A total of unrelated 938 northern Chinese were involved in this study, including 661 patients with IgAN proved by renal biopsy, and 277 geography-matched healthy controls with normal urine analysis and blood pressure. Patients with Henoch-schonlein purpura, systemic lupus erythematosus, and chronic hepatic diseases were excluded by detailed clinical and laboratory examinations. The mean ages were 31.2 ± 11.4 (patients) and 28.9 ± 8.2 (the controls) years. There were 82 female subjects in the control group and 280 female subjects in the IgAN patient group. Twenty-two individuals (15 patients who were recently diagnosed as primary IgAN and 7 controls) were selected for somatic mutation detection.
Clinical data of patients with IgAN, including age, course of kidney disease before renal biopsy, as well as blood pressure and the level of urine protein excretion at the time of renal biopsy, were collected. At the same time, the renal function was evaluated, including serum creatinine and estimated glomerular filtration rate (eGFR) calculated by the Modification of Diet in Renal Disease abbreviated equation .
The protocol for this study was approved by medical ethics committee of Peking University, and informed written consents for the study were obtained from all participants.
SNPs Discovery and Genotyping
Genomic DNA of subjects was extracted from the EDTA-anticoagulated whole blood samples by salting out procedure . The C1GALT1C1 gene was located in the chromosome X. Reference sequence of C1GALT1C1 gene (OMIM*300611, Version: NC_000023.9) was obtained from National Center for Biotechnology Information (NCBI) Gene database http://www.ncbi.nlm.nih.gov/entrez. Fifty-eight alleles from chromosome X were screened in all of the 46 individuals, including 27 unrelated patients with IgAN (6 females) and 19 unrelated healthy controls (6 females). The polymerase chain reaction (PCR) amplification regions included 5' untranslated regions and the upstream 1 kb from transcriptional initiation site. PCR primers were designed by Primer3 program . Target sequences were amplified by PCR from 50 ng genomic DNA in 20 μl of final reaction volume. DNA sequencing was performed on an ABI PRISM 3700 automated sequencer. The results of sequencing were analyzed by Phred/Phrap/Consed suite of software . The SNPs were described according to the Human Genome Variation Society (HGVS) nomenclature guidelines .
One single nucleotide polymorphism (SNP), c.-347-190G>A (rs3810744), was detected in the promoter region of the C1GALT1C1 gene. The MAF of c.-347-190G>A (rs3810744) was 48.48%. So the SNP was genotyped for further association analysis in all 938 subjects by the standard PCR-restriction fragment length polymorphism procedures. The genomic DNA samples were amplified by PCR using the following primers, forward: 5'-ACGCAGGGGTACATCAGAGAA-3', reverse: 5'-TGACCAGGCTGTTCTAGCTG-3'. The products of 420 base pairs (bps) were digested by restriction endonuclease Hpy8I (Fermentas International Inc., Hanover, USA). The genotypes weren't detected in three controls and one patient. Forty PCR products were sequenced for accuracy confirmation of PCR-restriction fragment length polymorphism analysis.
B lymphocyte DNA extraction and PCR amplification
Peripheral blood B lymphocytes from 22 participants (15 patients and 7 controls) were isolated by using lymphocyte isolation sterile solution (Amersham Biosciences, Uppsala, Sweden) and CD19 magnetic beads (Dynal Biotech ASA, OSLO, Norway), and then DNA of B lymphocytes was extracted by salting out procedure . The whole coding region was amplified by PCR with following primers, forward: 5'-GTTGTTGCAAAGTTCCAGTTTA-3', reverse: 5'-TTATACCAGTGCCACCAAATTA-3'. The length of PCR production was 1314 bps. B lymphocyte DNA (50 ng) was amplified in a final reaction volume of 50 μl, containing 10 pmol of each primer and 2U Pyrobest DNA polymerase (Takara Biotechnology, Japan). Touchdown PCR procedure was used as followings: the initial denaturation at 95°C was followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 68°Cfor 35 sec (the temperature decrease 0.5°C each circle), and elongation at 72°C for 100 sec, then 18 cycles of denaturation at 95°C for 30 sec, annealing at 53°C for 35 sec, elongation at 72°C for 100 sec, finally elongation at 72°C for 7 min.
Gene cloning and somatic mutation detection
PCR products from B lymphocyte DNA of 22 individuals were subcloned into PGEM-T vector (Promega Corporation, Madison, WI, USA) after purification and adding adenine to them. Then ligation productions were transformed to Ecoli Top 10 competent cells and cultured in Luria-Bertani (LB) solid medium for 14 hours at 37°C. More than 8 clones per individual were randomly selected and amplified in LB liquid medium for 14 hours at 37°C. Plasmids were extracted and digested with PST1 restriction enzyme (Promega Corporation, Madison, WI, USA) to verify the insertion of PCR productions. Total 202 clones, including 8 to 10 clones per individual, were directly sequenced to detect somatic mutation.
Observed genotype frequencies in female subjects for all case and control groups were tested for Hardy-Weinberg equilibrium using χ2 tests with 1 df. Data were expressed as percentages or mean ± standard deviation. Pearson's χ2 was used for categorical data. Continuous variables were tested in each group for normal distribution using the Kolmogorov-Smirnov test for one variable. Differences of the means between two groups were tested with Student's t test. The means among the three groups were compared by ANOVA analysis. Statistical analysis was performed by SPSS 10.0 program (SPSS Inc., USA). All tests were two-sided and a P value of less than 0.05 was considered statistically significant.