correspondence

have a different biologic behavior than adenocarcinomas. The vast majority of patients with gallbladder cancer in the ABC-02 population had adenocarcinomas. Table 1 of our article lists 2 adenosquamous (mixed) tumors and only 1 pure squamous-cell tumor, as compared with 377 adenocarcinomas. This proportion is likely to hold also among those patients classified as having carcinoma, type not specified (29 patients). It is reasonable therefore to conclude that any potentially different behavior of squamous-cell carcinomas is unlikely to have affected the outcome of this study. Hall points out that there was no cost-effectiveness analysis. Quality-of-life data were collect-

ed, and a cost-effectiveness analysis is the subject of ongoing work. The current article relates only to the efficacy and tolerability of the regimen. John Bridgewater, M.D., Ph.D.
University College London Cancer Institute London, United Kingdom j.bridgewater@ucl.ac.uk

Harpreet Wasan, M.D.

Imperial College Health Care Trust London, United Kingdom

Juan Valle, M.D.

Christie Hospital Manchester, United Kingdom Since publication of their article, the authors report no further potential conflict of interest.

Apolipoprotein C3 Gene Variants in Nonalcoholic Fatty Liver Disease

To the Editor: Petersen et al. (March 25 issue)1 report on a relationship between storage of tri glycerides in the liver and two variant alleles of the gene encoding apolipoprotein C3 (APOC3) that increased apolipoprotein C expression. Using proton nuclear magnetic resonance spectroscopy, they detected hepatic steatosis in variant-allele carriers but could not distinguish between nonalcoholic simple steatosis and nonalcoholic steatohepatitis. We compared apolipoprotein C expression with histologic findings in liver specimens obtained from 44 morbidly obese women of European descent and normal histologic findings in specimens obtained from a control group of 5 lean patients. Liver samples were obtained from the morbidly obese women during planned bariatric surgery and from the lean women by means of percutaneous biopsy. All biopsies were performed for clinical indications, and written informed consent was obtained from all patients. Histologic findings in the liver were classified as normal liver, simple steatosis, or steatohepatitis. Real-time polymerase-chain-reaction analysis showed that expression of apolipoprotein C messenger RNA (mRNA) in the liver was similar in the four groups of findings (the control findings and the three types of findings from the morbidly obese patients) (Fig. 1). We did not find any relationship between gene expression and plasma triglyceride concentrations or the insulin-resistance index determined by homeostasis model assessment. We were unable to establish any associations between APOC3 gene expression and types of nonalcoholic fatty liver disease. Cristobal Richart, M.D., Ph.D. Teresa Auguet, M.D., Ph.D. Ximena Terra, Ph.D.
Joan XXIII University Hospital Tarragona, Spain crichart.hj23.ics@gencat.cat

Relative mRNA Expression

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

Control (N=5)

Normal Liver (N=11)

Simple Steatosis (N=13)

NASH (N=20)

Morbidly Obese Women

Figure 1. Expression of Apolipoprotein C3 Messenger RNA in the Liver. No statistically significant differences among the groups were detected. The T bars indicate standard errors. MRNA denotes messenger RNA, and NASH nonalcoholic steatohepatitis.

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The

n e w e ng l a n d j o u r na l

of

m e dic i n e

Supported by a grant (SAF 2008-02278, to Dr. Richart) from the Ministerio de Ciencia e Innovacin of the government of Spain, a grant (PS09/01778, to Dr. Auguet) from the Fondo de Investigacin Sanitaria, and by funds from the Instituto en Salud Carlos III to the Centro de Investigacin Biomdica en Red en Obesidad y Nutricion. No potential conflict of interest relevant to this letter was reported.
1. Petersen KF, Dufour S, Hariri A, et al. Apolipoprotein C3

Silvia Fargion, M.D.

Universit degli Studi di Milano Milan, Italy No potential conflict of interest relevant to this letter was reported.
1. Romeo S, Kozlitina J, Xing C, et al. Genetic variation in

gene variants in nonalcoholic fatty liver disease. N Engl J Med 2010;362:1082-9.

To the Editor: Petersen et al. report an association between the C-428T and T-455C single-nucleotide polymorphisms (SNPs) in APOC3 and the risk of insulin resistance and nonalcoholic fatty liver disease among 258 healthy Indian men, but unfortunately, data on liver damage were not provided. Besides conferring a predisposition to insulin resistance, nonalcoholic fatty liver disease has become the most frequent and potentially progressive liver disease in industrialized countries. An important clinical challenge faced by hepatologists today is to distinguish patients with progressive nonalcoholic steatohepatitis from those with uncomplicated steatosis, and genetic factors may be early noninvasive markers of severe disease. In about 600 patients with nonalcoholic fatty liver disease, we recently observed that the SNP rs738409 in the gene encoding PNPLA3 (patatinlike phospholipase domaincontaining 3), which influences steatosis and levels of alanine amino transferase,1,2 and the SNPs in the genes encoding ectoenzyme nucleotide pyrophosphate phosphodiesterase (ENPP1), K121Q, and IRS-1 G972R , which influence hepatic insulin resistance, were independent predictors of the severity of fi brosis.3,4 It would be interesting to know whether APOC3 SNPs also were associated with levels of liver enzymes and the outcomes of liver-function tests in healthy Indian subjects, and with nonalcoholic steatohepatitis and fibrosis in large series of children and adult patients with nonalcoholic fatty liver disease studied by clinical research networks. Luca Valenti, M.D.
Universit degli Studi di Milano Milan, Italy luca.valenti@unimi.it

PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 2008;40:1461-5. 2. Kotronen A, Peltonen M, Hakkarainen A, et al. Prediction of non-alcoholic fatty liver disease and liver fat using metabolic and genetic factors. Gastroenterology 2009;137:865-72. 3. Valenti L, Al-Serri A, Daly AK, et al. Homozygosity for the patatin-like phospholipase-3/adiponutrin I148M polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease. Hepatology 2010;51:1209-17. 4. Dongiovanni P, Valenti L, Rametta R, et al. Genetic variants regulating insulin receptor signaling are associated with the severity of liver damage in patients with nonalcoholic fatty liver disease. Gut 2010;59:267-73.

To the Editor: Petersen et al. found that liver triglyceride content is associated with APOC3 gene variants in Asian Indian men. In the Discussion section of their article, they argue that their findings are of particular relevance because fatty liver plays an important role in causing hepatic insulin resistance. However, we suggest that although the increase in intrahepatic triglyceride content is directly related to the impairment of insulin action in the liver, skeletal muscle, and adipose tissue in obese subjects,1 it has been shown that fatty liver may exist without hepatic insulin resistance,2 indicating that liver fat is a marker of metabolic dysfunction, not a cause. Thus, considering the limited size of their population, the authors should have performed appropriate measurements of insulin resistance in the main sites of insulin action by using the hyperinsulinemiceuglycemic clamp coupled with tracer infusion.3 Without these data, we believe that it is not possible to draw any conclusion about the possible contribution of APOC3 gene variants to the pathogenesis of hepatic insulin resistance. Federico Salamone, M.D. Fabio Galvano, Ph.D. Giovanni Li Volti, M.D., Ph.D.
University of Catania Catania, Italy federicosalamone@yahoo.it No potential conflict of interest relevant to this letter was reported.
1. Korenblat KM, Fabbrini E, Mohammed BS, Klein S. Liver,

Valerio Nobili, M.D.

Bambin Ges Hospital Rome, Italy

muscle, and adipose tissue insulin action is directly related to intrahepatic triglyceride content in obese subjects. Gastroenterology 2008;134:1369-75. 2. Amaro A, Fabbrini E, Kars M, et al. Dissociation between intrahepatic triglyceride content and insulin resistance in familn engl j med 363;2 nejm.org july 8, 2010

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correspondence
ial hypobetalipoproteinemia. Gastroenterology 2010 March 18 (Epub ahead of print). 3. Bugianesi E, Gastaldelli A, Vanni E, et al. Insulin resistance in non-diabetic patients with non-alcoholic fatty liver disease: sites and mechanisms. Diabetologia 2005;48:634-42.

The Authors Reply: Richart and colleagues found no relationship between APOC3 mRNA expression and triglyceride content in the livers of morbidly obese women. This lack of a finding is not surprising, since hepatic steatosis will develop in most morbidly obese persons independent of any predisposing genetic factors. It is for this reason that we studied lean persons. As we suggest in our article, we believe that the APOC3 variant alleles (C-482T and T-455C) predispose lean persons (body-mass index [the weight in kilograms divided by the square of the height in meters], <25) to the development of nonalcoholic fatty liver disease associated with insulin resistance. We agree with Valenti and colleagues that it would be interesting to know whether the APOC3 variants that we describe also predispose persons Kitt Falk Petersen, M.D. to nonalcoholic steatohepatitis and fibrosis. We Gerald I. Shulman, M.D., Ph.D. are currently performing these analyses. Yale University School of Medicine We also agree with Salamone and colleagues New Haven, CT gerald.shulman@yale.edu that understanding whether fatty liver causes heSince publication of their article, the authors report no furpatic insulin resistance is critical. Our hypothesis ther potential conflict of interest. that increased liver fat plays a causal role in hepatic insulin resistance is supported by our pre- 1. Petersen KF, Oral EA, Dufour S, et al. Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodysvious studies that showed a strong relationship trophy. J Clin Invest 2002;109:1345-50. between liver-fat content and hepatic insulin re- 2. Petersen KF, Dufour S, Befroy D, Lehrke M, Hendler RE, Shulsistance in both humans and animal models of man GI. Reversal of nonalcoholic hepatic steatosis, hepatic insulin resistance, and hyperglycemia by moderate weight reduction nonalcoholic fatty liver disease with the use of in patients with type 2 diabetes. Diabetes 2005;54:603-8. the hyperinsulinemiceuglycemic clamp in com- 3. Savage DB, Petersen KF, Shulman GI. Disordered lipid mebination with glucose tracers.1-3 Furthermore, re- tabolism and the pathogenesis of insulin resistance. Physiol Rev 2007;87:507-20. versal of nonalcoholic fatty liver disease in pa- 4. Samuel VT, Liu ZX, Qu X, et al. Mechanism of hepatic insulin tients with type 2 diabetes by modest weight resistance in non-alcoholic fatty liver disease. J Biol Chem reduction2 and in patients with severe lipodystro- 2004;279:32345-53. 5. Samuel VT, Liu ZX, Wang A, et al. Inhibition of protein kiphy by leptin-replacement therapy1 normalized nase Cepsilon prevents hepatic insulin resistance in nonalcohepatic insulin responsiveness and fasting hy- holic fatty liver disease. J Clin Invest 2007;117:739-45.

perglycemia in these persons. With regard to the molecular mechanism, we have found that hepatic steatosis in rodents fed a diet high in fat is associated with increased hepatocellular diacylglycerol content, resulting in activation of protein kinase C epsilon isoform, reduced insulin signaling, and hepatic insulin resistance.3-5 Finally, we have now completed studies in transgenic mice, which overexpress human apolipoprotein C3, and we found that these mice have increased susceptibility to the development of hepatic steatosis and hepatic insulin resistance. These changes are associated with increased hepatic diacylglycerol content, activation of protein kinase C epsilon isoform, and reduced hepatic insulin signaling. These findings provide strong genetic evidence in support of our hypothesis that the APOC3 variant alleles (C-482T and T-455C) predispose lean persons to nonalcoholic fatty liver disease associated with hepatic insulin resistance.

Individual Genomes on the Horizon

To the Editor: In his editorial on individualized genomics, Lifton (April 1 issue)1 predicts that whole-genome sequencing could eventually be scaled to routine clinical practice, in part because innovation and intense competition will drive down testing costs. Unfortunately, this projection does not account for barriers related to genetic intellectual property. Approximately 20% of human genes are patented,2 and some claims also implicate large numbers of related oligonucleotide sequences3 or refer to genotypephenotype associations. Whole-genome testing would thus infringe on a wide variety of claims. The high cost of obtaining myriad third-party licenses to pro195

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