Amri Yessine, Fredj Sondess Hadj, Dabboubi Rym, Othmani Rym, Sahli Chaima, Baccouche Imen, Ouali Faida, Messaoud Taieb
Biochemistry Laboratory (LR00SP03), Bechir Hamza Children's Hospital, Bab Saadoun Square, Tunis, 1007, Tunisia.
Department of Educational Sciences, Higher Institute of Applied Studies in Humanity Le Kef, University of Jendouba, Kef, Tunisia.
Ann Hematol. 2025 Jun 9. doi: 10.1007/s00277-025-06320-2.
Alpha-thalassemia is a common inherited hemoglobin disorder caused by deletions or point mutations in the HBA1 and/or HBA2 genes, leading to reduced or absent synthesis of alpha-globin chains. Although deletional mutations are predominant, non-deletional mutations can also contribute to the clinical spectrum of the disease, with varying severity. This study aimed to characterize two novel mutations in the HBA1 gene, a 230 bp microdeletion and a missense mutation (NP_000549.1:p.Ala29Pro), identified in two unrelated Tunisian families. Additionally, we assessed the clinical, hematological, molecular, and in silico structural impact of these mutations. Peripheral blood samples were collected from affected individuals and family members. Hematological parameters were measured using automated cell counters, and hemoglobin fractions were analyzed by high-performance liquid chromatography (HPLC). Genomic DNA was extracted, and common α-thalassemia deletions were screened via gap-PCR. Negative cases underwent sequencing of HBA1 and HBA2 exons. The novel deletion was confirmed by gap-PCR, gel purification, and sequencing. The pathogenicity of the missense mutation was assessed using in silico tools (PolyPhen-2, SIFT, MutationTaster), and structural modeling was performed using Swiss-PdbViewer and DynaMut to evaluate protein stability and flexibility. In Family A, a 4-year-old proband exhibited mild anemia, microcytosis, hypochromia, and low ferritin levels unresponsive to iron supplementation. A novel heterozygous 230 bp deletion (NC_000016.10:g.176695_176925del), named Hemoglobin Ariana, was identified in the HBA1 gene, affecting the 5'UTR, exon 1, and part of intron 1. In Family B, a 4-month-old proband presented with co-inheritance of α-thalassemia and Hemoglobin C. Sequencing revealed a novel missense mutation (NM_000558.5:c.85G > C, NP_000549.1:p.Ala29Pro), designated Hemoglobin Tozeur. In silico analysis predicted the mutation as deleterious, with structural modeling indicating destabilization of the α-globin protein, reduced stability, and altered flexibility near the heme-binding region. This study identifies two novel HBA1 mutations associated with α-thalassemia in Tunisia. The combined clinical, molecular, and structural analyses highlight the importance of comprehensive genetic screening and in silico modeling for accurate diagnosis and improved management of thalassemia cases.
α地中海贫血是一种常见的遗传性血红蛋白疾病，由HBA1和/或HBA2基因的缺失或点突变引起，导致α珠蛋白链合成减少或缺失。虽然缺失突变占主导，但非缺失突变也可导致该病临床谱的变化，严重程度各异。本研究旨在对在两个不相关的突尼斯家庭中鉴定出的HBA1基因的两个新突变进行特征分析，一个是230 bp的微缺失，另一个是错义突变（NP_000549.1:p.Ala29Pro）。此外，我们评估了这些突变的临床、血液学、分子和计算机模拟结构影响。从受影响个体及其家庭成员采集外周血样本。使用自动血细胞计数器测量血液学参数，并通过高效液相色谱（HPLC）分析血红蛋白组分。提取基因组DNA，并通过缺口PCR筛选常见的α地中海贫血缺失。阴性病例对HBA1和HBA2外显子进行测序。通过缺口PCR、凝胶纯化和测序确认了新的缺失。使用计算机工具（PolyPhen-2、SIFT、MutationTaster）评估错义突变的致病性，并使用Swiss-PdbViewer和DynaMut进行结构建模，以评估蛋白质稳定性和灵活性。在A家庭中，一名4岁先证者表现为轻度贫血、小红细胞症、低色素血症和铁蛋白水平低，对铁补充无反应。在HBA1基因中鉴定出一个新的杂合230 bp缺失（NC_000016.10:g.176695_176925del），命名为血红蛋白阿丽亚娜，影响5'UTR、外显子1和内含子1的一部分。在B家庭中，一名4个月大的先证者表现为α地中海贫血和血红蛋白C的共同遗传。测序发现一个新的错义突变（NM_000558.5:c.85G＞C，NP_000549.1:p.Ala29Pro），命名为血红蛋白托泽尔。计算机分析预测该突变为有害突变，结构建模表明α珠蛋白不稳定，稳定性降低，血红素结合区域附近的灵活性改变。本研究在突尼斯鉴定出两个与α地中海贫血相关的HBA1新突变。临床、分子和结构分析相结合，突出了全面基因筛查和计算机模拟建模对地中海贫血病例准确诊断和改善管理的重要性。
