Activation of γ-globin expression by LncRNA-mediated ERF promoter hypermethylation in ß-thalassemia.

The mechanism that drives the switch from fetal to adult hemoglobin (Hb) provides a therapeutic target for ß-thalassemia. We have previously identified that hypermethylation of transcription factor ERF promoter reactivated γ-globin expression. To uncover the mechanism underlying the hypermethylation of ERF promoter, we performed RNA sequencing in ß0/ß0-thalassemia patients and identified an upregulated long noncoding RNA (RP11-196G18.23) associated with HbF production. RP11-196G18.23 bound to the ERF promoter and recruited DNA methyltransferase 3A to promote DNA hypermethylation-mediated ERF downregulation, thereby ameliorating ERF-induced γ-globin inactivation. The identification of RP11-196G18.23 provides an epigenetic mechanism for the reactivation of fetal γ-globin expression for ß-hemoglobinopathies.

Reference intervals for erythrocyte parameters and hemoglobin electrophoresis parameters for young children in Guangxi.

BACKGROUND: Erythrocyte parameter analysis is the important means for diagnosis and treatment of hematological diseases, which are essential for screening of thalassemia in southern China combined with hemoglobin electrophoresis analysis. But little is known regarding the reference intervals (RIs) in healthy pediatrics in these two areas. METHODS: 95% RIs of erythrocyte parameters were calculated from 853 healthy preschoolers, aged from 1 days to <6 years, according to the C28-A3C guidelines of the Institute of Clinical and Laboratory Standards. To express the magnitude of sex and age variation, standard deviation ratio (SDR) was calculated using ANOVA. Concurrently, we selected 3814 thalassemia carriers as carriers group and drew receiver operating characteristic (ROC) curves to establish the optimal cut-off values of hemoglobin electrophoresis parameters, which were used as the upper/lower limits of RIs to efficiently screen thalassemia. RESULTS: All parameters except red blood cell (RBC) required age partitioning, confirmed by SDRage above .4. There was no need for sex partitioning on all parameters, confirmed by SDRsex below .4. The optimal cut-off value of Hemoglobin A2 (Hb A2) in the four subgroups was <7.8% (Hb A), 2.3%-3.2%, 2.5%-3.6% and 2.6%-3.6%, respectively. CONCLUSION: In this study, the establishment of RIs improved the diagnostic efficiency of hematological disease (especially thalassaemia) for children in Guangxi. It provides reliable hematological references for the identification and diagnosis, treatment monitoring, and health screening of children's clinical diseases.

Molecular Characterization of α- and ß-Thalassaemia Among Children From 1 to 10 Years of Age in Guangxi, A Multi-Ethnic Region in Southern China.

Background: Thalassemia is one of the most common genetic diseases in southern China. Howerver, population in different regions or different population has their own spectrums of thalassemia. To investigate the prevalence and spectrum features of thalassemia among children in Guangxi. Hematology and genetic analysis were performed on 71,459 children aged 1-10 years in various regions of Guangxi. Results: A total of 11,821 children were diagnoses with thalassemia including 7,615 (10.66%) subjects of α-thalassemia, 3,507 (4.90%) subjects of ß-thalassemia, and 699 (0.98%) cases with both α- and ß-thalassemia. Nine α-thalassemia mutations and 30 genotypes were identified among the α-thalassemia children. The - -SEA and - -SEA/αα were the most frequent mutation and genotype, respectively. One α-thalassemia fusion gene and a rare 2.4 kb deletion both causing α+-thalassemia were identified, respectively. Thirteen ß-thalassemia mutations and 31 genotypes were characterized among the ß-thalassemia children, with the most common mutation CD41-42 (-CTTT) accounting for 46.05% of the ß-mutations. Two rare mutations IVS-II-5 (G>C), and IVS-I-2 (T>C) were firstly identified. Furthermore, 92 genotypes were identified among 699 children with both α- and ß-thalassemia. Conclusions: Our findings highlight the great heterogeneity and the extensive spectrum of thalassemia among children in Guangxi, which provide an available reference for prevention of thalassemia in this area.

Epigenetic inactivation of ERF reactivates γ-globin expression in ß-thalassemia.

The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of ß-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA+ erythroid cells from ß-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of ß-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in ß-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in ß-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34+ erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for ß-hemoglobinopathies.

DNA methylation patterns of ß-globin cluster in ß-thalassemia patients.

BACKGROUND: Reactivation of fetal hemoglobin (HbF, α2γ2) holds a therapeutic target for ß-thalassemia and sickle cell disease. Although many HbF regulators have been identified, the methylation patterns in ß-globin cluster driving the fetal-to-adult hemoglobin switch remains to be determined. RESULTS: Here, we evaluated DNA methylation patterns of the ß-globin cluster from peripheral bloods of 105 ß0/ß0 thalassemia patients and 44 normal controls. We also recruited 15 bone marrows and 4 cord blood samples for further evaluation. We identified that the CpG sites in the locus control region (LCR) DNase I hypersensitive site 4 and 3 (HS4-3) regions, and γ- and ß-globin promoters displayed hypomethylation in ß0/ß0-thalassemia patients, especially for the patients with high HbF level, as compared with normal controls. Furthermore, hypomethylations in most of CpG sites of the HS4-3 core regions were also observed in bone marrows (BM) of ß0/ß0-patients compared with normal controls; and methylation level of γ-globin promoter -50 and + 17 CpG sites showed lower methylation level in patients with high HbF level compared with those with low HbF level and a negative correlation with HbF level among ß0-thalassemia patients. Finally, γ-globin promoter + 17 and + 50 CpG sites also displayed significant hypomethylation in cord blood (CB) tissues compared with BM tissues from normal controls. CONCLUSIONS: Our findings revealed methylation patterns in ß-globin cluster associated with ß0 thalassemia disease and γ-globin expression, contributed to understand the epigenetic modification in ß0 thalassemia patients and provided candidate targets for the therapies of ß-hemoglobinopathies.

A novel splicing mutation in F8 causes various aberrant transcripts in a hemophilia A patient and identifies a new transcript from healthy individuals.

: Hemophilia A is an X-linked hemorrhagic disorder caused by deficiency or dysfunction of the coagulation factor VIII (FVIII), and a great variety of mutations in the factor VIII gene (F8) are identified. We aimed to identify the genetic defects of the F8 gene in a Chinese patient with moderate hemophilia A. We have identified a novel intronic variant in the hemophilia A patient by DNA sequence analysis, cDNA sequencing, and TA clone sequencing. An intronic variant, c.5816-1G>A, was identified and the cDNA sequencing confirmed the pathogenicity of the transition. TA clone sequencing showed that the splicing mutation produced two aberrant premRNA skipping exons (18 and exon 18 + 19, respectively). These aberrant mRNA forms maintain the reading frame and are predicted to code for deleted FVIII isoforms and the shorter abnormal transcript accounted for one-eighth of the total mRNA. There was a new unreported transcript with E22 spliced out in healthy individuals and our patient, whose specific functions need to be determined in further studies. Our study widens the mutation spectrum of the F8 gene. In addition, the study findings could provide the opportunity to reveal alternative splicing patterns.

A novel missense mutation in F9 gene causes hemophilia B in a family with clinical variability.

: Hemophilia B is an X-linked recessive bleeding disorder caused by diverse mutations throughout the F9 gene. The same F9 mutation may result in different degrees of clotting factor deficiency. The aim of this study was to investigate the pathogenesis of two hemophilia B patients with different severity in a family. A family with two hemophilia B patients was recruited in this study. Coagulation assays, activities of FVIII (FVIII:C) and FIX (FIX:C) were evaluated. All of the exons and intron exon boundaries of the F9 gene were amplified by PCR and analyzed by direct sequencing. The proband, 12-year-old boy with moderate bleeding history, had manifest prolonged activated partial thromboplastin time (98.1 s) and markedly decreased FIX activity (1%). His maternal uncle presented slightly prolonged activated partial thromboplastin time (48.2 s) and mildly decreased FIX activity (15.2%). Molecular genetic analysis of F9 revealed that they were hemizygous for a novel missense mutation, c.157G>C (p.Glu53Gln). Our study widens the mutation spectrum of the FIX gene. In addition, this report provides a specific case associated with genotype and phenotype heterogeneity of hemophilia B.

A novel 223 kb deletion in the beta-globin gene cluster was identified in a Chinese thalassemia major patient.

INTRODUCTION: Although mutations in the human beta-globin gene cluster are essentially point mutations, several large deletions have been described in recent years. METHODS: We have identified a novel 223 kb deletion in a Chinese patient by multiplex ligation-dependent probe amplification and characterized it by next-generation sequencing, Gap-PCR, and DNA sequence analysis. RESULTS: The deletion extends from the 3'UTR of the δ globin gene (HBD) to 215 kb downstream of the HBB. Compound heterozygous with the typical ß-thalassemia-CD41-42(-CTTT) mutation, the proband presented with microcytosis and hypochromic red cells, and required regulate transfusion. The patient was clinically diagnosed with thalassemia major. CONCLUSION: Our study widens the mutation spectrum of ß-thalassemia. In addition, this case may spark future studies of the regulatory regions of the beta-globin gene cluster.

Characterization of Hb Bart's Hydrops Fetalis Caused by - -SEA and a Large Novel α0-Thalassemia Deletion.

Hb Bart's hydrops fetalis is the most severe and generally fatal clinical phenotype of α-thalassemia (α-thal), which is due to the deletion of all four functional α-globin genes of hemoglobin (Hb), resulting in no α-globin chain production (- -/- -). Homozygosity for the - -SEA (Southeast Asian) α-globin gene deletion is the main cause of the Hb Bart's hydrops fetalis in Asia, especially South China. Occasionally, other α0-thal deletions can also be found. In this study, we report a case with an atypical form of Hb Bart's hydrops fetalis that was caused by - -SEA and a large novel α0-thal deletion (- -GX) (Guangxi). The fetus with Hb Bart's in our study presented fetal hydrops features in early gestation which was different from that of traditional Hb Bart's hydrops fetalis with a homozygous - -SEA deletion. The early onset of fetal hydrops is attributed to the decreased formation of embryonic Hb Portland (ζ2γ2), which is proposed as a candidate for reactivation in cases of severe α-thal. Our findings indicated that it was important to characterize new or rare mutations, and highlighted the significance of using ultrasonography to identify signs of Hb Bart's hydrops fetalis.

A novel variant of osteogenesis imperfecta type IV and low serum phosphorus level caused by a Val94Asp mutation in COL1A1.

Osteogenesis imperfecta (OI) is a rare congenital disorder characterized by bone fragility and fractures, and associated with bone deformity, short stature, dentin, ligament and blue­gray eye sclera. OI is caused by a heterozygous mutation in collagen α­1(I) chain (COL1A1) or collagen α­2(I) chain (COL1A2) genes that encode α chains of type I collagen. Collagen α chain peptide contains an N­propeptide, which has a role in assembly and processing of collagen. Point mutations in the N­propeptide domain appear to trigger OI. In the present study, a novel heterozygous missense mutation, c.281T>A (p.Val94Asp), was identified in the von Willebrand C domain of N­terminal of type I collagen in an individual with type IV OI. The majority of N­terminal mutations are associated with OI/Ehlers­Danlos syndrome (EDS); however, in the present study, the affected individual did not suffer from EDS and the level of serum phosphorus of the patient was low (0.67 mmol/l). A number of clinical phenotypes were observed at the same variation site or in the same region on the polypeptide chain of COL1A, which suggests that additional genetic and environmental factors may influence the severity of OI. The present study may provide insight into the phenotype­genotype association in collagen-associated diseases and improve clinical diagnosis of OI.

The prevalence and molecular characterization of (뫧)0 -thalassemia and hereditary persistence of fetal hemoglobin in the Chinese Zhuang population.

OBJECTIVE: To reveal the prevalence and molecular characterization of (δß)0 -thalassemia [(δß)0 -thal] and hereditary persistence of fetal hemoglobin (HPFH) in the Chinese Zhuang population. METHODS: A total of 105 subjects with fetal hemoglobin (Hb F) level ≥5% from 14 204 unrelated ones were selected for the study. Multiplex ligation dependent probe amplification was firstly used to analyze dosage changes of the ß-globin gene cluster for associated with (δß)0 -thal and HPFH mutations. The gap polymerase chain reaction was then performed to identify the deletions using the respective flanking primers. Hematologic data were recorded and correlated with the molecular findings. RESULTS: Twenty-one (0.15%) subjects were diagnosed with Chinese G γ(A γδß)0 -thal. Nine (0.06%) were diagnosed with Southeast Asia HPFH (SEA-HPFH) deletion. Seventy-five (0.53%) cases remained uncharacterized. Three genotypes for Chinese G γ(A γδß)0 -thal and SEA-HPFH deletion were identified, respectively. The genotype-phenotype relationships were discussed. CONCLUSION: Our study for the first time demonstrated that (δß)0 and HPFH were not rare events, and molecular characterized G γ(A γδß)0 -thal and HFPH mutations in the Chinese Zhuang population. The findings in our study will be useful in genetic counseling and prenatal diagnostic service of ß-thalassemia in this populations.

First Identification of the 3.5 kb Deletion (NC_000011.10: g.5224302-5227791del3490bp) on the ß-Globin Gene Cluster in a Chinese Family.

ß-Thalassemia (ß-thal) is one of the most common autosomal recessive disorders worldwide. It is caused mainly by point mutations or, more rarely, deletions on the ß-globin gene, leading to reduced (ß+) or absent (ß0) synthesis of the ß chains of hemoglobin (Hb). Molecular characterization of ß-thal is essential for the prevention of this disease in the population. In China, more than 46 different mutations have been found, while approximately five large deletional types of ß-thal have been reported. Here we described a large deletional mutation of the ß-globin gene cluster previously unreported in the Chinese population, the 3.5 kb deletion (NC_000011.10: g.5224302-5227791del3490bp) removing the ß-globin gene promoter and the whole ß-globin gene leading to a ß0-thal phenotype.

Characterization of a Large Novel α-Globin Gene Cluster Deletion Causing α0-Thalassemia in a Chinese Family.

We report a large novel α-globin cluster deletion that we named - -PG (NG_000006.1: g.93628_542759del450131), in a Chinese family. This large deletion is approximately 450 kb long, spanning from upstream of the PolR3k gene at the 5' end to the RAB11FIP3 gene at the 3' end of chromosome 16p13.3. This deletion removes all the globin distal regulatory elements as well as the whole α-globin gene cluster. Patients with heterozygous - -PG/αα had red blood cell (RBC) indices consistent with α-thalassemia (α-thal) trait, but no apparent increase in a cancer tendency or mental disability, microcephaly, relative hypertelorism, unusual facies or genital anomalies.

A Genetic Variant Ameliorates ß-Thalassemia Severity by Epigenetic-Mediated Elevation of Human Fetal Hemoglobin Expression.

A delayed fetal-to-adult hemoglobin (Hb) switch ameliorates the severity of ß-thalassemia and sickle cell disease. The molecular mechanism underlying the epigenetic dysregulation of the switch is unclear. To explore the potential cis-variants responsible for the Hb switching, we systematically analyzed an 80-kb region spanning the ß-globin cluster using capture-based next-generation sequencing of 1142 Chinese ß-thalassemia persons and identified 31 fetal hemoglobin (HbF)-associated haplotypes of the selected 28 tag regulatory single-nucleotide polymorphisms (rSNPs) in seven linkage disequilibrium (LD) blocks. A Ly1 antibody reactive (LYAR)-binding motif disruptive rSNP rs368698783 (G/A) from LD block 5 in the proximal promoter of hemoglobin subunit gamma 1 (HBG1) was found to be a significant predictor for ß-thalassemia clinical severity by epigenetic-mediated variant-dependent HbF elevation. We found this rSNP accounted for 41.6% of ß-hemoglobinopathy individuals as an ameliorating factor in a total of 2,738 individuals from southern China and Thailand. We uncovered that the minor allele of the rSNP triggers the attenuation of LYAR and two repressive epigenetic regulators DNA methyltransferase 3 alpha (DNMT3A) and protein arginine methyltransferase 5 (PRMT5) from the HBG promoters, mediating allele-biased γ-globin elevation by facilitating demethylation of HBG core promoter CpG sites in erythroid progenitor cells from ß-thalassemia persons. The present study demonstrates that this common rSNP in the proximal Aγ-promoter is a major genetic modifier capable of ameliorating the severity of thalassemia major through the epigenetic-mediated regulation of the delayed fetal-to-adult Hb switch and provides potential targets for the treatment of ß-hemoglobinopathy.