OBJECTIVETo detect rare types of thalassemia mutations among southern Chinese population.

METHODSPeripheral blood samples from 327 patients from various regions of southern China were collected. The patients were suspected as rare-type thalassemia for their inconsistency between hematological phenotypes and results of routine mutation screening. The samples were further analyzed with GAP-PCR and DNA sequencing.

RESULTSOne hundred and eight cases were diagnosed as rare types of thalassemia. Among whom 10 rare α-globin gene mutations including --THAI, HKα, αααanti3.7, αααanti4.2, -α2.8, -α27.6, CD74 GAC>CAC (Hb Q-Thailand), CD30 (-GAG), CD31 AGG>AAG and CD118 (+TCA), and 12 rare β-globin gene mutations including CD37 TGG>TAG, CD39 CAG>TAG/CD39 CAG>TAG, β II-2 (-T), -90(C>T), -31(A>C), -88(C>T), CD7(-A), CD138(+T), CD89-93 (--AGTGAGCTGCACTG), CD54-58 (-TATGGGCAACCCT), Chinese G γ +(A γδβ)0 and Vietnamese HPFH (HPFH-6) were identified. -88(C>T) (HBB: c.-138C>T) and CD39 CAG>TAG (HBB: c.118C>T) were discovered for the first time in Chinese population. CD7(-A) (HBB: c.23delA) and CD138(+T) (HBB: c.416_417insT) were new types of β-globin gene mutations.

CONCLUSIONThe present study have enriched the mutation spectrum of thalassemia in southern China, which has provided necessary information for its diagnosis.

Humans ; Mutation ; Thalassemia ; genetics ; alpha-Globins ; genetics ; beta-Globins ; genetics

Female ; Humans ; Mutation ; Pregnancy ; Prenatal Diagnosis ; alpha-Globins ; genetics

OBJECTIVETo study the effect of liposomal transfection of antisense oligonucleotide (ASON) on the erythroid cell alpha-globin gene in the patients with severe beta-thalassemia, and provide a new idea for beta-thalassemia gene therapy.

METHODSA highly effective ASON targeting alpha-globin gene was transfected into severe beta-thalassemic erythroid cells cultured in vitro by liposomal at an optimal concentration. The expression level of alpha, beta, gamma-globin gene, the level of hemoglobin, and the excess alpha-globin chains precipitates in ASON group and control group were carefully analyzed by quantitative real-time PCR(Q-RT-PCR), high performance liquid chromatography (HPLC), and electron microscope, respectively.

RESULTSThe mRNA expression of alpha-globin gene was significantly lower in ASON group (9.04 +/- 0.29) than in control group (24.23 +/- 0.29) (P<0.01). Simultaneously, the disequilibrium between alpha- and beta-, gamma-globin gene expression was partly modified by ASON, the ratios of ASON group and control group being 0.79 +/- 0.02 and 2.26 +/- 0.06 respectively (P<0.01). HPLC demonstrated that the levels of HbA2 and HbF increased with downregulation of alpha-globin gene in beta-thalassemic erythroid cells, particularly HbF. The precipitates of alpha-globin chains in ASON group were lessened under electron microscope, particularly in early erythroblast while no change in the control group.

CONCLUSIONThe high effective ASON contributes to inhibit the alpha-globin gene expression of severe beta-thalassemic erythroid cells, partly modify the disequilibrium between alpha-, beta- and gamma-globin gene expression and obviously reduce the precipitates of alpha-globin chains in erythroid cells. It might provide a new idea for gene therapy of beta-thalassemia.

Cells, Cultured ; Child ; Genetic Therapy ; Humans ; Liposomes ; Oligonucleotides, Antisense ; genetics ; Transfection ; alpha-Globins ; genetics ; metabolism ; beta-Globins ; metabolism ; beta-Thalassemia ; genetics ; metabolism ; therapy ; gamma-Globins ; metabolism

OBJECTIVETo establish a rapid and convenient method of reverse dot blot (RDB) analysis for detecting the point mutations of non-deletion alpha-thalassemia in Chinese.

METHODSLabel biotin to primers and amplify human alpha2 globin gene selectively, then hybridize PCR products with a set of oligonucleotide probes immobilized on strips, and develop colour to detect non-deletion alpha-thalassemia defects.

RESULTSThe PCR system using biotin-labeled primers could specifically amplify a 1085 bp fragment of human alpha2 globin gene which encompasses all six alpha-thalassemia mutations. After being hybridized with sequence-specific oligonucleotide probes and colour development, it could simultaneously identify all six types of non-deletion alpha-thalassemias encountered in Chinese.

CONCLUSIONThis method does not need semi-nested PCR, and the products amplified by biotinylated primers can be used directly to hybridize with the probes on strips under the identical conditions of hybridization. So, it is a specific and multiplex detection assay for screening non-deletion alpha-thalassemia defects in Chinese.

Humans ; Nucleic Acid Hybridization ; methods ; Point Mutation ; Reproducibility of Results ; alpha-Globins ; genetics ; alpha-Thalassemia ; diagnosis ; genetics

OBJECTIVEAlpha-thalassemia is one of the most common monogene disorders in the world. Most frequently, it is caused by deletions of alpha-globin gene (-alpha or --), and less commonly resulted from the non-deletional mutation (alpha(T)alpha). Hemoglobin H (HbH) disease is the most severe type among survivors of alpha-thalassemia. The clinical presentation of children with the disease was highly heterogeneous. The aim of this study was to investigate the effect of alpha-globin genotypes in the children with HbH disease on predicting the phenotypic severity and to define the factors involved in the disease progress.

METHODSForty-three children with the disease in Zhuhai area of Guangdong, China were examined by using established techniques to detect genotypes of alpha-globin and to determine all hematological parameters. All detailed clinical data of the cases were recorded. Then clinical and hematological findings, and the correlation with genotypes were evaluated.

RESULTSSix alpha-thalassemia mutations were detected and interacted to produce 5 HbH disease genotypes. Of these genotypes, -alpha(3.7)/--(SEA)(60%), -alpha(4.2)/--(SEA) (19%) and alpha(CS)alpha/--(SEA) (12%) HbH diseases were prevalent in the area. Compared with -alpha(3.7)/--(SEA) HbH disease, significantly lower red blood cell (RBC) count, hemoglobin (Hb), mean corpuscular hemoglobin (MCHC) and HbA(2) (P < 0.05, 0.01, 0.01 and 0.01, respectively), and significantly higher mean corpuscular hemoglobin volume (MCV) and HbH levels (both P < 0.01), and more severe clinical phenotypes were found in the HbH disease with alpha(T)alpha/--(SEA) genotype. While the differences were much more significant when compared with -alpha(3.7)/--(SEA) then compared with -alpha(4.2)/--(SEA) not only in the hematological parameters, but also in the severity of clinical phenotypes. In addition, HbH levels showed anegatively correlation with the RBC count (r = -0.39, P < 0.01).

CONCLUSIONThe phenotypes of HbH disease may be mainly related to the underlying genotypes. The children with alpha(T)alpha/--(SEA) genotype presented with more severe hematological and clinical phenotypes followed by the -alpha(4.2)/--(SEA) and then -alpha(3.7)/--(SEA) genotypes. But phenotypic severity was not simply related to the degree of alpha-globin deficiency. HbH levels were found to exacerbate anemia. These data might provide comprehensive and very valuable and basic information for the management of HbH disease, genetic counseling and prenatal diagnosis.

Child ; China ; Disease Progression ; Genotype ; Hemoglobin H ; genetics ; Humans ; Phenotype ; alpha-Globins ; genetics

OBJECTIVE: To study the hematologic and molecular features of 14 patients with hemoglobin (Hb) variants, so as to provide reference data for its laboratory screening. METHODS: A total of 1 029 samples were screened by high performance liquid chromatography (HPLC) on the Bio-Rad VariantⅡHPLC system. GAP-PCR and reverse dot blot (RDB) were used to detect common mutation of α and β globin gene in Chinese. DNA sequencing for α and β globin gene was simultaneously performed in samples with abnormal spectrum peak and negative thalassemia gene. RESULTS: In 1 029 samples, 10 types of structural Hb variants were detected in14 cases (1.36%), including 1 case of Hb E / β- thalassemia, 1 case of Hb E /α- thalassemia (HbH disease), 2 cases of HbG-Taipei, 2 cases of Hb Q-Thailand, 2 cases of Hb Youngstown, 1 case of Hb Guangzhou-Hangzhou, 1 case of Hb M-Boston, 1 case of Hb G-Siriraj, 1 case of Hb J-Baltimore, 1 case of Hb J-Sicilia and 1 case of Hb Tamano. CONCLUSION The occurrence of abnormal structural Hb variants with many genotypes in Shanghai is unique. Except for Hb E, Hb Youngstown, and Hb M-Boston, other types of heterozygous are normal in phenotypes, and symptoms such as hemolysis and anemia often occur when other diseases are combined.
China ; Genotype ; Hemoglobins, Abnormal/genetics* ; Humans ; Phenotype ; alpha-Thalassemia ; beta-Globins/genetics*

OBJECTIVE: To determine the composition and distribution of beta-thalassemia-associated genotypes in Liuzhou area of Guangxi, China. METHODS: From January to December 2017, 13 847 individuals who came for premarital examination, maternity examination or health check were recruited with informed consent. The subjects were analyzed by reverse dot blotting (RDB) for 17 common beta-thalassemia-associated variants among the Chinese population. Individuals with inconsistent results by blood test, electrophoresis, and RDB were subjected to Sanger sequencing to detect rare variants of the beta globin gene. RESULTS: In total 2098 individuals were found to harbor beta-thalassemia-associated variants, which included 2075 heterozygotes (98.90%), 12 compound heterozygotes (0.57%) and 11 homozygotes (0.52%). CD41-42 (48.43%) and CD17 (31.45%) were the most common variants. Three hundred and thirty eight-individuals were found to also carry heterozygous variants of the alpha globin gene, with the most common types being --SEA/aa, -a3.7/aa, aCSa/aa, -a4.2/aa. Through Sanger sequencing, rare genotypes such as beta-32/betaN, betaCD41-42/betaIVS-II-5 and betaCD30/betaN were detected. CONCLUSION Liuzhou area has a high incidence of beta-thalassemia, but with a complex variant spectrum and clinical phenotypes different from other regions. Genetic counseling and prenatal diagnosis for the carrier population is crucial for the reduction of the related birth defects. Our result may provide valuable information for the prevention and control of beta-thalassemia in this area.
China ; Female ; Genetic Counseling ; Genetic Variation ; Genotype ; Humans ; Mutation ; Pregnancy ; Prenatal Diagnosis ; alpha-Globins ; genetics ; beta-Globins ; genetics ; beta-Thalassemia ; diagnosis ; genetics

OBJECTIVETo establish a comprehensive and simple assay using denaturing high performance liquid chromatography (DHPLC) for the diagnosis of most common mutations and deletions of &alpha;-thalassemia gene in Southeast Asians and Southern Chinese.

METHODSThis assay has included a duplex polymerase chain reaction (PCR) followed by DHPLC analysis. An improved PCR was also performed followed by DHPLC analysis. With this assay, a blinded study of 160 samples was screened for three common mutations and three common deletions.

RESULTSThe duplex PCR-DHPLC combined with the improved PCR-DHPLC analysis has detected all mutations and the wild-type allele. The results were consistent with those by the original methods.

CONCLUSIONThis molecular assay may be used for the diagnosis of &alpha;-thalassemia patients from this geographical region. The method is accurate, rapid, semi-automatic and cost-effective, which makes it suitable for large-scale screening.

Chromatography, High Pressure Liquid ; methods ; DNA Mutational Analysis ; methods ; Gene Order ; Genotype ; Humans ; alpha-Globins ; genetics ; alpha-Thalassemia ; diagnosis ; genetics

The molecular genetic characteristics of a family with rare -88 C>G (HBB: c.-138 C>G) β-thalassemia gene mutation were studied using cohort study. The cohort study was conducted from June to August 2022 by Prenatal Diagnosis Center of Sanya Women and Children's Hospital Managed by Shanghai Children's Medical Center. The phenotype and genotype were analyzed by hematological cytoanalyzer, automatic electrophoretic analysis system, and next-generation sequencing (NGS). And then, Sanger sequencing was used to verify the rare gene results. The results showed that the proband, her father, her uncle and her younger male cousin had discrete microcytosis (MCV 70.1 fl, 71.9 fl, 73.1 fl and 76.6 fl, respectively) and hypochromia (MCH 21.5 pg,22.0 pg,22.6 pg and 23.5 pg, respectively), elevated hemoglobin A2 level (5.3%, 5.4%, 5.4% and 5.5%, respectively), slightly elevated or normal fetal hemoglobin (Hb F), but no anemia. The proband was identified to have co-inherited ɑ-thalassemia (Hb Westmead gene heterozygous mutation, ɑ^wsɑ/ɑɑ) and β-thalassemia with a rare -88 C>G (HBB: c.-138 C>G) heterozygous mutation (β^{-88 C>G}/β^N). Her mother had the same α-thalassemia as the proband. Her father, her uncle and her younger male cousin had the same rare -88 C>G heterozygous mutations as the proband. While her grandmother and younger brother were not carrier of thalassemia. In conclusion, 4 cases of rare -88 C>G(HBB:c.-138 C>G) heterozygous mutation had been detected in a Chinese family. Carriers of this beta-thalassemia are clinically asymptomatic. This study enriches the knowledge of the thalassemia mutation spectrum in Chinese people and provides valuable information for genetic counseling, prenatal diagnosis, and prevention of thalassemia, providing a scientific basis for improving the quality of birth population and preventing birth defects.
Female ; Humans ; Male ; alpha-Thalassemia/genetics* ; beta-Globins/genetics* ; beta-Thalassemia/diagnosis* ; China ; Cohort Studies ; Genotype ; Molecular Biology ; Mutation

OBJECTIVE: To explore the application value of next generation sequencing (NGS) in preimplantation genetic diagnosis of α/β complex thalassemia couple. METHODS: The coding regions of α-globin genes (HBA1, HBA2) and β-globin gene (HBB) were selected as the target regions. The high-density and closely linked single nucleotide polymorphism (SNP) sites were selected as the genetic linkage markers in the upstream and downstream 2M regions of the gene. After NGS, the effective SNP sites were selected to construct the haplotype of the couple, and the risk chromosome of the mutation carried by the couple was determined. The NGS technology was used to sequence the variations of HBA1, HBA2 and HBB directly and construct haplotype linkage analysis for preimplantation genetic diagnosis. RESULTS: Direct sequencing and haplotype linkage analysis of HBA1, HBA2 and HBB showed that two of the six blastocysts were α/β complex thalassemia, one was β-thalassemia heterozygote, two were α-thalassemias heterozygotes, and one was intermediate α-thalassemia. A well-developed embryo underwent preimplantation genetic diagnosis was implanted into the mother's uterus, and a healthy infant was born at term. CONCLUSION Preimplantation genetic diagnosis can be carried out by NGS technology in α/β complex thalassemia couples, and abortion caused by aneuploid embryo selection can be avoided.
Female ; High-Throughput Nucleotide Sequencing ; Humans ; Mutation ; Pregnancy ; Preimplantation Diagnosis ; alpha-Thalassemia ; beta-Globins/genetics* ; beta-Thalassemia/genetics*