Jill Kilner

Investigation of the association of BMP gene variants with nephropathy in Type 1 diabetes mellitus

Diabet. Med. 27, 624–630 (2010)

Genetic polymorphisms in nitric oxide synthase 3 gene and implications for kidney disease: a meta-analysis.

Background/Aims: The NOS3 gene is a biological and positional candidate for diabetic nephropathy. However, the relationship between NOS3 polymorphisms and renal disease is inconclusive. This study aimed to clarify the association of NOS3 variants with nephropathy in individuals with type 1 diabetes. Methods:We conducted a case-control study examining all common SNPs in the NOS3 gene by a tag SNP approach. Individuals with type 1 diabetes and persistent proteinuria (cases, n = 718) were compared with individuals with type 1 diabetes but no evidence of renal disease (controls, n = 749). Our replication collection comprised 1,105 individuals with type 1 diabetes recruited to a nephropathy case group and 862 control individuals with normal urinary albumin excretion rates. Meta-analysis was conducted for SNPs where more than three genotype datasets were available. Results: A novel association was identified in the discovery collection (rs1800783, pgenotype = 0.006, pallele = 0.002, OR = 1.26, 95% CI: 1.08–1.47) and supported by independent replication using a tag SNP (rs4496877, pairwise r2 = 0.96 with rs1800783) in the replication collection (pgenotype = 0.002, pallele = 0.0006, OR = 1.27, 95% CI: 1.10–1.45). Conclusion: The A allele of rs1800783 is a significant risk factor for nephropathy in individuals with type 1 diabetes, and further comprehensive studies are warranted to confirm the definitive functional variant in the NOS3 gene.

Resequencing of the CCL5 and CCR5 genes and investigation of variants for association with diabetic nephropathy

Chemokine (C–C motif) ligand 5 (CCL5) and chemokine (C–C motif) receptor 5 are implicated in the pathogenesis of diabetic nephropathy (DN). We hypothesize that variants in these genes may be associated with DN. The CCL5 and chemokine receptor type 5 (CCR5) genes were resequenced, variants identified (n=58), allele frequencies determined in 46 individuals (92 chromosomes) and efficient haplotype tag single-nucleotide polymorphisms (htSNPs) selected to effectively evaluate the common variation in these genes. One reportedly functional gene variant and eight htSNPs were genotyped in a case–control association study involving Caucasian individuals with type 1 diabetes (267 cases with DN and 442 non-nephropathic diabetic controls). Genotyping was performed using MassARRAY iPLEX, TaqMan, gel electrophoresis and direct capillary sequencing. After correction for multiple testing, there were no statistically significant associations between variants in the CCL5 and CCR5 genes and DN.

No support for association of protein kinase C, beta 1 (PRKCB1) gene promoter polymorphisms c.–1504C>T and c.–546C>G with diabetic nephropathy in Type 1 diabetes

1 New JP, Aung T, Baker PG, Yongsheng G, Pylypczuk R, Houghton J et al . The high prevalence of unrecognized anaemia in patients with diabetes and chronic kidney disease: a population-based study. Diabet Med 2008; 25 : 564–569. 2 Mostafa SA, Dave C, Burden AC. The prevalence of renal anaemia in diabetes. Diabet Med 2007; 24 (Suppl. 1): 76. 3 Baig S, Mostafa SA, Nwaomu SU, Patel N, Tagboto S, Davies SJ et al . Overrepresentation of diabetic patients with renal anaemia in primary care setting: could this extend above the new NICE cut-off? Diabet Med 2008; 25 (Suppl. 1): 120–121. 4 Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Ann Intern Med 1999; 130 : 461–470. 5 Vassalotti JA, Stevens LA, Levey AS. Testing for chronic kidney disease: a position statement from the National Kidney Foundation. Am J Kidney Dis 2007; 50 : 169–180. 6 Renal Association guidelines. Available at http://www.renal.org/ CKDguide/ckd.html 7 Mostafa SA, Burden AC, Tagboto S. Intravenous iron use in anaemia associated with chronic kidney disease: do patients with and without diabetes respond similarly? Diabet Med 2008; 25 (Suppl. 1): 120. 8 Winkler AS, Marsden J, Chaudhuri KR, Hambley H, Watkins PJ. Erythropoietin depletion and anaemia in diabetes mellitus. Diabet Med 1999; 16 : 813–819. 9 Dikow R, Schwenger V, Schomig M, Ritz E. How should we manage anaemia in patients with diabetes? Nephrol Dial Transplant 2001; 17 : 67–72.

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.

Design and Implementation of a Custom Next Generation Sequencing Panel for Selected Vitamin D Associated Genes

BackgroundBiologically active vitamin D has an important regulatory role within the genome. It binds the vitamin D receptor (VDR) in order to control the expression of a wide range of genes as well as interacting with the epigenome to modify chromatin and methylation status. Vitamin D deficiency is associated with several human diseases including end-stage renal disease.MethodsThis article describes the design and testing of a custom, targeted next generation sequencing (NGS) panel for selected vitamin D associated genes. Sequencing runs were used to determine the effectiveness of the panel for variant calling, to compare efficiency and data across different sequencers, and to perform representative, proof of principle association analyses. These analyses were underpowered for significance testing. Amplicons were designed in two pools (163 and 166 fragments respectively) and used to sequence two cohorts of renal transplant recipients on the Ion Personal Genome Machine (PGM)™ and Ion S5™ XL desktop sequencers.ResultsCoverage was provided for 43.8 kilobases across seven vitamin D associated genes (CYP24A1, CUBN, VDR, GC, NADSYN1, CYP27B1, CYP2R1) as well as 38 prioritised SNPs. Sequencing runs provided sufficient sequencing quality, data output and validated the effective library preparation and panel design.ConclusionsThis novel, custom-designed, validated panel provides a fast, cost effective, and specific approach for the analysis of vitamin D associated genes in a wide range of patient cohorts. This article does not report results from a controlled health-care intervention.