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. 2023 May 23;7(6):e0140.
doi: 10.1097/HC9.0000000000000140. eCollection 2023 Jun 1.

Circulating cell-free messenger RNA secretome characterization of primary sclerosing cholangitis

Naga Chalasani  1 , Raj Vuppalanchi  1 , Craig Lammert  1 , Samer Gawrieh  1 , Jerome V Braun  2 , Jiali Zhuang  2 , Arkaitz Ibarra  2 , David A Ross  2 , Michael Nerenberg  2 , Stephen R Quake  3   4 , John J Sninsky  2 , Shusuke Toden  2
Affiliations

Circulating cell-free messenger RNA secretome characterization of primary sclerosing cholangitis

Naga Chalasani et al. Hepatol Commun. .

Abstract

Background: Primary sclerosing cholangitis (PSC) is a rare chronic cholestatic liver disease characterized by multifocal bile duct strictures. To date, underlying molecular mechanisms of PSC remain unclear, and therapeutic options are limited.

Methods: We performed cell-free messenger RNA (cf-mRNA) sequencing to characterize the circulating transcriptome of PSC and noninvasively investigate potentially bioactive signals that are associated with PSC. Serum cf-mRNA profiles were compared among 50 individuals with PSC, 20 healthy controls, and 235 individuals with NAFLD. Tissue and cell type-of-origin genes that are dysregulated in subjects with PSC were evaluated. Subsequently, diagnostic classifiers were developed using PSC dysregulated cf-mRNA genes.

Results: Differential expression analysis of the cf-mRNA transcriptomes of PSC and healthy controls resulted in identification of 1407 dysregulated genes. Furthermore, differentially expressed genes between PSC and healthy controls or NAFLD shared common genes known to be involved in liver pathophysiology. In particular, genes from liver- and specific cell type-origin, including hepatocyte, HSCs, and KCs, were highly abundant in cf-mRNA of subjects with PSC. Gene cluster analysis revealed that liver-specific genes dysregulated in PSC form a distinct cluster, which corresponded to a subset of the PSC subject population. Finally, we developed a cf-mRNA diagnostic classifier using liver-specific genes that discriminated PSC from healthy control subjects using gene transcripts of liver origin.

Conclusions: Blood-based whole-transcriptome cf-mRNA profiling revealed high abundance of liver-specific genes in sera of subjects with PSC, which may be used to diagnose patients with PSC. We identified several unique cf-mRNA profiles of subjects with PSC. These findings may also have utility for noninvasive molecular stratification of subjects with PSC for pharmacotherapy safety and response studies.

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Conflict of interest statement

Jerome V. Braun, Jiali Zhuang, Arkaitz Ibarra, David A. Ross, Michael Nerenberg, John J. Sninsky, and Shusuke Toden are past or current employees at Molecular Stethoscope, Inc. Stephen R. Quake is a founder of Molecular Stethoscope, Inc. and a member of its scientific advisory board. Stephen R. Quake, John J. Sninsky, and Shusuke Toden have company stock options. Naga Chalasani has ongoing consulting agreements with Abbvie, Madrigal, Foresite, Zydus, ObsEva, and Galectin and research support from DSM, Exact Sciences, Zydus, and Intercept. Craig Lammert consults for Kezar. Stephen R. Quake advises, is employed, and owns stock in Molecular Stethoscope. Jerome V. Braun consults for Molecular Stethoscope. Samer Gawrieh consults for TransMedics and Pfizer and received grants from Zydus, Viking, and SonicIncytes. David A. Ross consults for Molecular Stethoscope. Michael Nerenberg owns stock in Molecular Stethoscope. John J. Sninsky is employed and owns stock in Molecular Stethoscope. The remaining author has no conflicts to report.

Figures

FIGURE 1
FIGURE 1
Identification of high levels of liver-specific genes in cf-mRNA of subjects with PSC. (A) Schematics of the study design. (B) A volcano plot showing the dysregulation of cf-mRNAs in subjects with PSC compared with that of healthy controls. (C) Top IPA canonical pathways for genes that are dysregulated in PSC; upregulated and downregulated genes are used as input. (D) Quantification of tissue-specific transcripts identified in cf-mRNA of subjects with PSC (mean ± SEM). (E) Quantification of genes that are associated with liver in genes that are differentially expressed in subjects with PSC compared with healthy controls (upregulated genes, top; downregulated genes, bottom). Reference data sets used to calculate the liver tissue and cell type specificities are HPA (left), GTEx (middle), and PanglaoDB (right). Abbreviations: cf-mRNA, cell-free messenger RNA; GTEx, genotype-tissue expression; HPA, Human Protein Atlas; IPA, ingenuity pathway analysis; PSC, primary sclerosing cholangitis.
FIGURE 2
FIGURE 2
Distribution of liver-specific genes. (A) Distribution of liver cell-type-specific genes that are differentially upregulated in subjects with PSC compared with healthy controls. (B) The abundance of liver cell-specific genes per sample based on cell types. (C) IPA was used to evaluate the upstream regulating genes (PSC genes that were upregulated compared with downregulated genes are used as the inputs). (D) The overlap between genes that were identified in cf-mRNA of subjects with PSC (TPM > 1) and genes identified in human EVs (ExoRBase used as the reference). (E) A table summarizing the liver-associated molecular characteristics of the top 10 most upregulated genes in cf-mRNA of subjects with PSC compared with healthy controls. Abbreviations: cf-mRNA, cell-free messenger RNA; EV, extracellular vesicle; IPA, ingenuity pathway analysis; PSC, primary sclerosing cholangitis; TPM, transcripts per million.
FIGURE 3
FIGURE 3
Cf-mRNA expression levels of liver cell-type-specific genes in subjects with PSC. (A) Gene expression correspondence of liver-specific genes between PSC and healthy controls (mean expression levels of individual genes were used for the correlation). (B) Schematics of liver cell-type-specific genes that may be involved in induction of sclerosis. (C) TPM levels of HRG, FGB, and FGA genes between PSC and healthy controls. (***p < 0.001) (D) TPM levels of HSC-associated genes NR1H4 and PPARA between PSC and healthy controls. (***p < 0.001) (E) TPM levels of KC-associated genes C1QA, C1QB, and C1QC between PSC and healthy controls. (***p < 0.001). (F) TPM levels of cholangiocyte cell-associated gene, LGALS4 and HNF1A between PSC and healthy controls. (***p < 0.001). Abbreviations: cf-mRNA, cell-free messenger RNA; FGA, fibrinogen alpha; FGB, fibrinogen beta; HRG, histidine-rich glycoprotein; PSC, primary sclerosing cholangitis; TPM, transcripts per million.
FIGURE 4
FIGURE 4
Identification of PSC-associated cf-mRNA gene clusters and subject stratification using cf-mRNA gene clusters. (A) Consensus matrix NMF clustering of genes that are differentially expressed in cf-mRNA of subjects with PSC compared with healthy controls. Seven major cluster subtypes are labeled. (B) Most significant canonical pathway identified for each of 7 major clusters (IPA gene set enrichment analysis). (C) The expression of the cluster 5 genes (total 161 genes) and cluster 1 genes (total 356 genes) and their tissue-specific gene expression using GTEx. (D) The individual levels of clusters 5 and 1 coefficient between healthy controls and subjects with PSC. (E) Unsupervised clustering of subjects with PSC using their cf-mRNA profile based on NMF clusters identified in (A) revealed 5 groups of subjects with PSC. Abbreviations; cf-mRNA, cell-free messenger RNA; GTEx, genotype-tissue expression; IPA, ingenuity pathway analysis; NMF, non-negative matrix factorization; PSC, primary sclerosing cholangitis.
FIGURE 5
FIGURE 5
Establishment of PSC diagnosis classifiers. (A) ROC curve of cf-mRNA PSC classifiers using liver-specific genes as input compared with healthy controls. (B) ROC curve of cf-mRNA PSC classifiers using all genes as input comparing to healthy controls. Abbreviations: cf-mRNA, cell-free messenger RNA; PSC, primary sclerosing cholangitis; ROC, receiver operating characteristic.
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