Tumor necrosis factor gene polymorphisms in adult patients with autoimmune hemolytic anemia

Sir, Autoimmune hemolytic anemia (AIHA) is defined as increased destruction of red blood cells (RBC) due to autoantibodies specific for RBC autoantigens. AIHA may occur as primary (idiopathic) or secondary to other lymphoproliferative diseases like chronic lymphocytic leukemia (CLL) or autoimmune diseases. The etiology of AIHA is unknown, but both genetic and environmental factors are thought to play role in the development of the disease. Tumor necrosis factors (TNF) are multifactorial cytokines that have potent proinflammatory effects, and are implicated in many inflammatory and autoimmune diseases, such as inflammatory bowel disease [1], rheumatoid arthritis [2], systemic lupus erythematosus [3], autoimmune thrombocytopenia, hemolytic anemia [4–6], and others. Single nucleotide polymorphisms (SNP) in the genes for tumor necrosis factor beta (TNFB+252G/A) and tumor necrosis factor alpha (TNFA-308G/A) have been associated with increased TNF production and interleukin-6 secretion [7,8]. Both genes are localized between HLA-B and HLA class III genes and that has raised the suspicion that TNF loci might play a role in the notable association between HLA and various autoimmune diseases [9]. The aim of our study was to investigate a possible association of two SNP in the genes for tumor necrosis factor beta (TNFB+252G/A) and tumor necrosis factor alpha (TNFA-308G/A) with primary and secondary AIHA. We have analyzed 80 adult patients with AIHA (50 patients with idiopathic AIHA and 30 patients with secondary AIHA and CLL) and 100 CLL patients without AIHA. Controls were 120 healthy, ageand sex-matched, control individuals. Median age of the patients was 63.6 10.8 years, 62% were men, and 38% were women. Diagnosis of AIHA and CLL was established on the bases of standard clinical and laboratory parameters. Diagnosis of AIHA was based on positive direct antiglobulin test (DAT) and laboratory signs of hemolysis (lower hemoglobin level and elevated bilirubin and reticulocyte count) [10]. Diagnosis of CLL was based on elevated lymphocyte count (>5 9 10/L) and flow cytometry analysis of lymphocytes confirming specific CLL immunophenotype according to WHO classification [11]. Informed written consent for genetic studies was obtained from all patients and controls. DNA was extracted from peripheral blood mononuclear cells with standard phenol–chloroform extraction. Genotyping was performed using PCR and restriction fragment length polymorphism (RFLP) method [7]. Specific primers and enzymes used for PCR-RFLP for TNFA-308G/A were as follows: (50-CAA AAG AAA TGG AGG CAA TAG GTT TTG AGG GCC AT-30 and 50-AGG GCG GGG AAA GAA TCA TTC AAC CAG CGG AAA AC-30; restriction enzyme NcoI) and for TNFB+252G/A:(50-CCG TGC TTC GTG TTT GGA CTA-30 and 50-AGA GGG GTG GAT GCT TGG GTT-30; restriction enzyme NcoI). Distribution of genotypes and allele frequencies were compared by chi-squared test using Microsoft Excel software. P < 0.05 was considered significant. Our results demonstrated significantly different distribution of the TNFB+252G/A genotypes among the patients with AIHA (n = 80; G/G = 18, A/G = 31, A/A = 31) compared with controls (n = 120; G/G = 16, A/G = 35, A/A = 69), P = 0.029 (Table 1). This difference was even more significant when only CLL patients with and without AIHA were compared, P = 0.018. We also found significantly different distribution of the TNFA308G/A genotypes in patients with AIHA (n = 80; G/G = 45, A/G = 28, A/A = 7) and controls (n = 120; G/G = 95, A/G = 23, A/A = 2), P = 0.002. This difference was most striking when we compared only CLL patients with and without AIHA, P = 0.0006. Our results demonstrated that the G allele of the TNFB+252G/A was more frequent among the patients with AIHA compared with controls (41.8% vs. 27.9%), P = 0.005 (Table 2). This difference was also significant when we compared only CLL patients with AIHA vs. CLL without AIHA, P = 0.003. We found that the A allele of the TNFA-308G/A was more common in patients with AIHA than in controls (26% vs. 11%), P = 0.0002. This