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.