Early-onset hepatic fibrosis linking to a novel PYROXD2 mutation: A case report

Abstract

BACKGROUND

Early-onset liver fibrosis is predominantly driven by inherited genetic mutations that disrupt critical metabolic or structural pathways in the liver. Pyridine nucleotide-disulfide oxidoreductase domain 2 (PYROXD2) (formerly named YueF) is a mitochondrial inner membrane/matrix-localized protein, regulating mitochondrial respiratory chain function. While emerging evidence highlights the role of PYROXD2 in mitochondrial redox homeostasis and respiratory chain integrity, its pathological contribution to early-onset liver fibrosis remains poorly characterized, particularly in the context of monogenic metabolic disorders and oxidative stress-mediated hepatocyte injury.

CASE SUMMARY

This report represents the first description of early-onset liver fibrosis in a patient harboring a heterozygous variant in the PYROXD2 gene. The patient presented with severe obesity, recurrent abnormalities in liver enzyme levels, and hyperuricemia, and demonstrated a suboptimal or absent response to hepatoprotective therapies. Whole-exome sequencing followed by Sanger validation identified a frameshift variant in the PYROXD2 gene (NM_032709.3, c.1082dupT, p.Phe361 Leu fs*50) in the affected patient. The variant was absent from population databases (gnomAD, 1000 Genomes) and had not been previously reported in the literature. Computational pathogenicity predictions consistently classified it as pathogenic. Protein modeling using SWISS-MODEL indicated that the variant induces deleterious conformational alterations.

CONCLUSION

Collectively, the present case highlights PYROXD2 as a novel candidate gene, contributing to early-onset liver fibrogenesis. The identification of this variant in the PYROXD2 gene in the patient demonstrates a previously unrecognized molecular pathway in juvenile hepatic fibrosis, with potential implications for personalized diagnosis and treatment.

Key Words: Early-onset liver fibrosis; PYROXD2; Obesity; Hyperuricemia; Frameshift mutation; Mitochondrial dysfunction; Metabolic disorder; Case report

Core Tip: For the first time, our study reports that a heterozygous frameshift mutation in the pyridine nucleotide-disulfide oxidoreductase domain 2 (PYROXD2) gene may underlie early-onset liver fibrosis. This variant impairs the function of mitochondrial oxidoreductases, ultimately triggering metabolic disturbances and the development of early liver fibrosis. Our findings uncover a novel pathogenic variation of PYROXD2 linking to early-onset liver fibrosis, thereby providing a new rationale for the genetic diagnosis of related disorders.

INTRODUCTION

Early-onset liver fibrosis, defined as progressive hepatic scarring occurring in childhood or adolescence, is predominantly driven by inherited genetic mutations that may disrupt critically metabolic or structural pathways in the liver. Key monogenic defects include mutations in SERPINA1 (causing alpha-1 antitrypsin deficiency), JAG1 (responsible for Alagille syndrome), ATP7B (linked to Wilson disease), and genes involved in regulating bile acid synthesis (e.g., HSD3B7), iron metabolism (e.g., HFE in hemochromatosis), and collagen processing (e.g., COL3A1 in vascular Ehlers-Danlos)[1-5]. These mutations trigger aberrant extracellular matrix deposition, hepatocyte injury, and activation of hepatic stellate cells, resulting in precocious fibrogenesis. This genetic predisposition highlights the importance of familial screening and personalized management to mitigate morbidity in affected young populations.

Clinically, patients present with heterogeneous manifestations, mainly emerging within the first two decades of their lives. Cardinal features include persistent jaundice, hepatosplenomegaly, and failure to thrive. Portal hypertension complicates advanced fibrosis, manifesting as ascites, recurrent gastrointestinal bleeding from esophageal varices, and hypersplenism-induced thrombocytopenia. Laboratory findings commonly demonstrate elevated transaminases, hyperbilirubinemia, and impaired synthetic function (e.g., coagulopathy). In the absence of appropriate intervention, these abnormalities mainly progress rapidly to cirrhosis and end-stage liver disease, thereby emphasizing the necessity of early genetic diagnosis to guide the initiation of targeted therapies, such as chelation, enzyme replacement, or transplantation.

Pyridine nucleotide-disulfide oxidoreductase domain 2 (PYROXD2) (formerly known as YueF) is a protein that interacts with the hepatitis B virus X protein that is highly expressed in hepatocytes. Previous studies have demonstrated that knockout of PYROXD2 gene increases mitochondrial reactive oxygen species (ROS) levels and the number of immature mitochondria[6]. However, the clinical significance of PYROXD2 in liver disease remains elusive. The first documented case of early-onset liver fibrosis associated with a novel heterozygous variant in PYROXD2 (NM_032709.3, c.1082dupT, p.Phe361 Leu fs*50) is reported in a 23-year-old man. Comprehensive clinical evaluations revealed the elevated liver enzymes, severe obesity, and hyperuricemia. Histopathological examination confirmed bridging fibrosis. Whole-exome sequencing excluded other known fibrogenic mutations. These findings demonstrated PYROXD2 dysfunction as a potential contributor to hepatic metabolic dysregulation. The findings revealed a novel mechanistic link between PYROXD2-mediated mitochondrial dysfunction and the progression of juvenile fibrogenesis, thereby broadening the genetic landscape of early-onset liver fibrosis.

CASE PRESENTATION

Chief complaints

A 23-year-old male patient with recurrent episodes of liver injury was admitted to our hospital.

History of present illness

His medical history indicated a 5-year history of metabolic dysfunction, which was characterized by radiologically confirmed hepatic steatosis, defined as ≥ 5% hepatocyte fat content for diagnostic purposes. Imaging examination revealed ≥ 20% fat infiltration.

History of past illness

Additionally, the patient exhibited persistent hyperuricemia since adolescence, with serum uric acid levels consistently exceeding 450 μmol/L, well above the diagnostic threshold of > 420 μmol/L in men and > 360 μmol/L in women.

Personal and family history

His father underwent blood test in a local hospital (Table 1). The results revealed elevated levels of alanine aminotransferase (ALT), aspartate transaminase (AST), fasting blood glucose, total cholesterol, total bilirubin (TBIL), indirect bilirubin (IBIL), and platelet count. Abdominal ultrasound and computed tomography (CT) scan displayed fatty liver. His fibrosis-4 (FIB-4) score was 1.46. CT value of 20 HU is diagnostic for fatty liver according to established criteria, and that the FIB-4 score of 1.46 indicates a risk for fibrosis.

Table 1 Abnormal results of the proband’s father’s blood test (laboratory test).

	Abnormal indicator	Reference range
ALT	61.7 U/L	0-40 U/L
AST	54.0 U/L	0-40 U/L
FBG	6.45 mmol/L	3.8-6.1 mmol/L
TC	3.12 mmol/L	0.57-1.46 mmol/L
TBIL	22.3 mmol/L	0-22 μmol/L
IBIL	18.7 mmol/L	0-17 μmol/L
PLT	259 × 109/L	100-300 × 109/L

Fibrosis-4 (FIB-4) index: FIB-4 = (age × aspartate transaminase)/(platelets × alanine aminotransferase). ALT: Alanine aminotransferase; AST: Aspartate transaminase; FBG: Fasting blood glucose; TC: Total cholesterol; TBIL: Total bilirubin; IBIL: Indirect bilirubin; PLT: Platelet.

Physical examination

Anthropometric measurements indicated obesity [body mass index (BMI): 31.5 kg/m², 95^th percentile for age and sex], and body composition analysis (In body analysis) revealed a body fat percentage of 31.1%.

Laboratory examinations

His biochemical testing results showed elevated levels of ALT, 150 U/L, reference range, 0-40 U/L, AST, 95 U/L, reference range, 0-40 U/L, γ-glutamyl transpeptidase, 102 U/L, reference range, 7-45 U/L, TBIL, 33.2 μmol/L, reference range, 0-22 μmol/L), direct bilirubin, 6.0 μmol/L, reference range, 0-4 μmol/L, IBIL, 27.2 μmol/L, reference range, 0-17 μmol/L, and uric acid, 706 μmol/L, reference range, 155-357 μmol/L. All hepatitis viral markers, autoimmune serologies (antinuclear antibody, anti-smooth muscle antibody, immunoglobulin G), and routine laboratory tests (complete blood count, coagulation, electrocardiogram) were within normal limits, excluding common secondary causes of hepatopathy.

Regarding the patient’s early-onset liver fibrosis, whole-exome sequencing (WES) was undertaken using a peripheral blood sample to identify potential pathogenic genetic variants that might underlie the condition. WES identified a frameshift variant in exon 11 of PYROXD2 gene (NM_032709.3, c.1082dupT, p.Phe361 Leu fs*50), which was subsequently confirmed by Sanger sequencing (Figure 1A and B). Sanger sequencing was subsequently performed to validate the variant in available family members. The results demonstrated a paternal inheritance of the variant, in which his father was heterozygous for PYROXD2 c.1082dupT, while his mother exhibited a wild-type genotype (see family pedigree, Figure 1C-F). No disease-causing variants in SERPINA1, JAG1, ATP7B, HSD3B7, HFE, and COL3A1 genes were found in neither patient and his father, as variations in these genes have been reported to be closely associated with the occurrence of early-onset liver fibrosis (Table 2). Consequently, the analysis concentrated on the PYROXD2 variant as the primary candidate mutation. Computational pathogenicity analysis using the rare disease data center prediction tool classified the PYROXD2 c.1082dupT variant as likely pathogenic. Protein modeling performed via SWISS-MODEL predicted that this variant could induce deleterious conformational changes, leading to protein truncation. Specifically, the c.1082dupT variant could cause a frameshift, resulting in a leucine-to-proline substitution at residue 362 (p.Leu362Pro), followed by the introduction of a premature termination codon 50 amino acids downstream. Located in exon 11/16, this variant truncates approximately 30% of the PYROXD2 protein (wild-type: 581 aa; mutant: 362 aa + 50 aa = 412 aa), potentially causing significant structural alterations in the native protein (Figure 2).

Figure 1 Gene mutation. A: Whole-exome sequencing results of a PYROXD2 pathogenic variant in the patient; B: Sanger sequencing of the targeted PYROXD2 variation in the patient. The arrow indicates the alteration in the PYROXD2 gene, specifically the c.1082dupT variant, identified in the patient; C: Sanger sequencing of the targeted PYROXD2 variation in his family members. His mother carried the wild-type genotype; D: His father harbored the same variant identified in the patient; E: Pedigree of PYROXD2 mutation in the proband’s family. The proband (II-1) and his father (I-1) carried the heterozygous PYROXD2 mutation c.1082dupT (p.Phe361 Leu fs*50) while his mother carried the wild-type genotype.

Figure 2 The 3D protein model, illustrating the location of the c.1082dupT variant in the PYROXD2 protein. A: Wild-type PYROXD2 protein structure predicted by computational modeling; B: The mutant PYROXD2 protein structure, In the red highlighted region, proline substituted for leucine, simulated using PyMol; C: Overlap of the protein structures, with the mutant PYROXD2 protein shown in red and the wild-type PYROXD2 protein in green; D: In the wild-type PYROXD2 protein, the amino acid at position 581 is methionine; E: In the mutant PYROXD2 protein, a frameshift mutation results in the substitution of leucine with proline at position 362, leading to the introduction of a premature stop codon at position 411 in the amino acid sequence.

Table 2 Whole-genome sequencing analysis of liver fibrosis-associated genes in the proband.

Gene name	Associated liver condition	Analysis result
SERPINA1	Alpha-1 antitrypsin deficiency	No rare coding variants identified
ATP7B	Wilson disease	No known pathogenic variants found
HFE	Hemochromatosis	No rare coding variants identified
PNPLA3	MASLD	No known pathogenic variants found
TM6SF2	MASLD	No rare coding variants identified
MBOAT7	MASLD	No rare coding variants identified
HSD17B13	MASLD	No rare coding variants identified
AGJ1	Alagille syndrome	No known pathogenic variants found
COL3A1	Vascular Ehlers-Danlos syndrome	No known pathogenic variants found

MASLD: Metabolic dysfunction-associated steatotic liver disease.

Imaging examinations

Fibroscan results indicated a controlled attenuation parameter of 347 dB/m and an E-value of 16.4 kPa. Hepatic fat proton density fat fraction analysis indicated a fat fraction of 15.3% in the left lobe, 17.4% in the right lobe, and an average fat fraction of approximately 16.3%, aligning with fatty liver (Figure 3) Histopathological examination of the liver biopsy revealed macrovesicular steatosis involving 30%-60% of hepatocytes, portal and lobular lymphocytic infiltration with 2-4 foci per 20 high-power fields, and disrupted lobular architecture with fibrous septa formation (Stage 3) (Figure 4). The diagnostic criteria for liver fibrosis employed in this study are based on the internationally recognized guidelines set forth by the European Association for the Study of the Liver (2016).

Figure 3 Imaging findings of the patient’s liver. A-C: Abdominal ultrasound showed fatty liver; D-F: Fat accumulation in the liver as detected by magnetic resonance imaging, with contrast to the spleen shadow.

Figure 4 Liver biopsy. A: Hematoxylin and eosin staining of the liver biopsy revealed disordered hepatic architecture with fibrosis, indicating fat vesicles and areas of inflammation (arrow) at 20 × magnification image; B: Panel A shows the image at 40 × magnification image (arrow); C: Masson’s trichrome staining of the liver biopsy demonstrates structural disruption and fibrosis of hepatic sinusoids (arrow) at 20 × magnification; D: Reticular fiber staining highlights liver fibrosis (arrow) at 20 × magnification.

FINAL DIAGNOSIS

Taken together, the clinical and genetic findings confirmed diagnosis of early-onset liver fibrosis secondary to a pathogenic PYROXD2 variant.

TREATMENT

It is recommended that: (1) Regular monitoring of hepatic and renal function, along with assessments of hepatic steatosis, be performed; (2) The current pharmacotherapy regimen (silymarin 140 mg three times daily, vitamin E 400 IU once daily, benzbromarone 50 mg once daily) be continued; and (3) Lifestyle modifications, including dietary optimization and structured physical activity for weight management, be implemented.

OUTCOME AND FOLLOW-UP

The patient was administered our combined therapeutic regimen encompassing pharmacotherapy, dietary intervention, and exercise intervention. We conducted regular follow-ups to monitor and optimize the patient’s dietary and exercise compliance, which led to a successful weight loss of approximately 15 kg and significant modification of their lifestyle habits. Noteworthy improvements were found after 7 months of outpatient monitoring, in which the patient’s hepatic function tests and hyperuricemia parameters exhibited gradual enhancement (Figure 5).

Figure 5 Changes in serum liver function, renal function, and creatine kinase level in the patient. A: The patient’s serum aspartate transaminase, alanine aminotransferase, γ-glutamyl transferase, and alkaline phosphatase levels; B: The patient’s serum direct bilirubin, indirect bilirubin, and total bilirubin levels; C: The patient’s serum uric acid level; D: The patient’s serum creatine kinase level. AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; GGT: γ-glutamyl transpeptidase; DBIL: Direct bilirubin; IBIL: Indirect bilirubin; TBIL: Total bilirubin CK: Creatine kinase; UA: Uric acid.

Following the diagnosis of significant liver fibrosis (F2-F3), the patient’s therapeutic regimen was intensively modified, incorporating a structured lifestyle intervention (calorie-controlled Mediterranean diet and supervised exercise program) combined with pharmacotherapy (vitamin E supplementation and optimized management of metabolic comorbidities). These comprehensive changes aimed to address the underlying metabolic drivers and oxidative stress, providing crucial context for interpreting the subsequent clinical course. The stabilization of non-invasive fibrosis markers observed during follow-up may be attributed, at least in part, to this optimized management strategy, highlighting the important interplay between genetic predisposition and modifiable risk factors in shaping disease progression.

DISCUSSION

We present the first report of liver fibrosis in an adult male patient harboring a heterozygous frameshift variant in the PYROXD2 gene (NM_032709.3, exon 11: C.1082dupT, p.Phe361 Leu fs*50), resulting in premature termination and truncation of approximately 30% of the protein. The sequencing methodology employed in this case was WES, a high-throughput approach based on standardized target region capture technology. The assay comprehensively covers approximately 20000 genes, including exonic regions with ± 20 bp of flanking intronic sequences, the full mitochondrial genome, as well as promoter regions, gene regulatory elements, and deep intronic regions containing known pathogenic variants (e.g., those documented in the ClinVar database). The sequencing yielded a data volume of 12.58 Gb, with 98.99% of the target regions achieving ≥ 20× coverage, ensuring high sensitivity for reliable variant detection. Moreover, an average sequencing depth of 131.75× provides strong confidence in the accuracy of variant calling. Bioinformatic analysis utilized a range of well-established tools and databases. This integrated methodological approach, combining robust wet-lab procedures with comprehensive bioinformatic analysis, ensures high-quality data generation and clinically relevant variant interpretation. Whole genome sequencing analysis revealed an East Asian population allele frequency of 0.001359 for this variant (gnomAD v4.0). In accordance with the American College of Medical Genetics and Genomics and the Association for Molecular Pathology joint guidelines[6], this variant fulfills the criteria for moderate pathogenicity-supporting evidence (PM2).

The mutation found in PYROXD2 gene (NM_032709.3, exon 11: C.1082dupT, p.Phe361 Leu fs*50) was predicted to disrupt the oxidoreductase domain, potentially impairing mitochondrial function. The patient presented with progressive metabolic dysregulation, including steatohepatitis, persistent transaminitis, and refractory hyperuricemia, indicating mitochondrial dysfunction. PYROXD2 encodes a mitochondrial oxidoreductase that interacts physically with cytochrome c oxidase subunit 5B (COX5B, complex IV) and plays a key role in regulating oxidative phosphorylation[7]. The clinical manifestations are correlated with three primary pathophysiological mechanisms: (1) Impaired energy metabolism due to disrupted PYROXD2-COX5B interaction and subsequent electron transport chain dysfunction; (2) Redox imbalance stemming from loss of oxidoreductase activity, resulting in oxidative stress; and (3) Systemic metabolic dysregulation, including perturbed purine metabolism and lipid homeostasis. This study reported the first documented association between PYROXD2 deficiency and human metabolic disorders. The finding that the proband’s father, who carries the same heterozygous PYROXD2 variant, exhibits a milder phenotype suggests variable expressivity. This difference in clinical severity may be influenced by a combination of genetic modifiers, environmental factors, or a differing burden of metabolic comorbidities, highlighting the complex interplay between genetic predisposition and other risk factors in the manifestation of liver fibrosis.

Notably, a recent Australian study identified biallelic PYROXD2 variants (compound heterozygous) in a patient exhibiting severe neuro-metabolic dysfunction, phenotypically resembling leigh syndrome. The pathogenic variants included a paternally inherited missense variant (NM_032709.3, c.1276G>A; p.Gly426Ser) and a maternally inherited frameshift variant (NM_032709.3, c.1490dupC, p.Val498Cysfs*79), in which disease progression resulted in fatal outcomes at 6.5 months postnatally[8]. Emerging evidence from a United States cohort revealed co-upregulation of PYROXD2 and AMPD3 in congestive heart failure-associated skeletal myopathy, with histopathological and biochemical hallmarks of mitochondrial impairment, reflecting a shared pathway in cardiometabolic dysfunction[9]. Notably, the patient presented with a significantly elevated serum creatine kinase (CK) level (2.3 × upper limit of normal). A significantly positive correlation was identified between patients’ serum CK levels and AST levels (Pearson’s coefficient, r = 0.621, P < 0.05). Creatine and its activated form, phosphocreatine, serve as essential “batteries” in the energy metabolism system and are primarily synthesized in the liver, kidneys, and pancreas[10]. CK and phosphocreatine together form the CK-phosphocreatine circuit (also known as the phosphocreatine shuttle), playing a pivotal role in energy conversion by catalyzing the reversible transfer of high-energy phosphate groups between creatine and adenosine triphosphate (ATP)[11]. Dysregulation of creatine metabolism has been implicated in obesity and tissue inflammation, in which it is strongly associated with the progression of liver cancer[12]. European genome-wide association studies have identified PYROXD2 polymorphisms as key regulators of trimethylamine metabolism, with specific single nucleotide polymorphisms at this locus, demonstrating a significant correlation with serum metabolome profiles, thereby highlighting a potential involvement in metabolic dysregulation associated with chronic kidney disease[13-15]. While the patient’s refractory hyperuricemia aligns with these findings, no direct evidence currently links PYROXD2 variants to renal metabolic dysfunction, necessitating further validation of this association.

Mitochondrial dysfunction can lead to abnormal fatty acid metabolism in the liver[16]. Abnormal mitochondrial function is noteworthy in obese individuals and those with metabolic-associated fatty liver disease, manifesting as mitochondrial ultrastructural lesions, decreased activity of respiratory chain complexes, and reduced ATP synthesis capacity[17]. Rodent studies demonstrated that mitochondrial dysfunction could induce remarkable hepatic metabolic disturbances, including increased hepatic free fatty acid level, elevated hepatic triglyceride level, escalated mitochondrial ROS production in liver cells, decreased hepatic mitochondrial cytochrome C content, increased uncoupling protein-2 level, and reduced ATP synthesis[18-21]. Previous studies have indicated that aberrant PYROXD2 expression correlates with elevated levels of mitochondrial ROS, and increased ROS is a key contributor to mitochondrial dysfunction. Collectively, these findings suggest that the metabolic syndrome observed in this case may stem from hepatocellular mitochondrial dysfunction induced by PYROXD2 deficiency. Further investigations are warranted to clarify the precise sub-mitochondrial localization of PYROXD2 and its additional regulatory functions, thereby evaluating its potential as a target for mitochondrial-targeted therapies. We have also generated rodent models using CRISPR/Cas9-edited mice harboring the human PYROXD2 mutation. These models will allow longitudinal investigation of phenotypic progression, tissue-specific effects, and potential therapeutic interventions, thereby bridging molecular observations to in vivo pathophysiology.

Nevertheless, the present study is not without limitations. For heterozygous mutations in the PYROXD2 gene, the possibility of incomplete penetrance warrants consideration. Additionally, future studies should screen for the incidence and phenotypic characteristics of this gene mutation in larger population cohorts. Finally, further development of animal models harboring PYROXD2 gene mutations is required to advance investigations into the progression of mutant phenotypes, tissue-specific effects, and potential therapeutic interventions.

In recent years, strategies targeting mitochondrial dysfunction have been increasingly developed and have yielded preliminary positive outcomes. A growing number of genes that regulate mitochondrial stress responses, metabolism, and biosynthesis have been identified and translated into pharmacological targets. Moreover, several more advanced interventions have been proposed and demonstrated promising efficacy, such as healthy mitochondrial transplantation and nucleic acid-based precision therapies[16,22]. Additionally, some clinically approved drugs have been shown to alleviate metabolic dysfunction-associated steatohepatitis by ameliorating mitochondrial dysfunction, including vitamin E and silymarin[23,24]. In this case, following the diagnosis of significant liver fibrosis (F2-F3), the patient's therapeutic regimen was intensively modified, incorporating a structured lifestyle intervention (calorie-controlled Mediterranean diet and supervised exercise program) combined with pharmacotherapy (vitamin E supplementation and optimized management of metabolic comorbidities). These comprehensive changes aimed to address the underlying metabolic drivers and oxidative stress, providing crucial context for interpreting the subsequent clinical course. The stabilization of non-invasive fibrosis markers observed during follow-up may be attributed, at least in part, to this optimized management strategy, highlighting the important interplay between genetic predisposition and modifiable risk factors in shaping disease progression.

CONCLUSION

In conclusion, a heterozygous PYROXD2 frameshift variant was identified in a patient with hyperuricemia and early-onset liver fibrosis. To date, no association between PYROXD2 variants and liver disease has been reported. Thus, the present study suggests a potential novel mechanism into the function of the PYROXD2 gene. Future research should prioritize comprehensive screening for metabolic disorders associated with pathogenic mitochondrial gene mutations and accelerate the development of targeted therapeutic strategies to correct mitochondrial dysfunction.

ACKNOWLEDGEMENTS

We would like to express our sincere gratitude to Dr. Peng-Hua Li for his invaluable support regarding the utilization of computer prediction software. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.