A fully automated method for FTO and ADRB3 genotyping in ... articol arsene cucu fto.pdf · A fully automated method for FTO and ADRB3 genotyping in syndromic (Prader-Willi Syndrome)

Romanian Biotechnological Letters Vol. 22, No. 6, 2016 Copyright © 2016 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER

Romanian Biotechnological Letters, Vol. 22, No. 6, 2016 12026

A fully automated method for FTO and ADRB3 genotyping in syndromic (Prader-Willi Syndrome) and common obesity

Received for publication, October 13th, 2015 Accepted, February 29th, 2016

ARSENE COSMIN1,2, URSU RADU3, VIOLETA DAN2, TRIFANESCU RALUCA1, BADIU CORIN1, NICULESCU MARIUS4,5, PUIU MARIA6, LYGIA ALEXANDRESCU7 AND CUCU NATALIA2,8*

1“Carol Davila” University of Medicine and Pharmacy, National Institute of Endocrinology “CI Parhon”, Department of Endocrynology, Bucharest, Romania 2 Association for Epigenetics and Metabolomics, Bucharest, Romania 3“Carol Davila” University of Medicine and Pharmacy, National Institute of Endocrinology “CI Parhon”, Department of Medical Genetics, Bucharest, Romania 4Titu Maiorescu University, Faculty of Medicine, Bucharest, Romania 5Colentina Clinical Hospital, Bucharest, Romania 6“Victor Babes” University of Medicine and Pharmacy, Deparment of Genetics, Timisoara, Romania 7 Association for Education in Nutrition, Bucharest, Romania 8 University of Bucharest, Faculty of Biology, Department of Genetics, Bucharest, Romania *Address correspondence to: University of Bucharest, Faculty of Biology, Department of Genetics, 1-3 Aleea Portocalilor, sector 5, Bucharest, 060101, Romania Tel.: +4021 318 15 65; e-mail: [email protected]

Abstract

FTO (fat mass and obesity associated gene) and ADRB3 (adrenal-receptor beta 3) genes are considered important candidates as obesity risk genes due to their association with the alteration of the energy balance. This paper describes the implementation of FTO and ADRB3 genotyping, using the fully automated system, based on the quenching probes high resolution melting analysis (HRMA). Detection of single nucleotide polymorphisms (SNPs) in FTO and ADRB3 are useful to guide different treatment decisions in obese patients, depending both on the presence of SNRPN altered imprinting and the mutant FTO and ADRB3 genotypes. Blood and saliva samples wereobtained from selected individuals and the DNA was extracted and prepared for Prader-Willi syndrome (PWS) molecular confirmation using MS-PCR for the promotor/exon1 SNRPN region; also DNA extracted during a previous research program, was selected for further genetic tests of FTO (rs9939609) and ADRB3 (rs4994). In total, samples from 61 individuals were studied, from which 36 had the common obesity diagnosis, and FTO was strongly associated (cca 88%), 4 had the molecular PWS confirmation,and FTO mutant was moderately associated (cca 50%), and 7 had negative molecular PWS test result, but mutant FTO was relatively highly associated (cca 70%); 20 control, nonobese individuals DNA samples were moderately associated also with the mutant FTO (cca 40%). ADRB3 mutant C allele incidence in the obese subjects was only 25%, in PWS patients it was 0% and in the case of clinically suspected patients it was recorded at 14.3%.

Key words: quenching probe method, obesity, Prader-Willi syndrome, single nucleotide polymorphisms

1. Introduction

FTO (fat mass and obesity associated gene) and ADRB3 (adrenal-receptor beta 3) genes are intensively studied because they are associated with the alteration of the energy balance, and they are important candidates among the so-called obesity genes (T. RANKINEN & al. [1], T.O. KILPELAINEN & al. [2], K.A. FAWCETT & al. [3], L. QUAN & al. [4]). The A/T polymorphism (rs9939609) in the fat mass and obesity associated (FTO) gene (which


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codes for the protein alphaketoglutarate-dependent dioxygenase, also known as fat mass and obesity-associated protein, FTO), has been initially associated with a high risk for obesity (T.O. KILPELAINEN & al. [2]). Subsequently, genome-wide association studies (GWAS) revealed the fact that this polymorphism has implications also in lowering the risk for obesity (T.M. FRAYLING & al. [5], A. SCUTERI & al. [6]). Studies in multiples obese cohorts revealed that the individuals homozygous for the A- allele, weigh on average more than those that are homozygous for the T allele (K.A. FAWCETT & al. [3]). ADRB3 rs4994 polymorphism is a controversial marker for obesity, because in some studies the CT-CC genotypes seems to be associated with the gain of weight, while other studies found no correlation between this genotypes and the body weight (K. CLEMENT & al. [7]; D.A. DE LUIS & al. [8], O.V. KOCHETOVA & al. [9]). ADRB3 gene remains an interesting obesity gene candidate, as its product - adrenergic-receptor beta3 (ADRB3) is located mainly on the surface of visceral and brown adipose cells and promotes lipolysis and thermogenesis by noradrenaline release from the sympathetic nerves, stimulated by cold temperature or food consumption (L. TAPPY [10], L. GIRARDIER & al. [11], P.A. INSEL [12]; M.A. SCOFIELD & al. [13]). The single nucleotide polymorphisms FTO rs9939609 and ADRB3 rs4994 are associated with abdominal obesity (E. WIDEN & al. [14], H. KIM-MOTOYAMA & al. [15]), weight gain (K. CLEMENT & al. [7]), and difficulty losing weight (T. YOSHIDA & al. [16], L. QUAN & al. [4]). Therefore, easy and rapid detection of SNPs in FTO or ADRB3 is needed to guide treatment decisions in obese patients. As previously reported, the fully automated system based on the quenching probe (QP) method is extremely effective in detecting mutations (R. TANAKA & al. [17], [N. URESHINO & al., [18], N. URESHINO & al. [19], H. TAKAHASHI & al. [20], TAKAHASHI & al. [21]). Following the addition of a QP and a primer set, a gene amplification response is generated, and the gene arrangement is quickly and easily detected by fluorimetry. In the present study, we will describe and validate analytically and clinically the QP method for detecting FTO or ADRB3 SNPs in different biological materials. 2. Materials and Methods

Patients and samples Samples from 61 individuals were studied, from which 36 with the common obesity

diagnosis, 4 with the molecular PWS confirmation and 7 with negative molecular PWS result; 20 control samples from individuals that did not present any obesity symptom were taken also into study as normal control. Recruitment of the patients and control individuals was based on their signed informed consent, whose protocol was approved by the Ethics Committee of the National Institute of Endocrinology “CI Parhon”, Bucharest. PWS clinically suspected individuals underwent molecular confirmation using methylation-specific PCR (MS-PCR) method for the promotor/exon1 SNRPN region. Venous blood samples were collected and used immediately or stored for DNA extraction, also saliva samples were collected using IsoHelix Buccal Swabs. The SNP analysis was made using as starting material DNA that was extracted from peripheral blood of the patients using DNeasy Blood & Tissue Kit (Qiagen), whole blood and saliva samples. 11 individuals (4 PWS confirmed and 7 non-confirmed individuals), were genotyped using not only DNA extracted from blood but also bucal swab and whole blood, to compare the results on different biological samples.

Detection of FTO and ADRB3 polimorphysms For the detection of FTO (rs9939609) and ADRB3 (rs4994) polymorphisms was used

a fully automated genotyping system (I-densy) (ARKRAY, Inc., Kyoto, Japan). The principle of this method is based on a quenching probe (QP) and the measurement of fluorescence

ARSENE COSMIN, URSU RADU, VIOLETA DAN, TRIFANESCU RALUCA, BADIU CORIN, NICULESCU MARIUS, PUIU MARIA, LYGIA ALEXANDRESCU, CUCU NATALIA


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intensity by the high resolution melting (HRM) analysis method. When the probe is hybridized to the target sequence it produces no fluorescence because the quenching probe contains a guanine quench fluorophore at its 3’ or 5’ end. As the base with the highest electron donating property, guanosine promotes the formation of charge transfer complexes between the fluorophores and nucleosides (C.A.M. SEIDEL & al. [22]; S. STEENKEN & V. JOVANOVIC [23]). The I-densy system uses forward and reverse primers and the guanine QP. The system can perform also the automated DNA extraction and purification from whole blood or saliva using 70 μL of the sample or it can run using as starting material extracted DNA in a volume of 4 μL. The DNA used in this test had the recommended concentration of 1 to 10ng/uL. The automatized platform can analyze 4 samples and 3 genetic targets in each sample at the same time. The multiplex ability is due to the fact that it has receptors for three types of flourophores that can be used simultaneously on three probes. After placing the necessary number of tips, reaction tubes and reagent packs, the system allows fully integrated automatic genotyping from sample pretreatment to gene amplification and signal detection in about 90-100 minutes. The platform incorporates also the Linux system computer processing so that the results can be measured and analyzed within the system. Therefore, after the measurement is complete, the results can be easily printed out and rapidly visualized in due time (Figure 1). For this study, the forward and reverse PCR primers and guanine QP (J-Bio21, Tokyo, Japan) were 5’-catcagttatgcatttagaatgtctgaattattattctag – 3’, 5’- ctccatttctgactgttacctattaaaactttagagtaac-3’ and 5’- TGTGAATTTaGTGATGCAC-3’, for FTO/rs9939609 and for ADRB3/rs4994 5’- gccaccgtgggaggcaacc-3’, 5’- ggctgcggccagcgaagtc-3’, 5’- ctcggagtccAggcg-3’. For FTO and ADRB3, PCR consisted of initial denaturation for 1 min at 95 °C, and 50 cycles of denaturation at 95 °C for 1 sec and annealing at 60 °C for 30 sec. After the PCR was complete, melting temperature (Tm) analyses were performed. The SNPs were identified based on differences in temperature and fluorescence.

Figure 1. Automatically printed out measurement results.

Figure 2. Differential wave pattern for FTO/rs9939609 and for

ADRB3/rs4994



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3. Results and Conclusions

By differentiating the fluorescence intensities by the temperature, the Tm was obtained within 100 min (Figure 2). All 61 individuals were successfully genotyped for FTO and ADRB3 polymorphisms. The results obtained by the QP based automated system, using whole blood, bucal swab, and DNA extracted from blood were identical. Using directly blood or saliva (diluted in distilled, purified water at 1;1 ratio) the time is significantly reduced, by elimination of the DNA extraction.

Table 1. Allele frequencies for FTO rs9939609 A and ADRB3 rs4994 in PWS, PWS suspected individuals not confirmed by the laboratory test, obese and normal individuals

FTO genotype ADRB3 genotype TA TT AA CT TT CC

PWS 50% 25% 25% 0% 100% 0% Non PWS 42.85% 14.3% 42.85% 42,85% 57.15% 0% Obese 41.7% 11.1% 47.2% 25% 75% 0% Normal 30% 60% 10% 16.07% 83.83% 0%

The incidence of FTO mutant A allele in the obese subjects was as high as 88.9%,

50% in PWS patients and 71.4%, in the case of clinically suspected patients that were not confirmed by the laboratory test, while in all the controls A allele was identified in just 40%. The incidence of the ADRB3 mutant C allele in the obese subjects was 25%, 0% PWS patients and 14.3% in the case of clinically suspected patients that were not confirmed by the laboratory test, while the controls revealed no C allele 0%. Detection peaks of FTO (rs9939609) and ADRB3 (rs4994) appear in wave 1 and 2, respectively (Figure 2). Detection peak of FTO (rs9939609) appears under 52°C for sample with homogeneous major only. When minor variant is detected, detection peak appeared at 58°C. Detection peak of ADRB3 (rs4994) is obtained only at 62°C for sample with homogeneous major only. When minor variant is detected, detection peak will appear at 52°C. For FTO genotype detection the wavelength was - 480nm and for ADRB3 detection wavelength was between 520 - 555nm. The efficiency of this method is supported by other studies in which they compared it with other methods like direct sequencing (DS); in that study the results obtained with the QP-system were completely identical to those examined by direct sequencing, thus, indicating the accuracy of the this system. Other good outcomes of the i-densyTM QP-system are linked with the short time it provides the results (within about only 90 min). The direct sequencing method, is more time and effort consuming as it requires several steps (DNA extraction, amplification and sequencing, as well as computational processing) (N. URESHINO & al. [19]). QP (quenching probe)-system using the i-densyTM is accurate, as with DS method, and is superior to the DS method in terms of convenience. Literature is abundant in articles describing oncological tests targeting KRAS mutations: the consistency of the KRAS mutations status in 133 of the 135 lung adenocarcinoma patients, and the positivity of the KRAS mutation with the QP method in two samples that contained few cancer cells, indicate the increased accuracy and sensitivity of the QP method compared with DS (N. URESHINO & al. [19]). This finding was similar to that obtained for the detection of another hematological important mutation: the JAK2 mutation. JAK2V617F was detected in 25 specimens by QP-system, while direct sequencing failed to detect JAK2V617F in 7 of the total of 25. The presence of JAK2V617F mutation in these 7 specimens was confirmed by allele-specific PCR. These findings indicated that QP-system is more sensitive and useful as compared with the direct sequencing for genotyping



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JAK2V617F. (R. TANAKA & al. [17]). Mitsuharu et al. (in S. SUZUKI & al. [24], H. MITSUHARU & al. [25]) developed a MBP-QP system which is about 10 times more sensitive than a traditional QP-system and the detection of the mutation is possible even when only 0.3% of the total number of molecules analyzed presents the modification. This principle is required in neoplastic cells diagnosis or pharmacogenetics when is needed to identify a very small population of cells that contains a genetic modification relevant for the treatment. In order to identify a much smaller number of mutated sequences, this method uses primers that are competitively hybridized to wild type and mutant type sequences. But to favor the amplification of mutant-type sequences, the primers that are reaching with their 3’ end the mutation site, have a number of characteristics: a) the mutant primer has a bigger length, b) the annealing temperature is optimal for the mutant primer. In addition to the MBP-QP system, the Wild Inhibition PCR-QP (WIP-QP) system is also sensitive, even when only 0.1% of the total number of molecules analyzed presents the modification, and can be applied to the detection of in-frame deletion in EGFR exon 19 (I. AKI [26]). Also, tumor derived plasma DNA can be detected using the MBP-QP method (N. SUEOKA-ARAGANE & al. [27]). The higher efficiency of mutant sequence amplification is due to the fact that the primers are paired with wild type DNA fragments complementary at the in-frame deletion site. The DNA fragments are not so tightly bound to the mutant sequence, favoring the amplification of the mutant sequence. This automated QP method proved to be rapid and convenient, reducing the analysis time significantly by automatically performing sample pretreatment to gene amplification and signal detection. When using blood samples or saliva collected on buccal swabs, it takes only 90-100 minute to obtain the result. With its high sensitivity, convenience and speed, the i-densyTM allows for the determination of FTO and ADRB3 genotypes in clinical settings. The FTO and ADRB3 genotyping demonstrated also that the studied FTO and ADRB3 mutants are not uniformly distributed in common obese, syndromal obese and non-obese individuals, at least in the Romanian populations (a total of 61 individuals), when the inclusion criteria were only considered the BMI values as essential and the clinical features of comorbities as secondary features. While the studied FTO rs was a relevant genotype associated with the obese state in terms of BMI, the studied ADRB3 rs was not relevant for the obesity association in the selected population. When the essential criteria were the metabolic syndrome features, like insulin resistance, trigliceride level, LDL cholesterol and HDL cholesterol levels, cardiovascular diseases risk both polymorphisms were relevant. These results were in accordance with the literature which reported studies on Italian, Spanish, South American and Asian populations (R. BRACALE & al.[28], E. WIDÉN & al. [29], A. E. MIRRAKHIMOV & al.[30], N.SAKANE & al.[31], S. KUMAR & al.[32]). However, the ADRB3 gene polymorphism relevance should be more extensively studied in the Caucasian populations in order that the correlations with the above listed comorbidities of obesity to be proved. The i-densyTM system may become an important tool, as it can be adapted theoretically for any mutation by exchanging reagent cartridges which contain specific PCR primers and QProbes for specific mutations, and will enable point-of-care testing in clinical laboratories and for patient-customized therapy for various diseases in the clinical field, such as decision of therapeutic strategy and selection of remedies, and could contribute to personalized medicine in the future. 4. Acknowledgements

This work received financial support through the projects entitled "CERO – Career profile: Romanian Researcher", grant number POSDRU/159/1.5/S/135760, cofinanced by the European Social Fund for Sectoral Operational Programme Human Resources Development



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2007-2013. This research was done in the Center of Genomic Medicine from the University of Medicine and Pharmacy “Victor Babes” Timisoara, POSCCE Project ID: 1854, cod SMIS:48749, “Center of Genomic Medicine v2”, contract nr. 677/09.04.2015. References

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A fully automated method for FTO and ADRB3 genotyping in ... articol arsene cucu fto.pdf · A fully automated method for FTO and ADRB3 genotyping in syndromic (Prader-Willi Syndrome)