This case-control study was conducted on 85 subjects: 55 patients with AA who were selected during the period from January 2018 to December 2018 in addition to 30 age- and sex-matched healthy controls.
Exclusion Criteria
Any patient with any dermatological disease other than AA, patients with other autoimmune or inflammatory diseases, and patients with recent infectious conditions were excluded.
All cases included were subjected to the following:
A complete history of the enrolled patients was taken and recorded, and they were subjected to general and dermatological examination. Clinical data regarding onset, course, duration of disease, and family of alopecia areata were collected.
Assessment of Disease Severity
- According to Kavak et al. [7]., patients were divided into three subgroups:
A1: mild, existence of three or less patches of alopecia with the largest diameter being of 3 cm or less or the disease limited to the eyelashes and eyebrows
A2: moderate, existence of more than three patches of alopecia or a patch larger than 3 cm at the largest diameter but not alopecia totalis or alopecia universalis
A3: severe, alopecia totalis, or alopecia universalis
- The Severity of Alopecia Tool (SALT) score set by the National Alopecia Areata Foundation Working Committee [8] was applied for all cases. Scalps were divided into four areas: the vertex 40%, the right side of scalp 18%, the left side of scalp 18%, and the posterior aspect of scalp 24%.
3 mm scalp punch biopsies, were taken from sites where the disease was active. Scalp biopsy specimens from healthy volunteers were included as controls.
All subjects included underwent real-time PCR estimation of ULPB3 mRNA expression levels in scalp tissue.
Sample Preparation
Scalp tissue samples were homogenized in RNeasy Lysis Buffer (RLT lysis buffer) (Qiagen RN ease mini kit, Helden, Germany), RNA extraction was done using Qiagen RNA easy mini kit (Helden, Germany) and stored at – 80°C till analysis time.
Assessing RNA quality and purity was determined by measurement of the absorbance at 260 nm (A260). Absorbance reading should be greater than 0.18 to ensure significance. Ratio greater than 1.6 was accepted (A260/A280). If the purity was lower than 1.5, it re-extraction was done. Total RNA concentration was determined using NanoDrop 2000 UV-Vis spectrophotometer (Thermofisher, Scientific Inc, USA). For cDNA synthesis, the extracted RNA sample was briefly incubated in a genomic DNA wipeout buffer for elimination of genomic DNA then, RNA extract was mixed with QuantiTect Reverse Transcription Kit, including Quantiscript Reverse Transcriptase, Quantiscript RT Buffer, and a unique RT Primer, incubated for 1h at 42°C to achieve reverse transcription then 5 min at 95°C to stop reverse transcription then 5 min at 4°C.
The mRNA level of ULBP3 and Glyceral-3 Phosphate Dehydrogenase (GAPDH) (as an endogenous control), were assessed by cDNA amplification using real-time PCR and Quanti Tect SYBR Green PCR kit with ready-made QuantiTect Primer Assay, Qiagen, Applied Biosystems, USA [9], cycling conditions were as follow: 95°C for 15 min 45 repeated cycles at 94°C for 15s, 55°C for 30s, 72°C for 30s , then 72°C for 10 min for final extension.
For relative quantitation of the results, the Cycle Threshold (the point at which PCR product is first detected above a fixed threshold) was determined for each sample [10]. Each run was completed using a melting curve analysis to confirm the specificity and absence of primer dimers. The analysis was done using applied Biosystem 7500, software version 2.0.1. GAPDH was included to monitor RT-PCR efficiency for all samples.
Statistical Analysis
Statistical analysis was conducted using a personal computer with Statistical Package of Social Science (SPSS) version 20 for windows (SPSS Inc., Chicago, o, Illinois, USA). P ≤ 0.05 is considered statistically significant. Descriptive statistics were conducted, in which quantitative data were presented in the form of Mean (X), Standard Deviation (SD), and range and were presented in the form of Numbers (N) and Percentages (%); analytical statistics were also conducted, including Mann–Whitney U test (U) and Spearman's correlation coefficient (r).