孕妇血浆中胎儿特异基因PLAC4和COL6A2等位基因杂合频率检测用于产前筛查21三体的可行性研究

Noninvasive prenatal screen of trisomy-21 using maternal plasma fetal free RNA allelic ratio

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（P0.05）。（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。

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 （P0.05）;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.