Association of CYP 2 C 19 genotype with type 2 diabetes

Background: CYP2C19 is a major isoform of cytochrome P450 that metabolizes a number of commonly prescribed drugs such as omeprazole, diazepam, tolbutamide and propranolol. Its expression is regulated by the constitutive androstane receptor (CAR), involved in glucocorticoids synthesis. Since a number of crosslinks have been described for CYPs and some hormones, an association of CYP2C19 with type 2 diabetes is likely. Methods: Two groups were studied, 352 diagnosed with type 2 diabetes patients and 342 healthy volunteers form Mexico City. Both groups were tested for CYP2C19*2 and *3 alleles. We carried out an allelic discrimination using TaqMan assay for *2, and used FRET sensor and anchor probes for *3. Results: Ninety one percent of the subjects had the wild type allele, 9% have the *2 allele; no subject presented the *3 allele. The CYP2C19*2 allele is associated with type 2 diabetes (p = 0.012). Admixmap program was used to correct the admixture of this population and get the correlation. This was further confirmed in a linear model with a 67% power and by the method of Strom and Wienker for association on subjects within the mean range of Amerindian ancestry only (60%). Conclusion: Type 2 diabetes patients have significatly more *2 allele than healthy volunteers, more evident for the patients with the homocygous genotype.


INTRODUCTION
The P450 Cytochrome gene super-family is involved in the synthesis of steroids, as well as in the metabolism of cholesterol [1].Cytochrome genes exhibit a number of mutations that alter their activity [2].In the case of CYP-2C19 the most common mutations *2 and *3 show no enzymatic activity.For this isoform, CYP2C19, known for metabolizing both, mephenytoin and omeprazole it has been demonstrated to have the greatest genetic variability among human populations [3].Some of them, Asians for example [4], present a very high frequency of mutations, Latin-Americans on the other hand, have a small frequency of inactive alleles [5,6], while Eskimo Inuits do not seem to present any mutations at all [7].
CYP2C19 metabolizes an estimated of 8% of all therapeutic drugs in current use [8], knowing the variants which do not present enzymatic activity is of huge interest for physicians prescribing drugs such as tolbutamide, omeprazole, proguanil, fluoxetine, citalopram, diazepam, propanolol, diclofenac, indomethacine and others.
Previous studies have identified the presence of CYP2C19*2 in Bolivians [5] and in Colombians [6].Whereas in Inuits no mutations have been identified [7].Asians have a high frequency of 2C19 mutations, actually they are so far the only population with 2C19*3 mutations.For this reason it is somehow obvious to look for the presence of this allele in our Mexican subjects since Amerindian populations came from Asia 30,000 to 45,000 years ago [9].In addition, characterizing our populations is an important advance towards a personalized medicine.
Nuclear receptors are transcription factors that modulate cellular responses to small lipophyllic molecules such as steroids and others.They also regulate the expres-sion of CYPs.Three of this nuclear receptors have been described to regulate the expression of CYP2C19, CXR (chicken xenobiotic receptor), PXR (pregnane X receptor) and CAR [10].All this elements are a linkage of CYP2C19 with its role on cholesterol elimination and on the physiology of other steroid hormones like cortisol.Both process are tightly involved in the development of obesity and type 2 diabetes.This regulation of the CYP2C19 is regulated by a complex crosslinking involving cholesterol levels, glucocorticoid activity and the action of other steroid hormones; therefore, the interest to look for a possible association of CYP2C19 with type 2 diabetes.

Subjects
We included 367 men and 330 women (age range: 18-76 years old; body mass index averages of 28 for males and 30 for females, Table 1) from the DNA library of the Biochemistry Research Unit of Medical Center 21 st century, IMSS, located in Mexico City.No relatives were included in the groups.The corresponding ethics and research committees approved the study.All participants signed the informed consent.About half of the subjects participating in this study were patients with type 2 diabetes, and because they were already part of other studies, we had previously characterized their population admixture.

Autosomal Ancestry-Informative Markers (AIMs)
Stratification in the subjects of this study was corrected using previously reported data [11], these markers have large frequency differences among populations of Native American (65%), European (30%), and West African (5%) ancestry.

Biochemical Profile
Clinical evaluation was made after 12 h overnight fasting.Blood samples were taken and used to measure glucose, triglycerides, total, high and low density cholesterol levels were assayed using an ILab 350 Clinical Chemistry System (Instrumentation laboratory, Mexico).All participants were interviewed by a physician who collected data about their weight, height, systolic and diastolic blood pressure.Quantitative measurements of fasting plasma insulin were carried out using the chemiluminescence's assay according to the manufacturer's instructtions (Immulite, France).

DNA Samples
DNA was extracted from blood cells using the Qiagen columns according to the manufacturer's recommendations (Qiagen, Chatsworth, CA, USA).Purity was verified by UV absorption at 260/280 nm and DNA integrity was checked by electrophoresis in 0.8% agarose gels, stained with ethidium bromide.

Allele-Specific TaqMan PCR for CYP2C19*2
DNA samples were analyzed for CYP2C19*2 using the TaqMan PCR assay to detect G > A polymorphism according to the manufacturer's instructions.Amplification and detection were made using the ABI PRISM 7000 (Applied Biosystems, USA) system with the following profile: a denaturing cycle of 95ºC for 10 min and 40 cycles of 92ºC for 15 s, extension phase of 60ºC for 1 min.Samples were judged positive for *2 when the value of the emitted fluorescence was greater than the threshold calculated by the instrument's software.Displayed as an allelic discrimination plot.Wild type allele CYP2C19*1 was labeled with VIC and *2 allele with FAM.VIC was for TTCCCGGGAACCCA and FAM for ATTTCCCAGGAACCCA (SNP showed in boldface).

CYP2C19*3 Evaluation by FRET Technology
This polymorphism was identified following the method described by Borlak and Thum [12] using fluorescence resonance energy transfer (FRET) to assay genomic G > A Sensor and anchor probes were labeled with fluoresceein and Red640, respectively.PCR was run with a 5 min denaturation step.Fifty cycles with 7 sec of annealing (48ºC) and 14 sec of extension (72ºC) in a Light Cycler (Model 1.2, Roche diagnostics, Germany).The melting curve was done from 40ºC (with 30 sec of previous stabilization) to 80ºC with a 0.1ºC/min slope.Tms were 61ºC for the wild CYP2C19*1 type and 67ºC for the CYP2 C19*3 genotype.Software used was Light Cycler (Roche, Version 4).Control probes for *1 and *3 were used as validation of fusion points.The assay was validated using standards provided by Roche.

Statistical Analysis
In order to correct the stratification of this population, ADMIXMAP program was employed to look for casecontrol (shown in Table 2) associations using logistic regression and to measure the Hardy-Weinberg equilibrium.Iterations were tested to 4000, burning was selected to 200, "every" parameter to 7. The rest of parameters were defaults.Co-variables were log of age and gender.Hardy-Weinberg equilibrium was also tested after the Sasieni method implemented by Strom and Wienker [13] (available at: http://ihg2.helmholtz-muenchen.de/ cgi-bin/hw/hwa1.pl)[14].Proportions of genotypes were compared by the Fisher exact test.Odds ratios and their corresponding 95% confidence intervals were also estimated.All p values < 0.05 were considered statistically significant.The association between total cholesterol, HDL-C, LDL-C levels with the CYP2C19*2 allele was analyzed by the type III linear model with and without co-variables, including the stratification of the population, health, age, gender, height, body mass, waist-hip index, and BMI.Linear models were also tested using model I for interaction, with an Amerindian index as co-variable as well as log of age and gender.In all tests significance was considered for values less than 0.05.

Characteristics of the Sample and Stratification of the Population
At total of 697 individuals were studied (Table 1).This sample of Mexico City population had been previously characterized for its ancestry informative makers by admixture mapping analysis, showing an estimated proportion of 65% component of Amerindian, 30% European and 5% West African.For this study, the stratification plays a critical role since the frequency of CYP2C19 is biased by ethnic admixture.

Genotypes
CYP2C19*2 genotypes were characterized for all subjects.In our control subjects, 0.6% were homozygous and 15.6% heterozygous for *2.In T2D patients, 1.4% were homozygous and 17.7% heterozygous for *2.These frequencies were in Hardy-Weinberg equilibrium after Fisher exact test (p = 1.000 for the control volunteers and p = 0.384 for the T2D group.CYP2C19*3 genotypes were not present in none of analyzed samples.The genotype frequencies are shown in Figure 1.

Allele Frequencies
Eight percent of the alleles in the control subjects, and 10% in T2D patients were CYP2C19*2 (Table 3).The odds ratio was 1.22, with 95% confidence interval [0.81-1.84],(p = 0.358), in a dominant model; and 2.57 with a 95% confidence interval of [0.44 -19.26], (p = 0.431) in a recessive model.The CYP2C19*3 allele was analyzed in 460 subjects: 198 control volunteers and 262 type 2 diabetes patients and all of them had the wild type form for the loci 636G > A (Table 2).

There is No Association between the CYP2C19*2 Allele and HDL-C, LDL-C and Total CYP2C19 Association with Type 2 Diabetes
Admixmap program revealed a significance of 0.012 for association of CYP2C19 genotypes with type 2 diabetes.The power for this test is indirectly estimated to be 0.95, using the G*power program.This was confirmed using the linear model I.In this case ethnicity measured as Amerindian content in a 1 to 4 scale.This index was calculated by scaling the results of Amerindian ancestry.Log of age and gender as co-variables were also included.
The significance was of 0.016 for subjects with recessive homozygous genotype.The statistical power for this test was 0.67.When subjects with Amerindian index of 3 were selected, an association was detected using the Sasieni procedure; however in this case type 2 diabetes subjects were not in Hardy-Weinberg equilibrium.Odds ratio for that group is shown in Table 4.
Classical approach using linear model also show the association for CYP2C19 with type 2 diabetes, see Table 2.In that case age and sex were taken as covariables, and the significance was 0.016.

DISCUSSION
Our finding of the *2 allele association with type 2 diab- etes is apparently due to the homozygous genotype group as seen in the raw database.Of six subjects presenting this genotype five were type 2 diabetes patients versus only one in the control group.The only healthy *2/*2 carrier was a young male (40 years) who is probably in high risk for developing type 2 diabetes.Type 2 diabetes is a multifactor disease; the role of CYP2C19 in this pathogenesis can be linked by its interaction with CAR and PXR [15,16].Interestingly Kohalmy [17] have described a CYP2C19 relationship with DHEA which at the same time is related to type 2 diabetes.Type 2 diabetes patients present increased CYP2E1 activity, measured as a decrease of the area under the curve after chlorzoxazone administration.CYP2E1 mRNA, in blood mononuclear cells, was found increased as well [18].On the other hand, it has been previously suggested that free radicals are a risk factor for type 2 diabetes.One source for the suppression of free radicals happens to be CYP activity.Among the involved isoforms, the CYP2C family can play a major role [19].
It has to be pointed out that CYP2C19*2 comprises an haplotype with at least four mutations: 99C > T; 681G > A; 990C > T; 991A > G [20].It has four variants: 2A, 2B, 2C and 2D; the last three, besides the mentioned four mutations also have other substitutions: 276 G > C, 481 G > C and 1213 G > A, respectively.For CYP2C19*2 the 681G > A, SNP was included in the taqman assay.CYP2C19*3 have two haplotypes: 3A (636G > A; 991A > G; 1251A > C) and 3B (636G > A; 991A > G; 1078G > A; 1251A > C) [20].For CYP2C19*3 the SNP of 636 G > A was the one included for the Light Cycler system.lation of Mexico City has the same frequencies for CY-P2C19 wild and *2 alleles than the populations reported in Bolivia and Colombia (Figure 2).The *3 allele was not detected in our population just as in other Latin-Americans.

Figure 1 .
Figure 1.Observed frequency of CYP2C19 genotypes on case (T2D) and control (healthy) groups.Note that the slight higher frequency of *1/*2 genotype together with the much higher frequency of *2/*2 in case group are responsible for the association of CYP2C19 genotype with type 2 diabetes (T2D).

Figure 2 .
Figure 2. Frequencies of main alleles of CYP2C19 on different populations.Colombians, Bolivians and Mexicans have the lower frequencies for *2 allele.But Inuits have no presence of them.While Asians have the greatest frequencies for the *2 and only they present *3.

Table 1 .
General characteristics of the group studied.
Values are Mean (Standard Deviation).

Table 2 .
Linear model for CYP2C19 associtation with type 2 diabetes.