Karyotype versus Microarray Testing for Genetic Abnormalities after Stillbirth

Article (PDF Available)inNew England Journal of Medicine 367(23):2185-2193 · December 2012with60 Reads
DOI: 10.1056/NEJMoa1201569 · Source: PubMed
Abstract
Background: Genetic abnormalities have been associated with 6 to 13% of stillbirths, but the true prevalence may be higher. Unlike karyotype analysis, microarray analysis does not require live cells, and it detects small deletions and duplications called copy-number variants. Methods: The Stillbirth Collaborative Research Network conducted a population-based study of stillbirth in five geographic catchment areas. Standardized postmortem examinations and karyotype analyses were performed. A single-nucleotide polymorphism array was used to detect copy-number variants of at least 500 kb in placental or fetal tissue. Variants that were not identified in any of three databases of apparently unaffected persons were then classified into three groups: probably benign, clinical significance unknown, or pathogenic. We compared the results of karyotype and microarray analyses of samples obtained after delivery. Results: In our analysis of samples from 532 stillbirths, microarray analysis yielded results more often than did karyotype analysis (87.4% vs. 70.5%, P<0.001) and provided better detection of genetic abnormalities (aneuploidy or pathogenic copy-number variants, 8.3% vs. 5.8%; P=0.007). Microarray analysis also identified more genetic abnormalities among 443 antepartum stillbirths (8.8% vs. 6.5%, P=0.02) and 67 stillbirths with congenital anomalies (29.9% vs. 19.4%, P=0.008). As compared with karyotype analysis, microarray analysis provided a relative increase in the diagnosis of genetic abnormalities of 41.9% in all stillbirths, 34.5% in antepartum stillbirths, and 53.8% in stillbirths with anomalies. Conclusions: Microarray analysis is more likely than karyotype analysis to provide a genetic diagnosis, primarily because of its success with nonviable tissue, and is especially valuable in analyses of stillbirths with congenital anomalies or in cases in which karyotype results cannot be obtained. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development.).
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original article
Karyotype versus Microarray Testing
for Genetic Abnormalities after Stillbirth
Uma M. Reddy, M.D., M.P.H., Grier P. Page, Ph.D., George R. Saade, M.D.,
Robert M. Silver, M.D., Vanessa R. Thorsten, M.P.H., Corette B. Parker, Dr.P.H.,
Halit Pinar, M.D., Marian Willinger, Ph.D., Barbara J. Stoll, M.D.,
Josefine Heim-Hall, M.D., Michael W. Varner, M.D., Robert L. Goldenberg, M.D.,
Radek Bukowski, M.D., Ph.D., Ronald J. Wapner, M.D.,
Carolyn D. Drews-Botsch, Ph.D., Barbara M. O’Brien, M.D.,
Donald J. Dudley, M.D., and Brynn Levy, Ph.D., for the NICHD Stillbirth
Collaborative Research Network*
From the Pregnancy and Perinatology
Branch, the Eunice Kennedy Shriver Na-
tional Institute of Child Health and Hu-
man Development (NICHD), National
Institutes of Health, Bethesda, MD
(U.M.R., M.W.); RTI International, Re-
search Triangle Park, NC (G.P.P., V.R.T.,
C.B.P.); University of Texas Medical
Branch at Galveston, Galveston (G.R.S.,
R.B.); University of Utah School of Medi-
cine and Intermountain Health Care, Salt
Lake City (R.M.S., M.W.V.); Brown Uni-
versity School of Medicine, Providence, RI
(H.P., B.M.O.); Emory University School
of Medicine and Children’s Healthcare of
Atlanta (B.J.S.) and Rollins School of Pub-
lic Health, Emory University (C.D.D.-B.)
both in Atlanta; University of Texas Health
Science Center at San Antonio, San Anto-
nio (J.H.-H., D.J.D.); and Columbia Uni-
versity Medical Center, New York (R.L.G.,
R.J.W., B.L.). Address reprint requests to
Dr. Reddy at the Pregnancy and Perinatol-
ogy Branch, NICHD, 6100 Executive Blvd.,
Rm. 4B03, MSC 7510, Bethesda, MD
20892-7510, or at reddyu@mail.nih.gov.
* The other members of the Eunice Ken-
nedy Shriver National Institute of Child
Health and Human Development
(NICHD) Stillbirth Collaborative Re-
search Network are listed in the Sup-
plementary Appendix, available at
NEJM.org.
N Engl J Med 2012;367:2185-93.
DOI: 10.1056/NEJMoa1201569
Copyright © 2012 Massachusetts Medical Society.
Abstr act
Background
Genetic abnormalities have been associated with 6 to 13% of stillbirths, but the true
prevalence may be higher. Unlike karyotype analysis, microarray analysis does not
require live cells, and it detects small deletions and duplications called copy-number
variants.
Methods
The Stillbirth Collaborative Research Network conducted a population-based study of
stillbirth in five geographic catchment areas. Standardized postmortem examina-
tions and karyotype analyses were performed. A single-nucleotide polymorphism
array was used to detect copy-number variants of at least 500 kb in placental or fetal
tissue. Variants that were not identified in any of three databases of apparently
unaffected persons were then classified into three groups: probably benign, clinical
significance unknown, or pathogenic. We compared the results of karyotype and
microarray analyses of samples obtained after delivery.
Results
In our analysis of samples from 532 stillbirths, microarray analysis yielded results
more often than did karyotype analysis (87.4% vs. 70.5%, P<0.001) and provided
better detection of genetic abnormalities (aneuploidy or pathogenic copy-number
variants, 8.3% vs. 5.8%; P = 0.007). Microarray analysis also identified more genetic
abnormalities among 443 antepartum stillbirths (8.8% vs. 6.5%, P = 0.02) and 67
stillbirths with congenital anomalies (29.9% vs. 19.4%, P = 0.008). As compared
with karyotype analysis, microarray analysis provided a relative increase in the di-
agnosis of genetic abnormalities of 41.9% in all stillbirths, 34.5% in antepartum
stillbirths, and 53.8% in stillbirths with anomalies.
Conclusions
Microarray analysis is more likely than karyotype analysis to provide a genetic di-
agnosis, primarily because of its success with nonviable tissue, and is especially
valuable in analyses of stillbirths with congenital anomalies or in cases in which
karyotype results cannot be obtained. (Funded by the Eunice Kennedy Shriver Na-
tional Institute of Child Health and Human Development.)
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S
tillbirth, which is defined as fetal
death at or after 20 weeks of gestation, oc-
curs in 1 of every 160 births in the United
States.
1
Despite extensive evaluation, 25 to 60% of
stillbirths remain unexplained.
2
Karyotypic abnormalities are detected in 6 to
13% of stillbirths with a successful karyotype
analysis.
3,4
Some stillbirths may have chromo-
somal imbalances below the resolution of con-
ventional cytogenetic analysis, which is typically
5 to 10 Mb. Single-nucleotide polymorphism (SNP)
oligonucleotide microarray analysis detects almost
all genomic imbalances recognized by karyotyp-
ing, as well as smaller deletions and duplications
in the kilobase range, termed copy-number vari-
ants. Microarray analysis can be performed on
DNA from nonviable, or even macerated, tissue.
We tested the hypothesis that microarray analy-
sis detects abnormalities in stillbirth samples more
often than karyotype analysis.
Methods
Study Design
From March 2006 through September 2008, the
Stillbirth Collaborative Research Network (SCRN)
conducted a population-based study of stillbirth
in a racially and ethnically diverse cohort in five
geographic catchment areas.
5
Induced abortions
of a live fetus were excluded.
The study was approved by the institutional
review board at each clinical site, the 59 partici-
pating hospitals, and the data-coordinating cen-
ter. An advisory board reviewed the progress and
safety of the study. We obtained maternal written
informed consent.
5
Full participation included a
maternal interview, chart abstraction, standard-
ized postmortem examination
6
and placental path-
ological examination,
7
karyotype analysis, and the
collection and testing of maternal and fetal bio-
specimens. Women could decline any one of these
components. Separate consent was obtained for
future genetic testing. Biospecimens included cord
blood, placental tissue, and fetal liver and muscle
tissue. Karyotypes were analyzed in university-
affiliated cytogenetic laboratories.
DNA was extracted with the use of established
methods (Puregene, Qiagen Systems). DNA from
placenta and cord blood was stored at −20°C for
2 to 5 years before microarray analysis, which
was performed at a single laboratory (Columbia
University Medical Center). DNA from stored
frozen muscle and liver specimens was extracted
immediately before microarray analysis.
Analysis of Copy-Number Variants
We analyzed samples using the Affymetrix Ge-
nomeWide Human SNP Array 6.0. Array data
were analyzed with the use of Chromosome Anal-
ysis Suite, version 1.0.1, and the NetAffx annota-
tion database, version 28 (Affymetrix), with data
aligned to the Human Genome release 18 (hg18).
Array data were analyzed to identify aneuploi-
dy, potential maternal–fetal contamination, and
sex discordance. We included all copy-number
variants of 500 kb or larger in our analysis. Cat-
egorization of variants was based on the Ameri-
can College of Medical Genetics standards and
guidelines for interpretation and reporting,
8
with
modifications. A copy-number variant was cate-
gorized as benign if its full length was listed in
any of three databases of apparently unaffected
persons: the Database of Genomic Variants,
9
the
benign database
10
of the International Standards
for Cytogenomic Arrays Consortium, or the Chil-
drens Hospital of Philadelphia database
11
con-
verted from hg17 to hg18.
9-13
The remaining copy-
number variants were classified as pathogenic,
probably benign, or of unknown significance.
Pathogenic variants had evidence of pathogenicity
according to the published literature, contained a
gene listed in the Online Mendelian Inheritance
in Man (OMIM) database that is known to cause
disease relevant to stillbirth or development,
14
or
were included in the pathogenic database
15
of the
International Standards for Cytogenomic Arrays
Consortium. For variants that were classified as
probably benign, the variant contained no genes
at all or evidence in the literature suggested that
the variants were benign. Variants that did not
meet the criteria for classification as pathogenic,
probably benign, or benign were classified as hav-
ing unknown significance. We considered a vari-
ant to be confirmed on observing a variant of
the same type and approximately the same size
in an independent DNA sample from the same
stillbirth.
Statistical Analysis
Individual stillbirths were the units of analysis.
Statistical analysis was performed with the use of
SAS software, version 9.2 (SAS Institute), or R soft-
ware, version 2.13.1 (www.r-project.org). Fisher’s
exact test was used to compare detection rates
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Karyotype vs. Microarray Testing after Stillbirth
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2187
across subgroups. We used McNemar’s test for
paired data to evaluate differences between karyo-
type analysis and microarray analysis in the de-
tection of variants. We combined two Wilson score
intervals to estimate confidence intervals for de-
tection rate ratios.
16
Results
Study Population
The study series (953 women with a stillbirth) is
described in Figure S1 in the Supplementary Ap-
pendix, available with the full text of this article
at NEJM.org. The 290 women who did not enroll
did not differ significantly from those who en-
rolled with respect to age, race or ethnic group,
insurance or method of payment, or gestational
age at delivery (Table S1 in the Supplementary
Appendix).
Comparison of Karyotype and Microarray
Analyses
We compared the performances of karyotype and
microarray analyses using samples obtained af-
ter delivery from 532 stillbirths in which both
karyotype and microarray testing were attempted.
These samples included tissue from 492 single-
ton stillbirths, 19 twin gestations with 1 still-
birth, 4 twin gestations with 2 stillbirths (but only
1 assessed by karyotype and microarray testing),
8 twin gestations with 2 stillbirths and both as-
sessed, and 1 triplet gestation with 1 stillbirth,
for a total of 524 pregnancies. A comparison of
the characteristics of the 524 pregnancies that were
included in the analysis and the 139 pregnancies
that were not included, among all women en-
rolled in the study, is provided in Table S1 in the
Supplementary Appendix.
We karyotyped both fetal and placental tissue
in 158 of 532 stillbirths (29.7%), fetal tissue only
in 309 stillbirths (58.1%), placental tissue only in
64 stillbirths (12.0%), and tissue of unknown type
in 1 stillbirth. If placental DNA was unavailable,
we used cord blood, fetal muscle, or fetal liver for
microarray analysis (106 cases, 19.9%).
Of the karyotype analyses we attempted, 375 of
532 (70.5%) yielded a result; 29.5% did not yield
a result in any tissues tested. Of karyotypes yield-
ing results, 31 of 375 (8.3%) were classified as ab-
normal (Fig. 1). Abnormalities included trisomy
21 in 9 stillbirths, trisomy 18 in 8 stillbirths, tri-
somy 13 in 2 stillbirths, monosomy X in 5 still-
births, other sex-chromosome abnormalities in
2 stillbirths, 46,XY,dup(2)(q37) in 1 stillbirth,
46,XY,del(18)(q22) in 1 stillbirth, and 3 stillbirths
with mosaic cell lines in the placenta (Table S3
in the Supplementary Appendix).
Microarray analysis that was performed on the
same samples yielded a result in 465 of 532 still-
births (87.4%), significantly more than were suc-
cessfully karyotyped (P<0.001) (Fig. 1). In 396 of
these 465 stillbirths (85.2%), we observed no
variants larger than 500 kb, benign variants, or
probably benign variants; 32 stillbirths (6.9%)
were aneuploid, 12 (2.6%) harbored a pathogenic
variant, and 25 (5.4%) harbored a variant of un-
known significance. Among the aneuploid still-
births, we observed trisomy 21 in 10 stillbirths,
trisomy 18 in 10 stillbirths, trisomy 13 in 2 still-
births, monosomy X in 8 stillbirths, and other
sex-chromosome abnormalities in 3 stillbirths
(with 1 stillbirth having both trisomy 21 and sex-
chromosome aneuploidy). Table S2 in the Sup-
plementary Appendix provides information that
was used to classify each of the 41 stillbirths with
variants meeting the criteria of 500 kb or more
that were not found in the three databases of
apparently unaffected persons. In samples from
37 stillbirths, there were 38 pathogenic variants
or variants of unknown significance. These ge-
nomic events included 10 deletions (584 kb to
25.3 Mb) and 28 duplications (500 kb to 2.8 Mb).
One stillbirth had a pathogenic deletion and a
duplication that were consistent with an unbal-
anced translocation (Fig. S2 in the Supplementary
Appendix).
On microarray analysis (but not on karyotype
analysis), we observed three copy-number vari-
ants in three stillbirths (one in each stillbirth) at
chromosome 22q11.2, a region disrupted in the
DiGeorge (also called velocardiofacial) syndrome.
Two of these variants were deletions typical of
those causing the DiGeorge syndrome, and one
was a duplication. One of the stillbirths carrying
a deletion had multiple cardiopulmonary anoma-
lies, abnormal facies, skeletal anomalies, a uro-
genital anomaly, and a hypoplastic thymus (Fig. S3
in the Supplementary Appendix).
Eight stillbirths with variants of unknown
significance had overlapping duplications of the
19p13.3 region, which is known to contain five
OMIM loci and many benign copy-number vari-
ants (
Table 1
). In these eight stillbirths, no con-
genital anomalies were noted on postmortem
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examination. However, these stillbirths had sub-
stantially abnormal placental findings, including
chronic deciduitis and villous infarction (in three),
chronic cytomegalovirus villitis (in one), villous
infarction (in two), and abruption (in two).
Microarray analysis provided improved detec-
tion of genomic abnormalities (aneuploidy plus
pathogenic variants), as compared with karyo-
type analysis (8.3% vs. 5.8%, P = 0.007), a 41.9%
increase (detection rate ratio, 1.42; 95% confidence
interval [CI], 1.07 to 1.89) (Fig. 1). When we in-
cluded variants of unknown significance in this
comparison, we observed an even greater detec-
tion of abnormalities with the use of microarray
analysis, as compared with karyotype analysis
(13.0% vs. 5.8%, P<0.001), a 122.6% increase
(detection rate ratio, 2.23; 95% CI, 1.63 to 3.04).
Of the 157 stillbirths for which karyotype analy-
sis failed to provide a definitive result, 79.6%
yielded a definitive microarray result: 73.9% were
normal or probably benign and 5.7% were ab-
normal (with aneuploidy or a pathogenic variant).
Table 1
shows microarray results for stillbirths
with aneuploidy, variants of unknown signifi-
cance, or pathogenic variants in which the
karyotype was normal or the test failed. Of the
44 stillbirths with aneuploidy or a pathogenic
variant detected on microarray analysis, 41% had
a normal karyotype or the test failed (Fig. 1).
We also assessed the ability of microarray
analysis to detect abnormalities identified by
karyotype analysis (Table S3 in the Supplemen-
Karyotype Results
(N=532)
Microarray Results
(N=532)
Normal 73.9% (N=116)
Failed
Failed
Microarray Results
(N=532)
Karyotype Results
(N=532)
VOUS
Pathogenic CNV
Aneuploidy
Normal
Abnormal
4.5% (N=7)
Normal
Failed
No CNV,
benign CNV,
or probably
benign CNV
Failed
Normal
Abnormal
VOUS
Pathogenic CNV
Aneuploidy
Pathogenic
CNV
2.6% (N=12)
Aneuploidy
6.9% (N=32)
Failed
Normal
Normal
Failed
Abnormal
Failed
40.0% (N=10)
VOUS
Pathogenic CNV
Aneuploidy
Normal
Abnormal
CNV of
unknown
significance
Failed
12.6%
(N=67)
Results
obtained
on 465
cases
Normal
74.4%
(N=396)
85.2%
(N=396/465)
Abnormal
8.3%
(N=44)
9.5%
(N=44/465)
VOUS
4.7%
(N=25)
5.4%
(N=25/465)
Failed
29.5%
(N=157)
Results
obtained
on 375
cases
Normal
64.7%
(N=344)
91.7%
(N=344/375)
Abnormal
5.8%
(N=31)
8.3%
(N=31/375)
0.0% (N=0)
4.0% (N=1)
56.0% (N=14)
20.5% (N=9)
20.5% (N=9)
59.1% (N=26)
0.5% (N=2)
70.2% (N=278)
29.3% (N=116)
3.0 % (N=2)
64.2% (N=43)
32.8% (N=22)
80.6% (N=25)
3.2% (N=1)
3.2% (N=1)
6.5% (N=2)
6.5% (N=2)
2.6% (N=9)
4.1% (N=14)
80.8% (N=278)
12.5% (N=43)
1.3% (N=2)
6.4% (N=10)
14.0% (N=22)
Figure 1. Performance of Karyotype and Microarray Analyses in Samples Obtained after Delivery in 532 Stillbirths.
On the left, karyotype results (blue) are categorized as failed, normal, or abnormal, as compared with findings from the same cases on
microarray analysis (orange). On the right, the microarray results are categorized as failed, normal, abnormal, or variants of unknown
significance (VOUS), with the corresponding karyotype results in the same cases. CNV denotes copy-number variant.
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Karyotype vs. Microarray Testing after Stillbirth
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2189
tary Appendix). Of the 31 stillbirths with abnor-
mal karyotypes, 29 had results when analyzed on
microarray analysis. A total of 25 stillbirths had
microarray results that were consistent with the
results obtained on karyotyping. Two low-level
mosaics with 10% or less abnormal cells on karyo-
typing were normal on microarray analysis (cases
1 and 2). Two stillbirths with abnormal karyotypes
had a different abnormality on microarray analy-
sis (cases 3 and 4), and another two with abnor-
mal karyotypes (trisomy 21 and a duplication) did
not yield microarray results because of DNA degra-
dation (cases 5 and 6). One of the stillbirths with
discordant results was a phenotypic male but
was 45,X according to karyotype. On the basis of
microarray results, much of the Y chromosome
was missing 46, XY del(Y)(q11.122 qter) but
the pseudoautosomal region down to and in-
cluding the sex-determining region was present.
Subgroup Analyses
We performed subgroup analyses for antepartum
stillbirths and stillbirths with structural anoma-
lies (
Table 2
). Karyotype analysis yielded a result
in 298 of 443 antepartum stillbirths (67.3%). Of
these 298 stillbirths, 29 (9.7%) were abnormal.
Microarray analysis yielded a result in 385 of the
443 antepartum stillbirths (86.9%). Of these 385
stillbirths, 31 (8.1%) were aneuploid, 8 (2.1%) had
pathogenic variants, and 24 (6.2%) had variants
of unknown significance. Microarray analysis de-
tected more abnormalities in the antepartum sub-
group than did karyotype analysis (8.8% vs. 6.5%,
P = 0.02), a 34.5% increase (detection rate ratio,
1.34; 95% CI, 1.01 to 1.78).
Of the 472 stillbirths with postmortem exami-
nations, 67 (14.2%) had structural anomalies.
Karyotype analysis yielded results in 45 of the 67
stillbirths (67.2%), of which 13 (28.9%) were abnor-
mal. Microarray analysis was successful in 60 of
the 67 stillbirths (89.6%), of which 17 (28.3%) had
aneuploidy, 3 (5.0%) had pathogenic variants,
and 3 (5.0%) had variants of unknown signifi-
cance. Microarray analysis detected more abnor-
malities in this group (in 20 of 67 stillbirths, or
29.9%) than did karyotype analysis (in 13 of 67
stillbirths, or 19.4%; P = 0.008), a 53.8% increase
(detection rate ratio, 1.54; 95% CI, 1.03 to 2.26).
Anomalous stillbirths were significantly more
likely than nonanomalous stillbirths to have ab-
normal results on microarray analysis and karyo-
type analysis (P<0.001 for both comparisons).
Discussion
Genomic techniques allow for the identification
of chromosomal abnormalities at high resolution.
The usefulness of these techniques has been shown
in children with unexplained developmental delay
or intellectual disability.
17-19
In addition, in this
issue of the Journal, Wapner and colleagues report
that microarray analysis improves the prenatal
detection of clinically relevant genetic abnormal-
ities.
20
Microarray analysis, as compared with con-
ventional karyotype analysis, has also increased
the detection of genetic abnormalities in preg-
nancy loss at a gestation of less than 20 weeks.
21-23
Two small studies have assessed the usefulness
of microarray analysis for the evaluation of still-
births. A study of 15 stillbirths with abnormali-
ties in two organs and either normal results on
karyotype analysis or failed karyotyping identi-
fied an instance of trisomy 21 and another in-
stance of an unbalanced translocation.
24
The
other study examined 29 unexplained stillbirths
and identified copy-number variants in 24 cases,
although only one variant was considered to be
causative of stillbirth.
25
The primary benefit of using microarray anal-
ysis over karyotype analysis is the greater likeli-
hood of obtaining a result because of the ability
to analyze nonviable tissue. We thus were able to
obtain a result in 90 more cases (24.0% more) than
we would have done using karyotype analysis
alone. Of the stillbirths for which definitive results
were obtained on either karyotype analysis or mi-
croarray analysis, the percentage of aneuploid
stillbirths was 7%, which is consistent with the
results of a large cytogenetic study.
4
Because of
the improved yield of results obtained on micro-
array analysis, the actual number of aneuploid
stillbirths detected on microarray analysis was
greater than that detected on karyotype analysis.
Moreover, as compared with karyotype analysis,
microarray analysis was more sensitive to the
presence of pathogenic variants. Some of the vari-
ants that were detected on microarray analysis
may represent unbalanced translocations, which
can be missed by karyotyping (Fig. S2 in the
Supplementary Appendix). Detection of inherited
translocations in stillbirths is important because
of future reproductive risks for the carrier parent.
Our results indicate that microarray analysis
identifies more abnormalities of unknown sig-
nificance than does karyotype analysis. A major
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challenge with microarray testing in stillbirths is
determining the clinical implications, since the
clinical relevance of many variants is unknown
(hence the classification of variant of unknown
significance). These unknown factors pose prob-
lems for genetic counseling. However, the chal-
lenge of counseling couples about unpredictable
outcomes is not new.
26
Table 1. Microarray Results for Stillbirths with Aneuploidy, Variants of Unknown Significance, or Pathogenic Variants
in Which Karyotyping Failed or Was Normal.
Microarray Result Case No. Observed Change on Microarray* Size
Failed karyotyping
Aneuploidy
Trisomy 18 1 18p11.32q23(1,543–76,116,030)×3 Chromosome
Trisomy 18 2 18p11.32q23(1,542–76,116,029)×3 Chromosome
Trisomy 21 3 21q11.q22.3(13,286,390–46,921,374)×3 Chromosome
Trisomy 21 and XXY 4 21p11.1q22.3(9,758,730–46,921,374)×3,
Xp22.33q28(2,401,346–154,843,252)×2,
19q13.12(41,553,395–42,220,583)×1
Chromosome
Chromosome
667 kb
Monosomy X 5 Xp22.33q28(108,464–154,849,094)×1 Chromosome
Monosomy X 6 Xp22.33q28(2,401,346–154,843,252)×1 Chromosome
Monosomy X 7 Xp22.33q28(108,464–154,849,094)×1 Chromosome
Pathogenic variant
Deletion 8 1q21.1(143,845,772–146,838,707)×1 4.0 Mb
Deletion 9 22q11.21q11.23(17,256,416–22,140,054)×1 4.9 Mb
Variant of unknown significance
Duplication 10 19p13.3(363,729–965,377)×3 602 kb
Duplication 11 19p13.3(441,414–965,377)×3 524 kb
Duplication 12 19p13.3(339,937–1,270,320)×3 931 kb
Duplication 13 19p13.3(441,414–1,261,136)×3 820 kb
Duplication 14 19p13.3(388,808 - 1,270,320)×3 882 kb
Duplication 15 19q13.12(57,198,183–57,722,222)×3 524 kb
Duplication 16 21q21.3(27,162,033–28,340,061)×3 1.2 Mb
Duplication 17 21q22.13(36,685,848–37,185,921)×3 500 kb
Duplication 18 Xq27.1(138,676,821–139,311,901)×3 635 kb
Duplication 19 5p15.2(10,908,334 - 11,459,739)×3 551 kb
Normal karyotyping
Pathogenic variant
Deletion 20 22q11.21(17,256,416–19,795,836)×1 2.5 Mb
Deletion 21 Xp22.31(6,903,881–7,774,557)×0 869 kb
Deletion 22 7q11.23(73,247,250–73,753,322)×1 506 kb
Unbalanced translocation: dele-
tion and duplication 23
4q32.3q35.2(165,903,367–191,254,120)×1
17p13.3(514–2,811,647)×3
25.3 Mb
2.8 Mb
Duplication 24 22q11.21(17,128,427–18,647,705)×3 1.5 Mb
Duplication 25 18p11.21(13,574,399–14,760,946)×3 1.2 Mb
Duplication 26 16p13.11p12.3(15,224,214–18,286,344)×3 3.0 Mb
Duplication 27 16p13.11p12.3(15,389,423–18,464,701)×3 3.1 Mb
Duplication 28 17q21.31(31,890,369–33,552,890)×3 1.7 Mb
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Karyotype vs. Microarray Testing after Stillbirth
n engl j med 367;23 nejm.org december 6, 2012
2191
We observed a recurrent variant of unknown
significance in a telomeric region of chromosome
19p13.3 (ranging in size from 632 to 930 kb) in
eight stillbirths. This region is known to contain
multiple benign variants as well as five loci in the
OMIM database that have been associated with
disease but not with stillbirth or developmental
disorders. The variant of unknown significance
that we observed in this region may be benign
or may confer a risk of stillbirth. Similarly, we
observed two deletions categorized as variants of
unknown significance in two stillbirths (one per
stillbirth) with major anomalies on postmortem
examination. Whether these variants are patho-
genic remains to be determined. Girirajan and
colleagues
18
have recently reported that children
who carry two large variants of unknown clinical
significance are eight times as likely to have de-
velopmental delay as are controls from the gen-
eral population.
We detected genomic imbalances in the 22q11.2
region in three cases on microarray analysis but
not on karyotype analysis. Microdeletions in the
22q11.2 region are associated with the DiGeorge
syndrome, and microduplications give rise to the
22q11.2 microduplication syndrome. The pheno-
type of both syndromes is variable, with shared
clinical anomalies that include heart defects, uro-
genital abnormalities, and velopharyngeal insuf-
ficiency.
27-29
The incidence of the DiGeorge syn-
drome is estimated to be 1 case in 4000 births.
30
The 22q11.2 microduplication syndrome appears
to be less prevalent. We detected three variants of
500 kb or more in the typical 22q11.2 region and
a typical DiGeorge deletion (2.8 Mb) in a stillbirth
that was not included in the primary analysis be-
cause karyotyping was not attempted. The three
stillbirths with a pathogenic variant in the 22q11.2
region represent an increase in the prevalence of
this abnormality by a factor of 22.6 (P = 3.5×10
−4
),
as compared with the frequency in the general
population (1 in 4000 births). If we count all
four variants in the 22q11.2 region and the 41
stillbirths that underwent microarray analysis
but not karyotype analysis, the prevalence is in-
creased by a factor of 27.3 (P = 1.5×10
−5
). These
results suggest that genomic imbalances in this
region may be associated with stillbirth. Identify-
ing the 22q11.2 variant in the stillbirth is impor-
tant because the DiGeorge syndrome is a haplo-
insufficiency disorder in which parental studies
are recommended.
30
The risk of recurrence in sub-
sequent pregnancies increases from less than
0.1% for genotypically normal parents to 50% if
a parent has the deletion.
30
In some cases, af-
fected offspring may serve as the index case
Table 1. (Continued.)
Microarray Result Case No. Observed Change on Microarray* Size
Variant of unknown significance
Deletion 29 Yq11.221(18,148,539–18,999,761)×0 850 kb
Deletion 30 1p35.3(28,444,904–28,952,754)×1 508 kb
Deletion 31 16p11.2(29,333,900 – 30,038,055)×1 704 kb
Duplication 32 19p13.3(373,237–1,261,136)×3 887 kb
Duplication 33 19p13.3(441,414–965,377)×3 524 kb
Duplication 34 19p13.3(392,194–972,725)×3 580 kb
Duplication 35 15q12q13.1(25,366,691–26,087,702)×3 721 kb
Duplication 36 19p12(23,613,361–24,388,578)×3 775 kb
Duplication 37 6p25.1p24.3(6,844,061–7,457,081)×3 613 kb
Duplication 38 10q23.31(90,658,193–91,207,964)×3 549 kb
Duplication 39 19q13.12(41,961,955–42,487,630)×3 526 kb
Duplication 40 8q24.23(137,042,624–139,247,552)×3 2.2 Mb
Duplication 41 3p21.31(45,806,446–46,455,963)×3 649 kb
Duplication 42 11p13(33,005,102–33,592,112)×3 587 kb
* For each observed change on microarray analysis, the times sign specifies the number of copies of the genomic region
indicated in the parentheses.
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The
ne w engl and jour na l
o f
medicine
n engl j med 367;23 nejm.org december 6, 2012
2192
leading to diagnosis in a parent with the 22q11.2
deletion who has a mild clinical phenotype.
30
There are limitations of microarray-based tech-
nology. Truly balanced rearrangements cannot be
detected on microarray analysis; however, they are
unlikely to cause stillbirth. In addition, low-level
mosaicism detected by means of karyotyping went
undetected on microarray analysis in our study,
although the clinical implications of this low-level
mosaicism are unclear.
Concurrent karyotype and microarray testing
on the same tissues would have been ideal. How-
ever, this was not possible because the study
design necessitated karyotyping in real time.
Another limitation was our inability to distin-
guish de novo from inherited variants owing to
the unavailability of parental DNA. De novo vari-
ants in clinically significant gene regions are more
likely to be causative. However, inherited patho-
genic variants should not be discounted as a cause
of stillbirth because of their variable expressivity
and incomplete penetrance.
8,26
Our ability to as-
sess confined placental mosaicism, in which the
fetus is genetically normal but the placenta is ge-
netically abnormal, was limited.
A major strength of our study was the large,
geographically and racially diverse, population-
based series of women with a stillbirth.
5
All the
stillbirths that were included in the analysis were
carefully phenotyped because the women provid-
ed consent for a complete evaluation, including
fetal postmortem examination, placental patho-
logical analysis, karyotyping, and maternal–fetal
testing.
31
Microarray analysis was performed at
an institution that was not part of the study, and
the researchers who performed the analysis were
unaware of the karyotyping results and the clini-
cal history.
In conclusion, we found that microarray analy-
sis could be useful in cases of stillbirth when
Table 2. Comparison of Karyotype Analysis and Microarray Analysis in the Diagnosis of Genetic Abnormalities in 532
Stillbirths, According to Subgroup.*
Stillbirth Subgroup
and Karyotype
Karyotype
Analysis Microarray Analysis
Failed
Normal or
Benign
Probably
Benign
Variant of
Unknown
Significance
Pathogenic
Variant Aneuploidy
number of stillbirths (percent)
Antepartum (N = 443)
Failed 145 (32.7) 22 (15.2) 104 (71.7) 0 10 (6.9) 2 (1.4) 7 (4.8)
Normal 269 (60.7) 35 (13.0) 214 (79.6) 2 (0.7) 13 (4.8) 5 (1.9) 0
Abnormal 29 (6.5) 1 (3.4) 2 (6.9) 0 1 (3.4) 1 (3.4) 24 (82.8)
Intrapartum (N = 89)
Failed 12 (13.5) 0 12 (100.0) 0 0 0 0
Normal 75 (84.3) 8 (10.7) 60 (80.0) 2 (2.7) 1 (1.3) 4 (5.3) 0
Abnormal 2 (2.2) 1 (50.0) 0 0 0 0 1 (50.0)
Anomalous (N = 67)
Failed 22 (32.8) 3 (13.6) 12 (54.5) 0 1 (4.5) 2 (9.1) 4 (18.2)
Normal 32 (47.8) 4 (12.5) 25 (78.1) 0 2 (6.2) 1 (3.1) 0
Abnormal 13 (19.4) 0 0 0 0 0 13 (100.0)
Nonanomalous (N = 405)
Failed 122 (30.1) 16 (13.1) 95 (77.9) 0 9 (7.4) 0 2 (1.6)
Normal 271 (66.9) 32 (11.8) 218 (80.4) 3 (1.1) 11 (4.1) 7 (2.6) 0
Abnormal 12 (3.0) 1 (8.3) 2 (16.7) 0 1 (8.3) 1 (8.3) 7 (58.3)
* Percentages for the karyotype analysis were calculated with the number of cases in the stillbirth subgroup (antepartum,
intrapartum, anomalous, or nonanomalous) as the denominator. Percentages for the microarray analysis were calculat-
ed with the number of cases in the karyotype subgroup (failed, normal, or abnormal) as the denominator. A total of
472 stillbirths underwent complete postmortem examination and were classified as either anomalous or nonanoma-
lous. The 60 stillbirths that did not undergo complete postmortem examination were not categorized.
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Karyotype vs. Microarray Testing after Stillbirth
n engl j med 367;23 nejm.org december 6, 2012
2193
karyotyping results cannot be obtained or in cases
in which there are congenital anomalies. Micro-
array analysis is more expensive than standard
karyotype analysis, although its cost is expected
to decrease
26
and may be offset by the higher
yield of genomic abnormalities.
The views expressed in this article are those of the authors
and do not necessarily reflect the views of the National Insti-
tutes of Health or the Eunice Kennedy Shriver National Institute
of Child Health and Development (NICHD).
Supported by grants (HD45925, HD45944, HD45952,
HD45953, HD45954, and HD45955) from the NICHD.
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
We thank the physicians, study coordinators, research nurses,
and patients who participated in the Stillbirth Collaborative Re-
search Network.
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    • "where microarray detected clinically significant abnormalities in 12.8% of specimens referred with normal or unknown karyotypes (Rosenfeld et al., 2015). Pathogenic results were found in 63.9% of copy-number variants, and the incidence of VOUS was overall 2.1% of all abnormal specimens and less than 1% of all analyzed specimens, consistent with the current literature (Reddy et al., 2012; Dhillon et al., 2014). We also analyzed our data by gestation age, and as expected, the proportion of abnormal results decreases with gestation age. "
    [Show abstract] [Hide abstract] ABSTRACT: Objective: To evaluate the performance of a laboratory protocol for direct genetic analysis performed on tissues obtained from miscarriages, stillbirth and postnatal death. Methods: Samples were collected between July 1st, 2011 and June 30th, 2014. QF-PCR analysis was the initial test followed by aCGH analysis performed on the normal QF-PCR specimens. Results: Of the 1195 submitted specimens, a total of 1071 samples were confirmed as true fetal. The failure rate was 1.4%. Of those, 30.8% yielded abnormal results. Of the latter, 57.6% had abnormal QF-PCR and 42.4% had abnormal microarray result. Autosomal trisomies were detected in 61.2%, triploidy in 7.6%, monosomy X in 9.1%, sex-chromosome aneuploidy (apart from monosomy X) in 1.5%, molar pregnancies in 5.8% and copy number variants in 14.2% including microdeletions/microduplications and cryptic unbalanced rearrangements. The highest diagnostic yield was observed in the 1st trimester specimens at 67.6%. We confirmed that maternal age correlates with the likelihood of autosomal trisomies but not with triploidy, sex chromosome aneuploidies, molar pregnancy, or CNVs. Conclusion: An efficient laboratory protocol, based on QF-PCR and aCGH of uncultured cells has replaced standard cytogenetic analysis in testing of tissue from all pregnancy losses in our center and resulted in reduced test failure rate and increased diagnostic yield.
    Full-text · Article · May 2016