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单倍体不足会导致 70-90% 的 Léri-Weill 软骨发育不良 (LWD) 和 2-10% 的特发性身材矮小 (ISS)。删除整个基因或增强子和编码区中的点突变是单倍体不足的公认原因。在对 ISS/LWD 患者进行诊断基因检测期间，除了经典的SHOX缺陷外，还有五个 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（不确定性变体）。本研究的目的是调查这些非编码变异在SHOX 中的作用单倍不足。测试了这些变体干扰受调节报告基因正确基因表达的能力（荧光素酶测定）。通过小基因剪接测定在体外测定了对计算机中预测的 c.-19G > A 的 mRNA 剪接的负面影响。荧光素酶测定表明，c.-51G > A、c.-19G > A 和 c.-9del 在纯合状态下显着降低荧光素酶活性 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。外显子捕获分析表明 c.-19G > A 决定了新分支位点的产生，从而导致异常的 mRNA 剪接。综上所述，这项研究使我们能够将 SHOX 诊断筛查过程中发现的两种 5'UTR 变异重新分类为可能致病，一种仍为 VUS，另两种可能为良性变异。这项分析首次扩大了遗传原因的范围SHOX单倍体不足对 5'UTR 中的非编码变异。