Ranaweera Dinali M, de Silva Deepthi C, Samarasinghe Duminda, Perera Shehan, Kugalingam Nirosha, Samarasinghe Sumudu R, Madushani Wadumesthri Y, Jayaweera Hiran H E, Gunewardene Siyath, Muneeswaran Kajan, Gnanam Vaz S, Chandrasekharan Naduviladath V
Department of Chemistry, University of Colombo.
Department of Physiology, Faculty of Medicine, University of Kelaniya.
Genes Genet Syst. 2025 May 21;100. doi: 10.1266/ggs.24-00142. Epub 2025 Jan 22.
A hemizygous 1.5-3.0-Mb microdeletion of human chromosome 22q11.2 with the loss of multiple genes including histone cell cycle regulator (HIRA) causes 22q11.2 deletion syndrome (22q11.2 DS), a common disorder with variable manifestations including congenital malformations affecting the heart, palate and kidneys in association with neurodevelopmental, psychiatric, endocrine and autoimmune abnormalities. The aim of this study was to develop a TaqMan-based dosage analysis PCR (TaqMan qPCR) for use as a rapid, cost-effective test for clinically suspected patients fulfilling previously described criteria for molecular diagnosis of 22q11.2 DS in a lower middle-income country where the cost of testing limits its use in routine clinical practice. Nineteen patients were recruited with informed consent following ethical approval from the Ethics Review Committee, Lady Ridgeway Hospital for Children, Colombo. Dosage analysis of extracted DNA was performed using a TaqMan qPCR assay by amplifying regions within the target (HIRA) and control (testin LIM domain protein (TES)) genes of suspected patient (P) and unaffected person (N) samples. For detection of a deletion, the normalized value (HIRA/TES dosage) of a P sample was compared with that of an N sample. A ratio of P:N of 0.5 confirmed the presence of a deletion while a ratio of 1.0 refuted this. Seven of the 19 patients were found to have a HIRA deletion, confirming the diagnosis of 22q11.2 DS, with these results being in complete agreement with those of fluorescence in situ hybridization (FISH) (performed in nine of the 19 cases) and whole-exome sequencing (all 19 samples tested). This TaqMan qPCR assay was able to reliably distinguish HIRA-deleted cases from non-deleted ones. The assay was both cheaper and faster compared to commercially available alternatives in our setting, including FISH and multiple ligation-dependent probe amplification.
人类22号染色体q11.2区域存在一个1.5 - 3.0兆碱基对的半合子微缺失，多个基因包括组蛋白细胞周期调节因子（HIRA）缺失，会导致22q11.2缺失综合征（22q11.2 DS），这是一种常见疾病，临床表现多样，包括影响心脏、腭和肾脏的先天性畸形，以及神经发育、精神、内分泌和自身免疫异常。本研究的目的是开发一种基于TaqMan的剂量分析聚合酶链反应（TaqMan qPCR），作为一种快速、经济高效的检测方法，用于临床疑似患者，这些患者符合先前描述的在中低收入国家分子诊断22q11.2 DS的标准，在这些国家检测成本限制了其在常规临床实践中的应用。在获得科伦坡莱迪·里奇韦儿童医院伦理审查委员会的伦理批准后，19名患者在知情同意的情况下被招募。通过TaqMan qPCR检测法对提取的DNA进行剂量分析，扩增疑似患者（P）和未受影响个体（N）样本的靶基因（HIRA）和对照基因（睾丸素LIM结构域蛋白（TES））内的区域。为了检测缺失情况，将P样本的标准化值（HIRA/TES剂量）与N样本的进行比较。P:N比值为0.5证实存在缺失，而比值为1.0则排除这种情况。19名患者中有7名被发现存在HIRA缺失，从而确诊为22q11.2 DS，这些结果与荧光原位杂交（FISH）（19例中的9例进行了此项检测）和全外显子测序（检测了所有19个样本）的结果完全一致。这种TaqMan qPCR检测法能够可靠地将HIRA缺失病例与未缺失病例区分开来。在我们的环境中，与包括FISH和多重连接依赖探针扩增在内的市售检测方法相比，该检测法既便宜又快速。
