Association the allelic variation and SNP rs12917707 genotyping with UMOD serum level among Iraqi patients infected with uro-pathogenic Escherichia coli

: The current study included 90 samples collected and divided into (45) Urinary tract infections of E. coli patients and (45) controls with different ages of both genders. Patient samples were collected from UTI patients admitted to AL-Yarmouk Teaching Hospital, AL-Karama Teaching Hospital and Al Kidney Teaching Hospital from November 2020 to March 2021. The current study measured Tamm Horsfall protein (THP) concentration in patients with Urinary tract infections and healthy groups. The study also included the Relationship of Umod rs12917707 genotype and Uromodulin level in patients and control using Nested T-ARMS PCR. Our study had two objectives: First, to address whether urinary uromodulin concentration is associated with urinary tract infection with E. coli in a community-based study, and second, to determine whether a single-nucleotide polymorphism (SNP) in the UMOD region, rs12917707, is associated with urinary uromodulin concentrations. After statistical analysis, the results showed that there could be an association between having mutant homozygous GG polymorphism in the UMOD gene and having UTI of E. coli . At the same time, the mutant homozygous TT represents a risk factor compared to other genotypes (ORs: 0.4, 95% CI (0.17 - 0.93 and ORs: 4.4, 95% CI (1.47-13.26) respectively. The re-sults also showed a significant decrease at P≤0.01 in the patients group with Urinary tract infection (1.38 ± 0.03) Ng/ml compared with the control sample, which was (1.83 ± 0.04) Ng/ml.


Introduction
Urinary tract infections (UTIs) are widely globally distributed health problems, and both men and women can be affected by UTIs, which usually present with different symptoms and prognoses.UTIs are serious public health issues and are responsible for nearly 150 million cases yearly worldwide. 1.Extraintestinal pathogenic E. coli (ExPEC) are facultative pathogens in normal human intestinal flora.However, their existence may be correlated with some infectious diseases, including neonatal meningitis E. coli (NMEC) and sepsis E. coli (SEPEC) 2 .In addition to the most prevalent infection, UTI, many are caused by a highly heterogeneous ExPEC group called uropathogenic E. coli (UPEC) 3 .
UPEC strains encode various virulence factors, which allow the microorganism to colonize the urinary tract and persist in the face of highly effective host defense 4 .UPEC isolates exhibit a high degree of genetic variety due to the possession of specialized virulence genes located on cellular genetic elements called pathogenicity islands 5 .Virulence factors of E. coli that have been potentially implicated as essential to establish UTI can be divided into two groups: The first group of virulence factors is associated with the surface of bacterial cells.In contrast, the second group of virulence factors is secreted and exported to the site of action 6 .Bacterial attachment to uroepithelial cells is essential for beginning and developing UTI 7 .Uromodulin (UMOD, previously known as (Tamm-Horsfall protein) is the most abundant protein excreted in the normal urine.This kidney-specific protein is essentially produced by the cells lining the thick ascending limb (TAL) of the loop of Henle and, to a much smaller extent, the initial segment of the distal convoluted tubule (DCT) (1).As a typical glycosylphosphatidylinositol-anchored protein, UMOD matures along the secretory pathway, becoming heavily glycosylated and sorted to the apical plasma membrane, where it is cleaved by the serine protease hepsin 8 .Once in the lumen, UMOD monomers assemble into homopolymeric filaments, encapsulating and aggregating uropathogens, such as type-1 fimbriated Escherichia coli, promoting their clearance in the urine 9 .Multilevel evidence supports the role of UMOD, the gene coding for UMOD, in a spectrum of kidney disorders.Rare missense mutations of UMOD are the most common cause of autosomal dominant tubulointerstitial kidney disease (ADTKD) 10 .In parallel, genome-wide association studies (GWAS) have consistently associated the UMOD locus with the eGFR and the risk of developing CKD in the general population. 11.The top UMOD promoter variant, rs12917707, was dosedependent with the urinary levels of UMOD, confirming the biological link between these variants and the expression of UMOD in kidney and urine 12 .

Material and Methods
Collection of Sample: 90 samples were collected and divided into (45) UTIs of E. coli patients and(45) controls with different ages of both genders.Patient samples were collected from UTI patients admitted to AL-Yarmouk Teaching Hospital, AL-Karama Teaching Hospital and Al Kidney Teaching Hospital from November 2020 to March 2021.Eight (8) ml of blood were obtained from each subject by vein puncture, two (2) ml were put into Ethylene Diamine Tetra Acetic Acid (EDTA) tubes, and the remaining six (6) ml were pushed slowly into disposable tubes containing separating gel.Blood in the EDTA tubes was stored in deep freeze (-40˚C ) in order to be used later in the genetic part of the study, while blood in the gelcontaining tubes was allowed to clot at room temperature for 30 minutes and then centrifuged at 2000×g for approximately 15 minutes then the sera were obtained and stored at -20˚C until analysis.

Determination of serum uromodulin: ELISA was used for estimating human UMOD (uromodulin) utilizing an ELISA kit based on the manufacturer's instructions (MyBioSource, USA).
Blood DNA Extraction: ACCORDING TO THE MANUFACTURER'S INSTRUCTIONS, the DNA was extracted from blood using a commercial wizard genomic DNA purification kit (Wizard-GenomicDNA-Purification Kit, Promega/ USA).After genomic DNA extraction, agarose gel electrophoresis has been adopted to confirm the presence and integrity of the extracted DNA (13).NanoDrop device (Thermo Fisher Scientific/USA) determined the concentrations and purity of extracted DNA.Also, the Genotyping of one common polymorphism (rs12917707) of the UMOD gene was conducted using Nested T-ARMS PCR SNP Genotyping Assays.

Nested T-ARMS PCR reaction:
A sterile 0.5-mL thin-walled amplification tube, or the well of a microtiter plate, was added and mixed in, as shown in Table 2.

Outer forward 1
Outer reverse 1

Table 2. Components of the Mixture of Nested T-ARMS PCR.T.
The first round of amplification was performed using denaturation, annealing, and polymerization times and temperatures listed in the following The second round of amplification was prepared in a sterile 0.5-mL thin-walled amplification tube and mixed as shown in Table 4:

Table 4. Components of the Mixture of Nested T-ARMS PCR.
When put in the machine on the fifth cycle, turn the machine off and start adding 1 micro of a mixture of outer forward and outer reverse.
The second round of amplification was performed using the denaturation, annealing, and polymerization times and temperatures listed in When put in the machine on the fifth cycle, turn the machine off and start adding 1 micro of a mixture of forward and outer reverse.Then, amplification results were analyzed by electrophoresis on agarose gel.outer

Detection of UROM gene polymorphisms
Total genomic DNA was extracted from the whole frozen blood using a genomic DNA purification kit; the DNA's concentration and purity were carried out using Nanodrop.Figure 1 showed sharp bands of chromosomal DNA, which were analyzed by gel electrophoresis using (1%) agarose gel and DNA Red safe dye at a voltage (70) for (30) minutes.A modified T-ARMS PCR technique was implemented in which a nested PCR product was introduced before the T-ARMS PCR for the human gene UMOD rs12917707 SNP.The amplified fragment result of the desired product with a molecular weight of 238 bp appeared sharp in the agarose gel, which represents the outer band PCR, which serves as a DNA template for the inner PCR (167 bp) for the allele (T) and (111 bp) for the allele (G).The genotype (GG) appeared with a band of (111 bp), while the heterozygous genotype (GT) showed two bands of (111bp and 167 bp), and the homogenate (TT) showed a band (of 167bp) as shown in Figure 2.

Genotyping Analysis Outputs of Umod rs12917707
Uromodulin (Umod) gene polymorphism analysis can provide vital information to optimize the individualized resistance and therapeutic approach.Therefore, determining the prevalence of functionally significant SNPs in uromodulin in patients is of great interest.Here, we evaluated the incidence of Umod rs12917707, individually and within patients, to detect the genotype carriers related to the risk of having the disease.The genotype frequencies of analyzed Umod rs12917707 SNP for healthy patients were as follows: 57.78% and 35.56% were G/G (wt/wt) as a wild type; 31.11% and 28.88% were G/T (wt/mt) as a heterozygous mutant and 11.11% and 35.56% were T/T (mt/mt) as a homozygous mutant, respectively.Statistical analysis of UMOD rs12917707 genotype and allele frequency showed a significant difference with high incidence in mutant homozygous TT and significance in GG as wild type.In order to estimate relative risk frequency between patients and control, we used odds ratio and 95% confidence interval (ORs and 95%CI) analysis to detect the genotype risk factors carriers for the development of UTI in the patients group compared with the control group.The odds ratio test in Table 6 showed evidence of an association between the GG genotype of uromodulin gen and the urinary tract infection toward protection (57.78% versus 35.56% for healthy subjects and urinary tract infections, respectively).The Umod rs12917707 mutant homozygous TT represents a risk factor for urinary tract infection incidence in Iraqi patients.(11.11% versus 35.56%) for healthy subjects and urinary tract infections, respectively).(ORs: 0.4, 95% CI (0.17 -0.93) and ORs: 4.4, 95% CI (1.47 -13.26), respectively.The frequencies of the G allele were 66% and 45% for healthy subjects and urinary tract infections, respectively).The frequencies of the T allele were 24% and 45% for healthy subjects and urinary tract infection, respectively).In the present study, the standard variant GG of (rs12917707) was associated with the disease of UTI in patients.People who carry TT have a four-fold risk of developing the disease compared to other genotypes.A previous study in Iraq was conducted by Ibraheem et al., who showed that the standard variant GG of (rs12917707) was associated with the development of albuminuria in diabetic patients, and the T minor allele had a protective factor in diabetes mellitus patients with macroalbuminuria.

Genotype
Other studies have suggested that UMOD mutations contribute to familial hereditary nephropathy, myeloid cystic kidney disease 16 and that promoter variants of the UMOD gene are associated with the estimated glomerular filtration rate (eGFR), blood pressure, plasma uric acid level and incidence of CKD 17 .The relationship between the rs12917707 variants with creatinine levels increasing was definitely in a meta-analysis of five European isolates (EUROPEAN) and at intervals of the sizeable European cluster 18 .

Uromodulin levels
The current study involved measuring the level of concentration of Tamm Horsfall protein (THP).The study showed a significant decrease at P≤0.01 in the patients group with Urinary tract infection (1.38 ± 0.03) Ng/ml compared with the control sample, which was (1.83 ± 0.04) Ng/ml as shown in Table 7.

Relationship of Umod rs12917707 genotype and Uromodulin level
The results in Table 7 revealed significant differences (P<0.05)among control and patients in the uromodulin level in all genotypes.All means of genotypes in control were significantly higher than those in patients.The potential mechanisms of the association of SNPs (including the top rs12917707) in the promoter of UMOD include potential cis-acting effects that could modulate the transcription of uromodulin by the TAL cells and, thus its excretion in the urine.

Discussion
There was a significant reduction in the glomerular filtration rate (GFR) in diabetic patients with macroalbuminuria that carrying a common variant GG genotype when compared with those having GT genotype 14 While, Adam and Elamin in Saudis who showed no significant association of rs12917707 with the Non-diabetic end-stage renal disease and neither of the SNP showed any association with the renal function determinants, serum albumin, and alkaline phosphatase enzyme; OR=1.73; 95%CI:0.50 -5.94, P value .38 for the T allele in the homozygous recessive model and OR=1.52; 95%CI: 0.36 -6.30, P value .56 for the T allele in the heterozygous model as compared to the homozygous model of the reference G allele 15 .This may reflect that the patients have more mutations as the mutation is one factor that influences the allele frequency (mutation, natural selection, migration (gene flow), genetic drift, evolution) 19 .The primary function of the UMOD protein is innate immunity; it blocks the adhesion of E. coli fimbriae to epithelial cells 20 .The T allele of rs12917707 within the promoter region of the UMOD gene is associated with a lower level of urinary uromodulin excretion 21 .So, it is expected to have a higher frequency of T alleles in UTI patients than controls.The high concentration of THP was similar to that obtained by Rajab and HassanWho mentioned that the high values of THP were found in the urine of patients with negative urine culture and control groups 22 .It was close enough to a study done by Khalif and Suleiman, who recorded that The current study involved measuring the level of concentration of Tamm Horsfall protein (THP).The study showed a significant decrease at P≤0.05 in (43%) and the rate of individuals (6.2 ± 38.3) Ng/ml compared with the control sample, which was (7.4 ± 256.2) Ng/ml 23 .Ibrahim et al. found that the level of uromodulin in serum was lowest in diabetic patients with macroalbuminuria, While Ibrahim and others found that microalbuminuria was higher in diabetic patients with normoalbuminuria and highest in non-diabetic control subjects 14 .Another study conducted by Sejdiu and Thorffvit mentioned that These cells produce Tamm-Horsfall protein (THP), which is a low excretion of THP associated with the development of renal insufficiency or cardiovascular disease 24 .On the other hand, lower THP values in the patient's group might be due to consumption of the THP urine content in defense against bacteria or fatigue of TAL cells due to higher bacterial numbers in urine 25,22 .This result was agreed with the findings of 26,27 .
The UMOD gene is exclusively transcribed in the cells lining the thick ascending limb of the loop of Henle and the early distal convoluted tubule. 28From here, uromodulin is cleaved into urine, which can be found free or as an aggregate.Other studies of individuals with rare monogenetic UMOD-related disease found substantially lower urinary uromodulin in patients compared with control subjects 25,29 .This may result from the suppression of wild-type uromodulin excretion by the harmful intracellular effects of mutant uromodulin 25 .In small, patient-oriented studies, lower concentrations of urinary uromodulin have also been observed in individuals with other forms of advanced kidney disease, and reduced urinary uromodulin concentrations have therefore been considered a marker of distal tubular cell damage 30 .A possible explanation for the discrepancy of our findings compared with patients with monogenetic UMOD disease is that different mutations in the UMOD gene may have different effects on the gene product, resulting in differential changes in urinary uromodulin concentrations.Evidence from monogenetic disease suggests that these mutations cause protein-processing defects, which may result in a harmful effect of mutated uromodulin retained in the kidney 31 .Higher amounts of urinary uromodulin, as we observed, could be due to increased transcription, faster protein maturation, less efficient binding of its anchor, or increased cleavage into the urine.The association of higher urinary uromodulin concentrations with incident CKD may indicate that higher uromodulin concentrations in urine are harmful or that wild-type uromodulin has a protective function inside the cell before its secretion.Alternatively, the genetic variant may result in altered glycosylation of uromodulin, which may affect some of the previously described protein functions, such as a role in innate immunity 21 .About 30% of the THP/ uromodulin molecule consists of carbohydrates, comprising at least five Nglycosidically bound sugar chains.In addition to the structural differences in THP, also affected by Sialic acid residues, it may be necessary for this action of THP to prevent UTIS and to inhibit stone formation.THP of healthy humans contains large amounts of sialic acid (about 5% of total molecular Weight).Therefore, the lower celiac acid leads to a defect in THP function 32 .These results were in accordance with the findings of 33.The amount of THP can be influenced by many physiological and pathological factors, which include urine volume, pregnancy, diabetes mellitus, diet, exercise, stone formation, and recurrent UTIs 34 .Through our current study, which included the study of the association of rs12917707 genotypes with urinary uromodulin levels, we observed that All means of genotypes in control were significantly higher than those in patients.The uromodulin level was not significantly decreased in patients with macroalbuminuria carrying GG genotype compared with patients carrying GT genotype 14 .The researcher Olden et al. (NO of research) noted that The G allele of rs12917707 was consistently associated with higher urinary uromodulin levels in an additive manner and with lower levels of GFR in CKD.The 2-fold increase in participants harboring the GG genotype was observed in the six cohorts investigated.This SNP was previously reported in association with GFR and CKD 35 .Previous GWAS have shown that promoter variants in the UMOD gene in high LD with our top rs12917707 SNP are associated with eGFR and CKD.A first GWAS with nearly 20,000 individuals from population-based studies in the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium identified rs12917707, whereby each copy of the minor allele T was associated with higher levels of eGFR and a lower risk of CKD 36 .Uromodulin Blocks E. coli adhesion proteins to prevent UTI infections, So lower uromodulin concentrations were noticed in UTI patients 37 .

Conclusion
Nested T-ARMS PCR is a suitable screening method to detect genetic variations in a uromodulin promoter region.
of Nested T-ARMS PCR for SNPrs12917707 of Urom gene.

Figure 1 .
Figure 1.Gel electrophoresis of genomic DNA in (1%) agarose gel at (70) volts for (30) min, stained with DNA dye, is Red Safe and visualized under UV.