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

> **NIH NIH R03** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2021 · $115,003

## Abstract

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.

## Key facts

- **NIH application ID:** 10285184
- **Project number:** 1R03DE031037-01
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Sally A. Camper
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $115,003
- **Award type:** 1
- **Project period:** 2021-08-04 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10285184

## Citation

> US National Institutes of Health, RePORTER application 10285184, High Throughput Functional Assessment SHH Signaling Variants Identified in Patients with Craniofacial Defects and Hypopituitarism (1R03DE031037-01). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10285184. Licensed CC0.

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