Am J Physiol Gastrointest Liver Physiol 320: G439–G449, 2021.

First published January 27, 2021; doi:10.1152/ajpgi.00397.2020

RESEARCH ARTICLE

Inflammation, Immunity, Fibrosis, and Infection

Noninvasive stratification of nonalcoholic fatty liver disease by whole

transcriptome cell-free mRNA characterization
Naga Chalasani,1 Shusuke Toden,2 John J. Sninsky,2 Richard P. Rava,2 Jerome V. Braun,2 Samer Gawrieh,1
Jiali Zhuang,2 Michael Nerenberg,2 Stephen R. Quake,3 and Tara Maddala2
1
Division of Gastroenterology and Hepatology, Department of Medicine, Indiana University School of Medicine, Indianapolis,
Indiana; 2Molecular Stethoscope, San Diego, California; and 3Departments of Bioengineering and Applied Physics, Stanford
University and Chan Zuckerberg Biohub, Stanford, California

Abstract
Hepatic fibrosis stage is the most important determinant of outcomes in patients with nonalcoholic fatty liver disease (NAFLD).
There is an urgent need for noninvasive tests that can accurately stage fibrosis and determine efficacy of interventions. Here,
we describe a novel cell-free (cf)-mRNA sequencing approach that can accurately and reproducibly profile low levels of circulat-
ing mRNAs and evaluate the feasibility of developing a cf-mRNA-based NAFLD fibrosis classifier. Using separate discovery and
validation cohorts with biopsy-confirmed NAFLD (n = 176 and 59, respectively) and healthy subjects (n = 23), we performed se-
rum cf-mRNA RNA-Seq profiling. Differential expression analysis identified 2,498 dysregulated genes between patients with
NAFLD and healthy subjects and 134 fibrosis-associated genes in patients with NAFLD. Comparison between cf-mRNA and liver
tissue transcripts revealed significant overlap of fibrosis-associated genes and pathways indicating that the circulating cf-mRNA
transcriptome reflects molecular changes in the livers of patients with NAFLD. In particular, metabolic and immune pathways re-
flective of known underlying steatosis and inflammation were highly dysregulated in the cf-mRNA profile of patients with
advanced fibrosis. Finally, we used an elastic net ordinal logistic model to develop a classifier that predicts clinically significant fi-
brosis (F2–F4). In an independent cohort, the cf-mRNA classifier was able to identify 50% of patients with at least 90% probabil-
ity of clinically significant fibrosis. We demonstrate a novel and robust cf-mRNA-based RNA-Seq platform for noninvasive
identification of diverse hepatic molecular disruptions and for fibrosis staging with promising potential for clinical trials and clini-
cal practice.
NEW & NOTEWORTHY This work is the first study, to our knowledge, to utilize circulating cell-free mRNA sequencing to de-
velop an NAFLD diagnostic classifier.

cell-free mRNA; classification biomarker; NASH; nonalcoholic fatty liver disease; nonalcoholic steatohepatitis

INTRODUCTION Although liver biopsy remains the preferred reference

Nonalcoholic fatty liver disease (NAFLD) is the most com-
mon liver disease in the world, affecting one-quarter of the
global population (1, 2). The more common form of NAFLD
is nonalcoholic fatty liver (NAFL) or simple steatosis,
whereas a subset of patients with NAFLD develop the pro-
gressive form, nonalcoholic steatohepatitis (NASH). NASH is
characterized by steatosis, lobular inflammation, and hepa-
tocyte ballooning and is associated with increasing levels of
liver fibrosis, which can subsequently progress to liver cir-
rhosis, liver failure requiring transplant, and hepatocellular
carcinoma (3). Identification of patients with NASH and
advanced hepatic fibrosis is critical due to their greater risk
for developing complications of chronic liver disease (4).
method for NASH and NAFLD diagnosis (5), biopsies are
invasive, expensive, subjective due to pathology review,
and prone to sampling error. In addition, although numer-
ous pharmacotherapies are in development, none is yet
approved for NASH, and many have failed in recent trials
(6–9). Intensive lifestyle intervention and bariatric surgery
have demonstrated reductions in steatosis and fibrosis but
would benefit from noninvasive efficacy metrics beyond
just weight loss, an imperfect correlate of liver steatosis
(10–13). Therefore, there is a critical need for accurate non-
invasive tests for NASH and advanced fibrosis.
Currently, there are two distinct approaches for noninva-
sive assessment of liver fibrosis, a “physical” approach based
on the assessment of liver stiffness using elastography

Correspondence: T. Maddala (tmaddala@molecularstethoscope.com); N. Chalasani (nchalasa@iu.edu).

Submitted 30 October 2020 / Revised 17 December 2020 / Accepted 8 January 2021

http://www.ajpgi.org 0193-1857/21 Copyright © 2021 the American Physiological Society G439

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 STRATIFICATION OF NAFLD BY CELL-FREE MRNA CHARACTERIZATION
techniques and a “biological” approach based on the quanti-
fication of biomarkers in serum samples (14). For imaging-
based approaches, vibration-controlled transient elastogra-
phy (VCTE) and magnetic resonance elastography are the
most widely used clinical tools (15–17). As for quantitative bi-
ological approaches, blood-based tests such as fibrosis 4
(FIB-4) index, which accounts for aspartate aminotransfer-
ase and alanine aminotransferase levels, the platelet count,
and age to give a fibrosis index, is used commonly in the
clinics (7, 18). Furthermore, several circulating protein-based
tests including the enhanced liver fibrosis (ELF) test as well
as NIS4, which combines protein markers and circulating
microRNA, miR-34a, levels, are also available (15, 19).
Although these diagnostic approaches have been useful to
estimate the severity of fibrosis in patients with NAFLD,
there are limited data on their utility in monitoring fibrosis
progression/regression (20).
Over the past decade, an increasing research focus on “liq-
uid biopsy” has resulted in the development of several circu-
lating nucleic acid-based diagnostic tests for multiple
diseases (21). Accordingly, various types of circulating
nucleic acids, including cell-free DNA (cf-DNA), methylated
cf-DNAs, and noncoding RNAs, especially microRNAs, have
been examined as potential noninvasive biomarker candi-
dates for NAFLD (22–25). In recent gene-expression profiling
studies of liver tissue acquired from patients with NAFLD/
NASH, gene-expression signatures for patients with NAFLD/
NASH and transcriptionally regulated pathways associated
with increased disease activity and fibrosis were discovered
(26). Therefore, messenger RNAs appear to be promising bio-
marker candidates for NAFLD/NASH diagnosis and monitor-
ing. Although quantification of cell-free messenger RNAs
(cf-mRNAs) was considered challenging due to their low
abundance in the circulation, we and others have developed
next-generation sequencing (NGS)-based platforms and
demonstrated that circulating cf-mRNA profiling may be
used for diagnosis and monitoring of multiple diseases (27–
29). Studies have shown that rather than cf-mRNA reflecting
debris from cellular apoptosis or necrosis as with cfDNA,
these selective actively released circulating nucleic acids
provide lines of intercellular communication to inform prox-
imal and distal cells of ongoing active transcriptional proc-
esses (30, 31). Furthermore, beyond a reflection of ongoing
cellular processes, cf-mRNA has been demonstrated to be
biologically functional such that cells that take up these
nucleic acids have altered transcription (32).
Here, using a robust NGS platform developed to measure
transcript levels in circulation, we sequenced the cf-mRNA
transcriptomes from the serum of 247 well-characterized
patients with NAFLD and 23 healthy control individuals. We
first established the potential of our technology to differenti-
ate NAFLD from healthy controls to identify dysregulated
pathological processes. Next, we identified cf-mRNA tran-
scriptomic signatures associated with the continuum of liver
fibrosis, many of which coincide with previously known bio-
logical processes of disease (33, 34). Using these differen-
tially expressed genes, we developed a cf-mRNA-based
classifier to stratify patients according to the severity of liver
fibrosis and assessed performance in an independent cohort
of patients with NAFLD. Our study demonstrates the poten-
tial of using cf-mRNA profiling as a “liquid-biopsy” to
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intercept the intercellular communication signals and in-
terrogate the molecular changes associated with NALFD
progression.
MATERIALS AND METHODS
Patients and Sample Information
Samples from two NAFLD cohorts (one retrospective, “dis-
covery cohort” and one prospective, “validation cohort”)
and one prospective healthy cohort were evaluated
(Supplemental Table S1 and S2; all supplemental material is
available at https://doi.org/10.5061/dryad.1jwstqjt1). A total
of 188 serum samples were retrospectively randomly selected
from the Indiana University School of Medicine biobank (IU)
to obtain a balanced representation of liver fibrosis (F0–F4),
of which 176 were evaluable and had matched liver biopsy
evaluated by central pathological reading. These 176 samples
were allocated as the “discovery cohort.” A total of 89 serum
samples were collected prospectively from IU and the biore-
pository at the University of Florida (UF), using a standar-
dized collection protocol. From UF, 59 were evaluable due to
having matched liver biopsy evaluated by a local pathologist.
Inclusion criteria for the both retrospective and prospective
cohort specified suspected NAFLD and NASH diagnosed via
biopsy, which was to be performed within 30 days of blood
collection. Patients were also required to fast for 8 h before
blood draw. Patients with other forms of liver disease (hepa-
titis B or C, alcoholic hepatitis, etc.), patients taking drugs
known to cause hepatic steatosis, and pregnant patients
were excluded. In brief, blood samples were collected in BD
Vacutainer clotting tubes (BD No. 367820) and processed
within 2 h after the blood draw. All samples were centrifuged
at 1,900 g for 10 min; then, serum was separated into new
tubes and stored at 80 C. All 59 biopsy-confirmed prospec-
tive NAFLD samples were used as the “validation cohort.” In
addition, serum samples from 23 control healthy individuals
were prospectively collected from the San Diego Blood Bank,
CA, using the same standardized protocol. Written informed
consent was obtained from all patients, and the study was
approved by the institutional review boards of all the partici-
pating institutions. Clinical data used in the study were
obtained from the individual institutions in March 2018 and
were collected and verified by each of the participating
institutions.
Histology and Biochemical Tests
All biopsies were routinely stained with hematoxylin
and eosin and Masson’s trichrome. Liver pathologists at
the individual institute scored stained sections using the
NASH Clinical Research Network scoring system (35). A
fasting blood sample was obtained at the time of biopsy,
and routine biochemical tests were performed using
standard methods and assays. Biochemical tests included
aminotransferase (ALT) and aspartate aminotransferase
(AST), and additional blood samples were drawn for se-
rum processing.
Library Preparation and Whole Transcriptome RNA-Seq
RNA was extracted using the QIAamp Circulating Nucleic
Acid kit (Qiagen) from up to 1 mL of serum and eluted in a
 STRATIFICATION OF NAFLD BY CELL-FREE MRNA CHARACTERIZATION
16 mL volume. Then, 1 mL of extracted RNA was analyzed on
an Agilent RNA 6000 Pico chip (Agilent Technologies) for
presence of observable RNA traces. In total, 5 mL of the
extracted RNA was then converted into a sequencing library
as described previously (30). Qualitative and quantitative
analyses of the NGS library preparation process were con-
ducted using a chip-based electrophoresis, and libraries
were quantified using a qPCR-based quantification kit
(Roche, Cat. No. KK4824). Sequencing was performed using
the Illumina NextSeq500 platform (Illumina Inc.), using
paired-end sequencing, 75-cycle sequencing. For all sequenc-
ing data, we obtained a median of 8.7 million pass-filter reads
per sample (range: 8.1–16.2 million reads). Base-calling was
performed on an Illumina BaseSpace platform (Illumina
Inc.), using the FASTQ Generation Application. For sequenc-
ing data analysis, adaptor sequences were removed, and low-
quality bases were trimmed using cutadapt (v1.11). Reads
shorter than 15 base-pairs were excluded from subsequent
analysis. Read sequences greater than 15 base-pairs were
compared with the human reference genome GRCh38 using
STAR (v2.5.2b) with GENCODE v24 gene models. Duplicated
reads were removed using the samtools (v1.3.1) rmdup com-
mand. Gene-expression levels were calculated from dedupli-
cated BAM files using RSEM (v1.3.0).
Multiplex qPCR
cDNA was generated by primer annealing 3 mL of RNA with
10 mM dNTPs (Thermo Fisher Scientific, 10297018) and ran-
dom hexamers (IDT) at 60 C for 5 min, followed by reverse
transcription using the SuperScript IV Reverse Transcriptase
kit (Invitrogen, 18090200) according to the manufacturer’s
instruction. Subsequently, cDNA was amplified using
Platinum Taq DNA polymerase (Invitrogen, 10966034)
and preamplification primers (Supplemental Table S3).
The cDNA samples were then treated with Exonucleus I
(Thermo Fischer Scientific, EN0582) to remove primers
used for the nested PCR reactions according to the manu-
facturer’s instruction. Multiplex qPCR was conducted
with the Fluidigm BioMark HD system (Fluidigm) using
96.96 Dynamic array IFC (Fluidigm) with SsoFast
EvaGreen Supermixes (Bio-Rad, 1725200) with target gene
primers (primer sequences are listed in Supplemental
Table S3).
Differential Expression Analysis and Correlation
Differential expression (DE) analysis was implemented
with DESeq2 (v1.12.4) (36) using read counts as input. Genes
with fewer than 200 total reads across the entire cohort were
excluded from subsequent analysis. Technical replicates
were averaged before the DE analysis, which applies a nega-
tive binomial model with raw read counts as the dependent
variable. For NAFLD versus healthy control analysis, the in-
dependent variables were NAFLD/healthy and sex. For anal-
ysis of fibrosis, fibrosis was treated as a continuous
variable. The Benjamini–Hochberg correction was used to
correct for multiple testing and to obtain adjusted P val-
ues in differential expression analysis. Gene Ontology
and Reactome Pathway enrichment analyses were con-
ducted using Metascape (37) using Homo sapiens as the
input and analysis species.
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Cf-mRNA Transcriptome Decomposition Using
Nonnegative Matrix Factorization
Nonnegative matrix factorization (NMF) was performed to
decompose normalized gene-expression profiles from cf-
mRNA into 12 components. In NMF decomposition, genes
sharing similar expression patterns across samples are
grouped together in an unsupervised manner. NMF decom-
position was performed using the scikit-learn Python
machine-learning library. Genes with >40% loading attrib-
utable to a particular component were considered enriched
in the component. For each component, we selected genes
enriched in the component and examined associated path-
ways and biological processes using Gene Ontology via
Metascape (37). To robustly perform Gene Ontology analysis,
we selected clusters with at least 200 genes as a criterion for
further analysis. We determined the title of the cluster by
preferentially using the top category identified in the Gene
Ontology biological processes. If the terminology provided
by the biological process category was nonspecific, we eval-
uated the top term identified in the pathway analysis and
named the cluster according to the pathway term.
Bioinformatics Analysis/Classifier
In each sample for each gene, we took the arithmetic aver-
age of the TPMs of the replicates. Because of sample size
limitations in the discovery cohort, fibrosis stage was aggre-
gated to three categories: F0/F1, F2, and F3/F4. We applied
an ordinal logistic regression model with an elastic net pen-
alty (equal weighting to ridge and lasso) to classify fibrosis
stage. Predictiveness curves were generated for the valida-
tion cohort. These curves depict the probability of clinically
significant fibrosis (F2–F4) on the y-axis and the percentile
of the biomarker score on the x-axis.
RESULTS
Cohort Descriptions
Evaluable samples from three cohorts of patients were
included in analyses: 176 NASH/NAFL patient samples from
a retrospective cohort (discovery cohort), 59 NASH/NAFL
patient samples from a prospective cohort (validation
cohort), and 23 healthy control samples collected prospec-
tively from the San Diego Blood Bank (Fig. 1 and
Supplemental Table S1). The full fibrosis spectrum of disease
was observed in both NAFLD cohorts (Supplementary Table
S2). The age and BMI distributions were reflective of the gen-
eral NAFLD population and were similar in both NAFLD
cohorts. Of note, the proportion of females was higher in the
discovery cohort compared with the validation and healthy
control cohorts (Supplemental Table S1). Furthermore, the
proportion of females increased with higher fibrosis in the
discovery cohort (Supplemental Table S2). The distributions
of some demographics and baseline factors were imbalanced
across cohorts and/or across fibrosis stage. Common comor-
bidities within this cohort were examined. In this study, 19%
of patients with NAFLD were confirmed diabetics, and the
median BMI among NAFLD patients was 34 kg/m2, which is
considered obese.
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 STRATIFICATION OF NAFLD BY CELL-FREE MRNA CHARACTERIZATION

RNA and subsequently used for multiplex qPCR profiling of

Cohort 96 genes. RNA-Seq TPM values inversely correlated with the
NAFL/NASH Enrolled (n = 247) qPCR cycle threshold (CT) value (Pearson’s correlation, r =
• Discovery (Retrospective samples, n = 188)
0.86), indicating a high degree of concordance between cf-
• Validation (Prospective samples, n = 59)
mRNA-seq and qPCR platforms (Fig. 2D). Collectively, these
data highlight the technical robustness of the cf-mRNA
Fibrosis Retrospective Prospective Total
RNA-Seq assay in individuals with NAFLD.
F0 53 9 62
F1 53 14 67
F2 38 11 49 Identification of Transcriptomic Signatures in
F3 24 9 33 Circulation Associated with NAFLD Compared with
F4 20 16 36 Healthy Controls
To evaluate dysregulated cf-mRNA transcripts that are
Excluded: Samples with distinct to patients with NAFLD, we conducted differential
hemolysis gene expression analysis between NAFLD (discovery cohort)
( n = 12 retrospective) and healthy controls. Applying the DEseq algorithm (using a
negative binomial model) to raw read counts (36), and
Analyzable data (n = 235)
adjusting for sex, we identified 2,498 differentially expressed
• Discovery (Retrospective samples, n = 176)
• Validation (Prospective samples, n = 59) genes [false discovery rate (FDR) < 0.05, Fig. 3A and
Supplemental File S1]. Of these genes, 1,527 genes were up-
Fibrosis Retrospective Prospective Total regulated and 971 genes were downregulated. Subsequently,
F0 51 9 60 using these dysregulated genes, we applied Gene Ontology
F1 49 14 63 and Reactome pathway analyses to identify pathways that
F2 35 11 46 are dysregulated in the cf-mRNA transcriptomes of patients
F3 22 9 31 with NAFLD. Gene Ontology analysis revealed dysregulation
F4 19 16 35 of immune system process and metabolic process as well
Figure 1. Consort diagram. Summary of patient samples that were col- as change in cellular component organization or biogene-
lected initially and subsequent samples that were used for the analysis. sis; all common processes that are associated with fibrosis
Samples that were excluded from the study are also summarized. (33) (Fig. 3B, Supplemental Fig. S2A, and Supplemental
File 1). Interestingly, we identified “localization” as the
most dysregulated pathway for both upregulated and
Technical Performance of cf-mRNA NGS Assay in
downregulated genes. Previous studies have indicated that
Human Serum Specimens
liver fibrosis dysregulates genes that are associated with
We have previously demonstrated the potential of cf- cellular localization (42, 43). Considering that dysregula-
mRNA as a platform for diagnosis and monitoring disease tion of genes that are associated with “localization” can be
status in patients with hematological cancers (30) and in overexpressed or inhibited in the liver, we speculate that
Alzheimer’s disease (38). We examined the robustness of the this GO term was identified in both groups. Similarly,
cf-mRNA platform in liver disease specifically, by evaluating Reactome pathway analysis identified dysregulation of fibro-
the technical reproducibility of libraries generated from cf- sis-associated pathways, including the adaptive immune sys-
RNA in all the serum samples in this study. For most sam- tem, Rho GTPase signaling, and angiogenesis (Supplemental
ples (239/247, 97%) multiple serum aliquots were available Fig. 2B and Supplemental File 1).
and technical replicates were analyzed. Whole transcriptome Next, to examine the overall biology of the cf-mRNA
comparison [transcripts per million (TPM)  10] of technical transcriptome in patients with NAFLD, we applied unsu-
replicates showed high correlation indicative of robust tech- pervised nonnegative matrix factorization (NMF) (27)
nical reproducibility (Pearson’s correlation  0.94 for 95% of decomposition of cf-mRNA transcriptomes using NAFLD
NAFLD samples) (Fig. 2A). In addition, the high correlation samples from the discovery cohort. Subsequently, we iden-
between observed versus expected number of extracellular tified 12 distinct gene clusters (Fig. 3C and Supplemental
RNA consensus consortium (ERCC) molecules demonstrated File S2). Of 12 clusters, we focused specifically on six clus-
high quantification accuracy (Fig. 2B and Supplemental Fig. ters where the number of genes in the cluster was greater
S1A), 95% of samples with Pearson’s correlation r  0.93) than 200 (Supplemental Fig. 2C). We then used Gene
(39). A median of 7,120 transcripts with  10 TPM were iden- Ontology to identify key biological processes and pathways
tified per sample (Fig. 2C), highlighting the diversity of the that are associated with individual clusters (Fig. 3D and
circulating mRNA transcripts quantified by the assay. To fur- Supplemental Fig. S2C). Functional analyses revealed iden-
ther validate the robustness of the cf-mRNA RNA-Seq assay, tification of the following clusters: cellular component or-
we compared the levels of circulating transcripts assessed by ganization, immune systems 1 and 2, metabolic process,
cf-mRNA RNA-Seq with qPCR. We used multiplex qPCR hemostasis, and cellular localization (Supplemental Fig.
(Fluidigm BioMark) to assess the expression of 96 gene tar- S2C). Interestingly, several clusters such as metabolic pro-
gets known to be expressed in healthy and NAFLD liver tis- cess, cellular component organization, and immune sys-
sue at different levels (40, 41) (Fig. 2D, Supplemental Table tems are well-recognized biological processes that are
3). Twelve plasma samples from the discovery cohort, sepa- associated with NAFLD, indicating that molecular altera-
rate aliquots to those used for RNA-Seq, were used to extract tions in the liver might be reflected in the circulation.

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 STRATIFICATION OF NAFLD BY CELL-FREE MRNA CHARACTERIZATION

A B
50
Sample number

40 20

30

Count
20 10

10 Figure 2. Cell-free (cf)-mRNA-based high-

throughput sequencing is an accurate, ro-
0 0 bust, and reproducible approach for charac-
0.88 0.92 0.96 1.0 0.900 0.925 0.950 0.975 terizing serum transcriptome. A: histogram
representing correlation between the cf-
Pearson Correlation Coefficient (r) Pearson’s correlation coefficient (r)
mRNA transcriptomes of technical replicates
Independent using Pearson’s correlation analysis (n =
RNA extraction 239). B: histogram compiling r values of
C D RNA NGS 96 genes was expected extracellular RNA consensus con-
Multiplex assessed in 12 sortium (ERCC) transcripts vs. observed
RNA qPCR
patients level of ERCC from each sample (n = 239).
C: histogram of number of transcripts
30 10000 detected (transcripts per million, TPM 10)
r = -0.81
per sample (n = 239). D: schematics for com-
1000 P < 0.0001
parison between next-generation sequenc-
Sample number

RNA-Seq (log TPM)

20 ing assay and qPCR (top). Correlation

100 between sequencing TPM against qPCR Ct
value (96 genes in total, n = 12) (bottom).
10
10
1

0.1
0
6000 7000 8000 0.01
5 10 15 20 25 30 35 40
Number of protein-coding genes
detected (TPM ≥ 10) qPCR (CT value)

Collectively, our data indicate that the cf-mRNA profile can metabolic processes and immune system processes, both
be used to noninvasively identify molecular alterations that well-known processes that are commonly dysregulated in
are specific to patients with NAFLD, and the gene-expres- patients with NAFLD (Fig. 4B). In addition, we used
sion profile of patients with NAFLD may reflect the molecu- Reactome pathway analysis to examine biological path-
lar characteristics of the disease. ways that are linked to cf-mRNA fibrosis-associated genes
(Supplemental Fig. 3B). The Reactome pathway analysis
Identification of Transcriptomic Signatures in identified pathways that are known to be associated with
Circulation Associated with Fibrosis Stage within the fibrosis and NAFLD, including notch pathways and lipid
NAFLD Cohort metabolism (44, 45). We then used publicly available liver
NAFLD-related transcriptomic changes in serum cf- tissue RNA sequencing data that were generated from
mRNA were evaluated in the discovery NAFLD cohort sam- patients with NAFLD and liver fibrosis (34) and compared
ples to identify markers of fibrosis. Applying the DESeq algo- genes that are associated with fibrosis between hepatic tis-
rithm to raw read counts and treating fibrosis as a sue and cf-mRNA. The tissue RNA-Seq data were gener-
continuous variable, we applied a less stringent threshold of ated from liver biopsy samples from 72 patients with
FDR < 0.1 due to the more challenging problem of identify- NAFLD, and ordinal regression was applied to identify
ing differentially expressed genes within the NAFLD patient genes that are associated with fibrosis stages (34). A total
cohort. We identified 134 differentially expressed genes of 4,010 genes were identified using FDR < 0.05 as the cut-
(FDR < 0.1, Fig. 4A, Supplemental File S2). The majority (79, off criterion. The comparison between differentially
59%) of genes were upregulated, whereas 55 (41%) genes expressed genes between tissues and cf-mRNA resulted in
were downregulated (Fig. 4A). The terms “upregulated” and identification of 43 common genes (32% of fibrosis associ-
“downregulated” are used to describe changes in the number ated cf-mRNA genes) (Fig. 4C). Furthermore, we evaluated
of RNA molecules in the circulation of higher fibrosis the common pathways that are dysregulated in fibrosis
patients. Next, we used Gene Ontology and Reactome path- among both tissue and cf-mRNA (Fig. 4C). We identified
way analysis algorithms to evaluate the functional roles and that of the 21 tissue and 17 cf-mRNA Gene Ontology biolog-
biological processes reflected by these differentially ical pathways identified, 15 pathways were common
expressed genes (Fig. 4B and Supplemental Fig. S3, A and between liver tissues and cf-mRNA (Fig. 4C). Collectively,
B). Using Gene Ontology enrichment analysis, cf-mRNA our data suggest that dysregulated genes in the cf-mRNA
genes that are associated with fibrosis stages were associ- reflect those of tissues and frequently display common
ated with biological processes of NAFLD, including dysregulated pathways.

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 STRATIFICATION OF NAFLD BY CELL-FREE MRNA CHARACTERIZATION

A B Most significant -log(p-value)

biological processes 0 20 40 60 80
Controls vs NAFLD localization
negative regulation of biological process

Upregulated
metabolic process
10.0 APOC1
multi-organism process
APOB cellular component organization or biogenesis
-log10(adjusted p-value)

HPD
response to stimulus
7.5 APOC3 CRTAC1 cellular process
immune system process
regulation of biological process
5.0 developmental process
LMBRD2

1 0 20 40 60 80
2.5
localization
Adj. p < 0.05 immune system process

Downregulated
0.0 response to stimulus
biological regulation
-2 0 2 multicellular organismal process
2
-log (fold change) biological adhesion
cellular component organization or biogenesis
cellular process
signaling
locomotion

C D Clusters
70
60

-log(p-value) 50
40
Hemostasis
30
20
10
0
cellular component organization or biogenesis

biological regulation

immune system process

metabolic process

localization

immune system process

Immune process 1
biological processes
Most significant

Metabolic process
Immune process 2 0.4
0.2
Cellular localization
0.0
-0.2
Cellular component -0.4
organization

Figure 3. Cell-free (cf)-mRNA profile shows distinct profile for patients with nonalcoholic fatty liver disease (NAFLD) compared with control subjects. A:
volcano plot depicting the cf-mRNA genes that are differentially expressed between patients with NAFLD and healthy controls (n = 176 and 23, respec-
tively, n = number of subjects). Significantly dysregulated genes are denoted in red, false discovery rate (FDR) < 0.05 was used as the cutoff criterion. B:
most significant biological pathways identified using upregulated (top) and downregulated genes (bottom) identified in A as inputs (Gene Ontology). C:
heat map showing nonnegative matrix factorization (NMF) gene clusters within patients with NAFLD. D: top Gene Ontology biological process categories
associated with each cluster.

Although many patients with NASH have advanced liver
fibrosis, NASH is a distinct histological entity that is diag-
nosed based on pattern recognition of steatosis, inflamma-
tion, and ballooning. We, therefore, examined cf-mRNA
genes that are dysregulated in patients with NASH compared
with in those with NAFL (Fig. 4D). We identified 167 differen-
tially expressed genes between NASH and NAFL (FDR < 0.10,
Fig. 4D, Supplemental File 3). Whereas 88 (53%) genes were
upregulated, 79 (47%) genes were downregulated (Fig. 4D).
Pathway analyses of these dysregulated genes revealed that

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 STRATIFICATION OF NAFLD BY CELL-FREE MRNA CHARACTERIZATION

A Fibrosis associated genes

B C Overlapping genes
5 -log10(p-value)
Most significant Tissue Cf-mRNA
FSCN1
biological processes 0 1 2 3 4 5 6
4 metabolic process 3967 43 91
-log10(adjusted p-value)

AKR1B10
PIGR
cellular component organization or biogenesis
multicellular organismal process Adj. p = 0.1
3 AZU1
BPI signaling Adj. p = 0.05
FCN3 immune system process
2
SLC2A1 AKAP12 TCIM
negative regulation of biological process Common biological
biological adhesion processes
developmental process
1 Adj. p < 0.1
Tissue Cf-mRNA
response to stimulus
biological regulation
6 15 2
0
-0.5 0 0.5 1
-log2(fold change)

D NASH vs. NAFL E F

6 -log10(p-value)
Most significant
biological processes 0 1 2 3 4 5 6 7 8
Overlapping genes
5 FCN3 metabolic process
-log10(adjusted p-value)

response to stimulus
4 Fibrosis NASH
developmental process
biological regulation
98 36 131
3 GLOD4 localization
TRPC1 MAN1A1 ADRM1
NACC2 IKZF3 regulation of biological process
2 FSCN1
cellular process
positive regulation of biological process
1 Adj. p < 0.1
negative regulation of biological process
detoxification
0
-2 -1 0 1 2 3
-log2(fold change)
Figure 4. Cell-free (cf)-mRNA profiling reveals key biological pathways and processes associated with advanced fibrosis. A: volcano plot depicting the
differential expression analysis associated with fibrosis in cf-mRNA (n = 176). Significantly dysregulated genes are denoted in red, false discovery rate
(FDR) < 0.10 was used as the cutoff criterion. B: most significantly enriched pathways identified using genes significantly dysregulated with fibrosis
stages (Gene Ontology). C: overlap of genes (top) and pathways (bottom) between tissue and cf-mRNA fibrosis stage-associated genes [tissue data
obtained from Hoang et al. (34)]. Pathway analysis was conducted using Gene Ontology (biological processes). D: volcano plot depicting the differential
expression analysis between nonalcoholic steatohepatitis (NASH) and NAFL in cf-mRNA (132 NASH vs. 44 NAFL). Significantly dysregulated genes are
denoted in green, FDR < 0.10 was used as the cutoff criterion. E: most significantly enriched pathways identified using genes significantly dysregulated
between NASH and NAFLD (Gene Ontology). F: overlap of differentially dysregulated genes between fibrosis and NASH vs. NAFLD comparison.

metabolic pathways were prominent (Fig. 4E and Supplemen- modeled fibrosis as an ordinal variable (F0/F1, F2, F3/F4)
tal Fig. 3C) for both Gene Ontology and Reactome (Fig. 4E and and applied an elastic net penalty. The resulting biomarker
Supplemental Fig. 3, C and D). In addition, we compared dys- score was applied to the independent prospective NAFLD
regulated genes between fibrosis and NASH versus NAFL com- cohort. Rather than reporting AUC, sensitivity, and specific-
parison. Interestingly, only 36 genes that were dysregulated in ity, which assume a clinically actionable risk threshold and
both analyses overlapped, indicating that the molecular profile are dependent on the cohort in which these parameters are
of NASH substantially differs from that of advanced fibrosis estimated, we display the predicted probability of having
and would require a separate set of genes to effectively identify clinically significant fibrosis (F2 to F4) on the y-axis as a
patients with NASH (Fig. 4F). function of the percentile of the biomarker score on
the x-axis (Fig. 5A). This predictiveness curve shows the
range and distribution of estimated risk levels associated
Stratification of NAFLD Fibrosis Stages
with the model when it is applied to the population from
Since biopsy-measured fibrosis is an imperfect gold stand- which the cohort was drawn (46). Although the predictive-
ard, we developed a classifier of fibrosis stage that yields the ness curve relates to classification performance, the curve
patient’s estimated probability of having F2–F4 fibrosis. To also displays essential information about risk that is not dis-
assess the feasibility of distinguishing fibrosis stages for played by the receiver operating characteristic curve and
detection, disease management, and therapeutic trials, we highlights the ability of the biomarker score to tease apart
developed this classifier based on the discovery cohort. We risk from the average risk in the cohort (40% in this cohort).

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 STRATIFICATION OF NAFLD BY CELL-FREE MRNA CHARACTERIZATION
A Fibrosis (F2/F3/F4)
1.00
Probability of fibrosis (F2/F3/F4)
0.75
0.50
0.25
0.00
0.00 0.25 0.50 0.75
Percentile of linear predictor
Figure 5. Performance of a cell-free (cf)-mRNA fibrosis classifier. A: predicted probability of clinically significant fibrosis (F2, F3, and F4) in the validation
set. B: predicted probability of advanced fibrosis (F3 and F4) in the validation set.
Therefore, the predictiveness curve reflects both risk model-
ing and population performance approach, providing a more
complete and comprehensive analysis than an evaluation of
classifier performance. The predictiveness curve depicted in
Fig. 5A shows 25% of patients, the majority of whom are la-
beled F0/1, with less than 25% probability of having F2–F4
disease. The figure also shows 40% of patients, the majority
of whom are labeled F2–F4, with higher than 90% probabil-
ity of having F2–F4 disease. Similarly, we developed a classi-
fier for advanced fibrosis (F3/F4). Thirty-four percent of
patients were classified with less than 25% probability of
having advanced fibrosis, a majority of whom were F0/1/2;
36% of patients had higher than 90% probability of having
advanced fibrosis, a majority of whom were F3–F4 (Fig. 5B).
Collectively, these data show that cf-mRNA transcriptome
can be used to develop effective classifiers for liver fibrosis.
DISCUSSION
NAFLD is a disorder that affects a quarter of the world-
wide population, resulting in a considerable global health
and economic burden (47). Although liver biopsy is currently
the reference method for NAFLD characterization, this inva-
sive approach has several key limitations. The biopsy sam-
ples are equivalent to only one fifty-thousandth of the liver
volume and may not reflect the pathological status of the
entire liver. Pathological grading of the biopsy samples
is subject to inter- and intraobserver variation (48, 49).
Furthermore, liver biopsies are expensive, are invasive, and
carry the risk, though infrequent, of serious complications,
thereby making them unsuitable for disease monitoring. For
these reasons, there is an urgent need for noninvasive tools
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B Advanced fibrosis (F3/F4)
1.00
Probability of advanced fibrosis (F3/F4)
0.75
0.50
0.25
0.00
1.00 0.00 0.25 0.50 0.75 1.00
Percentile of linear predictor
Fibrosis categories
F0/F1 F2 F3/F4
to diagnose and monitor the disease status of NAFLD, as
well as to identify novel therapeutic targets. Blood-based
noninvasive assays may potentially overcome many of the
limitations intrinsic to liver biopsy and enable better disease
screening, diagnosis, and monitoring (50). In the present
study, we utilized cohorts with biopsy-confirmed fibrosis
covering a broad spectrum of disease. Subsequently, we
identified cf-mRNA markers of NAFLD and NAFLD severity
that are common to those observed in liver tissue of patients
with NAFLD (34). We used two cohorts to demonstrate that
molecular alterations associated with liver fibrosis are
reflected in the circulation, and these transcriptional changes
may be used to develop classifiers to assess the severity and
perhaps ongoing diverse pathological changes with interven-
tion of liver fibrosis. We acknowledge that the present study
is a proof-of-concept study and has limitations in its design
that may have introduced selection bias as well as unmeas-
ured confounders. Further analytical and clinical develop-
ment and validation studies in the intended-use population
are needed as a next step to developing a robust clinical-
grade NASH fibrosis diagnostic.
Over the past decade, blood-based molecular biomarkers
have been identified as key candidates for the development of
clinically relevant noninvasive disease biomarkers. Accord-
ingly, several circulating DNA-based biomarkers have been
developed for prenatal diagnostics, transplant rejection, and
cancer monitoring (51–56). Recent gene-expression profiling of
liver tissues obtained from patients with NAFLD/NASH
revealed unique gene-expression alterations for patients with
NAFLD compared with those of healthy individuals (26). For
this reason, we believe that the signals observed from liver
reflect the expression of active processes and are reflective of
 STRATIFICATION OF NAFLD BY CELL-FREE MRNA CHARACTERIZATION
disease. In the present study, we demonstrated the utility of cir-
culating transcriptomic cf-mRNA profiling to evaluate molecu-
lar alterations associated with patients with NAFLD. Although
RNA-Seq-based cf-mRNA profiling has been used previously in
other diseases (27–29), this is the first study, to our knowledge,
to utilize the cf-mRNA RNA-Seq technology platform to com-
prehensively examine the transcriptional alterations in patients
with NAFLD and to evaluate the association of cf-mRNA pro-
files with severity of liver diseases. We showed that cf-mRNA
profiling reflected that of previously performed liver tissue
sequencing data and identified several pathways that were asso-
ciated with key biological processes that are linked to NAFLD,
including lipid metabolism and extracellular matrix dysregula-
tion. Our results demonstrate that cf-mRNA profiling can poten-
tially be used beyond simple disease diagnosis and fibrosis
assessment in liver disease by utilizing the genes and pathways
that are dysregulated in liver for therapeutic target identifica-
tion, patient selection, and the monitoring of efficacy of phar-
macotherapy, lifestyle intervention, and bariatric surgery.
With numerous drugs currently being developed for
NAFLD and a need to inform the encouragement of lifestyle
intervention or bariatric surgery, it is critical to identify the
stage of disease (57, 58). Although most current clinical trials
utilize a liver biopsy to identify a suitable patient population,
the invasive nature of biopsy limits its utility as a broader
patient-screening tool for clinical trials (50), patient monitor-
ing during trials, or for monitoring intervention efficacy. The
fibrosis-4 index (FIB4), NAFLD fibrosis score (NFS), NIS4, and
vibration-controlled transient elastography (VCTE) are cur-
rently available noninvasive tests to predict the presence and
severity of hepatic fibrosis in NAFLD (16, 17, 19, 50, 59).
However, these tests are based on simple variables such as
age, liver enzymes, platelets, and liver physical stiffness,
which unlike our cf-mRNA classifier are unlikely to reflect the
real-time dynamics of NAFLD pathophysiology or hepatic
transcriptome changes in response to therapeutic interven-
tions for NAFLD. We utilized whole transcriptome cf-mRNA
profiling to develop a classifier for fibrosis. The classifier was
able to identify a significant proportion of patients at either
very high or low probability of having clinically significant fi-
brosis (F2–F4) in the validation cohort. Since the classifier
includes multiple gene transcripts, it is more likely to reflect
the multiple underlying pathologies known to be involved in
this chronic complex disease. Because an expression-based
assay could be used to evaluate multiple aspects of the disease
and of patient response over time, our study demonstrates
the potential utility of cf-mRNA as a promising alternative to
liver biopsies, imaging, and conventional blood tests.
DATA AVAILABILITY
Sequencing data generated in this study was deposited in
Sequence Read Archive (SRA) under accession number
PRJNA701722 (https://www.ncbi.nlm.nih.gov/sra/PRJNA701722).
All data needed to evaluate the conclusions in the paper are
present in the paper and/or the supplementary materials.
SUPPLEMENTAL DATA
Supplemental Figures S1–3 and Tables S1–3: https://doi.org/10.
5061/dryad.1jwstqjt1)
AJP-Gastrointest Liver Physiol  doi:10.1152/ajpgi.00397.2020  www.ajpgi.org
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ACKNOWLEDGMENTS
We thank Teresa Wright and Guillermo Elias for conceptual dis-
cussions and critical reading of the manuscript; Neeraj Salathia,
Arkaitz Ibarra, John Aballi, Alex Acosta, Lucy Guo, Vera Huang,
Amy P Karns, Julianna R Parks, and Yue Zhao for technical input
and assistance; Kayla Gelow and Emily R Smith for sample collec-
tion and data management; Brooke McMillen Patz and the Clinical
and Translational Sciences Institute (CTSI) Biorepository group at
Indiana University, and Erin Kelly and the CTSI Biorepository
group at University of Florida, for sourcing patient samples; and
Ruben Rodrigues and Brian Read at the Development Research
Services for assistance with sourcing samples from the San Diego
Blood Bank.
DISCLOSURES
S.T., J.J.S., R.P.R., J.V.B., J.Z., M.N., and T.M. are current or past
employees at Molecular Stethoscope, Inc. S.T., J.Z., and M.N. are
named as inventors in patent applications related to the technolo-
gies used in this manuscript. S.T., J.Z., and M.N. are named as
inventors in pending patent applications related to the technolo-
gies used in this manuscript filed by Molecular Stethoscope
Inc. [WO2020092646A1 filed on October 30, 2019 (J.Z., M.N.),
WO2020087037A2 filed on October 25, 2019 (S.T., J.Z., M.N.),
and WO2019060369A1 filed on September 18, 2018 (M.N.)]. S.R.
Q. is a founder of Molecular Stethoscope, Inc., and a member of
its scientific advisory board. N.C. has ongoing consulting agree-
ments with Abbvie, Madrigal, Foresite, Zydus, ObsEva, and
Galectin and research support from DSM, Exact Sciences, Zydus,
and Intercept. At the time of this work, J.V.B. had ongoing consult-
ing agreements from Exact Sciences, CareDX, and Grail. These
outside interests are not directly or significantly related to this pa-
per. At the time of this work, T.M. had ongoing consulting agree-
ments and/or other compensation from CareDX, Delfi, Grail, and
Lexent Bio. These outside interests are not directly or significantly
related to this paper. S.G. consults for TransMedics and Pfizer and
receives research grant support from Zydus, Galmed and Viking.
None of these interests is related to this work. These outside
interests are not directly or significantly related to this paper. All
authors declare that they have no additional competing interests.
No external funding was used for this study.
AUTHOR CONTRIBUTIONS
N.C., S.T., M.N., and T.M. conceived and designed research;
N.C., S.T., J.J.S., R.P.R., J.V.B., J.Z., and T.M. analyzed data; N.C.,
S.T., J.J.S., R.P.R., J.V.B., S.G., J.Z., M.N., S.R.Q. and T.M. inter-
preted results of experiments; S.T., J.V.B., and J.Z. prepared fig-
ures; N.C., S.T., J.Z., and T.M. drafted manuscript; N.C., S.T., J.J.S.,
R.P.R., J.V.B., S.G., J.Z., M.N., S.R.Q., and T.M. edited and revised
manuscript; N.C., S.T., J.J.S., R.P.R., J.V.B., S.G., J.Z., M.N., S.R.Q.,
and T.M. approved final version of manuscript.
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