Maternal Mosaicism Is a Significant Contributor to Discordant Sex Chromosomal Aneuploidies Associated with Noninvasive Prenatal Testing Yanglin Wang, 1,2† Yan Chen, 2† Feng Tian, 3 Jianguang Zhang, 3 Zhuo Song, 3 Yi Wu, 1 Xu Han, 1 Wenjing Hu, 1 Duan Ma, 2 David Cram, 3* and Weiwei Cheng 2* BACKGROUND: In the human fetus, sex chromosome aneuploidies (SCAs) are as prevalent as the common autosomal trisomies 21, 18, and 13. Currently, most noninvasive prenatal tests (NIPTs) offer screening only for chromosomes 21, 18, and 13, because the sensitivity and specificity are markedly higher than for the sex chromosomes. Limited studies suggest that the re- duced accuracy associated with detecting SCAs is due to confined placental, placental, or true fetal mosa- icism. We hypothesized that an altered maternal karyo- type may also be an important contributor to discor- dant SCA NIPT results. METHODS: We developed a rapid karyotyping method that uses massively parallel sequencing to measure the degree of chromosome mosaicism. The method was validated with DNA models mimicking XXX and XO mosaicism and then applied to maternal white blood cell (WBC) DNA from patients with discordant SCA NIPT results. RESULTS: Sequencing karyotyping detected chromo- some X (ChrX) mosaicism as low as 5%, allowing an accurate assignment of the maternal X karyotype. In a prospective NIPT study, we showed that 16 (8.6%) of 181 positive SCAs were due to an abnormal maternal ChrX karyotype that masked the true contribution of the fetal ChrX DNA fraction. CONCLUSIONS: The accuracy of NIPT for ChrX and ChrY can be improved substantially by integrating the results of maternal-plasma sequencing with those for maternal-WBC sequencing. The relatively high fre- quency of maternal mosaicism warrants mandatory WBC testing in both shotgun sequencing– and single- nucleotide polymorphism– based clinical NIPT after the finding of a potential fetal SCA. © 2014 American Association for Clinical Chemistry 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 embry- onic 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 ab- normalities associated with early pregnancy failure (2– 4 ). A small proportion of these aneuploidies—such as tri- somy 21 (Down syndrome), trisomy 18 (Edwards syn- drome), 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 of fetal 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 im- portant aneuploidies in the established fetus, leading to a reduction in the number of children born with chromo- somal 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 af- 1 Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical College, Fudan University, Shanghai, China; 2 Prenatal Diagnosis Center, Inter- national Peace Maternity and Child Health Hospital, Shanghai Jiaotong Univer- sity, Shanghai, China; 3 Berry Genomics, Beijing, China. * Address correspondence to: D.C. at Berry Genomics, Beijing, Bldg. 9, Link Park, 6 Jingshun East St., Chaoyang District, Beijing 100015, China. Fax 86 –10- 84306824; e-mail david.cram@berrygenomics.com. W.C. at Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical College, Fudan University, Shanghai 200032, China. E-mail 18017316001@163.com. † Yanglin Wang and Yan Chen contributed equally to the work, and both should be considered first authors. Received September 1, 2013; accepted October 21, 2013. Previously published online at DOI: 10.1373/clinchem.2013.215145 4 Nonstandard abbreviations: SCA, sex chromosome aneuploidy; NIPT, noninva- sive prenatal test; CPM, confined placental mosaicism; ChrX, chromosome X; WBC, white blood cell. Clinical Chemistry 60:1 000 – 000 (2014) Molecular Diagnostics and Genetics 1 http://hwmaint.clinchem.org/cgi/doi/10.1373/clinchem.2013.215145 The latest version is at Papers in Press. Published November 5, 2013 as doi:10.1373/clinchem.2013.215145 Copyright (C) 2013 by The American Association for Clinical Chemistry