Shestak Anna G, Rumyantseva Victoria A, Zaklyazminskaya Elena V
Medical Genetics Laboratory, Petrovsky National Research Center of Surgery, Moscow, Russia.
Research Center for Medical Genetics, Moscow, Russia.
Front Genet. 2025 Jun 11;16:1571437. doi: 10.3389/fgene.2025.1571437. eCollection 2025.
Allelic dropout (ADO) is a common limitation of all PCR-based molecular diagnostic methods, leading to false-negative or false-positive results, depending on the allele that was dropped. We report a case of multiple locus-specific allele dropouts mediated by a common duplication beyond the primer-binding site of the endoglin gene. We observed a family with hereditary hemorrhagic telangiectasia (HHT) where the HHT diagnosis in the proband (female, 71 years old) and two family members was based on the Curaçao criteria. A nonsense heterozygous c.831C>A (p.Y277*) mutation and a common homozygous duplication c.991+21_26dup in exon 7 of the gene was revealed in the proband. Discrepancies were found between the obvious clinical HHT phenotypes of the two family members and the negative results of cascade familial screening based on capillary Sanger sequencing with classically designed oligoprimers. In addition, ADO was suspected due to the absence of c.991+21_26dup. We analyzed the primer-binding sites using gnomAD to reveal the cause of ADO. Amplicons with notable ADO were resequenced using alternative oligoprimers. Three primer pairs that were designed more distal (toward the 3'-end) after duplication were unable to amplify both alleles. Redesigning oligoprimers complementary to the narrow area successfully detected the heterozygous variant p.Y277* in two family members. The classical primer design for Sanger sequencing may lead to the inefficient amplification of exon 7 amplicons with duplications (up to 19% according to MAF in gnomAD). These results suggest that indels beyond the primer-binding sites may lead to allele loss and false-negative results in DNA diagnostics.
等位基因脱扣（ADO）是所有基于聚合酶链反应（PCR）的分子诊断方法的一个常见局限性，根据脱扣的等位基因不同，会导致假阴性或假阳性结果。我们报告了一例由内皮素基因引物结合位点以外的常见重复介导的多位点特异性等位基因脱扣病例。我们观察到一个患有遗传性出血性毛细血管扩张症（HHT）的家庭，其中先证者（71岁女性）和两名家庭成员的HHT诊断基于库拉索标准。在先证者中发现了一个无义杂合c.831C>A（p.Y277*）突变和该基因第7外显子中一个常见的纯合重复c.991+21_26dup。在两名家庭成员明显的临床HHT表型与基于经典设计寡核苷酸引物的毛细管桑格测序的级联家族筛查阴性结果之间发现了差异。此外，由于未检测到c.991+21_26dup，怀疑存在ADO。我们使用gnomAD分析引物结合位点以揭示ADO的原因。对具有明显ADO的扩增子使用替代寡核苷酸引物重新测序。在重复后设计得更远端（朝向3'端）的三对引物无法扩增两个等位基因。重新设计与狭窄区域互补的寡核苷酸引物成功检测到两名家庭成员中的杂合变体p.Y277*。用于桑格测序的经典引物设计可能导致第7外显子有重复的扩增子扩增效率低下（根据gnomAD中的MAF高达19%）。这些结果表明，引物结合位点以外的插入缺失可能导致DNA诊断中的等位基因丢失和假阴性结果。
