Association Between Polymorphism Of Dopamine D₂ Receptor Gene And Effects Of Domperidone In Functional Dyspepsia

PurposeFunctional dyspepsia (FD) is a common gastrointestinal disease, which refers toa clinical syndrome with the postprandial satiety, upper abdominal pains or burningsensation and is not explained by other pathologically based disorders. In Roma IIIprocess, FD is classified into two categories as postprandial distress syndrome andepigastric pain syndrome. Despite of intensive studies during the past few years, thepathogenesis of FD remains unclear. Recently, it is gradually recognized that FD isrelated with fasting gastric motor disorder, postprandial abnormal food distributionin stomach, abnormality of antrum-pylori tube-duodenal coordinate motor anddelayed gastric emptying. There is no specific medicine for the treatment of FD.Gastroprokinetic agents such as domperidone that specific aims at dopamine D2receptor is one of the most used drug for FD in clinic. However, the efficacy ofgastroprokinetic agents in patients with FD has been questioned. Although initialstudies showed a treatment benefit, more recent large trials did not show such abenefit. Other medications such as antacids, gastric mucosa protection, Helicobacter pylori eradication, and anti-anxiety agents have also been reported to be effective insome FD patients.Several evaluation methods have been developed to evaluate the therapeuticeffect of different medications in FD patients. Most of present methods areunpractical due to critical limitations such as being expensive, time-consuming, andlow rate of compliance. Therefore, it is necessary to develop an effective, economicand practical method to evaluate therapeutic effect of different medications in FDpatients. In this study, clinical symptom score combined with real-timeuntrasonography examination of antral motor index was applied to evaluate theeffects of dompridone therapy in FD patients. Furthermore, after the outcomes ofdompridone therapy was determined, single nucleotide polymorphism of dopamineD₂ receptor gene 141C Ins/Del, A-241G and TaqI were analyzed by ligase detectionreaction, in order to investigate potential reason for why therapeutic effects ofdompridone were varied among different FD patients. Materials and Methods1. SubjectsAll subjects were out-patients who visited Department of Gastroenterology, theFirst Affiliated Hospital, College of Medicine, Zhejiang University between June2006 and June 2007. The enroll criteria of FD specific to this study were describedas follow:(1) Age between 18 yr and 65 yr.(2) With symptoms of early satiation and vague abdominal discomfort formore than 4 weeks, with or without other complains described as nausea,occasional vomiting, postprandial fullness, epigastric pain, belching, orburning.(3) Exclusion of non-functional gastrointestinal disease, e.g. malignant tumor,peptic uncler, by gastrointestinal endoscopy examination and/or bariummeal examination in recent 4 weeks.(4) Exclusion of hepatobiliary and pancreatic disease by ultrasonographyexamination and blood biochemistry tests.(5) Abstain of the drugs those may influence the outcomes of the study, e.g.anticonvulsive drugs and any other kind of gastrokinetic compounds.2. Domperidone therapyAll FD patients enrolled in this study received a standard domperidone therapyfor 4 weeks (10 mg, tid, po).3. Observation items3.1 Clinical symptoms Clinical symptoms including early satiation, vague abdominal discomfort,nausea, occasional vomiting, epigastric pain, burning, belching, and postprandialfullness were recorded at the baseline and 4 weeks after domperidone therapy.3.2 Grade of symptomsGrade 0 (none): no symptom; grade 1 (mild): slight discomfortable, unaware ofthe symptoms unless specifically focused; grade 2 (moderate): the symptoms isobviously, but daily work is not influenced; grade 3 (severe): the symptoms is severeand daily work is influenced. As early satiation and vague abdominal discomfort aretwo main complains of FD, the scores for these symptoms are doubled. Totalameliorative rate of symptom (%) was calculated as follow: ameliorative rate =(baseline score - post-treatment score)/baseline score×100%.3.3 Outcomes of the therapy( 1 ) Fully recovery: the symptoms disappeared after the therapy;( 2 ) Marked effective: the symptom improved more than 2 grades;( 3 ) Effective: the symptom improved more than 1 grade;( 4 ) Ineffective: the symptom score was increased or unchanged after the therapy.3.4 Classification of the subjectsAll the FD patients whom received domperidone therapy for 4 weeks wereclassified into effective group and ineffective group according to the outcomes ofthe therapy. According to literatures and experiences, the patient with totalameliorative rate greater than 60% and/or the symptom improved more than 1 gradewas classified as effective group. The remaining patients were classified asineffective group. 4. Real-time ultrasonography examinationIn order to evaluate gastric emptying of FD patients, the following parametersof real-time ultrasonography were recorded:4.1 S₀:After abstained from gastroprokinetic agents for 3 days and an overnight fast,the subjects sat in a chair, leaned slightly backwards, an ultrasound probe waspositioned vertically to permit simulataneous visualization of the antrum, thesuperior mensenteric artery, and the abdominal aorta. Antral area was estimated bytracing the mucosal side of the antrum with the built-in caliper. The area wasdetermined as S₀.4.2â–³S:After ingested 500ml 10% glucose within 5 min, an ultrasound probe waspositioned vertically to permit simulataneous visualization of the antrum, thesuperior mensenteric artery, and the abdominal aorta. Immediately after ingestion,relaxed antral area and contracted antral area were estimated by tracing the mucosalside of the antrum with the built-in caliper for 6 times. The amplitude of contraction(â–³S) was calculated from the maximal reduction of the antral area for eachcontraction:â–³S=relaxed area-contracted area.4.3 F:The total times of antral contraction were recorded for 4 minutes. Thefrequency (F) of antral contractions was defined as the average number ofcontractions per min. 4.4 MI:Antral motor index (MI) was expressed as the multiplication of the meanamplitude and frequency of contractions: MI=â–³S×F. It is reported that MI is arealizing parameter for gastric empty by means of real-time untrasonography.Therefore, we applied this parameter in the present study to evaluate gastric emptyin FD patients.5. Determination of SNP sites of dopamine D2 receptor gene 141C Ins/Del,A-241G and TaqI..6. Primer and Probe6.1 Primer 6.2 Probe 7. Proposal for ligase detection reaction7.1 Separation of peripheral PMNC by Ficoll density gradient centrifugation.7.2 Genomic DNA isolationGenomic DNA was isolation from PMNC using DNA isolation kit (AXYGEN,AxyPrep-96 Blood Genomic DNA Kit). The DNA samples were run in 0.8%agarose gel by electrophoresis at 100-150V, and standardization to finalconcentration of 50ng/L.7.3 Materials used for ligase detection reaction( 1 ) Qiagen Hotstar Taq polymerase kit (5units/μl, buffer, Q-solution,Mg2+).( 2 ) 10M NaOH: NaOH 40g and add ddH₂O to final volume of 100ml.stored at 4℃for further use.( 3 ) 1MTris-HCl (PH8.0): Tris 12.1g and add ddH₂O to final volume of100ml, PH=8.0, stored at 4℃for further use.( 4 ) 0.5M EDTA (PH8.0): EDTA 18.61g and add ddH₂O to final volume of100ml, PH=8.0, stored at 4℃for further use.( 5 ) TE buffer: Tris-HCl 10mM, EDTA 1mM, PH=8.0, stored at 4℃forfurther use.( 6 ) 75% ethanol: 100% ethanol 75ml and add ddH2O to final volume of100ml, stored at 4℃for further use.( 7 ) 5×loading buffer: bromphenol blue0.125g, glycerine 32g and addddH2O to final volume of 50ml.( 8 ) EB (10mg/ml): EB 50mg and added 5ml DEPC H2O, stored away from light.( 9 ) 5×TBE: Tris 54g, boracic acid 27.5g, 0.5mol/L EDTA 20ml, addedddH2O to final volume of 1000ml, PH=8.0.7.4 Genotyping analysis of DRD2 141C Ins/Del, A-241G and TaqI( 1 ) Genomic DNA PCR reaction( 2 ) Detection of concentration, purity and integrality of genomic DNA:Genomic DNA was separated 0.8% agarose gel by electrophoresis at120V for 20-30min. The integrality of genomic DNA was detected inimage system.( 3 ) Ligase detection reaction.Step 1. Amplification: Amplify the fragment that contains the SNP sites byPCR using specific pairs of primers.1) PCR Master Mix was prepared and described as follows (20uLsystem)The following materials were added in to a 1.5ml eppendorf tube: 200ulPCR-buffer (10×), 60ul Mg2+ (100 mM), 200ul dNTP (20mM/each), 20ul Taqenzyme (5U/ul), 400ul Q-solution (4×), 40ul primer (5pM), and 980ul ddH₂O. Afterbeen mixed, 19ul of the mixtures and 1ul of DNA sample were added in to a 200ulPCR reaction tube. 2) The program for PCR reaction in Perkin-Elmer Gene Amp PCR Systems9600 was described as follow:3) After the amplification reaction, 2μl product was added to 3.0% agarose gel,and electrophoresis in 0.5×TBE buffer, to evaluate the result of PCR reaction.4) Remaining samples were stored in -20℃for further use.Step 2&3. Ligation and Detection: For each detection site, a pair ofallele-discrimination oligonucleotide detection probes and a single oligonucleotidedetection probe is designed. The discrimination oligonucleotide detection probes,containing 14 nucleotides that differ only at their 3' terminal nucleotides, shouldcomplement the corresponding SNP target sequence and contain a biotin label at its5' end. The detection probe, containing 14 nucleotides with the sequenceimmediately adjacent to SNP site, is labeled with a FITC at its 3' end andphosphorylated at its 5' end for ligation reaction. When the target DNA hybridizeswith the discriminating detection probes-detection probe, the ligation reactions startsimultaneously during the LDR cycles in the presence of a thermostable DNAligase.After denaturing the amplicon of PCR-LDR, the discrimination probe, thedetector, and the ligase product are separated from the target template. The ligaseproduct that has both biotin and FITC can form a complex with colored latexparticles through biotin-streptavidin interaction. The T-line has anti-FITC antibody,so as the compound moves down the lateral-flow DNA strip, those with both biotinand FITC will be captured at T-line to form a red line. In other words, perfect matchreaction and single-base mismatch reaction can be distinguished by the coloration at the T-line; therefore, the genotype of the SNP is easily identified.1) The same volume of ddH2O was added in to the PCR product after largescale amplification, the mixture was used as template for ligase reaction.2) Ligase reaction mix was prepared and described as follows (10ul system)The following materials were added in to a 1.5ml eppendorf tube: 100ul buffer(10×), 100ul Probe Mix, 5ulligase, 695ul dd H₂O. After been mixed, 9ul of mixtureand 1ul of PGR product were added in to a 200ul PCR reaction tube.3) The program for PCR reaction in Perkin-Elmer Gene Amp PCR Systems9600 was described as follow:4) 1μl LDR product, 1μl ABI GS-500 ROX marker, and 1μl deionizedformamide loading buffer were mixed, denaturing at 95℃for 2 min, cooledimmediately with iced-water, electrophoresis at 3000V for 2.5 h, and analysis wereperformed using the GENESCAN^TM672 software.5) Remaining samples were stored in -20℃for further use.8. Genemapper Data analysis:Gene types were determined by Genemapper data anylysis immediate diagram. Results1. Evaluation of therapeutic effect of domperidone1.1 Outcomes of domperidone therapyAccording to clinical symptom scores, a total of 74 FD patients were classifiedas effective group (21 males and 53 females, mean age 41.50±9.80 yr), and 73 FDpatients were classified as ineffective group (23 males and 50 females, mean age41.30±11.20yr).1.2 Real-time untrasonographyThe results of real-time untrasonography correlated with clinical symptomscores. In effective group, domperidone therapy significantly increased amplitude ofcontraction from 416.180±22.093 to 515.419±21.606 (P<0.01). The frequency ofcontraction was also significantly increased from 2.030±0.226 to 2.737±0.268(P<0.01). Most importantly, antral motor index was also significantly increasedfrom 847.061±119.978 to 1410.922±154.833 by domperidone therapy (P<0.01).In ineffective group, the amplitude of contraction, frequency of contraction, andMI was unaffected by domperidone therapy. The amplitude of contraction was416.269±23.046 pre-treatment and 416.838±20.294 post-treatment (P>0.05), thefrequency was 2.065±0.224 pre-treatment and 2.038±0.194 post-treatment(P>0.05), MI was 859.931±107.763 pre-treatment and 850.141±97.956post-treatment (P>0.05).2. Analysis of single nucleotide polymorphism2.1 Polymorphism of DRD₂-141C Ins/DelIn effective group, the genotype frequencies of "C", "C/-", and "-" were79.73%, 17.57%, and 2.70%, respectively; whereas 79.45%, 19.18%, and 1.37% in ineffective group (P>0.05). Also, the alleles frequencies were 88.51% and 11.49% ineffective group, whereas 89.04% and 10.96% in ineffective group (P>0.05).2.2 Polymorphism of DRD₂-A-241GIn effective group, the genotype frequencies of "A", "A/G" , "G" were58.11%, 37.84%, and 4.05%, respectively; whereas 61.64%, 35.62%, and 2.74% inineffective group. The alleles frequencies were 77.03% and 22.97% in effectivegroup, whereas 79.45% and 20.55% in ineffective group. The genotype or allelefrequencies were not statistical significantly different between two groups (P>0.05).2.3 Polymorphism of DRD₂-TaqIIn effective group, the genotype frequencies of "C", "C/T", and T were51.35%, 31.08%, and 17.57%, respectively, the allele frequencies were 66.89% and33.11%; whereas in ineffective group, the genotype frequencies of "C", "C/T", and"T" were 17.81%, 52.05%, and 30.14%, respectively, the allele frequencies were43.84% and 56.16%. The difference was statistically differently between the twogroups (P<0.01; Pearson x² test). Conclusions1,Clinical symptom scores combined with real-time ultrasonography iseffective to evaluate the therapeutic effect of domperidone in FD patients.2,The therapeutic effect of domperidone in FD patients was properlyassociated with polymorphism of dopamine D2 receptor gene (DRD2) TaqI,but not with the polymorphism of DRD2-141C Ins/Del and A-241G.