Persisting Hyperbilirubinemia in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Chronically Treated with Eculizumab: The Role of Hepatocanalicular Transport Variants

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is caused by an acquired mutation in the phosphatidylinositol (GPI)-anchors. The lack of GPI-anchored proteins on the surface of affected red blood cells (RBCs) results in complement mediated chronic intravascular hemolysis. The monoclonal anti-C5 antibody eculizumab protects PNH RBCs by blocking the terminal complement cascade. Eculizumab treated PNH patients show a dramatic decrease of LDH and bilirubin, however few patients remain hyperbilirubinemic, eventually indicating an inadequate response. Hepatocanalicular transporters are rate-limiting in the process of bile formation, and mutations in the genes encoding these transporters cause intrahepatic cholestasis.

PATIENTS and METHODS: Mutation analysis regarding hepatobiliary transporter and nuclear receptor defects (ABCB4, ABCB11, ATP8B1, NR1H4) were performed in eight [75% (6/8) male; mean age, 40 years (range 25 - 70 years)] out of the currently 141 patients with PNH /-clone at our department due to a persistent (isolated) increase in total serum bilirubin (median 4.5 mg/dL; range 3.0 - 8.5) during chronic treatment with eculizumab (LDH: median 274 U/L; range 172 - 836) and normal or slightly increased aminotransferases. Clinical symptoms resembled those of hyperbilirubinemia including jaundice. Median clone size of GPI-deficient granulocytes (FLAER) was 98.1% (range 79.0 - 99.5%), with a median observation time of 56.4 months (range 10.6 - 82.7 months). All patients were treated according to the current German PNH guidelines.

RESULTS: Both homozygous and heterozygous procholestatic variants in the ABCB4, ABCB11 and ATP8B1 genes, encoding canalicular transporters, were identified in the studied patients, and all patients carried the common ABCB4 c.787 A>T polymorphism (Table 1). None of the patients were tested positive for the common mutation c.-1g>t of the central bile acid sensor NR1H4.

Table 1: Hepatobiliary transporter gene variants in PNH patients (n=8)

Variant	Pat. 1	Pat. 2	Pat. 3	Pat. 4	Pat. 5	Pat. 6	Pat. 7	Pat. 8
ABCB4 c.504T>C (rs1202283)	HOM	HET	HET	HOM	neg.	neg.	HOM	neg.
ABCB4 c.787A>T (rs2109505)	HOM	HOM	HOM	HOM	HOM	HOM	HOM	HET
ABCB11 p.A444V (rs2287622)	HOM	HET	neg.	HET	neg.	neg.	HET	HOM
ABCB11 c.3084A>G (rs497692)	HET	HET	HET	neg.	HOM	HET	HOM	HET
ATP8B1 p.R952Q (rs12968116)	HOM	neg.	HET	HET	neg.	HET	neg.	neg.
NR1H4 (FXR) c.-1 g>t (rs56163822)	neg.	neg.	neg.	neg.	neg.	neg.	neg.	neg.

Pat.: Patient; HOM: homozygous; HET: heterozygous; neg.: negative

Routine sonographic follow-up examinations were performed in 75% of the patients (6/8) ruling out post-hepatic cholestasis. In three patients (pat. 2, 3 and 4), a cholecystectomy due to symptomatic cholelithiasis had been performed prior to diagnosis of PNH, whereas in one patient the diagnosis of a persistent, asymptomatic cholelithiasis (pat. 6) was made. A chronic (active) or recently acquired viral hepatitis was ruled out in all patients. Lab parameters were consistent with an isolated persistent increase in total serum bilirubin, despite normal (7/8) or slightly elevated transaminases (1/8; pat. 6) throughout observation time.

SUMMARY/CONCLUSIONS: This is the first report of mutations in hepatobiliary transporter genes predisposing to intrahepatic cholestasis in PNH patients. Therefore, persisting hyperbilirubinemia in PNH patients chronically treated with eculizumab might not be due to an insufficient response to eculizumab but due to bile acid transporter variants and ongoing extravascular hemolysis. Patients should be tested for and possible therapeutic agents established in the management include ursodeoxycholic acid, promoting bile flow, and rifampicin, accelerating hepatic detoxification and excretion of bilirubin and bile acids. Our data warrant further studies concerning the role of hepatobiliary transporter variants in PNH patients in order to determine their clinical significance.