Materials Methods
The study was performed at University Hospital of Saint Etienne in St. Etienne, France. This study included 44 patients with DS, 23 women (52.3%) and 21 men (47.7%) divided into two groups. All patients were over 7 years of age (range: 8-57 years), the age at which IgG4 reaches adult levels [13]. The first group comprised 23 patients with DS (11 males and 12 females) carrying severe low serum IgG4 (< 0.02 g /L), with 5 having an IgG4 level of 0 g/L and 21 DS subjects (10 males and 11 females) having normal serum level of IgG4 (level > 0.1 g/L). The patient group was compared with 38 healthy donors (controls) without DS. The IgG4 levels were analyzed in serum using Optilite (The Binding Site Group Ltd., 8 Calthorpe Road, Edgbaston, Birmingham, B15 1QT, UK) according to the manufacturer instructions and were measured in the Immunology Laboratory at the Hospital University of Saint-Etienne. Quantitative real-time polymerase chain reaction (qPCR) was carried out using The Step One Plus system (APPLIED Biosystems, Carlsbad, CA, version 2.3) to measure the number ofIGHG4 copies present and compare that number to a reference gene (36B4 ). The process was completed in 14.5μL mixtures containing Taq polymerase, buffer solution, SYBR Green (QuantiTect SYBR® Green PCR Kits, QIAGEN), and 5μL of extracted DNA 2ng/µl from whole blood samples with specific primers (IGHG4 -FOR,5’-aatcttctctctgcagagtccaaatatg-3’andIGHG4 -REV,5’gttggcttacctgggcatga-3’), which were designed to amplify IGHG4 gene exon2.
Because the structure of IGHG4 is homologous with immunoglobulin heavy constant G2 (IGHG2 ), we included this gene in this study using primers IGHG2 FOR,5’-caaatgttgtgtcgagtgccca-3’ andIGHG2 -REV, 5’-aagactgacggtcctgccac-3’
And tested other different structure in immunoglobin like immunoglobulin heavy constant E using primers(IGHE )IGHE -FOR,5’-gctgcaaaaacattccctcca-3’andIGHE -REV,5’gtggtggctggtaaggtcata-3’ as well as [14]. The reaction was performed in two steps, programmed for 45 cycles with primer hybridization at 60°C. The first step consisted of using primers of a reference gene, for which the number of copies is known. The second step consists of using the primers of the gene of interest,IGHG4 , for which the number of copies is desired. AnIGHG4/36B4 ratio was considered normal (two copies ofIGHG4 ) when the resulting values were between 0.8 and 1.2. To maintain the precision of the technique, electrical pipettes (Xplorer Eppendorf) were used, all DNA assays were carried out on the same day, and the same DNA dilutions were used for the 2 plates (36B4 andIGHG4) .
The data were analyzed with the Statistical Package for Social Studies (SPSS; IBM, Year). Continuous variables were expressed as means and standard deviations. Categorical variables were expressed as percentages. A set of t tests were used for continuous variables that exhibited normal distributions. Mann-Whitney U tests were used for nonparametric variables. The chi-square test and the Fisher exact test were used for categorical variables. The Shapiro-Wilk test was used to assess the normality of the data. A P value < 0.05 was considered statistically significant.