Based on the clinical use of two methods, a previous study demonstrated that differences between the two methods may lead to different results in the diagnosis of aneuploid chromosomes [6]. According to statistics, approximately 32% of fetuses with structural abnormalities have a clinically relevant abnormal karyotype, and an additional 6% have causative CNVs [7]. Therefore, it is necessary to evaluate more comprehensively the difference between karyotyping analysis and CMA and the combination of these two methods in the diagnosis of fetuses with structural abnormalities. Here, we explored the characteristics of chromosome abnormalities detected by conventional G-banding karyotyping and CMA in fetus cases and further compared the detecting results in a larger sample size using different testing techniques. Additionally, fluorescence in situ hybridization (FISH) analysis or CNVplex analysis was conducted to evaluate chromosome mosaicism. Those findings showed that the combination of conventional karyotyping analysis and CMA identified the additional, clinically significant cytogenetic information, indicating the value of the combination of the two methods.
A total of 157 cases showed abnormal CMA results but normal karyotype analysis. Additionally, we also found that 4 cases were chromosomal mosaicism (Table 3). In brief, the representative CAM results of case 15 represented the mosaic duplication of chromosomes (about 23.7%), indicating mosaicism for trisomy 2 (Figure 4). Correspondingly, CNVplex results showed that the signal of chromosome 2 in case 15 was between 2 and 3 before culture, suggesting mosaic trisomy 2. After culture, the signal of chromosome 2 was approximately equal to 2, suggesting that mosaic trisomy 2 was selective with no growth advantage in vitro (Figure 5).
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In China, a growing number of pregnant women are willing to accept additional molecular technology for prenatal diagnosis based on conventional karyotype analysis. Correspondingly, Shi et al. [10] have investigated the methods of prenatal diagnosis in the multicenter center and demonstrated that combined karyotype analysis and CMA are commonly used to assess pregnant women with other abnormal indications. Here, we found that conventional karyotype analysis and CMA can identify the cytogenetic differently information, which indicated the significant value of the combination of two methods for prenatal diagnosis.
According to statistics, approximately 6% of fetuses have chromosome abnormalities detected during amniocenteses, and the combined risk of a serious congenital anomaly in a fetus with translocations and inversions was estimated at 6.7% [11]. In the current study, karyotype abnormal results were detected in 201 (5.41%) of 3710 fetuses, which was similar to the proportion reported previously. Karyotype analysis is the gold standard for detecting chromosome abnormalities, but it is obviously inadequate for diagnosing chromosome mosaicism and estimating chromosome recombination. In 2013, the American College of Obstetrics and Gynecology (ACOG) recommended using CMA instead of traditional karyotype analysis when the fetus has one or more ultrasound abnormalities and requires invasive prenatal diagnosis [12]. Additionally, similar studies have confirmed that CMA is considered the first-line detection method for prenatal diagnosis [5, 13]. Our study found that CMA can detect 3.73% more chromosome abnormalities than karyotype analysis, which is consistent with previous findings [13]. However, several disadvantages of CMA have been pointed out. For example, a previous study demonstrated that CMA cannot detect low levels of mosaicism, leading to missed diagnoses [14]. Other studies have also pointed out that balanced chromosome rearrangement cannot be detected using CMA [15, 16]. On the other side, some chromosomal abnormalities would be missed if CMA was used alone. Briefly, a case diagnosed as Coffin-Lowry syndrome (disruption of CHD7) has normal CMA results but an abnormal karyotype result [17]. In our study, we also found that 12 cases had abnormal karyotype analysis, despite normal CMA results. Therefore, it is not appropriate for CMA to completely replace karyotyping in prenatal diagnosis, and a combination of the two methods is recommended.
Regarding chromosome mosaicism, FISH or CNVplex analysis was used to further evaluate the results of chromosome abnormalities in the current study. For instance, the karyotype result of case 1 was modified to 45, X, [73]/46, X, psu dic(Y) (q12) [10], based on a combination of G-banding and FISH analysis. FISH analysis, especially for the diagnosis of complex chromosome abnormalities, has set up a bridge between chromosome banding technology and molecular genetics [18]. Additionally, previous studies have demonstrated that FISH analysis can identify low-level mosaicism [19] and mosaicism for chromosomal rearrangement undetected by molecular cytogenetics [20]. Correspondingly, the combination of G-banding and FISH provides an efficient method for prenatal and postnatal chromosomal analysis [21]. CNVplex assay, a high-throughput multiplex CNVs analysis method developed by Genesky Biotechnologies, which can not only detect the abnormal number of 24 chromosomes, but also detect the deletion or duplication of the ends of chromosomes [22]. In terms of detection accuracy, CNVplex excludes possible maternal contamination, which decreases the false negative rate, thereby greatly enhancing the reliability of the results [23]. Importantly, the cost of its approach was relatively low. In our study, the CMA and CNVplex assays suggested the mosaicism for trisomy 2, indicating the value of CNVplex for prenatal diagnosis. Combined with those findings, we believed that FISH or CNVplex can enhance the reliability of prenatal and postnatal chromosomal analysis.
Conventional G-banding karyotyping and CMA have their own advantages and limitations (Supplementary Table 1), and it is also inappropriate that CMA completely replace karyotyping in prenatal diagnosis. Combined karyotype analysis and CMA for prenatal diagnosis can increase the reliability of detection. Additionally, FISH or CNVplex analysis was recommended to further evaluate the results when chromosomal mosaicism was detected. Those findings provided a better understanding of the clinically significant genetic variants.
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Overall, 63% of karyotyped POC were chromosomally abnormal on routine cytogenetics. The mean maternal age at the time of miscarriage was 37.2 years and the mean gestational age at the time of uterine evacuation was 8.75 weeks (see Table 1 for patient characteristics). The majority of abnormalities were due to autosomal trisomies (15 out of 19) and, not surprisingly, 14 of 15 of these were due to excess maternal chromosomes. In addition to the autosomal trisomies, there were two unbalanced translocations (one inherited and one de novo). There was one triploidy and one tetraploidy in our data set. All 46,XX results from cytogentics were confirmed to be POC samples as microarray analysis clearly detected one maternal and one paternal copy of each chromosome, ruling out maternal contamination. The cytogenetic karyotyping took, on average, 29 days to return results. In contrast, the microarray based karyotyping took 12 days for the initial research samples. The last eight microarray samples, run in a production setting, averaged only 7 days for turn around.
The application of informatics and microarray technology to analysis of miscarriage tissues gives fetal karyotype results in significantly less time than routine cytogenetic testing, and avoids the potential pitfalls of cell culture. While array comparative genomic hybridization (aCGH) has been reported in this context [10], [11], to our knowledge, this is the first application of SNP microarray technology to determining the karyotype of POC samples. The use of the informatics enhanced SNP microarray technique gives us additional information about parental source of aneuploidy, enables detection of uniparental disomy (UPD), and is able to differentiate POC from maternal DNA in the setting of a 46,XX result.
Errors involving structural chromosome rearrangement, such as balanced translocations and Robertsonian translocations, are found in approximately 0.5% of POC samples [15]. Note that a Robertsonian translocation is formally a balanced translocation, although in practice, the complementary chromosome made up of the two tiny p-arms found in acrocentric chromosomes is often lost after a few rounds of mitosis. The inability to detect balanced chromosomal structural rearrangements, or differentiate between actual trisomy due to three separate copies of the chromosome versus a specious trisomy resulting from an isochromosome or Robertsonian translocation, is a limitation inherent to all microarray testing [16]. These types of abnormalities can only be detected with traditional cytogenetic techniques where the chromosome itself and banding pattern can be viewed. A balanced translocation is not lethal and, therefore, would not be expected to cause a miscarriage. If the genetic material is not balanced, then the array would be able to identify any unbalanced karyotype. Couples at risk for having balanced structural rearrangements in either parent should undergo routing cytogentic analysis of peripheral blood to accurately identify whether a translocation is present. 2ff7e9595c
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