心境障碍与5-羟色胺转运体基因的连锁不平衡

维普资讯 http://www.cqvip.com ISSN 1671—5926 CN 21—1470／R w ．zgfc com kf23385083@sina．com 高树赛，等．心境障碍与 5一羟色胺转运体基因的连锁不平衡 DNA polymerase (1 U)， 2×GC buffer I (10 L)， and the two primers(both of 1 0 mmol／L)．The reaction was performed in 2 cy— cles of predenaturation at 94℃ for 3 minutes．94℃ for 30 seconds， 63℃ for 30 seconds．and 72℃ for 1 minute．2 cycles of 94℃ for 30 seconds，62℃ for 30 seconds，72℃ for 1 minute，and 34 cy— cles of 94 ℃ for 30 seconds． 61℃ for 30 seconds． 72℃ for 1 minute foIlowed by a final extension at 72℃ for 6 minutes．( 5一 HTrvN ITR loci：Th e total reaction system was 20 L containing DNA template(50 ng genomic DNA)，TaKaRa Taq polymerase(1 U)，two primers(both of 10 mmol／L)，and lOx PCR buffer(2 L)．After predenaturation at 95 oC for 3 minutes，the reaction was perform ed in 34 cycles of 95℃ for 1 minute．55℃ for 45 seconds．72℃ for 1 minute with a finaI extension at 72℃ for 10 minutes． ( 5一HTT一 3’UTRG／T Ioci： Th e totaI reacting system of 20 L¨ contained DNA template(50 ng genomic DNA)，Taq polymerase(1 U)，the primers (both of 10 mmol／L)， 10xPCR buffer (2 tLL)， and 1．5 mmo1／L MgC1’． Atier predenaturation at 95℃ for 3 minutes， the reaction was perform ed in 35 cycles of 94℃ for 30 seconds．56℃ for 1 minute． and 72℃ for 1 minute with a finaI extension at 72℃ for 6 minutes． MseI endonuclease was added for digestion at 37℃ in water baths overnight． Th e restriction enzyme system in a totaI volume of 1 0 L consisted of PCR product (2 L)， MseI (2 U)， 10xbuffer R+f1 IxL1 and adequate amount of ultrapure water． Gel electrophoresis and genotyping：The PCR product of the 3 loci fincluding the digestion products of 5一HTT3’UTRG／T Ioci1 under— went 6％ nondenaturing polyacrylamide gel electrophoresis(PAGE) for separation for 3-4 hours， and stained with silver nitrate． Th e genotypes of the 3 Ipci were determ ined according to the patterns of the bands displayed by electrophoresis． Statistical analysis：TDT was used for linkage disequilibrium analy— sis． In the core families comprising the patients and their parents， the probability of transmission of an allele from the parents wi th norm aI phenotype to their offsprings with mood discrder were eval uated with the parents as the internal contro1．In this study．extend． ed transmission／disequilibrium test伍TDrn software was adopted for statisticaI analysis of the association of each allele of the Ipci and the haploids with different combinations of the alleles with mood discrder[61． Th e laboratory experiment and part of the statistical analysis were completed in the Department of Forensic Medicine， College of Basic MedicaI Sciences．Sun Yat—sen University． RESULTS Quantitative analysis of participants Altogether 72 families， 247 subjects， were enrolled in this study including 65 COreS families containing 193 subjects 【patients and their parents， but 2 families only had a father)， 4 sibling families with 17 subjects(more than 2 siblings were affected)，and 3 high— risk family involving 37 subjects(over 3 members in 3 generations within two genealogies were affected)．Of these subjects，232 in 72 core families were eligible for hereditary correlation analysis and divided into④ patient group with 90 patients consisting of 5 1 male and 39 female patients aged 15-51 years with a mean of (24_+7)years，whose onset age varied from 12 to 35 years with an average of (20_+5) years， the course of illness ranged from 0．5 month to 1 8 years with the median of 4 years．In these patients，56 had bipolar disorder，and 34 had major depression(including sin— gle—episode depression and recurrent major depression)．( Parents group，including 142 parents consisting of 72 fathers and 70 moth— ers aged 36—78 years with an average of(5 l+8)years． Statistical inference ETDT analysis of 3 polymorphie loci in serotonin transporter gene (Table 1) Table 1 shows that in the 72 core families， the empirical P values 171 of the 3 loci did not exhibit statistical significance，suggesting that there was no linkage disequilibrium between these 3 loci and mood discrder． Table l Result of transmission／disequilibrium test of serotonin trans— porter gene f5一H1Tn allele in 72 mood disorder core families Transmission or non—transmission is the number of families in which the heterozygous parents tran s mitted the alleles to their offspring． The P value is the empirical value after 1 000 corrections with Monte Carlo sireulation； The detected 3 alIdes in 5．HT． TI|PR loci by PCR were s，L'L+ with length of 484 bp，528 bp，528+bp，respective— ly；alneligible for ETDT test． Analysis of haploids with different combination of the alleles at the loci of 5-HTT gene(Table 21 Table 2 Analysis of haploids with different combination of the alleles at the loci of serotonin transporter gene (5-HTT) gene in 72 mood disorder corefamilies H ⋯ aploids ． w ． ith di雎 ren T ransmissi0n Non．transmission P aLL eLe cotnblnatlons 5．H 【’rI R．VNTR S 【2 25 29 O_3O 0．58 S 【O 1O 8 O．22 O．71 L I2 2O 15 O．71 O-39 L 10 6 7 008 0 78 L 12 O 2 5．H‘ 【’rI R一3’UTRG／r 31 25 0．64 0．42 r 17 26 1．84 0．17 L ： 22 14 1．78 O．18 I』 r 4 7 O．82 O．36 L C O 2 5 H‘ rrVNTR．3’UTRG／r 1： !G 31 17 4．O8 0．04 1： 1T 14 24 2．63 O．11 1I )p 3 5 1I l1 13 O 17 0 68 5．H‘ 厂T1PH．VNTR．3’U．rRG／r S 2G 31 22 1．53 O．22 S 2T 12 21 2．46 0 12 S OG 4 1 S 0T 5 9 1 14 0．29 L 2G l9 l0 2．79 0．09 L 21、 1 5 L 0G 2 4 L 0T' 4 4 L l2Ga O 2 Transmission or non．trails mission was gous parents transmi tt~ the alleles in Ineligible for multi—allele TDT． the number of families in which the heterozy— different combinations to a haploid offspring．a Table 2 shows that of the 72 core families， haploid formed by the combination of 5．HTT VNTR and 3’UTRG／T was found connected with mood disorder =4．08，empirical P value of 0．04，with 1 000 corrections by Monte Carlo simulation)，but linkage disequilibrium could not be observed in the haploids with other loci(尸>0．05)． DISCUSSIoN Human 5一H I gene locates in the region 0f 17q11．1—12 coding f0r 5一H I protein， which pIavs a kev roIe in serotonin reuptake into the presynaptic neurons闱．OgiIvie e￡ⅡZ examined the reIationship between poIvmorp sm of VNTR in the sec0nd intr0n of 5一HTT 骶me and mood disorder，and f0r the 6rst time demonstrated the as— sociation between 5．HTT gene and maior depression．Lesch e￡ 【句 aIso studied the insertion／depIetion Ien甜h poIymorphism of a l4一 bp flra ent 1 kb upstream of 5一HTT#rene， and discovered that 5一 HTTLPR w_ds obvious connected with anxious personalitv． In a case．connl(】lled coITelation studv of mood dis再 er and 5．H I n．P carried out bv CoUier e￡ [7】 based on the data collected f om 3 维普资讯 http://www.cqvip.com 维普资讯 http://www.cqvip.com