Laboratorio di Genetica Umana, Dipartimento di Scienze della Salute, Università del Piemonte Orientale, Novara, Italy.
Endocrinologia Pediatrica, Dipartimento di Pediatria e Specialità Pediatriche, Ospedale Regina Margherita, Citta della Salute e della Scienza, Torino, Italy.
Eur J Hum Genet. 2021 Jan;29(1):110-121. doi: 10.1038/s41431-020-0676-y. Epub 2020 Jul 9.
SHOX haploinsufficiency causes 70-90% of Léri-Weill dyschondrosteosis (LWD) and 2-10% of idiopathic short stature (ISS). Deletions removing the entire gene or enhancers and point mutations in the coding region represent a well-established cause of haploinsufficiency. During diagnostic genetic testing on ISS/LWD patients, in addition to classic SHOX defects, five 5'UTR variants (c.-58G > T, c.-55C > T, c.-51G > A, c.-19G > A, and c.-9del), were detected whose pathogenetic role was unclear and were thus classified as VUS (Variants of Uncertain Significance). The purpose of the present study was to investigate the role of these noncoding variations in SHOX haploinsufficiency. The variants were tested for their ability to interfere with correct gene expression of a regulated reporter gene (luciferase assay). The negative effect on the mRNA splicing predicted in silico for c.-19G > A was assayed in vitro through a minigene splicing assay. The luciferase assay showed that c.-51G > A, c.-19G > A, and c.-9del significantly reduce luciferase activity by 60, 35, and 40% at the homozygous state. Quantification of the luciferase mRNA showed that c.-51G > A and c.-9del might interfere with the correct SHOX expression mainly at the post-transcriptional level. The exon trapping assay demonstrated that c.-19G > A determines the creation of a new branch site causing an aberrant mRNA splicing. In conclusion, this study allowed us to reclassify two of the 5'UTR variants identified during SHOX diagnostic screening as likely pathogenic, one remains as a VUS, and two as likely benign variants. This analysis for the first time expands the spectrum of the genetic causes of SHOX haploinsufficiency to noncoding variations in the 5'UTR.
SHOX 基因杂合性缺失导致 70-90%的 Léri-Weill 软骨发育不全症（LWD）和 2-10%的特发性身材矮小症（ISS）。缺失去除整个基因或增强子以及编码区的点突变是杂合性缺失的公认原因。在 ISS/LWD 患者的诊断性遗传检测中，除了经典的 SHOX 缺陷外，还检测到五个 5'UTR 变异（c.-58G>T、c.-55C>T、c.-51G>A、c.-19G>A 和 c.-9del），其致病作用尚不清楚，因此被归类为 VUS（意义未明的变异）。本研究旨在探讨这些非编码变异在 SHOX 基因杂合性缺失中的作用。通过荧光素酶测定法检测这些变异对受调控报告基因（荧光素酶测定法）正确表达的干扰能力。体外通过迷你基因剪接测定法对预测的 c.-19G>A 剪接的潜在负性影响进行了检测。荧光素酶测定法显示，c.-51G>A、c.-19G>A 和 c.-9del 在纯合状态下使荧光素酶活性分别显著降低 60%、35%和 40%。荧光素酶 mRNA 的定量表明，c.-51G>A 和 c.-9del 可能主要在转录后水平干扰正确的 SHOX 表达。外显子捕获测定法表明，c.-19G>A 导致新的分支位点的产生，从而引起异常的 mRNA 剪接。总之，本研究使我们能够重新将 SHOX 诊断筛查中发现的五个 5'UTR 变异中的两个归类为可能的致病性变异，一个仍然是 VUS，两个可能是良性变异。这项分析首次将 SHOX 基因杂合性缺失的遗传原因扩展到 5'UTR 的非编码变异。
