Whole-exome sequencing illuminates unexplained pediatric cholestatic liver disease

Abstract

Unexplained liver disease in infants and children remains a significant diagnostic challenge as the spectrum of noninfectious pediatric liver disease expands. Timely, accurate etiologic assignment remains central to optimal care and resource allocation. Advances in next-generation sequencing, particularly whole-exome sequencing (WES), have transformed evaluation by enabling rapid identification of monogenic disorders that previously eluded standard algorithms. In this editorial, we synthesize recent evidence showing that comprehensive genetic testing substantially improves etiologic diagnosis in pediatric hepatology. WES markedly increases diagnostic yield in infants with cholestasis and in children with cryptogenic aminotransferase elevations, directly influencing management and outcomes. We compare yields across international reports from Asia, North America, and Europe, illustrating how WES and broad gene panels uncover heterogeneous genetic contributors to pediatric liver disease. We also address practical issues, including decreasing costs, faster turnaround times, and the importance of integrating conventional investigations with modern genomics. Finally, we emphasize that while WES is a powerful tool to “decode” unexplained disease and guide precision therapies, it must complement, not replace, careful clinical assessment and, in selected cases, liver biopsy. An integrated approach is essential for navigating heterogeneity, avoiding unnecessary procedures, and advancing personalized medicine in pediatric hepatology worldwide to improve outcomes and equity.

Key Words: Pediatric cholestasis; Genetic diagnosis; Whole exome sequencing; Precision medicine; Alagille syndrome; Wilson disease; Inherited metabolic liver disease

Core Tip: The advent of whole-exome sequencing (WES) has transformed diagnosis of unexplained pediatric liver disease. In infants with cholestasis and children with cryptogenic hepatitis, comprehensive genomic testing markedly increases diagnostic yield, often exceeding 50% in recent cohorts, enabling earlier identification and management. Incorporating WES early can spare patients invasive procedures, including diagnostic liver biopsy, and facilitate timely, targeted interventions. Yet, given phenotypic heterogeneity, WES must be integrated with careful clinical assessment, family history, and selective traditional diagnostics. Falling costs and faster turnaround make WES a practical cornerstone of precision hepatology, while validation and multi-omics remain essential to realize its full potential.

INTRODUCTION

Pediatric hepatologists frequently encounter infants and children with liver dysfunction in whom infectious, autoimmune, or structural causes are absent[1,2]. Over the past decade, the share attributable to inherited or metabolic etiologies has increased, reflecting heightened awareness and improved diagnostics[2,3]. A singlecenter study from China reported that undiagnosed liver diseases constituted approximately one quarter of hospitalized pediatric liver cases in the 2010s[3]. Such “idiopathic” presentations often conceal treatable monogenic disorders, yet clinical manifestations overlap widely across entities. For example, neonatal cholestasis—conjugated hyperbilirubinemia in early infancy—may arise from defects in bile acid synthesis, canalicular transport, or bile duct development[4]. In older children, persistent aminotransferase elevation may reflect not only hepatic disease but also extrahepatic conditions (e.g., muscular dystrophies) that mimic hepatitis biochemically[5].

Nextgeneration sequencing (NGS) has been a watershed for pediatric hepatology. Traditional algorithms (e.g., NASPGHAN/ESPGHAN guidance) prioritized urgent exclusion of biliary atresia and infections, then pursued stepwise metabolic and imaging evaluations, often relegating genetics to the end[1]. By contrast, contemporary practice increasingly places whole-exome sequencing (WES) near the front of the pathway, because it interrogates thousands of genes simultaneously and detects unexpected diagnoses that singlegene tests might miss[5]. WES sequences all protein-coding regions of the genome (the exome), comprising about 1%-2% of the human genome, and thus captures the majority of known disease-causing variants while being faster and more cost-effective than whole-genome sequencing (WGS). However, because WES targets only exons, it may miss pathogenic changes in noncoding regions or structural variants; therefore, a negative WES result does not definitively exclude a genetic cause. In select cases—especially if clinical suspicion remains high—whole-genome sequencing can identify variants outside the exome that WES could not detect[6]. WES is particularly compelling in pediatrics, where phenotypes may be incomplete, and where a timely molecular diagnosis can avert invasive procedures.

In this issue, Chen et al[7] report WES in 80 children with unexplained liver disease, identifying pathogenic or likely pathogenic variants in over half and shaping management accordingly. Below, I synthesize their findings with multiregional evidence, emphasizing infantile cholestasis, pragmatic integration into clinical workflows, and priorities for the next phase of precision hepatology.

WES BOOSTS DIAGNOSTIC YIELD IN PEDIATRIC LIVER DISEASE

The study by Chen et al[7] adds to a growing body of evidence that comprehensive genomic sequencing can dramatically increase the diagnostic yield for pediatric liver diseases of unknown cause. In their cohort of 80 children (aged 1 month to 16 years) with unexplained liver injury, WES identified pathogenic or likely pathogenic variants in 52.5% of patients, with an additional 8.8% carrying variants of uncertain significance. In total, 46 of 80 children (57.5%) received a potential molecular diagnosis based on WES findings. This yield is strikingly consistent with a recent multicenter study from China, which reported a 55.8% diagnostic rate using NGS (either WES or targeted panels) in 172 children with presumed genetic liver disease. Notably, in that study by Fang et al[2], targeted gene panels performed as well as WES (62.0% vs 47.2% diagnostic rate, difference not significant), underscoring that both approaches can be effective when a broad range of genes are interrogated.

Table 1 summarizes selected studies that have applied NGS to pediatric liver disease cohorts. Across diverse populations, the implementation of WES or large gene panels has yielded diagnoses in roughly one-quarter to over one-half of cases, depending on the inclusion criteria and prior workup. For instance, in a large North American study of 2171 infants and children with cholestasis of unknown cause, a 66-gene sequencing panel provided a genetic diagnosis in 12% of patients[8]. The relatively lower yield in that study (compared to Chen et al[7] or Fang et al[2]) likely reflects its broad inclusion of any cholestatic child without an identified cause, a population that inevitably included some patients with multifactorial or non-genetic cholestasis (such as preterm infants with total parenteral nutrition-associated cholestasis). By contrast, studies focusing on stringently selected “idiopathic” cases after initial exclusions tend to report higher yields. Chen et al[7] excluded children with biliary atresia, viral hepatitis, drug injury, autoimmune liver disease, and other known causes, essentially enriching the cohort for genetic etiologies – a strategy that likely contributed to their high 57.5% yield[2]. Similarly, Togawa et al[9] in Japan achieved a 71% molecular diagnosis rate in a subset of infants classified clinically as “genetic cholestasis”, though their overall yield was 26% when including infants with cholestasis of unclear origin or confounded by other factors. As these studies demonstrate, WES is most powerful when applied to patients most likely to have a monogenic disorder, but even in unselected cholestatic cohorts it can uncover diagnoses that would otherwise be missed.

Table 1 Diagnostic yield of genetic testing in pediatric liver disease from selected studies.

Ref.	Population and method	Diagnostic yield	Key findings
Chen et al[7], 2025, China	80 children with unexplained liver disease; WES	57.50%	19 genes identified; 13 novel variants; WES guided therapy
Fang et al[2], 2021, China	172 children (cholestasis, elevated enzymes, hepatomegaly); WES or 62-gene panel	55.8% overall	25 genes identified; 46 novel mutations; highest yield (84.6%) in those with hepatosplenomegaly
Karpen et al[8], 2021, United States	2171 infants/children with cholestasis; 66-gene panel	12%	Top genetic diagnoses: Alagille syndrome (JAG1/NOTCH2), bile salt export pump deficiency (ABCB11), alpha-1 antitrypsin deficiency, MDR3 deficiency (ABCB4), POLG mitochondrial hepatopathy
Gürcan Kaya et al[4], 2025, Türkiye	378 neonates with cholestasis (1997-2024); targeted panel ± WES	28.00%	Genetic diagnoses increased from 18.2% before 2010 to 35.5% after 2010; common genes: ATP8B1, ABCB11, ABCB4, DCDC2, etc.
Togawa et al[9], 2016, Japan	109 infants with neonatal intrahepatic cholestasis; 18-gene panel	26% overall	71% yield in those with suspected genetic cholestasis; common diagnoses: Alagille syndrome, neonatal Dubin-Johnson syndrome, citrin deficiency, PFIC (low GGT types)
Ito et al[18], 2022, Japan	124 infants with cholestasis; expanded 61-gene panel (prospective)	26.60%	Top diagnoses: Alagille syndrome (JAG1/NOTCH2), neonatal Dubin-Johnson syndrome (ABCC2), neonatal citrin deficiency (SLC25A13) – together 78.8% of genetic cholestasis cases in Japan

WES: Whole-exome sequencing.

The consistently high yields (around 50%-60%) seen in studies like Chen et al[7] and Fang et al[2] are particularly noteworthy, as they rival the diagnostic rates achieved in other pediatric fields that have embraced WES (such as neurodevelopmental disorders and rare syndromes). In practical terms, this means that by using WES, clinicians can expect to find a genetic explanation in at least half of children who have gone through standard evaluations for liver disease without a diagnosis. This is a remarkable improvement over historical diagnostic algorithms that often left a large fraction of neonatal hepatitis or idiopathic hepatitis cases unresolved[1]. It also underscores an important point: Genetic causes are a major contributor to “idiopathic” pediatric liver disease. As Nicastro and D’Antiga[5] observed in 2018, nearly half of chronic liver disorders in children may have a genetic basis, and roughly 20% of pediatric liver transplantations are performed for inherited metabolic diseases. Applying NGS early can identify these conditions before they progress to end-stage liver disease, potentially obviating the need for transplant through targeted medical therapy or diet in many cases.

Furthermore, the breadth of genetic diagnoses emerging from WES reflects the extreme heterogeneity of pediatric liver diseases. In the cohort of Chen et al[7], 19 different disease genes were implicated among 46 diagnosed patients. These ranged from relatively common genes like ATP7B (Wilson disease) and JAG1 (Alagille syndrome) to rare ones like SLC25A13 (citrin deficiency) and ABCD3 (peroxisomal disorder)[2]. The identification of 13 novel (previously unreported) pathogenic variants in their study attests to the ability of WES to expand the mutational spectrum of known diseases and even hint at new disease mechanisms. For example, they report a child with cholestasis carrying compound heterozygous mutations in ABCD3 – a gene encoding a peroxisomal membrane transporter – which has been associated with a novel bile acid synthesis defect[7]. By capturing such findings, WES not only benefits individual patients but also contributes to medical knowledge by flagging candidates for new genetic disorders or new phenotypes of known genes.

It is also worth noting that WES can uncover genetic diagnoses beyond traditional “metabolic liver diseases”, catching systemic or neuromuscular conditions that present with liver enzyme abnormalities. Chen et al[7] highlight cases of muscular dystrophy (Duchenne/Becker, DMD gene; and limb-girdle muscular dystrophy, CAPN3 gene) that were initially investigated for liver disease due to elevated aminotransferases[10,11]. In these instances, the hypertransaminasemia was actually a secondary epiphenomenon of muscle disease (so-called “transaminase leakage” from dystrophic muscle) rather than intrinsic liver pathology. Identifying the true cause spared those children unnecessary liver-focused interventions and redirected care to the muscle disorder. Similarly, TNFAIP3 mutations were found in a child with elevated liver enzymes, leading to a diagnosis of autoinflammatory syndrome (A20 haploinsufficiency/Behçet-like disease) and appropriate immunosuppressive therapy[7]. These examples illustrate the diagnostic power of an agnostic genomic approach: WES does not require the clinician to correctly guess the organ of origin or the exact disease upfront – it can reveal an unexpected explanation that changes the entire management plan[12-15].

A FOCUS ON INFANTILE CHOLESTASIS: EARLY GENETIC TESTING PAYS OFF

Infantile cholestasis is one of the areas in pediatric hepatology where comprehensive genetic testing has proven most transformative. Cholestatic jaundice in infants has a broad differential diagnosis, but after excluding extrahepatic causes like biliary atresia, the majority of causes are genetic or metabolic in nature[4]. Classical teaching separated neonatal cholestasis into “giant cell hepatitis” (idiopathic neonatal hepatitis) and specific syndromes, but we now understand that many idiopathic cases have an underlying genetic defect[4]. Indeed, in the Turkish series by Gürcan Kaya et al[4], 28% of neonates with cholestasis had an identified genetic disorder, and the diagnostic rate doubled in the era after 2010 coincident with wider availability of genetic tests.

Crucially, an early genetic diagnosis in cholestasis can guide interventions that significantly alter outcomes. The paradigm example is Alagille syndrome, a multisystem condition caused by mutations in JAG1 (and less commonly NOTCH2). It is a leading genetic cause of neonatal cholestasis worldwide[8]. In Alagille syndrome, a liver biopsy classically shows paucity of intrahepatic bile ducts, but performing a biopsy on a newborn is invasive and may not always be diagnostic early in the disease course. If a cholestatic infant has clinical stigmata (cholestatic jaundice with cardiac murmur, butterfly vertebrae, posterior embryotoxon, dysmorphic facies), genetic testing can confirm Alagille syndrome without needing a liver biopsy[12,13]. Chen et al[7] identified JAG1 pathogenic variants in eight children, including three infants in their cholestasis group, confirming Alagille syndrome in each. Notably, they report that two of their Alagille patients had normal serum gamma-glutamyl transferase (GGT) levels despite having cholestasis[12,13]. Typically, Alagille syndrome is associated with markedly elevated GGT (since bile duct paucity leads to cholestatic pattern), and clinicians might be hesitant to diagnose Alagille in a low-GGT cholestasis case. However, as also observed by others[4], low or normal GGT does not entirely exclude Alagille syndrome – such atypical presentations do occur. The use of WES in those cases was pivotal, as it established the diagnosis and spared the families from a protracted diagnostic odyssey. It also spared the infants a biopsy; once the JAG1 mutation was known, the team could focus on supportive therapy (nutrition, fat-soluble vitamins, ursodeoxycholic acid, pruritus management) without needing histological confirmation[16].

Another common and treatable cholestatic condition is Progressive Familial Intrahepatic Cholestasis (PFIC). PFIC encompasses several autosomal recessive disorders of bile transport and presents in infancy with severe cholestasis. Depending on the gene affected (e.g., ATP8B1 for PFIC1, ABCB11 for PFIC2, ABCB4 for PFIC3), the biochemical phenotype varies – notably, PFIC1 and PFIC2 classically have normal or low GGT, whereas PFIC3 has high GGT[4]. In the study by Chen et al[7], one infant was diagnosed with PFIC1 due to ATP8B1 mutation and underwent liver transplantation at 1.5 years of age. Identifying PFIC early is critical because it enables consideration of emerging treatments (such as biliary diversion procedures or ileal bile salt transporter inhibitors) that can alleviate cholestasis and delay or prevent the need for transplant[15,17]. Genetic testing is the definitive way to diagnose PFIC, as phenotypic overlap with other causes of cholestasis can be misleading. For example, BSEP deficiency (PFIC2) can initially resemble idiopathic neonatal hepatitis or even biliary atresia (with high direct bilirubin and low GGT); only a gene test for ABCB11 confirms the diagnosis, allowing appropriate therapy (and avoiding unnecessary exploratory surgery for possible biliary atresia). Large panel testing has revealed that ABCB11 mutations (PFIC2) are among the more frequent findings in idiopathic cholestasis cohorts besides Alagille syndrome[8].

Beyond these, citrin deficiency (neonatal intrahepatic cholestasis caused by citrin deficiency, due to SLC25A13 mutations) is another important inherited cause of infant cholestasis, particularly in East Asia. Citrin deficiency often manifests with cholestatic jaundice in neonates which then resolves and can present later in life as failure to thrive or fatty liver. Ito et al[18] diagnosed infants with citrin deficiency using an expanded targeted next-generation sequencing gene panel, consistent with its status as a top genetic cholestasis cause in Asian populations. Early recognition of citrin deficiency is beneficial because management (dietary modifications to avoid hyperammonemia and ensure enough protein/fat intake) can improve outcomes and avoid misdiagnosis as idiopathic hepatitis.

It is instructive to compare the distribution of genetic diagnoses between infantile cholestasis and older pediatric patients with liver enzyme elevations. In Table 1, the North American panel study[8] which included many infants, found Alagille (JAG1/NOTCH2) as the number one genetic diagnosis, followed by ABCB11 (PFIC2), and SERPINA1 (alpha-1 antitrypsin deficiency). Alpha-1 antitrypsin deficiency is indeed a relatively common genetic liver disease in infancy that presents with cholestasis or elevated transaminases; it can be identified by a targeted blood test (low A1AT level and PiZZ genotype), but broader panels will also detect it. By contrast, Chen et al’s cohort had older children as well[7], and they observed Wilson disease (ATP7B mutations) to be the single most common cause, accounting for 15 of 80 cases (18.8%). Wilson disease typically presents in school-age children or adolescents with unexplained hepatitis, and it is highly endemic in certain regions (including China) due to autosomal recessive inheritance and founder mutations. The mean age of Wilson disease diagnosis in Chen et al[7] was 7.0 ± 3.5 years, notably younger than the 10.7 ± 4.2 years reported in a large French pediatric series[10]. This earlier diagnosis in the Chinese cohort likely reflects the use of WES to detect ATP7B mutations even before classic clinical signs (like Kayser-Fleischer rings or neuropsychiatric symptoms) fully develop. Early genetic diagnosis of Wilson disease is immensely valuable – it prompts timely initiation of chelation therapy and dietary copper restriction, which can normalize liver function and prevent progression to cirrhosis or brain damage[1]. In all 15 patients with Wilson disease identified by WES in Chen et al[7], follow-up showed normalization of liver enzymes with treatment. This illustrates precision medicine in action: A genetic test leading directly to a disease-specific therapy and excellent prognosis. It also underscores the point that even for diseases like Wilson disease which have conventional diagnostic tests (serum ceruloplasmin, copper studies), WES can expedite diagnosis, especially if suspicion is not initially high. For example, an asymptomatic 3-year-old with mild alanine transaminase (ALT) elevation might not be suspected of Wilson disease by a general pediatrician, but WES would still catch the ATP7B mutation and allow early intervention years before irreversible liver damage.

Chen et al[7] also shed light on several inherited metabolic disorders that cause liver disease and can be managed if diagnosed. They identified multiple cases of glycogen storage diseases (GSD) – specifically GSD type Ia (G6PC mutation), type III (AGL), type VI (PYGL), and type IX (PHKA2 X-linked)[7]. Each of these conditions can present with hepatomegaly and elevated transaminases in childhood, and they have overlapping clinical features (e.g., ketotic hypoglycemia or growth delays) that might not be obvious at initial presentation. Traditional diagnostic confirmation required liver biopsy for enzyme assays or single-gene testing guided by clinical suspicion. WES bypasses that by simultaneously screening for all GSD genes. In Chen et al’s cohort, 7 of 80 children (8.8%) had a GSD as the cause of their liver disease[7]. All were started on appropriate therapy such as frequent feeds and uncooked cornstarch to maintain euglycemia, and none progressed to acute liver failure after diagnosis[7]. This aligns with international guidelines that emphasize dietary management to prevent metabolic decompensation in hepatic GSDs[7]. The takeaway here is that recognizing an inherited metabolic liver disease early allows relatively simple interventions (dietary changes) to avert serious outcomes. WES provides a one-step way to screen for dozens of metabolic liver diseases at once, something that would be impractical with sequential biochemical tests.

Finally, WES in cholestatic infants can discover rare cholestasis syndromes which have specific management or implications. Chen et al[7] report one infant with Dubin-Johnson syndrome (mutation in ABCC2 encoding MRP2 transporter) who had intermittent conjugated hyperbilirubinemia. Dubin-Johnson is benign and mainly causes jaundice; knowing this diagnosis can avoid unnecessary treatments as the focus is on reassurance. Another infant had 45, X (Turner syndrome mosaic) identified via WES as the likely cause of neonatal cholestasis[1,16] – an intriguing association, since Turner syndrome can feature neonatal hepatitis. These diagnoses, while rare, highlight the breadth of conditions a comprehensive genetic test can capture in cholestasis beyond the usual suspects.

In sum, the experience from Chen et al[7] and others strongly supports incorporating WES or broad NGS panels early in the evaluation of infantile cholestasis, once surgical obstructions are ruled out. Doing so yields a diagnosis in a substantial fraction of cases (often 30%-50%+)[4], expedites appropriate therapy, and may preclude riskier procedures. Importantly, even as WES becomes routine, clinicians should continue to use phenotypic clues (such as GGT level, presence of heart murmurs or kidney anomalies, consanguinity history) to inform interpretation and guide interim management while genetic results are pending[4]. The partnership of astute clinical observation with unbiased genetic testing offers the best of both worlds – maximizing diagnostic speed and accuracy.

INTEGRATING GENOMICS WITH CLINICAL ASSESSMENT: THE NEED FOR A BALANCED APPROACH

While WES has proven to be a powerful diagnostic tool, it is not a panacea, and its optimal use is as part of an integrated diagnostic approach. Phenotypic heterogeneity in pediatric liver disease means that a thorough history, physical exam, and initial laboratory/imaging workup remain indispensable. One should not jump straight to WES without first excluding more common or urgent diagnoses via appropriate screening tests. A prime example comes from the context of pediatric non-alcoholic fatty liver disease, recently re-termed metabolic dysfunction-associated steatotic liver disease (MASLD). In overweight or obese children with elevated ALT and fatty liver on ultrasound, the index of suspicion for MASLD is high, but clinicians must still screen for other liver diseases (autoimmune hepatitis, viral hepatitis B/C, Wilson disease, etc.) because these have specific treatments and implications[19,20]. A large multicenter study conducted in North America involving 900 youths with presumed MASLD found that, after comprehensive evaluation, only 2% had alternative diagnoses (such as autoimmune or monogenic liver diseases); the vast majority were confirmed to truly have MASLD[19]. None were found to have Wilson disease or chronic viral hepatitis in that cohort, and only a handful had other conditions like alpha-1 antitrypsin deficiency. This tells us that even though WES could theoretically be applied to every child with fatty liver to look for rare genetic causes, the yield would be exceedingly low in that specific scenario. Instead, cost-effective care dictates using targeted tests (e.g., an ATP7B gene test or ceruloplasmin level for Wilson disease, autoantibodies for autoimmune hepatitis) based on clinical context before considering WES. In other words, a negative screening workup adds weight to pursuing WES, whereas a positive simple test (like high immunoglobulin levels suggesting autoimmune hepatitis) might direct the diagnosis down a different path without needing WES.

Another reason to maintain a balanced approach is the interpretation challenge of WES data. WES will invariably uncover numerous variants in each patient, many of which are variants of uncertain significance (VUS). Distinguishing a benign rare variant from the true pathogenic mutation requires clinical correlation and often additional studies (segregation analysis in family, functional assays, etc.)[5]. Laboratories typically apply the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines to aid variant interpretation, using standardized criteria that weigh multiple lines of evidence (e.g., allele frequency in populations, computational predictions, familial segregation, functional assays) to classify each sequence variant as benign, likely benign, VUS, likely pathogenic, or pathogenic[21]. In Chen et al’s study[7], 7 patients (8.8%) were left with VUS findings, no confirmed diagnosis. Some of these VUS might later be upgraded to likely pathogenic if, for example, they are found to segregate with disease in extended family testing or if new research links them to disease. But until such evidence emerges, clinicians must be cautious in over-interpreting WES results. The clinical picture should still “make sense” with the genetic result. For instance, if WES in a toddler with cholestasis reveals a single heterozygous variant in a recessive PFIC gene, that is likely not the cause unless a second variant is found or there’s some unusual mechanism. Such a case might still warrant a liver biopsy to look for diagnostic histology (e.g., giant cell transformation, iron or copper accumulation, etc.), or to search for an alternative explanation. Liver biopsy, despite being invasive, continues to have a role in selected scenarios: When a treatable metabolic disease is suspected but not confirmed by genetics, when assessing severity of damage (fibrosis) to guide therapy, or when genetics are inconclusive and one needs to rule out histopathological clues of an untested condition. For example, before genetic testing was readily available, an Alagille syndrome diagnosis was often confirmed by liver biopsy showing bile duct paucity; now, as discussed, a JAG1 mutation obviates the need for biopsy in classic cases[12,13]. However, consider neonatal hemochromatosis (a disorder of gestational alloimmune liver injury) – it causes cholestasis with iron overload in the liver and pancreas. If WES fails to identify a genetic cause in a cholestatic infant who has imaging or lab evidence of iron overload, an urgent biopsy might still be indicated to confirm neonatal hemochromatosis, because the treatment (exchange transfusion or IV immunoglobulin) is time-sensitive and not triggered by a gene result. In short, clinical judgment must guide the use of invasive diagnostics in the WES era.

Notably, the synergy between genomics and traditional workup is exemplified by the handling of overlapping or atypical phenotypes. Chen et al[7] mentioned four patients who were initially being evaluated for liver disease but turned out to have Duchenne muscular dystrophy or Becker muscular dystrophy mutations. The clue in such cases is often an isolated transaminase elevation out of proportion to any liver synthetic dysfunction, and perhaps mild motor symptoms. An experienced clinician might order a creatine kinase (CK) level to check for muscle injury, which would be massively elevated in dystrophies, thus revealing the true cause without WES. Conversely, a clinician focused on the liver might not think of checking CK; WES would then catch the diagnosis. Either pathway reaches the same conclusion, but integrating basic labs like CK into the initial workup of unexplained transaminase elevation is a low-cost step that can clarify whether the “hepatitis” is actually myositis. In an ideal scenario, a targeted lab panel (including CK, copper studies, A1AT level, etc.) is sent concurrently with WES, so that no time is lost and all possibilities are covered. The point is that WES complements – not replaces – the art of medicine. A negative WES does not mean the child is healthy; it simply refocuses the search to non-genetic causes or to genetic causes outside WES’s scope (e.g., deep intronic mutations, structural variants, epigenetic disorders).

Another consideration is cost and turnaround time. Fortunately, the cost of WES has plummeted in recent years, removing a major barrier to its use. Early in the 2010s, whole-exome sequencing could cost on the order of $5000 or more per test, which limited it to research or select cases[22]. Technological improvements have since driven the cost well below $1000 per exome, with some reports of costs in the few-hundred-dollar range in bulk settings[23]. This downward trend, coupled with the high diagnostic yield, makes WES a cost-effective choice for etiological diagnosis of rare diseases[5]. One analysis described NGS as a “highly cost-effective” approach to diagnosing monogenic diseases compared to traditional serial testing[5]. Turnaround times have also improved – for example, Karpen et al[8] reported a median of 21 days to results using their 66-gene cholestasis panel. Many clinical laboratories now offer rapid WES in 1-3 weeks, and ultra-rapid genomic sequencing in critically ill infants can even be done in a few days when needed. As these logistics continue to improve, genomic testing is moving from a last-resort option to a front-line diagnostic test in pediatrics.

LESSONS FROM GLOBAL STUDIES AND THE ROAD AHEAD

The collective experience from different countries underscores both the universal applicability of WES in pediatric hepatology and regional differences in disease prevalence. In North America and Europe, studies find Alagille syndrome and alpha-1 antitrypsin deficiency prominently among genetic cholestasis diagnoses[8], whereas in Asia, Wilson disease and citrin deficiency are relatively more prevalent[2]. Japan’s data highlight neonatal Dubin-Johnson syndrome (ABCC2 mutations) as a significant contributor to infant cholestasis, which is a reminder that ethnic founder mutations can influence the local spectrum (Japanese infants have higher rates of ABCC2 and citrin mutations, for instance)[18]. These differences reinforce the importance of sequencing panels or exomes that are broad and not narrowly targeted to one demographic’s “usual suspects”. WES inherently covers all genes, making it suitable for diverse populations and for discovering unexpected diagnoses across ethnic groups.

Another insight is the value of multidisciplinary collaboration in interpreting and acting on genetic findings. Chen et al[7] attribute the success of their program to a team that integrated pediatric hepatologists with geneticists/bioinformaticians. This model – sometimes termed “medical-engineering integration” – ensures that novel variants are rigorously analyzed and that variant calls are correlated with clinical data to avoid false positives. For example, distinguishing a truly pathogenic splice-site mutation from a benign polymorphism might require in silico modeling or even laboratory validation, which the genetics experts can provide, while the clinicians supply phenotypic context. Such teamwork is becoming standard in genomic medicine programs and is particularly crucial when dealing with the 10%-15% of cases that yield VUS or novel genes. In the future, moving from a genetic result to a confirmed disease mechanism might involve functional studies – for instance, introducing a novel mutation into cell lines or animal models (via CRISPR/Cas9 genome editing) to observe its effect[5]. Additionally, complementary omics technologies can help clarify uncertain cases: Transcriptomic analysis can reveal if a variant affects RNA splicing[5], and metabolomic or proteomic profiling might uncover a biochemical signature pointing to a metabolic pathway dysfunction even if the exact gene remains unknown. These research tools, while not yet routine clinically, are increasingly employed to support variant interpretation in difficult cases.

It is also essential to manage expectations: Even with WES, a subset of pediatric liver patients will remain without a clear diagnosis. In Chen et al’s study[7], about 42% had no genetic cause identified despite exhaustive testing. Some of these children might have non-genetic etiologies (e.g., an undocumented toxin exposure or an immune-mediated process that is not classically autoimmune hepatitis). Others might have genetic causes that WES cannot detect – for example, deep intronic mutations affecting gene regulation, copy-number variations, or structural genomic rearrangements. WGS could, in theory, pick up many of those lesions, and we may see WGS deployed in the future for WES-negative cases. Indeed, studies in NICU populations suggest WGS can diagnose a few extra cases missed by WES, albeit at greater cost and data analysis burden. For now, a practical approach for WES-negative pediatric liver disease is to revisit the clinical scenario: Consider a re-evaluation of family history, repeat some metabolic tests that might evolve over time, and perhaps reanalyze the exome data periodically as gene knowledge expands. Publishing and sharing these unsolved cases are equally important, as they fuel international collaboration to discover new liver disease genes.

CONCLUSION

Comprehensive genetic testing, particularly whole-exome sequencing, has fundamentally improved the diagnostic landscape of pediatric hepatology. The work by Chen et al[7] reinforces that when faced with a child who has liver injury without an obvious cause, one should strongly consider an unbiased genomic approach early in the workup. By doing so, more than half of such cases can now be explained – a quantum leap compared to previous eras. Crucially, establishing a genetic diagnosis is not mere academic exercise; it directly benefits patients through tailored treatments (chelators for Wilson disease, nutritional management for metabolic disorders, immunotherapy for certain inflammatory conditions) and spares them from ineffective or invasive interventions. In cholestatic infants, WES has allowed many to avoid diagnostic surgery or biopsy and instead receive appropriate medical care based on a molecular diagnosis. In older children, WES has unmasked unexpected diagnoses, redirecting care to the appropriate specialty or revealing familial conditions that warrant screening of relatives.

As we embrace WES as a routine tool, it must be wielded thoughtfully. The highest yields are achieved when WES is applied to patients after initial screening tests, in settings with multidisciplinary expertise to interpret results, and when integrated into a holistic understanding of the patient. The cost of genomic sequencing is no longer prohibitive, and its turnaround is compatible with clinical decision-making, making it an efficient route to precision medicine. However, clinicians should remain vigilant for conditions that elude WES and recognize that a normal WES result does not conclusively rule out all genetic contributions.

Looking ahead, continued improvements in sequencing technology (such as whole-genome sequencing and long-read sequencing) and analytic pipelines will likely further increase diagnostic rates. The discovery of novel disease genes will progressively shrink the proportion of “unknown” cases. Importantly, complementary research – functional assays for novel variants, and multi-omics integration to link genotype with phenotype – will be needed to keep pace with the deluge of data and to translate it into clinical action. The field of pediatric hepatology is entering an era where molecular diagnosis and therapy go hand in hand. With each success story, like those reported by Chen et al[7], we move closer to the ideal of every child’s liver disease being accurately diagnosed and optimally treated based on their individual genetic makeup. This is precision hepatology in action: Using the power of the genome to illuminate the path towards better outcomes for children with liver disease.