Submitting author:
Fakhra Naseem

Email address:
f.naseem@wlv.ac.uk

Stream:
Basic and Clinical science 

Category:
Diagnosis & Classification


Authors:
Mrs F Naseem1, Dr A Watkins2, Dr J E P Brown2, Dr A U Nayak3, Dr A Narshi1, Prof B M Singh1,4, Dr S J Dunmore1

Affiliations:
1Diabetes Research Group, PG Academic Institute of Medicine, University of Wolverhampton, Wolverhampton, UK,2Aston Research Centre for Healthy Ageing, Aston University, Birmingham, UK,3Department of Diabetes & Endocrinology, Royal Stoke University Hospital, Stoke-on-Trent, UK,4Wolverhampton Diabetes Centre, New Cross Hospital, Wolverhampton, UK   	  	  
  	 	 
Abstract title	  	  
SNPs in the Fructosamine-3-Kinase gene  as a possible explanation of variation in the glycation gap between individuals.	  	  
Abstract 

Background

We have previously shown that the Glycation gap(GGap), a discrepancy between average glycaemia calculated using HbA1c versus fructosamine and a consistent phenomenon in diabetic patients, may be explained by a substantially lower activity of the deglycating enzyme fructosamine-3-kinase (FN3K) in patients with positive compared with negative GGaps1. This difference may also account for a higher incidence of diabetic complications in the positive versus negative GGap groups as a result of increased production of advanced glycation endproducts (AGEs). We hypothesised that the difference in FN3K activity may result from characterised single nucleotide polymorphisms (SNPs) previously suggested to affect its activity. In view of the differences in AGE levels that we have identified between the GGap groups we also sought confirmation whether this was reflected in differing levels of receptors for AGEs which have been suggested to be associated with diabetic complications2.

Aims 

To explore frequency of specific FN3K polymorphisms (SNPs rs1056534 & rs1046896) in diabetic patients with positive and negative GGaps and to evaluate circulating levels of receptor for AGEs (RAGE) in these groups.


Methods 
Serum RAGE
We assayed serum RAGE from sub-groups of our diabetic patient cohort with high positive (>+0.5%,n=67) or negative (<-0.5%,n=77) G-Gaps respectively using sandwich ELISA (Human RAGE DuoSet ELISA Kit) . 
FN3K polymorphisms 
Genomic DNA was extracted from whole blood of diabetic patients with consistent positive (>+0.5%, n=38) and negative (>-0.5%, n=48) GGap (QIAamp DNA mini kit. The occurrence of two SNPs C/G (rs1056534) and C/T (rs1046896) of the FN3K gene were analysed using the TaqMan Gene Expression Assay (ThermoFisher Scientific) with provided primers and probes by RT-qPCR.  


Results

The proportion of patients with the C/G versus G/G SNP rs1056534  was very similar in positive (63.2% vs 36.8%) and negative (62.5% vs 37.5%) GGap groups. Only the C/T variant of SNP rs1046896 was found in both positive and negative GGap groups. Serum RAGE did not differ significantly between the GGap groups (p=0.345).
Discussion

Previous studies have suggested that certain SNPs in the FN3K gene may relate to levels of HbA1c in diabetic patients3,4. We have previously shown a 3-fold difference in FN3K activity in relation to FN3K protein expression between groups of diabetic patients with positive and negative GGaps1. In the present study however we found no relationship between prevalence of FN3K (rs1056534) and (rs1046896) polymorphisms with GGap. 

The serum RAGE level did not differ between positive and negative GGap subjects whereas we have previously found that serum AGE is higher in positive GGap so the latter is a more likely link between excessive glycation in subjects with a positive GGap which is likely to be a consequence of lower FN3K activity. 

The explanation for the very substantial difference in FN3K activity may lie in other SNPs or in alternative post-transcriptional splicing producing isoenzymes of FN3K with very different enzyme activities but similar immunoreactivity in the ELISA assay. To investigate this further we are undertaking a transcriptomic study to identify potential differences between GGap groups.

References
1.	Dunmore SJ et al. Evidence for the role of fructosamine-3-kinase in humans in the genesis of the glycation gap and the associated complications of diabetes. Diabetes; 2017 (in review)
2.	Tanji N et al. Expression of advanced glycation end products and their cellular receptor RAGE in diabetic nephropathy and nondiabetic renal disease. J Am Soc Nephrol.; 2000;11:1656–66. 
3.	Veiga da-Cunha M et al. Increased protein glycation in fructosamine 3-kinase-deficient mice. Biochem J 2006; 399: 257–64
4.	Mohás M et al. A polymorphism within the Fructosamine-3-kinase gene is associated with HbA1c levels and the onset of type 2 diabetes mellitus. Exp Clin Endocrinol Diabetes 2010; 118:209–12

SNPs in the fructosamine-3-Kinase gene as a possible explanation of variation in the glycation gap between individuals