Pitfalls in LightCycler diagnosis of the single-nucleotide polymorphism 13.9 kb upstream of the lactase gene that is associated with adult-type hypolactasia.

BACKGROUND: Patients presenting with symptoms of lactose intolerance are in some centres routinely tested for a single-nucleotide polymorphism C-13910T, which is located upstream of the lactase gene (LCT) and is tightly associated with genetically determined lactase persistence/non-persistence. Typing of this polymorphism enables differential diagnosis for genetic versus secondary causes of lactose intolerance. Several PCR-based methods have been established as tests for this SNP. In particular, automated genotyping assays conducted on LightCycler platforms provide a rapid, labour-saving means for routine high-throughput analysis of this variant. Recently, several novel allelic variants have been identified in non-European populations. Three of these variants occur in close proximity to C-13910T, but their effect on the genetic test is unknown. METHODS: Here we analyse whether the occurrence of C-13907G, T-13913C, and T-13915G, affect the diagnostic accuracy of C-13910T typings obtained using the LightCycler MutaREAL lactase real-time PCR kit. RESULTS: Genotyping of DNA samples harbouring respective variants or combinations thereof significantly influenced the LightCycler analysis. Some allelic combinations generated melting profiles that prevented the correct assignment of C-13910T. CONCLUSIONS: We conclude that genotyping of the C-13910T variant with the MutaREAL lactase real-time PCR kit in non-Europeans is prone to error and should be omitted.

Transforming growth factor-beta gene polymorphisms in sarcoidosis patients with and without fibrosis.

STUDY OBJECTIVES: Pulmonary fibrosis develops in approximately 25% of patients with chronic sarcoidosis. Transforming growth factor (TGF)-beta1 plays a central role in fibrosis, and accruing reports address the implication of TGF-beta2 and TGF-beta3 in this process. We determined whether single-nucleotide polymorphisms (SNPs) in the TGF-beta1, TGF-beta2, and TGF-beta3 genes might contribute to pulmonary fibrosis in sarcoidosis patients. SETTING: A hospital in the Netherlands. DESIGN: Five SNPs per TGF-beta gene were investigated. PATIENTS AND CONTROL SUBJECTS: Patients with either acute/self-remitting sarcoidosis (n = 50) and Löfgren syndrome (n = 46) or chronic disease with fibrosis (n = 24) and without fibrosis (n = 34) were assessed over a 4-year follow-up period. The control subjects included 315 individuals. MEASUREMENTS AND RESULTS: Polymorphism frequencies were not discordant between the patients and control subjects. The TGF-beta3 4875 A allele was significantly higher in fibrotic patients (carrier frequency, 0.29) than in patients with acute/self-remitting (0.06) and chronic (0.03) sarcoidosis combined (corrected p = 0.01; odds ratio [OR], 7.9). The TGF-beta3 17369 C allele carrier frequency was significantly higher in fibrotic patients (0.29) compared to acute/self-remitting (0.08) and chronic (0.06) patients combined (corrected p = 0.05; OR, 5.1). Although not significant after correction, the TGF-beta3 15101 G allele carrier frequency was lower in fibrotic patients (0.79) compared to acute/self-remitting (0.94) and chronic (1.00) patients combined (p = 0.02; corrected p = 0.1; OR, 0.15). The TGF-beta2 59941 G allele was more abundant in fibrotic patients (carrier frequency, 0.62) compared to patients with acute/self-remitting (0.41) and chronic sarcoidosis combined (0.28) [p = 0.04; corrected p = 0.2; OR, 2.9]. TGF-beta1 gene polymorphisms were not associated with fibrosis. CONCLUSIONS: This study is the first to suggest the implication of genetic variation of TGF-beta3 in the predilection for pulmonary fibrosis developing in sarcoidosis patients.

Transforming growth factor-beta1 gene polymorphisms are associated with progression of liver fibrosis in Caucasians with chronic hepatitis C infection.

AIM: Considerable attention is focused on polymorphisms in the gene encoding transforming growth factor-beta1 (TGF-beta1), a multifunctional cytokine that is in turn a potent growth inhibitor involved in wound healing and differentiation. In humans, it promotes the pathogenesis of organ fibrosis, atherosclerosis, cancer, autoimmune and inflammatory diseases, keloid disease, and hypertrophic scarring. For this reason, much emphasis has been placed on studies elucidating the impact of TGF-beta1 and its gene variations for the susceptibility and pathogenesis of these diseases. Unfortunately, some studies have serious limitations. METHODS: We have recently described a high-throughput method for investigation the Arg25Pro polymorphism of human TGF-beta1 gene and showed that the frequency of the Pro25 allele is significantly associated with hepatic fibrogenesis. In this report, we describe two novel LightCycler (LC) techniques that facilitate the examination of the two other known alterations in the coding region of TGF-beta1. We investigated whether these polymorphisms contribute to hepatitis-induced progression of fibrogenesis in Chinese and Caucasians. RESULTS: In the Chinese ancestry, the gene polymorphisms at codons 25 and 263 were not found and the genetic variant at codon 10 is unlikely to confer susceptibility to hepatic fibrosis. Contrarily, in Caucasians TGF-beta1 allelic variations are more frequent and the presence of prolines either in codon 25 or 10 is associated with the interindividual variability in developing more severe fibrosis during chronic hepatitis C infection. CONCLUSION: In summary, these results confirm the hypothesis that TGF-beta1 polymorphisms are associated with fibrosis progression in Caucasians chronically infected with hepatitis C.

Analysis of polymorphic TGFB1 codons 10, 25, and 263 in a German patient group with non-syndromic cleft lip, alveolus, and palate compared with healthy adults.

BACKGROUND: Clefts of the lip, alveolus, and palate (CLPs) rank among the most frequent and significant congenital malformations. Leu10Pro and Arg25Pro polymorphisms in the precursor region and Thr263Ile polymorphism in the prodomain of the transforming growth factor beta1 (TGF-beta1) gene have proved to be crucial to predisposition of several disorders. METHODS: In this study, polymorphism analysis was performed by real-time polymerase chain reaction (LightCycler) and TGF-beta1 levels determined by enzyme-linked immunosorbent assay. RESULTS: Only 2/60 Caucasian non-syndromic patients with CLP (3.3%) carried the Arg25Pro and another 2/60 patients (3.3%) the Thr263Ile genotypes, whereas, in a control group of 60 healthy Caucasian blood donors, these heterozygous genotypes were more frequent 16.7% having Arg25Pro (10/60; p < 0.035) and 10,0% having Thr263Ile (6/60), respectively. TGF-beta1 levels in platelet-poor plasma of heterozygous Arg25Pro individuals were lower than those of homozygous members (Arg25Arg) in the latter group, but this discrepancy narrowly failed to be significant. Although polymorphisms in codon 10 and 25 were associated with each other, no difference was found between patients and controls concerning the Leu10Pro polymorphism. CONCLUSIONS: The genetic differences in codons 25 and 263 suggest that TGF-beta1 could play an important role in occurrence of CLP, however, functional experiments will be required to confirm the mechanisms of disturbed development.

Analysis of the transforming growth factor-beta1 (TGF-beta1) codon 25 gene polymorphism by LightCycler-analysis in patients with chronic hepatitis C infection.

The polymorphism at position 25 of the gene encoding transforming growth factor-beta1 (TGF-beta1), which changes the amino acid sequence of the signal peptide sequence (arginine to proline), is causing a variation in TGF-beta1 production. The homozygous genotype (Arg25Arg) is associated with higher TGF-beta1 production than the heterozygous (Arg25Pro) genotype. Therefore, the possible involvement of this genetic variation in the TGF-beta1 gene for induction and progression of various diseases is under close investigation. At present, several labor-intensive established assays ranging from amplification refractory mutation system (ARMS)-PCR methodologies, sequence specific oligonucleotide probing (SSOP), restriction fragment length polymorphism (RFLP) analysis, 5' nuclease assays, and specialized fingerprinting protocols are applied to analyze the polymorphism in question. We developed a novel approach for analyzing this polymorphism in a LightCycler system and determined the allele frequency distributions between patients with different degrees of hepatic fibrosis induced by chronic hepatitis C virus infection. In patients with severe hepatic fibrosis (METAVIR-score 3-4), the Pro25 allele was twice as frequent compared to patients with mild fibrosis (METAVIR-score 0-2). However, we found no association of necroinflammatory activity and genotype distribution. This suggests that the stage of hepatic fibrosis, rather than the grade (inflammation), is influenced by the presence of proline at codon 25 in patients with chronic hepatitis C.