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Abstractβ-thalassemia (thal), hemoglobin (Hb) E, and high Hb F determinants, which are caused by mutations in the β-globin gene cluster, are common genetic disorders in Thailand and Southeast Asia. Prenatal diagnosis is essential for couples at risk to identify severe forms, including homozygous β-thal and Hb E/β-thal. Conventional methods, including reverse dot-blot hybridization and gap-polymerase chain reaction (PCR) for genotyping of point and large deletion mutations, require post-PCR steps, which are time-consuming and costly. This study aimed to develop a rapid and efficient method using monoplex high-resolution melting (HRM) analysis for genotyping of Hb E and 11 β-thal mutations; multiplex HRM analysis for identifying six deletional mutations, including two β0-thal mutations (3.5 and 45 kb deletion); and a novel method for detecting four high Hb F determinants, namely, δβ0-thal (12.5 kb deletion), HPFH6, Indian inv-del (Aγδβ)0-thal, and Thai del-inv-ins (Aγδβ)0-thal. The developed assays were validated using 182 blinded fetal DNA samples with 41 β-thal genotypes. Different HRM patterns were observed among wild-type, heterozygote, homozygote, and compound heterozygote genotypes. Six deletional mutations showed specific melt curves. This technique demonstrated 100% concordance with conventional methods. The assay showed 100% sensitivity, specificity, and positive and negative predictive values within the limit of detection at DNA concentrations of 8.0 ng/reaction. Finally, this developed assay was efficient in identifying both point mutations and large deletion, convenient, rapid, and cost-effective and did not require post-PCR steps. Thus, this technique has potential for application in prenatal diagnosis of thal and can inform prevention and control programs. [ABSTRACT FROM AUTHOR]

Whole blood transcriptome analysis is a valuable approachin medical research, primarily due to the ease of sample collection and the richness of the information obtained. Since the expression profile of individual genes in the analysis is influenced by medical traits and demographic attributes such as age and gender, there has been a growing demand for a comprehensive database for blood transcriptome analysis. Here, we performed whole blood RNA sequencing (RNA-seq) analysis on 576 participants stratified by age (20–30s and 60–70s) and gender from cohorts of the Tohoku Medical Megabank (TMM). A part of female segment included pregnant women. We did not exclude the globin gene family in our RNA-seq study, which enabled us to identify instances of hereditary persistence of fetal hemoglobin based on the HBG1 and HBG2 expression information. Comparing stratified populations allowed us to identify groups of genes associated with age-related changes and gender differences. We also found that the immune response status, particularly measured by neutrophil-to-lymphocyte ratio (NLR), strongly influences the diversity of individual gene expression profiles in whole blood transcriptome analysis. This stratification has resulted in a data set that will be highly beneficial for future whole blood transcriptome analysis in the Japanese population. [ABSTRACT FROM AUTHOR]

Hereditary Persistence of Fetal Hemoglobin is a rare case in community. This heterogeneous condition characterized by the production of fetal hemoglobin (Hb F) that persists into adulthood where it should be in adulthood that the production of adult hemoglobin (Hb A) dominates. HPFH is classified in two forms, namely deletion and non-deletion. HPFH can be considered a mild form of or thalassemia because failure of chain synthesis is compensated for by chains after the neonatal period. Hb F can be increased in adults due to hereditary disorders of hemoglobin synthesis or acquired conditions. from the differential diagnosis of other hemoglobin disorders. The diagnosis of HPFH is based on clinical conditions, routine blood tests, and special laboratory tests, namely by measuring Hb F levels and Hb F intercellular distribution. Method of analysis Hb F is used with hemoglobin electrophoresis methods. The Hb F examination aims to establish a diagnosis of hereditary globin chain disorders and other acquired conditions, in which case the Hb F level will increase. For this reason, it is necessary to accurately measure Hb F levels. [ABSTRACT FROM AUTHOR]

AbstractWe report the haematological parameters and molecular characterization of beta zero (β°) South East Asia (SEA) deletion in the HBB gene cluster with unusually high levels of Hb F compared to a classical heterozygous beta zero (β°)-thalassaemia.MethodsRetrospective study on 17 cases of (β°) South East Asia (SEA) deletion from 2016 to 2019 referred to Institute for Medical Research were conducted. The clinical information and haematological profiles were evaluated. The mutation was analyzed, and the results were compared with other β°-thalassaemia groups. For HBB gene genotyping, all the cases were subjected for multiplex gap-PCR, 5 cases were subjected for HBB gene sequencing for exclusion of compound heterozygous with other beta variants. Co-inheritance of α-thalassaemia were determined using multiplex gap-PCR and multiplex ARMS-PCR.ResultsSeventeen cases were positive for β°-thal SEA deletion. Fifteen cases were heterozygous and two were compound heterozygous for β°-thal SEA deletion. The results were compared with 182 cases of various heterozygous β° deletions and mutations. The mean Hb for heterozygous β°-thal SEA deletion (13.44 ± 1.45 g/dl) was normal and significantly higher than heterozygous IVS 1-1 and Codon 41/42 (post hoc test, p

Sickle cell disease (SCD) is a group of related yet genetically complex hemoglobinopathies. Universal newborn screening (NBS) for SCD is performed in the United States and many other nations. Classical, protein-based laboratory methods are often adequate for the diagnosis of SCD but have specific limitations in the context of NBS. A particular challenge is the differentiation of sickle cell anemia (SCA) from the benign condition, compound heterozygosity for HbS and gene-deletion hereditary persistence of fetal hemoglobin (HbS/HPFH). We describe a sequential cohort of 44 newborns identified over 4.5 years who had molecular genetic testing incorporated into NBS for presumed SCA (an “FS” pattern). The final diagnosis was something other than SCA in six newborns (12%). Three (7%) had HbS/HPFH. All had a final, correct diagnosis at the time of their first scheduled clinic visit in our center (median 8 weeks of age). None received initial counseling for an incorrect diagnosis. In summary, genetic testing as a component of NBS for SCD is necessary to provide correct genetic counseling and education for all newborns' families at their first visit to a sickle cell center. Genetic testing also permits the use of early, pre-symptomatic hydroxyurea therapy by preventing infants with HbS/HPFH from receiving unnecessary therapy. We argue that genetic testing should be incorporated into contemporary NBS for SCD.

Abstract Background Individuals with δβ-thalassemia/HPFH and β-thalassemia usually present with intermedia or thalassemia major. No large-scale survey on HPFH/δβ-thalassemia in southern China has been reported to date. The purpose of this study was to examine the molecular epidemiology and hematologic characteristics of these disorders in Guangzhou, the largest city in Southern China, to offer advice for thalassemia screening programs and genetic counseling. Methods A total of 125,661 couples participated in pregestational thalassemia screening. 654 subjects with fetal hemoglobin (HbF) level ≥ 5% were selected for further investigation. Gap-PCR combined with Multiplex ligation dependent probe amplification (MLPA) was used to screen for β-globin gene cluster deletions. Gene sequencing for the promoter region of HBG1 /HBG2 gene was performed for all those subjects. Results A total of 654 individuals had hemoglobin (HbF) levels≥5, and 0.12% of the couples were found to be heterozygous for HPFH/δβ-thalassemia, including Chinese Gγ (Aγδβ)0-thal, Southeast Asia HPFH (SEA-HPFH), Taiwanese deletion and Hb Lepore–Boston–Washington. The highest prevalence was observed in the Huadu district and the lowest in the Nansha district. Three cases were identified as carrying β-globin gene cluster deletions, which had not been previously reported. Two at-risk couples (0.0015%) were required to receive prenatal diagnosis. We also found 55cases of nondeletional-HPFH (nd-HPFH), including 54 with Italian nd-HPFH and one with the Aγ-197C-T heterozygous state. It is difficult to discriminate between Chinese Gγ (Aγδβ)0-thal and Italian nd-HPFH carriers using hemoglobin (Hb) analysis. Conclusions This study is the first to describe the familial prevalence of HPFH/δβ-thalassemia and the high-risk rate in Greater Guangzhou Area, and the findings will support the implementation of thalassemia screening for three common deletions by gap-PCR. We also presented a systematic description of genotype-phenotype relationships which will be useful for genetic counseling and prenatal diagnostic services for β-thalassemia intermedia.

Large deletions in the β-globin gene cluster lead to increased HbF levels by delaying the γ- to β-globin switch process. However, these deletions when inherited as a homozygous condition or when co-inherited with β-thalassemia result in variable clinical phenotypes. Individuals or families with a clinically presenting child, where the parents had HbF levels ≥ 10%, were further screened for the presence of large β-globin cluster deletions. Six deletions in the β-globin gene cluster were screened by GAP-PCR, and the uncharacterized deletions were further analyzed by gene dosage or by multiplex ligation-dependent probe amplification (MLPA). Among 192 individuals suspected for the inheritance of large deletions, 138 were heterozygous for large deletions, 45 were compound heterozygous of a large β-globin cluster deletion and β-thalassemia, and 9 were found to be homozygous for deletions. Among the heterozygotes, the Asian Indian inversion-deletion was found to be the most common deletion (39.9%), followed by the HPFH-3 deletion (30.0%). Other deletions 49.3 kb, δβ-thalassemia (21.2%), and 32.6 kb deletion (4.4%) were also found to be prevalent in our population. Patients compound heterozygous or homozygous for HPFH-3 and 32.6 kb deletions showed a milder clinical presentation, as compared with the patients compound heterozygous or homozygous for the Asian Indian inversion-deletion and 49.3 kb δβ-thalassemia. This comprehensive study highlights the mutation spectrum of large β-globin cluster deletions and the clinical heterogeneity in the patients homozygous or compound heterozygous with β-thalassemia, thus asserting the need for molecular characterization of these deletions. [ABSTRACT FROM AUTHOR]

Background: Individuals with δβ-thalassemia/HPFH and β-thalassemia usually present with intermedia or thalassemia major. No large-scale survey on HPFH/δβ-thalassemia in southern China has been reported to date. The purpose of this study was to examine the molecular epidemiology and hematologic characteristics of these disorders in Guangzhou, the largest city in Southern China, to offer advice for thalassemia screening programs and genetic counseling. Methods: A total of 125,661 couples participated in pregestational thalassemia screening. 654 subjects with fetal hemoglobin (HbF) level ≥ 5% were selected for further investigation. Gap-PCR combined with Multiplex ligation dependent probe amplification (MLPA) was used to screen for β-globin gene cluster deletions. Gene sequencing for the promoter region of HBG1 /HBG2 gene was performed for all those subjects. Results: A total of 654 individuals had hemoglobin (HbF) levels≥5, and 0.12% of the couples were found to be heterozygous for HPFH/δβ-thalassemia, including Chinese Gγ (Aγδβ)0-thal, Southeast Asia HPFH (SEA-HPFH), Taiwanese deletion and Hb Lepore–Boston–Washington. The highest prevalence was observed in the Huadu district and the lowest in the Nansha district. Three cases were identified as carrying β-globin gene cluster deletions, which had not been previously reported. Two at-risk couples (0.0015%) were required to receive prenatal diagnosis. We also found 55cases of nondeletional-HPFH (nd-HPFH), including 54 with Italian nd-HPFH and one with the Aγ-197C-T heterozygous state. It is difficult to discriminate between Chinese Gγ (Aγδβ)0-thal and Italian nd-HPFH carriers using hemoglobin (Hb) analysis. Conclusions: This study is the first to describe the familial prevalence of HPFH/δβ-thalassemia and the high-risk rate in Greater Guangzhou Area, and the findings will support the implementation of thalassemia screening for three common deletions by gap-PCR. We also presented a systematic description of genotype-phenotype relationships which will be useful for genetic counseling and prenatal diagnostic services for β-thalassemia intermedia. [ABSTRACT FROM AUTHOR]

Introduction: In sickle cell disease (SCD) patients, among the predictors of survival, HbF levels play a significant role in lowering the morbidity and mortality. Coinheritance of δβ thalassemia and hereditary persistence of fetal hemoglobin (HPFH) may contribute to variable HbF levels in SCD patients, thus influencing their clinicopathological profile. Such cases are sparsely documented in the literature and thus, we screened the presence of δβ thalassemia and HPFH in 126 cases of SCD with high HbF. Material and methods: A total 126 SCD individuals with raised HbF levels were the study subject. Capillary zone electrophoresis (CZE) was done for the quantitative assessment of hemoglobin variants. HbSC, HbSD, HbAS and HbSE cases were excluded. Asian Indian Gγ(Aγδβ)0-thal, δβ0-thal (Sicilian, 13.4 kb), (Chinese, 100 kb), HPFH-1 (Black, 106 kb), HPFH-2 (Ghanaian, 105 kb), HPFH-3 (Indian, 48.5 kb) were done by GAP-PCR. Results: Out of 126, 78 cases (62%) were homozygous for SCD. The remaining 48 cases suspected to be heterozygous were furthered screened and 6/48 cases (12.5%) were found to be compound heterozygous. Out of these 6 cases,4(66.66%) had HbS/ δβ- Gγ(Aγδβ)0 and 2(33%) had HbS/HPFH compound heterozygous condition. None of the patients had δβ0-thal (Sicilian, 13.4 kb), (Chinese, 100 kb), HPFH-1 (Black, 106 kb), HPFH-2 (Ghanaian, 105 kb). Conclusion: This study highlights the importance of understanding the complex patho-physiology of compound heterozygous cases of HbS/HPFH and HbS/δβ thalassemia, as these infrequent conditions lead to change in phenotype and clinical severity of the disease. Insight into more such cases will open the window to better analyze the disease pathogenesis in these rare compound heterozygous conditions, as this will be beneficial to formulate proper management protocol in these patients. [ABSTRACT FROM AUTHOR]

Background and objectives: Coinheritance of δβ thalassemia and HPFH with inherited factors is sparsely documented and may affect treatment modalities. So, we screened the presence of α deletion and β mutations in δβ thalassemia and HPFH disorders in 52 cases with high Hb F concentration. Material and methods: Fifty-two individuals with raised HbF levels were study subjects. CZE was done for quantitative assessment of hemoglobin variants. Asian Indian inversion deletion break point type A, B and HPFH-3 were done by GAP-PCR. Results: 18/52 cases of δβ Gγ (Aγδβ)0 thalassemia and 28/52 cases of HPFH-3 deletion were characterized. 6/52 patients with raised HbF levels were negative for δβ Gγ (Aγδβ)0 and HPFH-3 deletion. 9/18 (50%) were heterozygous for Gγ(Aγδβ)0 break point type A, 6/18 (33%) were heterozygous for break point type B and 3/18 (17%) were homozygous. Of the nine patients heterozygous for Gγ(Aγδβ)0 break point type A, three (33%) patients were double heterozygous with alpha 3.7 kb deletion and two (22%) patients showed compound heterozygosity with IVS 1-5(G-C) mutation. 4/9 (45%) patients were Gγ(Aγδβ)0 heterozygous. Discussion and conclusion: We found 5/18(27.β) δβ-thalassemia cases with co-inherited alpha 3.7 deletion and 3/18 (16β) cases with IVS 1-5(G-C) mutation. Patients showed features of thalassemia intermedia phenotype among which those with co-inherited IVS 1-5(G-C) mutation showed severe phenotype as compared to those with co-inherited alpha 3.7 deletion. So, we highlight importance of genotyping of patients with δβ thalassemia or HPFH and coinheritance with inherited factors which plays crucial role in clinicopathological profile and setting up prenatal diagnostic protocol. [ABSTRACT FROM AUTHOR]

• This study describes the molecular basis and genetic interactions of deletional high Hb F determinants in a large cohort of Thai individuals. • Eight different DNA deletions including a novel one causing HPFH and δβ0-thalassemia were described. • Genotype-phenotype interactions were reported. • Multiplex gap-PCR assays were described to detect these high Hb F determinants in routine setting. • Updated data on prevalence, molecular heterogeneity, and various genetic interactions were reported. High hemoglobin F determinants can be classified into hereditary persistence of fetal hemoglobin (HPFH) and δβ-thalassemia with different phenotype. We report the molecular basis and hematological features in a large cohort of deletional high Hb F determinants in Thailand. Subjects (n = 28,177) encountered during 2015–2022 were reviewed, and those with phenotypically suspected of having high Hb F determinants were selected. Combined PCR, multiplex ligation-dependent probe amplification, next-generation sequencing, and DNA sequencing were used to identify the mutations. Among 28,177 subjects investigated, 300 (1.06 %) were found to carry deletional high Hb F determinants in a total of 302 alleles, including heterozygote, compound heterozygote with β-hemoglobinopathies, and homozygote. DNA analysis identified eight different DNA deletions, including δβ0-thalassemia (12.6 kb deletion) (73.8 %), HPFH-6 (14.9 %), Indian deletion-inversion Aγδβ0-thalassemia (3.6 %), Thai deletion-inversion-insertion Aγδβ0-thalassemia (3.0 %), SEA-HPFH (3.0 %), Chinese Aγδβ0-thalassemia (1.0 %), Thai δβ0-thalassemia (11.3 kb deletion) (0.3 %), and a novel δβ0-thalassemia (137.1 kb deletion) (0.3 %). In addition, three novel genetic interactions, including Chinese Aγδβ0-thalassemia/Hb E, δβ0-thalassemia/Indian deletion-inversion Aγδβ0-thalassemia, and homozygous δβ0-thalassemia were found. Hematological features and Hb analysis results of 20 different genotypes were recorded. Multiplex gap-PCR assays for detection of these genetic determinants were described. Deletional high Hb F determinants are common and heterogeneous in Thailand. Data on the prevalence, molecular spectrum, phenotypic expression, and complex interactions of these genetic determinants should prove useful in the study and a prevention and control program of hemoglobinopathies in the region. [ABSTRACT FROM AUTHOR]

Hereditary persistence of foetal haemoglobin (HPFH) is a rare inherited haemoglobin disorders in India. We encountered five cases of HPFH-3 in heterozygous condition in a single family of western Odisha, India. All the cases had raised % HbF (26.1±3.23%) with pancellular distribution of HbF in erythrocytes. There were no abnormalities found in the red cell indices. All the cases were asymptomatic till date with normal growth and development. Molecular confirmation of this haemoglobin disorders is important for control and prevention of haemoglobinopathies in this region.

Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G) mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G) mutation. Furthermore, this is the first time that the -195 (C->G) mutation of the Agamma-globin gene has been evaluated by in vitro gene expression.

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Three major loci have been associated with HbF levels, including − 158C/T (XmnI) at HBG2 promoter region, and several polymorphisms at BCL11A intron-2 and HBS1L-MYB (HMIP) intergenic region. Mutations in the KLF1 gene were recently associated with increased HbF levels. This study aims to evaluate whether genetic variability at these loci influences HbF levels in β-thalassemia carriers and in normal individuals of Portuguese origin. Sixty five β-thalassemia carriers, HbF levels ranging from 0.2% to 9.5%, and 60 individuals with normal hematological parameters, HbF levels ranging from 0.2% to 7.4%, were selected for this study. In β-thal carriers linear regression models revealed a strong statistical significant association for HBG2 (XmnI) rs7482144 (β = 0.455; P = 5.858 × 10 − 7 ), and nominal significance for BCL11A rs766432 (β = 0.215; P = 0.029) and HMIP rs9399137 (β = 0.209; P = 0.011). In normal individuals, a case (HbF > 2%; n = 15) vs. control (HbF < 1.7%; n = 45) model, showed nominal significant associations for BCL11A SNPs rs11886868 (OR = 4; P = 0.001), rs766432 (OR = 3.7; P = 0.002) and rs7606173 (OR = 0.36; P = 0.032). KLF1 rs3817621 was not found associated with HbF levels. Our results suggest that in Portuguese β-thal carriers the HBG2 XmnI polymorphism is strongly associated with HbF levels. In normal individuals, BCL11A polymorphisms, but not HMIP or HBG2 (XmnI) loci , are nominally associated with HbF expression. [ABSTRACT FROM AUTHOR]

Hemoglobinopathies belong to the most common monogenic hereditary diseases worldwide. A particularly high prevalence is seen in the Mediterranean countries, in parts of Asia, the Middle East and West Africa. Nevertheless, due to migration hemoglobinopathies play an increasingly important role in Germany as well. Basic testing consists of blood count and hemoglobin differentiation. In addition, an iron deficiency should be excluded if necessary. Molecular genetic testing is used for the verification of hematologic findings and serves in the assessment of risk for a severe form of a hemoglobinopathy in offspring. In order to ensure efficient diagnostics, family history and previous findings of the patient should be communicated to the laboratory. This is especially crucial in the case of prenatal diagnostics. [ABSTRACT FROM AUTHOR]

Abstract: Background: Deletions represent about 5% of the mutations in the β-globin gene cluster. We report here the screening for such deletions in the two French urban areas of Paris and Lyon between 2003 and 2010. Methods: Semi-quantitative PCR methods were used for the first screening of deletions. Thereafter, a specific gap-PCR, eventually followed by DNA sequencing, was used for precise identification. Results: 285 patients bore a deletion or recombination event in the β-globin gene cluster. Hbs Lepore or anti-Lepore were detected in 99 patients. Among the remaining 186 patients, 132 bore a deletion that could be fully identified. The most prevalent deletions were the Ghanaian HPFH-2 (n=46), the Sicilian (δβ)0-thal (n=22) and the Spanish (δβ)0-thal (n=12). The other characterized deletions were the: HPFH-3, HPFH-1, Filipino, Senegalese, Corfu, Kabilian, −1.39kb, Indian −619bp and −468bp. Interestingly, three new deletions were fully characterized: a −7719bp deletion, a −27,825bp deletion with a 25bp insertion and a −125bp deletion. Conclusions: The present study emphasizes the importance to detect deletions in the β-globin gene cluster, particularly for at risk couples. The new −27,825bp deletion illustrates the complexity to understand the transcriptional regulation of fetal to adult hemoglobin switch. [Copyright &y& Elsevier]

ABSTRACT KLF1 encodes an erythroid transcription factor, whose essential function in erythropoiesis has been demonstrated by extensive studies in mouse models. The first reported mutations in human KLF1 were found in individuals with a rare and asymptomatic blood type called In( Lu). Here, we show that KLF1 haploinsufficiency is responsible for the In( Lu) blood type, after redefining this peculiar blood type using flow cytometry to quantify the levels of BCAM and CD44 on red blood cells. We found 10 (seven novel) heterozygous KLF1 mutations responsible for the In( Lu) blood type. Although most were obligate loss-of-function mutations due to the truncation of the DNA-binding domain of KLF1, three were missense mutations that were located in its DNA-binding domain and impaired the transactivation capacity of KLF1 in vitro. We further showed that the levels of the hemoglobin variants Hb F and Hb A2 were increased in the In( Lu) blood type, albeit differently. The levels of the membrane glycoproteins BCAM and CD44 were also differently reduced on In( Lu) red blood cells. This biochemical and genetic analysis of the In( Lu) blood type tackles the phenotypic outcome of haploinsufficiency for a transcription factor. [ABSTRACT FROM AUTHOR]

Hereditary persistence of fetal hemoglobin (HPFH) is a group of genetically heterogeneous conditions characterized by continued expression of fetal hemoglobin (HbF) in adulthood. HPFH may be due not only to point mutations or large deletions in different regions of the cluster β globin, but also to variations in several polymorphic sequences in this cluster. The objective of this work was to evaluate effects of polymorphic markers within cluster β globin on HbF expression. For the purpose, we have explored in this first study of Tunisian HPFH four polymorphic regions of β globin cluster in 68 healthy adults (34 subjects with high levels of HbF and 34 with normal HbF levels). Our results showed that the increase of HbF levels is associated with the −158 Gγ C → T polymorphism, the TGCGCACG, TC TGAG TGCG and TGCG configurations in the second intron of Gγ gene and the −540 β (AT)T and (AT)T repeated sequences. Among the 34 subjects with raised levels of HbF, approximately 97% carried one or more of these six markers. This study suggests that there is a significant association between certain polymorphic configurations of the β globin cluster and the increase of HbF levels in healthy individuals. [ABSTRACT FROM AUTHOR]

Impaired switching from fetal haemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal haemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate β-thalassaemia and sickle cell anaemia. Fetal haemoglobin levels are regulated by complex mechanisms involving factors linked or not to the β-globin gene ( HBB) locus. To search for factors putatively involved in the expression of the γ-globin genes ( HBG1, HBG2), we examined the reticulocyte transcriptome of three siblings who had different HbF levels and different degrees of β-thalassaemia severity although they had the same ΗBA- and ΗΒB cluster genotypes. By mRNA differential display we isolated the cDNA coding for the cold shock domain protein A (CSDA), also known as dbpA, previously reported to interact in vitro with the HBG2 promoter. Expression studies performed in K562 and in primary erythroid cells showed an inverse relationship between HBG and CSDA expression levels. Functional studies performed by Chromatin Immunoprecipitation and reporter gene assays in K562 cells demonstrated that CSDA is able to bind the HBG2 promoter and suppress its expression. Therefore, our study demonstrated that CSDA is a trans-acting repressor factor of HBG expression and modulates the HPFH phenotype. [ABSTRACT FROM AUTHOR]

β thalassemia major or Cooley's Anemia (CA) has been difficult to model in mice due to their lack of a fetal hemoglobin gene equivalent. This summary describes novel preclinical humanized mouse models of CA that survive on human fetal hemoglobin at birth and are blood-transfusion dependent for life upon completion of their human fetal-to-adult hemoglobin switch after birth. These CA models are the first to recapitulate the temporal onset of the disease in human patients. These novel humanized CA disease models are useful for the study of the regulation of globin gene expression, synthesis, and switching; examining the onset of disease pathology; development of transfusion and iron chelation therapies; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia. [ABSTRACT FROM AUTHOR]

Abstract: Despite the fact that mutations in the human β-globin gene cluster are essentially point mutations, a significant number of large deletions have also been described. We present here four new large deletions in the β-globin gene cluster that have been identified on patients displaying an atypical hemoglobin phenotype (high HbF) at routine analysis. The first deletion, which spreads over 2.0 kb, removes the entire β-globin gene, including its promoter, and is associated with a typical β-thal minor phenotype. The three other deletions are larger (19.7 to 23.9 kb) and remove both the δ and β-globin genes. Phenotypically, they look like an HPFH-deletion as they are associated with normal hematological parameters. The precise localization of their 5′ and 3′ breakpoints gives new insights about the differences between HPFH and (δβ)0-thalassemia at the molecular level. The importance of detection of these deletions in prenatal diagnosis and newborn screening of hemoglobinopathies is also discussed. [Copyright &y& Elsevier]

The human β globin locus consists of an upstream LCR and functional genes arranged sequentially in the order of their expression during development: 5′- HBE1, HBG2, HBG1, HBD, HBB-3′. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Here we show that the transcription factor Ikaros plays a major role in the formation of the β-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation, there is concomitant marked down-regulation of HBB, and up-regulation of HBG expression. We show for the first time Ikaros and its family member Eos, bind to critical cis elements implicated in haemoglobin switching and deletional hereditary persistence of fetal haemoglobin (HPFH). Chromatin conformation capture (3C) data demonstrated that Ikaros facilitates long-distance DNA looping between the LCR and a region upstream of HBD. This study provides new insights into the mechanism of stage-specific assembly of the β-globin ACH, and HPFH. [ABSTRACT FROM AUTHOR]

Background: Several previous studies reported that the Venezuelan Warao Indians presented unusual genetic characteristics. Aim: The present study checked previous reports of a high frequency of hereditary persistence of fetal hemoglobin (HPFH) and examined other hematological traits. Subjects and methods: Standard hematology, electrophoresis on cellulose acetate, fetal hemoglobin alkali denaturation, γ-globin chain, DNA amplification and sequencing, and denaturing gradient gel electrophoresis determinations were performed in 269 individuals living in two localities of the Orinoco River Delta. Results: Two βs genes, in apparently non-related individuals, were found. HPFH, detected in this same population of Warao Indians 25 years ago, was present in heterozygous form in five individuals from a large kindred, with hemoglobin F levels ranging from 3.7% to 8%, and with a pancellular distribution. The HPFH mutation was of the deletional type. β-globin gene haplotypes were determined by direct counting (through family studies) in 150 chromosomes; 26% of the 150 examined cluster presented haplotype 2, 22% haplotype 6, and 13% a new, Warao haplotype. Haplotype 3, of probable African origin, was also found with a frequency of 5%. Conclusions: The presence of the HPFH mutation was confirmed, and the new β-globin gene haplotype together with the presence of other rare variants indicates that the Warao are very distinctive in relation to other Native Americans. Evidence was also found of a slight admixture from Africa-derived subjects (Layrisse et al. 1988). [ABSTRACT FROM AUTHOR]

Researchers hope to increase γ-globin expression by controlling potential trans-acting factors that specifically activate the γ-globin gene in fetuses or silence this gene in adults to potentially treat sickle cell disease and β-thalassemias. To characterize genes encoding such factors, we analyzed the differential expression of mRNAs in erythroid induction cultures of CD34+ cells derived from normal adult bone marrow, umbilical cord blood, and bone marrow from a patient with heterocellular hereditary persistence of fetal hemoglobin. Using differential-display – reverse-transcription PCR analysis, we identified a number of genes with differential expression in the above-mentioned cells. The differential expression of some genes was also confirmed by quantitative real-time PCR. Our data provide important clues for identifying and validating trans-activators that activate the γ-globin gene in fetuses, and trans-acting factors involved in silencing the γ-globin gene in adults. [ABSTRACT FROM AUTHOR]

Abstract: The genetic mechanisms underlying the continued expression of the γ-globin genes during the adult stage in deletional hereditary persistence of fetal hemoglobin (HPFH) and δβ-thalassemias are not completely understood. Herein, we investigated the possible involvement of transcription factors, using the suppression subtractive hybridization (SSH) method as an initial screen to identify differentially expressed transcripts in reticulocytes from a normal and a HPFH-2 subject. Some of the detectable transcripts may participate in globin gene regulation. Quantitative real-time PCR (qRT-PCR) experiments confirmed the downregulation of ZHX2, a transcriptional repressor, in two HPFH-2 subjects and in a carrier of the Sicilian δβ-thalassemia trait. The chromatin remodeling factors ARID1B and TSPYL1 had a very similar pattern of expression with an incremental increase in HPFH and decreased expression in δβ-thalassemia. These differences suggest a mechanism to explain the heterocellular and pancellular distribution of fetal hemoglobin in δβ-thalassemia and deletional HPFH, respectively. Interestingly, α-globin mRNA levels were decreased, similar to β-globin in all reticulocyte samples analyzed. [Copyright &y& Elsevier]

Hereditary persistence of fetal hemoglobin (HPFH) is associated with a high level of hemoglobin F (HbF) synthesis in adult heterozygotes. In this study, 2 of 6 unrelated HPFH Thai families were found to be Southeast Asian-type HPFH (SEA-HPFH) by analyses of the hematologic data and Southern blot hybridization with polymerase chain reaction-amplified DNA probes. DNA mapping with a probe for a delta-globin fragment showed a 27-kb deletion of DNA that included the beta-globin gene and the 3' deoxyribonuclease I hypersensitive site 1 (3'HS1) sequence downstream. Deletion of the insulator, 3'HS1, and the juxta-position of the HPFH-3 core enhancer downstream to the 3' breakpoint have been postulated to be the cause of high HbF production in these individuals. To test this hypothesis, we transfected K562 cells with 4 different bacterial artificial chromosome constructs containing the enhanced green fluorescent protein (EGFP) gene at the position of the Agamma-globin gene (pEBAC/148beta:EGFP). Flow cytometry was used to compare EGFP expression from the pEBAC/148beta:EGFP construct with the HPFH-3 core enhancer immediately 5' to the SEA-HPFH breakpoint (pEnH), from the pEBAC/148beta:EGFP construct with 8 kb of the breakpoint sequence and the HPFH-3 core enhancer (pSEA-HPFH), and from the construct with 3'HS1 followed by the pSEA-HPFH sequence (pSEA-HPFH_3pHS1). The results show that high HbF production in SEA-HPFH occurs from a deletion of the 3'HS1 sequence and the juxtaposition of the HPFH-3 enhancer downstream to the delta-globin gene. [ABSTRACT FROM AUTHOR]

We recently described an erythroid e-globin gene repressor activity, which we named DRED (direct repeat erythroid-definitive). We show that DRED binds with high affinity to DR1 sites in the human embryonic (e-) and fetal (?-) globin gene promoters, b [ABSTRACT FROM AUTHOR]

The analysis of a number of cases of β-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the β-globin locus has led to the identification of several DNA elements that have been implicated in the switch from human fetal γ- to adult β-globin gene expression. We have tested this hypothesis for an element that covers the minimal distance between the thalassemia and HPFH deletions and is thought to be responsible for the difference between a deletion HPFH and δβ-thalassemia, located 5′ of the δ-globin gene. This element has been deleted from a yeast artificial chromosome (YAC) containing the complete human β-globin locus. Analysis of this modified YAC in transgenic mice shows that early embryonic expression is unaffected, but in the fetal liver it is subject to position effects. In addition, the efficiency of transcription of the γ-globin gene is decreased, but the developmental silencing of the -globin genes is unaffected by the deletion. These results show that the deleted element is involved in the activation of the β-globin gene perhaps through the loss of a structural function required for gene activation by long-range interactions. [ABSTRACT FROM AUTHOR]

We have examined factor binding to the distal human γ-globin CCAAT site and three naturally occurring hereditary persistence of fetal haemoglobin (HPFH) mutations of this site. Factor binding was examined using nuclear extracts from the erythroleukaemic cell lines K562 and MEL, and from A4 cells, a non-transformed mouse bone marrow stem cell line, using the electrophoretic mobility shift assay. Under standard binding conditions, in addition to the previously reported binding by a CCAAT factor (CP1) and GATA-1, the wild-type (wt) sequence bound high mobility factors which appeared to be GATA-2 isoforms. However, when the non-specific competitor conditions were varied, the binding profile with K562, but not MEL nuclear extract, was substantially altered. CP1 and GATA-1 were absent, and two new factors were detected, one of which bound preferentially to the Greek and Japanese non-deletion HPFH mutants. However, binding by the GATA-2 isoforms to the wt sequence was maintained with both cell types, as it was using the A4 cell line. With modified binding conditions, in A4 cells the two non-deletion and the Black deletion HPFH mutants each had a different protein binding profile which was lost on erythroid induction of the cells. We discuss the possiblity that the GATA-2 isoforms bound to the wt sequence may function to suppress wt γ gene expression in the bone marrow. Additionally, those factors which bind preferentially either to the deletion or non-deletion HPFH mutants may play positive roles in establishing an active chromatin structure. [ABSTRACT FROM AUTHOR]

We have analyzed the levels and composition of the fetal hemoglobin (Hb F) in several members of a Czechoslovakian family with a heterozygosity for a newly discovered β∘-thalassemia (codons 38/39; -C), or for a newly detected nondeletional hereditary persistence of fetal hemoglobin (a form of Swiss-HPFH with an A→C mutation at nucleotide −100 5′ to the Cap site of Gγ), or with a compound heterozygosity for these two conditions. The Hb F level in the β∘-thalassemia heterozygotes averaged ∼ 0.3% with low Gγ values (∼ 28%) and relatively high AγT values (∼ 50%), that in the two Swiss-HPFH heterozygotes averaged 0.8% with ∼95% Gγ, while that of the compound heterozygote was 3.1% with ∼ 95% Gγ. The low Hb F levels were determined with a recently published cation exchange high-performance liquid chromatography (HPLC) procedure that is accurate at the 0.1%-0.2% Hb F level [3]. This method, together with a reversed-phase HPLC procedure, made it possible to detect this unusual type of nondeletional Gγ-HPFH and provided the data indicating that the increased Hb F in the compound heterozygote was derived mainly from the chromosome with the HPFH determinant. [ABSTRACT FROM AUTHOR]

Objective: to determine the beneficial role of Fetal Hemoglobin (FHb) and alpha-thal on fetal and maternal morbidity during pregnancy in sickle cell patients., Study Site: the study was conducted at the sickle cell center of Kinshasa between 2008 and 2018., Setting and Study Population: this is a documentary and analytical study that included 980 deliveries of homozygous sickle cell patients., Methods: the diagnosis of SCD and the quantification of FHb were performed with the capillary electrophoresis technique. The molecular test confirmed the diagnosis of SCD. The diagnosis of alpha-thal was made with the multiplex ligation-dependent probe amplification (MLPA) technique. Sickle cell pregnancies were followed according to the protocol of care in force in the University of Kinshasa Hospital service. The variables of interest were: hematological variables, sickle cell crises during pregnancy, maternal and fetal complications., Statistics: statistical analyses were performed with SPSS 20.0 software. Means and standard deviations were compared with the Student's t and ANOVA tests. The value of p <0.05 was considered the significance level., Results: the Hb-SS / alpha-thal and HbSS / HPFH genotypes were observed in 101 and 121 women, respectively. Otherwise, 758 women had HbSS genotype. The morbidity related to sickle cell complications in the mother and fetus were less frequent in the Hb-SS / alpha-thal and HbSS / HPFH groups than in HB-SS group. The differences were statistically significant., Conclusion: this study showed a significant protective effect of alpha-thal and HPFH during pregnancy in sickle-cell pregnant women., Competing Interests: Competing interests: The authors have declared that no competing interests exist.

Hereditary persistence of foetal haemoglobin (HPFH) is a rare inherited haemoglobin disorders in India. We encountered five cases of HPFH-3 in heterozygous condition in a single family of western Odisha, India. All the cases had raised % HbF (26.1±3.23%) with pancellular distribution of HbF in erythrocytes. There were no abnormalities found in the red cell indices. All the cases were asymptomatic till date with normal growth and development. Molecular confirmation of this haemoglobin disorders is important for control and prevention of haemoglobinopathies in this region. [ABSTRACT FROM AUTHOR]

Elements within the γ-hemoglobin promoters ( HBG1 and HBG2 ) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the HBG1 promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous HBG promoters to de-repress fetal hemoglobin. Transfection of human CD34 + cells with TALEN mRNA resulted in indel generation in HBG1 (43%) and HBG2 (74%) including the 13-bp HPFH deletion (∼6%). Erythroid differentiation of edited cells revealed a 4.6-fold increase in γ-hemoglobin expression as detected by HPLC. Assessment of TALEN-edited CD34 + cells in vivo in a humanized mouse model demonstrated sustained presence of indels in hematopoietic cells up to 24 weeks. Indel rates remained unchanged following secondary transplantation consistent with editing of long-term repopulating stem cells (LT-HSCs). Human γ-hemoglobin expressing F cells were detected by flow cytometry approximately 50% more frequently in edited animals compared to mock. Together, these findings demonstrate that TALEN-mediated indel generation in the γ-hemoglobin promoter leads to high levels of fetal hemoglobin expression in vitro and in vivo , suggesting that this approach can provide therapeutic benefit in patients with SCD or β-thalassemia.

Fetal hemoglobin is a major genetic modifier of the phenotypic heterogeneity in patients with sickle cell disease and certain β-thalassemias. Normal levels of fetal hemoglobin postnatally are approximately 1% of total hemoglobin. Patients who have hereditary persistence of fetal hemoglobin, characterized by elevated synthesis of γ-globin in adulthood, show reduced disease pathophysiology. Hereditary persistence of fetal hemoglobin is caused by β-globin locus deletions (deletional hereditary persistence of fetal hemoglobin) or γ-globin gene promoter point mutations (non-deletional hereditary persistence of fetal hemoglobin). Current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin in adult life. To better understand these pathways, we generated new β-globin locus yeast artificial chromosome transgenic mice bearing the (A)γ-globin -175 T > C or -195 C > G hereditary persistence of fetal hemoglobin mutations to model naturally occurring hereditary persistence of fetal hemoglobin. Adult -175 and -195 mutant β-YAC mice displayed a hereditary persistence of fetal hemoglobin phenotype, as measured at the mRNA and protein levels. The molecular basis for these phenotypes was examined by chromatin immunoprecipitation of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2, and LDB1. In -175 HPFH versus wild-type samples, the occupancy of LMO2, TAL1 and LDB1 proteins was enriched in HPFH mice (5.8-fold, 5.2-fold and 2.7-fold, respectively), a result that concurs with a recent study in cell lines showing that these proteins form a complex with GATA-1 to mediate long-range interactions between the locus control region and the (A)γ-globin gene. Both hereditary persistence of fetal hemoglobin mutations result in a gain of (A)γ-globin activation, in contrast to other hereditary persistence of fetal hemoglobin mutations that result in a loss of repression. The mice provide additional tools to study γ-globin gene expression and may reveal new targets for selectively activating fetal hemoglobin., (© 2016 by the Society for Experimental Biology and Medicine.)

INTRODUCTION: Hereditary persistence of fetal hemoglobin (HPFH) is characterized by persistence of elevated levels of hemoglobin F in adult life in the absence of any anemia. Only a few cases are reported. During a routine antenatal screening for pregnancy this rare disorder was detected. CASE REPORT: A 25-year old female on a routine antenatal checkup was found to have mild jaundice and splenomegaly. Hematological investigations showed Hb 12 g/dL, MCV 75.3 fL, Retic 9.5%. Peripheral smear showed normocytic normochromic blood picture with polychromasia. Patient had a past history of recurrent attacks of jaundice. Similar complaints were present on the paternal side. HPLC showed HbF 100%. In view of high levels of HbF in the mother, son was investigated at 2 years of age. He was asymptomatic and hematological findings were Hb 13.2 g/dL, MCV 63 fL with microcytic hypochromic blood picture. HPLC showed HbF 23.6% and HbA2 2.2%. As the patient was clinically silent a final diagnosis of homozygous HPFH and heterozygous HPFH on the child was made. CONCLUSIONS: Hereditary persistence of fetal hemoglobin (HPFH) is a rare disorder with no apparent clinical abnormality. Therefore a careful screening by HPLC and molecular techniques are required to detect and confirm these disorders. [ABSTRACT FROM AUTHOR]