Original Article Non-invasive prenatal testing (NIPT) detected chromosome aneuploidies and beyond in a clinical setting

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1 Int J Clin Exp Med 2016;9(9): /ISSN: /IJCEM Original Article Non-invasive prenatal testing (NIPT) detected chromosome aneuploidies and beyond in a clinical setting Wei-Li Shi, Hui Zhang, Dong Wu, Yan Chu, Shi-Xiu Liao Medical Genetic Institute of Henan Province, Henan Provincial People s Hospital, People s Hospital of Zhengzhou University, Zhengzhou, PR China Received April 26, 2016; Accepted July 30, 2016; Epub September 15, 2016; Published September 30, 2016 Abstract: The discovery of circulating cell-free fetal DNA (cffdna) in maternal plasma opened up a new area in detecting fetal chromosome aneuploidies. Many studies have indicated that this technology had a high sensitivity and specificity. In this study, we have examined 4180 patients who underwent non-invasive prenatal testing (NIPT). Total of 36 cases of common trisomies (22 were trisomy 21, 8 were trisomy 18 and 6 were trisomy 13) were identified by NIPT; all were further confirmed by amniocentesis analysis except one case of trisomy 13. Moreover, four cases of subchromosome abnormalities also were identified by NIPT. Further investigations by array based comparative genomic hybridization (acgh) revealed that the abnormality was fetal origin in two, and maternal origin in two. In addition, in one case of negative NIPT, based on ultrasound abnormality, acgh showed a 2 M small deletion on chromosome 15. In conclusion, NIPT is a powerful tool in detecting fetal chromosome trisomy, even in subchromosome abnormalities. Keywords: Non-invasive prenatal testing (NIPT), chromosome aneuploidies, subchromosome abnormalities, genetic counseling Introduction The circulating cell-free fetal DNA (cffdna) in maternal plasma was first discovered more than one decade ago [1]. Since then, epigenetic differences between the placenta and maternal blood cells [2] and the ratio between alleles of a SNP in placental expressed PLAC4 mrna in maternal plasma [3, 4] were gradually clarified, and therefore were employed for non-invasive prenatal diagnosis. In particular, with the progress of next generation sequencing, massively parallel genomic sequencing of maternal plasma DNA was successfully used to detect of fetal chromosome trisomy [5] as well as other hereditary disease [6]. And this method, namely, non-invasive prenatal testing (NIPT), has shown high sensitivity and specificity for patients of fetal aneuploidies in many studies [7-10]. However, concerns on NIPT, such as false positive and false negative reports have also been discussed in other studies [11-13] pregnant of singleton pregnancy that came to our center for NIPT were recruited in our research. 22 cases of trisomy 21, 8 cases of trisomy 18 and 6 cases of trisomy 13 were identified by NIPT. All cases of trisomy were further confirmed by amniocentesis analysis except one case of trisomy 13. In addition, four cases of subchromosome abnormalities also were identified by NIPT. Furthermore, in one case of negative NIPT, based on ultrasound abnormality, acgh showed a 2 M microdeletion on chromosome 15. So far, fetal outcomes were ascertained in 3510 cases. Materials and methods Ethics statement Ethical approval was obtained from Ethics Committee of Henan Provincial People s Hospital, China. And all participants involved in this research have agreed and signed the written consents.

2 Table 1. Characteristic of the patients in our cohort Characteristic MA (years) (mean, range) 32 (20-44) GA (weeks) (mean, range) 20.1 (17-26) Serum biochemical Screening (High risk) 2006 (48.0%) Maternal age ( 35) 1714 (41.0%) Personal/family reason 201 (4.8%) Ultrasound findings 163 (3.9%) Not suitable for invasive tests 96 (2.3%) The average maternal age of the patient in the cohortis 32 (range from 20-44) years, and the average of gestation age is 20.1 (range from 17-26) weeks. The cohort can be sorted into positive serum biochemistry screening results, advanced maternal age ( 35 years of age at the due date in singleton pregnancies), personal/family reason, ultrasound findings, and not suitable for invasive tests at 48.0%, 41.0%, 4.8%, 3.9%, and 2.3% respectively. Study subjects We consecutively recruited consenting pregnant women from Medical Genetic Institute of Henan Province, Henan Provincial People s Hospital, from 24 April 2014 to 24 Dec All involved study subjects were singleton pregnancy. Sample preparation Maternal blood was collected at least at 12 weeks of gestation. Maternal plasma was separated by centrifuging the blood sample twice, and then plasma DNA was extracted using the protocol reported elsewhere [14]. 10 ml of amniotic fluid were collected from NIPT-positive pregnant, and then the standard karyotyping and acgh were performed. Plasma DNA sequencing, data align and analysis DNA libraries were prepared by using DNA extracted from the collected plasma (TruSeq DNA Sample Prep Kit, Illumina, Inc). Unique DNA adapter sequences were added to each sample so that pooled sequencing runs. Total of 96 different adapters were used to distinguish the individual maternal plasma sample. From DNA extraction to sequencing, each step has a quality control. Total of 4 lanes on flow cells were sequenced with Next Seq CN500 sequencer (Illumina) by standard single-end 45 bp reads (include 8 bp index). Q20, the sequencing quality value, was over 90% for each base. The amount of each sequencing reads was around million, and the GC content was around 39-42%. Sequences only mapped to one location in the repeat-masked reference human genome with no mismatch were analyzed by SOAP2 bioinformatic algorithm. The number of sequence reads that aligned to chromosome were counted, the z-score was further calculated with the formula described elsewhere [5]. Results and discussion Total of 4180 pregnant women were recruited in this research. The average of maternal age (MA) was 32 (ranging, 20-44) years old. The mean of gestation age (GA) was 20.1 (ranging, 17-26) weeks. The larg- est indication for testing is positive serum screening at 48.0%. This is followed by advanced maternal age at 41.0%, personal or family history at 4.8%, and ultrasound findings at 3.9% not suitable for invasive tests at 2.3%, respectively (Table 1). In this study, 22 cases of trisomy 21, 8 cases of trisomy 18 and 6 cases of trisomy 13 were identified by NIPT. The remains showed low-risk pregnancy (Table 2). Patients with a positive NIPT result were advised to undergo invasive testing for a prenatal karyotyping analysis. Karyotyping diagnostic tests confirmed 22 cases of trisomy 21, 8 of trisomy 18 and 5 of trisomy 13, while one case of trisomy 13 detected by NIPT showed negative karyotyping analysis. Further analysis revealed that this case of pregnant had maternal chromosome duplication on chromosome 13. This result suggested that NIPT is a reliable approach in clinical practice under a strict protocol and quality control. Beyond chromosome aneuploidy, NIPT also could detect genomic microdeletion or microduplication although sensitivity and specificity are still unclear. In this research, four cases of subchromosome abnormalities were detected by NIPT (Figure 1). Three cases were further confirmed by acgh although cytogenetic analysis showed negative results. The fourth cases showed positive results both in cytogenetic analysis and acgh analysis (Figure 2). Further acgh analysis showed that two cases of subchromosome abnormality were from maternal and two cases were from fetal. The examina Int J Clin Exp Med 2016;9(9):

3 Table 2. Results of the cohorts in NIPT, karyotyping and acgh NIPT Karyotyping acgh Trisomy Trisomy Trisomy A case with ultrasound findings Negative Normal 2 M Del on Chr 15 Microduplication 5 M Dup on Chr 13 Normal 1.2 M Dup on Chr 13 of maternal origin Microduplication 3 M Dup on Chr 4 Normal 0.5 M Dup on Chr 4 of fetal origin Microduplication 10 M Dup on Chr 15 46XY, dup (15) (q11.2q14) 11.8 M Dup on Chr 15 of fetal origin Microduplication 10 M Dup on Chr 16 Normal 1.5 M Dup on Chr 16 of maternal origin Both NIPT and karyotyping could effectively detect trisomy 21, trisomy 18 and trisomy 13. In addition, acgh detected a 2 M Del on Chr 15, while negative results were identidied by NIPTand karyotyping analysis in this case. Four cases of micro duplication were detected by NIPT, and acgh showed similar results. The remaining samples were not detected any anomalies by any of these three tests. -, not performed. Chr, chromosome. Dup, duplication. Del, deletion. Figure 1. Subchromosome abnormalities were detected by NIPT. A: Showed a 5 M duplication on chromosome 13. B: Showed a 3 M duplication on chromosome 4. C: Showed a 10 M duplication on chromosome 15. D: Showed a 10 M duplication on chromosome 16. Black arrow indicates subchromosome abnormalities on each chromosome detected by NIPT Int J Clin Exp Med 2016;9(9):

4 Figure 2. Copy number variations were examined by acgh. A: Confirmed a 1.2 M duplication on chromosome 13. B: Confirmed a 0.5 M duplication on chromosome 4. C: Confirmed a 11.8 M duplication on chromosome 15. D: Confirmed a 1.5 M duplication on chromosome 16. Red arrow indicates subchromosome abnormalities on each chromosome detected by acgh. tion of microdeletion/microduplication syndromes by NIPT would be another direction in the near future. In this study, there is a pregnant woman who received her negative NIPT report. With increasing gestation weeks, abnormal ultrasound of the fetal was found. Further invasive test was suggested to this patient. After the pregnant agreed to take invasive test, karyotyping analysis showed negative result, while acgh analysis of amniotic fluid showed a 2 M small deletion on chromosome 15 (Table 2), which was likely related to the fetal phenotype. Therefore, NIPT is not the best option for subtle anomalies in current. And post-test genetic counseling in clinical is essential even when a negative NIPT result was obtained. So far, fetal outcome of the NIPT-negative cases was ascertained in 3474 and NIPT-positive cases in 36, collectively accounting for 83.97% of the cohort. Overall, the positive rate was 0.53%, 0.19%, and 0.14% for trisomy 21, 18 and 13 respectively. Because one case of trisomy 13 was not confirmed, the false positive rate (FPR) was evaluated as 0.02% in detecting chromosome aneuploidy. The remains are still followed up in progress in current. Taken together, our data suggested that NIPT is a powerful tool in chromosome trisomy and has much potential in subchromosome abnormalities. But it is still a screening tool and should not be used as a diagnostic method. To be noted, post-test genetic counseling in clinical utilization of NIPT must be provided to pregnant women. Acknowledgements We wish to thank all the members in our lab for their discussions on this project. This work is supported by grants from Natural Science Foundation of China (SXL) and (SXL), and a grant of Science and Technology Development Program of Henan Science and Technology Department 1224 (SXL). Disclosure of conflict of interest None. Address correspondence to: Shi-Xiu Liao, Medical Genetic Institute of Henan Province, Henan Provincial People s Hospital, People s Hospital of Zhengzhou University, Zhengzhou, PR China. ychsliao@126.com References [1] Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW and Wainscoat JS. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350: Int J Clin Exp Med 2016;9(9):

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