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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

Author: YiPeng
Tutor: XuXiangMin
School: Southern Medical University,
Course: Cell Biology
Keywords: Dominant inheritance β-thalassemia Frameshift mutations Intermediate beta thalassemia Haplotype β-globin gene Nonsense mutation -mediated mRNA decay
CLC: R556
Type: Master's thesis
Year: 2008
Downloads: 72
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Abstract

Background and Purpose beta thalassemia (βthalassemia, referred to as β-thalassemia) [MIM ??141900] is one of the world's most common single gene disorders, β-thalassemia geographic distribution in the world's tropical and sub-tropical malaria-prone areas, including North Africa , the Mediterranean region, the Middle East, the Indian subcontinent, Southeast Asia and southern China. The frequency of gene carriers of the disease in the population of southern China a high incidence of 0.5-6%, which is staggering, especially in Guangxi, Guangdong and Hainan provinces (regions), β-thalassemia major population of these regions (including intermediate the incidence of thalassemia) is estimated at 0.4 ‰. Beta thalassemia is very common in southern China, but the different regions and ethnic groups thalassemia mutation spectrum yet to be confirmed, especially rare lead to the identification of the β-globin gene mutations. Improve China's southern population beta thalassemia mutation spectrum is important for guiding the clinical diagnosis and genetic counseling, and the development of effective crowd prevention programs. β-thalassemia is generally considered an autosomal recessive genetic disease, β-globin gene, homozygous or double heterozygous can lead to disease. However, caused by the β-globin gene mutation heterozygous dominant inheritance with β thalassemia rare cases have been found in multiple ethnic groups, such as β-thalassemia dominant mutations in the British, Italian, French, Czechs, Spaniards, Koreans and Japanese have been reported. Dominantly inherited β thalassemia major by the β-globin gene frameshift mutations caused by missense mutations and nonsense mutations. Mutations have been reported in a total of 40 kinds. Among them, there are 29 kinds in exon 3,7 located in exon 2, and the rest located in exon 1 and exon intron splicing region. Dominantly inherited β thalassemia clinical performance variation can all exist to transfusion dependence symptoms from mild clinical symptoms. A Hmong family in Guizhou Province of β-thalassemia mutation screening process identified a cause dominantly inherited β thalassemia new frameshift mutation-cd53 (-T), as well as two new RFLP single haplotype. Based on pedigree analysis, real-time fluorescence quantitative reverse transcription PCR technology, we analyzed the mRNA level of this new mutation of the gene transcriptional effects. Materials and methods proband is a 22-year-old young woman, typical face of anemia, yellowish discoloration of skin, abdominal palpation, enlargement of the liver, there is no history of blood transfusion. Proband and her family members (53, 3 generation) are native to the Hmong people of Anshun in Guizhou Province in China. Routine inspection of the proband small cell hypochromic anemia, Hb content 9.9g/dl, MCV and MCH reduce, HbA2 increased. 24 kinds of β-thalassemia point mutations, known to the Chinese people using reverse dot blot screening, no positive results. Signed informed consent, our investigation of the pedigree of the proband and his 17 family members. 17 family members, 6 of whom were first-degree relatives of probands, 11 were two relatives of the proband. For various reasons, the rest of the family members did not participate in the study. Peripheral blood genomic DNA samples using phenol / chloroform extraction. Reverse dot blot technique for Chinese people known 24 β-thalassemia point mutation genotyping. Gap-PCR technology for the Chinese people known common alpha thalassemia deletion mutation (- ^{SEA /-α ^{3.7 / and-α ^{4.2 /) analysis; reverse dot blot technique for Chinese people known alpha thalassemia point mutation (α ^{, cd30 α / alpha ^{cd31 α /, α ^{cd59 experienced α / α ^{QS α /, α ^{CS α / and α ^{WS α /) diagnosis. These nine mutations account for 98% of the known Chinese human α-thalassemia mutations. Evaluation of β-thalassemia mutations in the β-globin gene expression levels, we have designed a real-time quantitative reverse transcription PCR experiments using quantitative analysis based on the double-standard curve method, the pedigree of six new mutation carriers and 6 normal mRNA gene expression levels of detection. And independent samples t-test to analyze the relative mRNA content of the difference between the average mutation group and normal control group. To investigate the molecular genetics background of new mutations, we chose the β-globin gene cluster of seven classic restriction sites (ie Hinc Ⅱ-5'ε, Hind Ⅲ-Gγ, Hind Ⅲ-Aγ, Hinc Ⅱ-Ψβ, Hinc Ⅱ -3'Ψβ, Ava Ⅱ-β, and Bam HI-3'β) used to build the β-globin gene PCR-based RFLP haplotypes. Results and discussion the proband and the family members of the β-globin gene is fragment was amplified by PCR and PCR direct DNA sequencing analysis, we found that the proband and six of its phenotype positive family members , there are a mutant CD53 (-T). Literature search in PubMed the query globin gene Services Web site (http://globin.cse.psu.edu/), were not found in this type of mutation. Confirmed that this mutation is dominantly inherited β thalassemia cases reported for the first time in the first reported β-thalassemia mutation type, but also the Chinese people crowd. By observing the pedigree of 53 people, can be seen in the parents of a β thalassemia patients, only one party involved. And the intermediate β thalassemia symptoms appeared in every generation of the family. The The Genealogy characteristics consistent with autosomal dominant inheritance passed. Through genetic analysis, the pedigree exclude the possibility of the the combined Chinese people known α-thalassemia mutation; quantitative real-time reverse transcription PCR analysis showed that the β-globin gene expression levels of the case group and the control group no significant difference. At present, the world found that 40 kinds can cause dominantly inherited the B thalassemia gene mutations. There are four types as follows: ① missense mutation at the molecular level, the genetic defect; ② complete codon, deletion or insertion; ③ nonsense mutations; ④ generated with the exception of carboxy-terminal extension or truncation protein frameshift mutation and abnormal shear point mutations. Have been reported in the β-globin gene second exon frameshift mutations can cause dominantly inherited β thalassemia. These four kinds of mutations: CD91 (-T), CD94 (TG), CD100 (- the CTT TCTGAGAACTT) and cd104 (-G), respectively, lead containing 156,156,158 and 156 amino acids of the extension of the β-globin mutation was generated. Cd53 (-T) led to contain 59 amino acids of the β-globin variants, so this is the first in the lead on the second exon truncated β-globin variants and cause dominantly inherited β thalassemia a frameshift mutation. β-globin gene cd53 missing nucleotide T, resulting in a premature termination codon sub (PTC) at its downstream 19bp. Most nonsense mutations and frameshift mutations can trigger nonsense-mediated mRNA decay (NMD), to clear the PTC-containing mRNA. However, the study suggests that the frameshift mutation in the β-globin gene exon to cause disease is not significantly reduce the level of mRNA transcription. The possible reason is the \If the mutation and downstream of the AUG codon is close enough, the translation reinitiation mechanism able to eliminate the role of the NMD. This the theory has triosephosphate isomerase gene and β-globin gene Guozha described. Since codons 73/74 also has one of the AUG translation reinitiation mechanism will also be explained cd53 (-T) \Abnormal β-globin mRNA can be translated to generate a large number of truncated β-globin, and abundant α-globin deposited simultaneously within red blood cells, resulting in red blood cells of invalid generate. This mechanism could explain cd53-T mutation carriers showed intermediate β-thalassemia phenotype. Further research is needed to clarify the basis of cd53 (-T) mutation produces abnormal mRNA stability and translation of molecular biology. Miao is China's fourth largest ethnic minority population of 8.94 million. Half of them living in Guizhou Province, the rest distributed in several other provinces of southern China. The the Miao nation in the history of long-term migration in Vietnam, Thailand, Laos, the United States, Canada, France and Australia, there are over 300 million population distribution. China's Miao beta thalassemia carriers is still under investigation, this study is the new beta globin gene mutations found in the nation for the first time. Because most of the Hmong population living in areas of high incidence of thalassemia, so for a large number of the population of the incidence of thalassemia and mutational spectrum of research, the formulation of the thalassemia community-based prevention programs. In addition to new mutations cd53 (-T), we also identified two new beta globin gene haplotype, ie Miao A \Miao A, haplotypes exist in all seven new mutation carriers, linked inheritance cd53 (-T). Miao B, haplotype exists only among the members of a family, passed by her mother to come. The Hmong population beta globin gene single haplotype new knowledge, will contribute to the study of gene flow, crowd kinship and ethnic origin.}}}}}}}}}

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