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