Dear editor: With considerable interest we’ve read the brief communication of Sabbagh et al. [1], recently published in this journal. In their paper, the authors report on the presence of a

mosaic intragenic deletion in the Duchenne muscular dystrophy (DMD) gene, detected by chromosomal microarray (CMA) analysis, and validated by MLPA analysis, in DNA isolated from a chorionic

villus (CV) sample. Because of the mosaic nature of the deletion in the CV, DNA isolated from amniotic fluid was studied subsequently, to determine whether the deletion was also present in

the foetus itself. No deletion was detected and a healthy boy was born. In their discussion, Sabbagh et al. conclude that their case “_emphasizes the importance of conducting amniocentesis

referred statement. CV consist of two cell-layers, with a different embryonic origin, being the syncytio- and cytotrophoblast (CTB), derived from the trophoblast of the blastocyst, and the

mesenchymal core (MC), derived from the inner cell mass (ICM) of the blastocyst (supplemental Figure 1). CPM is a well-known phenomenon that refers to the presence of genetically abnormal

cells only present in the CV and not in the foetus, resulting from post-zygotic cell division errors. Based on the affected cell lineage, CPM can be categorised into CPM type I, with the

genetic abnormality only present in the CTB, CPM type II, with the abnormality only present in the MC, and CPM type III, with the abnormality in both cell-layers [2, 3]. Cells from the MC

cell-layer are more related to the foetus due to their common embryonic origin, the ICM (supplemental Figure 1), and the genotype of these cells is more representative for the actual foetal

genotype than the genotype of cells from the CTB layer. Therefore, in the cytogenetic society, it is common practice to dissociate both cell layers [4] and to analyse their genetic

constitution separately. Originally this was done by preparing short-term cultures (STC) of the CTB and long-term cultures (LTC) of the MC. If a chromosome aberration is found to be

restricted to the CTB only (with normal MC), the diagnosis of CPM type I with a normal foetus is by far the most likely and follow-up investigations in amniotic fluid can be ommitted [5, 6].

Only a chromosomally abnormal MC needs a secondary amniocentesis for verification of the foetal karyotype. The original Specific Constitutional Cytogenetic Guidelines [7] stated that _“If

an initial cytogenetic diagnosis is made on short-term preparations, a long term culture should be available for confirmation, in order to minimise problems of interpretation_” and

“_Analysis solely on short-term incubation preparations is not recommended”_. The updated version of these guidelines [8] also recommend to dissociate the cell layers, in order to “_allow

for mosaicism exclusion when needed”_. To minimize work load, nowadays in cytogenetic diagnostic laboratories it is common practice to only analyse DNA from the MC (not the CTB). By doing

so, CPM type I, accounting for ~40% of all CPMs [6], will not be noticed but still CPM types II and III, that require follow-up testing in amniotic fluid, will be revealed. For their

analysis, Sabbagh et al. used DNA extracted from an uncultured, undigested CV sample, and thus analysed DNA from a mixture of cells from the CTB and the MC. If mosaicism is detected in such

a mixture it is unknown which cell layer is affected and follow-up investigations in amniotic fluid are indicated to verify the foetal karyotype. This leads to unnecessary secondary invasive

procedures in all cases of CPM type I, causing stress and a delay of a prenatal diagnosis. As in all genetic fields, also in prenatal cytogenetic diagnosis, there is a shift from

conventional cytogenetic analyses towards more molecular analyses. We certainly encourage and embrace this _molecularization_, and notice that the introduction of such molecular techniques

goes along with a more pronounced collaboration with molecular laboratories in prenatal genetic diagnostics. But historically, the involvement of molecular laboratories in prenatal

diagnostics has mainly been restricted to the analysis of familial variants, for which dissociation of the cell-layers is less crucial. Therefore, these laboratories often use DNA isolated

from the total, undissociated CV for prenatal diagnosis. From routine cytogenetic studies focussing on microscopically visible chromosome aberrations, it is known that CPM affects 1-2% of

all CV samples [9]. Percentages including submicroscopic Copy Number Variations (CNVs) vary and increase up to 4% [10]. When analysing DNA from undissociated CV, the detection of mosaicism

should warrant confirmatory studies in amniotic fluid in order to differentiate between CPM and generalized mosaicism also affecting the foetus. We thus do agree with the statement of the

authors that “_From a broader level, this report emphasizes the benefit of amniocentesis following detection of placental mosaicism for chromosomal rearrangements in order to reasonably rule

out CPM and prevent unnecessary medical termination of pregnancy_”. However, we strongly feel there should be an additional message in the paper, being that the risk of detecting mosaicism

caused by CPM type 1 should be avoided as much as possible, since it causes stress and delays a final prenatal diagnosis. This can simply be achieved by dissociating the CTB and MC

cell-layers, as recommended in the European Guidelines for Constitutional Cytogenomic Analysis [8], and subsequently analysing DNA isolated from the MC only (or at least separately), instead

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not receive any financial assistance. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Clinical Genetics, Erasmus MC, University Medical Center, Rotterdam, the Netherlands Diane

Van Opstal * Department of Human Genetics, Radboud university medical center, Nijmegen, The Netherlands Brigitte H. W. Faas Authors * Diane Van Opstal View author publications You can also

search for this author inPubMed Google Scholar * Brigitte H. W. Faas View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS DVO and BF contributed

equally in concepting and writing the correspondence. CORRESPONDING AUTHOR Correspondence to Brigitte H. W. Faas. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

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