Hemochromatosis gene mutations in patients with alcoholic cirrhosis.

To the Editor: Oxidative stress, which can be induced by iron overload, may play a pivotal role in the pathogenesis of alcoholic liver disease (ALD) which is characterized by fatty liver, hepatitis, fibrosis, cirrhosis and hepatocellular carcinoma (1). Increasing evidence indicates that ALD is a multifactorial disease in which both environmental and multiple genetic factors play a role. The hemochromatosis (HFE) protein is involved in iron metabolism and the synergistic effect between iron and alcohol is suggested in the progression of alcoholic cirrhosis (2). Therefore, it has been speculated that homoor heterozygous mutations in the HFE gene which increase serum iron levels might contribute to the pathological process. There have been five published reports investigating the relationship between HFE gene mutations and alcoholic cirrhosis in white populations and all of them have failed to detect any association between the C282Y mutation and susceptibility to ALD (3–7). On the other hand, the H63D mutation was investigated in only three studies, with contradictory results (5– 7). A control group of alcoholics without liver disease has not been included in most of the studies cited. The effect of the third common mutation, S65C, on disease susceptibility has not been investigated. The aim of our study was to investigate the contribution of these three HFE genetic polymorphisms to susceptibility for ALD in a sample of Croatian and Slovenian patients. The study included 147 patients with alcoholic cirrhosis and two control groups: 66 alcoholics without cirrhosis and 350 healthy blood donors. The alcoholic groups were well matched for age, sex, origin, and alcohol intake (all consumed more than 80 g of ethanol per day for 5 or more years). There were no significant differences in the sex ratio and age between the patients and controls (p . 0.05). Patients were recruited from the internal clinics of University Medical Centers in Rijeka (Croatia) and Ljubljana (Slovenia). The diagnosis of cirrhosis was based on the clinical, biochemical, and ultrasonographic features. These patients had severe liver disease: all had decompensated, with mean prothrombin time prolongation of 4.5 s on presentation, increasing the risk of liver biopsy. Patients with other causes of cirrhosis and also patients with anemias with ineffective erythropoeisis and hemolysis were excluded by appropriate serologic and biochemical investigations. Alcoholics without cirrhosis had no clinical signs of cirrhosis and had normal plasma levels of bilirubin, albumin, aspartate aminotransferase, and alanine aminotransferase and normal prothrombin time. On ultrasound examination, their liver was normal. They were attending alcoholic support groups in Rijeka and Ljubljana. Healthy control subjects were clinically unaffected individuals (without liver disease or anemia). The study design was approved by the institutional ethics commitees of both centers and informed consent was obtained from all subjects. Genotyping of HFE gene mutations was performed by the polymerase chain reaction/ restriction fragment length polymorphism method as described previously (8, 9). Differences in the frequencies of various alleles between patients with alcoholic cirrhosis and control subjects were performed using the chi-square test and Fisher’s exact test. Table 1 shows the distribution of HFE genotypes and allele frequencies in ALD patients and in both control groups. There were no significant differences in the frequencies of the C282Y and S65C mutations between the ALD patients and control groups. On the other hand, the frequency of H63D heterozygotes was significantly higher (p 1⁄4 0.0019) in ALD patients (31.98%) than in healthy controls (19.14%). The frequency of H63D heterozygotes was also higher in ALD patients (31.98%) than in alcoholics without cirrhosis (22.73%) but failed to reach statistical significance possibly due to the limited number of alcoholics without cirrhosis. There were no