Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011
A precise guideline establishing chromosomal microarray analysis (CMA) applications and platforms in the prenatal setting does not exist. The controversial question is whether CMA technologies can or should soon replace standard karyotyping in prenatal diagnostic practice. A review of the recent literature and survey of the knowledge and experience of all members of the Italian Society of Human Genetics (SIGU) Committee were carried out in order to propose recommendations for the use of CMA in prenatal testing. The analysis of datasets reported in the medical literature showed a considerable 6.4% incidence of pathogenic copy number variations (CNVs) in the group of pregnancies with sonographically detected fetal abnormalities and normal karyotype. The reported CNVs are likely to have a relevant role in terms of nosology for the fetus and in the assessment of reproductive risk for the couple. Estimation of the frequency of copy number variations of uncertain significance (VOUS) varied depending on the different CMA platforms used, ranging from 0–4%, obtained using targeted arrays, to 9–12%, obtained using high-resolution whole genome single nucleotide polymorphism (SNP) arrays. CMA analysis can be considered a second-tier diagnostic test to be used after standard karyotyping in selected groups of pregnancies, namely those with single (apparently isolated) or multiple ultrasound fetal abnormalities, those with chromosomal rearrangements, even if apparently balanced, and those with supernumerary marker chromosomes. Copyright © 2012 ISUOG. Published by John Wiley & Sons, Ltd.
In the last few years chromosomal microarray analysis (CMA) technology (array comparative genomic hybridization, aCGH; single nucleotide polymorphism array, SNP array) has acquired increasing relevance, becoming a fundamental diagnostic tool in medical genetics. In fact, technological evolution and experimental optimization have resulted in a notable simplification of analytic protocols, leading to a decrease in costs and enabling the progressive spread of this technology in many laboratories all over the world. Encouraging results, in terms of detection rate, were obtained in patients affected by unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD) or multiple congenital anomalies (MCA), in whom the diagnostic yield was improved over that obtained by karyotyping by an estimated 10–20%1–3. Accurate evaluation of the gene content of the imbalanced genomic regions, together with comparison with data collections present in publicly available repository databases (DGV, http://projects.tcag.ca/variation/; DECIPHER, http://decipher.sanger.ac.uk/; OMIM, http://www.ncbi.nlm.nih.gov/omim), enabled detection of critical regions related to known syndromes, allowing genotype–phenotype correlations in several cases. For such reasons, in 2010 the Italian Society of Human Genetics (SIGU) Committee proposed a national document in which, based on the literature and on the experience of all participating institutions, CMA was recommended as the first-tier diagnostic test in the postnatal setting for patients with DD/ID, ASD or MCA (http://www.sigu.net).
The advantages offered by CMA technology have opened up new avenues regarding its possible application in prenatal diagnosis, where traditional karyotyping is still considered the gold standard method for all indications for invasive testing. Compared with conventional karyotyping, CMA can rapidly detect imbalances with a resolution of up to a few Kb using standardized protocols4.
A precise guideline establishing CMA applications and platforms in the prenatal setting does not exist and this situation has led to debates and controversies5–11 concerning whether CMA technology can or should replace standard karyotyping in prenatal diagnostic practice. Considering the limited knowledge in this field, the SIGU Committee has focused on disadvantages related to this technology and currently advises against its unlimited and unselected application in routine prenatal diagnosis. Without strict guidelines for the use of CMA in prenatal diagnosis, it could potentially be more harmful than it is useful when applied during prenatal life, because of the unclear results it can provide. Current knowledge has gaps regarding the clinical interpretation of copy number variations (CNVs). This is because of the possibility of detecting an imbalance not previously described, the lack of knowledge about the function of many genes, our relatively poor understanding of gene–gene and gene–environment interactions, and the role of epigenetic modifications in modulating the penetrance and expressivity of CNVs12–14. There are additional questions related to the detection during the prenatal diagnostic period of variations of uncertain significance (VOUS), which have no known predictive value with regards to fetal and future health, and can thus cause increased parental anxiety7, 15. In addition, the diagnostic yield of CMA in the prenatal setting has not been established clearly in all categories of indications because the majority of published papers included selected cases with fetal abnormalities detected by ultrasound and an apparently normal karyotype. In this group of pregnancies the CMA detection rate is, on average, 6.4% (range, 0–15.6%) (Table 1). Datasets reported in the medical literature clearly show a significant incidence of pathogenic CNVs in this group of pregnancies and these detected CNVs are likely to have a relevant role in terms of nosology for the fetus and for the assessment of reproductive risks for the couple16–32. In cases with sonographic fetal abnormalities, the sum of the detection rates of conventional cytogenetic analyses (28% for chorionic villi and 12% for amniotic fluid: ∼ 20% on average)33 and CMA (6.4%), i.e. combining the first-tier karyotype with the second-tier CMA, provide an overall detection of ∼ 27%.
Table 1. Incidence of pathogenic variations and unclear results from published studies regarding use of chromosomal microarray analysis (CMA) in prenatal diagnosis
|Le Caignec et al.16||49||1 (2.0)||49||4 (8.2)|
|Vialard et al.18||39||NR||37||4 (10.8)|
|Van den Veyver et al.20||300||3 (1)||84||5 (6)|
|Shaffer et al.21||151||1 (0.7)||110||2 (1.9)|
|Coppinger et al.22||213||1 (0.5)||155||6 (3.9)|
|Kleeman et al.23||50||1 (2)||50||0 (0)|
|Tyreman et al.24||106||13 (12.3)||106||10 (9.4)|
|Valduga et al.25||50||NR||50||5 (10)|
|Faas et al.26||38||3 (7.9)||32||3 (9.4)|
|Maya et al.27||269||0 (0)||102||2 (2)|
|Evangelidou et al.28||25||1 (4)||15||1 (6.6)|
|Gruchy et al.29||38||0 (0)||38||3 (7.9)|
|D'Amours et al.30||49||6 (12.2)||49||4 (8.2)|
|Zuffardi et al. (ISPD 2010)†||63||1 (1.6)||63||5 (9.5)|
|De Toffol et al.31||32||1 (3.1)||32||5 (15.6)|
|Leung et al.32||48||NR||48||6 (12.5)|
|Overall||1520||32 (2.3)||1020||65 (6.4)|
Frequencies of VOUS seem to be difficult to assess due to the different CMA platforms used in the various studies, and range from 0–4% when assessed by targeted arrays to ∼ 9–12% when assessed by high resolution whole genome SNP arrays (Table 1)16–32. In contrast, the rate of detection of known, disability-causing pathogenic CNVs by CMA in all pregnant women has been estimated to be between 0.16% and 0.3%6. Analysis of the proportion of ambiguous findings compared to pathogenic CNVs shows that using CMA technology in the prenatal setting without a specific clinical indication is not justified at present.
Another important limitation related to the application of CMA as a first-tier test is represented by the impossibility of detecting balanced rearrangements i.e. those without genetic losses or gains. This would lead to underestimation of the risks of phenotypic consequences related to: (i) disruption or modulation of the expression of gene(s) located at the breakpoint(s); (ii) inactivation (position effect) of gene(s) at the breakpoint region(s); and (iii) missing the opportunity to investigate and detect uniparental disomy conditions related to imprinting syndromes in cases involving imprinted chromosomes34–36. SNP array has the advantage of being able to detect long continuous stretches of homozygosity (LCSH), representing whole chromosomal or segmental uniparental isodisomies (a duplicate of one chromosome from a parent and no chromosome from the other parent). It cannot, however, detect heterodisomies (the most common form of uniparental disomy, in which both chromosomes in a pair are inherited from one parent) without testing parents in conjunction with the fetal specimen. In addition, SNP array provides consanguinity information (occurrence of incest) that raises important ethical issues; therefore, its use in terms of LCSH may be limited37. Finally, polyploidies and mosaicisms lower than 30%, that are relatively common findings in chorionic villi and amniotic fluid samples33, cannot currently be detected by aCGH38, 39.
On the other hand, CMA is useful to clarify abnormal karyotype results. In cases with supernumerary marker chromosomes, CMA can aid in their classification and characterization, improving the diagnostic accuracy and allowing specific genetic counseling to be offered to the couple40–42. The role of CMA prenatally in cases with de novo apparently balanced chromosomal rearrangements has not been studied extensively; however, in postnatal datasets of patients with de novo apparently balanced chromosomal rearrangements and an abnormal phenotype, CMA detects cryptic imbalances in 35–40% of samples with reciprocal translocations and in 72–75% of samples with complex rearrangements43–45.
Recommendations for Microarray Application in Prenatal Diagnosis
The SIGU Committee members belong to both public and private institutions. Based on review of the recent literature and the knowledge and experience of all members of the committee, we recommend the use of CMA in prenatal testing:
- 1)never as a substitute for conventional karyotyping;
- 2)for specific diagnostic purposes in selected pregnancies and not for general screening in all pregnancies;
- 3)only in prenatal cases with specific indications, such as:
- i)single (apparently isolated) or multiple sonographic fetal abnormalities;
- ii)de novo chromosomal rearrangements, even if apparently balanced, detected by standard karyotyping, to investigate the possible presence of cryptic imbalance(s) related to the structural chromosome abnormality;
- iii)supernumerary marker chromosomes in order to characterize their origin and genetic content.
In these groups of pregnancies we recommend the application of a genome-wide, and not a targeted, platform enriched with probes containing dosage-sensitive and disease-causing genes with an average spatial resolution of at least 250 Kb with calls in the backbone (the regions between known disease-causing regions) of at least 500 Kb. When an uncommon CNV is found, parental testing is needed to help in the interpretation of genotype–phenotype correlations.
Further data are needed on the application of CMA in other groups of pregnancies, such as those with:
abnormal maternal serum screening with an increased risk for Down syndrome and normal karyotype;
one or more soft markers (e.g. choroid plexus cysts, intestinal hyperechogenicity, renal pyelectasis, single umbilical artery, echogenic cardiac foci);
intrauterine growth restriction and/or amniotic fluid volume alteration without major structural abnormalities (e.g. cardiac malformations, diaphragmatic hernia, central nervous system abnormalities).
Robust genotype–phenotype correlations collected from large-scale research studies are necessary before future introduction of this technique in all pregnancies as a screening tool and in place of standard karyotyping.
Presently, CMA analysis can be considered a second-tier diagnostic test which can complement, but not replace, standard karyotyping in a selected group of pregnancies.
Laboratories providing CMA-based analysis are encouraged to participate in an external quality assessment program and in proficiency testing among laboratories to monitor their performance.
All SIGU Committee members dedicate this document to the memory of our colleague Claudio Castellan, MD. The authors would like to thank the SIGU Past President Prof. Lidia Larizza, the current President Prof. Giovanni Neri and the members of the SIGU Managing Committee, Dr Antonio Amoroso, Prof. Luciana Chessa, Dr Corrado Romano, Dr Marco Seri and Dr Francesca Torricelli, for their support during the processing of this position paper. The Committee would like to thank the following SIGU members for their contribution in debating this issue during the meetings: Carmen Ardisia, Giuseppe Barrano, Paola Battaglia, Romina Bonora, Dagna Francesca Bricarelli, Rosa Busuito, Anna Carmela Capalbo, Massimo Carella, Rossella Caselli, Girolama Casile, Simona Cavani, Bommina Celso, Alessandro Civolani, Domenico Coviello, Maria Ida Corpaccioli, Ambra Cortesi, Gianfranco Croci, Elvira D'Alessandro, Domenico Dell'Edera, Monia Letizia Di Maria, Marco Fichera, Lonardo Fortunato, Francesca Forzano, Fabrizia Franchi, Alessandra Friso, Sara Ghezzo, Silvia Guarducci, Silvana Guerneri, Nadia Iacobelli, Carmelo Laganà, Salvatrice Antonia Lauricella, Rosetta Lecce, Elisabetta Lenzini, Elisabetta Lippi, Ermanna Lisi, Ilaria Longo, Giuseppina Marseglia, Francesca Malvestiti, Anna Montaldi, Roberta Murru, Gianfranco Nocera, Anna Lucia Nutini, Daniela Orteschi, Marilena Pantaleo, Antonio Pedicini, Chiara Pescucci, Antonella Pettinari, Barbara Pivetta, Antonio Pizzuti, Gisa Police, Diana Postorivo, Lucia Prima, Orsola Privitera, Erica Racalbuto, Alessandra Renieri, Simona Rossi, Guglielmo Sabbadini, Sandra Santucci, Elisa Savin, Lorenzo Sinibaldi, Iolanda Spasari, Antonella Tanzariello, Romano Tenconi, Barbara Torres, Annalisa Vetro, Sara Zanchetti, Anna Zilio and Marcella Zollino. We are grateful to Prof. Agatino Battaglia, MD for critical revision and improvements to the manuscript.