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

摘要 前脑、中脑、后脑、五个面神经和脑垂体在第4-7周之间发育。 人类的怀孕破坏这些过程的遗传缺陷会导致一系列发育障碍， 具有终身后果的疾病，严重程度从前脑无裂畸形（HPE）到视隔畸形 垂体激素缺乏症（先天性垂体功能减退症，CH）。HPE患者具有可变的 前脑、眼睛和脑垂体的缺陷，严重者可导致胚胎死亡。三要素 SOD的诊断包括视神经发育不全，中线脑异常和CH。 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是高度异质性的， 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、GLI 2、GLI 3、HHIP、SHH、SIX 3和TGIF 1。我们筛选了约200名 与CH和各种相关特征无关的先证者，并确定了罕见的，可能的致病变异， 转录因子GLI 2和SIX 3中的不确定意义变体（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， 分析，分别。VUS是实现分子遗传检测承诺的主要障碍 诊断，这是令人生畏的个别实验室，以建立各种功能测试测定 遗传异质性疾病所必需的。我们建议创建一个目录的功能效果 GLI 2和SIX 3中所有可能的变体，使用变体效应的多重测定（MAVE）。这种方法 扩大我们已经开发的用于一次测试一种变体的检测方法， 可以同时进行测试，产生定量功能信息，将变体分配为增益， 这种高通量系统解决了变异的问题， 通过提供有关单核苷酸表型后果的可比信息来解释 变异，这将改善遗传信息转化为诊断。MAVE已应用于 了解不同基因的功能和致病类型，从剪接到氨基酸取代 从信号通路到转录因子。 功能、耐受性或功能丧失。 完成这些目标将进一步加深我们对GLI 2的了解 和SIX 3的结构和功能在疾病中的作用，并为使用MAVE生成 在SHH途径中引起颅面缺陷的其他基因的功能注释。

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.