Multiplex ligation dependent probe amplification (MLPA) for rapid distinction between unique sequence positive and negative marker chromosomes in prenatal diagnosis

Background: Small supernumerary marker chromosomes (sSMC) are extra structurally abnormal chromosomes that cannot be unambiguously identified with conventional chromosome banding techniques. These marker chromosomes may cause an abnormal phenotype or be harmless depending on different factors such as genetic content, chromosomal origin and level of mosaicism. When a sSMC is found during prenatal diagnosis, the main question is whether the sSMC contains euchromatin since in most cases this will lead to phenotypic abnormalities. We present the use of Multiplex Ligation Dependent probe Amplification (MLPA) for rapid distinction between non-euchromatic and euchromatic sSMC. Results: 29 well-defined sSMC found during prenatal diagnosis were retrospectively investigated with MLPA with the SALSA MLPA centromere kits P181 and P182 as well as with the SALSA MLPA telomere kits P036B and P070 (MRC Holland BV, Amsterdam, The Netherlands). All unique-sequence positive sSMC were correctly identified with MLPA, whereas the unique-sequence negative sSMC had normal MLPA results. Conclusions: Although different techniques exist for identification of sSMC, we show that MLPA is a valuable adjunctive tool for rapidly distinguishing between unique-sequence positive and negative sSMC. In case of positive MLPA results, genetic microarray analysis or, if not available, targeted FISH can be applied for further identification and determination of the exact breakpoints, which is important for prediction of the fetal phenotype. In case of a negative MLPA result, which means that the sSMC most probably does not contain genes, the parents can already be reassured and parental karyotyping can be initiated to assess the heritability. In the mean time, FISH techniques are needed for determination of the chromosomal origin

Comparable low-level mosaicism in affected and non affected tissue of a complex CDH patient

In this paper we present the detailed clinical and cytogenetic analysis of a prenatally detected complex Congenital Diaphragmatic Hernia (CDH) patient with a mosaic unbalanced translocation (5;12). High-resolution whole genome SNP array confirmed a low-level mosaicism (20%) in uncultured cells, underlining the value of array technology for identification studies. Subsequently, targeted Fluorescence In-Situ Hybridization in postmortem collected tissues demonstrated a similar low-level mosaicism, independently of the affected status of the tissue. Thus, a higher incidence of the genetic aberration in affected organs as lung and diaphragm cannot explain the severe phenotype of this complex CDH patient. Comparison with other described chromosome 5p and 12p anomalies indicated that half of the features presented in our patient (including the diaphragm defect) could be attributed to both chromosomal areas. In contrast, a few features such as the palpebral downslant, the broad nasal bridge, the micrognathia, microcephaly, abnormal dermatoglyphics and IUGR better fitted the 5p associated syndromes only. This study underlines the fact that low-level mosaicism can be associated with severe birth defects including CDH. The contribution of mosaicism to human diseases and specifically to congenital anomalies and spontaneous abortions becomes more and more accepted, although its phenotypic consequences are poorly described phenomena leading to counseling issues. Therefore, thorough follow-up of mosaic aberrations such as presented here is indicated in order to provide genetic counselors a more evidence based prediction of fetal prognosis in the future

Abnormal non-invasive prenatal test results concordant with karyotype of cytotrophoblast but not reflecting abnormal fetal karyotype

We present a unique case in which non-invasive and invasive prenatal diagnoses showed abnormal, but discordant, results. A patient with abnormal non-invasive prenatal test (NIPT) results, indicating a 99% risk for monosomy X, was referred to our center for genetic counseling and confirmatory studies. Cytogenetic analysis of uncultured mesenchymal core of chorionic villi (CV) revealed a mosaic male karyotype consisting of two abnormal cell lines: one with monosomy X and the other with an isodicentric chromosome Y. Array analysis of the trophoblast confirmed the NIPT results. Based on the CV results, the patient opted for termination of pregnancy. After extensive counseling by a clinical geneticist about the possible outcomes and by a gynecologist about the risk of a second-trimester abortion procedure, the patient agreed to undergo early amniocentesis. Amniocentesis confirmed that the fetus had a male karyotype with an isodicentric chromosome Y, and the single nucleotide polymorphism (SNP) array profile suggested absence of the monosomy X cell line. The male infant was expected to be infertile. The patient finally decided to continue the pregnancy. Our case confirms that NIPT results are comparable with those of short-term cultured CV investigating the cytotrophoblast. Our patient was not aware that the NIPT results reveal the placental karyotype, which sometimes may be different from the fetal karyotype. Pretest counseling and providing the risk figures for false-positive and false-negative NIPT results are of great importance in order to discourage women from terminating pregnancies based on NIPT results alone. Copyright (C) 2014 ISUOG. Published by John Wiley & Sons Ltd