Autosomal Dominant Hypocalcemia (ADH) Cell Models for Research

Autosomal Dominant Hypocalcemia (ADH) is a rare genetic disorder characterized by low blood calcium levels (hypocalcemia) due to activating mutations in the calcium-sensing receptor (CASR) gene or, less commonly, in GNA11. The exact prevalence is unknown, but it is estimated to affect 1 in 70,000 individuals. ADH type 1 (ADH1) is caused by gain-of-function mutations in CASR, while ADH type 2 (ADH2) results from activating mutations in GNA11. The condition presents with mild to severe hypocalcemia, often leading to seizures, paresthesias, and muscle cramping. In some cases, it can cause basal ganglia calcification and nephrocalcinosis. The clinical severity varies, and some patients may be asymptomatic. There is no cure, and treatment focuses on managing symptoms with calcium and vitamin D supplements, which can be challenging due to the risk of hypercalciuria and nephrocalcinosis. The disease serves as a model for understanding calcium homeostasis and the role of the calcium-sensing receptor in parathyroid and renal function.

ADH is an ideal model for studying G-protein-coupled receptor (GPCR) signaling, particularly the calcium-sensing receptor (CASR). The disease is monogenic, making it amenable to gene editing to create isogenic cell lines that recapitulate the disease phenotype. These models are valuable for:

• • Elucidating the molecular mechanisms of CASR activation and signaling.
• • Investigating the differential effects of CASR mutations on receptor function.
• • Screening for compounds that modulate CASR activity, which could lead to new therapeutics.
• • Understanding the pathophysiology of hypocalcemia and related disorders.

Public datasets, such as ClinVar, provide a wealth of mutation data, while DepMap offers functional genomics data on cell lines, though ADH-specific cell lines are limited. Gene-edited cell models can fill this gap, providing controlled systems to study the disease.

The primary pathway involved in ADH is the calcium-sensing receptor (CASR) signaling pathway. CASR is a G-protein-coupled receptor that senses extracellular calcium levels and maintains calcium homeostasis. In ADH, activating mutations increase the receptor's sensitivity to calcium, leading to:

1. Increased signaling through the Gq/11-phospholipase C (PLC) pathway, resulting in inositol trisphosphate (IP3) production and intracellular calcium release.

2. Enhanced activation of the mitogen-activated protein kinase (MAPK) pathway, particularly ERK1/2.

3. Modulation of the parathyroid hormone (PTH) secretion and renal calcium reabsorption.

In ADH2, mutations in GNA11, which encodes the Gα11 subunit, also lead to constitutive activation of the same downstream pathways.

These alterations result in:

• • Suppressed PTH secretion from parathyroid chief cells.
• • Increased renal calcium excretion due to altered CASR signaling in the thick ascending limb of the loop of Henle.
• • The net effect is hypocalcemia and hypercalciuria.

Data from ClinVar and literature. The majority of CASR mutations are missense mutations located in the extracellular domain or transmembrane regions, leading to increased receptor activity. GNA11 mutations are less frequent but also result in constitutive activation.

The key deregulated signaling networks in ADH include:

• • Gq/11-PLC-IP3 pathway: Activation leads to intracellular calcium release and PKC activation.
• • MAPK/ERK pathway: Enhanced ERK1/2 phosphorylation, affecting cell proliferation and differentiation.
• • PI3K/AKT pathway: Crosstalk with CASR signaling, influencing cell survival.
• • β-arrestin signaling: Altered receptor desensitization and internalization.

Key nodes:

• • CASR
• • Gα11
• • PLCβ
• • IP3 receptor
• • PKC
• • ERK1/2
• • AKT

Organoids derived from patient tissues are emerging as more physiologically relevant models. They can be generated from induced pluripotent stem cells (iPSCs) carrying ADH mutations, providing a platform to study tissue-specific effects.

Animal models for ADH are limited, but include:

• • Genetically engineered mouse models (GEMMs): Mice with knock-in mutations in Casr or Gna11 that mimic human ADH mutations. These mice exhibit hypocalcemia and hypercalciuria.
• • Induced models: Use of pharmacological agents to activate CASR, such as calcimimetics, to mimic the hyperactive receptor.
• • Patient-derived xenografts (PDX): Not applicable for ADH as it is not a cancer, but xenografts of parathyroid tissue could be used.

These models are useful for studying systemic effects and testing therapeutic interventions.

CRISPR-based gene editing enables the creation of isogenic cell lines with specific ADH mutations. These models are essential for dissecting the functional consequences of individual mutations in a controlled genetic background. Examples include:

• • CASR knockout cell lines: Complete loss of CASR function, useful for studying the receptor's role in calcium sensing.
• • CASR gain-of-function knock-in lines: Introduction of specific ADH mutations (e.g., p.Arg185Gln) into a wild-type background, recapitulating the disease phenotype.
• • GNA11 knock-in lines: Similar approach for ADH2 mutations.

These gene-edited cell lines are commercially available from various sources, ensuring sequence verification and quality. They are invaluable for drug screening, functional genomics, and mechanistic studies. For instance, a CASR knockout line can be used as a negative control, while a knock-in line with an activating mutation can be used to screen for negative allosteric modulators (calcilytics) that could treat ADH.

Gene-edited cell lines are used to validate the function of CASR and GNA11 mutations. By introducing specific mutations, researchers can:

• • Assess the impact on receptor signaling using calcium mobilization assays.
• • Determine the effect on downstream pathways such as MAPK/ERK.
• • Study the interaction of mutant receptors with ligands and allosteric modulators.

For example, a knock-in cell line with a known ADH mutation can be compared to the isogenic wild-type to identify differentially expressed genes and pathways.

Isogenic pairs (wild-type vs. mutant) are powerful tools for drug screening. They allow:

• • High-throughput screening of compounds that modulate CASR activity.
• • Identification of calcilytics that inhibit the overactive receptor.
• • Testing of resistance mechanisms: cells can be exposed to drugs to select for resistant clones, revealing secondary mutations.

For ADH, such screens could lead to novel therapeutics that normalize calcium levels without causing hypercalciuria.

CRISPR-based screens, such as synthetic lethality screens, can identify genes that are essential for the survival of cells with ADH mutations. This could reveal novel therapeutic targets. Additionally, gene-edited cells can be used to identify biomarkers of disease progression or response to treatment.