Genetic factors are implicated in a significant proportion of pediatric cholestatic liver diseases, with estimates suggesting that at least 45% of cases have a genetic basis [1]. Progressive familial intrahepatic cholestasis (PFIC) is a rare group of autosomal recessive liver disorders that cause intrahepatic cholestasis in children. It typically presents in infancy with jaundice, pruritus, failure to thrive, and often progresses to end-stage liver disease. The disease is genetically heterogeneous, with multiple subtypes caused by mutations in different genes affecting bile formation and secretion. These subtypes show overlapping clinical features but may vary in severity, age of onset, and prognosis [1,2,3]. In the Online Mendelian Inheritance in Man (OMIM) database (https://www.omim.org/), bi-allelic (homozygous or compound heterozygous) variants in 12 genes have been associated with PFIC [4]. Worldwide, PFIC has an estimated incidence of 1 per 50,000–100,000 live births [5]; however, the exact incidence in Pakistan is unknown.

Clinical presentations and laboratory findings in PFIC patients often show overlapping features among various PFIC types. While it is possible to suspect and differentiate between different PFIC types based on clinical presentation and basic diagnostic tests, confirmatory diagnosis usually relies on genetic testing. Pakistani hospitals have implemented whole genome sequencing (WGS) as part of routine diagnostics for patients with cholestatic liver disease. This study aims to assess the clinical and laboratory diversity, as well as the overlap in these diagnostic findings, among various types of PFIC in a large cohort of Pakistani children at the time of their genetic confirmation. The study also evaluates the clinical outcomes of these patients.

In our study, we aimed to determine the distribution, disease characteristics, and clinical outcomes of patients with PFICs in Pakistan. In our cohort, PFIC3 emerged as the predominant type, accounting for nearly half of the cases (44.8%). This is followed by PFIC2, PFIC1, PFIC4, and PFIC5, which comprise 24.1%, 16.4%, 12.9%, and 1.7% of the cases, respectively. Earlier studies included diverse patient populations from France [8], Germany [9], India [10], without specifying the ethnicity [11], and the United States, encompassing Caucasian, African-American, Japanese, and Korean origins [12]. In these studies, PFIC1 and PFIC2 were the most frequent types, followed by PFIC3; PFIC4 and PFIC5 were much rarer. However, PFIC3 was the most common type (59.5%) in the study from Saudi Arabia, followed by PFIC2 (34.2%), PFIC1 (5.1%), and PFIC4 (1.3%) [5].

The clinical and laboratory profiles of PFIC types have been extensively documented in case reports and systematic reviews collating data from numerous publications on PFICs. The clinical presentations and laboratory findings in our study largely align with those reported previously. However, several features in our cohort diverge from previous reports and may reflect population-specific characteristics.

Generally, PFIC1 and PFIC2 manifest within the first year of life, while PFIC3 presents over a broader age range from infancy to early adulthood. PFIC4 and PFIC5 typically show symptoms in early childhood, although the exact age of onset can vary due to their rarity and clinical variability [2,5,13,14]. In our study, at the time of genetic diagnosis, PFIC3 patients were the oldest (median age—98 months) compared to other PFIC types diagnosed within 40–48 months of age (p-value < 0.001), and this finding aligns with previous studies. However, our results suggest that PFIC genetic diagnosis in Pakistan may be delayed by several months, highlighting the need for increased awareness and timely diagnostic protocols to improve early detection and management.

In our study, the majority of patients in all PFIC groups reported a family history of liver diseases. Almost all PFIC variants, with only one exception, were homozygous rather than compound heterozygous. This suggests a highly consanguineous population within our cohort. Generally, consanguineous marriages in Pakistan are reported to range from 46–98%, depending on the region, which correlates with high rates of genetically inherited diseases and infant mortality [15].

Jaundice, due to elevated conjugated bilirubin, is present in all PFIC patients. Conjugated bilirubin was most elevated in PFIC4 and less elevated in PFIC3, with a significant difference. This disparity is likely due to the underlying genetic mutations and their impact on bile secretion pathways. TJP2 is essential for maintaining the integrity of tight junctions in hepatocytes. Loss of TJP2 function leads to impaired tight junction sealing in the bile canaliculi, resulting in severe bile leakage and paracellular reflux of bile constituents, including conjugated bilirubin, back into the bloodstream. This disruption of bile flow at the cellular junctional level leads to marked cholestasis and high levels of conjugated bilirubin, even early in the disease course [16,17,18]. In contrast, PFIC3 results from mutations in the ABCB4 gene, which encodes the MDR3 protein responsible for transporting phosphatidylcholine into bile. While this defect leads to toxic bile composition and progressive cholangiopathy, the onset is typically later and progression is more gradual, resulting in less dramatic elevations in conjugated bilirubin at the time of diagnosis [16,19,20].

Acholic stool is a common cholestatic symptom. The proportion of patients with PFICs affected and the severity of this symptom can vary depending on bile secretion and flow [2,21]. In our study, acholic stool was observed in 23.1% to 46.4% of patients across different PFIC types, without a statistically significant difference between the groups.

Pruritus is commonly regarded as a hallmark symptom of cholestasis, including in patients with PFICs [3,22,23]. In this study, we observed that around a quarter of PFIC1 and PFIC3 patients, and a third of PFIC2 and PFIC4 patients, did not report pruritus. Previous studies indicate that pruritus remains a significant and distressing symptom in PFICs, particularly in PFIC1 and PFIC2, where it is often severe and nearly universal; PFIC3 patients tend to experience milder pruritus, while PFIC4 and PFIC5 patients have fewer comprehensive data but include pruritus as a symptom for most patients [13,24,25]. However, pruritus may be underreported in preverbal children, leading to potential underestimation of its prevalence [26]. This underscores the need for increased clinician awareness of pruritus to ensure timely and accurate diagnosis and management.

Splenomegaly and portal hypertension were less frequent in PFIC1 patients in our study compared to other PFIC types. This observation aligns with previous findings on the distinct nature of genetic mutations and their impact on liver pathology. PFIC1, caused by mutations in the ATP8B1 gene, leads to a unique progression of liver disease characterized primarily by issues with bile acid transport and liver cell damage, rather than extensive bile duct proliferation and fibrosis. This results in a less frequent progression to cirrhosis and portal hypertension in the early stages of the disease course. In PFIC2 and PFIC3 types, mutations in the ABCB11 and ABCB4 genes, respectively, often lead to more severe bile secretion defects, increased bile duct proliferation, and quicker progression to liver fibrosis and portal hypertension [22,27].

The higher prevalence of diarrhea in PFIC1 patients compared to other PFIC types can be attributed to the unique pathophysiology associated with mutations in the ATP8B1 gene. PFIC1 involves defects in the FIC1/ATP8b1 protein, which is crucial for maintaining the balance of bile acids and other lipids in the liver and intestines. This defect leads to steatorrhea and diarrhea due to impaired bile acid reabsorption in the intestines. Additionally, mutations in the ATP8B1 gene can affect pancreatic function, resulting in exocrine pancreatic insufficiency and subsequent diarrhea. These extrahepatic manifestations are distinct in PFIC1. In contrast, the lower rates of diarrhea observed in PFIC2, PFIC3, and PFIC4 align with their pathophysiological mechanisms, which primarily involve isolated hepatic involvement without significant extrahepatic effects [13,14,16,22].

In our study, 15.8% of PFIC1 and 13.3% of PFIC4 patients developed hearing loss. Previous studies have documented instances of hearing loss in patients with PFIC1 and PFIC4, linking these symptoms to specific genetic mutations. In PFIC1, the ATP8B1 gene product, the FIC1/Atp8b1 protein, is specifically localized in the stereocilia of cochlear hair cells. The mechanosensory function and integrity of these hair cells critically depend on ATP8B1 activity, which maintains lipid asymmetry in the cellular membranes of the stereocilia. Disruptions in this function due to ATP8B1 mutations can lead to hearing loss [22,28]. Similarly, mutations in the TJP2 gene, which encodes tight junction protein 2 (TJP2), can be associated with PFIC4. The TJP2 protein is crucial for maintaining cell-cell adhesion and the integrity of tight junctions in the inner ear, and mutations in TJP2 can disrupt these structures, leading to hearing loss [29]. Hearing loss in one out of 52 patients with PFIC3 is likely not related to the PFIC disease.

Previous studies have shown that among PFIC types, PFIC3 is characterized by high levels of GGT; in contrast, PFIC1 and PFIC2 typically present with normal or low GGT levels [2,13,14,16,22]. PFIC4 can also exhibit elevated GGT levels, but this is not as consistent as in PFIC3 [17]. However, in our study, about half of the PFIC1 and PFIC2 patients exhibited elevated GGT levels, though these increases were mild to moderate (<2 ULN). The observed correlations suggest that in PFIC1, elevated GGT does not reflect more severe liver injury. In PFIC2, the strong positive correlation between GGT and bilirubin confirms its association with cholestatic injury. Additionally, other factors, such as comorbidities (e.g., viral hepatitis, pancreatitis, nonalcoholic fatty liver disease, diabetes mellitus) [30] or medications such as rifampicin can contribute to GGT elevation [31,32]. Therefore, GGT elevation in PFIC1 and PFIC2 may reflect multifactorial influences beyond the primary genetic defect.

Increases in ALT, AST, and ALP, as well as impaired blood coagulation tests, have been well-documented in all types of PFIC. The predominant increase in one or more of these markers among patients with different types of PFIC in our cohort can likely be attributed to the specific set of patients presenting at various stages and severities of the disease at the time of genetic diagnosis across the PFIC groups. This variation reflects the heterogeneous nature of PFIC progression and the liver’s response to ongoing cholestasis and hepatocellular damage.

The literature suggests that PFIC2, PFIC3, and PFIC4 carry an elevated risk for the development of liver tumors [33]. However, we did not observe any liver tumors in our cohort, including patients beyond their second decade of life. AFP testing was performed in only seven patients, with only one demonstrating a normal level. While AFP > 400 mcg/L is considered suggestive of HCC in the appropriate context [34], moderate elevations (<400 mcg/L) may occur in other liver conditions, including PFIC [22,23,35]. Although not specific for HCC, such findings warrant further evaluation, particularly as HCC often remains undiagnosed until advanced stages, and is sometimes only detected post-mortem [36]. This highlights the need for systematic tumor surveillance in PFIC patients.

Anemia in PFIC patients can result from various factors related to liver dysfunction and disease impact, and usually develops at the advanced stage of the disease. Anemia in PFIC patients arises from several factors: chronic liver disease disrupts iron metabolism and protein production; splenomegaly can lead to hypersplenism, causing excessive red blood cell destruction; nutritional deficiencies due to malabsorption further contribute to anemia; increased bleeding risk through reducing clotting factor synthesis can lead to chronic blood loss; chronic inflammation can suppress bone marrow function, reducing red blood cell production [2,3,14,22,23,37]. Additionally, frequent blood draws during hospital stays exacerbate the condition, particularly in children [38]. A significant majority of PFIC patients in our cohort (84.2–89.3%) presented with anemia, indicating that most patients received their PFIC genetic diagnosis at the advanced stage of the disease. This delay is likely due to limited awareness of genetic cholestasis among general pediatricians and restricted access to WGS, which is mostly available in tertiary centers. As a result, many children face prolonged diagnostic delays. To improve early detection, we recommend national guidelines with clear referral criteria and standardized use of genetic testing, along with targeted training for healthcare providers.

A common feature among all PFIC types in our cohort was a poor prognosis, with a low cumulative survival rate of 20% (95% CI: 5–41%) over 20 years. Consistent with previous reports, untreated PFIC1 and PFIC3 typically progress to end-stage liver disease and death within 10–20 years, with an even worse prognosis in cases of PFIC2 [2,16,39]. A review of 10 studies [14] showed mortality rates as high as 87%, with the median age at death being 4 years; survival rates for patients who did not undergo biliary surgery or LT dropped to 50% by age 10, with virtually no survivors by age 20. This high mortality rate underscores the frequent need for LT in PFIC patients, with transplantation rates as high as 65% [5].

The literature indicates that PFIC1, PFIC2, PFIC4, and PFIC5 are particularly aggressive in their disease course, often rapidly progressing to end-stage liver disease [23]. This aligns with our observations: PFIC3 exhibited a less aggressive course compared to PFIC1, PFIC2, and PFIC4 in the first decade.

Truncated variants, such as frameshift, nonsense, stop gain, splice, and start loss mutations, lead to the production of dysfunctional or absent proteins, exacerbating the pathophysiological mechanisms of PFIC. Consequently, patients with these variants often present with more severe symptoms, faster disease progression, and poorer outcomes compared to those with non-truncated variants, such as missense and substitution mutations [13,24,40,41,42]. In our cohort, patients with non-truncated variants showed better, though not statistically significant, cumulative survival (Figure 1D). The increased aggressiveness of the disease in patients with truncated variants is also associated with poorer responses to conventional treatments and a higher necessity for LT. These findings underscore the importance of early genetic diagnosis and personalized treatment approaches to improve outcomes for PFIC patients.

This study acknowledges certain limitations. There is a possibility that patients with a late onset of symptoms, particularly in their second decade, were not captured in our study. The study did not include patients with cholestasis and negative genetic results, preventing the analysis and comparison of genetic and idiopathic forms of PFIC. An important limitation is that the analysis of mutation type (truncated vs. non-truncated) concerning survival outcomes was performed across the full PFIC cohort and not stratified by individual PFIC types due to limited sample sizes. This may have obscured subtype-specific genotype-phenotype correlations, particularly in PFIC2, which warrants focused analysis in future studies. While WGS offers broader and more complete variant detection than WES or targeted panels, it may still miss certain types of genetic variation (e.g., complex structural rearrangements), and its diagnostic accuracy depends heavily on the robustness of bioinformatic pipelines and expert interpretation. Another limitation of this study is the absence of data on age at symptom onset, which relied solely on parental recall and was often inconsistently documented. Given the lack of standardized criteria for defining PFIC onset, ranging from mild pruritus to overt liver symptoms, we excluded this variable to avoid introducing bias. Instead, we reported age at genetic diagnosis, which was uniformly available and reflects diagnostic delays. Future studies should aim to capture onset data prospectively using clear clinical definitions. While our cohort was not based on a pre-defined sample size, it includes all consecutively diagnosed PFIC cases over four years, providing a real-world snapshot; nonetheless, future studies with larger samples and standardized inclusion criteria are warranted to validate these findings.

The clinical applicability of our findings lies in their potential to inform earlier diagnosis and management strategies in pediatric patients with cholestatic liver disease. WGS not only enables precise diagnosis but also guides clinical decision-making, including timely referral for liver transplantation in severe cases. In regions with high consanguinity and limited access to advanced diagnostics, such as Pakistan, our data underscore the importance of integrating genetic testing into routine hepatology care. Implementation of early genetic screening, particularly for high-risk families, may significantly improve outcomes and reduce mortality in PFIC patients.

This study represents the first comprehensive analysis of PFIC in Pakistani children, providing valuable insights into the genetic and phenotypic diversity of this condition. All PFIC types demonstrated significant clinical and laboratory overlap, making differentiation based on these results challenging. WGS proved to be a simple diagnostic tool, crucial for accurate diagnosis.

The clinical presentations, laboratory findings, and outcomes in our study largely align with those reported previously. However, we identified some distinguishing features. In our cohort, PFIC3 emerged as the most prevalent type, followed by PFIC2, PFIC1, PFIC4, and PFIC5. The increased GGT in half of the PFIC1 and PFIC2 patients may reflect multifactorial influences beyond the primary genetic defect. Anemia and abnormal coagulation profiles in nearly all patients with all PFIC types at the time of genetic diagnosis can be attributed to delayed PFIC genetic diagnosis in Pakistani children. This highlights the need for the implementation of genetic testing protocols.

The decrease in the proportion of patients with pruritus and the absence of liver tumors in the large cohort may indicate the need for the implementation of additional protocols for pruritus diagnosis in preverbal children, as well as the implementation of a screening and monitoring system for liver tumors in these patients in Pakistan.

Patients with PFIC1, PFIC2, PFIC4, and/or truncated variants exhibited poorer outcomes, underscoring the importance of early genetic diagnosis and personalized treatment approaches, early consideration for LT in particular, to improve outcomes.