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: 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
• 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
• 来源: https://reporter.nih.gov/project-details/10461927
• 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; 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.

抽象的 前脑，中脑，后脑，五个面部突出和垂体在WK 4-7之间发展 人类的妊娠。遗传缺陷破坏这些过程会导致一系列发展 终身后果的疾病，从全休脑脑（HPE）到Septo-Optic的严重程度范围 垂体激素缺乏症（先天性下降症，CH）的发育异常（SOD）。 HPE患者有变量 前脑，眼睛和垂体和严重病例的缺陷是胚胎致死的。特征三合会 SOD的诊断包括视神经发育不全，中线脑异常和CH。患者被诊断出 使用CH，但不是HPE或SOD，有时具有与这些疾病有关的功能，包括视觉， 听力和/或大脑异常。这些疾病的遗传原因是高度异质的， 重叠。遗传原因中突出的是几个影响声音刺猬（SHH）的基因 信号传导，包括CDON，GLI2，GLI3，HHIP，SHH，SIX3和TGIF1。我们筛选了〜200的队列 具有CH和各种相关特征的无关概率，并确定了罕见的，可能的致病性变体， 转录因子GLI2和SIX3的不确定意义（VU）的变体。我们证实了致病性 使用SHH信号传感器细胞系测定和瞬态转染的几种Gli2和Six3变体中 分别分析。 VUS是实现分子基因检测承诺的主要障碍 诊断，单个实验室建立各种功能测试测定的种类是令人生畏的 对于遗传异质疾病所必需的。我们建议创建一个目录的目录 使用变体效应（MAVE）的多重测定法（Maves）中的GLI2和SIX3中的所有可能变体。这种方法 扩展我们已经开发用于一次测试一个变体的测定法 可以同时测试，得出定量功能信息，将变体分配为 这个高吞吐量系统解决了变体的问题 通过提供有关单核苷酸表型后果的可比较信息来解释 变体将改善遗传信息转化为诊断。师范已应用于 了解从剪接到氨基酸取代的各种基因和类型的致病性功能 从信号通路到转录因子。 功能，容忍或功能丧失。 完成这些目标将进一步我们对GLI2的了解 疾病中的Six3结构和功能，并为使用Maves生成目录的阶段奠定了基础 SHH途径中其他基因引起颅面缺陷的功能注释。

项目成果

Disruption of RNA Splicing Is an Important Contributor to Congenital Hypopituitarism and Other Human Genetic Diseases.

RNA 剪接的破坏是先天性垂体功能减退症和其他人类遗传疾病的重要原因。

• DOI: 10.1210/endocr/bqad039
• 发表时间: 2023
• 期刊: Endocrinology
• 影响因子: 4.8
• 作者: Camper,SallyA;Smith,Cathy;Kitzman,JacobO
• 通讯作者: Kitzman,JacobO

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency

• DOI: 10.1016/j.ajhg.2021.06.013
• 发表时间: 2021-08-05
• 期刊: AMERICAN JOURNAL OF HUMAN GENETICS
• 影响因子: 9.8
• 作者: Gergics, Peter;Smith, Cathy;Camper, Sally A.
• 通讯作者: Camper, Sally A.