Snp genotyping within ins, insr, tcf7l2 and ppr- y genes associated with type 2 diabetes in india
Alireza Shahrjerdi,
1,* Mohsen najafi,
2
1. Razi vaccin &Serum Research Institute, Agricultural Research, Educatuion and Extension (AREEO), Tehran, Iran
2. School of Medicine, Faculty of Medical Sciences, IAU, Sari, Iran
Abstract
Introduction
A pilot population-based study was carried out to evaluate linkage between singlenucleotide
polymorphisms within the known risk candidate genes of type 2 diabetes in
western indian population. the study population was comprised of 25 normal glucosetolerant
individuals (12 males and 13 females) and 25 type 2 diabetes patients (13 males
and 12 females). the genes and their corresponding single-nucleotide polymorphisms that
we screened were ins (rs5505), insr (rs10500204), tcf7l2 (rs7903146) and ppar-g
(rs1801282). the risk alleles were more frequent in the subjects with type 2 diabetes.
copyright ª 2013, jpr solutions; published by reed elsevier india pvt. ltd. all rights
reserved.
Methods
2.1. human subjects
the study subjects were a part of an ongoing insulin resistance
study being undertaken by department of life sciences,
university of mumbai in association with medical genetics
study centre, geneombio technologies, india. the study was
directed towards a sub-population living in the state of
maharashtra (india) and suffering from t2d. blood, serum and
dna samples of 25 t2d cases (13 males and 12 females) and 25
normal glucose tolerant (ngt) (12 males, 13 females) individuals
were studied. all blood samples were obtained at the
baseline visit and all participants provided a written informed
consent for investigations.
the recruited members of the resource population were
above the age of 25 years with an average of (mean sd)
44.6 10.42 and 49.6 12.5 years for control and t2d group
respectively. the diagnosis of t2d was confirmed by
analyzing medical records for symptoms, use of medication,
and measuring of fasting glucose levels following the guidelines
of american diabetes association (diabetes care,
december 29, 2009; january 2010, supplement).
primary inclusion criteria comprised a medical record
indicating either 1) a fasting plasma glucose level of 126 mg/
dl or 7.0mmafter aminimum of 12 h fasting or 2) a 2-h postglucose
level [2-h oral glucose tolerance test (ogtt)] of
200 mg/dl or 11.1 mm on more than one occasion with
symptoms of diabetes.
impaired glucose tolerance was defined as a fasting plasma
glucose level of 100 mg/dl (5.6 mm) but 126 mg/dl (7.0 mm)
or a 2-h ogtt of 140 mg/dl (7.8 mm) but 200 mg/dl
(11.1 mm).
in cases where a medical report was not readily available,
self-reported t2d cases were confirmed by performing a 2-h
ogtt. the 2-h ogtts were performed according to the
who criteria (75 g oral load of glucose). body mass index (bmi)
was computed as weight (kg)/height (meter) while waist-tohip
ratio (whr) was calculated as the ratio of abdomen or
waist circumference to hip circumference. details of the ngt
and t2d population mentioned in tables 1 and 2.
the ngt subjects that participated in this study were from
the same subpopulation group from maharashtra. all protocols
were reviewed and approved by the project authorities
at geneombio technologies in pune and a memorandum of
understanding and material transfer agreements for sample
sharing were signed between the two collaborating institutes.
2.2. metabolic assays
quantification of hba1c was done from whole blood. hba1c
levels were determined by turbidometric inhibition immunoassay
(tina quant; roche).
the homeostatic model assessment (homa) was used to
quantify insulin resistance and beta-cell function. homa-ir
value of t2d population was 4.6 0.75 as compared to control
group 2.7 0.44. the homa-b mean value in control and
diabetic population was 196.6 180.17 and 28.7 7.15
respectively. thus indicating insulin resistance and reduction
in beta-cell function in t2d population.
2.3. snp genotyping
dna was extracted from blood cells using standardized
sdsephenol/chloroform method described by sambrook et al
(1989).7 genotyping of samples for single-nucleotide polymorphisms
(snps) within ins (rs5505), insr (rs10500204),
tcf7l2 (rs7903146) and ppar-g (rs1801282) were done according
to halsall et al (2004), bennermo et al (2004), marquezine
et al (2008), and romeo et al (2001) respectively.8e11 for quality
control, 2 replicates of positive controls and 1 replicate of
negative controls were included in each pcr run to match the
concordance. the discrepancy in the concordance was <0.01%.
genotyping success ratewas 100%for all the investigated snps. statistical analysis
the hardyeweinberg equilibrium was used with a one-degree
of freedom goodness-of-fit test separately among cases and
controls with the help of the pearson chi-square test. allelic
frequencies between test and control samples were done
using the chi-square test or the fisher exact probability test,
wherever appropriate.
unconditional logistic regression was used, before and
after adjusting for gender, age and other variants for statistical
analysis of genetic effects measured by the odds ratio (or) and
its corresponding 95% confidence limits. association analyzes
were performed for each polymorphism using the ‘snpassoc’
software.
Results
All samples, including those with t2d (n ¼ 25) and normal
glucose tolerant (n ¼ 25), were genotyped for 4 snp within 4
genes of interest.
a total of 4 genes and 4 snps were identified for genotyping
analysis within each of the samples from the resource population. the details of the gene name, snp identification
number (reference snp or the ‘rs’ number), position of the snp
on the chromosome as indicated by genome build version
37.1 (the fasta sequence of the human chromosomes; build
37; national council of biological information, usa) and frequency
of occurrence of each of the snps in the resource
population are summarized in tables 3 and 4.
the genes and their snps indicate strong association with
conditions of t2d. ins: rs5505 with risk allele ‘t’ was observed
in the present study population. the risk allele ‘t’ was found
58% in t2d cases (or ¼ 1.52) compared to 38% in the control
group thus showing a strong link with decreased insulin level.
among the t2d group, 13 cases had the risk allele ‘t’ as
compared to only 5 cases in control group. same risk allele ‘t’
was also reported by boesgaard et al (2010) in danish and
czech populations.13 the insulin gene variable number tandem
repeat (insevntr) has been extensively studied and is
proposed to exert pleiotropic effects on birth weight and diabetes
susceptibility.14 however, evidence for this has been
conflicting and a role for ins in type 2 diabetes predisposition
has not been definitively established.
Conclusion
The present study provided insight into the association of
snps linked tot2d. the above findings suggest that there is a
co-relation between the risk alleles and susceptibility to t2d
in the present pilot study population. the data raises the
prospects of developing an snp-based genetic prediction test
for detecting genetic predisposition towards this important
lifestyle disease and aid to design better management ideas to
defer or prevent the onset of t2d.
Keywords
Snp
ins
insr
tcf7l2
ppar-y