Stimulation of PSTPIP1 to trigger proinflammatory responses in asymptomatic SARS-CoV-2 infections.

Background: A hyperinflammatory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection gravely worsens the clinical progression of coronavirus disease 2019 (COVID-19). Although the undesirable effects of inflammasome activation have been correlated to the severity of COVID-19, the mechanisms of this process in the asymptomatic infection and disease progression have not yet been clearly elucidated. Methods: We performed strand-specific RNA sequencing in 39 peripheral blood mononuclear cell (PBMC) samples from asymptomatic individuals（n = 10）, symptomatic patients（n = 16） and healthy donors（n = 13）. Results: Dysregulation of pyrin inflammasomes along with the proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene was identified in SARS-COV-2 infection. Notably, the PSTPIP1 expression level showed a significant negative correlation with an adjacent long-noncoding RNA (lncRNA) RP11-797A18.6 in the asymptomatic individuals compared with the healthy controls. In addition, a decline in the nuclear factor kappa B subunit 1 (NFKB1) gene expression was observed in asymptomatic infection, followed by a rise in the mild and moderate disease stages, suggesting that altered NFKB1 expression and associated proinflammatory signals may trigger a disease progression. Conclusions: Overall, our results indicate that PSTPIP1-dependent pyrin inflammasomes-mediated pyroptosis and NF-κB activation might be potential preventive targets for COVID-19 disease development and progression.

Identification and characterization of CHD4-associated eRNA as a novel modulator of fetal hemoglobin levels in ß-thalassemia.

Fetal-to-adult hemoglobin switching is controlled by programmed silencing of γ-globin while the re-activation of fetal hemoglobin (HbF) is an effective strategy for ameliorating the clinical severity of ß-thalassemia and sickle cell disease. The identification of enhancer RNAs (eRNAs) related to the fetal (α2γ2) to adult hemoglobin (α2ß2) switching remains incomplete. In this study, the transcriptomes of GYPA+ cells from six ß-thalassemia patients with extreme HbF levels were sequenced to identify differences in patterns of noncoding RNA expression. It is interesting that an enhancer upstream of CHD4, an HbF-related core subunit of the NuRD complex, was differentially transcribed. We found a significantly positive correlation of eRNA-CHD4 enhancer-gene interaction using the public database of FANTOM5. Specifically, the eRNA-CHD4 expression was found to be significantly higher in both CD34+ HSPCs and HUDEP-2 than those in K562 cells which commonly expressed high level of HbF, suggesting a correlation between eRNA and HbF expression. Furthermore, prediction of transcription binding sites of cis-eQTLs and the CHD4 genomic region revealed a putative interaction site between rs73264846 and ZNF410, a known transcription factor regulating HbF expression. Moreover, in-vitro validation showed that the inhibition of eRNA could reduce the expression of HBG expression in HUDEP-2 cells. Taken together, the findings of this study demonstrate that a distal enhancer contributes to stage-specific silencing of γ-globin genes through direct modulation of CHD4 expression and provide insights into the epigenetic mechanisms of NuRD-mediated hemoglobin switching.

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.

Analysis of Hematological Indices and Splenectomy Rates in 2,130 Patients with Hemoglobin H Diseases or ß-Thalassemia.

INTRODUCTION: Splenomegaly and hypersplenism are common complications of thalassemia patients due to the excessive clearance of defective red blood cells from the spleen. To date, splenectomy has been considered one of the most effective treatments for splenomegaly, reducing clinical severity among thalassemia patients. Thus, we aim to investigate the differences in splenectomy rates and hematological indices among thalassemia patients with different genotypes. METHOD: In this study, we analyzed the clinical data of thalassemia in 2,130 patients admitted to the 923rd Hospital of the People's Liberation Army from January 2006 to December 2020, and the statistical software SPSS 26.0 was applied to analyze the data. RESULT: Of the 2,130 patients with thalassemia, 265 patients underwent splenectomy. It was determined that significantly more patients with hemoglobin H (HbH) disease, a form of α-thalassemia, have undergone splenectomy than ß-thalassemia patients (20% vs. 7%). Further, HbH disease patients were diagnosed at a significantly older age than ß-thalassemia patients. CONCLUSION: The greater probability of HbH disease patients undergoing splenectomy is likely influenced by multiple factors, including their lower dependency on transfusion, leading to high spleen compensatory stress on the spleen, and the destruction of defective erythrocytes. In contrast, ß-thalassemia is clinically more severe and less tolerant of hemoglobin fluctuations. Based on these findings, clinicians are suggested to pay more attention to HbH disease patients as many of them are still under-transfused, which could lead to chronic hemolysis and more severe hepatosplenomegaly. These results might offer insight for improving the clinical management of patients with different types of thalassemia.

LC-MS/MS-Based Absolute Quantitation of Hemoglobin Subunits from Dried Blood Spots Reveals Novel Biomarkers for α-Thalassemia Silent Carriers.

Identification of α-thalassemia silent carriers is challenging with conventional phenotype-based screening methods. A liquid chromatography tandem mass spectrometry (LC-MS/MS)-based approach may offer novel biomarkers to address this conundrum. In this study, we collected dried blood spot samples from individuals with three α-thalassemia subtypes for biomarker discovery and validation. We observed differential expression patterns of hemoglobin subunits among various α-thalassemia subtypes and normal controls through proteomic profiling of 51 samples in the discovery phase. Then, we developed and optimized a multiple reaction monitoring (MRM) assay to measure all detectable hemoglobin subunits. The validation phase was conducted in a cohort of 462 samples. Among the measured hemoglobin subunits, subunit µ was significantly upregulated in all the α-thalassemia groups with distinct fold changes. The hemoglobin subunit µ exhibits great potential as a novel biomarker for α-thalassemia, especially for silent α-thalassemia. We constructed predictive models based on the concentrations of hemoglobin subunits and their ratios to classify the various subtypes of α-thalassemia. In the binary classification problems of silent α-thalassemia vs normal, non-deletional α-thalassemia vs normal, and deletional α-thalassemia vs normal, the best performance of the models achieved average ROCAUCs of 0.9505, 0.9430, and 0.9976 in the cross-validation, respectively. In the multiclass model, the best performance achieved an average ROCAUC of 0.9290 in cross-validation. The performance of our MRM assay and models demonstrated that the hemoglobin subunit µ would play a vital role in screening silent α-thalassemia in clinical practice.

Quantification of human embryonic ζ-globin chains in Southeast Asian deletion (--SEA) carriers.

AIMS: Reactivation of embryonic ζ-globin is a promising strategy for genetic treatment of α-thalassaemia. However, quantification of ζ-globin as a quantitative trait in α-thalassaemia carriers and patients remains incompletely understood. In this study, we aimed to set up a reliable approach for the quantification of ζ-globin in α-thalassaemia carriers, followed by a population study to investigate its expression patterns. METHODS: ζ-globin was purified as monomers from cord blood haemolysate of a Hb Bart's fetus, followed by absolute protein quantification, which was then tested by in-house ELISA system and introduced as protein standard. It was then used for large-scale quantification in peripheral blood samples from 6179 individuals. Finally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) introduced as an independent validating approach by measuring ζ-globin expression in a second cohort of 141-SEA/αα carriers. RESULTS: The ELISA system was proved sensitive in distinguishing individuals with varied extent of ζ-globin. Large scale quantitative study of this --SEA/αα carrier cohort indicated the high diversity of ζ-globin expression ranging from 0.00155 g/L to 1.48778 g/L. Significant positive correlation between ELISA and LC-MS/MS (R=0.400, p<0.001) was observed and it is more sensitive in distinguishing the samples with extreme expression of ζ-globin (R=0.650, p<0.001). CONCLUSION: Our study has reported reliable approaches for the quantification of ζ-globin and presented the expression patterns of ζ-globin among the --SEA/αα carrier population, which might lay a foundation on subsequent genotype-phenotype studies on mechanisms of delayed haemoglobin switch in α-thalassaemia.

A stepwise haematological screening and whole-exome sequencing reveal multiple mutations from SUPT5H causing an elevation of Hb A2 from a cohort of 47336 individuals.

INTRODUCTION: Though an increase in Hb A2 is one of the most key markers of ß-thal carriers, a few independent cases are reported to show elevated Hb A2 levels caused by mutations in other genes beyond ß-globin gene. METHODS: We reviewed the haematological indices of 47336 individuals to analyse the phenotype-genotype correlation and identified 1439 individuals (3.04%) positive in the elevation of Hb A2 . Globin and KLF1 genes analysis was performed, and further whole-exome sequencing was carried to dissect the genetic causes of those positive samples without ß-thalassemic or KLF1 mutations. RESULTS: Of these 1439 individuals with elevated Hb A2 , 1381 had a molecular defect in globin genes, and most were ß-thalassemic mutation; 10 had a molecular defect in KLF1 gene. Finally, among the 38 individuals without ß-thalassemic or KLF1 mutations, 7 were identified to carried a loss-of-function mutation in SUPT5H. CONCLUSION: This study has provided a mutation spectrum of SUPT5H in a cohort screening leading to the elevation of Hb A2 . According to the previous observations that individuals with a combination of ß-thal mutation and a SUPT5H variant might present moderate ß-thaelassemia, these findings emphasized the importance of comprehensive molecular diagnosis to prevent birth defects of ß-thaelassemia caused by rare mutations from modifier genes.

TEA domain transcription factor 4 modulates repression of fetal haemoglobin by direct binding to the γ-globin gene promoters.

Re-activation of fetal haemoglobin (HbF) has been proved to be an effective strategy for the treatment of ß-haemoglobinopathies. In this study, we identified TEA domain transcription factor 4 (TEAD4) as a new potential regulator of HbF by integrating public data sets with quantitative polymerase chain reaction analysis in ß-thalassaemia patients. Significant negative correlation was observed between the expression of TEAD4 and HbF levels in ß-thalassaemia patients. Functional validations of TEAD4 inhibition in both ß-thalassaemia CD34+ cells and HUDEP-2 cells indicated that depletion of TEAD4 led to a significant increase of HbF. Finally, we identified a binding motif of TEAD4 on γ-globin gene promoters; its disruption consistently led to de-repression of HbF. Taken together, these results demonstrate that TEAD4 could act as a transcriptional inhibitor of the γ-globin gene through direct binding on its promoter. Our findings demonstrate a novel role of TEAD4 on the regulation of HbF, which may benefit patients with ß-haemoglobinopathies.

GATA zinc finger domain-containing protein 2A (GATAD2A) deficiency reactivates fetal haemoglobin in patients with ß-thalassaemia through impaired formation of methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex.

Reactivation of fetal haemoglobin (HbF) expression is an effective way to treat ß-thalassaemia and sickle cell anaemia. In the present study, we identified a novel GATA zinc finger domain-containing protein 2A (GATAD2A) mutation, which contributed to the elevation of HbF and ameliorated clinical severity in a patient with ß-thalassaemia, by targeted next-generation sequencing. Knockout of GATAD2A led to a significant induction of HbF in both human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) and human cluster of differentiation (CD)34+ cells with a detectable impact on erythroid differentiation. Furthermore, heterozygous knockout of GATAD2A impaired recruitment of chromodomain helicase DNA-binding protein 4 (CHD4) to the methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex. Our present data suggest that mutations causing the haploinsufficiency of GATAD2A might contribute to amelioration of clinical severity in patients with ß-thalassaemia.

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.

A Novel Hemoglobin Variant Hb Liaobu [α107(G14)Val→Leu, HBA2: c.322G>C] Detected by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry.

We here describe a novel hemoglobin (Hb) variant, Hb Liaobu [α107(G14)Val→Leu, HBA2: c.322G>C], in a Chinese family. The structurally abnormal α chain variant could not be detected using capillary electrophoresis (CE) and was subsequently characterized by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS), and further confirmed by reversed phase high performance liquid chromatography (HPLC). Sanger sequencing revealed a novel base mutation on the α2-globin gene and RNA analysis by reverse transcription polymerase chain reaction (RT-PCR) showed the presence of an abnormal HBA transcript. The isopropanol stability test indicated the stable state of this structural Hb variant. In conclusion, a new Hb variant, Hb Liaobu, was discovered and characterized. It was proven to be a nonpathogenic variant. Our study resolved the confusion in the clinical diagnosis of individuals with this novel Hb variant in this family.

A natural DNMT1 mutation elevates the fetal hemoglobin level via epigenetic derepression of the γ-globin gene in ß-thalassemia.

DNA methyltransferase 1 (DNMT1) is a major epigenetic regulator of the formation of large macromolecular complexes that repress human γ-globin expression by maintaining DNA methylation. However, very little is known about the association of DNMT1 variants with ß-thalassemia phenotypes. We systematically investigated associations between variants in DNMT1 and phenotypes in 1142 ß-thalassemia subjects and identified a novel missense mutation (c.2633G>A, S878F) in the DNMT1 bromo-adjacent homology-1 (BAH1) domain. We functionally characterized this mutation in CD34+ cells from patients and engineered HuDEP-2 mutant cells. Our results demonstrate that DNMT1 phosphorylation is abrogated by substituting serine with phenylalanine at position 878, resulting in lower stability and catalytic activity loss. S878F mutation also attenuated DNMT1 interactions with BCL11A, GATA1, and HDAC1/2, and reduced recruitment of DNMT1 to the γ-globin (HBG) promoters, leading to epigenetic derepression of γ-globin expression. By analyzing the F-cell pattern, we demonstrated that the effect of DNMT1 mutation on increased fetal hemoglobin (HbF) is heterocellular. Furthermore, introduction of S878F mutation into erythroid cells by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) recapitulated γ-globin reactivation. Thus, the natural S878F DNMT1 mutation is a novel modulator of HbF synthesis and represents a potential new therapeutic target for ß-hemoglobinopathies.

Characterization of two novel Alu element-mediated α-globin gene cluster deletions causing α0-thalassemia by targeted next-generation sequencing.

α-thalassemia is an inherited blood disorder commonly caused by deletions or point mutations involving one or both α-globin genes. Recent studies shed new light on the critical role of upstream enhancers multi-species conserved sequences (MCSs) in the ordered regulation of α-globin gene expression. Herein, we reported two unrelated probands with deletions in α-globin genes and MCSs, respectively. The proband from Family A is a compound heterozygote carrying a known α+ mutation (-α3.7) and a novel 60.2 kb deletion causing the absence of both α-globin genes. The proband from Family B, on the other hand, is a compound heterozygote with a known α0 mutation (--SEA) and a novel deletion involving only upstream regulatory elements MCS-R1, R2 and R3, while the α-globin genes remain intact. Notably, both these two patients suffered varied extent of anemia, indicating that the loss of enhancer elements could equally lead to reduced synthesis of α-globin. Upon these observations, we then confirmed the exact breakpoints of these two novel deletions using a targeted next-generation sequencing (NGS) previously established by our group, which may enable further elucidation of the rearrangement mechanisms on these deletions and functional dissection of MCSs. Taken together, our study reports a reliable NGS-based molecular screening approach for accurate identification of copy number variations (CNVs) in the α-globin cluster and the genetic diagnosis of these two probands may help to extend the spectrum of α-thalassemia mutations in Chinese population.

LOVD-DASH: A comprehensive LOVD database coupled with diagnosis and an at-risk assessment system for hemoglobinopathies.

Hemoglobinopathies are the most common monogenic disorders worldwide. Substantial effort has been made to establish databases to record complete mutation spectra causing or modifying this group of diseases. We present a variant database which couples an online auxiliary diagnosis and at-risk assessment system for hemoglobinopathies (DASH). The database was integrated into the Leiden Open Variation Database (LOVD), in which we included all reported variants focusing on a Chinese population by literature peer review-curation and existing databases, such as HbVar and IthaGenes. In addition, comprehensive mutation data generated by high-throughput sequencing of 2,087 hemoglobinopathy patients and 20,222 general individuals from southern China were also incorporated into the database. These sequencing data enabled us to observe disease-causing and modifier variants responsible for hemoglobinopathies in bulk. Currently, 371 unique variants have been recorded; 265 of 371 were described as disease-causing variants, whereas 106 were defined as modifier variants, including 34 functional variants identified by a quantitative trait association study of this high-throughput sequencing data. Due to the availability of a comprehensive phenotype-genotype data set, DASH has been established to automatically provide accurate suggestions on diagnosis and genetic counseling of hemoglobinopathies. LOVD-DASH will inspire us to deal with clinical genotyping and molecular screening for other Mendelian disorders.

A Cell-free DNA Barcode-Enabled Single-Molecule Test for Noninvasive Prenatal Diagnosis of Monogenic Disorders: Application to ß-Thalassemia.

Noninvasive prenatal testing of common aneuploidies has become routine over the past decade, but testing of monogenic disorders remains a challenge in clinical implementation. Most recent studies have inherent limitations, such as complicated procedures, a lack of versatility, and the need for prior knowledge of parental genotypes or haplotypes. To overcome these limitations, a robust and versatile next-generation sequencing-based cell-free DNA (cfDNA) allelic molecule counting system termed cfDNA barcode-enabled single-molecule test (cfBEST) is developed for the noninvasive prenatal diagnosis (NIPD) of monogenic disorders. The accuracy of cfBEST is found to be comparable to that of droplet digital polymerase chain reaction (ddPCR) in detecting low-abundance mutations in cfDNA. The analytical validity of cfBEST is evidenced by a ß-thalassemia assay, in which a blind validation study of 143 at-risk pregnancies reveals a sensitivity of 99.19% and a specificity of 99.92% on allele detection. Because the validated cfBEST method can be used to detect maternal-fetal genotype combinations in cfDNA precisely and quantitatively, it holds the potential for the NIPD of human monogenic disorders.

miR-326 regulates HbF synthesis by targeting EKLF in human erythroid cells.

Haploinsufficiency of erythroid Krüppel-like factor (EKLF/KLF1) has been shown recently to ameliorate the clinical severity of ß-thalassemia by increased expression levels of fetal hemoglobin (HbF). The underlying mechanisms for role of EKLF in regulating HbF are of great interest but remain incompletely understood. In this study, we used a combination of in silico, in vitro, and in vivo approaches to identify microRNAs (miRs) involved in EKLF regulation and to validate the role of miR-326 in HbF modification. We found that miR-326 suppresses EKLF expression directly by targeting its 3' untranslated region. miR-326 overexpression in K562 cells or CD34+ hematopoietic progenitor cells resulted in reduced EKLF protein levels and was associated with elevated expression of γ-globin, whereas inhibition of physiological miR-326 levels increased EKLF and thus reduced γ-globin expression. Moreover, miR-326 expression is positively correlated with HbF levels in ß-thalassemia patients. Our results suggest that miR-326 plays a key role in regulating EKLF expression and in modifying the HbF level, which may provide a new strategy for activating HbF in individuals with ß-thalassemia or sickle cell disease.

Role of tissue-specific promoter DNA methylation in regulating the human EKLF gene.

Erythroid Krüppel-like factor (EKLF/KLF1) is an erythroid-specific transcription factor whose activity is essential for erythropoiesis. The underlying mechanisms for EKLF specifically restricted to erythroid cells are of great interest but remain incompletely understood. To explore the epigenetic regulation of EKLF expression by promoter DNA methylation, we investigated the methylation status of the EKLF promoter and EKLF gene expression from a panel of human tissues. We observed that erythroid-specific hypomethylation of the EKLF promoter in adult erythroid cells was positively associated with EKLF expression. Demethylation of the EKLF promoter by 5-aza-2'-deoxycytidine led to elevated EKLF expression in non-erythroid cells. We further uncovered that EKLF promoter DNA methylation reduced the binding affinity for the transcription factors GATA1 and c-myb (MYB), which in turn silenced EKLF expression. These results suggest that hypomethylation of the EKLF promoter has functional significance in the establishment and maintenance of erythroid-specific gene expression.

Clinical efficacy of low-temperature radiofrequency ablation of pharyngolaryngeal cyst in 84 Chinese infants.

The aim of the study is to investigate the advantages and disadvantages of low-temperature radiofrequency ablation of pharyngolaryngeal cyst.The study population was composed of 84 children diagnosed with pharyngolaryngeal cyst who underwent surgical treatment at the Department of Otolaryngology, Wuhan Children's Hospital, Wuhan, China, between January 1984 and December 2013. All patients were operated using a self-retaining laryngoscope and were divided into 3 groups: traditional cystectomy group (N = 9), dynamic cutting system group (N = 18), and low-temperature radiofrequency ablation group (N = 57). Clinical outcomes were analyzed to assess the efficacy of low-temperature radiofrequency ablation in treatment of pharyngolaryngeal cyst.Compared with traditional cystectomy group or dynamic cutting system group, operation time was shorter, bleeding was less and one-year recurrence rate was much lower in low-temperature radiofrequency ablation group. However, operation time and bleeding was not statistically different between traditional cystectomy and dynamic cutting system group.Low-temperature radiofrequency ablation may be an effective substitute for treating pharyngolaryngeal cyst.

Rapid Targeted Next-Generation Sequencing Platform for Molecular Screening and Clinical Genotyping in Subjects with Hemoglobinopathies.

Hemoglobinopathies are among the most common autosomal-recessive disorders worldwide. A comprehensive next-generation sequencing (NGS) test would greatly facilitate screening and diagnosis of these disorders. An NGS panel targeting the coding regions of hemoglobin genes and four modifier genes was designed. We validated the assay by using 2522 subjects affected with hemoglobinopathies and applied it to carrier testing in a cohort of 10,111 couples who were also screened through traditional methods. In the clinical genotyping analysis of 1182 ß-thalassemia subjects, we identified a group of additional variants that can be used for accurate diagnosis. In the molecular screening analysis of the 10,111 couples, we detected 4180 individuals in total who carried 4840 mutant alleles, and identified 186 couples at risk of having affected offspring. 12.1% of the pathogenic or likely pathogenic variants identified by our NGS assay, which were undetectable by traditional methods. Compared with the traditional methods, our assay identified an additional at-risk 35 couples. We describe a comprehensive NGS-based test that offers advantages over the traditional screening/molecular testing methods. To our knowledge, this is among the first large-scale population study to systematically evaluate the application of an NGS technique in carrier screening and molecular diagnosis of hemoglobinopathies.