Noninvasive prenatal testing detects microdeletion abnormalities of fetal chromosome 15

Abstract Objective Noninvasive prenatal testing (NIPT) is widely used in clinical detection of fetal autosomal duplications or deletions. The aim of this study was to investigate the clinical application of NIPT for detection of chromosomal microdeletions. Methods Microdeletions of about 5 Mb in the long arm of chromosome 15 (q11.2‐q12) were detected by NIPT and were confirmed by karyotype analysis and copy number variation (CNV) analysis based on high‐throughput sequencing technology. Results The CNV results of prenatal diagnosis showed that there were approximately 4.96 Mb of microdeletions in 15q11.2‐q13.1, which was consistent with the NIPT results. The karyotype analysis showed no abnormalities. Conclusion In this study, the microdeletion fragment of fetal chromosome 15 was successfully detected and diagnosed using NIPT. This suggests that NIPT is an efficient method to gain genetic information about chromosomal abnormalities.


| INTRODUC TI ON
Birth defects comprise one of the three major categories of diseases that endanger human health. 1 Fetal chromosomal abnormality is one of the most important causes of neonatal birth defects. The most common type of abnormality is chromosomal aneuploidy, which includes trisomy 21 (Down's syndrome), trisomy 13 (Patau syndrome), trisomy 18 (Edward's syndrome), and sex chromosome aneuploidy.
Noninvasive prenatal testing (NIPT) is widely used in the clinical detection of common fetal aneuploidy due to its high specificity and sensitivity [2][3][4] ; however, reports on chromosomal deletions are rare. 5 In our clinical prenatal screening, we found a case of a mid-pregnancy patient with an abnormal chromosome 15 deletion. The NIPT results showed a 5-Mb microdeletion on chromosome 15. Karyotype analysis and CNV test were used to confirm the clinical value of NIPT in chromosome microdeletion.

| Noninvasive prenatal testing analysis
Operating according to standard procedures (CapitalBio, China), 5 ml of peripheral blood was collected from the pregnant woman and processed within 8 hours. After two low-temperature centrifugations, the obtained plasma was transferred to a new 2.0 ml-centrifuge tube and stored at −80°C. The frozen plasma was later reconstituted at room temperature and thoroughly mixed for use. After the DNA extraction, sequencing library preparation, sequencing reaction, enrichment, sequencing, and other processes, the data were analyzed to obtain the Z value. Sequencing was performed using an Ion Proton Sequencing System (Life Technologies). For the test results, Z ≥ 4 was judged as high-risk; 1.96 < Z < 4 was critical, and amniocentesis was used for a clear diagnosis; and Z ≤ 1.96 was low-risk and allowed to proceed with the routine pregnancy check process.

| Chromosome karyotype analysis
Following the principle of informed and voluntary, the NIPT results suggest that amniocentesis should be performed for high-risk pregnant women, whose condition was judged to be critical, to verify the fetal karyotype analysis. For this case, the amniocentesis was performed under the guidance of ultrasound in the pregnant woman, and 15-20 ml of amniotic fluid was taken. The karyotype analysis of fetal amniotic fluid exfoliated cells was performed by visual identification.

| CNV analysis based on high-throughput sequencing technology
Operating according to standard procedures (CapitalBio, China), 10 ml fetal amniotic fluid or 2 ml of uncultured peripheral blood samples were collected, and DNA extraction, sequencing library preparation, sequencing, and other processes were performed as described. Sequencing was performed using an Ion Proton Sequencing System (Life Technologies). The results were analyzed using the chromosome Analysis software. Analyze genotypes non-type correlations and interpret the data through public databases (decipher, ISCA, omim, Clinva, ucsc, ncbi).

| Noninvasive prenatal testing
Noninvasive prenatal testing results gave a Z-score for chromosome 15 of 2.315 and showed that there was about a deletion of 5 Mb at 24 Mb-28 Mb (Figure 1). Because these scores indicated that chromosome 15 had a deletion of fetal DNA fragments, the NIPT results were verified with CNV.

| Copy number variation analysis
The CNV analysis results were seq[hg19] 15q11.

| Karyotype analysis
Amniotic fluid karyotype analysis showed no obvious abnormalities in fetal chromosome structure (Figure 3). over a wide range of gestational weeks, requiring less clinical information, being a simple process, and having relatively easy quality control. 9 NIPT is becoming more accepted by clinicians and patients.

| D ISCUSS I ON
Several studies have confirmed that high-throughput sequencing of fetal-free DNA in maternal plasma has high detection sensitivity and specificity for noninvasive prenatal testing of common fetal chromosome aneuploidy. 3,10,11 The detection rates of chromosome 21, 18, and 13 aneuploidies by NIPT are 99.2%, 96.3%, and 91.0%, respectively. 9 However, there are few reports of detecting chromosome microdeletions and duplications.
Studies found that NIPT technology can detect microdeletions and microduplications greater than 300 Kb in fetal genomes. 12,13 This study successfully used NIPT to detect microdeletions of about 5 Mb in fetal chromosome 15, which is consistent with the literature. CNV was used to further pinpoint the specific missing regions, confirming the results of NIPT. There was no abnormality observed in the karyotype analysis, which appears to be inconsistent with the NIPT and CNV results. However, identifying chromosome deficiencies and duplications of less than 5 Mb by amniotic fluid karyotype analysis is difficult and they may often be missed. and 2%-4% by a single gene defect. 16 The clinical manifestations are mental retardation, severe speech disorder, facial deformity, secondary cerebellar malformation, ataxia, epilepsy, and abnormal behavior, such as easy-to-cause laughter.
Prader-Willi syndrome (PWS, OMIM 176270) is a multi-system disease, of which 65%-75% of cases are caused by microdeletion of 15q11-q13 in the paternal line, 20%-30% by maternal diploidy (UPD) in the 15q11-q13 region, and 1%-3% by a single gene defect. 17 The clinical manifestations are growth retardation, difficulty feeding in infancy, obesity due to high appetite and drowsiness in childhood, mental retardation, low muscle tone, short stature, small hands and feet, horseshoe kidney, and genital dysplasia due to hypogonadism.
In the case presented here, the microdeletion mutation in the 15q11.2-q13.1 region is expected to cause a disease. We verified the parents of the fetus, and the parent's research indicated that the deletion of this region of the fetal chromosome 15 was not inherited from the parents. The chromosomes of their other children had no abnormalities. The abnormality in the present case may be due to the age of the parents, but further research would be needed to establish this.

| CON CLUS ION
This report shows that NIPT can detect fetal chromosome 15 abnormalities, including microdeletions. The clinical application of NIPT screening can reduce the number of invasive prenatal diagnoses, F I G U R E 3 Karyotype analysis of maternal amniotic fluid showing no significant fetal chromosomal abnormalities reduce the incidence of related iatrogenic abortion, and significantly improve the detection rate of fetal chromosomal abnormalities, including microdeletions, which is more sensitive than karyotype analysis.

ACK N OWLED G M ENTS
We thank patients, family members, and all researchers for their contributions to this research.