A Novel 5 kb Deletion in the ß-Globin Gene Cluster Identified in a Chinese Patient.

ß-Thalassemia (ß-thal), a highly prevalent disease in tropical and subtropical regions of Southern China, is caused mainly by point mutations in the ß-globin gene cluster. However, large deletions have also been found to contribute to some types of ß-thal. We identified a novel 5 kb deletion in the ß-globin cluster in a Chinese patient using multiplex ligation-dependent probe amplification (MLPA), and characterized it with single molecule real-time (SMRT) sequencing, gap-polymerase chain reaction (gap-PCR) and Sanger sequencing. The deletion was located between positions 5226189 and 5231091 on chromosome 11 (GRCh38), extending from 4 kb upstream of the 5' untranslated region (5'UTR) to the second intron of the ß-globin gene. The patient with this deletion presented with microcytosis and hypochromic red cells, as well as relatively high Hb F and Hb A2 levels. Our research indicated that SMRT sequencing is a useful tool for accurate detection of large deletions. Our study broadens the spectrum of deletional ß-thalassemias and provides a perspective for further study of the function of the ß-globin cluster.

[Differential Diagnosis of Three Commonest Deletion ß-Thalassemia in Chinese].

OBJECTIVE: To analyze the hematological characteristics of Chinese Gγ+(Aγδß)0-thalassemia，SEA-HPFH and Taiwan type ß-thalassemia. METHODS: Hemoglobin electrophoresis and blood routine test were used to analyze the hematological indexes of all peripheral blood samples，PCR-Flow fluorescent hybridization and Gap-PCR were used to detect the globin gene mutations and the data were analyzed statistically. RESULTS: The 3 types of deletion ß- Thalassemia patients were showed as hypochromic small cell anemia. The MCH and MCV values of Taiwan type ß-thalassemia patients were the lowest. The results of hemoglobin electrophoresis showed that the increasing of HbF was found in all of the 3 types. Except for the decreasing of Hb A2 in Chinese Gγ+(Aγδß)0-thalassemia，the levels of Hb A2 in the other two deletion ß-thalassemia patients were significantly increased. Except for Hb, there were significant differences in MCV, MCH, Hb A2 and HbF between Chinese Gγ+(Aγδß)0-thalassemia and SEA-HPFH(P<0.001). CONCLUSION: Through analyze the hematological characteristics, it can be provide that the guidance for the differential diagnosis and genetic consultation of the three commonest deletion ß-thalassemia in Chinese.

[Genetic Effect Analysis of ß-globin Gene 3'UTR+101G>C (HBB:c. *233G>C) Variant].

OBJECTIVE: To investigate whether ß-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 ß-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 ß-thalassemia, and 3 cases also carried abnormal hemoglobin variation, but did not have hematological characteristics of ß-thalassemia. CONCLUSION: The study shows that HBB:c.*233G > C variant has no obvious genetic effect and should be a benign polymorphism.

[Hematological Analysis and Diagnosis of Two Rare Abnormal Hemoglobin].

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 ß-thalassemia, which also showed mild anemia. Hemoglobin electrophoretogram showed an abnormal hemoglobin variant peak at Hb A2 zone. 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.

[Gene Diagnosis and Phenotypic Analysis of ß-Thalassemia Caused from a Rare Synonymous Mutation CD29 (C>T)].

OBJECTIVE: To investigate the gene diagnosis and phenotypes analysis for a couple with ß-thalassemia suspected from of blood routine test and hemoglobin electrophoresis, as well as the prenatal gene diagnosis of the fetus. METHODS: The gene mutation of ß-globin in the samples of peripheral blood of pregnant woman and her husband, as well as amniotic fluid of pregnant woman were analyzied and identified by using PCR-RDB and Sanger sequencing. RESULTS: The detection showed that the heterozygote mutation of IVS-Ⅱ-654 (C>T), which is common mutation of ß-globin gene, existed in pregnant woman, while her husband carried a rare mutation CD29 (c.90 C>T) of ß-globin gene. The prenatal diagnosis indicated that the fetus inherited with mutation from the parents, fetus genotype was ßIVS-Ⅱ-6541/ßCD29. CONCLUSION: The CD29(C>T) mutation of ß-globin gene has been identified in Chinese population first. Although this mutation type is symonymous mutation, but its carrier displays phenotype of ß-thalaessmia. Therefore, the attention to this mutation should be paid considering the genetic risk. It contributes to genetic counseling and prenatal gene diagnosis.

[Genetic Analysis, Prenatal Diagnosis and Preimplantation Genetic Diagnosis of Taiwanese Deletion ß-Thalassemia].

OBJECTIVE: To perform genetic analysis, prenatal diagnosis and preimplantation genetic diagnosis (PGD) in a family with a rare deletional ß- thalassemia. METHODS: Hematological parameters of the peripheral blood collected from all the family members were analyzed by whole blood cell analysis and capillary zone electrophoresis (CZE). Polymerase chain reaction-reverse dot blot (PCR-RDB) was used to identify 17 common ß- thalassemia gene mutations, the multiplex ligation-dependent probe amplification (MLPA) and gap-polymerase chain reaction (gap-PCR) were used to identify ß- globin gene cluster deletions. Chorionic villus sample or umbilical cord blood was obtained for prenatal diagnosis. Oligo-cells from blastocyst biopsy were collected for preimplantation genetic diagnosis by whole genome amplification and next generation sequencing. RESULTS: The proband was a carrier of Taiwanese deletion ß- thalassemia, two fetuses were both thalassemia majors. The PGD results showed that 6 of 11 tested embryos could be choose for transplantation. CONCLUSION: The Taiwanese deletion is a rare type deletion of ß- globin gene cluster, and it can lead to thalassemia intermedia or thalassemia major when compounded with other ß- globin gene mutation. PGD is another choice for thalassemia couples.