Proband and nephrolithiasis family description
A elder man presented with post-polio syndrome of right foot claudication,20 years history of renal calculi, repeated bilateral back pain, urgent micturition, hematuria and dysuria. The patient suffered from hypertension almost 10 years, admitted oral antihypertensive drugs, and also complained with intermittent voiding stones in urine 3 times, but was not treated. He had noticed with aggravation of back pain and voiding stones in urine again 1 month ago, which make them admitted to our Urology Department. Physical examination revealed a temperature of 36.3 °C, pulse of 102, blood pressure (BP) of 109/88 mmHg, left flank tenderness and a palpable left kidney but was otherwise normal. Laboratory testing indicated that creatinine concentration was 89.9 umol/L, the serum calcium was 2.26 mmol/L, uric acid was357 umol/L, serum phosphorus was 1.06 mmol/L, and serum magnesium was 0.78 mmol/L. The serum electrolytes were within normal limits. Urinalysis showed specific gravity of 1.02, PH 6.5, no protein, and 6 to 10 white blood cells and red blood cells (RBCs) per high-power field without casts or crystals. Abdominal ultrasound showed multiple cystic dark areas with the maximum diameter of 2.2 cm, clear boundaries and regular morphology. Ultrasonography and computed tomography (CT) of left kidney demonstrated hydronephrosis, no marked parenchymal atrophy, and multiple stones in therenal pelvis. There was a cystic dark area, diameter of 1.1 cm, in right kidney with regular morphology and clear boundary (Additional file 1: Figure S1).The patient has endured family history of nephrolithiasis (Fig. 1).
Nine hundred ninety three cases of nephrolithiasis (NL) and 1314 controls were obtained from the Second Affiliated Hospital of Third Military Medical University during the period of 2013–2016. Diagnostic evaluations were performed separately in person using standardized criteria for diagnosing nephrolithiasis. Two independent raters collected family history from the proband, each participating parent, and a nursing sister recorded pedigree information during the clinical interview. In addition, 328 cases patients with simple cyst and 622 matched (1:2) controls were recruited for PDE1A genotype analysis. All of the cases were diagnosed by spiral computed tomography (CT), ultrasonography or X ray. Control inclusion criteria: ①Screen for a lifetime absence of NL or cyst using CT, ultrasonography or X ray. ② Excluded significant complication disease of NL or cyst. This study was conducted with the approval of the ethics committee of Third Military Medical University. whether the participants had the capacity to consent was evaluated by the following criteria: Firstly, patients have the ability to understand and reasoning; Secondly, patients have the ability to make rational decisions. All participants gave written informed consent; parental consent for children who were younger than 18 years old was obtained.
DNA and RNA extraction and quality controls
Genomic DNA was extracted from peripheral blood cells using TIANamp Blood DNA Kit (TIANGEN BIOTECH, BEIJING) following as the manufacturer’s instructions. Total RNA was extracted from whole blood with MagMAX™ for Stabilized Blood Tubes RNA Isolation Kit following the manufacturer’s instructions. Quality control of DNA or RNA were conducted by Agarose gel electrophoresis and λDNA-Hind III digest band (Additional file 1: Figure S2).
Captured library construction, Clustering & Sequencing
Each sequenced sample was prepared according to the Illumina protocols. Briefly, onemicrogram of genomic DNA was fragmented by nebulization, the fragmented DNA isrepaired, an ‘A’ is ligated to the 3′ end, Illumina adapters are then ligated to the fragments, and the sample was size selected aiming for a 350–400 base pair product. The size selectedproduct was PCR amplified, and the final product was validated using the Agilent Bioanalyzer. Two steps of hybridization and wash were needed for construction. PCR was used in order to amplify the enriched DNA library for sequencing. PCR was performed with the same PCR primer cocktail used in TruSeq DNA Sample Preparation. Axeq Technologies performs procedures for quality control analysis on the sample library and quantification of the DNA library templates. Illumina utilizes a unique “bridged” amplification reaction that occurs on the surface of the flow cell. A flow cell containing millions of unique clusters was loaded into the HiSeq 2004 for automated cycles of extension and imaging. Following cluster generation, 151 nt paired-end sequencing was performed using the standard Illumina protocols.
Quality control for sequencing results and variants calling
Quality control of raw data was conducted by FastQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were mapped to a custom hg19 build using Burrows-Wheeler Alignment tool (BWA).The duplicate reads were flagged using Picard-tools (http://broadinstitute.github.io/picard/). GATK IndelRealigner module was used to realign reads around insertion/deletion (Indel) sites. Individual sequence data (in BAM format) was preprocessed as whole NGS community suggests which mainly include local Indel realignment, PCR duplicates removal and base quality recalibration. Read qualities were recalibrated using GATK Table Recalibration. GATK unified Genotyper module was then used to call variants (both SNVs and Indels) from multiple samples simultaneously, which create a single Variant Call Format (VCF) file. Raw read data were visualized using the Integrative Genome Viewer (IGV).Individual level quality control was conducted on raw and clean variant to make sure avoiding false positive variants. A suite of per-individual metrics, which included the total number of alternate alleles, dbSNP coverage (build137), and Transition/Transversion (Ti/Tv) ratio, and variant quality recalibration (VQSR) were calculated. From available exome data, we extracted common variants and estimated per-individual heterozygosity (~inbreeding), pairwise relatedness, and sex-check using PLINK (Additional file 2: Table S1). Variants quality control was conducted by software KGGSeq (http://statgenpro.psychiatry.hku.hk/limx/kggseq/ doc/UserManual.html), which were carefully designed to filter and prioritize gene variants in exome sequencing of rare Mendelian and common complex disorder.
Candidate mutation from the next generation sequencing were tested using standard Sanger sequencing on an ABI 3730xl DNA Analyzer to validate the reality, by designing custom primers (Sigma) based on ~200 bp of genomic sequence flanking each variant (Additional file 1
: Figure S3).
Variance quality control and candidate gene filtering
Variants were kept if ① the minimum overall sequencing quality scores ≥ 50 (−-seq-qual 50) and the minimum overall mapping quality score ≥ 20 (−-seq-mq 20);②The minimal genotyping quality per genotype ≥ 30 (−-gty-qual 30) and the minimal read depth per genotype ≥ 30 (−-gty-qual 30); ③The fraction of the reads carrying alternative allele ≤ 5% at a reference-allele homozygous genotype (−-gty-af-ref. 0.05), the fraction of the reads carrying alternative allele ≥ 25% at a heterozygous genotype (−-gty-af-het 0.25), and the fraction of the reads carrying alternative allele ≥ 75% at an alternative-allele homozygous genotype (−-gty-af-alt 0.75); ④ Minimal observed number of non-missing genotypes in all samples as 50 (−-min-obs 50).
Candidate variants were kept if ① mutation present in patients with nephrolithiasis, but not in family members. ② Nonsynonymous; All mutations were annotation by Annovar, we ignored synonymous variants because nucleotide substitution of these kind variants does not change amino acid. ③ Predict damaging; All of nonsynonymous variants that met any of the following criteria were considered potentially damaging: frameshift, nonsense, stoploss, stopgain, splicing and missense mutation with Polyphen score≧0.90 and/or SIFT p≦0.05 and/or Grantham score≧100 and/or phyloP score≧2.0. ④ SNP or In/Del within a gene, which have been proved with the cause of nephrolithiasis or related pathway in previous study.
Six SNPs were chosen for further validation which based on the prediction scores, minor allele frequency (MAF) and function annotation (Additional file 2
: Table S2). The SNP genotyping was performed using an improved multiplex ligation detection reaction (iMLDR) technique or TaqMan, which was newly developed by Genesky Biotechnologies, Inc. (Shanghai, China). We designed primers and probes for TaqMan genotyping assays for SNP rs182089527. Each genomic DNA sample (20 ng) was amplified with TaqMan universal PCR master mix reagent (Applied Biosystems, Foster City, CA) combined with the specific TaqMan SNP genotyping assay mixture, corresponding to the SNP to be genotyped. The iMLDR technique was applied for genotyping remaining SNPs which follow the instruction.
The sequencing of PDE1A designed primers as following:
Construction of mutation expression plasmid
Blood samples were collected from all family members. RNA were extracted and was converted into complementary DNA (cDNA) using a Reverse Transcription System Kit (Invitrogen, Carlsbad, CA, USA). Wild-type and mutant-type of p.M1R in PDE1A9 were harvested from 6 unaffected members and 4 affected patients.
cDNAs were PCR-amplified with forward primerand reverse primer (5- TACCGGACTCAGATCTCGAGCGCCACCAGGGGCAAAAAGATAAACAAAC-3 and 3-GATCCCGGGCCCGCGGTACCGTCTGATGAATAAACTCACACTTCTG-5). Human wild-type and mutant PDE1A9 inserted into the GV320 vector (SHANGHAI GENECHEM CO., LTD.). The viruses were collected on Day 3 after the transfectionand were concentrated by ultracentrifugation.Transport plasmid with human wild-type and mutant PDE1A9 constructs were transiently or stably transfected intoTubular epithelial cells (ATCC, Manassas VA, USA; CCL-93). All constructs were verifiedby sequencing.
All the tubular epithelialcell lines were provided by prof. Jing Zhang and her student Yi Gong (Department of Nephrology, The Third Millitary University) and cultured in DMEM supplemented with 10% fetal bovineserum, streptomycin (100 mg/ml) and penicillin (100 U/ml). Cells wereserum deprived for 6 h before treatment with transport plasmid andthen cultured in DMEM containing 0.5% fetal bovine serum (conditionedmedium).
Protein was extracted from cultured tubular epithelial cell lines after washed with PBS and lysed in RIPA buffer (Beyotime, Shanghai, China). Protein concentration was measured using the RCDC method (BIO-RAD, USA). Total protein extracts of 50 μg were separated using 8–10% SDS-PAGE gels (Beyotime, Shanghai, China) and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, USA). After blocked with 5% BSA at room temperature for 2 h and the membraneswere incubated with primary antibodies against PDE1A9at 4 °C overnightfollowed by the secondary antibodies. Proteins were visualized using ECL (Millipore, Bradford, MA, USA) and detected using Image Quant LAS-4000 (Fujifilm, Tokyo, Japan) BioImaging System.
Cultured tubular epithelial cells were lysed on the ice for 20 min. cDNAs were synthesized by Sensiscript RT Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The primers was the same as above description. The thermocycling program consisted of 94 °C for 1 min, 60 °C for 30 s and 72 °C for 1 min (40 cycles).The PCR products were visualized using 2% agarose gel electrophoresis followed by GoldView (SBS Genetech Co., Beijing, China) staining.
Clinical characteristics are presented as means ± SD. The chi-squared test or Fisher’s exact test with Bonferroni correction was used for the analysis of contingency tables depending on the sample size. Monte Carlo simulation was employed to calculate the difference between the NL patients with the rs182089527 mutation and healthy control group. All statistical analyses were conducted using R software (http://www.R-project.org/).