Unidade de Genética Molecular, Centro de Genética Médica Jacinto de Magalhães (CGM), Centro Hospitalar Universitário do Porto (CHUPorto), Porto, Portugal.
Unidade Multidisciplinar de Investigação Biomédica (UMIB), Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Laboratory for Integrative and Translational Research in Population Health (ITR) Universidade do Porto (UP), Porto, Portugal.
Sci Rep. 2021 Jul 19;11(1):14676. doi: 10.1038/s41598-021-93473-5.
Over 100 X-linked intellectual disability genes have been identified, with triplet repeat expansions at the FMR1 (FRAXA) and AFF2 (FRAXE) genes being the causative agent in two of them. The absence of FRAXE pathognomonic features hampers early recognition, delaying testing and molecular confirmation. Hence, our laboratory uses a multiplex PCR-based strategy to genotype both FRAXA and FRAXE. However, AFF2 expansions are missed giving rise to an uninformative result in around 20% of female samples. To rule out undetected expansions and confirm homozygosity Southern blot analysis is performed being labour- and resource-intensive. The aim of this study is to develop a timely and economic triplet-primed amplification (TP-PCR) screening strategy to size the AFF2 GCC repeat and accurately assess homozygosity as well as pinpoint multiplex-PCR false negatives in female samples. In order to achieve this, validation was performed in a cohort of 500 females with a previous uninformative FRAXE PCR result. Interestingly, the presence of a T > C SNP (rs868949662), contiguous to the GCC repetitive tract, allows triplet primer binding in two additional repeats, increasing the discrimination power of the TP-PCR assay in heterozygous and homozygous samples. Twelve alleles outside the normal range were recognized: eight intermediate and four premutated, which seems relevant considering the rarity of the AFF2 expansions. All genotypes are concordant with that obtained by Southern blotting, confirming this as a strict, reproducible and low-cost homozygosity screening strategy that enables the identification of small expanded alleles missed by the routine multiplex-PCR due to allele dropout. Overall, this assay is capable of spotting multiplex-PCR false negatives besides identifying alleles up to > 80 GCC repeats. Furthermore, the occurrence of intermediate repeat sizes with unexpected frequency, introduces new areas of clinical research in this cohort in understanding these less explored AFF2 repeat sizes and newly associated phenotypes.
已经鉴定出超过 100 个 X 连锁智力障碍基因，其中 FMR1（FRAXA）和 AFF2（FRAXE）基因中的三核苷酸重复扩展是其中两个的致病因素。由于缺乏 FRAXE 的特征性表现，早期识别受到阻碍，导致检测和分子确认延迟。因此，我们的实验室使用基于多重 PCR 的策略对 FRAXA 和 FRAXE 进行基因分型。然而，AFF2 扩展会被遗漏，导致大约 20%的女性样本结果无信息。为了排除未检测到的扩展并确认纯合子，需要进行Southern 印迹分析，但这种方法劳动强度大且资源密集。本研究旨在开发一种及时且经济的三核苷酸引物扩增（TP-PCR）筛选策略，以确定 AFF2 GCC 重复的大小，并准确评估纯合子状态以及确定女性样本中多重 PCR 的假阴性。为了实现这一目标，我们在先前 FRAXE PCR 结果无信息的 500 名女性队列中进行了验证。有趣的是，紧邻 GCC 重复序列的 T>C SNP（rs868949662）的存在允许三核苷酸引物在另外两个重复序列中结合，从而提高了 TP-PCR 检测在杂合子和纯合子样本中的区分能力。在正常范围之外识别出 12 个等位基因：8 个中间型和 4 个前突变型，考虑到 AFF2 扩展的罕见性，这似乎很重要。所有基因型与 Southern 印迹获得的结果一致，证实了这是一种严格、可重复且低成本的纯合子筛选策略，可识别由于等位基因缺失而被常规多重 PCR 遗漏的小扩展等位基因。总的来说，该检测除了能够发现多重 PCR 的假阴性外，还能够识别高达 80 个以上 GCC 重复的等位基因。此外，中间重复大小的出现频率出乎意料，为该队列中理解这些研究较少的 AFF2 重复大小和新关联表型的新临床研究领域开辟了道路。
