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Maternal mosaicism is a significant contributor to discordant sex chromosomal aneuploidies associated with noninvasive prenatal testing.

Chromosomal aneuploidy in the human originates from either gamete meiotic or mitotic cleavage-stage errors in the early preimplantation embryo (1). The vast majority of these aneuploidies are lethal, either by causing embryonic growth arrest before implantation or by spontaneous abortion of the developing fetus during the first trimester of pregnancy. Autosomal trisomies, polyploidies, and monosomy X are the main groups of chromosomal abnormalities associated with early pregnancy failure (2-4). A small proportion of these aneuploidies--such as trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), monosomy X (Turner syndrome), XXY (Klinefelter syndrome), XXX (triple X syndrome), and XYY (Jacob syndrome)--are more tolerated, for reasons still unknown, in the second and third trimester offetal development and thus present as chromosome disease in approximately 0.3% of live births (5). For more than 30 years, the practice of prenatal diagnosis has enabled early identification of clinically important aneuploidies in the established fetus, leading to a reduction in the number of children born with chromosomal diseases (6-8). Prenatal diagnosis of the common trisomies 21, 18, and 13 has proved highly accurate, thanks to maternal-serum screening, ultrasound, and follow-up chorionic villus sampling or amniocentesis, in combination with fetal karyotyping (6,9). In contrast, apart from monosomy X, the diagnosis of fetuses with sex chromosome aneuploidies (SCAs) [4] or their mosaic SCA variants remains more problematic, because most affected pregnancies do not show any overt clinical signs or ultrasound abnormalities (10,11).

With the clinical introduction of massively parallel sequencing of maternal plasma, pregnant women can now choose to have a noninvasive prenatal test (NIPT) for the clinically important trisomies 21, 18, and 13, as well as for SCAs, which represent approximately half the clinically important chromosome abnormalities seen in the fetus (12). Current data from prospective NIPT studies (13-15) have shown remarkably high sensitivities and specificities, approaching 100% for the 3 fetal trisomies. False-positive and false-negative NIPT results do occur, albeit at very low frequency (<0.1%). Limited case study follow-up of discordant NIPT results via fetal karyotyping and placental cytogenetic analysis has shown that confined placental mosaicism (CPM) (16-18) and placental mosaicism (1821 ), in which differences occur in the distribution and proportion of euploid and aneuploid cells, are important biological factors that either increase the effective fetal DNA fraction to yield a false-positive fetal aneuploidy or decrease the effective DNA fraction to yield a false-negative aneuploidy. Although NIPT results are also highly accurate, slightly lower sensitivities and specificities have been consistently reported for SCAs in blinded studies of known SCAs and euploid samples (22, 23) and in prospective studies (13, 15). Even with improvements in the identification of homologous chromosome X (ChrX) and ChrY sequences (24) that lead to more accurate calculations of ChrX and ChrY z scores, it is now widely believed that the vast majority of discordant SCA NIPT results are also caused by either CPM or placental mosaicism, with true fetal mosaicism being an additional contributing factor (9).

An altered or mosaic maternal karyotype represents another possible cause of discordant SCAs, because the effective fraction of ChrX DNA in the maternal plasma would be dramatically different from that of typical maternal 46,XX somatic cells and thus affect the calculation of the true fraction of fetal ChrX DNA. We recently reported a discordant NIPT case of trisomy 18 due to 6% maternal mosaicism for this aneuploidy (15), thus demonstrating in principle that an altered maternal-DNA plasma can substantially skew the final fetal z score for the involved chromosome. Furthermore, another unusual case report found (upon substantive clinical follow-up) that the source of a trisomy 13 and monosomy 18 false-positive NIPT result ultimately was due to a metastatic tumor in the mother (25). It is well known that there exists a small percentage of otherwise healthy, fertile women who have maternal SCA mosaicism, or occasionally a full-blown SCA such as XO or XXX, and who can conceive healthy euploid offspring (11). In addition, some women of advanced reproductive age have been reported to undergo gradual and preferential loss of the X chromosome that undergoes X inactivation, thus converting their blood karyotype from XX to an XO/XX mosaic (26). Indeed, a discordant NIPT result in 1 isolated report was attributed to low-level maternal 45,XO mosaicism (18). We therefore speculated that a small but noteworthy proportion of women presenting for NIPT have an altered ChrX karyotype that would skew the maternal ChrX DNA fraction and thus manifest as discordant fetal SCAs. The present study aimed to develop a rapid and accurate method for maternal SCA detection and to determine the frequency of discordant SCA NIPT results due to an altered maternal karyotype.



For NIPT, we collected 10-mL samples of peripheral blood from pregnant Chinese women (12-16 weeks' gestation) in a Cell-Free DNA BCT(tm) tube (Streck) and sent them to Berry Genomics, Beijing, for processing and sequencing. The plasma and white blood cells (WBCs) from 2.3 mL of blood were separated by 2 rounds of centrifugation to produce an approximately 1.3-mL plasma fraction and an approximately 1.0-mL fraction of WBCs. DNA was extracted from a 1.0-mL plasma aliquot with the QIAamp Circulating Nucleic Acid Kit (Qiagen). Genomic DNA was purified from 0.2-mL aliquots of the maternal-WBC fractions. For the sensitivity and reproducibility analysis, we processed 2-mL blood samples from a healthy male (46,XY) and from a patient with triple X syndrome (47,XXX) in the same manner. We karyotyped amniocytes or maternal WBCs via G-banding analysis of metaphase chromosome spreads at 450-band resolution.


We constructed plasma DNA libraries and performed massively parallel sequencing on the Illumina HiSeq 2000 platform, as previously described (15,27). We generated approximately 8 X [10.sup.6] single-end reads of 36 bp from each library and aligned them to the unmasked human genome sequence (28). We counted uniquely mapped reads and then calculated z scores for each chromosome after GC normalization for Chr13, Chr18, Chr21, and ChrX (27). Chromosome z score values less than -3.0 or greater than + 3.0 were classified as abnormal (15).


For each sample, we used 50 ng of WBC genomic DNA with Nextera DNA Sample Preparation Kits (Epicentre/Illumina) to construct the sequencing library. Multiple libraries were indexed and pooled into a single lane. Library fragments were sequenced to 43 bp (with 7 bp being the index sequence) on a HiSeq 2000 instrument. Sequencing reads were analyzed according to the data analysis pipeline summarized in Fig. 1. First, sequencing reads were aligned to the unmasked human genome sequence (hg19). Second, we calculated the read density by dividing each chromosome into contiguous 20-kb bins. For each bin, i, of a given sample, the reads that were uniquely and perfectly mapped to that bin ([RNB.sub.i]) were counted and normalized to the total read number for the sample and the total possible unique 36-bp fragment numbers within bin i ([U.sub.i]), according to the following equation: NRNB, = RNB, X (8 X [10.sup.6])/(total read number of the sample X [U.sub.i]), where [NRNB.sub.i] is the normalized read number uniquely and perfectly mapped to bin i. For any given bin i of the reference samples, we calculated the median ([[mu].sub.i]) of NRNB,. We assumed that changes in abnormal copy numbers among test samples are rare for each bin; therefore, the median of NRNB, represents the normal copy number. For any bin, of a test sample, we calculated the ratio ([ratio.sub.i]) between the test sample and the normal chromosome copies according to the following equation: [ratio.sub.i] = [NRNB.sub.i]/[[mu].sub.i];. We then plotted [log.sub.2]([ratio.sub.i]) values for all of the bins for a given sample to generate the copy number values along the length of each chromosome. The gain or loss of chromosome regions was detected via the fused lasso algorithm, as described previously (29). Lastly, to determine the level of chromosome mosaicism, we calculated the normalized chromosome representations ([NCR.sub.j]) for each chromosome of any given test sample, j, and the reference samples ([NCR.sub.f]) with the method described previously (27). The percentage of chromosomal mosaicism was then calculated according to the following equation: ([NCR.sub.j] - [NCR.sub.f])/[NCR.sub.f].




To determine the effect of any level of maternal mosaicism on the interpretation of NIPT fetal results for SCAs, we deemed it imperative to develop a rapid and accurate method. We speculated that separation of the WBC fraction from maternal blood samples at the same time as the plasma DNA would most likely represent the best and most accurate source of cells for analysis, because WBCs are believed to be the primary source of cells contributing to the steady-state fraction of maternal DNA in the plasma (9). We hypothesized that the DNA-sequencing strategy used for NIPT could be applied equally to the mother's WBCs for rapid identification of an altered karyotype and/or to various levels of maternal mosaicism of the sex chromosomes. To test this hypothesis, we designed a genomic DNA-mixing model in which we purified DNA from a 47,XXX patient and a 46,XY male control to mimic various levels of maternal ChrX mosaicism. In this experimental design, we mixed known 46,XY and 47,XXX genomic DNAs in various ratios, starting with pure 46,XY (equivalent to 45,XO in this context) DNA and incrementally increasing the proportion of 47,XXX DNA (to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95%) to create samples mimicking increasing proportions of XO in a background of XX, a balanced XX sample (no ChrX change), samples with increasing proportions of XXX in a background of XX, and then finishing with pure XXX. We sequenced the samples and calculated the percentage gains in copy number for ChrX.

Examples of ChrX-sequencing plots for XO (100%), XO (80%)/XXX (20%), XO (50%)/XXX (50%), XO (20%)/XXX (80%), and XXX (100%) samples are shown in Fig. 2. All levels of "XO mosaicism" and "XXX mosaicism" in an XX background determined by sequencing were virtually identical to the expected values. To assess the reproducibility of the sequencing method, we performed the experiment with 3 independent technical replicates (prepared by 3 different technicians) at each level of XO and XXX mosaicism. We then plotted the actual XO/XXX ratios (experimental) calculated by sequencing against the expected XO/XXX ratios (theoretical) for all increased proportions of ChrX in the 3 independent sample replicates (Fig. 3). The actual and expected XO/XXX ratios were linearly correlated ([r.sup.2] = 0.99893). Equally important, we were able to detect XO mosaicism levels of <5% accurately, indicating that our "sequencing karyotyping" method was highly sensitive. As expected, the mean [log.sub.2] values for all autosomes in these ChrX mosaic DNA samples showed no significant deviations from zero (data not shown), indicating normal copy numbers.


In routine NIPT, we identified 3 samples with unusually high ChrXz scores (greater than + 5) and 3 samples with unusually low ChrX z scores (less than -5) that could not be accounted for statistically as a fetal SCA (Table 1). We suspected that the abnormally skewed ChrX z scores were caused by a deviation in the effective ChrX maternal-plasma DNA fraction produced by an altered WBC karyotype. To investigate this possibility further, we applied our newly developed sequencing karyotyping strategy to the WBC fractions derived from the original NIPT blood samples. Sequencing ChrX karyotyping profiles for 4 of the 6 maternal WBC samples are depicted in Fig. 4. According to the percentage increase in ChrX (Table 1), NIPT samples 1, 4, and 6 with high z scores were associated with a 100% XXX, a 48% XXX mosaic, and a 94% XXX mosaic maternal karyotype, respectively. In contrast, according to the percentage loss of ChrX, samples 2, 3, and 5 with low z scores were associated with a 5%, 20%, and 15% XO mosaic maternal karyotype, respectively. In all 6 samples, the degree of change in the presumed fetal ChrX DNA fraction was generally correlated with the degree of ChrX change in the maternal-WBC DNA. Given that fetal DNA constitutes only a minor fraction of the maternal plasma DNA, we concluded that the altered maternal karyotype was the predominant contributor to the final fetal ChrX z score (Table 1). We also karyotyped the maternal WBCs by G-banding analysis as a confirmatory step to validate the sequencing maternal karyotypes (Table 1). Apart from sample 2, the karyotypes determined by G-banding analysis and those determined by sequencing were highly concordant. Follow-up amniocentesis and G-banding karyotyping revealed that all 6 fetuses were euploid (Table 1).


To determine the frequency of maternal mosaicism contributing to discordant SCAs, we surveyed NIPT SCAs during a 5-month period of clinical NIPT, from December 2012 to April 2013. During this time, only trisomies 21, 18, and 13 were reported. A total of 446 abnormal NIPT samples were identified, of which 259 (58.1%) were trisomy 21, 18, or 13; 187 (41.9%) of these samples were SCAs. Sequencing analysis for the maternal karyotype of the 187 patients with a positive SCA NIPT result (including the 6 patients described above) identified 16 patients (8.56%) with an altered or mosaic maternal karyotype: 6 (9.52%) of 63 patients had an abnormal ChrX gain, and 10 of 124 patients (8.06%) had an abnormal ChrX loss (Table 2). For the remaining 259 patients with a fetal trisomy result, maternal-DNA sequencing revealed typical 46,XX karyotypes in all cases.



The limited follow-up studies of discordant NIPT results for trisomy 21 (17,20, 21), trisomy 18 (20), and trisomy 13 (19) with placental analyses have revealed the biological phenomena of CPM and/or placental mosaicism to be important contributing factors. Therefore, CPM/placental mosaicism and perhaps true fetal mosaicism (9) have also been assumed to be the major causes of discordant SCAs. In the present study, we used a novel maternal-WBC sequencing strategy with high sensitivity and specificity to analyze 187 cases with abnormal NIPT results and identified 16 samples (8.5%) in which the discordance was directly attributable to either an altered or a mosaic maternal karyotype. This relatively high frequency of discordant re suits due to an altered maternal karyotype suggests that an abnormal maternal ChrX fraction of the plasma DNA is an important cause of discordant SCAs. This finding is the first to explain, in part, the consistently lower sensitivities and specificities that have been reported for SCAs in the clinical setting (13, 15). Largescale patient follow-up studies in which fetal karyotyping of abnormal SCA NIPT results are combined with targeted placental analyses, which were not possible in the present study, are now needed to determine the relative contributions of altered maternal karyotype, CPM/placental mosaicism, and true fetal mosaicism to discordant SCA NIPT results.


The study highlights the value of determining the maternal karyotype in increasing the accuracy of reporting NIPT results for chromosomes X and Y. For this purpose, we specifically developed and validated a rapid sequencing karyotyping method that uses the maternal-WBC fraction from the same blood sample used to isolate the maternal-plasma DNA. For 5 of the 6 SCA-discordant samples, we showed that the degree of loss or gain of ChrX as determined by sequencing is an accurate predictor, as confirmed by conventional G-banding karyotyping. For the remaining sample (no. 2; Table 1), G-banding analysis revealed a higher proportion of 45,XO cells than sequencing. Because the ChrX NIPT z score of -7.95 was more consistent with the relatively small loss of ChrX predicted by sequencing, we concluded that the discordant G-banding result was possibly caused by other factors known to be associated with karyotyping, such as suboptimal culture conditions, pseudomosaicism for 45,XO in extended culture (30), and even cell-counting errors. The sequencing karyotyping method specifically developed in this study also has other useful advantages over conventional karyotyping. First, it is rapid, allowing identification of a maternal karyotype that can be used to interpret the NIPT result. Second, being a molecular assay, the method is scalable. Third, the method is highly sensitive, with a capacity to detect not only an altered maternal ChrX karyotype but also mosaicism levels of <5%. In rare cases, the method should also be able to identify changes in the autosomal karyotype. Given these advantages, we therefore recommend that the maternal karyotype be determined after identification of any abnormal NIPT result. This order of analysis would improve the accuracy of reporting NIPT ChrX and ChrY results.


The findings of the study also indicate that determination of the maternal karyotype will substantially decrease the rate of discordant SCAs and in effect increase the overall sensitivity and specificity of NIPT for SCAs. Nevertheless, difficulties remain for NIPT cases in which the woman has low-level mosaicism for ChrX. If women with an altered karyotype can conceive healthy euploid babies, they can also conceive babies with SCAs, because this mosaicism may also be present in the germ line (11, 31). Although the true fetal ChrX measurement will be masked in most cases by the dominant maternal ChrX change, laboratories would still be alerted to a potential fetal SCA simply because of an altered maternal karyotype. It is possible in a very small number of cases, however, that a fetal SCA will be missed when the fetal SCA is in balance with the maternal X mosaicism. The following hypothetical scenario illustrates this possibility. If the fetus is XXX and the mother is 10% XO and 90% XX, and if the fetal DNA fraction is approximately 10%, the NIPT result will appear normal in the absence of knowledge of the maternal karyotype. In cases of knowledge of a presumably normal NIPT result but an altered maternal karyotype, however, the patient can be identified immediately and referred for amniocentesis and fetal karyotyping to clarify the NIPT result. If an independent and reliable method for calculating the fetal fraction from genomic sequencing data were available (other than that associated with ChrX and ChrY), this method could eventually be used as an alternative to maternal DNA sequencing in a more holistic approach for identifying whether a ChrX abnormality is of maternal or fetal origin.

On the basis of our finding that a high percentage of the discordant NIPT SCA results are due to maternal mosaicism, we recommend that ifNIPT is offered clinically via either shotgun sequencing-based or single-nucleotide polymorphism-based approaches, the maternal karyotype should be tested for all samples that appear abnormal. This recommendation may raise ethical concerns, particularly in cases in which the mother has not previously been karyotyped and is unaware of having an altered X karyotype. Given that possibility, NIPT consent forms would need to be modified to state that a small number of fetal SCAs are attributable to maternal mosaicism. In addition, any report of such a case should also recommend genetic counseling for the mother to discuss any future reproductive issues and any potential effects of the altered karyotype on her health and well-being. One of the largest challenges remaining for NIPT is discordant trisomy and SCA results caused by placental mosaicism that either increase or decrease the effective fetal DNA fraction for the involved chromosome. Although false-positive results can be identified via amniocentesis and karyotyping to avoid termination of pregnancies with an otherwise healthy fetus, false-negative results remain more problematic, particularly for SCAs that usually do not show any overt clinical signs during pregnancy.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: F. Tian, Berry Genomics; Z. Song, Berry Genomics; D. Cram, Berry Genomics.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: Shanghai Committee of Science and Technology, China (grant no. 134119a4600).

Expert Testimony: None declared.

Patents: None declared.

Role of Sponsor: The funding organizations played a direct role in the design of study, choice of enrolled patients, review and interpretation of data, and preparation and final approval of the manuscript.


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Yanlin Wang, [1,2] ([dagger]) Yan Chen, [2] ([dagger]) Feng Tian, [3] Jianguang Zhang, [3] Zhuo Song, [3] Yi Wu, [2] Xu Han, [2] Wenjing Hu, [2] Duan Ma, [1] David Cram, [3] * and Weiwei Cheng [2] *

[1] Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical College, Fudan University, Shanghai, China; [2] Prenatal Diagnosis Center, International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai, China; [3] Berry Genomics, Beijing, China.

[4] Nonstandard abbreviations: SCA, sex chromosome aneuploidy; NIPT, noninvasive prenatal test; CPM, confined placental mosaicism; ChrX, chromosome X; WBC, white blood cell.

([dagger]) Yanlin Wang and Yan Chen contributed equally to the work, and both should be considered first authors.

* Address correspondence to: D.C. at Berry Genomics, Beijing, Bldg. 9, Link Park,

Received September 1, 2013; accepted October 21, 2013.

DOI: 10.1373/clinchem.2013.215145

[6] Jingshun East St., Chaoyang District, Beijing 100015, China. Fax +86-10 84306824; e-mail W.C. at Prenatal Diagnosis Center, International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai 200030, China. E-mail
Table 1. Detailed analysis and follow-up of NIPT results
with unusually high or low ChrX z scores.

NIPT      NIPT       Calculated         Fetal
sample   ChrX z      NIPT fetal     karyotype (a)
no.       score    ChrX gain/loss

1         51.55       + 79.31%          46,XN
2         -7.95        -13.46%          46,XN
3        -21.84        -31.99%          46,XN
4         29.34%       +48.95%          46,XN
5        -10.53%       -11.82%          46,XN
6         62.80        +87.03%          46,XN

NIPT       Maternal            Maternal
sample     WBC ChrX           karyotype
no.        gain/loss       by G-banding (b)
         by sequencing

1           +103.93%      47,XXX
2             -4.82%      45,X[64]/46,XX[36]
3            -19.97%      45,X[10]/46,XX[40]
4            +47.18%      47,XXX[60]/46,XX[40]
5            -14.35%      45,X[6]/46,XX[78]
6            +93.69%      47,XXX

(a) N, ChrX or ChrY.

(b) Square brackets indicate the number of metaphases analyzed.

Table 2. Contribution of an abnormal ChrX maternal karyotype
in a prospective study of 187 discordant SCAs.

Clinical    NIPT findings

NIPT        Abnormal NIPT for SCA, n
follow-up   Normal maternal karyotype, n
            Altered maternal karyotype, n
            Maternal mosaicism rate

Clinical    NIPT ChrX    NIPT ChrX    Total
              gain         loss

NIPT           63           124        187
follow-up      57           114        171
                6            10         16
                9.52%         8.06%      8.56%
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Title Annotation:Molecular Diagnostics and Genetics
Author:Wang, Yanlin; Chen, Yan; Tian, Feng; Zhang, Jianguang; Song, Zhuo; Wu, Yi; Han, Xu; Hu, Wenjing; Ma,
Publication:Clinical Chemistry
Geographic Code:1USA
Date:Jan 1, 2014
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