Pediatric Cholestatic Liver Disease

Bile Acid Synthetic Defects

Bile acid synthetic defect (BAD) diseases constitute the first general category of genetic cholestatic diseases in which the mechanism is impairment in bile acid production. The bile acids are produced in the hepatocyte and drive more than 60% of bile flow. They are synthesized from cholesterol by 14 enzymatic steps, all of which are coded for by a specific gene. There are seven known gene defects in this pathway, which are described in Chapter 3 . These diseases cause hepatocellular cholestasis due to toxicity of retained abnormal bile acid intermediates and low production of normal bile acids with resultant insufficient bile flow for normal function. Progressive liver damage is then inevitable. Clinical presentation varies among the seven disorders; however, jaundice, cholestasis, elevated transaminases, fat-soluble vitamin deficiency associated with low γ-glutamyl transferase (GGT), and low serum bile acids are the hallmarks of the disease. Diagnosis of BAD is made by testing the urine for normal and abnormal bile acid species with use of fast atom bombardment spectroscopy (FABS), which can identify the “fingerprint” of the inborn error by the pattern of bile acids present.

Defects in Membrane Transporters

The normal mechanisms of bile formation are described in Chapter 3 . Specifically, bile formation is dependent on the interaction of the bile acid transporters and solute carrier systems on the basolateral membrane of the hepatocyte and the mostly ATP-dependent transporters (ABC transporters) located on the canalicular membrane ( Fig. 70.1 ). Bile flow begins at the basolateral membrane with uptake and exchange of solute from the portal blood, which does not require active transport. The basolateral transporters include the superfamily of organic ion transport proteins, which allow organic anion uptake in exchange for bicarbonate or glutathione. The sodium-dependent taurocholate cotransporter (NTCP) transports only bile acids coupled with sodium. The multidrug resistance-associated proteins—MRP3 ⁷ and MRP4(Abcc4)—function as basolateral efflux pumps and are upregulated under cholestatic conditions to export bilirubin conjugates (by the former) and bile acids and glutathione (by the latter). There are no described genetic disorders of cholestasis related to basolateral membrane transporters.

The canalicular membrane transporters reside in the canalicular membrane, which is rich in cholesterol and sphingomyelin (see Fig. 70.1 ). This membrane is very metabolically active, containing many ATP-dependent solute transport proteins. It also houses ion and water exchangers, vesicle fusion proteins (i.e., soluble N -ethylmaleimide-sensitive-factor [NSF] attachment protein [SNAP] or NSF attachment protein receptor [SNARE]), skeletal proteins (i.e., villin), and tight-junction proteins. Of particular importance as membrane transporters are FIC1 ( ATP8B1 ), which is an aminophospholipid translocase; the bile salt export pump (BSEP) ( ABCB11 ), which mediates conjugated bile acid transport; and the multidrug-resistant protein MDR3 ( ABCB4 ), a flippase of phosphatidylcholine. Defects in each of these transporters have been linked to inherited cholestatic diseases that were commonly identified as PFIC types I, II, and III, respectively ( Table 70.2 ). The three other forms of PFIC ( TJP2, NR1H4, and MY05B ) are not specific canalicular transporter defects but are listed here for ease of grouping the conditions.

Progressive Familial Intrahepatic Cholestasis

Our understanding of the family of conditions that make up PFIC and the genotypic and phenotypic differences among them is the result of functional and genetic mapping studies that have identified the genes and their functions over the last 15 years. Initially PFIC was clinically identified by the presence of hepatocellular cholestasis, low serum levels of GGT activity, and autosomal recessive inheritance. PFIC has now been redefined into six separate and distinct diseases with their own specific gene defects and distinct clinical profiles. Each of these genes has a role in normal bile production and excretion (see Table 70.2 ).

PFIC has evolved from its origins as a constellation of symptoms seen in the Amish who were descended from a single ancestor, Jacob Byler, when it was labeled “Byler disease.” Subsequently, numerous phenotypically similar non-Amish patients were reported, and then the term Byler syndrome was used to describe these patients. Later, the term PFIC was applied to all Byler-like patients—however, the patients were sorted into two distinct subtypes: low-GGT PFIC (PFIC-1,2, 4, 5, and 6) and high-GGT PFIC (PFIC-3). It is now the custom to refer to these diseases by their gene defect, that is, PFIC-1 as FIC1 disease, or by the transporter defect (see Table 70.2 )— ATP8B1 (FIC1 ), ABCB11 (BSEP), ABCB4 (MDR3), TJP2, NR1H4, and MYO5B . ^{, ,} Despite their genetic distinctness, there are many clinical similarities between the PFIC subtypes, especially between PFIC-1 and PFIC-2. PFIC is clinically characterized by chronic cholestasis that begins in early childhood and usually progresses to cirrhosis within the first decade of life. The average age at onset is 3 months, and the disease may progress rapidly and result in cirrhosis during infancy or be slowly progressive with minimal scarring well into the teenage years. Pruritus is the dominant feature of cholestasis in the majority of patients. The pruritus is often misdiagnosed as skin disease because of the intense itching, which is unexplained; liver disease is not considered because of the disproportionately low level of jaundice in this condition. Patients begin to present with generalized mutilation of skin, usually most severe on the extensor surfaces of the arms and legs and on the flanks of the back, due to the disabling pruritus, which does not usually respond to medical therapies.

Severe episodes of recurrent epistaxis, perennial asthma-like disease, and growth failure are common associated problems. PFIC patients are described as “stocky” because of a high prevalence of short stature (95% of patients have stature below the fifth percentile), with often normal weight for height. Without treatment, there is often delayed onset of puberty and sexual development. Intellectually, these patients are equal to their peers if their pruritus is treated. Without treatment, their scholastic achievement can be compromised by inability to focus or concentrate and loss of sleep due to constant pruritus. Complications of cholestasis such as fat-soluble vitamin deficiencies are prevalent in untreated patients and discussed elsewhere in the text. Most patients will develop hepatic fibrosis and eventually cirrhosis, which is associated with the findings of hepatomegaly and sometimes splenomegaly. Unlike other cholestatic disease such as ALGS, PFIC patients do not develop xanthomas. In a recent clinical study comparing the presentation and course of PFIC-1 and PFIC-2 patients, several key differences were noted, which included that PFIC-2 (BSEP) patients exhibited more severe hepatobiliary disease compared to PFIC-1 (FIC-1) patients, whereas PFIC-1 patients had greater evidence of extrahepatic disease with diarrhea, pancreatic disease, pneumonia, abnormal sweat tests, hearing impairment, and poor growth. Patients with PFIC-1 are more likely to have associated watery diarrhea, some of which is severe. This secretory diarrhea may persist after liver transplantation.

Hallmark laboratory findings in PFIC-1, -2, -4, -5, and -6 are low GGT and normal or near-normal serum cholesterol, but markedly elevated levels of serum bile acids. In contrast, the GGT in PFIC-3 is elevated. Other serum values of liver-related enzymes such as alkaline phosphatase, aminotransferases, bilirubin, and bile salts are not distinct from those seen in several other cholestatic disorders.

The histopathologies of PFIC-1 and PFIC-2 are similar at the light microscopy level ( Figs. 70.2 and 70.3 ). Uniformly there is the presence of hepatocellular and canalicular cholestasis with pseudoacinar transformation consistent with cholate injury. The presence of multinucleated giant cell transformation is most commonly seen in infancy and has been recently reported to be more commonly seen in PFIC-2 (BSEP-deficiency) than PFIC-1 (FIC-1 deficiency). Degeneration of bile ducts may be seen with apoptotic changes of the biliary epithelium consisting of pyknotic nuclei (small and hyperchromatic) and attenuated cytoplasm and loss of duct lumina. Inflammation is absent. Bile duct paucity develops in 70% of older children because of these changes. In advanced fibrosis, there may be bile ductules at the edge of the portal tract. Lacy lobular fibrosis typically develops early and progresses to portal to central bridging and eventually to cirrhosis (see Figs. 70.2 and 70.3 ). The rate of progression of the fibrosis is highly variable but correlates loosely with the severity of the clinical disease. The major distinguishing histologic finding is seen at the level of electron microscopy with the presence of coarse granular bile in canalicular spaces of PFIC-1 patients, labeled “Byler bile.” PFIC-2 patients have a more filamentous morphology than the bile seen in the canalicular spaces. PFIC-2 (BSEP disease) has been associated with multiple cases of gallstones and hepatocellular carcinoma.

In PFIC-1 and PFIC-2, there is also an abnormal distribution both quantitatively and qualitatively of bile acids in serum and bile. Total serum bile acid concentrations are markedly elevated (usually greater than 200 μmol/L, normal less than 10) with an elevated ratio of chenodeoxycholic acid to cholic acid conjugates, usually greater than 10:1. The total biliary bile acid concentrations are low (0.1 to 0.3 mmol/L, normal above 20) even in comparison to other cholestatic syndromes such as ALGS, with a predominance of cholic acid conjugates.

As indicated by its name, PFIC is a progressive disease that culminates in cirrhosis and end-stage liver disease in the majority of patients. Medical treatment has consisted of the usual supportive care of cholestatic disease with fat-soluble vitamin supplementation and the use of ursodeoxycholic acid (ursodiol, 20 to 30 mg/kg/day). Although there is evidence that ursodiol may enhance bile flow, there is no evidence that it alters disease progression overall.

Surgical therapy consisting of the partial external biliary diversion (PEBD) has been used for the last two decades to treat PFIC and ALGS. PEBD involves the surgical placement of an enteric conduit between the gallbladder and the skin through which bile flow is partially diverted away from the enterohepatic circulation. It typically results in an approximately 50% diversion of bile flow, which amounts to ∼30 to 120 mL of bile per day that drains into the ostomy bag and is discarded. PEBD has been effective in improving chronic cholestasis and its associated complications in both ALGS and PFIC. ^, PEBD has become a standard intervention for PFIC and can slow or halt the progression of liver disease in this condition, which usually progresses to cirrhosis and end-stage liver disease if untreated. ^, After diversion, the bile salt pool converts to predominantly cholic acid conjugates, which has been associated with histologic and clinical improvement of the liver disease. PEBD may not always affect the natural progression of either ALGS or PFIC, and at present, no clinical parameters have been defined that predict patients who are likely to respond to biliary diversion procedure. Although PEBD has been shown in some cases to halt or reverse disease progression, it is generally ineffective in patients with established cirrhosis. Rare patients develop “watery” bile output after PEBD, with severe electrolyte losses that need to be monitored and replaced. Twenty years of experience with PEBD and PFIC has demonstrated variable relief of pruritus, improvement in liver histology, improved growth, and improvement in bile acid content of bile. ^{, , ,} A variation of PEBD is the limited ileal diversion, in which the distal 20% to 25% of the ileum is removed from the intestinal mainstream and made into a self-emptying blind loop, which results in loss of bile salts similar to PEBD. Ileal diversion was originally reserved for patients who have had a cholecystectomy, as it tends to become less effective over time. In some cases, PEBD or ileal diversion does not significantly affect the progression to cirrhosis, and liver transplantation becomes necessary. ^{, , ,}

PFIC-2: BSEP Disease

The PFIC-2 gene is located at chromosome 2q24. ^{, ,} It codes for an ABC bile salt transporter, also called the BSEP. The PFIC-2 gene is analogous to the rat sister gene of p-glycoprotein (S-PGP), which, in rats, has been shown to be important in bile salt transport. Studies of liver tissue from patients with mutation of BSEP have revealed lack of canalicular BSEP expression by immunohistochemistry ( Fig. 70.4 ). ^, This finding has clarified that PFIC-2 patients may have limited or no BSEP protein and therefore have a primary inability to transport bile salt.

Benign Recurrent Intrahepatic Cholestasis

The condition benign recurrent intrahepatic cholestasis (BRIC) presents very similarly to PFIC, with cholestasis, pruritus, low GGT level, and high serum bile acid level; however, the hallmark of this condition is intermittent episodes of cholestasis without progression to liver failure and later onset than PFIC. ^, Patients are totally asymptomatic, both clinically and biochemically, in between the episodes of cholestasis. BRIC-1 and BRIC-2 share the same locus with PFIC-1 ( ATP8B1 mutation) and PFIC-2 [ ABCB11 (BSEP)] respectively, but the mutations cause only partially impaired protein synthesis (see Table 70.2 ). ^{, ,} The cholestasis episodes have been treated by temporary biliary diversion using nasobiliary tube drainage of bile during the episode with some success in relieving the pruritus. However, in select cases, surgical biliary diversion has also been used for cases of BRIC with frequent debilitating attacks, or when there appears to be progression to permanent cholestasis. The clinically more severe disease identified as Greenland Eskimo infantile cholestasis is seemingly also a variant of FIC1 disease.

Hereditary Cholestasis with Lymphedema: Aagenaes Syndrome

Aagenaes syndrome is identified as a genetic form of cholestasis associated with lymphedema that is mapped to chromosome 15q. It was initially reported in Norwegian patients; however, subsequently there have been reports in Italian children, in Japanese children, and in siblings with French/German heritage. The inheritance appears to be autosomal recessive. Studies are underway to identify the gene locus for this disease using linkage disequilibrium.

Clinically, jaundice in the first weeks of life with acholic stools may be the first manifestation of the disease. Overall, the cholestatic liver disease tends to improve with age such that bilirubin and aminotransferase levels may be normal by school age. Cholestasis occurs episodically in older children with cholestatic periods lasting 2 to 6 months. The liver disease tends to be mild in most patients, but several older children and adults have progressed to cirrhosis. Both puberty and pregnancy have been associated with transient increases in cholestasis.

The liver histopathology in early childhood shows massive giant cell transformation of hepatocytes and intracellular retention of bile pigment. Patients in clinical remission may have liver morphology close to normal. Some patients may have bile plugs and a slight increase in portal fibrosis. Four of 26 patients reported by Aagenaes et al. have developed biopsy-proven cirrhosis. Treatment is that of cholestasis, utilizing fat-soluble vitamin supplementation and choleretic agents. The lymphedema usually appears in the lower extremities in early childhood and has been attributed to lymphatic vessel hypoplasia. The greater clinical problem tends to be the lymphedema, which can become disabling. Patients are offered physical therapy and restrictive wraps to limit the fluid accumulation and prevent skin breakdown.

Arthrogryposis Multiplex Congenita, Renal Dysfunction, and Cholestasis Syndrome

Arthrogryposis multiplex congenita, renal dysfunction, and cholestasis syndrome (ARC) is an autosomal recessive multisystemic disorder associated with the germline mutation VPS33B that is mapped to chromosome 15q26. In the largest study of ARC patients published to date involving 66 patients, the most prevalent clinical features described were failure to thrive, the presence of neonatal cholestasis with low GGT level, platelet dysfunction with high risk of hemorrhage with liver biopsy and spontaneous bleeding, renal tubular leak, and hypotonia with arthrogryposis. Less frequent clinical features include small for gestational age, dysmorphic features (lax skin, low-set ears, high arched palate, and cryptorchidism), ichthyosis, metabolic acidosis, nephrogenic diabetes insipidus, recurrent infections, recurrent febrile illnesses, and diarrhea. Survival beyond a year of age is unusual. If the patient survives infancy, cerebral manifestations including severe developmental delay, hypotonia, nerve deafness, poor feeding, microcephaly, and defects of the corpus callosum may become evident.

VPS33B is a vacuolar sorting protein involved in the regulation of vesicular membrane fusion and protein sorting by interacting with SNARE protein on membranes such as the canalicular membrane. Patients with ARC have evidence of abnormal polarized membrane protein trafficking by immunostaining of renal and liver biopsies that show mis-localization of several apical membrane proteins in the liver and kidney ( Fig. 70.5 ). In the zebrafish knockdown of the vps33b ortholog, bile duct paucity and impaired intestinal lipid absorption were observed. These findings phenocopied the digestive disease seen in ARC patients and suggest that VPS33B may play a role in primary bile duct development. VPS33B is also required for megakaryocyte and platelet alpha-granule biogenesis. Defects in platelets of patients with ARC have been documented and are associated with increased platelet size, a pale appearance in blood films, and a decrease or absence in alpha-granules. This defect causes platelet dysfunction that clinically has been manifested as increased incidence of bleeding with liver biopsy and spontaneous bleeding.

Severe cholestasis may occur in association with arthrogryposis multiplex congenita and renal disease. The cholestatic liver disease is usually present at birth, and paucity of intrahepatic bile ducts and multinucleate transformation of hepatocytes are the predominant features. Lipofuscin deposition has been described in several cases of ARC, and pigmentary change, bile duct paucity, and giant cell transformation may coexist in some patients. Patients with ARC rarely survive long enough to develop cirrhosis. Causes of death have been reported as infection, bleeding complications, or metabolic derangements.