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首页> 美国卫生研究院文献>Journal of the Endocrine Society >MON-717 Novel GLI2 Mutations Identified in Pediatric Patients with Combined Pituitary Hormone Deficiency: One Gene Various Genotypes
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MON-717 Novel GLI2 Mutations Identified in Pediatric Patients with Combined Pituitary Hormone Deficiency: One Gene Various Genotypes

机译：在垂体垂体激素缺乏组合的小儿患者中鉴定Mon-717新型Gli2突变：一种基因各种基因型

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Combined pituitary hormone deficiency (CPHD) is an important clinical problem caused by mutations in more than 30 different genes. Six genes in the Sonic Hedgehog (SHH) signalling pathway are reported to cause CPHD. SHH signaling is essential to induce pituitary cell identity in cells of Rathke’s pouch by stimulating expression of the transcription factors Lhx3 and Lhx4. In the absence of SHH signaling, a repressive isoform of the transcription factor GLI2 (Gli-Kruppel family member 2) suppresses gene expression. In the presence of SHH signaling, the activating form of GLI2 gains access to the nucleus and induces expression of downstream target genes. Heterozygous GLI2 loss of function mutations are found in patients with holoprosencephaly (HPE), HPE-like phenotypes associated with pituitary anomalies, and combined pituitary hormone deficiency with or without other extra-pituitary findings. We sought to identify the cause of CPHD in 171 unrelated patients diagnosed with or without extra-pituitary manifestations that were recruited from several Argentinean medical centers. We conducted panel sequencing, and identified GLI2 heterozygous variants that were rare and predicted to be deleterious in two unrelated patients, (p.L761P and p.1404Lfs) and a single, heterozygous, rare, likely deleterious GLI2 variant identified by exome sequencing (p.A203T). p.L761P and p.A203T variants were previously reported as candidates for HPE/CPHD, no functional studies were carried out to determine the effect of the variants on the gene function. We performed functional analysis of these variants using a mammalian cell line (NIH/3T3-CG) previously engineered to be a sensor for SHH signaling. It was stably transfected with a reporter gene that expresses GFP in response to GLI2 activation by a SHH agonist. We modified this cell line to assay GLI2 variants. We created a homozygous knock out of both endogenous Gli2 genes using CRISPR-Cas9 editing, and individual cell clones were selected for loss of GFP expression in response to SHH agonist treatment by FACS. We verified that transfecting the knockout cells with wild type Gli2 restored SHH responsive GFP expression. We assayed the ability of three patient GLI2 variants to rescue GFP expression and SHH agonist responsiveness and found that all three failed to fully rescue to wild type levels. This supports the hypothesis that the GLI2 variants in three CPHD patients are likely pathogenic. Thus, we identified three likely pathogenic GLI2 mutations in CPHD patients from Argentina. The variable phenotype of patients with GLI2 mutations worldwide could be caused by variation in other genes, environmental exposures, maternal effects, and/or epigenetic factors.
机译：组合的垂体激素缺乏（CPHD）是由30多种不同基因突变引起的重要临床问题。据报道，Sonic Hedgehog（SHH）信号通路中的六个基因导致CPHD。 SHH信号传导至关重要，以通过刺激转录因子LHX3和LHX4的表达来诱导Rathke小袋细胞中的垂体细胞同一性。在没有SHH信号传导的情况下，转录因子GLI2（GLI-KRUPPEL家族2）的抑制同种型抑制基因表达。在SHH信号的存在下，GLI2的激活形式获得对细胞核的进入并诱导下游靶基因的表达。杂合Gli2失去功能突变的损失是在患有与垂体异常相关的HPE样表型的患者中，以及组合垂体激素缺乏或没有其他垂体调查结果。我们试图确定诊断有或没有从几个阿根廷医疗中心招募的额外垂体表现的171名无关患者中CPHD的原因。我们进行了面板测序，并鉴定了稀有的Gli2杂合的变体，并预测在两个无关患者中有害，（P.L761P和P.1404LF）和由Exome测序鉴定的单一，杂合，罕见的罕见的Gli2变体（P .A203T）。 P.L761P和P.A203T变体先前报告为HPE / CPHD的候选者，未进行功能性研究以确定变体对基因功能的影响。我们使用先前设计的哺乳动物细胞系（NIH / 3T3-CG）对这些变体进行功能分析，以成为SHH信号传导的传感器。用记者基因稳定转染，报告基因响应于SHH激动剂的Gli2活化而表达GFP。我们将该细胞系修改为测定Gli2变体。我们使用CRISPR-CAS9编辑创建了一种纯合的GLI2基因，并且选择单个细胞克隆以响应FACS的SHH激动剂治疗而丧失GFP表达。我们验证了用野生型GLI2恢复SHH响应GFP表达转发敲除细胞。我们测定了三个患者GLI2变体拯救了GFP表达和SHH激动剂反应性的能力，发现所有三个未能完全救出野生类型。这支持三个CPHD患者的Gli2变体可能致病的假设。因此，我们确定了阿根廷的CPHD患者中的三种可能的致病性Gli2突变。全世界GLI2突变患者的可变表型可能是由其他基因，环境暴露，母体效应和/或表观遗传因素的变化引起的。

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