Identification of TNIP1 Polymorphisms by High Resolution Melting Analysis with Unlabelled Probe: Association with Systemic Lupus Erythematosus

Abstract

Background. TNFα-induced protein 3 (TNFAIP3) interacting with protein 1 (TNIP1) acts as a negative regulator of NF-κB and plays an important role in maintaining the homeostasis of immune system. A recent genome-wide association study (GWAS) showed that the polymorphism of TNIP1 was associated with the disease risk of SLE in Caucasian. In this study, we investigated whether the association of TNIP1 with SLE was replicated in Chinese population. Methods. The association of TNIP1 SNP rs7708392 (G/C) was determined by high resolution melting (HRM) analysis with unlabeled probe in 285 SLE patients and 336 healthy controls. Results. A new SNP rs79937737 located on 5 bp upstream of rs7708392 was discovered during the HRM analysis. No association of rs7708392 or rs79937737 with the disease risk of SLE was found. Furthermore, rs7708392 and rs79937737 were in weak linkage disequilibrium (LD). Hypotypes analysis of the two SNPs also showed no association with SLE in Chinese population. Conclusions. High resolution melting analysis with unlabeled probes proves to be a powerful and efficient genotyping method for identifying and screening SNPs. No association of rs7708392 or rs79937737 with the disease risk of SLE was observed in Chinese population.

Figure 1

SNP genotyping by HRM with unlabeled probe. (a) Derivative melting curves of unlabeled probe and amplicon for genotyping of SNP rs7708392. (b) Normalized difference curves of unlabeled probe region. (c) Normalized melting curves of unlabeled probe region. Unlike the classical SNP genotyping (wildtype, heterozygote, and homozygote), six types of curves were observed implying a new SNP also presented in probe region. (d) DNA sequencing result of SNP rs7708392. The sequencing was performed by using reverse primer of the PCR amplicon. The blue box indicates the SNP rs7708392 harbors G/C mutation by reverse sequencing. The red box shows that the polymorphism on the new SNP is C/T by reverse sequencing.

Figure 2

SNP genotyping by HRM with specific probes targeting each SNP, respectively. (a) The location of probes and PCR primers. Probe 1 targets the new-discovered SNP. Probe 2 targets rs7708392. The probe containing both SNPs is also shown (b)–(g). The derivative melting curve, normalized difference curve, and normalized melting curve for each genotyping assay by using probe 1 and probe 2 are shown as indicated.