Trichohepatoenteric Syndrome (THES) Cell Models for Research

Trichohepatoenteric syndrome (THES), also known as syndromic diarrhea, is an ultra-rare autosomal recessive disorder with an estimated incidence of 1 in 500,000 to 1 in 1,000,000 live births (WHO, 2023). The condition is characterized by severe intractable diarrhea in infancy, leading to profound failure to thrive, facial dysmorphism, hair abnormalities, and immune dysfunction. Prognosis is poor, with many patients succumbing to complications in early childhood; however, some survive into adolescence with aggressive nutritional support and infection management (NCI, 2023). The rarity of THES poses significant challenges for clinical research, but its well-defined genetic basis makes it an attractive model for studying intestinal epithelial homeostasis, immune regulation, and RNA metabolism.

THES is caused by mutations in two genes, TTC37 and SKIV2L, which encode components of the human SKI complex, a cytoplasmic RNA exosome cofactor. This pathway is evolutionarily conserved and plays a critical role in RNA surveillance and degradation. Studying THES provides insights into fundamental cellular processes such as mRNA turnover, ribosome quality control, and the unfolded protein response. Moreover, the intestinal phenotype offers a unique opportunity to investigate epithelial barrier function and innate immunity. Public datasets from the NCBI Gene and ClinVar provide detailed mutation information, and the availability of patient-derived cell lines and induced pluripotent stem cells (iPSCs) facilitates mechanistic studies. However, isogenic gene-edited models are essential to dissect the specific contributions of TTC37 and SKIV2L mutations in a controlled genetic background.

The SKI complex, comprising TTC37 and SKIV2L, is a cytoplasmic cofactor of the RNA exosome, which degrades aberrant mRNAs and regulates RNA processing. Loss-of-function mutations in TTC37 or SKIV2L impair RNA surveillance, leading to accumulation of defective mRNAs and activation of stress responses. Key pathways affected include:

• • RNA exosome-mediated mRNA decay: The SKI complex unwinds RNA secondary structures and recruits the exosome to degrade mRNAs with premature stop codons or other errors.
• • Nonsense-mediated decay (NMD): The SKI complex interacts with NMD factors to eliminate transcripts containing nonsense mutations, preventing the production of truncated proteins.
• • Ribosome-associated quality control (RQC): The SKI complex is involved in the degradation of mRNA after ribosome stalling, ensuring proper protein synthesis.
• • Unfolded protein response (UPR): Accumulation of aberrant mRNAs can trigger endoplasmic reticulum stress and activate the UPR, leading to apoptosis or inflammation.

These disruptions result in intestinal epithelial cell dysfunction, immune dysregulation, and the characteristic clinical features of THES.

Data from ClinVar and the Human Gene Mutation Database (HGMD) indicate that most THES-associated mutations are loss-of-function, leading to complete or partial loss of SKI complex activity. The mutational spectrum is diverse, with no clear genotype-phenotype correlation, underscoring the need for functional validation of specific variants.

The SKI complex is a central regulator of RNA metabolism, and its dysfunction affects multiple signaling pathways:

• • Innate immune signaling: Accumulation of viral or endogenous double-stranded RNA can activate RIG-I-like receptors and the type I interferon response, contributing to immune dysregulation.
• • Inflammatory bowel disease (IBD)-like pathways: Intestinal inflammation in THES may involve activation of NF-κB and cytokine production, similar to IBD.
• • Epithelial barrier integrity: Disrupted RNA surveillance affects tight junction proteins and mucosal homeostasis, leading to diarrhea and malabsorption.
• • Apoptosis and cell survival: ER stress and UPR activation can trigger apoptosis in intestinal epithelial cells.

Key nodes in these networks include RIG-I, MDA5, NF-κB, and the unfolded protein response sensors IRE1, PERK, and ATF6.

Organoids derived from patient intestinal biopsies or iPSCs recapitulate the intestinal epithelium and are valuable for studying THES pathophysiology. However, they are limited by genetic heterogeneity and difficulty in genetic manipulation. Isogenic gene-edited cell lines provide a more controlled system.

• • Zebrafish models: Knockdown of ttc37 or skiv2l in zebrafish recapitulates intestinal defects and immune abnormalities.
• • Mouse models: Germline knockout of Ttc37 or Skiv2l in mice leads to embryonic lethality, so conditional knockouts are needed. Induced models using CRISPR/Cas9 are being developed.
• • Patient-derived xenografts (PDX): Not applicable for THES as it is not a cancer, but organoid xenografts can be used for drug testing.

These models are useful for studying disease mechanisms but are limited by species differences and the complexity of the intestinal phenotype.

CRISPR-Cas9 gene editing enables the creation of isogenic cell lines with precise mutations in TTC37 or SKIV2L. These models are essential for functional studies because they allow direct comparison of mutant and wild-type cells in the same genetic background. Examples include:

• • TTC37 knockout cell lines: Generated by introducing frameshift mutations in exon 2 or 3, leading to loss of protein expression.
• • SKIV2L point-mutation knock-in lines: Mimic patient-specific missense mutations, such as p.Arg888Trp, to study their functional impact.

These cell lines are commercially available from several vendors and are sequence-verified. They are ideal for studying RNA metabolism, immune signaling, and drug responses. Using such models accelerates research by providing reproducible, well-characterized tools.

Gene-edited cell lines are used to validate the functional consequences of THES-associated mutations. For example, TTC37 knockout cells can be used to assess the impact on RNA exosome activity, mRNA decay rates, and the expression of downstream target genes. Similarly, SKIV2L knock-in cells can be used to study the effect of specific mutations on helicase activity and protein-protein interactions. These models enable high-throughput screens to identify genetic modifiers and synthetic lethal partners.

Isogenic pairs of wild-type and mutant cells are ideal for drug screening. For THES, there are no targeted therapies, but gene-edited cells can be used to screen for compounds that rescue the cellular phenotype, such as small molecules that enhance RNA surveillance or reduce ER stress. Additionally, these models can be used to test drug resistance mechanisms, as the SKI complex may influence the response to chemotherapeutic agents.

CRISPR screens using gene-edited cells can identify genes that, when knocked out, suppress or exacerbate the THES phenotype. This approach can reveal novel biomarkers and therapeutic targets. For example, a genome-wide CRISPR screen in TTC37 knockout cells could identify genes whose loss rescues cell viability, providing insights into compensatory pathways.