中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2015年
8期
568-575
,共8页
许雅娟%翟闪闪%罗晓华%张莹莹%冉利敏%任利单
許雅娟%翟閃閃%囉曉華%張瑩瑩%冉利敏%任利單
허아연%적섬섬%라효화%장형형%염리민%임리단
唐氏综合征%产前诊断%妊娠蛋白质类%胶原Ⅵ型%多态性,单核苷酸%聚合酶链反应
唐氏綜閤徵%產前診斷%妊娠蛋白質類%膠原Ⅵ型%多態性,單覈苷痠%聚閤酶鏈反應
당씨종합정%산전진단%임신단백질류%효원Ⅵ형%다태성,단핵감산%취합매련반응
Down syndrome%Prenatal diagnosis%Pregnancy proteins%Collagen type Ⅵ%Polymorphism,single nucleotide%Polymerase chain reaction
目的：通过对孕妇血浆中胎儿特异基因PLAC4和COL6A2等位基因杂合频率的检测，探讨其用于产前筛查21三体的可行性。方法随机选取2013年6至12月在郑州大学第三附属医院体检门诊进行身体健康检查的河南籍汉族人群500例（男、女各250例）为健康体检组，采用DNA测序和PCR-限制性片段长度多态性（RFLP）技术检测健康体检组受检者外周血中PLAC4和COL6A2基因单核苷酸多态性（SNP）位点的杂合频率，并将SNP位点杂合频率检测结果与美国国家生物技术信息中心（NCBI）数据库中相对应的杂合频率进行差异性比较；随机选取同期在产科门诊行孕期检查的健康孕妇30例为健康妊娠组，采用实时荧光定量逆转录PCR技术检测健康妊娠组孕妇孕8周、10周、12周、14周、16周外周血中PLAC4和COL6A2 mRNA表达水平；选取同期40例行羊水胎儿细胞染色体核型分析的标本，其中21三体阳性20例、阴性20例为21三体验证组，采用逆转录-多重连接探针扩增（RT-MLPA）技术筛查21三体验证组胎儿的21三体。结果（1）健康体检组受检者SNP位点等位基因杂合频率：对PLAC4和COL6A2基因10个SNP位点的基因型及杂合频率检测显示，杂合频率较高的位点分别为rs7717、rs559、rs1044598、rs59066201、rs1042917，人群覆盖率为98%。rs59066201位点等位基因杂合频率在NCBI数据库未见；rs8130833、rs9977003、rs7844位点等位基因杂合频率分别与NCBI数据库中相对应的杂合频率比较，差异有统计学意义（P<0.05）；其余6个SNP位点等位基因杂合频率分别与NCBI数据库的杂合频率比较，差异无统计学意义（P>0.05）。（2）健康妊娠组不同孕周PLAC4和COL6A2 mRNA的表达水平：在孕8周、10周、12周、14周、16周的孕妇外周血中PLAC4 mRNA的水平分别为7.22±1.05、8.02±1.41、9.51±1.69、11.33±2.11、13.31±2.58，其表达水平随着孕周的增加而升高，其中，孕8周与孕10周比较，差异无统计学意义（P>0.05）；其他各孕周的表达水平相互间比较，差异均有统计学意义（P<0.05）。COL6A2 mRNA在孕8周、10周、12周、14周、16周的表达水平分别为8.95±1.28、11.19±1.36、15.00±1.58、16.87±1.72、18.96±2.79，其表达水平随着孕周的增加而升高，各孕周表达水平相互间比较，差异均有统计学意义（P<0.05）。（3）21三体验证组产前筛查21三体：据健康体检组受检者SNP位点等位基因杂合频率，选取rs7717、rs559、rs1044598、rs59066201、rs1042917共5个位点筛查21三体验证组，17例21三体标本和18例阴性标本被准确检出，筛查敏感度85%（17/20），筛查特异度90%（18/20）；1例21三体标本和2例阴性标本的5个SNP位点均为纯合子，2例21三体标本只有1个杂合子，此5例标本无法有效筛查，筛查准确率达100%（35/35）。结论胎儿特异基因PLAC4和COL6A2 mRNA在不同孕周的孕妇外周血中均有表达，其表达水平随孕周的增加而升高；其中rs7717、rs559、rs1044598、rs59066201、rs1042917等5个SNP位点杂合频率较高，且与NCBI数据库中相对应的杂合频率有所不同；RT-MLPA技术是一种快速、有效、无创、费用低的产前筛查21三体的方法。
目的：通過對孕婦血漿中胎兒特異基因PLAC4和COL6A2等位基因雜閤頻率的檢測，探討其用于產前篩查21三體的可行性。方法隨機選取2013年6至12月在鄭州大學第三附屬醫院體檢門診進行身體健康檢查的河南籍漢族人群500例（男、女各250例）為健康體檢組，採用DNA測序和PCR-限製性片段長度多態性（RFLP）技術檢測健康體檢組受檢者外週血中PLAC4和COL6A2基因單覈苷痠多態性（SNP）位點的雜閤頻率，併將SNP位點雜閤頻率檢測結果與美國國傢生物技術信息中心（NCBI）數據庫中相對應的雜閤頻率進行差異性比較；隨機選取同期在產科門診行孕期檢查的健康孕婦30例為健康妊娠組，採用實時熒光定量逆轉錄PCR技術檢測健康妊娠組孕婦孕8週、10週、12週、14週、16週外週血中PLAC4和COL6A2 mRNA錶達水平；選取同期40例行羊水胎兒細胞染色體覈型分析的標本，其中21三體暘性20例、陰性20例為21三體驗證組，採用逆轉錄-多重連接探針擴增（RT-MLPA）技術篩查21三體驗證組胎兒的21三體。結果（1）健康體檢組受檢者SNP位點等位基因雜閤頻率：對PLAC4和COL6A2基因10箇SNP位點的基因型及雜閤頻率檢測顯示，雜閤頻率較高的位點分彆為rs7717、rs559、rs1044598、rs59066201、rs1042917，人群覆蓋率為98%。rs59066201位點等位基因雜閤頻率在NCBI數據庫未見；rs8130833、rs9977003、rs7844位點等位基因雜閤頻率分彆與NCBI數據庫中相對應的雜閤頻率比較，差異有統計學意義（P<0.05）；其餘6箇SNP位點等位基因雜閤頻率分彆與NCBI數據庫的雜閤頻率比較，差異無統計學意義（P>0.05）。（2）健康妊娠組不同孕週PLAC4和COL6A2 mRNA的錶達水平：在孕8週、10週、12週、14週、16週的孕婦外週血中PLAC4 mRNA的水平分彆為7.22±1.05、8.02±1.41、9.51±1.69、11.33±2.11、13.31±2.58，其錶達水平隨著孕週的增加而升高，其中，孕8週與孕10週比較，差異無統計學意義（P>0.05）；其他各孕週的錶達水平相互間比較，差異均有統計學意義（P<0.05）。COL6A2 mRNA在孕8週、10週、12週、14週、16週的錶達水平分彆為8.95±1.28、11.19±1.36、15.00±1.58、16.87±1.72、18.96±2.79，其錶達水平隨著孕週的增加而升高，各孕週錶達水平相互間比較，差異均有統計學意義（P<0.05）。（3）21三體驗證組產前篩查21三體：據健康體檢組受檢者SNP位點等位基因雜閤頻率，選取rs7717、rs559、rs1044598、rs59066201、rs1042917共5箇位點篩查21三體驗證組，17例21三體標本和18例陰性標本被準確檢齣，篩查敏感度85%（17/20），篩查特異度90%（18/20）；1例21三體標本和2例陰性標本的5箇SNP位點均為純閤子，2例21三體標本隻有1箇雜閤子，此5例標本無法有效篩查，篩查準確率達100%（35/35）。結論胎兒特異基因PLAC4和COL6A2 mRNA在不同孕週的孕婦外週血中均有錶達，其錶達水平隨孕週的增加而升高；其中rs7717、rs559、rs1044598、rs59066201、rs1042917等5箇SNP位點雜閤頻率較高，且與NCBI數據庫中相對應的雜閤頻率有所不同；RT-MLPA技術是一種快速、有效、無創、費用低的產前篩查21三體的方法。
목적：통과대잉부혈장중태인특이기인PLAC4화COL6A2등위기인잡합빈솔적검측，탐토기용우산전사사21삼체적가행성。방법수궤선취2013년6지12월재정주대학제삼부속의원체검문진진행신체건강검사적하남적한족인군500례（남、녀각250례）위건강체검조，채용DNA측서화PCR-한제성편단장도다태성（RFLP）기술검측건강체검조수검자외주혈중PLAC4화COL6A2기인단핵감산다태성（SNP）위점적잡합빈솔，병장SNP위점잡합빈솔검측결과여미국국가생물기술신식중심（NCBI）수거고중상대응적잡합빈솔진행차이성비교；수궤선취동기재산과문진행잉기검사적건강잉부30례위건강임신조，채용실시형광정량역전록PCR기술검측건강임신조잉부잉8주、10주、12주、14주、16주외주혈중PLAC4화COL6A2 mRNA표체수평；선취동기40례행양수태인세포염색체핵형분석적표본，기중21삼체양성20례、음성20례위21삼체험증조，채용역전록-다중련접탐침확증（RT-MLPA）기술사사21삼체험증조태인적21삼체。결과（1）건강체검조수검자SNP위점등위기인잡합빈솔：대PLAC4화COL6A2기인10개SNP위점적기인형급잡합빈솔검측현시，잡합빈솔교고적위점분별위rs7717、rs559、rs1044598、rs59066201、rs1042917，인군복개솔위98%。rs59066201위점등위기인잡합빈솔재NCBI수거고미견；rs8130833、rs9977003、rs7844위점등위기인잡합빈솔분별여NCBI수거고중상대응적잡합빈솔비교，차이유통계학의의（P<0.05）；기여6개SNP위점등위기인잡합빈솔분별여NCBI수거고적잡합빈솔비교，차이무통계학의의（P>0.05）。（2）건강임신조불동잉주PLAC4화COL6A2 mRNA적표체수평：재잉8주、10주、12주、14주、16주적잉부외주혈중PLAC4 mRNA적수평분별위7.22±1.05、8.02±1.41、9.51±1.69、11.33±2.11、13.31±2.58，기표체수평수착잉주적증가이승고，기중，잉8주여잉10주비교，차이무통계학의의（P>0.05）；기타각잉주적표체수평상호간비교，차이균유통계학의의（P<0.05）。COL6A2 mRNA재잉8주、10주、12주、14주、16주적표체수평분별위8.95±1.28、11.19±1.36、15.00±1.58、16.87±1.72、18.96±2.79，기표체수평수착잉주적증가이승고，각잉주표체수평상호간비교，차이균유통계학의의（P<0.05）。（3）21삼체험증조산전사사21삼체：거건강체검조수검자SNP위점등위기인잡합빈솔，선취rs7717、rs559、rs1044598、rs59066201、rs1042917공5개위점사사21삼체험증조，17례21삼체표본화18례음성표본피준학검출，사사민감도85%（17/20），사사특이도90%（18/20）；1례21삼체표본화2례음성표본적5개SNP위점균위순합자，2례21삼체표본지유1개잡합자，차5례표본무법유효사사，사사준학솔체100%（35/35）。결론태인특이기인PLAC4화COL6A2 mRNA재불동잉주적잉부외주혈중균유표체，기표체수평수잉주적증가이승고；기중rs7717、rs559、rs1044598、rs59066201、rs1042917등5개SNP위점잡합빈솔교고，차여NCBI수거고중상대응적잡합빈솔유소불동；RT-MLPA기술시일충쾌속、유효、무창、비용저적산전사사21삼체적방법。
Objective Through the detections of the heterozygote frequencies tests of fetal specific genes PLAC4 and COL6A2 mRNA alleles in plasma of pregnant women, to explore its possibility of application in the noninvasive prenatal screenings of trisomy-21. Methods A toltal of 500 cases (males and females 250 cases respectively)of Han ethnic groups with Henan Provice of China who were subject to the physical checkup clinic of the Third Affiliated Hospital,Zhengzhou University from June to December, 2013 were selected as the healthy physical checkup group, and such techniques as DNA sequencing and PCR-restriction fragment length polymorphism (RFLP) were adopted to the determinations of the heterozygote frequencies of the single nucleotide polymorphism(SNP)of the PLAC4 and COL6A2 genes in the maternal peripheral blood in the healthy physical checkup group, and the differential comparisons of the determination results of the SNP heterozygote frequencies and the corresponding heterozygote frequencies in the National Center for Biotechnology Information (NCBI) database;30 cases of healthy pregnant women who spontaneously underwent pregnancy checkups at the maternity clinic were randomly selected as the healthy pregnancy group, and real-time fluorescence quantitative reverse transcription-PCR technique was adopted for determining the expression levels of PLAC4 and COL6A2 mRNA in the peripheral blood of pregnant women of 8 weeks, 10 weeks, 12 weeks, 14 weeks and 16 weeks;40 cases of the same phase were selected for acting as the specimens for the karyotype analyses of the amniotic fluid cells, among which 20 cases were trisomy-21, and the 20 cases of the negative control group, and reverse transcription-multiplex ligation dependent probe amplification (RT-MLPA) technique was adopted for screening the fetal trisomy-21. Results (1) The allele heterozygote frequencies of the SNP of the healthy physical checkup group:determinations of the genotypes and hybrid rates of the 10 SNP sites of the PLAC4 and COL6A2 genes indicated that those with higher heterozygote frequencies were respectively rs7717, rs559, rs1044598, rs59066201 and rs1042917, with population coverage of 98%. Among them, the allele hybrid rates of rs59066201 were never seen in the NCBI database;in the respective comparisons of the allele hybrid rates of rs8130833, rs9977003 and rs7844 with the hybrid rates of the NCBI database, the variations had statistical significance (P<0.05). (2) The expression levels of PLAC4 and COL6A2 mRNA of the different pregnancy weeks of the healthy pregnancy group: the levels of PLAC4 mRNA in the peripheral blood of women of 8 weeks, 10 weeks, 12 weeks, 14 weeks and 16 weeks of pregnancy were respectively 7.22 ± 1.05, 8.02±1.41,9.51±1.69,11.33±2.11 and 13.31±2.58, with their expression levels rising along with the increase of the pregnancy weeks; among them, the comparison of pregnancy 8 weeks and pregnancy 10 weeks, the variations had no statistical significance (P>0.05);in the mutual comparisons among the expression levels of the various pregnancy weeks, the variations had statistical significance (P<0.05). The expression levels of COL6A2 mRNA in 8 weeks, 10 weeks, 12 weeks, 14 weeks and 16 weeks were respectively 8.95 ± 1.28, 11.19 ± 1.36,15.00 ± 1.58,16.87 ± 1.72 and 18.96 ± 2.79, with their expression levels rising along with the increase of the pregnancy weeks, and in the mutual comparisons between the expression levels of the various pregnancy weeks, the variations all had statistical significance (P<0.05). (3) Prenatal screenings of trisomy-21 in the validation group of the trisome:a total of 5 sites of rs7717, rs559, rs1044598, rs59066201 and rs1042917 were selected from the allele heterozygote frequencies of SNP sites were selected from the subjects of the healthy physical checkup group, and 10 cases of trisomy-21 specimens and 10 cases of negative CTR specimens were accurately determined, with the sensitivity reached 80%(17/20), and the specificity reached 90%(18/20). One case of the trisomy-21 and two negative cases were both homozygotes, and among the trisomy-21 specimens of two cases, only one SNP was a heterozygote, and it was impossible to conduct screenings on these 5 cases, with the screening accuracy reaching 100%(35/35). Conclusions Fetal specific genes PLAC4 and COL6A2 mRNA are expressed in the peripheral blood of pregnant women in different gestational age;its expression level increases with the increase of gestational age. Among them, five SNP including rs7717, rs559, rs1044598, rs59066201 and rs1042917 show highest heterogeneity rate, which is different from the corresponding heterogeneity rate in NCBI database. RT-MLPA technology is a rapid, effective, noninvasive and low cost method of prenatal screening 21 trisomy.
