NASH AS PART OF THE METABOLIC SYNDROME 59 The main source of increased lipid turnover in NAFLD patients is not clear. However, an important role for visceral adiposity has been proposed. It is generally accepted that visceral adipose tissue is more insulin-resistant than subcutaneous adipose tissue [17], and people with increased visceral fat are char- acterized by a more severe deterioration of their lipid profile [18]. The association of NAFLD with cent- ral adiposity and increased lipolysis, as assessed by anthropometric measurements [10,14], needs to be confirmed by a quantitative nuclear magnetic reson- ance (NMR) assessment of visceral fat. Finally, the role of insulin resistance in NAFLD is supported by pilot therapeutic studies. Troglitazone, an insulin-sensitizing drug, significantly reduces tran- saminase levels, with inconclusive results on liver his- tology [19] (but see Chapter 24). In an animal model of NASH in obese leptin-deficient mice [20] and in humans [21], metformin reduces transaminase levels, which return to normal in approximately 50% of cases. Metformin also improves other metabolic abnormalities associated with the insulin resistance syndrome [22]. In summary, a large body of evidence indicates that NAFLD may stem from a defect of insulin activ- ity, involving both glucose and lipid metabolism, which explains the link with the associated metabolic disorders. In the scenario of metabolic and liver dis- ease, NAFLD looks very much like type 2 diabetes and obesity, but also shares features common to more advanced liver disease (Table 5.3). However, the defects are not necessarily linked to the presence of obesity and diabetes. Lean subjects with normal fasting gluc- ose and normal glucose tolerance may also present with NAFLD. These subjects are nevertheless charac- terized by enlarged waist girth, and possibly belong to The pattern of insulin resistance so far described in NAFLD patients is more like that observed in patients with type 2 diabetes or in their relatives, than in patients with cirrhosis. Non-diabetic patients with cirrhosis are characterized by hyperinsulinaemia, both in the fasting state and following glucose load, but basal endogenous glucose production is normal, and is normally suppressed by insulin. In contrast, both dia- betic and NAFLD patients have a blunted insulin- mediated suppression of hepatic glucose production and decreased rates of both oxidative and non-oxidative (glycogen synthesis) glucose metabolism. The derangement in lipid metabolism so far described is to be expected in NAFLD patients with obesity or hypertriglyceridaemia, but it is still present when these confounding factors are absent. In a selected popula- tion of lean NAFLD patients with normal glucose tolerance and lipid levels, lipolysis was increased by approximately 40% in the basal state and less effici- ently inhibited after insulin administration. Although the percentage decrease of glycerol turnover was comparable to controls after insulin administration (–62%), its absolute value remained higher in NAFLD patients (Bugianesi et al., personal communication). Likewise, lipid oxidation was higher in the basal state and less efficiently inhibited by insulin. The pattern of metabolic defects in non-obese non-diabetic NAFLD patients is thus consistent with accelerated lipolysis athe immediate result of insulin resistance in adipose tissueabeing responsible for the increased FFA supply and their oxidative use at the whole body level. The finding of a tight correlation between lipid oxidation and glucose production/disposal, may suggest that the hepatic and peripheral insulin resistance in these NAFLD patients was primarily the consequence of insulin resistance in fat tissues. Table 5.3 Metabolic features of insulin resistance in various clinical disorders. NAFLD Cirrhosis Obesity Type 2 diabetes Total glucose disposal ↓↓ ↓↓ Glucose oxidation ↓↔↓↓ Non-oxidative glucose disposal ↓↓ ↓↓ Suppression of hepatic glucose output ↓ ↔ ↔ ↓ ↓ Suppression of lipolysis ↓↔↓↓ Insulin secretion ↑↑ ↑↓↑ CHAPTER 5 60 An increased peripheral iron burden has also been reported in other conditions characterized by insulin resistance. In males, hypertension is characterized by a higher prevalence of increased iron stores and metabolic abnormalities that are part of the IRHIO syndrome [26]. The prevalence of IRHIO among type 2 diabetic patients is as high as 40%, and can be associated with a higher prevalence of steatosis and inflammation [27]. Iron depletion improves metabolic control and insulin sensitivity [28]. A similar improvement in insulin sensit- ivity has been observed in obese subjects with impaired glucose tolerance [29]. The hypothesis that iron might be the cause of NASH has received much attention, but available data do not completely support this conclusion (Table 5.4). A large proportion of NAFLD patients have no evid- ence of hepatic iron overload, and no differences are present in clinical features in relation to iron status [30]. In addition, iron status does not classify patients according to the histological severity of their liver dis- ease [31], and serum indices of iron overload do not correlate with measures of insulin sensitivity [14]. However, recent data do suggest that iron may have a role; iron depletion to a level of near-iron deficiency by quantitative phlebotomy produces a near normaliza- tion of alanine aminotransaminase and a marked reduc- tion of fasting and glucose-stimulated insulin. Also, HOMA values were reduced in most cases, but did not return to normal values [29]. (The potential role of iron as a factor determining fibrotic severity of NASH is discussed in Chapters 1 and 7.) Insulin resistance, oxidative stress and cytokines The role of iron, if present, might be mediated by oxidative stress, which might also be generated by dif- ferent conditions. Insulin resistance is an atherogenic state, characterized by oxidative changes of circulat- ing low-density lipoprotein (LDL) cholesterol par- ticles, induced by an excessive activity of free radicals [32], and a role for hyperinsulinaemia is suggested. Quinones-Galvan et al. [33] demonstrated that acute physiological hyperinsulinaemia enhances the oxid- ative susceptibility of LDL-cholesterol particles and reduces the vitamin E content in the LDL molecule. These changes are well characterized in type 2 diabetes, but they may also be present in hyperinsulinaemic the subgroup of normal-weight metabolically obese patients (usually with central obesity, see Chapter 18), a phenotype more frequently observed in subjects of Asian descent. Considering the importance of lifestyle behaviours in the pathogenesis of metabolic disorders, lean NAFLD patients might be subjects with a primary (genetic?) defect of insulin activity, where healthy lifestyles have not yet permitted the expression of the usual phenotype of the insulin resistance syndrome. Iron and the insulin resistance syndrome Iron deposition has long been known to cause clinical and laboratory findings similar to those observed in the insulin resistance syndrome. Moirand et al. [23] described a syndrome characterized by increased serum iron and liver iron deposition, associated with abnormal glucose tolerance, overweight or obesity, dyslipidaemia and insulin resistance. Patients were predominantly male and middle-aged, with a slightly increased preval- ence of the compound heterozygote HFE mutation C282Y/H63D. Steatosis was present in 25% of patients and NASH in 27%. Portal fibrosis (grades 0–3) was present in 62% of patients (grade 2 or 3 in 12%) in association with steatosis, inflammation and increased age. This syndrome, insulin resistance-associated hep- atic iron overload (IRHIO), occurs both in the absence and in the presence of increased transferrin saturation and serum ferritin and is frequently associated with NASH [24]. This association stimulated research on the possible role of iron in the pathogenesis of NASH. Iron is an ideal culprit for fatty liver disease. Iron deposition in genetic haemochromatosis is associated with insulin resistance and diabetes mellitus. Iron is a potent oxid- ative agent and might trigger oxidative stress with resultant liver injury. The relationship between hepatic iron overload and hyperinsulinaemia and/or insulin resistance may be twofold. Transferrin receptors, glucose transporters and insulin-like growth factor II receptors co-localize in cultured adipocytes, and are simultaneously regulated by insulin. Any genetic or acquired condition characterized by increased serum and liver iron is expected to downregulate glucose transporters, leading to hyperinsulinaemia and insulin resistance. Alternatively, if insulin resistance and hyper- insulinaemia were the primary defects, alterations in iron metabolism would be expected [25]. NASH AS PART OF THE METABOLIC SYNDROME 61 These features are independently related to cardiovas- cular mortality, which has given rise to the name of ‘deadly quartet’ for this syndrome [36]. In 1988, Gerald Reaven proposed the term ‘syn- drome X’ [37] to define the contemporary presence of diabetes and/or impaired glucose tolerance, hyper- triglyceridaemia, low HDL-cholesterol and hyperten- sion. He pointed out the role of hyperinsulinaemia and insulin resistance in the pathogenesis of the disease [37]. The metabolic disorder is probably much wider and other features might be added. Most subjects have evidence of additional metabolic disorders (elev- ated urate concentrations, impaired fibrinolysis and endothelial dysfunction). The primary role of hyperinsulinaemia is supported by several cross-sectional and longitudinal studies [38]. Central obesity, type 2 diabetes, hyperlipidaemia and hypertension are all characterized by raised insulin concentrations, and elevated insulin levels predict the development of the metabolic disorder [39]. Accord- ingly, DeFronzo and Ferrannini [40] proposed the term ‘insulin resistance syndrome’ to define this clustering of diseases. The borders of the syndrome remain difficult to define. The critical number of metabolic disorders to define the syndrome has not been specified; the dis- orders may progressively develop over the course of time, with obesity usually occurring first, followed by hyperlipidaemia and diabetes. Hypertension may fre- quently be present independently from other compon- ents. In addition, the ‘normal’ limits for the individual normoglycaemic conditions, such as obesity, essential hypertension and dyslipidaemia. Human liver biopsy specimens, when assessed for lipid peroxidation by staining for 3-nitrotyrosine, showed higher levels of lipid peroxidation in NASH relative to fatty liver and controls [15]. The levels of thiobarbituric acid reactive substances (TBARS), a gross measure of lipid peroxida- tion, also are increased in NAFLD. Finally, insulin resistance might stem from cytokine activation. In animal models, the chronic activation of IKKβ, the kinase that activates nuclear factor β, is associated with the presence of insulin resistance. Conversely, the administration of salicylate to inhibit IKKβ abolishes lipid-induced insulin resistance in the skeletal muscle of animals [34]. Cytokines might represent the link between insulin resistance and oxidative stress. Oxidant and inflammatory stresses are powerful activators of the IKKβ pathway, possibly via tumour necrosis factor α (TNF-α), suggesting a direct link between oxidative stress and insulin resis- tance. Whether treatment with antioxidants (e.g. vita- min E) might improve insulin sensitivity remains to be proven. Definition of the metabolic syndrome The clustering of metabolic disorders had been known for a long time before Avogaro et al. [35] first reported the association of obesity, hyperlipidaemia and dia- betes in 1967. Hypertension is also frequently present. Table 5.4 Pros and cons for a role of iron in the insulin-resistance syndrome and NASH. Pros Cons 1 Iron overload is associated with insulin-resistance 1 A poor correlation exists between HFE mutations and iron stores 2 A link at subcellular level connects transferrin 2 In NASH, iron overload is not associated with a receptors and glucose transporters higher prevalence of features of the metabolic syndrome 3 Serum and liver iron are frequently increased 3 Iron status does not classify patients according in NASH patients to the severity of liver disease 4 Serum iron is increased in hypertension and 4 Indices of iron overload do not correlate with diabetes quantitative measures of insulin resistance 5 Iron depletion improves diabetes control 6 Iron depletion reduces transaminase levels in obese subjects with impaired glucose tolerance CHAPTER 5 62 and 83% of males, and three criteria were fulfilled in 60% of females and 30% of males (Fig. 5.1). The prevalence of the metabolic syndrome increased with increasing BMI, from 18% in normal-weight subjects to 67% in obese subjects. The presence of the metabolic syndrome was significantly associated with female gender (OR, 3.08; 95% CI, 1.57–6.02) and age (OR, 1.54; 1.23–1.93 per 10 years) after adjustment for BMI class. The presence of impaired fasting glucose (blood glucose ≥ 110 mg/dL) disorders have been repeatedly changed in the last few years, so as to prevent a clear-cut assessment. The first attempt to define the metabolic syndrome came from the World Health Organization (WHO). The expert committee setting new criteria for the definition of diabetes proposed a classification based on the presence of one out of two necessary conditions (altered glucose regulation and insulin resistance), coupled with two additional features (Table 5.5) [41]. These criteria may be easily applied to diabetic popu- lations, but are not useful in a general setting. The assessment of insulin resistance requires complex tech- niques. Surrogate markers (fasting insulin, HOMA values), although validated by correlation analysis [42], have no defined ‘normal’ limits. New criteria were defined by the European Group for Insulin Resistance in 1999, limiting the syndrome to non-diabetic subjects [12], but the critical problem of insulin resistance was not set. In 2001 a new proposal by the Third Report of the National Cholesterol Education Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III, ATPIII) [43] provided a working definition of the meta- bolic syndrome, based on a combination of five categ- orical and discrete risk factors, which can easily be measured in clinical practice, and are suitable for epidemiological purposes. The limits for individual components (central obesity, hypertension, hypertrigly- ceridaemia, low HDL-cholesterol and hyperglycaemia) are derived from the guidelines of the international societies or the statements of WHO [1,41,43,44]. It is important to note that the anthropometric criteria vary between ethnic groups, with values being sub- stantially lower among Asians (see Chapter 18). NASH as part of the metabolic syndrome A very recent study with a large number of NAFLD patients was specifically aimed at assessing the preval- ence of the metabolic syndrome in relation to liver histology. In 304 consecutive NAFLD patients with- out overt diabetes, Marchesini et al. [45] defined the metabolic syndrome according to the ATPIII proposal. The population had a mean age of 41 years and a BMI of 27.5, but nearly 80% were overweight or obese. Over 80% were males. At least one criterion for the metabolic syndrome was present in 96% of females Table 5.5 Comparison of different diagnostic criteria for the metabolic syndrome. WHO proposal (1998, revised 1999) [41] Altered glucose regulation or insulin resistance plus two of the following: 1 Obesity (BMI ≥ 30 kg /m 2 or WHR > 1.0 [M] or > 0.9 [F]) 2 High triglycerides (> 150 mg /dL) or low HDL-cholesterol (< 35 mg /dL [M] or < 39 mg/dL [F]) 3 Hypertension (≥ 140/90 mmHg) 4 Microalbuminuria (> 30 µg /min) EGIR proposal (1999) [12] No diabetes Hyperinsulinaemia or insulin resistance plus two of the following: 1 Impaired fasting glucose (glucose, 110–126 mg /dL) 2 Hypertension (≥ 140/90 mmHg) 3 High triglycerides (> 175 mg /dL) or low HDL-cholesterol (< 39 mg /dL), independently of gender 4 Central obesity (waist girth ≥ 94 cm [M] or ≥ 80 cm [F]) ATP III proposal (2001) [43] Three of the following: 1 Waist girth (> 102 cm [M] or > 88 cm [F]) 2 Arterial pressure (≥ 130/85 mmHg) 3 Triglycerides (≥ 150 mg /dL) 4 HDL-cholesterol (< 40 mg /dL [M] or < 50 mg/dL [F]) 5 Glucose (≥ 110 mg /dL) BMI, body mass index; F, female; HDL, high-density lipoprotein; M, male; WHR, waist : height ratio. NASH AS PART OF THE METABOLIC SYNDROME 63 was the most predictive criterion for the metabolic syndrome (OR, 18.9; 6.8–52.7) also in this non- diabetic population. Insulin resistance (HOMA method) was significantly associated with the metabolic syn- drome (OR, 2.5; 1.5–4.2; P < 0.001). Liver biopsy was available in over 50% of cases, and histology was diagnostic for NASH in 74% of cases. At least one criterion for the metabolic syndrome was fulfilled in 88% of NASH patients and only in 67% of fatty liver (P = 0.004; Fisher’s exact test). This agrees almost exactly with an earlier study in which 85% of patients with histological NASH had WHO criteria for the metabolic syndrome [10]. NASH patients were characterized by more severe liver cell necrosis, measured by 20% higher alanine and aspartate aminotransferase levels. Of the five criteria for the metabolic syndrome, only hyperglycaemia and/or diabetes was significantly associated with NASH after correction for age, gender and obesity, but the simultaneous presence of three or more criteria (a defined metabolic syndrome) was associated with a different histopathological grading, including a higher prevalence (94% versus 54%) and severity of fibrosis (P = 0.0005) as well as of necroinflammatory activity (97% versus 82%; P = 0.031), without differences in the degree of fat infiltration (Fig. 5.2). Logistical regression analysis showed that the presence of the metabolic syndrome was associated with a high risk of NASH among NAFLD subjects (OR, 3.2; 1.2–8.9; 12345 No. of positive criteria Prevalence (%) Fig. 5.1 Frequency of criteria for the metabolic syndrome (ATPIII proposalaExpert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults [43]) in NAFLD patients according to gender. Note that the presence of three or more criteria defines the metabolic syndrome. P = 0.026), after correction for sex, age and body mass. In particular, the metabolic syndrome was associated with a high risk of severe fibrosis (bridging or cirrhosis: OR, 3.5; 1.1–11.2; P = 0.032), without differences in the degree of steatosis and necroinflammatory activity. The study indicates that the presence of multiple metabolic disorders is associated with a potentially progressive, more severe liver disease. Conclusions The increasing prevalence of obesity, coupled with diabetes, dyslipidaemia, hypertension and ultimately the metabolic syndrome puts a very large population at risk of developing liver failure in the coming decades. All these diseases have insulin resistance as a common factor, and are associated with atherosclerosis and cardiovascular risk. The occurrence of diabetes may be prevented by adequate lifestyle interventions [46–48], and recent evidence indicates that the progression of the disease and its complications may also be reduced by these same lifestyle interventions [49]. Additional studies are now needed to verify the effectiveness of lifestyle changes in the progression of fatty liver to NASH and/or cirrhosis. Pilot studies support a bene- ficial effect [50] (and see Chapter 24). References 1 World Health Organization. Preventing and Managing the Global Epidemic: Report of a WHO Consultation. World Health Organization, WHO Technical Report Series 894, 2000. 2 Mokdad AH, Ford ES, Bowman BA et al. Prevalence of obesity, diabetes, and obesity-related health risk factors, 2001. J Am Med Assoc 2003; 289: 76–9. 3 Marceau P, Biron S, Hould FS et al. Liver pathology and the metabolic syndrome X in severe obesity. J Clin Endocrinol Metab 1999; 84: 1513–7. 4 Ratziu V, Giral P, Charlotte F et al. 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Necroinflammation: dashed area, no necroinflammation; open area, occasional ballooned hepatocytes and no or very mild inflammation; grey area, ballooning of hepatocytes and mild to moderate portal inflammation; black area, intra-acinar and portal inflammation. The significance of differences is reported (Fisher’s exact test). NASH AS PART OF THE METABOLIC SYNDROME 65 and its association with fasting insulin and central and overall obesity in a general population. Metabolism 1996; 45: 699–706. 39 Haffner SM, Valdez RA, Hazuda HP et al. Prospective analysis of the insulin-resistance syndrome (syndrome X). Diabetes 1992; 41: 715–22. 40 DeFronzo RA, Ferrannini E. Insulin resistance: a multi- faceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardio- vascular disease. Diabetes Care 1991; 14: 173–94. 41 WHO Consultation. Definition, diagnosis and classifica- tion of diabetes mellitus and its complications. 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Hepatology 2003; 37: 917–23. 46 Pan XR, Li GW, Hu YH et al. Effects of diet and exercise in preventing NIDDM in people with impaired glucose tolerance: the Da Qing IGT and Diabetes Study. Diabetes Care 1997; 20: 537–44. 47 Tuomilehto J, Lindstrom J, Eriksson JG et al. Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance. N Engl J Med 2001; 344: 1343–50. 48 Knowler WC, Barrett-Connor E, Fowler SE et al. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002; 346: 393–403. 49 Gaede P, Vedel P, Larsen N et al. Multifactorial interven- tion and cardiovascular disease in patients with type 2 diabetes. N Engl J Med 2003; 348: 457–9. 50 Ueno T, Sugawara H, Sujaku K et al. Therapeutic effects of restricted diet and exercise in obese patients with fatty liver. J Hepatol 1997; 27: 103–7. 24 Mendler MH, Turlin B, Moirand R et al. Insulin resist- ance-associated hepatic iron overload. Gastroenterology 1999; 117 : 1155–63. 25 Ferrannini E. Insulin resistance, iron, and the liver. Lancet 2000; 355: 2181–2. 26 Piperno A, Trombini P, Gelosa M et al. Increased serum ferritin is common in men with essential hypertension. J Hypertens 2002; 20: 1513–8. 27 Turlin B, Mendler MH, Moirand R et al. Histologic fea- tures of the liver in insulin resistance-associated iron over- load: a study of 139 patients. Am J Clin Pathol 2001; 116: 263–70. 28 Fernandez-Real JM, Penarroja G, Castro A, Garcia- Bragado F, Hernandez-Aguado I, Ricart W. Blood letting in high-ferritin type 2 diabetes: effects on insulin sensitiv- ity and β-cell function. Diabetes 2002; 51: 1000–4. 29 Facchini FS, Hua NW, Stoohs RA. Effect of iron depletion in carbohydrate-intolerant patients with clin- ical evidence of non-alcoholic fatty liver disease. Gastro- enterology 2002; 122: 931–9. 30 George DK, Goldwurm S, McDonald GA et al. Increased hepatic iron concentration in non-alcoholic steatohepatitis is associated with increased fibrosis. Gastroenterology 1998; 114: 311–8. 31 Matteoni CA, Younossi ZM, Gramlich T et al. Non- alcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 1999; 116: 1413– 9. 32 Bucala R, Makita Z, Koschinsky T, Cerami A, Vlassara H. Lipid advanced glycosylation: pathway for lipid oxidation in vivo. Proc Natl Acad Sci USA 1993; 90: 6434–8. 33 Quinones-Galvan A, Sironi AM, Baldi S et al. Evid- ence that acute insulin administration enhances LDL cholesterol susceptibility to oxidation in healthy humans. Arterioscler Thromb Vasc Biol 1999; 19: 2928–32. 34 Yuan M, Konstantopoulos N, Lee J et al. Reversal of obesity- and diet-induced insulin resistance with salicy- lates or targeted disruption of IKKβ. Science 2001; 293: 1673–7. 35 Avogaro P, Crepaldi G, Enzi G, Tiengo A. Associazione di iperlipemia, diabete mellito e obesità di medio grado. Acta Diabetol Lat 1967; 4: 572–90. 36 Kaplan NM. The deadly quartet: upper-body obesity, glucose intolerance, hypertriglyceridemia, and hyperten- sion. Arch Intern Med 1989; 149: 1514–20. 37 Reaven GM. (Banting Lecture 1988) Role of insulin resis- tance in human disease. Diabetes 1988; 37: 1595–607. 38 Schmidt MI, Watson RL, Duncan BB et al. Clustering of dyslipidemia, hyperuricemia, diabetes, and hypertension 66 Abstract While the vast majority of individuals with obesity and type 2 diabetes mellitus will have steatosis, only a minority will ever develop non-alcoholic steatohep- atitis (NASH), fibrosis and cirrhosis. Family studies suggest that genetic factors are important in disease progression, although dissecting genetic factors having a role in NASH and fibrosis from those influencing the development of its established risk factors is clearly difficult. A variety of approaches can be used to look for genetic factors having a role in NASH. In future, genome-wide single nucleotide polymorphism (SNP) scanning of cases and controls may become feasible. However, to date studies have relied on candidate gene, case–control allele association methodology. Investigators using this approach must take care to avoid a number of pitfalls in study design likely to lead to spurious results. If these can be avoided, our increased understanding of disease pathogenesis sug- gests a variety of candidate genes worthy of study as susceptibility factors. Recent, and as yet preliminary studies, have reported associations between steatosis severity, NASH and fibrosis with genes whose prod- ucts are involved in lipid metabolism, oxidative stress and endotoxin–cytokine interactions. If confirmed, NASH is a genetically determined disease Christopher P. Day & Ann K. Daly 6 Key learning points 1 Only a minority of patients with risk factors for non-alcholic fatty liver disease (NAFLD) develop non- alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Family studies suggest that genetic factors may have a role in determining susceptibility to advanced disease. 2 Candidate gene, case–control allele association-based approaches are currently the best methods avail- able for the detection of susceptibility genes, although in future genome-wide scanning may be technically and economically feasible. 3 In future, the choice of candidate genes worthy of study seems likely to be guided by tissue expression profiling and mouse mutagenesis approaches. 4 Recent studies have reported associations between steatosis severity, NASH and fibrosis with genes encoding proteins involved in lipid metabolism, oxidative stress and endotoxin-cytokine interactions. 5 If confirmed, these associations will greatly enhance our understanding of disease pathogenesis and, accordingly, our ability to design effective therapies. Fatty Liver Disease: NASH and Related Disorders Edited by Geoffrey C. Farrell, Jacob George, Pauline de la M. Hall, Arthur J. McCullough Copyright © 2005 Blackwell Publishing Ltd NASH IS A GENETICALLY DETERMINED DISEASE 67 these associations will greatly enhance our understand- ing of disease pathogenesis and, accordingly, our abil- ity to design effective therapies. Introduction Obesity and insulin resistance are undoubtedly associ- ated with the whole spectrum of non-alcoholic fatty liver disease (NAFLD), with the degree of obesity and the severity of insulin resistance increasing the risk of advanced disease (see Chapters 3 and 4). However, despite these strong associations, it is clear that while the majority of individuals with these risk factors will have steatosis, only a minority will ever develop NASH. An autopsy study in 351 non-drinking individuals reported that, while more than 60% of obese patients with type 2 diabetes mellitus had steatosis, only 15% had NASH [1], and a recent analysis of the Third National Health and Nutritional Examination Survey (NHANES III) database reported that only 10.6% of obese individuals with type 2 diabetes mellitus had any elevation of serum alanine aminotransferase [2]. These studies suggest that while obesity and/or insulin resist- ance are undoubtedly involved in the pathogenesis of steatosis and NASH, some other environmental and/or combination of genetic factors is required for progres- sion to NASH and fibrosis. This is analogous to the situation in alcoholic liver disease (ALD) where excess- ive drinking leads to steatosis in the majority of indi- viduals, but other, largely unknown, factors determine why only a minority of heavy drinkers develop hepat- itis and cirrhosis [3]. With respect to environmental factors influencing the risk of NASH, diet, exercise and possibly small bowel bacterial overgrowth are obvious candidates, with the latter contributing to increased hepatic levels of tumour necrosis factor-α (TNF-α) [4]. A role for genetic factors in NASH is suggested by two recent reports of family clustering. Struben et al. [5] reported the coexistence of NASH and cryptogenic cirrhosis in seven out of eight kindreds studied, while Willner et al. [6] found that 18% of 90 patients with NASH had an affected first-degree relative. The absence of these genetic factors presumably explains the benign prognosis of simple non-alcoholic fatty liver [7,8]. It remains to be determined whether this clustering of cases is simply a reflection of the well-established heritability of the established risk factors for NAFLDa obesity and insulin resistance. Methodology for studying genes involved in NASH susceptibility If it is assumed that there is a genetic component to NASH, what methodology is currently available to search for genetic factors predisposing to this un- doubtedly polygenic disease? Methods fall into three broad and overlapping categories: family-based link- age analysis, candidate gene studies and genome-wide SNP scanning. Family-based studies Allele-sharing methods involve studying affected relat- ives in a pedigree to determine how often a particular copy of a chromosomal region is shared identical- by-descent (IBD). The frequency of IBD sharing at a particular locus can then be compared with random expectation. Typically, this has involved linkage ana- lysis in large cohorts of affected sibling pairs using widely spaced multiallelic markers such as microsatel- lites to identify chromosomal regions. Unfortunately, linkage analysis, which has been so successful in iden- tifying genes responsible for single gene disorders, has (with few notable exceptions [9]) been generally disap- pointing when applied to polygenic diseases, probably because of its limited power to detect genes of moderate effect [10]. Candidate gene studies An alternative methodological approach involves the study of candidate genes. In this method, a polymorph- ism (or polymorphisms) is identified by various means in a ‘functional’ candidate gene (a gene whose pro- duct is thought to have a role in disease pathogenesis). The polymorphism is then examined for association with disease using one of two approaches: intrafamilial allelic association studies and case–control associ- ation studies. The most commonly used test for familial association is the transmission disequilibrium test (TDT), which compares the frequency with which the allele under study is transmitted to affected offspring by each parent with the frequency expected by random transmission [11]. The major limitation of TDT testing is that the index case must have at least one surviving parent from whom to collect DNA, although vari- ations on the TDT using siblings of affected individuals (discordant sibship) have recently been proposed [12]. CHAPTER 6 68 The value of this test is still unclear, but, as with the TDT, when the allele frequency is low, achieving stat- istical significance requires very large numbers of fam- ilies. Even if large enough numbers of NASH families could be collected, investigators using family-based approaches to study genetic susceptibility to NASH face two further problems: 1 Since there is currently no reliable non-invasive way of accurately determining the presence or severity of NAFLD, family members will ideally require liver biopsy for definitive diagnosis. 2 Relatives must be discordant for the established risk factors for NASH, otherwise any association with NASH observed may simply reflect an association with obesity or diabetes. In view of these difficulties, it is perhaps not surpris- ing that, as with ALD, studies using the candidate gene approach to look for genetic factors in NASH have thus far relied on case–control methodology [12]. In this method, the frequency of the allele(s) under study is compared in cases and controls to see whether it is associated with disease. When applying this methodo- logy to studies in NAFLD, phenotype definition in the cases and controls is particularly important because it seems highly likely that different genetic factors will determine the development of steatosis, steatohepat- itis and fibrosis. For studies specifically on NASH, controls should be individuals with steatosis only, ideally matched for body mass index (BMI), age, dia- betes or insulin resistance and ethnic origin to index cases. If appropriate cases and controls can be collected, a number of criteria should be applied in selecting candidate genes worthy of study: 1 The gene product must be considered to have a key role in disease pathogenesis. 2 The polymorphism must be reasonably common, occurring in at least 1 in 20 individuals in the normal ‘background’ population. 3 Ideally, an effect of the polymorphism on gene expression or protein structure and/or function should be established. 4 The function of the gene product and the alteration attributable to the polymorphism should lead to a plausible a priori hypothesis explaining a link between the polymorphism and disease pathogenesis. Once a candidate gene polymorphism has been selected, an adequate number of cases and controls should be recruited to give the study sufficient power to detect a predetermined magnitude of difference in allele frequencies between cases and controls. Fin- ally, whenever possible, plans should be made to seek replication of any significant associations in a distinct set of cases and controls to reduce the risk of reporting spurious or ‘chance’ associations [13]. Novel approaches to candidate gene selection Two novel approaches to identifying candidate genes worthy of study in NAFLD/NASH have recently been described. The first utilizes oligonucleotide microarray (‘chip’) methodology to examine global gene expres- sion in liver biopsies from patients with NAFLD. Two groups have recently presented preliminary data that several genes involved in oxidative stress, lipid metabol- ism and fibrosis are either up- or downregulated in patients with NASH compared to steatosis only [14], or in NASH-related cirrhosis compared to other causes of cirrhosis [15]. Whether these changes in gene expres- sion are a primary or secondary phenomenon is, as yet, unknown. However, these studies have already sug- gested a number of novel candidate genes worthy of subjecting to proximal promoter SNP screening strate- gies. The second approach, which has yet to be applied to NAFLD, is that of phenotype-driven mouse mut- agenesis [16]. In this technique, male mice are treated with the mutagen ethyl nitrosourea and their progeny are screened for dominant mutations giving rise to the phenotypical change of interest. The mutation is then mapped to a specific gene and the human homo- logue is screened for SNPs that are subsequently tested for disease association using standard case–control methodology. Whole genome scanning An alternative to this ‘hypothesis-driven’ methodology based on careful selection of candidate genes, driven by the availability of a comprehensive human SNP map [17], is the possibility of looking for disease asso- ciations in polygenic diseases by performing a genome- wide survey [10]. At present, genome-wide scanning is extremely expensive, but costs may fall in the future as more efficient genotyping technologies are developed and the number of SNPs requiring genotyping falls because of the availability of haplotype maps [18]. Haplotypes are defined by multiple SNPs that co- segregate (are inherited together more often than expected by chance) and are in so-called linkage dis- equilibrium (LD). Recent studies of haplotype structure [...]... 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