Identification Of A Novel Frameshift Mutation At Codon 53 (-T) In The β Globin Gene Causing Dominantly Inherited β Thalassemia In A Chinese Miao Family

Background and Objective:βthalassemia is one of the most common inherited disorders of hemoglobin synthesis in the world. The geographic distribution ofβthalassemia is in the tropical and subtropical areas with a high incidence of malaria, including North Africa, Mediterranean region, the Middle East, the Indian subcontinent, Southeast Asia and South China. About 0.5-6% of Chinese living in the areas with a high incidence ofβthalassemia are mutation carriers, p thalassemia of Guangxi, Guangdong and Hainan are more severe and the incidence rate ofβthalassemia major and intermedia is 0.4%o.βthalassemia is very common in south China, but the mutations in different regions and different ethnic groups have not been elucidated completely, especially in the minor ethnic group living in the remote areas.βthalassemia is generally considered as an autosomal recessive disorder because homozygote or double heterozygote is necessary to produce clinical feature. However, dominantly inheritedβthalassemia caused by a mutantβglobin gene has been found in different ethnic groups such as English, Italian, French, Czech, Spanish, Korean and Japanese. Molecular basis of dominantly inheritedβthalassaemia is mostly due to frameshift mutations, missense mutations and nonsense mutations, with a total of 40 causing-mutations previously reported. Among them, 29 are located in exon 3 and the remaining mutations occur at other sites including 7 in exon 2 of theβglobin gene. Severity of disease within this form of thalassemia ranges from a just detectable clinical phenotype to transfusion dependency.Based on population screening for heterozygous traits aimed at prevention of thalassemia major, we identified a novel frameshift mutation at cd53 (-T) in exon 2 of theβglobin gene resulting in dominantly inheritedβthalassemia and two novel RFLP haplotypes in a Chinese Miao family from Guizhou Province in Southwestern China. The functional effect of this mutation was accessed at the mRNA level by real-time quantitative reverse-transcript PCR (RT-PCR) technology.Materials and methodsThe proband was a 22-year-old woman with a typical anemia facies and had never been transfused. Splenomegaly and jaundice were noted. The woman and all her family members (53 subjects in three generations) belonged to the Miao population (a minor ethnic group in China) and were aborigines who lived in Guizhou Province in southwestern China. Analysis of her hematological parameters showed hypochromic microcytic anemia with reduced hemoglobin level, reduced mean corpuscular volume, reduced mean corpuscular hemoglobin and raised hemoglobin A2 concentration.βglobin gene mutation analysis for the proband was performed by using PCR-based reverse dot blot (RDB) hybridization technique. But after an investigation of 24 known mutations in Chinese population, no mutation was found. Therefore, this patient and her 17 family members were referred to our laboratory for further unknown mutation investigation, and all of them gave informed consent. Among the 17 family members, 6 and 11 were first and second degree relatives of the proband, respectively. The remaining members were not available for investigation.Genomic DNA was extracted from peripheral blood by standard phenol/chloroform method. The 24 knownβglobin mutations in Chinese were scanned by RDBhybridization technique. The common a thalassemia deletional mutations (--^SEA/,-α^3.7/ and -α^4.2/) were typed by gap PCR, and 6 non-deletional mutations (α^cd30α/,α^cd31α/,α^cd59α/,α^QSα/,α^XSα/ andα^WSα/) were scanned by RDB assay in all 18 samples tested. These nine mutations account for >98% of all a thalassemia chromosomes in the Chinese population.We design a real-time quantitative reverse-transcript PCR assay to evaluate the functional effect of this novel mutation on mRNA level. On the basis of two standard curves method, we calculate the mean mRNA relative concentration in 6 mutation carriers and 6 normal individuals.Theβglobin haplotype associated with the novel mutation in this family was ascertained by PCR-RFLP technique and family linkage analysis. Seven classical polymorphic restriction enzyme sites selected for haplotype analysis were Hinc II-5'ε, HindⅢ-Gγ, HindⅢ-Aγ, HincⅡ-Ψβ, HincⅡ-3'Ψβ, AvaⅡ-βand Bam HI-3'β.The difference of mean mRNA relative concentration between mutation carriers and normal individuals were analyzed by the independent samples t test using statistical software SPSS, version 13.0.Results and discussion Direct DNA sequencing of the entire humanβglobin gene was performed in the 18 family members and mutation cd53 (-T) was identified in the proband and the other 6 family members. 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 novelβthalassemia mutation, which had never been reported.Acording to the pedigree of this Miao family, every mutation carrier only had one affected parent, andβthalassemia intermedia phenotype appeared in every generation. Thus, the disorder was transmitted in an autosomal dominant fashion in this family.Up to now, about 40 dominantly inheritedβthalassemia alleles have been described in patients with thalassemia intermedia from dispersed ethnic origins. At the molecular level, they fall into four groups: missense mutations, minor deletions or insertions of intact codons, single base substitutions leading to premature termination, and mutations causing frameshifts or aberrant splicing resulting in elongated or truncatedβglobin variants with abnormal carboxy terminal ends. To our knowledge, four frameshift mutations located in exon 2 of theβglobin gene were previously reported to produce dominantly inheritedβthalassemia. These four mutations, cd91 (-T), cd94 (+TG), cd100 (-CTT, +TCTGAGAACTT) and cd104 (-G) lead to 156, 156, 158 and 156 amino acids extendedβglobin variants, respectively. Because cd53 (-T) results in a 59 amino acidsβglobin variant, it is the first frameshift mutation reported in exon 2 leading to a truncatedβ-globin variant and producing dominantly inheritedβthalassemia.The deletion of a nucleotide T at cd53 within exon 2 causes a frameshift mutation that can result in a premature termination code (PTC) 19 bp downstream. In most of cases with nonsense or frameshift mutations, nonsense-mediated mRNA decay (NMD) will be triggered to degrade mRNA containing PTC. However, in our study we found there is no statistical difference of theβglobin mRNA level between heterozygotes with cd53 (-T) mutation and normal individuals. The possible reason is that the novel mutation may be NMD-resistant. Translation reinitiation can abrogate NMD if the mutation is close enough to the downstream AUG codon. Such translation reinitiation model has been described in the triosephosphate isomerase gene and theβglobin gene. Thus, this is a possible explanation for NMD resistance of cd53 (-T) mutation since there is an AUG codon located at codons 73/74 of theβglobin gene. Translation of the mutantβglobin mRNA could give rise to truncated p chains which precipitate in the erythrocyte with the concomitant redundant a chains and lead to increased ineffective erythropoiesis. This mechanism could explain thalassemia intermedia phenotype observed in heterozygotes with cd53 (-T) mutation from the Miao family. Our work is the first one showing that frameshift mutation in exon 2 of theβglobin gene can give rise to almost normal mRNA level and further research is needed to demonstrate molecular basis including mRNA stability and its effect on translation. Miao population is the fourth largest minor ethnic group in China, with a total population of 8.94 million. Half of them live in Guizhou Province and the remains scatter over south China. Because of a long time migration of Miao population, there are over 3 million people living in Vietnam, Thailand, Laos, USA, Canada, France and Australia. The carrier rate ofβthalassemia in the Miao population is currently under investigation and it is the first time to identify a novel mutation in this ethnic group based on such genetic epidemiology survey. Studies on the prevalence and spectrum ofβthalassemia in a large population are very important for developing a community-based prevention program in the areas where the Miao people live in with high frequency of thalassemia alleles. In addition to this novel frameshift cd53 (-T) mutation, we ascertained two unreportedβglobin haplotypes in this Miao family,which are designated as haplotype Miao A for "----+-+"and haplotype MiaoB for "--+++-+". Haplotype Miao A was observed in all seven heterozygotesassociated with the novel mutation cd53 (-T) and Haplotype Miao B only existed in one family member III 9 inherited from his mother. Knowledge aboutβglobin haplotypes in the Miao people could be useful in determining the pattern of gene flow, population affinities, and origin of this ethnic group.