Patients and clinical data
In this prospective study, children diagnosed with CAKUT (age group: newborn -18 years) were enrolled for the study from pediatric nephrology and surgery units between Jan 2016 and Jan 2019. Both incident and prevalent cases with follow up for at least 6 months were considered for recruitment. Institutional Ethics Committee approved the study (IEC Ref No- 163/2016) and all participants were recruited after informed consent. The diagnosis of CAKUT was made by pediatric nephrologists or surgeon on the basis of the following: ultrasonography (USG) of kidney and urinary tract, DMSA (Dimercaptosuccinic acid) scan and Micturating Cysto-urethrogram (MCUG), to identify any abnormality of number, size, shape, or anatomic position of the kidneys or other parts of the urinary tract. It included at least one of the following: vesicoureteral reflux (VUR) with or without hypodysplasia, posterior urethral valve (PUV), uretero–pelvic junction obstruction (UPJO), vesico-ureteric junction obstruction (VUJO), multicystic dysplastic kidney (MCDK), isolated renal hypodysplasia which is characterized by small kidney size with altered echogenicity, unilateral renal agenesis, duplex collecting system, megaureter and horseshoe kidney (6,11). Both, syndromic (defined as CAKUT with extra renal features like optic nerve coloboma, ear fistula, pinna deformation, hearing loss, cardiac, gastrointestinal, neurological or skeletal defects) and non-syndromic cases (defined as CAKUT without extra renal features) were included in the study. Patients with a known causative genetic or chromosomal abnormality, isolated neurogenic bladder, and those who have undergone nephrectomy for medical reasons were excluded from the study. Clinical data, laboratory and imaging details were documented. Glomerular filtration rate (GFR) was estimated using revised Schwartz equation. Hypertension, chronic kidney disease (CKD) and ESKD was defined as per standard criteria (12). For children less than 2 years, age-specific normal ranges for GFR was used to diagnose CKD (13).
Next generation sequencing
Blood sample (EDTA 5ml) collected from patients at the time of recruitment was further processed for extraction of DNA. Genomic DNA was extracted from leukocytes using QIAamp blood mini kit (Qiagen, Hilden, Germany) as per manufacturer's instructions. Quantity of extracted DNA was estimated using Qubit fluorometric assay (Thermofisher scientific, MA, USA).
Genes for the customized panel were selected through a literature search in various public databases such as OMIM (Online Mendelian Inheritance in Man) database (http://omim.org/) and PUBMED (http://www.ncbi.nlm.nih.gov/pubmed/) (Dec 2015). The following keywords were used: CAKUT, urinary tract anomalies or abnormalities, multicystic kidney dysplasia, vesico-ureteral reflux, duplex collecting system, posterior urethral valve, uretero–pelvic junction obstruction and renal/kidney diseases, genetic testing and mutations in CAKUT. Thirty-one genes associated with CAKUT were finally selected after a review of published studies in different ethnicities. Customized gene panel consisting of 31 genes was designed using Ion-Ampliseq primer designer (Life Technologies), targeting the coding regions of the genes (Additional file 1.docx Genes included in the customized CAKUT NGS panel). The panel consisted of 825 primers (3 primer pools) with amplicon size ranging from 125 to 375 bp, targeting the exonic regions (221.38 kb, 449 exons) with coverage of 98.99%. The uncovered regions were mainly repeat rich region making primer designing difficult.
Sixty-nine patients (3 syndromic children) with complete clinical and follow up data and representing various CAKUT phenotypes were selected for sequencing. The barcoded libraries were prepared using 20ng DNA and HQ-Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific Inc. USA) as per the manufacturer’s instructions. Library quantity and quality was determined using Qubit fluorometric assay and Agilent BioAnalyzer High-Sensitivity DNA kit (Agilent Technologies, CA, USA), respectively. The libraries were amplified using Ion-Ampliseq Hi-Fi PCR mix, partially digested and phosphorylated and barcoded using Unique-Ion express barcodes. The barcoded libraries were pooled and enriched using Ion PGM Template OT2 400 kit (Thermo Fisher Scientific Inc. USA) according to the manufacturer’s protocol. Next generation sequencing was carried out using the Ion PGM Hi-Q Sequencing Kit (318 Chip, Thermo Fisher Scientific Inc. USA) on Ion Torrent Personal Genome Machine sequencer (Thermo Fisher Scientific Inc. USA) as per the manufacturer’s protocol.
NGS data analysis:
Analysis was carried out using Ion Torrent Suite™ Browser version 5.0 and Ion Reporter™ version 5.0. Torrent Suite™ Browser was used to perform initial quality control including chip loading density, base calling, alignment (hg19/GRCh37), median read length and number of mapped reads, assembly, coverage analysis and variant calling. Variants were identified by Ion Reporter using in house developed and validated filtering criteria: variant coverage >20x,variant type and effect (non-synonymous, frame-shift, nonsense), location (to detect splice site variants) and variants with a minor allele frequency (MAF) <1% in public databases (ExAc and 5000 Exome (http://evs.gs.washington.edu/EVS/)(14). All frame-shift variants and variants affecting stop codons were retained irrespective of their MAF. Synonymous variants except those located ±2 bp off exon boundaries and intronic variants >10 bp from exon boundaries were removed. Variants with a minor allele frequency (MAF) higher than 0.01 in online databases ExAC and 1000 genome, were excluded. The filtered variants were manually inspected with a high-performance visualization tool - Integrative Genomics Viewer (IGV)(15)to filter out variants with possible strand-bias and variants within homopolymeric region. The functional significance of the filtered variants were evaluated in-silico using online prediction software SIFT(16),Polyphen-2(17)and MutationTaster (18). Variants of interest were classified as pathogenic, likely pathogenic, variants of uncertain significance (VUS), likely benign, or benign using American College of Medical Genetics and Genomics (ACMG) guidelines and Sherloc guidelines by Nykamp et.al, which was a refinement of ACMG guidelines(19,20). A scoring system developed by Karbassi et al. was used to determine the pathogenicity of VUS (21). Causal variants were confirmed by Sanger sequencing using variant specific primers in patients as well as in 30 healthy individuals. The details of the primers are provided in the Additional file 2: Table 1 S1.xlsx- Details of primers used for Sanger sequencing to validate the variants identified by next generation sequencing