锌指转录因子的Gli家族调节SonicHedgehog(Shh)信号通路,该通路在早期垂体和腹侧前脑发育中起关键作用。在全前脑畸形(HPE)中已经报道了人类的杂合GLI2功能缺失突变,与垂体异常相关的HPE样表型和垂体激素缺乏伴或不伴其他垂体外发现。
本研究的目的是在一组意大利CPHD患者中寻找GLI2突变,并通过体外研究评估鉴定出的变异的致病作用。
从不同的意大利中心招募了一百四十五名诊断有或没有垂体外表现的无关CPHD患者。
GLI2突变筛选通过所有13个外显子和内含子-外显子边界的直接测序进行。进行荧光素酶报告基因测定以评价检测到的错义变体的作用。
在5名患者中鉴定出5种不同的新型杂合非同义GLI2变体。突变是三个错义(p。Pro386Leu,p.Tyr575His,p.Ala593Val),一个移码(p。Val1111Glyfs*19)和一个废话(p。Arg1226X)。后两种突变体可能是致病性的,因为它们导致截短的蛋白质。携带三个错义变体中的两个(即p.Tyr575His和p.Ala593Val)的质粒的体外功能研究显示转录活性显着降低。
总之,在患有CPHD的个体中对GLI2的分析导致了对GLI2蛋白可能具有负面影响的五种变异的鉴定,证实GLI2是CPHD的重要致病基因。对错义变异进行的功能体外研究分析有助于加强致病性的假设。
The Gli-family of zinc-finger transcription factors regulates the Sonic Hedgehog (Shh) signalling pathway that plays a key role in early pituitary and ventral forebrain development. Heterozygous GLI2 loss of function mutations in humans have been reported in holoprosencephaly (HPE), HPE-like phenotypes associated with pituitary anomalies and combined pituitary hormone deficiency with or without other extra-pituitary findings.
The aim of this study was the search for GLI2 mutations in a cohort of Italian CPHD patients and the assessment of a pathogenic role for the identified variants through in vitro studies.
One hundred forty-five unrelated CPHD patients diagnosed with or without extra-pituitary manifestations were recruited from different Italian centres.
The GLI2 mutation screening was carried out through direct sequencing of all the 13 exons and intron-exon boundaries. Luciferase reporter assays were performed to evaluate the role of the detected missense variants.
Five different novel heterozygous non-synonymous GLI2 variants were identified in five patients. The mutations were three missense (p.Pro386Leu, p.Tyr575His, p.Ala593Val), one frameshift (p.Val1111Glyfs*19) and one nonsense (p.Arg1226X). The latter two mutants are likely pathogenic since they lead to a truncated protein. The in vitro functional study of the plasmids bearing two of the three missense variants (namely p.Tyr575His and p.Ala593Val) revealed a significant reduction in transcriptional activity.
In conclusion, the analysis of GLI2 in individuals with CPHD led to the identification of five variations with a likely negative impact on the GLI2 protein, confirming that GLI2 is an important causative gene in CPHD. The functional in vitro study analysis performed on the missense variations were useful to strengthen the hypothesis of pathogenicity.