Molecular Basis and Hematologic Phenotype of Hemoglobin H Disease Combined with Two Rare ß-Globin Mutations.

In area where α-thalassemia and ß-thalassemia are prevalent, the coinheritance of hemoglobin H disease (Hb H disease) and ß-thalassemia are not uncommon and could result in complex thalassemia intermedia syndromes. In this study, we investigate the hematological and molecular characteristics of two previously undescribed cases that co-inherited Hb H disease and rare ß-globin gene (HBB) mutations found in Chinese populations. Proband I was a boy with Hb H disease in association with IVS-II-5(G > C) (HBB:c0.315 + 5G > C) mutation. Proband II was a boy with a combination of Hb H and Hb Zengcheng [ß114(G16) Leu > Met; HBB:c.343C > A]. Both of them had mild hypochromic microcytic anemia, and neither had ever received a blood transfusion. In both cases, the level of Hb A2 was within normal range, and no Hb H was detected, but a small amount of Hb Bart's was observed in proband I. Routine DNA analysis detected the deletional Hb H disease in both cases. IVS-II-5(G > C) (HBB:c0.315 + 5G > C) and Hb Zengcheng (HBB:c.343C > A) mutations were found by DNA sequencing of ß-globin gene. The co-inheritance of Hb H disease with rare ß-thalassemia may result in an atypical pattern of Hb H disease, and further investigation of rare genotypes should be conducted to avoid missed diagnosis.

Analysis of circRNAs and circRNA-associated competing endogenous RNA networks in ß-thalassemia.

The involvement of circRNAs in ß-thalassemia and their actions on fetal hemoglobin (HbF) is unclear. Here, the circRNAs in ß-thalassemia carriers with high HbF levels were comprehensively analyzed and compared with those of healthy individuals. Differential expression of 2183 circRNAs was observed and their correlations with hematological parameters were investigated. Down-regulated hsa-circRNA-100466 had a strong negative correlation with HbF and HbA2. Bioinformatics was employed to construct a hsa-circRNA-100466­associated competing endogenous RNA (ceRNA) network to identify hub genes and associated miRNAs. The hsa-circRNA-100466▁miR-19b-3p▁SOX6 pathway was identified using both present and previously published data. The ceRNA network was verified by qRT-PCR analysis of ß-thalassemia samples, RNA immunoprecipitation of K562 cell lysates, and dual-luciferase reporter analysis. qRT-PCR confirmed that hsa-circRNA-100466 and SOX6 were significantly down-regulated, while miR-19b-3p was up-regulated. Hsa-circRNA-100466, miR-19b-3p, and SOX6 were co-immunoprecipitated by anti-argonaute antibodies, indicating involvement with HbF induction. A further dual-luciferase reporter assay verified that miR-19b-3p interacted directly with hsa-circRNA-100466 and SOX6. Furthermore, spearman correlation coefficients revealed their significant correlations with HbF. In conclusion, a novel hsa-circRNA-100466▁miR-19b-3p▁SOX6 pathway was identified, providing insight into HbF induction and suggesting targets ß-thalassemia treatment.

Placental transcriptome sequencing combined with bioinformatics predicts potential genes and circular RNAs associated with hemoglobin Bart's hydrops fetalis syndrome.

AIM: Hemoglobin Bart's hydrops fetalis syndrome (BHFS) is the most severe form of α-thalassemia. Histological alternations can be observed in placenta, but placental transcriptome profile and circular RNAs have not been studied in this disease. The aim of this study was to define the placental transcriptional changes and find relevant circular RNAs in BHFS. METHODS: We performed high-throughput RNA sequencing to detect placental samples from fetuses affected by BHFS (n = 5) and normal fetuses (NF, n = 5), quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Sanger sequencing to validate the differentially expressed circRNAs and their potentially related miRNAs (BHFS, n = 22; NF, n = 11). Bioinformatics methods were performed for further analysis. RESULTS: Our results showed 152 differentially expressed genes (DEGs), 112 circRNAs, and 45 microRNAs that were differentially expressed. DEGs were found to be involved in Gene Ontology terms related to gas transport, cell adhesion, oxidative stress, organ development, hemopoiesis, and others. RT-qPCR results showed that hsa_circ_0003961 and hsa_circ_0006687 were upregulated (p < 0.05). The competing endogenous RNA and co-expression networks showed that hsa_circ_0003961 and hsa_circ_0006687 were connected with 3 miRNAs and some DEGs, including cell adhesion genes (e.g., CLDN19), hemoglobin related genes (e.g., SOX6 and HBZ) and angiogenesis related genes (e.g., EPHB2). Downregulations of hsa-miR-1299 and hsa-miR-625-5p in ceRNA network were also validated by RT-qPCR. Gene set enrichment analysis results for the two circRNAs showed that some gene sets associated with cell adhesion, hematopoietic system and apoptosis were significantly enriched. CONCLUSIONS: Our study characterized the placental transcriptome of BHFS. The circRNAs hsa_circ_0003961 and hsa_circ_0006687 in placenta may be relevant to BHFS.

Invasive prenatal diagnosis of α-thalassemia to control Hb Bart's hydrops fetalis syndrome: 15 years of experience.

PURPOSE: The aim of the present study was to report experiences with invasive prenatal diagnosis of α-thalassemia for the prevention of Hb Bart's hydrops fetalis syndrome in the Guangxi Zhuang Autonomous Region, China. METHODS: Pregnant women and their partners who tested positive for α0-thalassemia or were diagnosed with HbH diseases were counseled and suggested to undergo a prenatal diagnostic procedure for α-thalassemia. Fetal material was obtained by chorionic villus sampling (CVS) between 9 and 13 weeks of gestation, by amniocentesis between 16 and 24 weeks of gestation and by cordocentesis after 24 weeks of gestation. The α0-thalassemia gene types were detected by gap polymerase chain reaction (Gap-PCR). All results were finally confirmed by DNA analysis after delivery or termination of pregnancy. RESULTS: An invasive prenatal α-thalassemia diagnosis was performed in 3155 cases at risk for Hb Bart's hydrops fetalis syndrome at our hospital from 2002 to 2016. CVS was performed in 1559 cases (49.4%), amniocentesis in 1240 cases (39.3%) and cordocentesis in 356 cases (11.3%). In total, 786 fetuses were diagnosed as Hb Bart's hydrops fetalis syndrome. Among these cases, the α-thalassemia genotype was --SEA/--SEA in 784 cases and --SEA/--THAI in 2 cases. All affected pregnancies were terminated in time. CONCLUSIONS: This extensive experience suggests that carrier screening, molecular diagnostics, genetic counselling, and prenatal diagnosis are effective measures to prevent Hb Bart's hydrops fetalis syndrome.

A Novel Variant with Positive Natural Selection Influenced Hb A2 Levels in Chinese Individuals with ß-Thalassemia.

ß-Thalassemia (ß-thal) is the most common inherited hemolytic anemia worldwide. Elevated Hb A2 is a mark of ß-thal carriers. The aim of this study was to identify the pathogenic variants associated with the Hb A2 levels. One thousand and thirty ß-thal carriers were recruited for this study. Using positive natural expression quantitative trait loci (eQTL) analysis, a significant variant was selected. Genotyping for the rs231841 polymorphism was performed by the Sequenom MassARRAY IPLEX platform. All genetic association analyses were performed with the PLINK program. The linear regression analysis showed that rs231841 in the intron region of the potassium voltage-gated channel subfamily Q member 1 (KCNQ1) gene on chromosome 11p15 was significantly associated with Hb A2 levels. The presence of the C allele was associated with elevated Hb A2 levels. Our results suggest that rs231841 on the KCNQ1 gene with positive natural selection is related to Hb A2 levels in Chinese ß-thal carriers, and KCNQ1 is probably associated with the expression of the ß-like globin gene cluster.

Genome-wide analysis of aberrantly expressed lncRNAs and miRNAs with associated co-expression and ceRNA networks in ß-thalassemia and hereditary persistence of fetal hemoglobin.

The implications of lncRNAs regarding fetal hemoglobin (HbF) induction in hemoglobin disorders remain poorly understood. In this study, microarray analysis was performed to profile lncRNAs, miRNAs and mRNAs in individuals with hereditary persistence of fetal hemoglobin (HPFH), ß-thalassemia carriers with high HbF levels and healthy controls. The results show aberrant expression of 862 lncRNAs, 568 mRNAs and 63 miRNAs in the high-HbF group compared with the control group. Altered NR_001589, NR_120526, T315543, miR-486-3p, miR-19b-1-5p and miR-20a-3p expression was confirmed by quantitative reverse transcription-polymerase chain reaction, and Spearman correlation coefficients revealed significant positive correlations with HbF. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed the hematopoietic cell lineage and apoptosis to be most significantly dysregulated in HbF induction. We analyzed coding genes near the lncRNAs and constructed a coding-noncoding co-expression network. Based on the results, lncRNAs likely contribute to increased HbF levels by activating expression of HBE1 and hematopoietic cell lineage-inducible molecules and by inhibiting that of apoptosis-inducible molecules. Finally, through construction of a competing endogenous RNA network, we found that 6 lncRNAs could bind competitively with miR-486-3p, resulting in increased HbF levels. Taken together, our findings provide new insights into the mechanisms of HbF induction and potentially provide new targets for the treatment of ß-thalassemia major.

The prevalence of thalassemia in mainland China: evidence from epidemiological surveys.

Comprehensive data regarding the epidemiology and prevalence of thalassemia in mainland China are lacking. To assess the prevalence of thalassemia, we performed a meta-analysis including 16 articles published from 1981 to 2015. The overall prevalence of α-thalassemia, ß-thalassemia and α + ß-thalassemia was 7.88%, 2.21% and 0.48%, respectively. Trends in thalassemia prevalence in mainland China were not steady; a prevalence map based on a geographic information system (GIS) showed that the geographic distribution of thalassemia was highest in the south of China and decreased from south to north. Additionally, the most common α- and ß-globin gene mutation was --SEA and CD41/42, respectively. The current study provides valuable information regarding epidemiology and intervention and supports the planning, implementation and management of prevention programmes for public health.