Holoprosencephaly 3 (HPE3) Cell Models for Research

Holoprosencephaly (HPE) is the most common structural malformation of the forebrain in humans, with an estimated prevalence of 1 in 250 during embryogenesis and 1 in 10,000 to 20,000 at birth. HPE3 is a genetic subtype associated with mutations in the Sonic Hedgehog (SHH) gene. The clinical spectrum ranges from severe alobar HPE, which is often fatal, to milder microforms with subtle facial dysmorphism. According to the World Health Organization (WHO), congenital anomalies account for approximately 240,000 neonatal deaths within the first 28 days of life globally, and HPE contributes to this burden. The National Cancer Institute (NCI) does not track HPE as it is not a cancer, but the genetic pathways involved are relevant to cancer research. The 5-year survival for severe forms is low, but for milder forms, survival into adulthood is possible with appropriate management.

HPE3 is an ideal model for studying the SHH signaling pathway, which is critical in embryonic development and also dysregulated in various cancers, including medulloblastoma and basal cell carcinoma. The availability of patient-derived mutations and the well-characterized SHH pathway make HPE3 a valuable system for mechanistic studies. Public datasets, such as those from the International HPE Consortium and ClinVar, provide a wealth of genetic information. Open questions include the role of modifier genes, the impact of specific SHH mutations on protein function, and the development of therapeutic strategies to modulate SHH signaling.

• • Although HPE3 is a developmental disorder, the SHH pathway is a major oncogenic pathway in several cancers. The pathway is activated by the binding of SHH ligand to the Patched (PTCH1) receptor, which relieves inhibition of Smoothened (SMO), leading to activation of GLI transcription factors. In cancers, mutations in PTCH1, SMO, or SUFU can lead to constitutive activation. The pathway can be described in steps:

1. SHH ligand binds to PTCH1.

2. PTCH1 inhibition of SMO is relieved.

3. SMO activates GLI proteins.

4. GLI translocates to the nucleus and regulates target genes involved in cell proliferation and survival.

In HPE3, the primary genetic alterations are loss-of-function mutations in the SHH gene. According to ClinVar and the Human Gene Mutation Database, over 100 pathogenic variants have been identified. The following table summarizes common mutation types based on data from ClinVar and COSMIC (for cancer-related SHH mutations):

• • The SHH pathway is a key signaling network deregulated in HPE3. Key nodes include:
• • SHH ligand: mutations reduce its availability or affinity.
• • PTCH1: loss-of-function mutations increase pathway activity.
• • SMO: gain-of-function mutations lead to constitutive signaling.
• • GLI transcription factors: mutations affect their activity or stability.
• • SUFU: mutations impair negative regulation of GLI.
• • Additionally, cross-talk with other pathways such as WNT, FGF, and Notch is important in brain development and cancer.

Common cell lines used to study SHH signaling include:

Organoids derived from patient iPSCs with SHH mutations can recapitulate forebrain development and are valuable for studying HPE3 pathology.

• • Animal models for HPE3 include:
• • Shh knockout mice: exhibit holoprosencephaly and are used to study the role of SHH in development.
• • Conditional knockouts: tissue-specific deletion of Shh in the forebrain.
• • Patient-derived xenografts (PDX): not typical for HPE3 but used for SHH-driven cancers.
• • Genetically engineered mouse models (GEMM) with SHH mutations: used to study pathway activation in cancer.

CRISPR-based gene editing has enabled the creation of isogenic cell lines with specific SHH mutations. For example, a SHH knockout cell line can be generated in a human cell line (e.g., HEK293T) to study loss-of-function effects. Similarly, a knock-in of a pathogenic SHH point mutation (e.g., p.Cys183Phe) can be introduced to model the disease. These sequence-verified models are commercially available and accelerate research by providing consistent, reproducible systems for drug screening and functional studies.

Gene-edited cell lines are used to validate the function of SHH and other pathway components. For example, SHH knockout cells can be used to confirm the loss of downstream signaling by measuring GLI1 expression. Knock-in of specific mutations can help determine the impact on protein function and pathway activity.

Isogenic pairs (wild-type vs. SHH knockout) are valuable for drug screening. For instance, they can be used to identify compounds that selectively kill SHH-deficient cells, which is relevant for cancer therapy. Additionally, resistance to SMO inhibitors (e.g., vismodegib) can be studied by introducing secondary mutations in SMO or SUFU.

CRISPR-based synthetic lethality screens can identify genes that are essential in SHH-mutant cells but not in wild-type cells. This can lead to the discovery of novel biomarkers and therapeutic targets.