Identification Of 2 Novel Mutation Genes Causing Thalassemia In Chinese People

Background: Thalassemia caused by unbalance of alpha and beta haemoglobin chains and decrease of haemoglobin is the most common monogenic inherited disease in the tropical and semitropical regions of the world. It is a autosomal recessive inheritance disease and is defined by alpha thalassemia and beta thalassemia according to the damage of alpha or beta haemoglobin synthesization. The genotype of alpha/beta thalassemia is different from each other in different areas of the world. In china, the most common genotypes of alpha thalassemia are -SEA/aa -a3.7/aa, and -a4.2/aa, which caused by deletion, and aCSa/aa aQSa/aa, which caused by point mutation. And the most common genotypes of beta thalassemia are CD41/42(-TCTT), rVS-2-654(C-T), -28(A-G), CD71/72(+A), BE :CD26(G-A), CD17(A-T), which are all point mutations and offer up 90% of the all beta thalassemia genotypes in southern china. However there are still some novel or rare mutations occurred in alpha or beta haemoglobin gene being found frequently, such as -a2.7/aa found in the alpha globin gene cluster and CD37 TGG-TGA found in beta globin gene. These novel or rare mutations are of supplement to the common mutations and importance for mapping the whole mutations of thalassemia of a certain areas, which benefit to gene diagnosis. Identification of novel or rare mutations of thalassemia is a long-term and seriate work, which needs chance, keenness of researchers and hard work.Test patients with thalassemia trait and screening the people in communities are two ways to identify the novel or rare mutation genes causing thalassemia. Recently, we perform a epidemiological investigation of alpha and beta thalassemia in five cities by phenotype method and gene diagnosis and a locale investigation in sihui city bythe same method. In these works, we got some samples with alpha or beta thalassemia trait but no known mutations been found in these samples. Then we used DNA mapping method such as southern blot, gap-PCR and DNA sequencing to identify the potential novel mutations. Semiquantitative method RT-PCR is used to measure the expression of these novel mutation genes in mRNA level after they are identified.Design and method: cluster sampling is used to collect samples. About 5606 cord blood samples and 7792 peripheral blood samples were used in this study. We perform haematological analysis by blood cell counting and haemoglobin electrophoresis and genotyping by gap-PCR and RDB aimed at the common alpha and beta thalassemia mutations in south china. The sample which haematological phenotype is abnormal but genotype is 'normal' is tested further. That is ,cord blood samples are tested by southern blot for the novel deletion and sequenced for the point mutation occurred in the alpha 1 and alpha 2 globin genes. And peripheral blood samples are tested by sequencing the whole beta globin gene for possible mutations. The novel mutations identified in this study are tested further for their effect on transcript levels by RT-PCR.Result and discussion: we found a novel 11.1kb deletion occurred in alpha globin gene cluster and a novel mutation(C-T) in the position of -90 in the beta globin gene. These two novel mutation genes are registered in the GeneBank, the GeneBank Accessions are AY342392 and AY260740 respectively. RT-PCR analysis shows the alpha mRNA levels of patients with -11.1/aa is the same as that of -SEA/aa patients, but obviously decreases compared with that of the normal persons. Also the beta mRNA of the patients with -90 mutation decreases compared with that of normal persons. Besides, we identified anothertwo mutations occurred in alpha globin gene cluster. One is +TCA insert in position 118 of the alphal gene(GeneBank Accession: AY261678) and the other is --THAI/aa mutation that is the first case reported in the mainland of China.Our discoveries perform a new insight to the thalassemia in China and in the world. The mutations identified in the study are the bases of gene diagnosis and furthermore they are potential research materials for human mol...