Humans ; Hemoglobinopathies ; Hemoglobins, Abnormal/genetics*

Humans ; Hemoglobinopathies ; Hemoglobins, Abnormal/genetics* ; Oxygen

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
Blastocyst ; CRISPR-Cas Systems ; Hemoglobins, Abnormal ; genetics ; metabolism ; Humans ; Zygote

OBJECTIVE: To explore the hematological phenotype and genotype of hemoglobin Q-Thailand in Fujian area. METHODS: Genomic DNA was extracted from peripheral venous blood samples of patients. Suspected samples were screened by hematological parameters analysis and verified with DNA sequencing. RESULTS: In 35 patients suspected with Hb Q-Thailand, 20 were confirmed, which included one case compounded with heterozygous β mutation and one compounded with Hb New York. CONCLUSION Analysis of hematological phenotype and genotype of Hb Q-Thailand can faciliate genetic counseling for patients from Fujian area.
China ; Genotype ; Hemoglobins, Abnormal ; genetics ; Heterozygote ; Humans ; Mutation ; Phenotype

OBJECTIVE: To explore the genetic basis for a child with Hemoglobin Santa Ana (Hb Santa Ana). METHODS: The child was admitted to the Children's Hospital of Chongqing Medical University on August 4, 2013 due to anemia, splenomegaly and deepening urine color for 5 years. His clinical data were collected. Peripheral blood samples of the child and his parents were collected for routine blood test. The erythrocyte parameters of the child and his parents were determined with an automatic hemocyte analyzer. The hemoglobin components of the child and his parents were detected by high-performance liquid chromatography (HPLC). Common mutations associated with thalassemia were detected by Gap-PCR and PCR-reverse dot blotting (PCR-RDB). Rare variants of the globin genes were detected by next generation sequencing (NGS), and the result was verified by Sanger sequencing. RESULTS: The child has shown mild to moderate hemolytic anemia. Routine blood test showed that he had lower hemoglobin (90 g/L) and mean corpuscular hemoglobin concentration (267 g/L) but a higher reticulocyte ratio (0.141), which indicated hypopigmented hyperplastic anemia. Analysis of hemoglobin component showed that his hemoglobin F was elevated to 10.7%, which indicated abnormal synthesis of β globin peptide chain. HPLC analysis showed an abnormal peak accounting for 4.5% of the total area. Neither of his parents was found to have abnormal results for routine blood test and hemoglobin component analysis. No common globin gene variant was detected in the child. Gene sequencing revealed that the child has harbored a heterozygous variant of HBB: c.266T>C, which was de novo in origin. Based on the guidelines of American College of Medical Genetics and Genomics, the variant was predicted to be pathogenic. CONCLUSION The heterozygous HBB: c.266T>C variant probably underlay the hemolytic anemia associated with Hb Santa Ana in this patient.
Child ; Humans ; Male ; Chromatography, High Pressure Liquid ; Hemoglobins, Abnormal/genetics*

OBJECTIVE: To investigate whether β-globin gene 3'UTR+101G>C (HBB:c.*233G>C) variant has genetic effect and provide basis for gene diagnosis and genetic counseling. METHOD: Whole blood cell analysis and capillary zone electrophoresis (CZE) were used to analyze the hematological indexes. The most frequent 23 mutations in southern Chinese individuals were routinely measured by PCR-flow fluorenscence immunmicrobeads assay. Sanger sequencing was used to detect the other variants of β-globin gene (HBB). RESULTS: In 463 cases, a total of 7 cases with HBB:c.*233G>C variant were detected, among them 4 cases carried other pathogenic variants of HBB gene (2 cases were in trans, 2 cases were in cis), who had typical hematological characteristics of mild β-thalassemia, and 3 cases also carried abnormal hemoglobin variation, but did not have hematological characteristics of β-thalassemia. CONCLUSION The study shows that HBB:c.*233G > C variant has no obvious genetic effect and should be a benign polymorphism.
3' Untranslated Regions ; Hemoglobins, Abnormal/genetics* ; Humans ; Mutation ; beta-Globins/genetics* ; beta-Thalassemia/genetics*

OBJECTIVETo analyze the relationship between genotype and phenotype of homozygous hemoglobin Constant Spring (Hb CS) in Guangxi province, and to explore the reasons of missed diagnosis and the methods for screening and diagnosing.

METHODSScreening Hb CS by acetate fibrous membrane electrophoresis with benzidine staining. Gene mutation of homozygous Hb CS by polymerase chain reaction (PCR) and DNA sequencing.

RESULTSOut of the 9 patients, 4 had no clinical symptoms, 2 showed mild anemia and splenomegaly, 3 were jaundice. Hemoglobin levels were normal or mild anemia. MCVs were normal or reduced. Peripheral blood smear of all the patients displayed hypochromia, anisocytosis, poikilocytosis and target cells. The quantities of HbA(2) + Hb CS were 4.3% - 6.72%, while HbA(2) < 2%. Gene analysis confirmed the diagnosis of homozygous Hb CS.

CONCLUSIONThere was quite different in clinical symptoms, hematological parameters and hemoglobin quantifications for patients with homozygous Hb CS. They might have no clinical and hematological signs and looked like the phenotype of Hb H disease. Homozygous Hb CS was very easy to miss diagnosis. Gene analysis can be helpful.

Adult ; Child ; Child, Preschool ; Female ; Hemoglobins, Abnormal ; genetics ; Humans ; Male ; Mutation ; alpha-Thalassemia ; genetics

OBJECTIVE: To analyze the hematological phenotypes of Hb J-Bangkok and concomitant thalassemia. METHODS: In total 72 397 samples were screened by using capillary electrophoresis. Samples with Hb J-Bangkok were identified by DNA sequencing and analysis of red blood cell parameters. Gap-PCR and PCR-reverse dot blotting (PCR-RDB) were used for analyzing the thalassemia genes. RESULTS: Thirty one cases of Hb J-Bangkok were identified, all of which were heterozygotes. The hematological phenotype index (Hb, mean corpuscular volume, mean corpuscular hemoglobin, Hb J-Bangkok, Hb A CONCLUSION Hb J-Bangkok heterozygotes have normal hematological phenotypes, though they may show different hematological characteristics when concomitant with different types of thalassemia, for which genetic counseling should be provided accordingly.
Female ; Hemoglobins, Abnormal/genetics* ; Heterozygote ; Humans ; Male ; Phenotype ; Thailand ; beta-Thalassemia/genetics*

OBJECTIVE: To analyze the hematological characteristics of Hb Broomhill and Hb Hornchurch, and prenatal diagnosis should be carried out in two families. METHODS: RBC parameters and hemoglobin electrophoretogram were analyzed on the peripheral blood of all patients, and amniotic fluid was collected for prenatal diagnosis. PCR-Flow fluorescent hybridization and Sanger sequencing were performed for gene diagnosis of thalassemia. RESULTS: Three cases of Hb Broomhill were detected, including 2 cases with common SEA α-thalassemia, which was characterized by hypochromic microcytic mild anemia, the capillary electrophoregram revealed a tiny shoulder peak before the Hb A peak; 1 case was diagnosed as Hb Hornchurch combined with β-thalassemia, which also showed mild anemia. Hemoglobin electrophoretogram showed an abnormal hemoglobin variant peak at Hb A CONCLUSION The carriers of Hb Broomhill and Hb Hornchurch do not have microcytic hypochromic anemia, which do not aggravate the hematological symptoms, such as anemia when being combined with thalassemia of the same type.
Anemia, Hypochromic ; Hemoglobins, Abnormal/genetics* ; Heterozygote ; Humans ; alpha-Thalassemia/genetics* ; beta-Thalassemia

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*