Rickets Cell Models for Research

Rickets is a childhood bone disease characterized by impaired mineralization of the growth plate, leading to skeletal deformities, growth retardation, and muscle weakness. The global incidence varies, but nutritional rickets remains a public health concern in low- and middle-income countries, with a prevalence of up to 10% in some regions (WHO, 2023). In developed countries, inherited forms such as X-linked hypophosphatemic rickets (XLH) affect approximately 1 in 20,000 individuals (NCBI). The clinical impact includes significant morbidity, with complications like bowed legs, bone pain, and dental abnormalities. Early diagnosis and treatment are critical to prevent long-term disability.

Rickets serves as an ideal model for studying bone metabolism, calcium-phosphate homeostasis, and vitamin D signaling. The disease encompasses multiple subtypes with distinct genetic etiologies, including nutritional, vitamin D-dependent, and hypophosphatemic forms. These subtypes provide a spectrum of molecular perturbations that can be dissected using gene-edited cell models. Public datasets, such as those from the NCBI Gene Expression Omnibus (GEO), offer transcriptomic profiles of bone and kidney tissues from rickets patients, enabling researchers to identify novel pathways and therapeutic targets. Open questions include the precise molecular mechanisms linking FGF23 to phosphate regulation and the role of vitamin D receptor (VDR) in non-classical tissues.

Rickets arises from disruptions in the vitamin D endocrine system, phosphate homeostasis, or calcium metabolism. Key pathways include:

• • Vitamin D signaling: Vitamin D is hydroxylated in the liver (CYP2R1) to 25-hydroxyvitamin D, then in the kidney (CYP27B1) to the active form 1,25-dihydroxyvitamin D. This binds to the vitamin D receptor (VDR), regulating gene expression involved in calcium and phosphate absorption.
• • FGF23 signaling: Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone that inhibits renal phosphate reabsorption and suppresses CYP27B1 activity. Excess FGF23 leads to hypophosphatemia, as seen in XLH and autosomal dominant hypophosphatemic rickets (ADHR).
• • Calcium-sensing receptor (CaSR) pathway: CaSR modulates parathyroid hormone (PTH) secretion, which influences renal calcium and phosphate handling. Mutations in CaSR can cause familial hypocalciuric hypercalcemia and rickets-like phenotypes.
• • Phosphate regulating endopeptidase homolog, X-linked (PHEX): PHEX is a metalloprotease that regulates FGF23 expression. Inactivating mutations in PHEX cause XLH, the most common inherited form of rickets.

Data from TCGA and COSMIC for somatic mutations are not applicable as rickets is primarily a germline disease. Frequencies are based on ClinVar and literature.

Rickets involves crosstalk between several signaling networks:

• • FGF23-Klotho axis: FGF23 binds to FGFR1 and co-receptor Klotho, activating MAPK/ERK pathways in the kidney, leading to downregulation of sodium-phosphate cotransporters (NPT2a/NPT2c) and CYP27B1.
• • Vitamin D-VDR signaling: VDR heterodimerizes with RXR, binding to vitamin D response elements (VDREs) to regulate genes like TRPV6, calbindin, and osteocalcin. Mutations in VDR or CYP27B1 disrupt this axis.
• • PTH-PTH1R signaling: PTH activates PTH1R on bone and kidney, increasing calcium reabsorption and phosphate excretion. In rickets, secondary hyperparathyroidism often occurs due to hypocalcemia.
• • Wnt/β-catenin pathway: This pathway is critical for osteoblast differentiation and bone mineralization. Altered FGF23 signaling can modulate Wnt activity, contributing to bone defects.

Organoids derived from patient-derived induced pluripotent stem cells (iPSCs) offer a more physiologically relevant model. For example, kidney organoids can recapitulate proximal tubule function and allow study of phosphate handling. Bone organoids are emerging as tools to investigate mineralization defects.

Animal models for rickets include:

• • Hyp mouse: A spontaneous PHEX mutant mouse model that recapitulates XLH, characterized by hypophosphatemia, rickets, and osteomalacia.
• • VDR knockout mouse: Exhibits alopecia, hypocalcemia, and rickets, mimicking VDDR-II.
• • CYP27B1 knockout mouse: Shows impaired calcium absorption and rickets, similar to VDDR-I.
• • FGF23 transgenic mouse: Overexpressing FGF23 leads to hypophosphatemia and rickets.
• • Diet-induced rickets: Rodents fed a low-phosphate or low-vitamin D diet develop nutritional rickets.

Patient-derived xenografts (PDX) are less common for rickets due to the non-oncogenic nature, but bone and kidney tissue from patients can be used for ex vivo studies.

CRISPR-Cas9 technology enables the creation of isogenic cell lines with precise genetic modifications to study rickets-associated genes. For example:

• • PHEX knockout cell lines: Generated in HEK293 or HK-2 cells to study FGF23 regulation and phosphate transport.
• • CYP27B1 knockout cell lines: Used to investigate vitamin D metabolism and its downstream effects.
• • VDR knockout cell lines: Help elucidate VDR-mediated gene regulation and resistance to vitamin D.
• • FGF23 knock-in cell lines: Introducing gain-of-function mutations (e.g., R176Q) allows study of FGF23 processing and signaling.

These sequence-verified models are commercially available from various sources, ensuring reproducibility and accelerating research. They are essential for functional validation of genetic variants and drug screening.

Gene-edited cells are used to validate the functional impact of genetic variants identified in rickets patients. For instance, introducing a specific PHEX mutation into a wild-type cell line and assessing FGF23 expression confirms causality. Similarly, knocking out CYP27B1 in kidney cells can demonstrate the role of this enzyme in converting 25(OH)D to 1,25(OH)2D. These models allow high-throughput screening of potential therapeutic compounds.

Isogenic cell line pairs (wild-type vs. knockout) are powerful tools for drug screening. For example, a VDR knockout cell line can be used to test the efficacy of vitamin D analogs in a background lacking VDR, revealing off-target effects. Resistance mechanisms can be studied by exposing cells to escalating doses of drugs and selecting for resistant clones, then identifying genetic changes via sequencing.

CRISPR-based synthetic lethality screens can identify genes that, when knocked out, are lethal only in the context of a specific rickets mutation. This approach can uncover novel therapeutic targets. Additionally, gene-edited cells can be used to discover biomarkers by comparing secretomes or transcriptomes of mutant vs. wild-type cells, leading to potential diagnostic markers.