Multiplex Genetic Test in Liver Detoxification Function for Predicting Liver Disease Progression Ran Oren, Hava Peretz, Sigal Fishman, Guy Rosner, Zamir Halpern

Liver Biopsy Is the gold standard for the diagnosis Who should we biopsy? When should we biopsy? Can we biopsy everybody?

L'appareil Probe 3.5 MHz Specific electronic equipment Ultrasound acquisition chip Digital signal processing Integrated computer Patient data base

The examination The examination can be made following the ultrasound scan, with, the patient lying down in the same position The time needed for examination is less than 5 minutes. The examination can be carried out by qualified hospital staff other than doctors : nurses, technicians

Objective Up to 33% of Hepatitis C Patients progress to cirrhosis in less than 20yUp to 33% of Hepatitis C Patients progress to cirrhosis in less than 20y It is of great importance to find reliable predicting markers of cirrhosis progressionIt is of great importance to find reliable predicting markers of cirrhosis progression Previous studies have shown an association between CYP2D6 polymorphism and liver cirrhosisPrevious studies have shown an association between CYP2D6 polymorphism and liver cirrhosis

Background The human cytochrome P450 (CYP) superfamily comprises 57 genesThe human cytochrome P450 (CYP) superfamily comprises 57 genes These code for enzymes having role in drug metabolism, foreign chemicals, arachidonic acid, cholesterol metabolism, generation of ROS in the liver, etc.These code for enzymes having role in drug metabolism, foreign chemicals, arachidonic acid, cholesterol metabolism, generation of ROS in the liver, etc. Studies have shown that a number of gene polymorphisms influence the progression of fibrosis in patients with various liver diseasesStudies have shown that a number of gene polymorphisms influence the progression of fibrosis in patients with various liver diseases –Alcoholic liver disease : CYP2E1 –NASH : HFE

Pathogenesis of Liver Fibrosis

Role of CYP in liver fibrosis - Hypothesis Hepatic cyctochrome P450 generates ROS -> activation of HSCs-> -> FibrosisHepatic cyctochrome P450 generates ROS -> activation of HSCs-> -> Fibrosis Arachidonic Acid Arachidonic Acid Collagen type 1 CYP2 family degradation

Objective CYP2D6*4 is the most common ‘poor metabolizer’ variantCYP2D6*4 is the most common ‘poor metabolizer’ variant Prevalence in the Caucasian population - 23%Prevalence in the Caucasian population - 23%

Aim The aim of the study was to elucidate the impact of CYP2D4*6 on fibrosis progression rate The aim of the study was to elucidate the impact of CYP2D4*6 on fibrosis progression rate

Patients and methods 75 Caucasians patients with chronic hepatitis C75 Caucasians patients with chronic hepatitis C Other liver diseases were excludedOther liver diseases were excluded Blood of twenty healthy Caucasian neonates served as controlBlood of twenty healthy Caucasian neonates served as control

Patients and methods Histopathology: Histopathology: Stage and grade were assessed according to the Batts and Ludwing system Stage and grade were assessed according to the Batts and Ludwing system

Patients and methods Definition of “fast” and “slow” fibrosers Poynard’s fibrosis progression model based on age of exposure and duration of Infection J Hepatol May;34(5):730-9

Adapted from Poynard T et al. J Hepatol. 2001;34: Probability of Fibrosis Progression to F4 According to Age at Infection >50 41–50 31–40 21–30 <21 Duration of Infection (y) Probability

Patients and methods Infection in age of less than 20 y- cirrhosis after 40yInfection in age of less than 20 y- cirrhosis after 40y Infection in 3 rd and 4 th decades – cirrhosis after 30yInfection in 3 rd and 4 th decades – cirrhosis after 30y Infection in 5 th decade – cirrhosis after 20yInfection in 5 th decade – cirrhosis after 20y Infection after the age of 50y- cirrhosis after 15 yearsInfection after the age of 50y- cirrhosis after 15 years

Patients and methods Non biopsied patients: Clinical diagnosis of cirrhosis (signs &imaging)Clinical diagnosis of cirrhosis (signs &imaging) Patients with no date of exposure : Cirrhotic young patients - not expected to reach cirrhosis by the modelCirrhotic young patients - not expected to reach cirrhosis by the model

Patients and methods CYP2D6 assay: CYP2D6 assay: Genomic DNA was extracted from peripheral blood by a salting-out procedure.Genomic DNA was extracted from peripheral blood by a salting-out procedure. Cytochrome P450-2D6*4 mutation was detected by real time PCR method, using Fluorecent hybridization probes in the lightCycler instrumentCytochrome P450-2D6*4 mutation was detected by real time PCR method, using Fluorecent hybridization probes in the lightCycler instrument

Demographic and clinical characteristics of the two groups P-value Slow fibrosers (33) Fast fibrosers (42) (57.6) 26 (61.9) Male (%) < (16.8) 43 (9.4) Mean Age(SD) (11.3) 24.6 (11.3) Mean age of exposure(SD) < (12.4) 18 (7.9) Mean duration of infection:years (SD)

Demographic and clinical characteristics of the two groups P-velue Slow fibrosers (17) Fast fibrosers (33) < (21) Liver Transplatation(%) < (1.5)3.9(0.32) Mean stage of fibrosis by biopsy (SD) < (0.05) 0.06(0.05) 30.26(0.1) Mean value of fibrosis progression rate per year (SD)

CYP2D6*4 and fibrosis progression CYP2D6*4 prevalence: Controls -12%Controls -12% Slow fibrosers-15%(10/66)Slow fibrosers-15%(10/66) Fast fibrosers-34.5% (29/84)Fast fibrosers-34.5% (29/84) P value=0.007 P value=0.007

The prevalence of homozygotes, heterozygotes and carriers in the ‘fast’ and ‘slow’ fibrosers Slow fibrosers Fast fibrosers CYP2D6*4 carrier status 1(3.0)7(16.7) Homozygote (%) 1 8(24.2)15(35.7) Heterozygote (%) 9(27.3) 1 22(52.4) Homozygote or heterozygote (%) 24(72.7)20(47.6) None (%) 1 P-value= P-value = 0.06

CYP2D6*4 carrier state predicts fast progression of fibrosis significance Odd ratio Independent variant CYP2D6P*4 carrier state Duration of infection

Summary CYP2D6*4 allele is associated with fast progression to cirrhosisCYP2D6*4 allele is associated with fast progression to cirrhosis CYP2D6*4 allele frequency is significantly higher in the ‘fast fibrosers’ than in the ‘slow fibrosers’CYP2D6*4 allele frequency is significantly higher in the ‘fast fibrosers’ than in the ‘slow fibrosers’

Conclusions Genetic variations influencing hepatic fibrogenesis is a topic of scientific interest Liver fibrosis progression rate may be predicted CYP2D6*4 might serve as a genetic non invasive marker for ‘fast fibrosers’CYP2D6*4 might serve as a genetic non invasive marker for ‘fast fibrosers’

Subject of the Discussion To develop a commercial kit for predicting the progression of liver disease in chronic liver disease patients The kit will be composed of: –Various CYP2D6 alleles (*1, *4, *3, *5, *6) –Other genetic variants can be studied (TNF alpha, IL10, etc) Target population: –Chronic liver disease patients (hepatitis C, hepatitis B, fatty liver, …)

Biological kit for predicting liver disease progression The project has to include: –Founder –Consultant –Laboratory –Technicians