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.

Full text

J Clin Lab Anal. 2019;33:e22911.  
|
1 of 5
https://doi.org/10.1002/jcla.22911
wileyonlinelibrary.com/journal/jcla
1 | INTRODUCTION
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 in-
cludes 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 de-
tection of common fetal aneuploidy due to its high specificity and
sensitivity2-4; 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 chro-
mosome microdeletion.
2 | CASE INTRODUCTION
A 38-year-old pregnant woman, pregnancy 2, parturition 1, ges-
tational age 18 weeks, was sent to the Third Affiliated Hospital
of Guangxi Medical University (Nanning, China). The woman was
161 cm tall and weighed 67 kg, and fetal developmental mileage was
normal. Ultraso nography at 17 weeks of ges tational age had reve aled
a low placenta and placental maturity level 0. Since pregnant women
who are older mothers do not have routine serological screening,
NIPT was selected to screen for fetal chromosomal abnormalities.
Received:13March2019
|
Revised:17April2019
|
Accepted:18April2019
DOI: 10.100 2/jcla. 22911
CASE RE PORT
Noninvasive prenatal testing detects microdeletion
abnormalities of fetal chromosome 15
Lianli Yin1 | Yinghua Tang2 | Qing Lu3 | Mingfang Shi1 | Aiping Pan2 |
Danyun Chen1
This is an op en access article under t he terms of the Creat ive Commo ns Attri bu tion License, whi ch permits use, distrib ution an d reprod uctio n in any medium,
provide d the orig inal work is proper ly cited .
© 2019 The Auth ors. Journal of Clinical Laboratory Analysis Published by W iley Peri odicals, Inc.
1Department of Clinical Laboratory, Nanning
Second People's Hospital, The Third
Affiliated Hospital of Guangxi Medical
University, Nanning, China
2Department of Clinical Laboratory, Guangxi
Hospital Of Traditional Chinese Medicine,
The First Affiliated Ho spital of Guang xi
University of Chinese Medicine, Nanning,
China
3Department of genetic counseling, Nanning
Second People's Hospital, The Third
Affiliated Hospital of Guangxi Medical
University, Nanning, China
Correspondence
Yinghu a Tang, Depar tment of Clinical
Labor atory, The First Affiliated Hospital of
Guangxi University of Chinese Medicine,
No. 89-9 Dongge Road, Nanning 530023,
Guangxi, China.
Email: 271101521@qq.com
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 approxi-
mately 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 effi-
cient method to gain genetic information about chromosomal abnormalities.
KEY WORDS
chromosome, copy number variation, deletion, noninvasive prenatal testing

2 of 5
|
YIN et al.
The NIPT results showed that there was a microdeletion of about
5 Mb in the long arm of chromosome 15 at q11.2-q12. Amniotic fluid
was taken for prenatal diagnosis, including karyotype and copy num-
ber variation (CNV). The CNV results showed that there were about
4.96 Mb of microdeletions in 15q11.2-q13.1 (23.62-28.58), while the
karyot ype analysis showed no significant abnormalities. The predi-
agnosis CNV was consistent with the NIPT results.
3 | MATERIALS AND METHODS
3.1 | Noninvasive prenatal testing analysis
Operating according to standard procedures (CapitalBio, China), 5 ml
of peripheral blood was collected from the pregnant woman and pro-
cessed within 8 hours. After t wo 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, en-
richment,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.
3.2 | Chromosome karyotype analysis
Following the principle of informed and voluntary, the NIPT results
suggest that amniocentesis should be per formed for high-risk preg-
nant women, whose condition was judged to be critical, to verify the
fetal karyotype analysis. For this case, the amniocentesis was per-
formed 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.
3.3 | 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 de-
scribed. Sequencing was performed using an Ion Proton Sequencing
System (Life Technologies). The results were analyzed using the
chromos ome Analysis sof tware. A nalyze genot ypes non‐typ e cor-
relations and interpret the data through public databases (decipher,
ISCA, omim, Clinva, ucsc, ncbi).
4 | RESULTS
4.1 | 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 chro-
mosome 15 had a deletion of fetal DNA fragments, the NIPT results
were verified with CNV.
4.2 | Copy number variation analysis
The CNV analysis results were seq[hg19] 15q11.2-q13.1
(23.62 Mb-28.58 Mb)x1, indicating a deletion of about 4.96 Mb on
chromosome 15q11.2-q13.1 (Figure 2). CNV analysis of the chromo-
somes of both parents and their other children showed no obvious
abnormalities.
4.3 | Karyotype analysis
Amniotic fluid kar yotype analysis showed no obvious abnormalities
in fetal chromosome structure (Figure 3).
5 | DISCUSSION
Fetal chromosomal abnormalities are among the most common
types of birth defects seen in clinical practice, with 95% of chro-
mosomal abnormalities being aneuploidies. Common aneuploidies
include trisomy 21, trisomy 18, trisomy 13, and sex chromosome
abnormalities.6,7 Some chromosome abnormalities are due to mi-
crodeletions or duplications. At present, the detection techniques
for chromosome deletions mainly include karyot ype analysis, FISH,
FIGURE 1 A NIPT study of maternal plasma showing a Z-score of 2.315 for fetal chromosome 15 and a deletion of approximately 5 Mb
from the 24 Mb-28 Mb region

|
3 of 5
YIN et al .
and CNV. However, the high cost of detection and the invasiveness
of the detection technology make these techniques difficult to use
as clinical screening tools, and there is a 1%-3% risk of abortion in
interventional prenatal diagnosis.8 NIPT, as a new prenatal screen-
ing method, has many advantages, such as being noninvasive, having
a high detec tion rate and a low false-positive rate, being applicable
FIGURE 2 Copy number variation study of maternal amniotic fluid showing that the long arm of fetal chromosome 15 has a deletion of
approximately 4.96 Mb (15q11.2-q13.1; 23.62 Mb-28.58 Mb)

4 of 5
|
YIN et al.
over a wide range of gestational weeks, requiring less clinical infor-
mation, being a simple process, and having relatively easy quality
control.9 NIPT is becoming more accepted by clinicians and patients.
Several studies have confirmed that high-throughput sequencing of
fetal-free DNA in maternal plasma has high detection sensitivit y and
specificity for noninvasive prenatal testing of common fetal chromo-
some aneuploidy.3,10,11 The detection rates of chromosome 21, 18,
and 13 aneuploidies by NIPT are 99.2%, 96.3%, and 91.0%, respec-
tively.9 However, there are few reports of detecting chromosome
microdeletions and duplications.
Studies found that NIPT technology can detect microdeletions
and microduplications greater than 30 0 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 litera-
ture. CNV was used to fur ther pinpoint the specific missing regions,
confirming the results of NIPT. There was no abnormalit y obser ved
in the kar yotype analysis, which appears to be inconsistent with the
NIPT and CNV results. However, identifying chromosome deficien-
cies and duplications of less than 5 Mb by amniotic fluid karyotype
analysis is difficult and they may often be missed.
Most children with chromosomal microdeletions can survive
normally, but they may exhibit various degrees of physical or mental
developmental abnormalities after birth. The results of the CNV in
the study showed that there was a microdeletion of 4.96 Mb in the
position of q11.2-q13.1 (23.62 Mb-28.58 Mb) of autosomal chro-
mosome 15 in the sample (Figure 1). The deletion of this region of
chromosome 15 covers the reported regions of Angel syndrome and
Prader-Willi syndrome (15:23619912-28438266, about 4.82 Mb),
including the pathogenic genes Ube3a of Angel syndrome,14 and
SNRPN and NDN of Prader-Willi syndrome.15
Angelman syndrome (AS, OMIM 105830) is a neurodevelop-
mental disorder syndrome, of which 70%-90% of cases are caused
by microdeletion of 15q11-q13 in the maternal line, 3%-7% by pa-
ternal diploidy (uniparental disomy, UPD) in the 15q11-q13 region,
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, difficult y 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 dele-
tion of this region of the fetal chromosome 15 was not inherited from
the parents. The chromosomes of their other children had no abnor-
malities. The abnormality in the present case may be due to the age of
the parents, but further research would be needed to establish this.
6 | CONCLUSION
This report shows that NIPT can detect fetal chromosome 15 abnor-
malities, including microdeletions. The clinical application of NIPT
screening can reduce the number of invasive prenatal diagnoses,
FIGURE 3 Karyotype analysis
of maternal amniotic fluid showing
no significant fetal chromosomal
abnormalities

|
5 of 5
YIN et al .
reduce the incidence of related iatrogenic abortion, and significantly
improve the d etection rate of fe tal chromosoma l abnormalities , includ-
ing microdeletions, which is more sensitive than karyotype analysis.
ACKNOWLEDGMENTS
We thank patients, family members, and all researchers for their
contributions to this research.
ORCID
Lianli Yin https://orcid.org/0000-0003-4425-1592
Yinghua Tang https://orcid.org/0000-0002-5655-9650
REFERENCES
1. Copeland GE, Kirby RS. Using birth defects registr y data to eval-
uate infant and childhood mortality associated with birth defect s:
an alternative to traditional mortality assessment using underly-
ing cause of death statistics. Birth Defec ts Res A Clin Mol Teratol.
2007;79(11):792-797.
 2. NorwitzER,LevyB.Noninvasiveprenataltesting:thefutureisnow.
Rev Obstet Gynecol. 2013;6(2):48-62.
3. Dan S, Wang W, Ren J, et al. Clinical application of massively par-
allel sequencing-based prenatal noninvasive fetal trisomy test for
trisomies 21 and 18 in 11,105 pregnancies with mixed risk factors.
Prenat Diagn. 2012;32(13):1225-1232.
 4. NshimyumukizaL,MenonS,HinaH,RousseauF,Reinharz D.Cell‐
free DNA noninvasive prenatal screening for aneuploidy versus
conventional screening: a systematic review of economic evalua-
tions. Clin Genet. 2018;94(1):3-21.
5. Wang T, Duan C, Shen C , et al. Detection of complex deletions in
chromosomes 13 and 21 in a fetus by noninvasive prenatal testing.
Mol Cytogenet. 2016;9:3.
6. Bryndor f T, Lundsteen C, Lamb A, Christensen B, Philip J. Rapid
prenatal diagnosis of chromosome aneuploidies by interphase
fluorescence in situ hybridization: a one‐year clinical experience
with high-risk and urgent fetal and postnatal s amples. Acta Obstet
Gynecol Scand. 2000;79(1):8-14.
7. Witters I, Dev riendt K , Legius E, et al. Rapid prenatal diagno-
sis of trisomy 21 in 5049 consecutive uncultured amniotic fluid
samples by fluorescence in situ hybridisation (FISH). Prenat Diagn.
2002;22(1):29-33.
8. Valayatham V, Subramaniam R , Juan YM, Chia P. Indications
for invasive prenatal diagnostic procedures at a dedicated fetal
medicine centre: an 8 year audit 2003–2010. Med J Malaysia.
2013;68(4):297-300.
 9. Gil MM, Que zada MS, Revello R, Akolekar R, Nicolaides KH.
Analysis of cell-free DNA in maternal blood in screening for fet al
aneuploidies: updated meta-analysis. Ultrasound Obstet Gynecol.
2015;45(3):249-266.
10. Bianchi DW, Platt LD, Goldberg JD, Abuhamad AZ, S ehner t AJ,
Rava RP. Genome-wide fetal aneuploidy dete ction by maternal
plasma DNA sequencing. Obstet Gynecol. 2012;119(5):890-901.
11. Jiang F, Ren J, Chen F, et al. Noninvasive Fetal Trisomy (NIFT Y) test:
an advanced noninvasive prenatal diagnosis methodology for fetal
autosomal and sex chromosomal aneuploidies. BMC Med Genomics.
2012;5:57.
12. Srinivas an A, Bianchi D, Huang H, Sehnert A , Rava R. Noninvasive
detection of fetal subchromosome abnormalities via deep sequenc-
ing of maternal plasma. Am J Hum Genet. 2013;92(2):167-176.
13. Lo YM, Chan KC, Chiu RW. Noninvasive fet al trisomy 21 detec-
tion using chromosome-selective sequencing: a variation of the
molecular counting theme. Exper t Rev Mol Diagn. 2012;12(4):
329-331.
14. Chamberlain SJ, Lalande M. Angelman syndrome, a genomic
imprinting disorder of the brain. J Neurosci. 2010;30(30):
9958-9963.
15. Cheon CK. Genetics of Prader-Willi syndrome and Pr ader-Will-Like
syndrome. Ann Pediatr Endocrinol Metab. 2016;21(3):126-135.
16. Neubert G, von Au K, Drossel K, et al. Angelman syndrome and
severe infections in a patient with de novo 15q11.2-q13.1 de-
letion and maternally inherited 2q21.3 microdeletion. Gene.
2013;512(2):453-455.
17. Angulo MA , Butler MG, Catalet to ME. Prader-Willi syndrome: a re-
view of clinical, genetic, and endocrine findings. J Endocrinol Invest.
2015;38(12):1249-1263.
How to cite this article: Yin L, Tang Y, Lu Q, Shi M, Pan A,
Chen D. Noninvasive prenatal testing detects microdeletion
abnormalities of fetal chromosome 15. J Clin Lab Anal.
2019;33:e22911. https ://doi.org/10.1002/jcla.22911