Institute of Genetics and Biophysics "Adriano Buzzati Traverso" (IGB-ABT, CNR), National Research Council, 80131 Napoli, Italy.
Department of Precision Medicine, University of Campania L. Vanvitelli, 80138 Napoli, Italy.
Int J Mol Sci. 2023 Jan 30;24(3):2577. doi: 10.3390/ijms24032577.
α-thalassemia is characterized in about 80% of cases by deletions generated by the presence of duplications and interspersed repeated sequences in the α-globin gene cluster. In a project on the molecular basis of α-thalassemia in Southern Italy, we identified six families, showing an absence of the most common deletions, and normal α-globin gene sequences. Multiplex Ligation-dependent Probe Amplification (MLPA), qRT-PCR, and the sequencing of long-range PCR amplicon have been used for the identification and characterization of new deletions. MLPA analysis for the identification of α- and β-globin rearrangement revealed the presence of five new α-thalassemia deletions. The set-up of qRT-PCR allowed us to delimit the extent of the deletions ranging from about 10 kb to more than 250 kb, two of them being of the telomeric type. The long-range PCR generated a specific anomalous fragment in three deletions, and only several unspecific bands in the other two deletions. The sequencing of the anomalous amplicons revealed the breakpoints of two deletions: the --PA, 34 kb long, identified in two families, and the telomeric --AG, 274 kb long. The anomalous fragment containing the breakpoint of the deletion --FG was partially sequenced, and it was not possible to identify the breakpoints due to the presence of several repetitive Alu sequences. The analysis of the breakpoint regions of the --Sciacca and --Puglia, respectively, are about 10 and 165 kb long, and revealed the presence of repeats that most likely impaired the amplification of a specific fragment for the identification of the breakpoint. MLPA, in association with qRT-PCR and long-range PCR, is a good approach for the identification and molecular characterization of rare or new deletions. Breakpoint analysis confirms that Alu sequences play an important role in favoring unequal crossing-over. Southern Italy shows considerable genetic heterogeneity, as expected with its central position in the Mediterranean basin, favoring migratory flows.
α-地中海贫血症的特征在于，大约 80%的病例是由α-珠蛋白基因簇中重复序列的存在而导致的缺失。在意大利南部α-地中海贫血症分子基础的研究项目中，我们发现了六个家族，它们表现出最常见缺失的缺失，以及正常的α-珠蛋白基因序列。多重连接依赖性探针扩增（MLPA）、qRT-PCR 和长距离 PCR 扩增子的测序已被用于鉴定和描述新的缺失。用于鉴定α-和β-珠蛋白重排的 MLPA 分析显示存在五种新的α-地中海贫血缺失。qRT-PCR 的建立使我们能够确定缺失的范围，从大约 10kb 到 250kb 以上，其中两个是端粒型。长距离 PCR 在三种缺失中产生了一个特定的异常片段，而在另外两种缺失中只产生了几个非特异性片段。异常扩增子的测序揭示了两个缺失的断点：在两个家庭中发现的 34kb 长的--PA，以及 274kb 长的端粒--AG。异常片段包含缺失--FG 的断点部分进行了测序，但由于存在多个重复 Alu 序列，因此无法确定断点。--Sciacca 和--Puglia 的断点区域分析分别长约 10kb 和 165kb，并且存在重复序列，这很可能会影响用于识别断点的特定片段的扩增。MLPA 与 qRT-PCR 和长距离 PCR 相结合，是鉴定和分子描述罕见或新缺失的良好方法。断点分析证实 Alu 序列在促进非均等交叉方面起着重要作用。意大利南部显示出相当大的遗传异质性，这与其在地中海盆地的中心位置以及促进迁徙流有关。
