A Preliminary Study on Single Nucleotide Polymorphisms (SNPs) in SPP1 Gene with Higher Threat of Urolithiasis in Victims from West Bengal, India

in SPP1 Gene with Higher Threat of Urolithiasis in Victims from West Bengal, India Manalee Guha † , Hritwick Banerjee † , Dilip Kumar Pal & Madhusudan Das 1 Department of Zoology, University of Calcutta, Kolkata-700 019, India 2 Department of Biomedical Engineering, National University of Singapore, Singapore117583, Singapore 3 Department of Urology, Institute of Post Graduate Medical Education & Research, Kolkata700 020, India † Authors equally contributed towards this manuscript. *For correspondence. E-mail: guha.manalee@gmail.com; hritwick_banerjee@mtech2012.iitgn.ac.in. Urolithiasis is a painful experience associated with hematuria, damage to kidney tissue and renal failure. It is a multi-factorial disorder while in India, its prevalence is rapidly increasing imposing a large burden for both healthcare and economy globally. In this article, we aimed to evaluate the association between genetic defects in SPP1 gene and urolithiasis from East Indian patients.75 urolithiasis patients were recruited from SSKM Hospital & Institute of Post Graduate Medical Education & Research (IPGME&R), Kolkata, India while 75 healthy controls were recruited from the same community. SNPs based areas of SPP1 gene were analyzed by direct sequencing to identify genetic defects.We identified 3 polymorphisms one synonymous and two 3’UTR variants rs1126616: p.Ala250Ala, rs1126772: 7315 a>g, rs9138: 7471 a>c in SPP1 gene in study individuals. Genotype and allele frequency analysis of these SNPs revealed that, rs9138 SNP was significantly associated with urolithiasis risk in East Indian patients. To our knowledge this is the first study reporting the role of the gene with urolithiasis in the population of West Bengal, India.


Introduction
In dearth attempt in understanding pathogenic mechanisms of urolithiasis causes a crucial barrier to its early detection and treatment.In spite of being a major issue, fragmented studies have conducted towards renal stones in northern Indian region [1][2] , while our published data on genetic association of renal stones in calcium homeostasis 3 is believed to be the first published report in East Indian population.In the realm of renal stones, secreted-phosphoprotein 1(SPP1) is reported to be an important modulator [4][5] , whereas rs9138 (7471 a>c) of SPP1 gene is established to be highly polymorphic [6][7][8][9] .Thus, the rationale of our present study is to analyse association of SPP1 gene variations with urolithiasis in West Bengal, eastern part of India study group population which potentially believed to be the first ever report herein.Urolithiasisis a common painful ailment which forms in kidneys when normal substances of urine become too concentrated 10 .Based on our recent work on genetic association of renal stones in calcium homeostasis pathway, we reported calcification procedure in which genotype frequency of rs9138 polymorphism was considerably connected with the risk of renal stone formation (p-0.001) 3 .To continue the discussion further in the realm of genetically natural inhibitor, references can be made where SPP1 acts as a natural inhibitor of abnormal calcification in the kidneys including crystallization, crystal retention and crystal congregation [11][12][13] .To investigate the connection between SPP1 with West Bengal, India population, a preliminary study of 75 patients having renal stone with age and sex matched 75 healthy individuals is being conducted.Three variants of SPP1 gene reported (rs1126616: p.Ala250Ala, rs1126772: 7315 a>g, rs9138: 7471 a>c) here in our preliminary study.As a future scope, we are continuing our work to establish the correlation between SPP1 gene variants and urolithiasis in larger sample size to understand better of useful early genetic markers.2.

Study participants
The

Genotyping
Blood specimens were drawn from the urolithiasis patients and healthy subjects in ethylenediaminetetraacetic acid (EDTA) anticoagulated vial.Genomic DNA was extracted using the isolation kit (QIAamp Blood Kit, QIAGEN,Germany).SNPs based areas were amplified by PCR using a gradient thermal cycler due to their reported association.
Primer details were: P1: 5'-Forward TACCATATTCCCATCCCTAGCC P1: 5'-Reverse GGAGTTTCCATGAAGCCACAA P2: 5'-Forward CCAAAGTCAGCCGTGAAT P2: 5'-Reverse AAACATCACACCGTACCC The cycling conditions that we followed is as herein: (i) preliminary denaturation was conducted at 95°C for 3min trailed by 42 cycles for 30s, (ii) following denaturation was annealing procedure conducted at 58°C-64°C for 45s, extension at 72°C for 45s,and (iii) finally it was extension undergone at 72°C for 5min.The Cycle sequencing PCR products were fulfilled using the Big dye terminator kit v 3.1 (Applied Biosystems, Foster City, USA) on an ABI prism DNA sequencer (Model 3700; Applied Biosystems, Foster City, USA).Finally, sequencing was performed in both forward and reverse directions and sequences were aligned in between case and control individuals using ClustalW program.

Statistical methods
Comparison of mean values of continuous self-determining variables (age, serum creatinine, calcium) between cases and controls were done using t-test.The genotype and allele frequencies in cases were compared with control subjects using the chi-square test.Chi-squared or Fisher exact test were taken when desirable for examining the association of each SNP.Chisquare tests were used to determine whether individual SNP are in Hardy-Weinberg equilibrium (p<0.001).Mann-Whitney U test was done to investigate nonparametric variables.
Association analyses for dominant, recessive and co-dominant effect of each of the polymorphisms were measured using Graphpad Instat software (SanDiego, CA).

Results and Discussion
Our  SPP1 as a regulator of mineralization is highly related with calcification process especially in the matrix of renal stones [14][15] .It was reported that the concentrations of SPP1was dropping in urine in stone formers 16 .Genetic association studies with SPP1 gene have been studied by various researchers in various populations either with urolithiasis or various malignant disorders or inflammation, leukocyte recruitment, cell survival and wound healing which paved away for our present study further [17][18][19] .In an earlier published report it was elucidated that SNPs in SPP1 gene poses nontrivial influence in patients with recurrent urolithiasis 20 .These relevant earlier results demonstrate a podium for further investigation of SPP1 significance in urolithiasis.In this realm, our preliminary result on Genotype and allele frequency analysis demonstrates that, rs9138 in exon 7 is expressively associated with urolithiasis while serum creatinine and 24 hours urinary calcium excretion revealed substantial difference between study groups (p<0.001).Together these findings strongly indicate, a proper power calculation with larger sample size should be used for future research direction with a more robust correlation between SPP1 and urolithiasis therein.

Table 1 . Clinical variables of urolithiasis patients and controls
Kolkata, India with participant's written consent.*p-values given are averages ± standard deviation

preprints.org) | NOT PEER-REVIEWED | Posted: 19 November 2018 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 19 November 2018 doi:10.20944/preprints201811.0440.v1 increased
study has been conducted with total 75 urolithiasis patients including 51(68%) risk (OR= 2.94; 95% CI=1.56-5.55;p=0.001) in urolithiasis patients.The allele frequencies of rs1126616 and rs1126772 in the present study were quiet similar compared to European and Asians (NCBI Hapmap project).Towards the end, allele frequencies of rs9138 male and 24(32%) female participants.Mean age with standard deviation (SD) for the case group was 39.32±10.19years.Our study population is unbiased in terms of age and sex.Males have higher frequency of manifestation of stones than female participants (2:1).Compared to control individuals, there was no significant difference documented about BMI at the time of sampling.In our study serum creatinine, serum calcium and urinary calcium excretion in both cases and controls were evaluated.The analysis released that levels of serum creatinine [(case-The risk allele frequency of rs1126616 (T) were 38% and 28% for cases and controls respectively (OR = 1.58; 95% CI= 0.97-2.56;p=0.086).G allele of rs1126772 had 2.94 times Preprints (www.

Table 2 . Allele frequencies of SPP1 gene polymorphisms of urolithiasis patients and controls
*p-values for chi squared test.OR, odds ratio; CI, confidence interval Preprints (www.