1. Susceptibility to Alcoholic Liver Disease
Chris Day
Newcastle University UK

2. Alcohol and cirrhosis deaths in Europe: a significant problem
45, 000 deaths per annum in the EU due to alcoholic cirrhosis
65% of ALL cirrhosis deaths
25% of all alcohol-related deaths

3. FATTY LIVER

4. ALCOHOLIC
HEPATITIS

5. ALCOHOLIC
CIRRHOSIS

6. Prevalence of disease stages in unselected heavy drinkers
Normal
(0-30%)
Fatty Liver (60-100%)
WHY??
Steatohepatitis
(20-30%)
Highlight declining % get each lesion
Majority get fat - mechanisms well understood
Increased supply, esterfiication and impaired export
Key question is what determines who goes on to inflammation and fibrosis - what\is mechanism of damage?
Feature that distinguishes liver damage from other forms of liver damage is the STEATO hepatitis
?Does FAT must have something to do with the pathogenesis of more progressive disease
Cirrhosis
(<10%)

7. Non-genetic factors Dose & pattern of alcohol intake Dietary factors
Specific nutrients
High fat (?unsaturated) Rotily 1990
Low carbohydrate Rotily 1990
? Anti/pro-oxidants
General excess (obesity)
(Exercise)
Smoking Becker 2002, Klatsky 2006
Coffee drinking Klatsky 2006
Importantly these are not mutually exclusive 13

8. ALD & Dose: population level
Cirrhosis death rate correlates
with per capita alcohol intake
Leon et al Lancet 2006

9. ALD & Dose: individual level
% of category
Little doubt that there is an association between daily intake and risk of disease. Confirmed in Bellentani recent study. Threshold 21 drinks per week. Also shown in Becker prospective study 1996.
Importantly, in these 2 studies less than 10% of individuals in highest categories were cirrhotic and less than 20% had any evidence of disease at all.
Drinks per day
Bellentani et al 1997 1

10. ALD risk and pattern of intake
Risk of ALD increased by:
Drinking outside meal-times
Risk : 3.4[ ] Bellentani 1997
Drinking beer/spirits rather than wine
Risk : 2.5[ ] Becker 2002
Daily versus weekend drinking
Risk : 2.5 [ ] Corrao 2000

11. Is wine vs beer effect due to dietary factors?
Johansen, Friis, Skovenborg, Gronbaek BMJ 2006

12. Diet and ALD: the role of obesity
1604 heavy drinkers
Overweight = minimum BMI>25 women, >27 men in previous 10yr
In multivariate analysis obesity/glucose best predictors of cirrhosis (Risk  > x 2 for obesity)
Naveau et al 1997
Raynard et al 2002

13. Risk of ALD and coffee drinking
Klatsky et al Arch Intern Med 2006
Cohort study
sample
330 developed cirrhosis to 2001
Confirmed previous risk factors
BMI
Smoking

14. Coffee intake Relative Risk of ALD Never/seldom 1.0 <1
0.7 [ ]
1-3
0.6[ ], p<0.001 ≥4
0.2[ ], p<0.001
Per cup/day
0.8[ ], p<0.001
Adjusted for age, weight, smoking, gender, ethnicity

15. Gender and ALD Females develop ALD at “lower” intake
Explanation:  blood alcohol concentration for = dose/Kg (body fat) Marshall 1983
Estrogen sensitises liver to effects of gut bacteria (animal models) Thurman 1999
In most (not all) studies reported females develop ALD at a lower intake.
Studies done in fasting state with amounts of alcohol < 1 unit. Not confirmed in conditions of normal social drinking.
More likely either underreporting or women are smaller 10

16. Genetic factors: non sex-linked
Concordance rates (%) among twin pairs
Alcoholism genetic
ALD genetic
Risk “over-and-above” the risk of alcoholism
Hrubec & Omenn 1981
+ Ethnic variation in susceptibility
Caetano & Clark 1998, Stinson 2001, Klatsky 2006 19

17. Benefits of elucidating genetic factors involved in ALD susceptibility
Prevention
Understanding disease mechanisms
Rationale for design of new treatments
?Mechanism-specific therapy

18. Use of genetics to influence treatment strategies
Patients with apparently identical disease with 3 different genetic profiles and 4 alternative treatments A, B, C or D

19. X Polymorphism (“SNP”): common (>5%), inherited
variation in this code X
All cells have same DNA but only some genes expressed as proteins in a particular cell – thats what makes skin cell look different from heart cell. Also in a cell some genes permanently on and other on and off in response to signals

20. Effect of “functional“SNP
Promoter “switch” Coding
region region
ATTGC..
..GCCT
Transcription
Factor
Protein
Effects protein
QUANTITY
Effects protein
STRUCTURE &
FUNCTION 28

21. Finding the genes in ALD
Hypothesis free
Whole genome scanning X
Hypothesis-driven
“Candidate” gene studies 

22. Selection of candidate genes
Hypothesis-driven approaches
Gene product ? plays a role in disease pathogenesis
Gene knockout/over-expression in animal models influences disease development
Hypothesis-free approaches
Gene expression is altered in microarray studies of tissue from patients/animal models
Mutation of the gene leads to relevant phenotype in mouse random (ENU) mutagenesis studies

23. Selection of candidate genes
Hypothesis-driven approaches
Gene product ? plays a role in disease pathogenesis
Gene knockout/over-expression in animal models influences disease development
Hypothesis-free approaches
Gene expression is altered in microarray studies of tissue from patients/animal models
Mutation of the gene leads to relevant phenotype in mouse random (ENU) mutagenesis studies

24. Selection of candidate genes
Hypothesis-driven approaches
Gene product ? plays a role in disease pathogenesis
Gene knockout/over-expression in animal models influences disease development
Hypothesis-free approaches
Gene expression is altered in microarray studies of tissue from patients/animal models
Mutation of the gene leads to relevant phenotype in mouse random (ENU) mutagenesis studies
ERAB funded

25. Tilg & Day Nat Clin Practice 2006

26. Current “State-of-the-art”
1st report in 1991: Day, Crabb, James et al
All in the audience!
Only 2 replicated associations
ADH2*2 in East Asian populations
 acetaldehyde
<5% in Caucasians Zintzaras Hepatology 2006
TNFa Grove (UK) 1997 & Pastor (Spain) 2006
Plausible associations reported from single, well designed studies awaiting replication:
IL Grove 2000
Gut bacteria receptor (CD14) Lindross 2001

27. Gene (micro) array technique
Looks at level of expression of ALL gene “messages” (mRNA) in a tissue sample
Have used liver biopsies from patients with ALD at different stages
Analysed on CodeLink Human Whole Genome Bioarrays
Codelink Gene Expression Systems

28. Codelink Bioarray

29. Fold change scatter plot: advanced versus mild scarring1
0.4
0.5
0.6
0.7
0.8 2 3 4
Condition 2: Fibrosis N (normalized)

30. Conclusions Susceptibility due to a combination of genes & environment
Several important (modifiable) environmental risk factors such as diet/exercise/coffee identified
New “hypothesis free” methods of candidate gene selection opening up whole new directions for studies that are already yielding significant associations
These associations are likely to have major implications for preventative and therapeutic strategies for ALD

31. Acknowledgements The funding: The people:
European Research Advisory Board (ERAB)
European Association for the Study of the Liver (EASL)
Commonwealth Fellowship Programme
The people:
Helen Reeves, Luca Miele (Arrays, KLF’s)
Patrick Chinnery, Nimantha de Alwis (Mitos)
Pro