Lipoid Congenital Adrenal Hyperplasia (LCAH) Cell Models for Research

Lipoid Congenital Adrenal Hyperplasia (LCAH) is a rare autosomal recessive disorder characterized by impaired steroidogenesis, leading to adrenal insufficiency and disorders of sex development. The exact incidence is unknown, but it is more prevalent in Japanese and Korean populations due to founder mutations. According to the WHO, adrenal disorders contribute to significant morbidity if untreated. The NCI does not track LCAH specifically, but it is a model for understanding adrenal physiology and steroid hormone biosynthesis. Early diagnosis and glucocorticoid/mineralocorticoid replacement therapy improve outcomes, but long-term complications include adrenal crises and infertility.

LCAH is an ideal model for studying steroidogenesis, cholesterol transport, and mitochondrial function. The disease is primarily caused by mutations in the STAR gene (steroidogenic acute regulatory protein) and less frequently in CYP11A1 (cholesterol side-chain cleavage enzyme). These genes are critical for the first step of steroidogenesis. Research focuses on understanding the molecular mechanisms of these mutations, developing gene therapies, and screening for drugs that can bypass the block. Public datasets from ClinVar and gnomAD provide variant information, but functional studies require cellular models.

LCAH is not a cancer, but the pathways involved are relevant to adrenal tumors and steroidogenic disorders. The key pathway is the steroidogenesis pathway, which involves:

1. Cholesterol uptake and transport to the inner mitochondrial membrane by STAR.

2. Conversion of cholesterol to pregnenolone by CYP11A1.

3. Subsequent enzymatic steps to produce cortisol, aldosterone, and androgens.

Mutations in STAR or CYP11A1 disrupt this pathway, leading to accumulation of cholesterol esters and cellular damage. This can trigger oxidative stress and apoptosis in adrenocortical cells.

Data from ClinVar and literature. The most common STAR mutation is c.772C>T (p.Arg258Trp) in Japanese patients.

The primary deregulated network is the steroidogenesis pathway. Key nodes include:

• • STAR: regulates cholesterol import into mitochondria.
• • CYP11A1: catalyzes side-chain cleavage.
• • CYP17A1, CYP21A2, CYP11B1, CYP11B2: downstream enzymes.
• • ACTH receptor (MC2R) and its signaling cascade (cAMP/PKA).

Disruption of this pathway leads to compensatory upregulation of ACTH, causing adrenal hyperplasia. Additionally, oxidative stress and ER stress pathways are activated due to lipid accumulation.

Organoids derived from adrenal tissue or iPSCs are emerging as more physiologically relevant models, allowing long-term culture and differentiation.

• • Star knockout mice: exhibit lipid accumulation in adrenal glands and die shortly after birth due to adrenal insufficiency.
• • Cyp11a1 knockout mice: similar phenotype, but also show sex reversal in males.
• • Induced models using CRISPR in zebrafish or rats are being developed.
• • PDX models are not common for LCAH due to its non-cancerous nature.

CRISPR-Cas9 gene editing enables the creation of isogenic cell lines with specific STAR or CYP11A1 mutations. For example:

• • STAR knockout cell lines: generated in NCI-H295R or SW-13 cells to model loss of function.
• • CYP11A1 point mutation knock-in lines: introduce pathogenic variants (e.g., p.Arg258Trp) to study their impact.

These models are commercially available from various sources and are sequence-verified. They are essential for studying the molecular consequences of mutations and for drug screening.

Knockout and knock-in lines are used to validate the functional impact of STAR and CYP11A1 variants. For example, STAR knockout cells show reduced pregnenolone production, confirming the gene's role. Knock-in of a pathogenic mutation can recapitulate the disease phenotype, allowing mechanistic studies.

Isogenic pairs (wild-type vs. mutant) are used to screen for compounds that can restore steroidogenesis. For instance, drugs that enhance cholesterol transport or bypass STAR function can be tested. Resistance to treatments can also be modeled by exposing cells to increasing concentrations of drugs.

CRISPR synthetic lethality screens can identify genes that, when knocked out, kill STAR or CYP11A1 mutant cells but not wild-type cells. This can reveal potential therapeutic targets. Additionally, transcriptomic and proteomic analyses of mutant cells can identify biomarkers for early diagnosis.