This case control study was performed at Ege University Pediatric Rheumatology Department between 2019 and 2022. All procedures were conducted according to the principles of the Declaration of Helsinki and human and animal rights. This study was approved by the Ege University Medical School Hospital Ethics Committee (reference no 23-5.1T/39). Written, informed consent was obtained from the presented patients and their parents to participate in the study.
A total of 53 newly diagnosed IgAV patients and 50 healthy controls who were younger than 19 years old were enrolled. The diagnosis of IgAV was based on 2008 EULAR/PRINTO/PRES criteria as the presence of palpabl purpura with lower limb predominance associated with at least one of the following criteria: diffuse abdominal pain, arthritis or arthralgia, renal involvement (hematuria, proteinuria), or biopsy with predominant IgA deposition [1]. Arthritis was described with inflammatory joint involvement (swelling or pain with limitation of motion). Gastro intestinal (GI) involvement was defined if there is an existence of abdominal pain, bowel intussusception or bleeding as hematochezia, melena or occult blood in stool. Renal manifestations were charecterized by hematuria (≥5 red blood cells/hpf) and/or proteinuria (24 hours urinary protein >4 mg/m2/h), with nephrotic range (≥40 mg/m2/h,) or non-nephrotic range (4 - 40 mg/m2/h). Severity criteria for IgAVN was based on SHARE recommendation which was defined for mild nephritis (normal glomerular filtration rate (GFR) and mild proteinuria), and for moderate nephritis (with <50% crescents on renal biopsy and impaired GFR or severe persistent proteinuria) and for severe nephritis (with >50% crescents on renal biopsy and impaired GFR or severe persistent proteinuria) [24].
All of the patients were diagnosed as IgAV for the first time and followed-up in the out-patient clinic at least one year after the initial diagnosis. The exclusion criteria were thrombocytopenia (platelet number <100,000/mm3) and being diagnosed with any systemic or autoimmune disease. In addition, 50 healthy controls were selected while routine health checkup in out-patient clinic who do not have any systemic disease or history of skin rash. Patients were evaluated through extensive medical history including demographic data and clinical characteristics. The demographical and clinical data of study group are listed in Table-1.
Baseline laboratory studies such as complete blood cell count (CBC), serum immunoglobulins G-A-M (by nephelometry), erythrocyte sedimentation rate (ESR), C- reactive protein (CRP), serum amyloid-A protein (SAA), serum complement components C3 and C4, MEFV gene sequence analysis, fecal occult blood and microscopic urine analyse and protein/creatinin ratio in spot urine were performed on the same day at admission. In addition, one more blood sample was collected in EDTA-containing tube for each patient and healthy control and immediately centrifuged (10 min at 2000xg) and stored at 4 °C until genetic analysis. To assess possible infectious triggers, we analysed the throat swab culture, antistreptolysin O (ASO) titre for upper respiratory tract infections and also we asked for diarrhea to learn if the patient had acute gastroenteritis before IgAV attack. ASO titres of 200 Todd units or more were considered as positive according to the manufacturer’s manual. Skin biopsy was performed when the patients presented with atypical symptoms, for excluding other forms of vasculitis. If the patients suffered from the persistent or nephrotic range proteinuria, renal biopsy was performed.
Patients and healthy controls were genotyped for the PTPN22 +788 G>A (rs33996649), TGF-β-509C>T (rs18004069), IL-1β-511C>T (rs16944), IL-5-746C/T (rs2069812), ACE I/D (rs4646994) polymorphisms to understand genotype frequencies and we evaluated if there is an association between them and IgAV predisposition. Furthermore, in order to examine even if the selected SNPs might influence the different clinical manifestations of the disease, patients were examined also in subgroups according to presence or absence of nephritis and arthritis, and GI involvement. In addition, patients were divided into two different age onset disease groups as 2-10 years and older than 10 years and they were also compared for severity, renal involvement and gene polymorphisms.
We used five different genetic model for comparing analyses based on a major allele (A) and a minor allele (a); “Genotype” (AA versus Aa versus aa), “Allele” (A versus a), “Dominant” (AA versus Aa + aa), “Recessive” (AA + Aa versus aa) and “over-dominant” (Aa versus AA + aa) models [25]. Subsequently, we investigated IL-1β, IL-5 and TGF-β mRNA expression levels in order to learn if they were affected by related polymorphisms.
Genotyping: Molecular analysis was carried out on genomic DNA extracted from Ethylenediaminetetraacetic acid anticoagulated venous blood using QiAamp DNA Blood Mini kit (QIAGEN GmbH, Hilden, Germany). For all genes primers, designed Exon Primer-Primer Design program obtained from the Helmholz Institute of Human Genetics (Munich, Germany) were used. All synthetic oligonucleotide primers synthesized and purchased by Invitrogen Ltd (Invitrogen, Carlsbad, CA) as the high-performance liquid chromatography purification grade. PCR amplification was carried out on Veriti Gradient Thermal Cycler in a 25-lL reaction mixture in 0.2 mL thin-wall PCR strip tubes (Axygen Scientific, Inc, Union City, CA) containing 1 lL genomic DNA solution, 1.0 U Platinum TAQ with Enhancer Buffer (Invitrogen Ltd) 50 lmol/L each of the dGTP, dATP, dTTP and dCTP (Promega, Madison, WI), 5 pmol each forward and reverse primers. The cycling conditions comprised a hot start at 95C for 10 min, followed by 35 amplification cycles at gradient program.
mRNA isolation and measurement method; Peripherial venous blood with EDTA anticoagulant obtained from patients has stored in the Ambion RNAlater Stabilization solution (Thermo Fisher Scientific Waltham, MA USA). mRNA was extracted using TRIzol-Chloroform (Thermo Fisher Scientific, USA) treatment followed by Invitrogen PureLink RNA Mini (Thermo Fisher Scientific, USA) by isolation procedure.
The purity of RNA was determined using Qubit® 2.0 Fluorometer with dsDNA HS Assay Kit for Qubit (Life Technologies, USA). After isolation and quantification, cDNA synthesis was performed immediately. The RNA was reverse transcribed into complementary DNA (cDNA) using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA). The reaction was performed with amplification parameters of 10 min. at 25°C, 120 min. at 37°C, 5 min. at 85°C, ∞ at +4°C on Thermal Cycler with 1-cycle reaction program. The cDNA concentration was measured with NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, USA). Real-Time PCR was performed to observe the changes in the mRNA expression of target genes between the groups expressed as the patient group and healthy control. ACTB gene was used as the control house -keeping gene.
Real-Time PCR reactions were carried out with ABI PRISM™ 7000 Sequence Detection Systems (Thermo Fisher Scientific, USA). The reaction was performed with total reaction volume of 25 μl with 12.5 μl Taqman 2X PCR Master Mix, two separate gene specific 2 μl forward, 2 μl reverse primers, 0.5 μl probe set, 5-7 μl (avg. 4ng/µl) of cDNA templates and 1-3 μl of dH2O. Standard Real-Time PCR program was applied.
The results were calculated with the comparative CT (2-∆∆CT) method.
Statistical analysis; Data were statistically processed and analyzed by SPSS program version 25.0. Kolmogorov Smirnov and Shapiro Wilk tests were used to determine normal distribution. Continuous variables were defined as the mean ± standard deviation (SD) and median with interquartile ranges (IQR) (IQR;75th-25th percentiles values) and categorical variables were defined by number and percent. For independent group comparisons, we used independent samples t-test or One-Way ANOVA when parametric test assumptions were met. The Mann-Whitney U test and Kruskal Wallis Variance Analysis were used when parametric test assumptions were not met. The genotypes of PTPN22 rs33996649, TGF-β rs18004069, IL-1β rs16944, IL-5 rs2069812, ACE rs4646994 were examined for deviation from the Hardy–Weinberg equilibrium (HWE). All SNPs were evaluated independently, while the wild-type genotype was accepted as reference. In addition to genotype, alternative genetic models (dominant, recessive and over-dominant) were compared with chi-square test between the patients and healthy controls as well as patient subgroups. Strength of association was estimated using odds ratios (OR) and 95 % confidence intervals (CI) using logistic regression. P values lower than 0.05 were considered statistically significant.