TLR4 Gene Variant as a Risk Marker of Diabetic Foot Ulcers 

Asad Mehmood Raja1, Muhammad Mobeen Zafar1, Muhammad Aamir Riaz2, Jamal Zafar3, Muhammad Saqlain1, Abid Mahmood1, Majid Mahmood2, Ghazala Kaukab Raja1
1 University Institute of Biochemistry and Biotechnology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi
2 Department of Zoology, University of Poonch Rawalakot
3 Department of General Medicine, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad

ABSTRACT
Background
Diabetes and secondary complications are the fastest growing chronic diseases worldwide. Diabetic foot ulcers are severe forms of diabetes complications resulting in limb amputation, severe health issues or even early death. The lifetime risks of diabetes and diabetic foot ulcers are on rapid rise in Pakistan. Though large data exists regarding environmental and genetic risk factors of diabetes, genetic causes of diabetic foot ulcers are yet to be identified. Toll Like Receptor 4 (TLR4) gene encodes a receptor with role in innate immunity A number of Single Nucleotide Polymorphisms (SNPs) in TLR4 gene are found associated with infectious diseases and other inflammatory diseases. Toll like receptor (TLR4) play very vital role in regeneration, immunity and tissue repair. Significant associations of toll like receptor 4, single nucleotide polymorphisms (SNPs) rs4986790, rs4986791, rs11536858 (fused into rs10759931), rs1927911, and rs1927914, have been reported with increased risk of foot ulcer in type 2 diabetes mellitus patients. 
AIM:
The present study was designed to identify risk allele frequency of rs4986791 (C/T or Thr399Ile) TLR4 SNP in Pakistani diabetic subjects with foot ulcer and to explore its association with disease. 
METHODS:
The anthropometric parameters and clinical tests data were obtained from a total of 260 subjects, including 110 diabetic patients with foot ulcer, 50 diabetic patients without foot ulcer, and 100 healthy controls. For microbiota identification, pus samples from the infected foot parts were collected and cultured subsequently. Allele specific Polymerase Chain Reaction (AS-PCR) was performed for the genotyping of rs498679 SNP. 
RESULTS AND DISCUSSION:
The frequency of risk allele T was 0.48 in both diabetic and control subjects and 0.46 among diabetic foot ulcer patients. The T allele of rs4986791 SNP significantly increased the risk of foot ulcers among diabetic subjects (OR=2.59 CI= 1.24-5.40). Of diabetic foot ulcer microbiota Staphylococcus aureus was found highly frequent (26.34%) while Amikacin as highly susceptible antibiotic in our population. The prominent environmental risk factors identified in diabetic foot ulcers were; poor glycemic control, advanced age, male gender, over-weight, higher HbA1c levels, and family history of diabetes, , foot deformities are significantly associated with disease.

TLR4 gene variant as a risk marker of diabetic foot ulcers