Development of next generation sequencing panel for UMOD and association with kidney disease

Chronic kidney disease (CKD) has a prevalence of approximately 10% in adult populations. CKD can progress to end-stage renal disease (ESRD) and this is usually fatal unless some form of renal replacement therapy (chronic dialysis or renal transplantation) is provided. There is an inherited predisposition to CKD with several genetic risk markers now identified. The UMOD gene has been associated with CKD of varying aetiologies. An AmpliSeq next generation sequencing panel was developed to facilitate comprehensive sequencing of the UMOD gene, covering exonic and regulatory regions. SNPs and CpG sites in the genomic region encompassing UMOD were evaluated for association with CKD in two studies; the UK Wellcome Trust Case-Control 3 Renal Transplant Dysfunction Study (n = 1088) and UK-ROI GENIE GWAS (n = 1726). A technological comparison of two Ion Torrent machines revealed 100% allele call concordance between S5 XL™ and PGM™ machines. One SNP (rs183962941), located in a non-coding region of UMOD, was nominally associated with ESRD (p = 0.008). No association was identified between UMOD variants and estimated glomerular filtration rate. Analysis of methylation data for over 480,000 CpG sites revealed differential methylation patterns within UMOD, the most significant of these was cg03140788 p = 3.7 x 10-10

Comparison of suitability of PGM and S5XL next generation sequencers for analysis of UMOD.

<p>Comparison of suitability of PGM and S5XL next generation sequencers for analysis of UMOD.</p

Haplotype blocks.

<p>Haplotype blocks with population frequencies displayed next to each haplotype. Lines represent the most common crossings between block (thicker lines are more common) beneath which the value of multiallelic D’ is shown, measuring the level of linkage disequilibrium (LD) between blocks. Greater recombination is denoted by a value closer to zero.</p

UMOD transcripts.

<p>Three UMOD transcripts (3'-5') from NCBI gene website [last accessed: 17/10/2015]. <i>Green blocks indicate the position of exons on the sequence with arrows denoting the direction of expression</i>.</p

Chromosome location and alleles associated with multiple transcripts and associated gene locations for non-synonymous SNP rs55772253.

<p>Chromosome location and alleles associated with multiple transcripts and associated gene locations for non-synonymous SNP rs55772253.</p

Ampliseq design.

<p>The UMOD gene region plus 2 kb upstream and downstream is highlighted by the blue line, coverage by Ampliseq amplicons is shown in green and regions not covered by the optimised assay design is shown in red. The structure of UMOD from RefSeq genes is represented in dark blue.</p