thalassaemia. The presence of abundant splice variants in normal controls suggests that β globin gene is regulated by multiple promoters and other regulatory elements and identification of these sequences will help in understanding the molecular basis of human globin gene regulation.