Ultrasound Obstet Gynecol 2015; 46: 650–658 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/uog.14880
Genomic microarray in fetuses with increased nuchal translucency and normal karyotype: a systematic review and meta-analysis M. GRANDE*, F. A. R. JANSEN†, Y. J. BLUMENFELD‡, A. FISHER§, A. O. ODIBO¶, M. C. HAAK† and A. BORRELL* *Department of Maternal-Fetal Medicine, Institute of Gynecology, Obstetrics and Neonatology, Hospital Clinic of Barcelona, Catalonia, Spain; †Leiden University Medical Center, Department of Obstetrics and Fetal Medicine, Leiden, The Netherlands; ‡Department of Obstetrics & Gynecology, Stanford University School of Medicine, Stanford, CA, USA; §Elliot Health System, Manchester, NH, USA; ¶Department of Obstetrics & Gynecology, Division of Maternal Fetal Medicine, University of South Florida, Tampa, FL, USA
K E Y W O R D S: copy number variants; cystic hygroma; genomic microarray; increased nuchal translucency; prenatal diagnosis
ABSTRACT Objective To estimate the incremental yield of detecting copy number variants (CNVs) by genomic microarray over karyotyping in fetuses with increased nuchal translucency (NT) diagnosed by first-trimester ultrasound. Methods This was a systematic review conducted in accordance with PRISMA criteria. We searched PubMed, Ovid MEDLINE and Web of Science for studies published between January 2009 and January 2015 that described CNVs in fetuses with increased NT, usually defined as ≥ 3.5 mm, and normal karyotype. Search terms included: fetal or prenatal, nuchal translucency or cystic hygroma or ultrasound anomaly, array comparative genomic hybridization or copy number variants, with related search terms. Risk differences were pooled to estimate the overall and stratified microarray incremental yield using RevMan. Quality assessment of included studies was performed using the Quality Assessment tool for Diagnostic Accuracy Studies (QUADAS-2) checklist. Results Seventeen studies met the inclusion criteria for analysis. Meta-analysis indicated an incremental yield of 5.0% (95% CI, 2.0–8.0%) for the detection of CNVs using microarray when pooling results. Stratified analysis of microarray results demonstrated a 4.0% (95% CI, 2.0–7.0%) incremental yield in cases of isolated NT and 7.0% (95% CI, 2.0–12.0%) when other malformations were present. The most common pathogenic CNVs reported were 22q11.2 deletion, 22q11.2 duplication, 10q26.12q26.3 deletion and 12q21q22 deletion. The pooled prevalence for variants of uncertain significance was 1%.
Conclusion The use of genomic microarray provides a 5.0% incremental yield of detecting CNVs in fetuses with increased NT and normal karyotype. Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.
INTRODUCTION The risk of genetic syndromes or neurodevelopmental delay in fetuses with increased nuchal translucency (NT) at 11–13 weeks and normal karyotype is not well established yet. The prevalence of genetic disorders when NT is > 3.5 mm or > 99th centile has been reported previously to be as low as 0.5% and as high as 6.6%1 . More than 50 genetic conditions have been described in association with an increased NT, although some, such as spinal muscular atrophy, have not been confirmed in larger studies2,3 . The most commonly found disorders are congenital adrenal hyperplasia, 22q11 microdeletion syndrome, Noonan syndrome, Smith–Lemli–Opitz syndrome, fetal akinesia deformation sequence (Pena–Shokeir), multiple pterygium syndrome (Escobar syndrome), Fanconi pancytopenia syndrome, campomelic dysplasia and VACTERL association4 – 9 . Genomic microarrays, also known as chromosomal microarray analysis (CMA) or molecular karyotyping, include array comparative genomic hybridization (aCGH) and single-nucleotide polymorphism array (SNP array). These techniques can detect copy number variants (CNVs) within the genome that are undetectable using conventional karyotyping. However, since microarrays do not typically detect monogenic disorders, only one of the nine genetic disorders listed above is detected typically by microarrays (22q11 deletion syndrome). Most
Correspondence to: Dr A. Borrell, Department of Maternal-Fetal Medicine, Institute of Gynecology, Obstetrics and Neonatology, Hospital Cl´ınic Barcelona, Sabino de Arana 1, Barcelona 08028, Catalonia, Spain (e-mail:
[email protected]) Accepted: 10 April 2015
Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.
SYSTEMATIC REVIEW
Microarray and increased nuchal translucency
651
Search in PubMed, Ovid MEDLINE and Web of Science January 2009–January 2015 (n = 529 titles)
Excluded based on title/abstract (n = 472) • Case reports • Technique other than array (MLPA, CGH, BACs on beads) • Postnatal samples • No genetic research
Articles reviewed (n = 57)
Excluded (n = 40) • Review/opinion • No increased NT/CH group described • Full data unavailable • Overlapping populations
Articles on array in fetal increased NT or CH included (n = 17) • Part of a cohort with multiple malformations (n = 14) • Focussed on array in fetuses with increased NT or CH (n = 3)
Figure 1 Flowchart showing inclusion of studies in the review. BAC, bacterial artificial chromosome; CGH, comparative genomic hybridization; CH, cystic hygroma; MLPA, multiplex ligation-dependent probe amplification; NT, nuchal translucency.
prenatal microarray studies provide overall detection rates for fetuses presenting with different kinds of ultrasound anomalies10 – 31 , and only a few specify the incremental yield over conventional karyotyping in fetuses with increased NT32 – 37 . In addition, many reported CNVs in fetuses with increased NT are from case reports, such as 16p13.11 microdeletion or Mowat–Wilson syndrome38,39 . In this systematic review, we estimate the incremental yield of detecting CNVs using microarray in fetuses with increased NT and normal karyotype (normal fluorescence in-situ hybridization (FISH) or quantitative fluorescence polymerase chain reaction (QF-PCR) in certain studies) in order to assist clinicians in antenatal counseling. As the term ‘cystic hygroma’ is still used in first-trimester fetuses to describe markedly increased NT, when septations are evident40,41 , we included these cases in our review.
METHODS A literature review was performed according to PRISMA guidelines for conducting systematic reviews. A systematic search of studies performing microarray analysis on prenatal cases presenting with increased NT (defined by The Fetal Medicine Foundation as NT ≥ 3.5 mm, as this measurement corresponds to the 99th percentile in the general population) or cystic hygroma was conducted. Electronic searches were performed of PubMed, Ovid MEDLINE and ISI Web of Knowledge (Web of Science)
Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.
databases from January 2009 to January 2015. Search terms included the following: ‘fetal’ or ‘prenatal’, ‘nuchal translucency’ or ‘cystic hygroma’ or ‘ultrasound anomaly’, ‘array comparative genomic hybridization’ or ‘copy number variants’, with related search terms (the complete search string is available in Appendix S1). There were no language restrictions to our search. Included articles were selected in a two-step process: initially, all titles and abstracts were reviewed by two authors (M.G. and A.B.) and full-text articles of those deemed potentially eligible were extracted. Only original research articles describing the use of microarrays on prenatal samples with abnormal ultrasound findings were reviewed for the full text. Case reports and studies using conventional comparative genomic hybridization (CGH), the technique preceding aCGH that reveals chromosomal regions in which there are relative gains or losses of DNA sequence with about 3-Mb resolution, were excluded. We analyzed the references of eligible articles for further inclusions. Studies including only fetuses with an increased NT and normal karyotype and those including any type of ultrasound findings were identified. When karyotyping was not performed prior to microarray (because it was performed simultaneously or not performed), genomic imbalances > 5 Mb were considered detectable by the karyotype and, therefore, not included. We did, however, include cases from Shaffer et al.36 using a 10 Mb cut-off, and some cases for which size was not specified, if the authors in the cases involved considered the imbalances
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Table 1 Summary of findings from the 17 studies included in this review of incremental yield of microarray over karyotyping in fetuses with increased nuchal translucency (NT) Non- Criteria for Isolated isolated subsequent NT (n) NT (n) microarray
Study
Inclusion criteria for original study
Tyreman (2009)20
Major ultrasound abnormalities or multiple soft markers
18
Leung (2011)37
NT > 3.5 mm
38
10
Shaffer (2012)36 Srebniak (2012)19 Faas (2012)17
Abnormal ultrasound findings
568
144
Fetal ultrasound abnormalities (NT > 3.5 mm) Fetal structural anomalies
15
6
Armengol (2012)18
Fiorentino (2013)15
Rooryck (2013)35 Yatsenko (2013)16
Scott (2013)14
Hillman (2013)13 Evangelidou (2013)12 Huang (2014)33 Oneda (2014)10
Donelly (2014)32
Brady (2014)11 Lund (2015)34
1
Microarray type
Normal G-banded karyotype and 22q11.2 Normal karyotype
All
28
Normal RAD (FISH, MLPA or QF-PCR) Normal QF-PCR
Abnormal ultrasound findings, altered biochemical screening, family history of chromosomopathy or other genetic condition, advanced maternal age, other exceptional conditions Advanced maternal age, parental anxiety, abnormal results in maternal serum screening, abnormal ultrasound findings, abnormal fetal karyotype, history of genetic abnormality, cell culture failure Abnormal ultrasound findings and karyotyping anomalies (NT > 3.5 mm)
25
All
Prenatally detected ultrasound abnormalities, history of genetic abnormality, abnormal maternal serum screening Advanced maternal age, elevated aneuploidy risk on combined first-trimester screening, fetal ultrasound abnormality, family history of genetic abnormality, parental concern, fetal demise (NT > 3.5 mm) Fetal anomaly detected on ultrasound (NT > 3.5 mm) Abnormal ultrasound findings or apparently balanced structural aberration Increased fetal NT (NT ≥ 3.5 mm)
23
Advanced maternal age, abnormal maternal serum screening, abnormal ultrasound findings, NT ≥ 3.0 mm, genetic condition, parental anxiety Advanced maternal age, positive aneuploidy screening result, structural anomalies detected by ultrasound, family history of congenital disorder (NT > 3.5 mm) Multiple abnormalities or isolated abnormality observed on ultrasound Increased NT (NT ≥ 3.5 mm)
25
2
57
16
42
35
1
16
203
Affymetrix Genomewide Human SNP Nsp/Sty 6.0 array: 700 b Agilent Fetal DNA Chip high-resolution 44 k oligonucleotide CGH array: 100 kb SignatureChip Whole Genome (Coppinger) Illumina HumanCytoSNP-12 array Affymetrix GeneChip 250 k (NspI) SNP array platform: 150–200 kb Targeted microarray developed in the scope of the project
All
CytoChip Focus Constitutional BAC microarray: 1 Mb/100 kb
Normal FISH and conventional karyotyping Normal FISH analysis (chr 13, 18, 21, X and Y)
Agilent Oligonucleotide 8 × 60 k CGH array: 400 kb
All
Agilent ISCA 8 × 60 k CGH array: 70 kb
Normal QF-PCR
CytoChip Focus Constitutional BAC microarray: 2 Mb/200 kb CytoChip ISCA 105 k array: 150 kb
All
Agilent whole genome 105 k or Roche 135 k oligonucleotide microarray
12
Normal karyotype with conventional cytogenetic analysis Normal results on conventional karyotyping
Customized CGH 44 K Fetal Chip v1.0 and CGH + SNP array 8 × 60 k Fetal Chip v2.0 Whole Genome 2.7 M SNP array or Cytoscan HD SNP array: 20–100 kb
186
48
Normal results on conventional karyotyping
Agilent 4-plex or Affimetrix Genome-Wide Human SNP Array 6.0: 75 kb
19
11
Normal FISH or QF-PCR
CytoSure Syndrome Plus 105 k CGH array and CytoSure Syndrome Plus 180 k CGH array Agilent SurePrint G3 Human CGH microarray 180 k: 80 kb
53
94
Normal QF-PCR
Only first author of each study is given. BAC, bacterial artificial chromosome; CGH, comparative genomic hybridization; FISH, fluorescence in-situ hybridization; MLPA, multiplex ligation-dependent probe amplification; QF-PCR, quantitative fluorescence polymerase chain reaction; RAD, rapid aneuploidy detection; SNP, single-nucleotide polymorphism.
Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.
Ultrasound Obstet Gynecol 2015; 46: 650–658.
Microarray and increased nuchal translucency
RESULTS The search revealed 464 articles, of which the full text of 57 was reviewed. Forty studies were excluded because they did not include an increased NT group, populations overlapped, or we could not obtain data required for the analysis. A total of 17 met the inclusion criteria and were included in the meta-analysis10 – 20,32 – 37 (Figure 1). Table 1 displays the characteristics of the 17 studies including the inclusion criteria for the initial karyotyping and the criteria for (subsequent) microarray, sample size and microarray type. The NT cut-off was provided in nine studies: 3.5 mm was used in eight13,14,19,32 – 35,37 and 3 mm in one10 . Three studies included only fetuses with increased NT33,34,37 , eight included different ultrasound anomalies11 – 13,17,19,20,35,36 and the remaining six also considered pregnancies with advanced maternal age or abnormal maternal serum screening10,14 – 16,18,32 . In 12 studies, genomic microarray testing was performed after a normal result was obtained by karyotyping, FISH or QF-PCR analysis, whereas karyotype and microarray analysis were performed
Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.
Flow and timing Reference standard Index test QUADAS-2 domain
to be undetectable by karyotyping. Data on inclusion criteria, microarray resolution, fetal ultrasound findings (whether the NT was isolated or associated with other anomalies) and CNV description were extracted from the studies. Nine authors were contacted to obtain missing information. Details of reported microarray anomalies were reviewed independently by two authors (M.G. and A.B.) to evaluate clinical significance. Incremental yield (risk difference) of microarray was defined as the yield over karyotyping for each prenatal series. When possible, cases were stratified according to whether the enlarged NT was an isolated finding or associated with other ultrasound anomalies. Risk differences from each study were pooled to estimate an overall and stratified incremental yield of microarray using RevMan version 5.3.4 (Review Manager, The Cochrane Collaboration, Copenhagen, Denmark) and corresponding forest and funnel plots were constructed. Confidence intervals (95% CI) were computed. Studies with fewer than 15 cases were excluded from the meta-analysis. Statistical heterogeneity was examined using Higgins’ I2 (quantitative) test. To take into account the low statistical power of tests of heterogeneity, we considered statistically significant heterogeneity using Cochran’s Q test with a P < 0.1 or I2 > 30%. A random-effects model was used when there was significant heterogeneity. We assessed publication bias graphically using funnel plots (Figure S1). Quality assessment of included studies was performed using the Quality Assessment tool for Diagnostic Accuracy Studies (QUADAS-2) checklist, and risk of bias and applicability was assessed according to patient selection, index test, reference standard and flow and timing42 . Finally, the reported rates of pathogenic CNVs and variants of unknown significance (VOUS) in each study were listed in two separate tables.
653
Patient selection 0
20
40
60
80
100
Proportion of studies with low, high or unclear risk of bias (%)
Reference standard
Index test
Patient selection 0
20
40
60
80
100
Proportion of studies with low, high or unclear concerns regarding applicability (%)
Figure 2 Summary of quality assessment of included studies using Quality Assessment tool for Diagnostic Accuracy Studies (QUADAS-2) checklist. , low; , high; , unclear.
simultaneously in three studies12,15,18 , or as standalone test in two14,36 . Quality assessment of the included studies using QUADAS-2 is displayed in Figure 2. The forest plot with details of the 17 included studies and the pooled results from the meta-analysis are shown in Figure 3, indicating that the incremental yield of microarray detecting CNVs after karyotyping was 5% (95% CI, 2.0–8.0%). Five studies contained cases with isolated increased NT10,12,18,34,35 , nine included cases with both isolated and associated increased NT11,13,15,16,19,32,33,36,37 , two provided information only about isolated increased NT cases17,20 , while no data were available on associated findings in one study14 . Pooled results of 16 studies indicate an incremental yield of 4% (95% CI, 2.0–7.0%) for isolated cases of increased NT (Figure 4), and pooled results of 10 studies indicate an incremental yield of 7% (95% CI, 2.0–12.0%) for cases of increased NT with associated ultrasound abnormalities (Figure 5). Only 38 of the 76 pathogenic CNVs identified were specified by the authors and are listed in Table 2. The most common CNVs found were 22q11.2 deletion, 22q11.2 duplication, 10q26.12q26.3 deletion and 12q21q22 deletion. The size of the pathogenic CNVs identified ranged between 50 kb and 16 Mb. The reported prevalence of VOUS in all included studies ranged from 0.5% to 7.7%, with a pooled prevalence of 0.8% (95% CI, 0.4–1.3%; Table 3).
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Study or subgroup Tyreman (2009)20 Leung (2011)37 Shaffer (2012)36 Srebniak (2012)19 Faas (2012)17 Armengol (2012)18 Scott (2013)14 Yatsenko (2013)16 Evangelidou (2013)12 Hillman (2013)13 Rooryck (2013)35 Fiorentino (2013)15 Brady (2014)11 Donelly (2014)32 Huang (2014)33 Oneda (2014)10 Lund (2015)34
aCGH Events Total
Karyotype Events Total
1 4 26 2 0 1 2 4 1 2 3 3 2 12 0 4
19 48 712 21 28 25 42 39 16 36 57 27 30 234 215 53
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
19 48 712 21 28 25 42 39 16 36 57 27 30 234 215 53
3.2 5.5 10.5 2.8 6.8 4.5 6.1 4.5 2.6 5.3 6.9 3.3 4.4 9.7 10.7 6.0
0.05 (–0.08, 0.19) 0.08 (–0.00, 0.17) 0.04 (0.02, 0.05) 0.10 (–0.05, 0.24) 0.00 (–0.07, 0.07) 0.04 (–0.06, 0.14) 0.05 (–0.03, 0.12) 0.10 (–0.00, 0.21) 0.06 (–0.09, 0.22) 0.06 (–0.03, 0.14) 0.05 (–0.01, 0.12) 0.11 (–0.02, 0.24) 0.07 (–0.04, 0.17) 0.05 (0.02, 0.08) 0.00 (–0.01, 0.01) 0.08 (–0.00, 0.15)
9
94
0
94
7.2
0.10 (0.03, 0.16)
1696
100.0
0.05 (0.02, 0.08)
Total (95% CI)
1696
Risk difference M–H, random, 95% CI
Risk difference Weight (%) M–H, random, 95% CI
Total events 76 0 Heterogeneity: τ2 = 0.00; χ2 = 104.08, df = 16 (P < 0.00001); I2 = 85% Test for overall effect: Z = 3.63 (P = 0.0003)
–0.50
–0.25
0
0.25
0.50
Incremental yield
Figure 3 Forest plot of incremental yield by microarray over karyotyping in fetuses with isolated increased nuchal translucency with or without associated ultrasound abnormalities. Only first author of each study is given. aCGH, array comparative genomic hybridization; M–H, Mantel–Haenszel.
Study or subgroup Tyreman (2009)20 Leung (2011)37 Shaffer (2012)36 Srebniak (2012)19 Armengol (2012)18 Faas (2012)17 Rooryck (2013)35 Evangelidou (2013)12 Yatsenko (2013)16 Fiorentino (2013)15 Hillman (2013)13 Brady (2014)11 Oneda (2014)10 Huang (2014)33 Donelly (2014)32 Lund (2015)34
aCGH Events Total 1 2 18 1 1 0 3 1 1 3 1 2 4 0 7 9
18 38 568 15 25 28 57 16 23 25 35 19 53 203 186 94
Karyotype Events Total 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
18 38 568 15 25 28 57 16 23 25 35 19 53 203 186 94
Risk difference M–H, random, 95% CI
Risk difference Weight (%) M–H, random, 95% CI 2.8 5.7 13.0 2.2 4.4 7.3 7.4 2.4 4.0 2.8 6.5 2.3 6.3 13.3 11.6 7.8
0.06 (–0.09, 0.20) 0.05 (–0.03, 0.14) 0.03 (0.02, 0.05) 0.07 (–0.10, 0.23) 0.04 (–0.06, 0.14) 0.00 (–0.07, 0.07) 0.05 (–0.01, 0.12) 0.06 (–0.09, 0.22) 0.04 (–0.07, 0.16) 0.12 (–0.02, 0.26) 0.03 (–0.05, 0.10) 0.11 (–0.06, 0.27) 0.08 (–0.00, 0.15) 0.00 (–0.01, 0.01) 0.04 (0.01, 0.07) 0.10 (0.03, 0.16)
Total (95% CI) 100.0 0.04 (0.02, 0.07) 1403 1403 Total events 54 0 Heterogeneity: τ2 = 0.00; χ2 = 70.58, df = 15 (P < 0.00001); I2 = 79% Test for overall effect: Z = 3.18 (P = 0.001)
–0.50
–0.25
0
0.25
0.50
Incremental yield
Figure 4 Forest plot of incremental yield by microarray over karyotyping in fetuses with isolated increased nuchal translucency. Only first author of each study is given. aCGH, array comparative genomic hybridization; M–H, Mantel–Haenszel.
Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.
Ultrasound Obstet Gynecol 2015; 46: 650–658.
Microarray and increased nuchal translucency
Study or subgroup Tyreman (2009)20 Leung (2011)37 Shaffer (2012)36 Srebniak (2012)19 Yatsenko (2013)16 Hillman (2013)13 Fiorentino (2013)15 Huang (2014)33 Donelly (2014)32 Brady (2014)11
aCGH Events Total 0 2 8 1 3 1 0 0 5 0
1 10 144 6 16 1 2 12 48 11
Karyotype Events Total 0 0 0 0 0 0 0 0 0 0
1 10 144 6 16 1 2 12 48 11
655
Weight (%)
Risk difference M–H, random, 95% CI
0.3 3.1 49.9 1.9 5.2 0.3 0.7 9.7 20.5 8.5
0.00 (–0.85, 0.85) 0.20 (–0.08, 0.48) 0.06 (0.02, 0.09) 0.17 (–0.19, 0.53) 0.19 (–0.02, 0.40) 1.00 (0.15, 1.85) 0.00 (–0.60, 0.60) 0.00 (–0.15, 0.15) 0.10 (0.01, 0.20) 0.00 (–0.16, 0.16)
Total (95% CI) 251 251 100.0 Total events 20 0 Heterogeneity: τ2 = 0.00; χ2 = 10.38, df = 9 (P = 0.32); I2 = 13% Test for overall effect: Z = 2.83 (P = 0.005)
Risk difference M–H, random, 95% CI
0.07 (0.02, 0.12)
–1.0
–0.5
0
0.5
1.0
Incremental yield
Figure 5 Forest plot of incremental yield by microarray over karyotyping in fetuses with increased nuchal translucency and associated ultrasound abnormalities. Only first author of each study is given. aCGH, array comparative genomic hybridization; M–H, Mantel– Haenszel.
DISCUSSION The results of this systematic review and meta-analysis support the use of microarray analysis in karyotypically normal fetuses with increased NT, and indicate that microarray yields additional clinically valuable information over conventional karyotyping in 5.0% (95% CI 2.0–8.0%) of these fetuses, with a minimal burden of 0.8% VOUS. The microarray can be performed as a first tier in cases with increased NT or, alternatively, after the most common aneuploidies have been ruled out by normal FISH or QF-PCR results, or even after a normal karyotype. Previous reports investigating the potential value of microarray in fetuses with increased NT and normal karyotype present with conflicting results. Although a recent study which focused on 215 pregnancies with increased fetal NT33 did not find any pathogenic CNVs, other recent studies, including a range between 38 and 56834 – 37 fetuses with isolated increased NT, reported incremental yields over karyotyping that ranged from 3% to 10%. The 4.0% incremental yield of pathogenic CNVs in cases of isolated NT observed in our meta-analysis is slightly higher than the 3.1% (5/162) obtained in the only systematic review reported previously that analyzed NT separately from other ultrasound findings43 . Our review not only extends from 162 cases in four series to 1696 cases in 17 series, but we also performed a meta-analysis. The finding of about a 1% pathogenic CNV rate in the general pregnant population15,24 makes the assumption of Huang et al.33 (that CNVs are related to the associated structural anomalies rather than to NT itself) counterintuitive since, in that case, increased NT would be protective rather than a risk marker of genetic disease. In addition, the majority of microdeletion syndromes are not expected to present with structural anomalies at prenatal ultrasound examination. Differences in the
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incremental yield could be explained by the different type (targeted or whole genome oriented) and resolution of the microarrays performed, different previous testing (full karyotype or five chromosome testing by FISH or QF-PCR) or the small cohort numbers. We were not surprised by the fact that the CNV frequency observed in fetuses with increased NT was between the 1% rate observed in the general population (advanced maternal age or abnormal maternal serum screening are not risk factors for microdeletion or microduplication syndromes) and the 6–9% rate observed in cases with ultrasound anomalies10 – 20,32,35,36 . According to Fetal Medicine Foundation data, the karyotypic abnormality rate increases from 19% to 39% in fetuses with an increased NT, depending on whether the 95th or the 99th percentile is used for the NT cut-off. In this population, QF-PCR or FISH would miss only 5% of these chromosomal anomalies44 . Pathogenic imbalances reported in this systematic review indicate that the most frequently found is 22q11.2 deletion, causing 22q11.2 microdeletion syndrome, also named DiGeorge syndrome. Given that this microdeletion accounts for 13% (5/38) of pathogenic genomic imbalances (CNVs), which are present in 5.0% of fetuses with increased NT and normal karyotype, we can conclude that 0.66% of these fetuses are affected by 22q11 microdeletion syndrome. This microdeletion is 10 times more frequent in fetuses with cardiac defects and a normal karyotype (7%), as shown in a recent systematic review45 . In this systematic review, only one of the five fetuses’ 22q11.2 microdeletion syndrome is described to present with an associated cardiac defect (tetralogy of Fallot). The second most frequently found CNV is 22q11.2 duplication (4/38), causing 22q11.2 duplication syndrome, with a variable phenotype ranging from mild learning disabilities as the only symptom, to congenital malformations leading to early death46 . Other recurrent
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Table 2 Pathogenic copy number variants (CNV) and size identified by microarray in fetuses with increased nuchal translucency (NT) and normal karyotype Pathogenic CNV type
CNV size
arr1q21.1q21.2 loss (1q21.1 deletion syndrome) arr1p36.23p36.13 loss, 14q11.2q12 loss, 14q11.2 loss (1p36 syndrome) arr4pter16.3 loss, 7pter-p21.3 gain arr5p14.1 loss arr5p14.3p14.2 gain, 15q25.2q25.3 loss arr5q35.2q35.3 loss (Sotos syndrome) arr6q25.3q27 gain, 10q26.12q26.3 loss* arr6q22.32 loss* arr8p23.3p23.1 loss, 8p22p21.1 gain arr8p23.1 gain, 8p23.1 loss (8p23.1 del/dup syndrome) arr9p21.1, 21q22.3, 9p21.13 9q22.2 loss arr9q31.2q33.1 loss, 9q33.1 loss arr10q26.12q26.3 loss, 16q23.1q24.3 gain arr11q24.2q25 gain arr12p13.33p11.1 gain (Pallister–Killian syndrome)* arr12q21q22 loss arr12q21.2q22 loss arr15q11.2 loss arr15q12 gain* arr15q26.3 loss arr16p13.11p12.3 gain (16p13.11 micro-dup syndrome) arr16q22 loss arr17q12 loss* arr18q23 loss, 22q13.1q13.33 gain arr19q13.41q13.42 gain arr21q22.2q22.3 loss arr22q11.21 loss*(22q11.2 del syndrome) arr22q11.21 loss*(22q11.2 del syndrome) arr22q11.21-q11.22 loss (22q11.2 del syndrome) arr22q11.21 loss (22q11.2 del syndrome) arr22q11.21 loss (22q11.2 del syndrome) arr22q11.21 gain (22q11.2 dup syndrome) arr22q11.21 gain (22q11.2 dup syndrome) arr22q11.21 gain (22q11.2 dup syndrome) arr22q11.21 gain(22q11.2 dup syndrome) arrXp11.23p11.22 loss arrXp21.2p21.2 loss arrXp22.31 loss (ichthyosis X-linked)
3.3 Mb 9.2 Mb, 4.0 Mb, 578 Kb 1.8 Mb, 16 Mb 1.7 Mb 2.4 Mb, 2.2 Mb 1.7 Mb 12.5 Mb, 12.2 Mb 7.9 Mb 6.5 Mb, 14.6 Mb 1.8 Mb, 1.8 Mb 200 kb 8.9 Mb, 273 kb 13.6 Mb, 14.6 Mb NS NS 13.42 Mb 15.7 Mb 0.9 Mb 4.9 Mb 624 kb 1.9 Mb 1.2 Mb 1.4 Mb 0.4 Mb, 11 Mb 529 kb 5.5 Mb 0.6 Mb 2.5 Mb 1 Mb 2.5 Mb 2.6 Mb NS 2.5 Mb 2.8 Mb 2.4 Mb 3.3 Mb 50 kb 1.7 Mb
Study† 34 10 11 37 12 15 16 37 15 34 14 35 15 13 13 35 16 19 37 10 34 37 16 34 20 34 19 16 14 35 34 18 10 34 34 10 11 34
*Identified in case with increased NT and associated ultrasound anomalies. †Reference number of study in which the CNVs were detected. del, deletion; dup, duplication; NS, not stated.
pathogenic CNVs are 10q26.12q26.3 deletion (2/38) and 12q21q22 deletion (2/38). Among the remaining syndromes, 1p36 deletion syndrome and Sotos syndrome (caused by 5q35 microdeletion in 10–40% of cases) were observed. Although more than half of the included CNVs were less than 5 Mb in size, some CNVs were > 10 Mb and were overlooked by karyotyping. The microarray detection of Pallister–Killian syndrome that was missed by karyotyping13 may be explained by the preferential growth of the normal cell line after culture that masked the mosaicism. Unexpectedly, we did not find any relationship between higher resolution of the microarray, or the year in which the microarray was performed, and higher incremental yield for pathogenic CNVs or VOUS. The incremental yield for pathogenic CNVs during four consecutive time periods (2009–2011, 2012, 2013 and 2014–2015) was 7%, 4%, 7% and 4%, respectively (data not shown). The
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0.8% rate of VOUS found in the overall series and the rates after stratified analyses (0.9% for isolated NT and 1.2% for associated NT (data not shown)) are similar to the 1.4% reported in a recent meta-analysis of microarray in cases with abnormal ultrasound findings13 . Performing microarray rather than karyotyping could lead to a decrease in undiagnosed genetic disorders in fetuses with an increased NT. In a publication by Senat et al., in which a cohort of 171 fetuses with an increased NT above the 99th percentile were followed for up to 2 years of age, an 8.8% rate of genetic disorders (15/171), including musculoskeletal disorders, was observed6 . It was concluded that at 20–22 weeks’ gestation, there was a 13% (2/15) risk of undiagnosed syndromes in fetuses with persistent nuchal fold and no other ultrasound findings (Simpson–Golabi syndrome and Miller–Dieker syndrome were diagnosed after termination and at birth, respectively). Among fetuses with no ultrasound
Ultrasound Obstet Gynecol 2015; 46: 650–658.
Microarray and increased nuchal translucency Table 3 Prevalence of variants of unknown significance (VOUS) in fetuses with increased nuchal translucency and normal karyotype Study
VOUS (n (%)) (2009)20
Tyreman Leung (2011)37 Shaffer (2012)36 Srebniak (2012)19 Faas (2012)17 Armengol (2012)18 Fiorentino (2015)15 Rooryck (2013)35 Yatsenko (2013)16 Scott (2013)14 Hillman (2013)13 Evangelidou (2013)12 Huang (2014)33 Oneda (2014)10 Donelly (2014)32 Brady (2014)11 Lund (2015)34 Pooled prevalence (% (95% CI))
NS 1/48 (2.1) NS NS 1/28 (3.6) NS NS NS 3/39 (7.7) NS NS NS 1/215 (0.5) 4/53 (7.5) NS 1/30 (3.3) 3/94 (3.2) 14/1696 (0.8 (0.4–1.3))
Only first author of each study is given. NS, not stated.
abnormalities at 20–22 weeks and with no previous history of genetic disorders, the corresponding risk was 2% (2/105) (two livebirths with 2q11 microdeletion syndrome and cephalo-oculofascioskeletal syndrome). One of the conditions in each of the two groups (Miller–Dieker and 22q11 microdeletion) would have been detected if a microarray had been carried out, which would have halved residual risk rates (6% and 1%) if microarray rather than karyotype had been performed. The major limitation of our meta-analysis is the high heterogeneity found in the overall series (I2 = 85%) and after stratified analysis (isolated NT, I2 = 79%), possibly due to the different microarray platforms and filters applied, together with the clinical design (microarray performed simultaneously to karyotyping, after a normal karyotype result or after normal FISH or QF-PCR results). We are aware that in the presence of high heterogeneity, pooled rates have to be considered with caution. The strengths of our study are that it is the first meta-analysis on the incremental yield of microarray over karyotyping in fetuses with increased NT, and is the largest systematic review on this topic, given that it includes data on 17 studies and 1696 pregnancies. In conclusion, microarray analysis has a 5.0% incremental yield for detecting CNVs in fetuses with increased NT and normal karyotype.
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SUPPORTING INFORMATION ON THE INTERNET The following supporting information may be found in the online version of this article: Appendix S1 Complete search string used in systematic literature search Figure S1 Funnel plots of studies reporting on incremental yield of microarray over karyotyping in fetuses with: (a) isolated or non-isolated increased nuchal translucency (NT); (b) isolated increased NT; and (c) increased NT with associated ultrasound anomalies.
This article has been selected for Journal Club. A slide presentation, prepared by Dr Maddalena Morlando, one of UOG's Editors for Trainees, is available online. Chinese translation by Dr Yang Fang. Spanish translation by Dr Masami Yamamoto.
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Ultrasound Obstet Gynecol 2015; 46: 650–658.
