High Throughput Functional Assessment SHH Signaling Variants Identified in Patients with Craniofacial Defects and Hypopituitarism

高通量功能评估在颅面缺陷和垂体机能减退患者中鉴定出 SHH 信号变异

基本信息

批准号：
10461927
负责人：
Sally A. Camper
金额：
$ 11.49万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-04 至 2024-07-31
项目状态：
已结题

项目摘要

Abstract The forebrain, midbrain, hindbrain, five facial prominences, and pituitary gland develop between wk 4-7 of gestation in humans. Genetic defects that disrupt these processes cause a spectrum of developmental disorders with life-long consequences that range in severity from holoprosencephaly (HPE) to septo-optic dysplasia (SOD) to pituitary hormone deficiency (congenital hypopituitarism, CH). HPE patients have variable defects in forebrain, eyes, and pituitary, and severe cases are embryonic lethal. The triad of features diagnostic of SOD include optic nerve hypoplasia, midline brain abnormalities, and CH. Patients diagnosed with CH, but not HPE or SOD, sometimes have features associated with those disorders, including vision, hearing, and/or brain anomalies. The genetic causes of these disorders are highly heterogeneous and overlapping. Prominent amongst the genetic causes are several genes that affect sonic hedgehog (SHH) signaling, including CDON, GLI2, GLI3, HHIP, SHH, SIX3, and TGIF1. We screened a cohort of ~ 200 unrelated probands with CH and various associated features and identified rare, likely pathogenic variants and variants of uncertain significance (VUS) in the transcription factors GLI2 and SIX3. We confirmed pathogenicity of several GLI2 and SIX3 variants using a SHH signaling sensor cell line assay and transient transfection assay, respectively. VUS are a major impediment to delivering on the promise of genetic testing for molecular diagnosis, and it is daunting for individual laboratories to establish the variety of functional testing assays necessary for genetically heterogenous disorders. We propose to create a catalog of the functional effects of all possible variants in GLI2 and SIX3 using multiplexed assays of variant effects (MAVEs). This approach scales up the assays we have already developed for testing one variant at a time so that thousands of variants can be tested simultaneously, yielding quantitative functional information that assigns variants as gain of This high throughput system addresses the problem of variant interpretation by providing comparable information about the phenotypic consequences of single nucleotide variants, which will improve the translation of genetic information into diagnosis. MAVEs have been applied to understand the function of diverse genes and types of pathogenicity, from splicing to amino acid substitution and from signaling pathways to transcription factors. function, tolerated, or loss of function. Completing these aims will further our knowledge of GLI2 and SIX3 structure and function in disease and set the stage for using MAVEs to generate catalogs of functional annotation for other genes in the SHH pathway that cause craniofacial defects.

抽象的前脑、中脑、后脑、五个面部突起和垂体在出生后第 4-7 周之间发育。人类妊娠。破坏这些过程的遗传缺陷会导致一系列发育障碍具有终生后果的疾病，其严重程度从前脑无裂畸形 (HPE) 到中隔视神经发育不良（SOD）至垂体激素缺乏（先天性垂体功能低下，CH）。 HPE 患者的情况各不相同前脑、眼睛和垂体有缺陷，严重的情况下会导致胚胎死亡。特征三元组 SOD 的诊断包括视神经发育不全、中线脑异常和 CH。确诊患者患有 CH，但不是 HPE 或 SOD，有时具有与这些疾病相关的特征，包括视力、听力和/或大脑异常。这些疾病的遗传原因具有高度异质性，重叠。遗传原因中最突出的是影响音刺猬 (SHH) 的几个基因信号传导，包括 CDON、GLI2、GLI3、HHIP、SHH、SIX3 和 TGIF1。我们筛选了约 200 人的队列与 CH 无关的先证者和各种相关特征，并鉴定出罕见的、可能的致病变异和转录因子 GLI2 和 SIX3 中的不确定意义变异 (VUS)。我们确认了致病性使用 SHH 信号传导传感器细胞系测定和瞬时转染检测几种 GLI2 和 SIX3 变体分别进行测定。 VUS 是实现分子基因检测承诺的主要障碍诊断，对于各个实验室来说建立各种功能测试分析是令人畏惧的遗传异质性疾病所必需的。我们建议创建一个功能效果目录使用变异效应多重分析 (MAVE) 检测 GLI2 和 SIX3 中所有可能的变异。这种做法扩大我们已经开发的用于一次测试一种变体的检测方法，以便能够检测数千种变体可以同时进行测试，产生定量的功能信息，将变体分配为增益这种高通量系统解决了变异问题通过提供有关单核苷酸表型后果的可比较信息来进行解释变异，这将改善遗传信息到诊断的转化。 MAVE 已应用于了解不同基因的功能和致病性类型，从剪接到氨基酸取代以及从信号通路到转录因子。功能、耐受性或功能丧失。完成这些目标将加深我们对 GLI2 的了解和 SIX3 在疾病中的结构和功能，并为使用 MAVE 生成疾病目录奠定基础 SHH 通路中导致颅面缺陷的其他基因的功能注释。