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

• 批准号: 10285184
• 负责人: Sally A. Camper
• 金额: $ 11.5万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-04 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10285184
• 关键词: Address; Affect; Agonist; Alleles; Amino Acid Substitution; Amino Acids; Argentina; Binding; Binding Proteins; Biological Assay; Blindness; Brain; CDON gene; CRISPR/Cas technology; Caring; Cataloging; Catalogs; Cell Line; Cessation of life; Clinical; Congenital Abnormality; Consumption; Craniofacial Abnormalities; Defect; Diagnosis; Diagnostic; Disease; Disease Progression; DsRed; Embryo; Engineering; Enhancers; Eye; Face; Family; GLI2 gene; GLI3 gene; Gene Expression; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genetic Translation; Green Fluorescent Proteins; Hearing; Holoprosencephaly; Human; Hypopituitarism; Individual; Intellectual functioning disability; Knowledge; Laboratories; Letters; Life; Mammalian Cell; Mediating; Midbrain structure; Molecular Diagnosis; Mutagenesis; Mutate; Mutation; NIH 3T3 Cells; Neonatal; Neuraxis; Ophthalmology; Optic Nerve; Pathogenicity; Patients; Phenotype; Pituitary Gland; Pituitary Hormones; Predictive Value; Pregnancy; Process; Property; Prosencephalon; RNA Splicing; Reporter; Repression; Risk; SHH gene; Septo-Optic Dysplasia; Severities; Signal Pathway; Signaling Protein; Single Nucleotide Polymorphism; Sonic Hedgehog Pathway; Structure; System; Testing; Therapeutic; Time; Transactivation; Transfection; Triad Acrylic Resin; United States National Institutes of Health; Variant; Vision; base; brain abnormalities; cohort; craniofacial; craniofacial development; deafness; developmental disease; disorder risk; exome sequencing; gain of function; gene panel; genetic corepressor; genetic disorder diagnosis; genetic information; genetic testing; genome sequencing; hindbrain; hormone deficiency; improved; inhibitor/antagonist; loss of function; loss of function mutation; multiplex assay; proband; promoter; protein expression; rare variant; response; scale up; sensor; smoothened signaling pathway; transcription factor; variant of unknown significance; whole genome

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.

摘要 前脑、中脑、后脑、五个面部隆起和脑垂体腺在第4-7周期间发育。 人类的受孕。破坏这些过程的遗传缺陷会导致一系列的发育 具有终生后果的疾病，其严重程度从无前脑畸形(HPE)到视间隔 发育不良(SOD)到垂体激素缺乏症(先天性垂体功能减退症，CH)。HPE患者有不同的 前脑、眼睛和脑下垂体的缺陷和严重的病例都是胚胎致死的。三位一体的特征 超氧化物歧化酶的诊断包括视神经发育不全、中线脑异常和脑出血。被诊断为 患有CH，但不是HPE或SOD，有时会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。在遗传原因中，突出的是几个影响音速刺猬(SHH)的基因 信令，包括CDON、GLI2、GLI3、HHIP、SHH、Six3和TGIF1。我们筛选了大约200人的队列 无血缘关系的先证者CH和各种相关特征，并确定罕见的，可能的致病变异和 转录因子GLI2和Six3中的不确定意义变体(VUS)。我们确认了致病性 应用SHH信号传感细胞系检测和瞬时转染法检测几种GLI2和Six3变异体 分别进行了测定。VUS是实现分子遗传检测承诺的主要障碍 诊断，对于单个实验室来说，建立各种功能测试分析是令人望而生畏的 对于遗传性异质性疾病来说是必要的。我们建议创建一个目录的功能效果 GLI2和Six3中所有可能的变异使用多路变异效应分析(MAVE)。这种方法 扩大我们已经开发的一次测试一个变种的化验方法，以便数千个变种 可以同时进行测试，产生量化的功能信息，将变体指定为 这种高吞吐量系统解决了变种的问题 通过提供关于单核苷酸表型后果的可比信息进行解释 变异，这将改善遗传信息到诊断的转换。MAVES已被应用于 了解不同基因的功能和致病类型，从剪接到氨基酸替换 并从信号通路到转录因子。 功能、容忍或丧失功能。 完成这些目标将进一步加深我们对GLI2的了解 和Six3在疾病中的结构和功能，为利用MAVES生成疾病目录奠定了基础 SHH途径中导致头面部缺陷的其他基因的功能注释。