Detection of T.N.F-α (238 G/A) Polymorphism by Real-time PCR

Abstract:

This laboratory report outlines the methodology for detecting T.N.F-α (238 G/A) gene polymorphism using TaqMan probe SNP assay and real-time PCR. The study also includes the assessment of cardiovascular liability index, anthropometric characteristics, and medical characteristics among the subjects. The analysis was conducted on 50 psoriatic subjects and 50 controls. The results provide insights into the genetic variations associated with psoriasis and its potential correlation with cardiovascular risk factors.

1. Introduction:

Psoriasis is a chronic autoimmune skin disease that has been associated with various genetic factors.

T.N.F-α (238 G/A) gene polymorphism is one such factor of interest. This report presents the methodology for its detection and explores the relationship between genetic variations and cardiovascular risk factors.

2. Materials and Methods:

2.1 DNA Extraction and Purification:

Genomic DNA was extracted from stored samples using DNA purification kits following the manufacturer's protocol. The following steps were involved:

2.2 Detection of T.N.F-α (238 G/A) Polymorphism by Real-time PCR:

The assay is based on the TaqMan probe SNP assay method. It utilizes a probe containing a fluorescent editor dye and a quencher dye to detect genetic variations.

The following steps outline the process:

Principle of the Assay:

The TaqMan probe contains a fluorescent editor dye at the 5' end and a quencher dye at the 3' end. When the probe is intact, fluorescence is inhibited. During PCR, the probe is cleaved, separating the dyes, allowing detection of edited dye fluorescence (F.A.M. for the T allele and V.I.C. for the G allele).

Step-by-Step Process:

An oligonucleotide probe is used with edited fluorescent and quencher dyes. The probe is cleaved during PCR, allowing detection of fluorescence.

Cleavage of Probe:

The probe is cleaved, releasing the edited dye, leading to increased fluorescence for G and A alleles of the T.N.F-α gene.

The amplified products are analyzed using TaqMan probes for G and A alleles, with fluorescence intensities used for detection.

2.3 Cardiovascular Liability Index:

The cardiovascular liability index was determined using various parameters:

• Body Mass Index (BMI)
• Waist Circumference
• Lipid Profile
• Fasting Blood Sugar
• Electrocardiography (ECG)
• Carotid Duplex to measure Carotid Intima-Media Thickness (CAIMT)

2.4 PASI (Psoriasis Area and Severity Index) Score:

The PASI score was calculated to evaluate the severity of psoriasis. It considers the extent of erythema, thickness, and scaling at four anatomic sites and assigns numerical values to these parameters.

Formula:

PASI = (0.1 * (Eh + Ih + Dh) * Ah) + (0.2 * (Eu + Iu + Du) * Au) + (0.3 * (Et + It + Dt) * At) + (0.4 * (El + Il + Dl) * Al)

Where E = erythema, I = infiltration, D = desquamation, A = area, h = head, t = trunk, u = upper extremities, and l = lower extremities.

2.5 Dermatology Life Quality Index (DLQI):

The DLQI was used to assess the impact of psoriasis on the quality of life. It involves a scoring system based on the patient's responses to specific questions.

2.6 Statistical Analysis:

Data were analyzed using SPSS software. Descriptive statistics, t-tests, chi-square tests, and Fisher exact tests were used for data analysis.

3. Results:

The study included 50 psoriatic subjects and 50 controls. Key findings are summarized below:

3.1 Demographics:

The mean age of psoriatic subjects was 39.75 years, with a male-female ratio of 52% to 48%. Family history of psoriasis was present in 12% of cases.

3.2 Anthropometric Characteristics:

Anthropometric characteristics of the psoriatic subjects were as follows:

3.3 Medical Characteristics:

Medical characteristics among psoriatic subjects included carotid duplex measurements, blood pressure, hypertension, diabetes, and ECG results.

4. Discussion:

The results of this study indicate a potential association between T.N.F-α (238 G/A) gene polymorphism and psoriasis. Furthermore, cardiovascular risk factors, including BMI, waist circumference, and blood pressure, were observed among psoriatic subjects. These findings suggest the importance of monitoring cardiovascular health in psoriasis patients.

5. Conclusion:

This laboratory report highlights the methodology for detecting T.N.F-α (238 G/A) gene polymorphism in psoriatic subjects and the assessment of cardiovascular liability index. The study provides valuable insights into the genetic and cardiovascular factors associated with psoriasis, contributing to a better understanding of the disease.