荧光定量PCR技术在母体细胞污染鉴定和快速产前诊断中的应用

Application of QF-PCR in maternal cell contamination identification and prenatal diagnosis

摘要

目的 评价荧光定量PCR(QF-PCR)技术在母体细胞污染(MCC)鉴定和常见染色体非整倍体快速产前诊断中的临床应用价值。方法 建立MCC浓度梯度QF-PCR检测模型,分析不同比例MCC下的短串联重复序列(STR)图谱特点。回顾性分析577例产前羊水样本中MCC发生率,通过与羊水核型分析结果比对,评估QF-PCR对13、18、21、X及Y染色体非整倍体的检测效能。结果 在10%及以上的MCC模型中,STR图谱出现明显可辨别“污染峰”特征。577例羊水样本中,4例存在MCC(0.69%,4/577);QF-PCR检出常见染色体异常倍性60例,包括21-三体24例、18-三体11例、XO单体3例、XXX综合征4例、XXY综合征6例和XYY综合征6例,提示倍性异常6例。羊水核型分析检出异常69例,两种检测结果比较显示,QF-PCR对52例染色体非整倍体的检出与核型分析完全一致;2例核型分析结果为Y染色体结构异常的样本,QF-PCR判定为XYY;6例性染色体嵌合的样本,QF-PCR仅能提示倍性异常而无法确定具体构成;另有9例染色体平衡重组和片段异常由于QF-PCR试剂盒的局限性未检出。结论 荧光定量PCR技术可有效识别≥10%的母体细胞污染,对常见染色体非整倍体的诊断具有快速和高敏感性的优势,但对染色体嵌合组成、平衡易位和拷贝数变异等检出存在局限性,建议将其作为产前常见染色体非整倍体诊断的辅助技术手段。

Abstract

Objective To evaluate clinical application value of quantitative fluorescent polymerase chain reaction (QF-PCR) for maternal cell contamination (MCC) identification and rapid prenatal diagnosis of common chromosomal aneuploidies. Methods MCC concentration-gradient detection models were established to analyze characteristics of short tandem repeats (STR) profiles at different proportions of MCC. The incidence of MCC in 577 amniotic fluid samples was retrospectively analyzed, and detection efficacy of QF-PCR for chromosomal aneuploidies (13, 18, 21, X, and Y) was evaluated through comparative analysis with amniotic karyotyping results. Results In 10% or above MCC models, obviously “contamination peak” characteristics appeared the STR profiles. MCC was identified in 4 of 577 amniotic fluid samples (0.69%, 4/577). Totally, 60 chromosomal aneuploidies were identified out by QF-PCR, including 24 cases of trisomy 21, 11 cases of trisomy 18, 3 cases of monosomy X (XO), 4 cases of triple X (XXX) syndrome, 6 cases of Klinefelter syndrome (XXY) and 6 cases of XYY syndrome, as well as 6 additional cases of suggestive ploidy abnormalities. 69 chromosomal aneuploidies were identified out by amniotic karyotyping analysis. Comparative analysis of results by QF-PCR with those by amniotic karyotyping revealed full concordance for 52 cases of non-mosaic aneuploidies, and the discrepancies included: two cases of Y-chromosome structural rearrangements were misclassified as XYY by QF-PCR; for six sex chromosome mosaicism cases, QF-PCR suggested ploidy anomalies only but failed to resolve mosaic composition; and nine undetected cases of balanced rearrangements and deletions/duplications attributed to QF-PCR kit limitations. Conclusion QF-PCR can effectively identify out MCC at ≥10% thresholds and exerts such advantages as rapid diagnosis and high sensitivity for common chromosomal aneuploidies. However, limitations persist in detecting chromosomal mosaicism composition, balanced translocations and copy number variations (CNVs). So, QF-PCR could be used as an adjunctive screening tool for prenatal aneuploidy diagnosis in clinic.

关键词

母体细胞污染 / 荧光定量PCR / 产前诊断 / 染色体非整倍体

Key words

maternal cell contamination / quantitative fluorescent polymerase chain reaction / prenatal diagnosis / chromosomal aneuploidy

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基金资助

上海市松江区科技攻关项目(2020SJ356)