CHST3 Human

What is the molecular function of CHST3 in human biology?

CHST3 (Carbohydrate Sulfotransferase 3), also known as chondroitin 6-sulfotransferase (C6ST), catalyzes the transfer of sulfate to the C-6 position of N-acetylgalactosamine residues in chondroitin chains. This enzyme is critical for proper glycosaminoglycan modification, creating specific sulfation patterns that influence the structural and functional properties of the extracellular matrix. The sulfation process creates negative charges on glycosaminoglycan surfaces, which is essential for their biological functions in tissue development and homeostasis .

CHST3 belongs to the CHST family, which includes 14 enzymes that transfer sulfate onto various positions of GalNAc, Gal, and GlcNAc residues on glycoproteins, proteoglycans, and glycolipids. These modifications create specific epitopes recognized by extracellular matrix proteins and cell surface receptors, enabling complex cellular signaling processes .

Where is CHST3 primarily expressed in human tissues?

CHST3 exhibits a diverse tissue expression pattern, being highly expressed in heart, skeletal muscles, placenta, and thymus . Immunohistochemical analysis has detected CHST3 in the cytoplasm of endocrine cells in human pancreatic tissue, demonstrating tissue-specific localization patterns . This expression profile corresponds to the various physiological roles of CHST3 in different organ systems.

Methodologically, CHST3 detection in tissues requires specific approaches such as immunohistochemistry with heat-induced epitope retrieval and appropriate staining protocols, as demonstrated in studies examining pancreatic tissue sections .

How is CHST3 regulated during development and aging?

CHST3 expression and activity undergo significant regulation throughout the lifespan. Notably, epigenetic mechanisms play a crucial role in CHST3 regulation during aging. Research in murine models has revealed a decrease in H3K4me3 (three methyl groups at lysine 4 on histone H3 proteins) associated with the promoter region of the CHST3 gene specifically in non-neuronal (NeuN-negative) cells of aging brains, but not in neuronal (NeuN-positive) cells .

This epigenetic dysregulation corresponds with decreased CHST3 expression in aged brains (22-26 months old). Experimental analyses comparing young (2-3 month) and older (>30 month) mice demonstrated that while general extracellular matrix genes show minor changes in expression, CHST3 exhibits significant downregulation with age . These findings suggest that age-related epigenetic mechanisms specifically target CHST3, potentially contributing to altered extracellular matrix composition in aging tissues.

What pathogenic variants in CHST3 have been identified and how are they characterized?

Multiple pathogenic variants in the CHST3 gene have been identified in patients with spondyloepiphyseal dysplasia with congenital joint dislocations (SEDCJD). These include:

Characterization of these variants typically involves whole-exome sequencing followed by validation through Sanger sequencing and segregation analysis in family members. Bioinformatic analysis incorporates evidence from multiple sources including clinical databases (ClinVar, OMIM), population frequency databases, and in silico prediction tools .

What inheritance patterns are observed with CHST3-related disorders?

CHST3-related spondyloepiphyseal dysplasia follows an autosomal recessive inheritance pattern. Individuals with the condition carry two mutated copies of the CHST3 gene, one from each parent. Carriers with heterozygous mutations typically do not exhibit skeletal abnormalities or other manifestations of CHST3 dysfunction .

In consanguineous families, homozygous mutations are more common, as demonstrated in a Chinese family where affected individuals carried a homozygous mutation (c.626C>A; p.Pro209Gln) . In non-consanguineous families, compound heterozygosity is frequently observed, where individuals inherit two different pathogenic variants. For example, one case identified compound heterozygous variants c.491C>T (p.Pro164Leu) and c.500A>G (p.His167Arg) with maternal and paternal inheritance respectively .

For prenatal diagnosis, methodologies such as chorionic villus sampling followed by Sanger sequencing can determine fetal genotype for known familial CHST3 variants, facilitating informed reproductive decisions .

What is the clinical spectrum of CHST3-related skeletal dysplasias?

CHST3 mutations cause spondyloepiphyseal dysplasia with congenital joint dislocations (SEDCJD), characterized by several distinctive clinical features:

• Severe short stature, often with prenatal onset

• Multiple joint dislocations, particularly of the knees

• Progressive scoliosis

• Club foot (talipes equinovarus)

• Deformed feet

• Short and stubby long bones (femur, tibia-fibula, and radius-ulna)

Radiographic findings typically include abnormal epiphyseal development and characteristic skeletal abnormalities visible on prenatal ultrasound in severe cases. The condition results from impaired chondroitin 6-sulfation, which affects cartilage development and joint formation .

How does phenotypic variability manifest in patients with CHST3 mutations?

Significant phenotypic variability exists among individuals with CHST3 mutations, even those sharing identical genetic variants. This variability extends to specific features like hearing loss, which is present in some patients but absent in others from the same geographic or ethnic background .

For example, in a study of Pakistani families with biallelic CHST3 variants (c.590T>C, c.603C>A, and c.661C>T), none of the affected individuals exhibited hearing loss, contrary to previous reports of hearing impairment in patients with CHST3-related disorders . This observation suggests that additional genetic, environmental, or epigenetic factors modify the clinical expression of CHST3 mutations.

Another dimension of phenotypic variability involves extra-skeletal manifestations. Some patients with CHST3 variants demonstrate multiple heart valve deformities, while others show exclusively skeletal phenotypes . This heterogeneity complicates genotype-phenotype correlations and necessitates comprehensive clinical evaluation alongside genetic testing.

What methodologies are most effective for studying CHST3 expression and function?

Multiple complementary approaches can be employed to investigate CHST3 expression and function:

Protein Detection Techniques:

• Immunohistochemistry (IHC): Effective for localizing CHST3 in tissue sections, as demonstrated in studies of human pancreas where specific staining was identified in the cytoplasm of endocrine cells. Optimal protocols involve heat-induced epitope retrieval, primary antibody incubation (e.g., using Rat Anti-Human CHST3 Monoclonal Antibody), and appropriate detection systems .

• Enzyme-Linked Immunosorbent Assay (ELISA): Direct ELISAs can detect recombinant human CHST3, with validated antibodies showing specificity without cross-reactivity to related proteins like CHST1 .

Genetic and Epigenetic Analysis:

• Fluorescence-Activated Cell Sorting (FACS) followed by cell type-specific analysis: This approach has been used to separate neuronal (NeuN-positive) from non-neuronal (NeuN-negative) cells to study cell type-specific epigenetic regulation of CHST3 .

• Chromatin Immunoprecipitation (ChIP): Essential for assessing histone modifications (e.g., H3K4me3) at the CHST3 promoter region to understand epigenetic regulation mechanisms .

Animal Models:

• Experimental design considerations: Studies in mice have utilized age-matched cohorts (e.g., 2-3 month vs. 22-26 month animals) to investigate age-related changes in CHST3 expression. Cognitive assessment followed by molecular analysis allows correlation between behavioral and biochemical parameters .

How can CHST3 be implicated in age-related musculoskeletal disorders?

Recent transcriptomic analyses have identified CHST3 as one of three key genes (along with PGBD5 and SLIT2) with potential diagnostic value for osteoporosis and sarcopenia . The research methodology integrating multiple approaches has yielded several insights:

• Differential Expression Analysis: Using LIMMA, WGCNA, and DEseq2 packages to identify consistently dysregulated genes across multiple datasets for osteoporosis and sarcopenia .

• Machine Learning Validation: Three distinct machine learning methods were employed to determine the final common diagnostic genes, with receiver operating characteristic (ROC) curves used to evaluate their predictive performance .

• Functional Characterization: Gene Set Enrichment Analysis (GSEA) revealed that CHST3 is primarily involved in pathways related to cell cycle regulation, fatty acid metabolism, DNA replication, and carbohydrate synthesis .

• Immune System Involvement: Immune infiltration abundance calculations demonstrated that CHST3 participates in immune response pathways, which may contribute to the inflammatory component of age-related musculoskeletal degeneration .

Experimental validation using RT-PCR and Western blotting has confirmed the dysregulation of CHST3 in these conditions, supporting its potential use as a diagnostic biomarker and therapeutic target .

How do epigenetic mechanisms regulate CHST3 expression in aging and disease?

Epigenetic regulation of CHST3 has emerged as a significant area of research, particularly in the context of aging. Experimental evidence from mouse models demonstrates selective epigenetic dysregulation of CHST3 in aging brains. Specifically, a decrease in H3K4me3 (a histone modification associated with active transcription) was observed at the CHST3 promoter region in non-neuronal cells from aged mice (22-23 months old), but not in neuronal cells .

This cell type-specific epigenetic regulation corresponds with the observed downregulation of CHST3 expression in aging brains. The experimental approach to establish this finding involved:

• Age-stratified cohorts of mice (2-3 months vs. 22-23 months)

• FACS sorting to separate neuronal (NeuN-positive) from non-neuronal (NeuN-negative) cells

• Analysis of histone modifications associated with the CHST3 promoter

• Correlation with gene expression data

These findings suggest that epigenetic mechanisms target specific genes like CHST3 during aging, potentially contributing to age-related changes in extracellular matrix composition and function.

What strategies are being explored for therapeutic targeting of CHST3-related disorders?

While no approved therapies specifically target CHST3 dysfunction, several approaches show promise:

Computational Drug Repurposing:
The Connectivity Map (CMap) database has been used to identify potential therapeutic compounds that may reverse gene expression signatures associated with CHST3 dysregulation in osteoporosis and sarcopenia. These predicted therapeutics have undergone preliminary validation through techniques like RT-PCR and Western blotting .

Genetic Approaches:
For monogenic CHST3-related disorders like spondyloepiphyseal dysplasia, genetic interventions hold promise. Current strategies include:

• Prenatal genetic diagnosis: Chorionic villus sampling followed by Sanger sequencing allows identification of CHST3 variants in utero, enabling informed reproductive decisions .

• Potential gene therapy approaches: Given the recessive nature of CHST3-related disorders, gene replacement strategies could theoretically restore enzyme function.

Targeting Downstream Pathways:
Understanding the molecular pathways affected by CHST3 dysfunction provides opportunities for intervention at downstream points. GSEA analysis has identified several pathways influenced by CHST3, including cell cycle regulation, fatty acid metabolism, and immune responses . These pathways represent potential targets for therapeutic development.

Advances in understanding the structure-function relationships of CHST3 and the specific mechanisms by which its dysfunction leads to disease will be essential for developing targeted therapeutic approaches for CHST3-related disorders.