Zeng Y, Huang S
Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, 24/1400 West Beijing Road, 200040, Shanghai, China.
Clin Chim Acta. 2001 Nov;313(1-2):107-11. doi: 10.1016/s0009-8981(01)00660-x.
In the past two decades, a large-scale survey of hemoglobinopathies and thalassemia was carried out in mainland China, involving nearly one million people in 28 provinces. The incidences of hemoglobin (Hb) variants, alpha-thalassemia and beta-thalassemia were 0.33%, 2.64% and 0.66%, respectively. The chemical structural analysis identified 67 Hb variants. Among them, 20 are new variants. The analysis of the alpha-globin gene organization in 111 HbH patients showed 76 cases (68.5%) were of the deletion type, 8 had Hb Constant Spring and the other cases were of non-deletion type. The results of the molecular characterization of more than 200 beta-thalassemia alleles showed that the most common types of beta-thalassemia mutations in China are CD 41/42 (-4 bp), IVS-II-nt.654 C-->T, CD 17 A-->T, CD 71/72 (+A) and -28 A-->G.
To explore the simple method for molecular diagnosis of beta-thalassemia, multiplex allele-specific amplification (MAS-PCR) was used that could simultaneously detect the above five common types of beta-thalassemia mutations.
Based on the molecular analysis of beta-thalassemia intermedia, beta-thalassemia homozygotes or compound heterozygotes combined with alpha-thalassemia, as well as the conjunctive abnormalities of beta-thalassemia heterozygote with triplicated haplotype of alpha-globin genes, were the most common cause of thalassemia intermedia in China. We also used the RT-PCR quantitation method to show that the most common beta-thalassemia allele, IVS-II-nt.654 C-->T, still produced a small amount (about 15%) of normally spliced beta-globin mRNA, therefore, causing beta+-thalassemia. In clinical trials of hydroxyurea (HU) treatment for beta-thalassemia patients, we found that HU may enhance the expression of the beta-globin gene in some patients, leading to an alleviation of clinical symptoms. In the studies of the reversal of aberrant splicing of IVS-II-nt.654 C-->T allele by the antisense approach, we constructed a mammalian expression vector that can produce an antisense RNA targeting against the aberrant splice sites of IVS-II-nt.654 C-->T pre-mRNA.
The results indicated that the antisense RNA produced from the vector could efficiently suppress the aberrant splicing pattern and restore the correct splicing pathway in vitro and in vivo, leading to the improvement of globin chain biosynthesis in thalassemia cells.
在过去二十年中，中国大陆开展了一项针对血红蛋白病和地中海贫血的大规模调查，涉及28个省份近100万人。血红蛋白（Hb）变异体、α地中海贫血和β地中海贫血的发病率分别为0.33%、2.64%和0.66%。化学结构分析鉴定出67种Hb变异体。其中，20种为新变异体。对111例HbH患者的α珠蛋白基因结构分析显示，76例（68.5%）为缺失型，8例有Hb Constant Spring，其他病例为非缺失型。对200多个β地中海贫血等位基因的分子特征分析结果表明，中国β地中海贫血最常见的突变类型为CD 41/42（-4bp）、IVS-II-nt.654 C→T、CD 17 A→T、CD 71/72（+A）和-28 A→G。
为探索β地中海贫血分子诊断的简便方法，采用多重等位基因特异性扩增（MAS-PCR）技术，可同时检测上述五种常见的β地中海贫血突变类型。
基于对中间型β地中海贫血、β地中海贫血纯合子或复合杂合子合并α地中海贫血以及β地中海贫血杂合子与α珠蛋白基因三联单倍型联合异常的分子分析，发现这些是中国中间型地中海贫血最常见的病因。我们还采用RT-PCR定量方法表明，最常见的β地中海贫血等位基因IVS-II-nt.654 C→T仍能产生少量（约15%）正常剪接的β珠蛋白mRNA，因此导致β+地中海贫血。在羟基脲（HU）治疗β地中海贫血患者的临床试验中，我们发现HU可能会增强某些患者β珠蛋白基因的表达，从而减轻临床症状。在通过反义方法逆转IVS-II-nt.654 C→T等位基因异常剪接的研究中，我们构建了一个哺乳动物表达载体，该载体可产生针对IVS-II-nt.654 C→T前体mRNA异常剪接位点的反义RNA。
结果表明，该载体产生的反义RNA在体外和体内均可有效抑制异常剪接模式并恢复正确的剪接途径，从而改善地中海贫血细胞中的珠蛋白链生物合成。
