核型分析、BoBs、CNV和Y染色体AZF微缺失检测在羊水染色体鉴定中的应用

Application of Karyotype Analysis,BoBs,CNV and Y Chromosome AZF Microdeletion Detection in the Identification of Amniotic Fluid Chromosomes

目的探讨染色体核型分析、细菌人工染色体标记-微球鉴别/分离法(bacterial artificial chromosome on beads,BoBs)、基因拷贝数目变异检测(copy number variations,CNV)和Y染色体无精子症因子(azoospermia factor,AZF)微缺失联合检测在孕妇羊水染色体鉴定中的应用价值。方法选取2021年7月至2022年12月于皖南医学院第一附属医院(弋矶山医院)产前诊断中心就诊中符合产前诊断指征的孕妇507例,抽取孕16~25 w羊水,分别进行细胞培养染色体核型分析,提取DNA进行BoBs检测,对其中1例21-三体综合征和1例标记染色体进行CNV验证,2例Y染色体进行AZF微缺失验证,统计结果。结果507例羊水穿刺指征统计,以唐筛高风险占比最高,达39.1%(198/507);NIPT高风险组仅占总检查孕妇的14.0%(71/507),但核型分析和BoBs异常结果占比最高,分别占全部异常结果的40.3%(23/57)和47.7%(21/44)。507例羊水标本一共检出异常结果59例(11.6%),其中染色体核型分析检出异常57例(11.2%),常染色体数目异常26例(5.1%),常染色体结构异常14例(2.8%);性染色体数目异常13例(2.6%),性染色体结构异常4例(0.7%)。BoBs检出异常44例(8.7%),其中常染色体数目异常26例(5.1%),性染色体数目异常14例(2.8%),性染色体部分缺失2例(0.4%),检出46,XN,22q11重复2例(0.4%)。BoBs与核型分析结果比对,常染色体和性染色体数目异常结果符合率分别为100.0%和99.8%。另外BoBs将其中1例46,X,del(Y)(q11)判读为45,XO,1例47,XN,+mar[50]/46,XN[10]判读为46,XN,其余染色体结构异常不在BoBs检测范围。CNV验证致病性拷贝数变异1例,临床意义未明拷贝数变异1例;Y染色体AZF微缺失验证2例Y染色体为SRY+,存在AZFb+c缺失。结论羊水染色体核型分析能够发现染色体数目和结构异常核型,对于未知来源的标记染色体和<10 Mb的微缺失/微重复缺乏优势。BoBs对于常染色体数目检测和微缺失/微重复检测具有优势,能提示性染色体片段缺失,但要注意对性染色体的误判,常染色体结构异常不在BoBs检测范围。CNV对于全基因组微缺失和微重复检测具有优势。Y染色体AZF微缺失检测可对Y染色体进行验证。联合应用上述检测技术,能对羊水染色体数目和结构异常提供多方位诊断,值得推广应用。

Objective To investigate the application value of a combined approach,including chromosome karyotype analysis,bacterial artificial chromosome on beads(BoBs),copy number variations(CNV),and azoospermia factor(AZF)on the Y chromosome,for identifying chromosomal abnormalities in amniotic fluid samples from pregnant women.Methods From July 2021 to December 2022,507 pregnant women who met the indications of prenatal diagnosis were selected from the prenatal diagnosis center of the First Affiliated Hospital of Wannan Medical College(Yijishan Hospital).Amniotic fluid of 16-25 w of pregnancy was extracted,and cell culture chromosome karyotype analysis was performed,and DNA was extracted for BoBs detection.CNV verification was performed in 1 case of trisomy 21 syndrome and 1 case of labeled chromosome,and AZF microdeletion verification was performed in 2 cases of Y chromosome.Results Among the 507 cases,the highest indication for amniotic fluid puncture was due to a high-risk of Down′s syndrome,accounting for 39.1%(198/507);however,the majority of abnormal cases were detected in the non-invasive prenatal testing high-risk group,which only accounted for 14%of all pregnant women,with abnormalities found by both karyotype analysis(23/57)and BoBs(21/44).Abnormal results were found in 59(11.6%)of 507 amniotic fluid samples.Out of these cases,57 cases were abnormal by chromosome karyotype analysis(11.2%),including 26 cases with abnormal autosomal numbers(5.1%),14 cases with autosomal structural abnormalities(2.8%),13 cases with abnormal sex chromosome numbers(2.6%),and 4 cases with structural abnormalities of sex chromosomes(0.7%).BoBs detected 44 abnormal cases(8.7%),including 26 cases with abnormal autosomal numbers(5.1%),14 cases with abnormal sex chromosome numbers(2.8%),2 cases with partial deletions of sex chromosomes(0.4%),and 2 cases with 46,XN,22q11 duplication(0.4%).The coincidence rates of abnormal autosomal numbers and sex chromosomes numbers between BoBs and karyotype analysis were 100.0%and 99.8%,respectively.However,1 case of 46,X,del(Y)(q11)was misjudged as 45,XO and 1 case of 47,XN,+mar[50]/46,XN[10]was misjudged as 46,XN by BoBs.Chromosome structural abnormalities were not within the detection range of BoBs.One case of pathogenic copy number variation and one case of unknown copy number variation were verified by CNV.Two cases of Y chromosomes were positive for SRY+and AZFb+c deletion confirmed by AZF.Conclusion chromosome karyotype analysis can detect abnormal chromosome number and structure in amniotic fluid,but may not be able to detect marker chromosomes of unknown origin or microdeletions or microrepeats smaller than 10 Mb.BoBs are advantageous in detecting chromosome numbers and microdeletions or microrepeats,and can also indicate the loss of sex chromosome fragment deletion.However,the misjudgment of sex chromosomes should be taken into consideration,and BoBs cannot detect autosomal structural abnormalities.CNV has advantages in detecting genome-wide microdeletions or microrepetitions,and AZF can verify Y chromosomes.A combination of these techniques can provide a multidirectional diagnosis of chromosomal abnormalities in amniotic fluid.