Identification Of The De Novo Mutation At The α₂-globin Gene And SEA Type Of α-thalassmia Deletion Causing Hemoglobin H Disease

Background and ObjectiveThe inherited hemoglobin disorders are one of the most common human monogenic diseases worldwide. Among them,α-andβ-thalassemia are two most common forms mainly occurred in the tropical and sub-tropical regions of the world, which mostly result from the gross deletions ofα-globin gene cluster and the point mutations in theβ-globin gene (HBB), respectively. Both nondeletional a-thalassemia mutations and gross deletions of P-thalassemia are relatively uncommon. In addition to this, rare de novo alterations leaded to these two disorders have been characterized in some unusual cases withα-orβ-thalassemia phenotypes, which included a few de novo deletions located on theα-globin locus, particularly those abnormality causing ATR-16 syndrome and a group of spontaneous mutations occurred within the P-globin gene identified in various populations.α-thalassemia is one of the most common hereditary disorder in which a-globin chain synthesis is either decreased or absent. The resulting imbalance of globin chain synthesis leads to the accumulation of excess y-globin or P-globin chains which eventually causes the destruction of affected red blood cells. According to the epidemiologic data, the incidence of this disease screened by electrophoresis in seven provinces of China was calculated to be 2.64%. The data show that the incidence of a-thalassemia in Guangdong province is 4.11%.Hemoglobin (Hb) H disease is a moderate form of a-thalassaemia resulting from various genetic defects and it is particularly prevalent in Southeast Asia and in southern China. There is the heterogeneity of Hb H disease genotypes in the different endemic populations. Interaction of common types ofα0-thalassemia deletions (such as--SEA/allele) with the silentα+-thalassemia deletions (such as-α3.7/or-α4.2/allele) or nondeletionalα-thalassemia mutations in theα2-globin gene (HBA2) (such asαConstant Springα/allele)have been extensively demonstrated to be causes of giving rise to a Hb H disease phenotype. Only one case with Hb H disease attributed to the phenotype of (--de novo/-α3.7) involving a de novo deletion of the entireξ-αglobin gene cluster was firstly reported by Waye.In this report, we identified a novel de novo frameshift mutation (-C) occurred at the HBA2 gene in a Chinese boy with Hemoglobin H disease. We have performed establishment of a genotype-phenotype correlation in a Chinese family with a-thalassemia involving this de novo mutation defined as a cause of Hb H disease in our study. To our knowledge, this is the first time the de novo single-base lesion causing a-thalassemia has been reported to occur in human a-globin gene.Materialss and MethodsSubjectsThe proband, a 2-year-old boy who was the offspring of unrelated parents that originated from Guilin city, Guangxi Province of southern China. The pregnancy was uneventful, and he was delivered vaginally with a birth weight of 3650g. There was no history of jaundice at birth and no obvious retardation of growth. He looked pale when he was 6 months old. He was first diagnosed as Hb H disease at age one year according to the clinical phenotype of moderate anemia and mild hepatomegaly, as well as a typical hematological features of hypochromic microcytosis with Hb level of 8.0g/dL, a mean cell volume (MCV) of 56.3fL, and a mean cell hemoglobin (MCH) of 17.8 pg. Analysis of his hemoglobin showed Hb H band. Iron deficiency was excluded.During the following one year, his anemia had not worsened and he never received a blood transfusion up till now. He was referred for molecular test for confirmatory diagnosis at age of 2 years. He and his family member's peripheral blood samples were collected using EDTA as anticoagulant after informed consent was obtained.Hematologic MethodsHematological parameters were measured by an automated cell counting (Model Sysmex F-820; Sysmex Co Ltd, Kobe, Japan) and quantification of hemoglobin were conducted on the capillary electrophoresis (CE) device (Capillarys, Sebia, Montpellier, France).DNA analysis ofα-andβ-globin geneGenomic DNA was extracted from peripheral blood leucocytes by standard phenol/chloroform method. The genotype of known types of defects in Chinese population both in a-globin and in P-globin genes was performed using our protocols as described previously. Direct DNA sequencing of the entire both a-globin genes (α1 andα2, GenBank Accession NG.000006) was performed for identifying the unknown mutation as previously described. To exclude the possibility of a somatic mosaicism of a point mutation in the HBA2 gene, that is a phenomenon previously defined as a cause of thalassemia intermedia (TI), we performed a sequencing of a2-globin gene of the patient's genomic DNAs derived from lymphocytes, hair root and oral mucosal exfoliated cells, respectively. Their paternity tests were conducted by using a set of fifteen short tandem repeat (STR) markers and amelogenin gene.RNA analysisWe design a real-time quantitative reverse-transcript PCR asaay to evaluate the expression level ofα-globin gene mRNA. On the basis of two standard curves method, the gene expression at the mRNA level of both the mutant and wild-type a-globin alleles were measured by using SYBR Green-based relative quantitative RT-PCR method and theβ-globin gene served as a control for assessment of equivalent RNA loading as our previous protocol. Four independent tests for each of samples with three different a-globin genotypes (the proband, his mother and a normal people) were conducted in order to calculate the mean mRNA concentration.ResultsHematological data analysisThe proband (Ⅲ:1) was found to have a Hb H disease phenotype with a hypochromic microcytic anemia (Hb 8.4g/dL, MCV 60.7 fL and MCH 15.9 pg), 3.2%Hb H,5.4%HbBart's. His mother (Ⅱ:1) also have a hypochromic microcytic anemia. In addition, his three other family members (Ⅰ:1,Ⅱ:2,Ⅱ:3) showed normal red-cell parameters and hemoglobin electrophoretic profile. DNA analysisDirect DNA sequencing of the entire human a-globin gene was performed and mutation cd44 (-C) was identified in the proband. After retrieving PubMed and online database of human hemoglobin variants and thalassamias on the Globin Gene Server website (http://globin.cse.psu.edu/), it was confirmed to be a novelα-thalassemia mutation, which had never been reported. Results of molecular studies showed that the proband's mother carries the Southeast Asianα-thalassemia deletion, which the proband inherited, whereas his father does not have any commonα-thalassaemia deletions or point mutations. The sequence results of the proband's DNA derived from lymphocytes were completely consistent with that of testing his hair root and oral mucosal exfoliated cells.thereby excluding the possibility of a somatic mosaicism of a point mutation in the HBA2 gene. The results of the DNA paternity testing proved that the alleged father is the biological father of the proband and the probability of paternity is 99.99%. RNA analysisWe determined that the mean relative a-globin mRNA levels of 0.0856±0.01 in the proband versus of 0.598±0.069 in his mother (a SEA deletion carrier) when the normal standard was defined as 1.0.DiscussionAlthough parentally inherited alterations involved in a-globin genes are responsible for a large majority of cases, a few de novo abnormalities can result in a-thalassemia phenotypes, particularly de novo gross deletions defined as the cause of ATR-syndrome or a very rare form of Hb disease. In addition to this, only a few cases of spontaneous mutations identified at both a-globin genes (α1 andα2), that give rise to the structural Hb variants were previously reported in very small individuals. We report here for the first time, in a Chinese patient, a de novo single mutation event at the a-globin gene detected in a nondeletional Hb disease.According to the data observed from our family study, we report a proband with Hb H disease who is constitutionally heterozygous for the SEA type ofα0-thalassemia deletion inherited from his mother. The proband's father shows a normal both in the hematological phenotype and a-globin genotype, thereby indicating that the abnormality arose as de novo events. Based on the results of excluding the possibility of a somatic mosaicism of a point mutation in the HBA2 gene, it is suggested that the de novo single-base deletion should have arisen during the spermatogenic process or earlier embryonic stage. Thus, the effects on expression of this de novo mutant allele should correspond to that of an inherited mutation. Therefore, interaction of this de novo frameshift mutation (-C) at theα2-globin gene with the SEA type ofα0-thalassemia deletion in the proband could give rise to a Hb H disease phenotype). Our observation that the clinical manifestation of classic Hb H disease presented in the boy aged 2 may be classified into anemia of moderate severity known as the nondeletional form of Hb H disease.We determined that the mean relative a-globin mRNA levels of 0.0856±0.01 in the proband versus of 0.598±0.069 in his mother (a SEA deletion carrier) when the normal standard was defined as 1.0. Thus indicating that this de novo frameshift mutation in the HBA2 gives rise to a significantly reduced the mRNA levels ofα2-globin gene expression. As the frameshift mutation generates a premature translation termination codon at position 48, it most likely that the mechanism of a nonsense-mediated mRNA decay (NMD), that had been demonstrated previously by the very similar case with the inherited microdeletion (-C) at the a2-globin gene, should be responsible for the reduced mRNA levels although we could not directly investigate the effect of the nonsense codon on theα2-globin gene expression.