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Evaluation of two aneuploidy screening tests for chorionic villus samples: Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization
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Evaluation of two aneuploidy screening tests for chorionic villus samples: Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization Tonghua Wu, Yuanchang Zhu, Ling Hong, Qi Lin, Chunmei Chen, Jing Yang, Lijun Ye, Wensi Huang, Yong Zeng* Shenzhen Key Laboratory for Reproductive Immunology of Preimplantation, Shenzhen Zhongshan Institute for Reproduction and Genetics, Fertility Center, Shenzhen Zhongshan Urology Hospital, Shenzhen, Guangdong, 518045, China
ARTICLE INFO
ABSTRACT
Keywords: Multiplex ligation-dependent probe amplification (MLPA) Fluorescence in situ hybridization (FISH) Fetal chromosomal abnormality Mosaicism Pregnancy loss
The vast majority of first-trimester pregnancy losses are the consequence of numerical aberrations in fetal chromosomes, which may involve nearly all chromosomes. Although commercial probes for all chromosomes are available for multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH) analyses, their use has rarely been reported for screening all 24 chromosomes for early fetal demise, especially by FISH. Here, we validated the ability of MLPA and FISH techniques as two low-cost aneuploidy screening methods for 24 chromosomes in 165 chorionic villus samples (CVSs). The results obtained by two methods were compared by the Chi-square test and the Kappa agreement test. Both methods gave conclusive results for all CVSs tested and showed highly consistent results (kappa = 0.890, p < 0.001). There was no statistically significant difference between the aneuploidy rate of the CVSs tested by the two methods (p = 0.180). Most of the samples showed fully concordant molecular karyotyping results (81.21%) between the two analytical methods, 10.91% had incompletely concordant results, and 7.88% had discordant results. The inconsistencies included segmental abnormalities, mosaicism, and polyploidy. Both assays used to screen 24 chromosomes were powerful techniques for detecting aneuploidy in CVSs. In terms of cost-effectiveness and diagnostic accuracy, the combination of subtelomeric (P036, P070) and centromeric (P181) MLPA assays is the better analytic strategy and follow-up analysis by FISH is recommended for MLPA-negative samples.
1. Introduction Pregnancy loss (PL), the most common pregnancy-related complication, occurs in about 10%–15% of clinically recognized pregnancies [1]. Its etiology is heterogeneous. The vast majority of first-trimester PLs (50%–60%) are the consequence of fetal chromosomal abnormalities (FCAs), which can be of parental origin or de novo [2]. The spectrum of FCAs consists of numerical abnormalities (86%), structural abnormalities (6%), and other abnormalities (8%) [3]. Identifying the cause of a miscarriage plays an important role in patient management and next pregnancy guidance. Therefore, an effective testing approach to detect FCAs associated with PL is valuable. Conventional G-banding karyotyping, which has been considered as the standard reference test to detect FCAs, is hampered by culture failure, poor metaphase chromosome quality, and maternal cell contamination (MCC). Furthermore, it is time consuming and labor-
*
intensive [4–6]. Molecular approaches have been swiftly developed to detect aneuploidies without depending on cell culture, aimed at overcoming the intrinsic obstacles of karyotyping. DNA-based tests are mainly classified into two categories, whole genome techniques and chromosome region-specific techniques. The former refers to chromosomal comparative genomic hybridization (CGH), chromosomal microarray analysis (CMA) and next-generation sequencing (NGS), while the latter includes fluorescence in situ hybridization (FISH), quantitative fluorescent-polymerase chain reaction (QF-PCR), multiplex ligation-dependent probe amplification (MLPA), and bacterial artificial chromosomes (BACs)-on-Beads (BoBs). High-resolution CMA and low-pass NGS have become first-tier tests for most pediatric and prenatal diagnoses owing to their abilities to simultaneously detect numerical chromosomal abnormalities, submicroscopic chromosomal imbalances, and loss of heterozygosity at the entire genome-wide level. These two techniques are starting to be used
Corresponding author. E-mail address: [email protected] (Y. Zeng).
https://doi.org/10.1016/j.mcp.2019.101422 Received 26 May 2019; Received in revised form 9 July 2019; Accepted 15 July 2019 0890-8508/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Tonghua Wu, et al., Molecular and Cellular Probes, https://doi.org/10.1016/j.mcp.2019.101422
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Table 1 FISH probes for 24 chromosomes. Type
Label color
Target chromosome
Centromeric probes
Spectrum Spectrum Spectrum Spectrum Spectrum
CEP 1, CEP 2, CEP 3, CEP 16, CEP 17, CEP 20, CEP Y CEP 4, CEP 6, CEP 7, CEP 8, CEP 9, CEP 10, CEP 11, CEP 12, CEP 15, CEP X CEP 18, TelVysion 13q, TelVysion 14q, TelVysion 21q, TelVysion 22q TelVysion 5p, TelVysion 19p
Telomeric probe
Orange Green Aqua Orange Green
in the genetic evaluation of miscarriage cases, and approximately 6% additional findings over successful karyotyping have been reported [7–9]. The incremental diagnostic yield is mainly attributed to copy number variations (CNVs). Since it is not possible to identify the phenotype of a CNV found in an early abortus, whether these CNVs are causative of PLs remains uncertain and more large cohort studies are required to determine their pathogenicity [4,8]. In addition to the challenge of interpreting CNVs, the high testing fees and the professional data processing capabilities of these whole genome approaches currently remain prohibitive for routine application in the etiological diagnosis of PLs, especially in developing countries. Compared to whole genome techniques, chromosome region-specific techniques are widely applied as rapid aneuploidy tests for prenatal diagnosis because of their numerous advantages, which include ease-of-handling, rapid and sensitive screening, simple result interpretation, and relatively low cost [10,11]. The distribution of aneuploid chromosomes, however, is strikingly different between prenatal samples and miscarriage samples [12,13]. A limited probe panel for chromosomes 13, 18, 21 and sex chromosomes, which is an efficient and rapid aneuploidy test used for prenatal diagnosis, was reported to detect only around 20% of the FCAs in first trimester PLs, and the use of a panel for chromosomes 13, 16, 18, 21, 22, and sex chromosomes only detected about 60% [14]. Since numerical aberrations may involve nearly all chromosomes during the first trimester [15], only the assays providing information on all chromosomes are valid for testing miscarriage products. Though MLPA and FISH have commercially available probes for all chromosomes, the FCA analysis of early fetal demise using the commercial probes for all chromosomes has rarely been reported in the literature, especially for FISH. We, therefore, investigated the detectability and limitation of these two methods as aneuploidy screening tests for 24 chromosomes in chorionic villus samples (CVSs). We further proposed an economical and accurate testing strategy that can be widely applied to clinical testing.
samples were screened by MLPA with SALSA P036, P070 and P181 probemixes (MRC-Holland, The Netherlands) following the manufacturer's protocols. The PCR products were separated by capillary electrophoresis (ABI 3500 Genetic Analyzer, Applied Biosystems, USA) and the results were analyzed with Coffalyser. Net software (MRC-Holland, The Netherlands). Dosage quotients (DQ) = 0, 0.4 < DQ < 0.65, 0.8 < DQ < 1.2, 1.3 < DQ < 1.65, and 1.75 < DQ < 2.15 were interpreted as homozygous deletions, heterozygous deletions, normal, heterozygous duplications, and homozygous duplications, respectively. All other ambiguous DQ values were considered as mosaicism. CNVs for all the subtelomeric and subcentromeric probes of any individual chromosome indicated whole chromosome aneuploidy. Increased or decreased DQ at subtelomeric or/and subcentromeric probes of one chromosomal arm indicated segmental aneuploidy. 2.3. FISH The CVSs collected for FISH analysis were first incubated in 7 ml 75 mM KCl at 37 °C for 30 min, then were prefixed by adding 3 ml of 3:1 fixative (methanol: acetic acid) to the hypotonic solution for 5 min at room temperature. After the solution was decanted, the cells were fixed in 8 ml of 3:1 fixative (methanol: acetic acid) three times. The villi cells with fresh fixative were dropped onto three oblique slides. In order to ensure that all chromosomes were detected, each slide was divided into four isolated regions and each region was processed for interphase FISH detection using 2 to 3 different color probes for different chromosomes. The chromosomal FISH probes were commercially available from Vysis (Abbott Molecular, USA) and the details are listed in Table 1. Hybridization was performed overnight in a humidified HYBrite (Vysis, USA). The calculation and analysis of the hybridization signals for each probe were conducted using a fluorescent microscope (Nikon, Japan). According to the Chinese expert's consensus on the application of FISH technique in prenatal diagnosis, a minimum of 100 interphase nuclei per probes were randomly scored. The percentage of abnormal cells, scored as < 10%, 10%–60%, and > 60% indicated normal, mosaicism, and aneuploidy, respectively [16,17].
2. Materials and methods 2.1. Sample collection and preparation
2.4. Statistical analysis
Terminated singleton pregnancies, performed by dilation and curettage (D&C) in the first trimester at Shenzhen Zhongshan Urology Hospital and of women who agreed to chromosomal analysis of the abortuses, were included in the present study. The study was approved by the Research Ethics Committee of Shenzhen Zhongshan Urology Hospital and informed consent was obtained from each patient prior to study initiation. The products of conception obtained by D&C were collected into sterile falcon flasks containing normal saline. The CVSs were dissected carefully from the tissue and washed thoroughly in aseptic normal saline solution to minimize any MCC. The obtained samples were divided into two parts for MLPA and FISH analyses.
The results between the two methods were compared by the McNemar Chi-squared test and the Kappa agreement test using IBM SPSS Statistics 23 software. The level of statistical significance was set at p < 0.05. 3. Results A total of 165 samples were successfully collected for this study, including 135 cases of PL and 30 cases of induced abortion (IA). Both methods gave conclusive results for all the tested CVSs. The molecular karyotyping results were categorized as euploidy, one chromosome aneuploidy, one chromosome segmental abnormality, multiple chromosome abnormality, polyploidy, and mosaicism. The categorial proportions of the molecular karyotyping results detected by the MLPA and FISH assays are shown in Fig. 1. Among the 165 samples, a normal pattern for all probes was obtained in 72 (46 PL, 26 IA) cases by MLPA, and in 77 (50 PL, 27 IA) cases by FISH. Whether the specimen was
2.2. MLPA DNA of the CVSs was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer's instructions and was quantified by spectrophotometer (Shimadzu, Japan). The subtelomeric and subcentromeric regions of each chromosome in the DNA 2
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aneuploidies by MLPA. The median mosaic percentage detected by FISH in the former 10 chromosomes and the latter four chromosomes was 21.17% (range 11.04%-30.25%) and 49.08% (range 27.63%60.00%) respectively, while that of the 6 chromosomes identified as mosaicism by both methods was 51.42% (range 46.00%-60.18%). In addition, trisomy 21 mosaicism was detected by MLPA in one sample (No. PL-106), where FISH only detected the normal clone. And in another case (sample No. PL-63), MLPA identified mosaic trisomy 20, whereas FISH only detected the trisomy 20. Two cases showed discordant results between FISH and MLPA testing because of polyploidy. In one sample, pure tetraploidy (92, XXXX) detected by FISH was predictably identified as normal by MLPA. The other case was an aneuploidy occurring on a triploid background (71, XXX, +8, +8), which was recognized by FISH. The aneuploidy was detected as trisomy 8, while the baseline polyploidy karyotype was not recognized by MLPA in this instance. 4. Discussion The chromosome region-specific technique with a complete set of 24 FISH probes has been rarely applied to genetic diagnosis in spontaneous miscarriage in the past decades. Some studies have reported the evaluation of MLPA as a technique to detect aneuploidies in miscarriages products, but nearly all these studies used P095 aneuploidy probemix for 5 chromosomes (Chr. 13, 18, 21, X, and Y) or P036/P070 subtelomeric probemix for all chromosomes [18–21]. The combination of subtelomeric (P036, P070) and subcentromeric (P181) MLPA assays for 24 chromosomes has not yet been subjected to methodological evaluation. In our study, we evaluated the performance of these two molecular approaches for aneuploidy detection of all chromosomes in 165 CVSs samples. Both methods successfully detected aneuploidy, provided quick results (normally available in 2–3 days), and detected a wide variety of FCAs. The study results suggested that both methods overcame the main disadvantages of conventional cytogenetic analysis, including culture failure (20%) and time consumption (10–21 days) [22]. Furthermore, the MLPA technique was much less expensive (just 1/5 the cost of FISH), less labor-intensive (manual operation time, 2–3 h/run vs. 6–8 h/run for FISH), and had higher throughput capability (maximum sample size, 32 CVSs/run/technician vs. 3 CVSs/run/ technician for FISH) than FISH. Both methods presented excellent detection performance and a highly consistency for all euploidies and non-mosaic whole-chromosome aneuploidies identified. One case (sample No. PL-72) was misdiagnosed as monosomy 6 by FISH initially because of the poor signal. Thus, strictly following the guidelines for FISH issued by the American College of Medical Genetics (ACMG) is critical for guaranteeing the accuracy of FISH results. One such guideline states that the results of FISH analysis should be confirmed by at least two experienced technologists [23]. One remarkable advantage of MLPA analysis compared to FISH is the ability to detect unbalanced structural abnormalities, because it analyzes several loci of each chromosome simultaneously. Of the 17 segmental aneuploidies detected by MLPA in the present study, 11 CNVs were subtelomeric, 2 CNVs were subcentromeric, and the rest were subtelomeric and subcentromeric concurrently. However, the exact size of the imbalance was unknown due to the low number of MLPA probes. Three subcentromeric duplications were not revealed by centromeric FISH probes. This might be attributed to the imbalance fragment, which was smaller than the FISH probe size. Laboratory technicians should be aware that duplication or deletion affecting only one subtelomeric probe of a chromosomal arm is more likely to be considered a copy number polymorphisms. This is particularly true for the subtelomeric polymorphisms quoted in the product descriptions on the MRC-Holland website [24]. For the sake of avoiding false positive results, it is therefore strongly recommended to use both P036 and P070 probemixes to confirm subtelomeric CNVs, since all the probes of two
Fig. 1. Stacked column diagram of molecular karyotyping results analyzed by MLPA and FISH.
determined as euploid or not by these two methods was highly consistent (kappa = 0.890, p < 0.001). There was no statistically significant difference between the aneuploidy rate of the CVSs tested by these two methods (p = 0.180). The results of the MLPA and FISH analyses were assorted in the following manner. Fully concordant cases were defined as those with agreement between the MLPA and FISH results. Incompletely concordant cases referred to those where consistent abnormalities were detected by MLPA and FISH but one test contained more clinically useful information than the other. Discordant cases referred to those where the molecular karyotype results of the two methodologies were different. All findings in these three categories are summarized in Table 2. A fully concordant molecular karyotyping result was identified in most samples (81.21%), while 10.91% and 7.88% of the cases showed incompletely concordant and discordant results respectively. There were three main types of inconsistencies between the MLPA and FISH results: segmental abnormalities, mosaicisms, and polyploidies. Segmental aneuploidy was the principal cause of the inconsistent results (n = 17). Despite the fact that the precise size of the duplications or deletions was not clear, the 21 chromosomes with segmental abnormalities observed in MLPA testing (4 samples with 2 chromosomes involved) could represent reliable CNVs. This is because the subtelomeric CNVs were identified by both MLPA probemixes P036 and P070 while the subcentromeric CNVs were confirmed by retesting. Two duplications [chromosome (Chr.) 13 in samples No. PL-65 and No. PL28] were identified three FISH signals, while the rest of the CNVs had two FISH signals. The locus of the FISH probe that was matched to or outside the CNV region could reasonably explain the two FISH-detectable CNVs and 16 FISH-undetectable CNVs, but it was not applicable to the three FISH-undetectable centromeric duplications detected by centromeric FISH probes (Chr.7 in samples No. PL-31 and No. PL-32, Chr.11 in sample No. PL-50). Fifteen of the cases with inconsistent results were attributed to the presence of mosaicism. Thirteen cases with 14 chromosomes were identified as mosaic by FISH, but ten of the 14 mosaic chromosomes were identified as disomies and four were identified as nonmosaic 3
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Table 2 Molecular karyotyping results of 165 CVSs analyzed by both MLPA and FISH. Category Fully concordant (n = 134) Normal (n = 70) Abnormality (n = 64) Incompletely concordant (n = 18) Mosaicism (n = 10)
Segmental abnormality (n = 7)
Compound (n = 1) Discordant (n = 13) Polyploidy (n = 2) Mosaicism (n = 2) Segmental abnormality (n = 7)
Compound (n = 2)
ID no.
MLPA results
FISH results
(too many to list) (too many to list) IA-3 PL-30 PL-52 PL-75 PL-87 PL-97 PL-142 PL-41 PL-63 PL-67 PL-28 PL-31 PL-32 PL-33 PL-50 PL-65 PL-156 PL-104
Trisomy 16 Trisomy 8 Trisomy 22 Trisomy 13 Trisomy 15 Trisomy 15 Trisomy 22 Trisomy 15 Mos trisomy 20 Trisomy 15, trisomy 16, trisomy 21 Trisomy 4, dup 13q subtel, dup 12p subtel Monosomy X, dup 7p subtel, dup 7 subcen Trisomy 22, dup 7p subtel, dup 7 subcen Monosomy X, del 13q subcen Monosomy X, trisomy 5, dup 11 subcen Dup 13q subtel, dup 13 subcen Trisomy 21, del 13q-c subtel Trisomy 3, dup Xp subtel
Trisomy 16, mos trisomy 5 (20.63%) Trisomy 8, mos monosomy 5 plus mos trisomy 5 (30.25%) Trisomy 22, mos trisomy 9 (22.4%) Trisomy 13, mos trisomy 7 (13.71%) Trisomy 15, mos monosomy 17 (11.04%) Trisomy 15, mos trisomy 21 (12.28%) Mos trisomy 22 (60.00%), mos trisomy 8 (25.50%) Mos trisomy 15 (27.63%) Trisomy 20 Mos trisomy 15 (44.50%), trisomy 16, trisomy 21 Trisomy 4, trisomy 13 Monosomy X Trisomy 22 Monosomy X Monosomy X, trisomy 5 Trisomy 13 Trisomy 21 Mos trisomy 3 (53.66%)
PL-137 PL-114 IA-10 PL-106 IA-1 IA-28 PL-85 PL-99 PL-122 PL-147 PL-72 PL-48 PL-79
Normal Trisomy 8 Normal Mos trisomy 21 Del 19q subtel Dup Xq subtel Dup 7q subtel, dup 2q subtel Dup 19q subtel Del 10q subtel, dup 12q subtel Del 4p subtel Del 9p subtel, dup 9p subcen Monosomy X, dup 5q subtel, del 6q subtel Dup Xq subtel
Tetraploidy (92, XXXX) Near triploidy (71, XXX, +8, +8) Mos trisomy 5 (24.24%) Normal Normal Normal Normal Normal Normal Normal Normal (misidentified as monosomy 6 initially) Monosomy X, mos trisomy 9 (21.71%) Mos trisomy 13 (17.36%)
IA, induced abortion; PL, pregnancy loss; mos, mosaic; dup, duplication; del, deletion; subtel, subtelomere; subcen, subcentromere.
kits are different, or to employ other techniques (e.g., CMA) for defining the CNV sizes. In addition, parental karyotypes and trio MLPA tests could be helpful to clarify the clinical significance of segmental abnormalities. Compared to FISH, which seems to be an effective mosaic diagnostic method for obtaining the precise percentage of each cell line, the MLPA technique is a less sensitive method for discriminating mosaicism. Our study revealed that the capability of MLPA to detect mosaicism was dependent on the prevalence of aneuploid cell lines, with a threshold around 25%. This means that cases with low levels of mosaicism (10%–25%) would be missed by the MLPA assay. Besides the mosaic level, multiple aneuploid cell lines of the same chromosome existing simultaneously may affect the MLPA results. This occurred in sample No. PL-30, where the complementarity of monosomy 5 cells and trisomy 5 cells made the MLPA DQ of Chr. 5 close to normal, thereby leading to false negative MLPA results for mosaicism. In some cases of mosaicism, discrepancies between MLPA and FISH results were likely due to the different sampling sites of the CVSs. Even though only tiny amounts of tissues are required for molecular approaches, multi-point dissection of CVSs should be advocated in order to improve the detection rate of mosaicism. The published literatures have developed a consensus that ploidy status can be easily detected by performing FISH but is unable to be detected by MLPA [4], which implies that the MLPA method would miss the correct diagnosis in about 7% of miscarriages [4,22]. As mentioned above, nearly all the published MLPA studies chose subtelomeric probe sets (P036/P070) to screen all chromosomes [20,21]. The subtelomeric probes for the X and Y chromosome are identical as they detect sequences in the pseudoautosomal regions (PAR1 and
PAR2) which are identical in the X and Y chromosomes. This explains their inability to diagnose polyploidies since the results would show normal patterns from all probes. Different from the P036 and P070 kits, the P181 kit includes subcentromeric probes for each chromosome, except for the Y chromosome. Therefore, 69, XXY, 69, XYY and 92, XXXY, which comprise nearly 50% of polyploid abortuses [25,26], can actually be detected by MLPA because their DQs for sex chromosomes were similar in P036/P070 but different in P181 to that in a normal male reference. Moreover, in order to avoid missing the identification of the remaining half of the polyploidies (69, XXX, 92, XXXX and 92, XXYY), dual- or tri-color FISH with probes for any two or three different autosomes is recommended as an auxiliary test of the CVSs if no abnormalities are found by subtelomeric and subcentromeric MLPA screening [27]. In summary, both MLPA and FISH assays are powerful techniques for screening all 24 chromosomes on CVSs for aneuploidy detection, although each one has its own methodological weaknesses. Most of the FCAs, except for certain types of polyploidy and low percentage (< 25%) of mosaicism, can be discerned by MLPA easier and at a lower cost than FISH. Because of the prominent advantages of MLPA, we conclude that the joint application of P036, P070, and P181 MLPA kits is the preferred strategy for FCA detection and we recommended that FISH is used as a supplementary examination for MLPA-negative samples. This allows for the most economical and fastest detection while increasing the probability of identifying most aneuploidies. Declaration of interest None. 4
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Funding [13]
This work was supported by the Basic Research Program of Shenzhen [JCYJ20160427113223456] and Sanming Project of Medicine in Shenzhen [SZSM201502035], China.
[14]
Acknowledgements
[15]
The authors acknowledge the participants in this study. The authors thank the Molecular Biology team and the Cytogenetics team at the Fertility Center of Shenzhen Zhongshan Urology Hospital for assisting with sample preparation.
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Evaluation of two aneuploidy screening tests for chorionic villus samples: Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization Tonghua Wu, Yuanchang Zhu, Ling Hong, Qi Lin, Chunmei Chen, Jing Yang, Lijun Ye, Wensi Huang, Yong Zeng* Shenzhen Key Laboratory for Reproductive Immunology of Preimplantation, Shenzhen Zhongshan Institute for Reproduction and Genetics, Fertility Center, Shenzhen Zhongshan Urology Hospital, Shenzhen, Guangdong, 518045, China
ARTICLE INFO
ABSTRACT
Keywords: Multiplex ligation-dependent probe amplification (MLPA) Fluorescence in situ hybridization (FISH) Fetal chromosomal abnormality Mosaicism Pregnancy loss
The vast majority of first-trimester pregnancy losses are the consequence of numerical aberrations in fetal chromosomes, which may involve nearly all chromosomes. Although commercial probes for all chromosomes are available for multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH) analyses, their use has rarely been reported for screening all 24 chromosomes for early fetal demise, especially by FISH. Here, we validated the ability of MLPA and FISH techniques as two low-cost aneuploidy screening methods for 24 chromosomes in 165 chorionic villus samples (CVSs). The results obtained by two methods were compared by the Chi-square test and the Kappa agreement test. Both methods gave conclusive results for all CVSs tested and showed highly consistent results (kappa = 0.890, p < 0.001). There was no statistically significant difference between the aneuploidy rate of the CVSs tested by the two methods (p = 0.180). Most of the samples showed fully concordant molecular karyotyping results (81.21%) between the two analytical methods, 10.91% had incompletely concordant results, and 7.88% had discordant results. The inconsistencies included segmental abnormalities, mosaicism, and polyploidy. Both assays used to screen 24 chromosomes were powerful techniques for detecting aneuploidy in CVSs. In terms of cost-effectiveness and diagnostic accuracy, the combination of subtelomeric (P036, P070) and centromeric (P181) MLPA assays is the better analytic strategy and follow-up analysis by FISH is recommended for MLPA-negative samples.
1. Introduction Pregnancy loss (PL), the most common pregnancy-related complication, occurs in about 10%–15% of clinically recognized pregnancies [1]. Its etiology is heterogeneous. The vast majority of first-trimester PLs (50%–60%) are the consequence of fetal chromosomal abnormalities (FCAs), which can be of parental origin or de novo [2]. The spectrum of FCAs consists of numerical abnormalities (86%), structural abnormalities (6%), and other abnormalities (8%) [3]. Identifying the cause of a miscarriage plays an important role in patient management and next pregnancy guidance. Therefore, an effective testing approach to detect FCAs associated with PL is valuable. Conventional G-banding karyotyping, which has been considered as the standard reference test to detect FCAs, is hampered by culture failure, poor metaphase chromosome quality, and maternal cell contamination (MCC). Furthermore, it is time consuming and labor-
*
intensive [4–6]. Molecular approaches have been swiftly developed to detect aneuploidies without depending on cell culture, aimed at overcoming the intrinsic obstacles of karyotyping. DNA-based tests are mainly classified into two categories, whole genome techniques and chromosome region-specific techniques. The former refers to chromosomal comparative genomic hybridization (CGH), chromosomal microarray analysis (CMA) and next-generation sequencing (NGS), while the latter includes fluorescence in situ hybridization (FISH), quantitative fluorescent-polymerase chain reaction (QF-PCR), multiplex ligation-dependent probe amplification (MLPA), and bacterial artificial chromosomes (BACs)-on-Beads (BoBs). High-resolution CMA and low-pass NGS have become first-tier tests for most pediatric and prenatal diagnoses owing to their abilities to simultaneously detect numerical chromosomal abnormalities, submicroscopic chromosomal imbalances, and loss of heterozygosity at the entire genome-wide level. These two techniques are starting to be used
Corresponding author. E-mail address: [email protected] (Y. Zeng).
https://doi.org/10.1016/j.mcp.2019.101422 Received 26 May 2019; Received in revised form 9 July 2019; Accepted 15 July 2019 0890-8508/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Tonghua Wu, et al., Molecular and Cellular Probes, https://doi.org/10.1016/j.mcp.2019.101422
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Table 1 FISH probes for 24 chromosomes. Type
Label color
Target chromosome
Centromeric probes
Spectrum Spectrum Spectrum Spectrum Spectrum
CEP 1, CEP 2, CEP 3, CEP 16, CEP 17, CEP 20, CEP Y CEP 4, CEP 6, CEP 7, CEP 8, CEP 9, CEP 10, CEP 11, CEP 12, CEP 15, CEP X CEP 18, TelVysion 13q, TelVysion 14q, TelVysion 21q, TelVysion 22q TelVysion 5p, TelVysion 19p
Telomeric probe
Orange Green Aqua Orange Green
in the genetic evaluation of miscarriage cases, and approximately 6% additional findings over successful karyotyping have been reported [7–9]. The incremental diagnostic yield is mainly attributed to copy number variations (CNVs). Since it is not possible to identify the phenotype of a CNV found in an early abortus, whether these CNVs are causative of PLs remains uncertain and more large cohort studies are required to determine their pathogenicity [4,8]. In addition to the challenge of interpreting CNVs, the high testing fees and the professional data processing capabilities of these whole genome approaches currently remain prohibitive for routine application in the etiological diagnosis of PLs, especially in developing countries. Compared to whole genome techniques, chromosome region-specific techniques are widely applied as rapid aneuploidy tests for prenatal diagnosis because of their numerous advantages, which include ease-of-handling, rapid and sensitive screening, simple result interpretation, and relatively low cost [10,11]. The distribution of aneuploid chromosomes, however, is strikingly different between prenatal samples and miscarriage samples [12,13]. A limited probe panel for chromosomes 13, 18, 21 and sex chromosomes, which is an efficient and rapid aneuploidy test used for prenatal diagnosis, was reported to detect only around 20% of the FCAs in first trimester PLs, and the use of a panel for chromosomes 13, 16, 18, 21, 22, and sex chromosomes only detected about 60% [14]. Since numerical aberrations may involve nearly all chromosomes during the first trimester [15], only the assays providing information on all chromosomes are valid for testing miscarriage products. Though MLPA and FISH have commercially available probes for all chromosomes, the FCA analysis of early fetal demise using the commercial probes for all chromosomes has rarely been reported in the literature, especially for FISH. We, therefore, investigated the detectability and limitation of these two methods as aneuploidy screening tests for 24 chromosomes in chorionic villus samples (CVSs). We further proposed an economical and accurate testing strategy that can be widely applied to clinical testing.
samples were screened by MLPA with SALSA P036, P070 and P181 probemixes (MRC-Holland, The Netherlands) following the manufacturer's protocols. The PCR products were separated by capillary electrophoresis (ABI 3500 Genetic Analyzer, Applied Biosystems, USA) and the results were analyzed with Coffalyser. Net software (MRC-Holland, The Netherlands). Dosage quotients (DQ) = 0, 0.4 < DQ < 0.65, 0.8 < DQ < 1.2, 1.3 < DQ < 1.65, and 1.75 < DQ < 2.15 were interpreted as homozygous deletions, heterozygous deletions, normal, heterozygous duplications, and homozygous duplications, respectively. All other ambiguous DQ values were considered as mosaicism. CNVs for all the subtelomeric and subcentromeric probes of any individual chromosome indicated whole chromosome aneuploidy. Increased or decreased DQ at subtelomeric or/and subcentromeric probes of one chromosomal arm indicated segmental aneuploidy. 2.3. FISH The CVSs collected for FISH analysis were first incubated in 7 ml 75 mM KCl at 37 °C for 30 min, then were prefixed by adding 3 ml of 3:1 fixative (methanol: acetic acid) to the hypotonic solution for 5 min at room temperature. After the solution was decanted, the cells were fixed in 8 ml of 3:1 fixative (methanol: acetic acid) three times. The villi cells with fresh fixative were dropped onto three oblique slides. In order to ensure that all chromosomes were detected, each slide was divided into four isolated regions and each region was processed for interphase FISH detection using 2 to 3 different color probes for different chromosomes. The chromosomal FISH probes were commercially available from Vysis (Abbott Molecular, USA) and the details are listed in Table 1. Hybridization was performed overnight in a humidified HYBrite (Vysis, USA). The calculation and analysis of the hybridization signals for each probe were conducted using a fluorescent microscope (Nikon, Japan). According to the Chinese expert's consensus on the application of FISH technique in prenatal diagnosis, a minimum of 100 interphase nuclei per probes were randomly scored. The percentage of abnormal cells, scored as < 10%, 10%–60%, and > 60% indicated normal, mosaicism, and aneuploidy, respectively [16,17].
2. Materials and methods 2.1. Sample collection and preparation
2.4. Statistical analysis
Terminated singleton pregnancies, performed by dilation and curettage (D&C) in the first trimester at Shenzhen Zhongshan Urology Hospital and of women who agreed to chromosomal analysis of the abortuses, were included in the present study. The study was approved by the Research Ethics Committee of Shenzhen Zhongshan Urology Hospital and informed consent was obtained from each patient prior to study initiation. The products of conception obtained by D&C were collected into sterile falcon flasks containing normal saline. The CVSs were dissected carefully from the tissue and washed thoroughly in aseptic normal saline solution to minimize any MCC. The obtained samples were divided into two parts for MLPA and FISH analyses.
The results between the two methods were compared by the McNemar Chi-squared test and the Kappa agreement test using IBM SPSS Statistics 23 software. The level of statistical significance was set at p < 0.05. 3. Results A total of 165 samples were successfully collected for this study, including 135 cases of PL and 30 cases of induced abortion (IA). Both methods gave conclusive results for all the tested CVSs. The molecular karyotyping results were categorized as euploidy, one chromosome aneuploidy, one chromosome segmental abnormality, multiple chromosome abnormality, polyploidy, and mosaicism. The categorial proportions of the molecular karyotyping results detected by the MLPA and FISH assays are shown in Fig. 1. Among the 165 samples, a normal pattern for all probes was obtained in 72 (46 PL, 26 IA) cases by MLPA, and in 77 (50 PL, 27 IA) cases by FISH. Whether the specimen was
2.2. MLPA DNA of the CVSs was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer's instructions and was quantified by spectrophotometer (Shimadzu, Japan). The subtelomeric and subcentromeric regions of each chromosome in the DNA 2
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aneuploidies by MLPA. The median mosaic percentage detected by FISH in the former 10 chromosomes and the latter four chromosomes was 21.17% (range 11.04%-30.25%) and 49.08% (range 27.63%60.00%) respectively, while that of the 6 chromosomes identified as mosaicism by both methods was 51.42% (range 46.00%-60.18%). In addition, trisomy 21 mosaicism was detected by MLPA in one sample (No. PL-106), where FISH only detected the normal clone. And in another case (sample No. PL-63), MLPA identified mosaic trisomy 20, whereas FISH only detected the trisomy 20. Two cases showed discordant results between FISH and MLPA testing because of polyploidy. In one sample, pure tetraploidy (92, XXXX) detected by FISH was predictably identified as normal by MLPA. The other case was an aneuploidy occurring on a triploid background (71, XXX, +8, +8), which was recognized by FISH. The aneuploidy was detected as trisomy 8, while the baseline polyploidy karyotype was not recognized by MLPA in this instance. 4. Discussion The chromosome region-specific technique with a complete set of 24 FISH probes has been rarely applied to genetic diagnosis in spontaneous miscarriage in the past decades. Some studies have reported the evaluation of MLPA as a technique to detect aneuploidies in miscarriages products, but nearly all these studies used P095 aneuploidy probemix for 5 chromosomes (Chr. 13, 18, 21, X, and Y) or P036/P070 subtelomeric probemix for all chromosomes [18–21]. The combination of subtelomeric (P036, P070) and subcentromeric (P181) MLPA assays for 24 chromosomes has not yet been subjected to methodological evaluation. In our study, we evaluated the performance of these two molecular approaches for aneuploidy detection of all chromosomes in 165 CVSs samples. Both methods successfully detected aneuploidy, provided quick results (normally available in 2–3 days), and detected a wide variety of FCAs. The study results suggested that both methods overcame the main disadvantages of conventional cytogenetic analysis, including culture failure (20%) and time consumption (10–21 days) [22]. Furthermore, the MLPA technique was much less expensive (just 1/5 the cost of FISH), less labor-intensive (manual operation time, 2–3 h/run vs. 6–8 h/run for FISH), and had higher throughput capability (maximum sample size, 32 CVSs/run/technician vs. 3 CVSs/run/ technician for FISH) than FISH. Both methods presented excellent detection performance and a highly consistency for all euploidies and non-mosaic whole-chromosome aneuploidies identified. One case (sample No. PL-72) was misdiagnosed as monosomy 6 by FISH initially because of the poor signal. Thus, strictly following the guidelines for FISH issued by the American College of Medical Genetics (ACMG) is critical for guaranteeing the accuracy of FISH results. One such guideline states that the results of FISH analysis should be confirmed by at least two experienced technologists [23]. One remarkable advantage of MLPA analysis compared to FISH is the ability to detect unbalanced structural abnormalities, because it analyzes several loci of each chromosome simultaneously. Of the 17 segmental aneuploidies detected by MLPA in the present study, 11 CNVs were subtelomeric, 2 CNVs were subcentromeric, and the rest were subtelomeric and subcentromeric concurrently. However, the exact size of the imbalance was unknown due to the low number of MLPA probes. Three subcentromeric duplications were not revealed by centromeric FISH probes. This might be attributed to the imbalance fragment, which was smaller than the FISH probe size. Laboratory technicians should be aware that duplication or deletion affecting only one subtelomeric probe of a chromosomal arm is more likely to be considered a copy number polymorphisms. This is particularly true for the subtelomeric polymorphisms quoted in the product descriptions on the MRC-Holland website [24]. For the sake of avoiding false positive results, it is therefore strongly recommended to use both P036 and P070 probemixes to confirm subtelomeric CNVs, since all the probes of two
Fig. 1. Stacked column diagram of molecular karyotyping results analyzed by MLPA and FISH.
determined as euploid or not by these two methods was highly consistent (kappa = 0.890, p < 0.001). There was no statistically significant difference between the aneuploidy rate of the CVSs tested by these two methods (p = 0.180). The results of the MLPA and FISH analyses were assorted in the following manner. Fully concordant cases were defined as those with agreement between the MLPA and FISH results. Incompletely concordant cases referred to those where consistent abnormalities were detected by MLPA and FISH but one test contained more clinically useful information than the other. Discordant cases referred to those where the molecular karyotype results of the two methodologies were different. All findings in these three categories are summarized in Table 2. A fully concordant molecular karyotyping result was identified in most samples (81.21%), while 10.91% and 7.88% of the cases showed incompletely concordant and discordant results respectively. There were three main types of inconsistencies between the MLPA and FISH results: segmental abnormalities, mosaicisms, and polyploidies. Segmental aneuploidy was the principal cause of the inconsistent results (n = 17). Despite the fact that the precise size of the duplications or deletions was not clear, the 21 chromosomes with segmental abnormalities observed in MLPA testing (4 samples with 2 chromosomes involved) could represent reliable CNVs. This is because the subtelomeric CNVs were identified by both MLPA probemixes P036 and P070 while the subcentromeric CNVs were confirmed by retesting. Two duplications [chromosome (Chr.) 13 in samples No. PL-65 and No. PL28] were identified three FISH signals, while the rest of the CNVs had two FISH signals. The locus of the FISH probe that was matched to or outside the CNV region could reasonably explain the two FISH-detectable CNVs and 16 FISH-undetectable CNVs, but it was not applicable to the three FISH-undetectable centromeric duplications detected by centromeric FISH probes (Chr.7 in samples No. PL-31 and No. PL-32, Chr.11 in sample No. PL-50). Fifteen of the cases with inconsistent results were attributed to the presence of mosaicism. Thirteen cases with 14 chromosomes were identified as mosaic by FISH, but ten of the 14 mosaic chromosomes were identified as disomies and four were identified as nonmosaic 3
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Table 2 Molecular karyotyping results of 165 CVSs analyzed by both MLPA and FISH. Category Fully concordant (n = 134) Normal (n = 70) Abnormality (n = 64) Incompletely concordant (n = 18) Mosaicism (n = 10)
Segmental abnormality (n = 7)
Compound (n = 1) Discordant (n = 13) Polyploidy (n = 2) Mosaicism (n = 2) Segmental abnormality (n = 7)
Compound (n = 2)
ID no.
MLPA results
FISH results
(too many to list) (too many to list) IA-3 PL-30 PL-52 PL-75 PL-87 PL-97 PL-142 PL-41 PL-63 PL-67 PL-28 PL-31 PL-32 PL-33 PL-50 PL-65 PL-156 PL-104
Trisomy 16 Trisomy 8 Trisomy 22 Trisomy 13 Trisomy 15 Trisomy 15 Trisomy 22 Trisomy 15 Mos trisomy 20 Trisomy 15, trisomy 16, trisomy 21 Trisomy 4, dup 13q subtel, dup 12p subtel Monosomy X, dup 7p subtel, dup 7 subcen Trisomy 22, dup 7p subtel, dup 7 subcen Monosomy X, del 13q subcen Monosomy X, trisomy 5, dup 11 subcen Dup 13q subtel, dup 13 subcen Trisomy 21, del 13q-c subtel Trisomy 3, dup Xp subtel
Trisomy 16, mos trisomy 5 (20.63%) Trisomy 8, mos monosomy 5 plus mos trisomy 5 (30.25%) Trisomy 22, mos trisomy 9 (22.4%) Trisomy 13, mos trisomy 7 (13.71%) Trisomy 15, mos monosomy 17 (11.04%) Trisomy 15, mos trisomy 21 (12.28%) Mos trisomy 22 (60.00%), mos trisomy 8 (25.50%) Mos trisomy 15 (27.63%) Trisomy 20 Mos trisomy 15 (44.50%), trisomy 16, trisomy 21 Trisomy 4, trisomy 13 Monosomy X Trisomy 22 Monosomy X Monosomy X, trisomy 5 Trisomy 13 Trisomy 21 Mos trisomy 3 (53.66%)
PL-137 PL-114 IA-10 PL-106 IA-1 IA-28 PL-85 PL-99 PL-122 PL-147 PL-72 PL-48 PL-79
Normal Trisomy 8 Normal Mos trisomy 21 Del 19q subtel Dup Xq subtel Dup 7q subtel, dup 2q subtel Dup 19q subtel Del 10q subtel, dup 12q subtel Del 4p subtel Del 9p subtel, dup 9p subcen Monosomy X, dup 5q subtel, del 6q subtel Dup Xq subtel
Tetraploidy (92, XXXX) Near triploidy (71, XXX, +8, +8) Mos trisomy 5 (24.24%) Normal Normal Normal Normal Normal Normal Normal Normal (misidentified as monosomy 6 initially) Monosomy X, mos trisomy 9 (21.71%) Mos trisomy 13 (17.36%)
IA, induced abortion; PL, pregnancy loss; mos, mosaic; dup, duplication; del, deletion; subtel, subtelomere; subcen, subcentromere.
kits are different, or to employ other techniques (e.g., CMA) for defining the CNV sizes. In addition, parental karyotypes and trio MLPA tests could be helpful to clarify the clinical significance of segmental abnormalities. Compared to FISH, which seems to be an effective mosaic diagnostic method for obtaining the precise percentage of each cell line, the MLPA technique is a less sensitive method for discriminating mosaicism. Our study revealed that the capability of MLPA to detect mosaicism was dependent on the prevalence of aneuploid cell lines, with a threshold around 25%. This means that cases with low levels of mosaicism (10%–25%) would be missed by the MLPA assay. Besides the mosaic level, multiple aneuploid cell lines of the same chromosome existing simultaneously may affect the MLPA results. This occurred in sample No. PL-30, where the complementarity of monosomy 5 cells and trisomy 5 cells made the MLPA DQ of Chr. 5 close to normal, thereby leading to false negative MLPA results for mosaicism. In some cases of mosaicism, discrepancies between MLPA and FISH results were likely due to the different sampling sites of the CVSs. Even though only tiny amounts of tissues are required for molecular approaches, multi-point dissection of CVSs should be advocated in order to improve the detection rate of mosaicism. The published literatures have developed a consensus that ploidy status can be easily detected by performing FISH but is unable to be detected by MLPA [4], which implies that the MLPA method would miss the correct diagnosis in about 7% of miscarriages [4,22]. As mentioned above, nearly all the published MLPA studies chose subtelomeric probe sets (P036/P070) to screen all chromosomes [20,21]. The subtelomeric probes for the X and Y chromosome are identical as they detect sequences in the pseudoautosomal regions (PAR1 and
PAR2) which are identical in the X and Y chromosomes. This explains their inability to diagnose polyploidies since the results would show normal patterns from all probes. Different from the P036 and P070 kits, the P181 kit includes subcentromeric probes for each chromosome, except for the Y chromosome. Therefore, 69, XXY, 69, XYY and 92, XXXY, which comprise nearly 50% of polyploid abortuses [25,26], can actually be detected by MLPA because their DQs for sex chromosomes were similar in P036/P070 but different in P181 to that in a normal male reference. Moreover, in order to avoid missing the identification of the remaining half of the polyploidies (69, XXX, 92, XXXX and 92, XXYY), dual- or tri-color FISH with probes for any two or three different autosomes is recommended as an auxiliary test of the CVSs if no abnormalities are found by subtelomeric and subcentromeric MLPA screening [27]. In summary, both MLPA and FISH assays are powerful techniques for screening all 24 chromosomes on CVSs for aneuploidy detection, although each one has its own methodological weaknesses. Most of the FCAs, except for certain types of polyploidy and low percentage (< 25%) of mosaicism, can be discerned by MLPA easier and at a lower cost than FISH. Because of the prominent advantages of MLPA, we conclude that the joint application of P036, P070, and P181 MLPA kits is the preferred strategy for FCA detection and we recommended that FISH is used as a supplementary examination for MLPA-negative samples. This allows for the most economical and fastest detection while increasing the probability of identifying most aneuploidies. Declaration of interest None. 4
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Funding [13]
This work was supported by the Basic Research Program of Shenzhen [JCYJ20160427113223456] and Sanming Project of Medicine in Shenzhen [SZSM201502035], China.
[14]
Acknowledgements
[15]
The authors acknowledge the participants in this study. The authors thank the Molecular Biology team and the Cytogenetics team at the Fertility Center of Shenzhen Zhongshan Urology Hospital for assisting with sample preparation.
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