Federico Maggi

Fetoplacental mosaicism: potential implications for false-positive and false-negative noninvasive prenatal screening results

Purpose:Noninvasive prenatal screening for fetal aneuploidy analyzes cell-free fetal DNA circulating in the maternal plasma. Because cell-free fetal DNA is mainly of placental trophoblast origin, false-positive and false-negative findings may result from placental mosaicism. The aim of this study was to calculate the potential contribution of placental mosaicism in discordant results of noninvasive prenatal screening.Methods:We performed a retrospective audit of 52,673 chorionic villus samples in which cytogenetic analysis of the cytotrophoblast (direct) and villus mesenchyme (culture) was performed, which was followed by confirmatory amniocentesis in chorionic villi mosaic cases. Using cases in which cytogenetic discordance between cytotrophoblast and amniotic fluid samples was identified, we calculated the potential contribution of cell line–specific mosaicism to false-positive and false-negative results of noninvasive prenatal screening.Results:The false-positive rate, secondary to the presence of abnormal cell line with common trisomies in cytotrophoblast and normal amniotic fluid, ranged from 1/1,065 to 1/3,931 at 10% and 100% mosaicism, respectively; the false-negative rate was calculated from cases of true fetal mosaicism, in which a mosaic cell line was absent in cytotrophoblast and present in the fetus; this occurred in 1/107 cases.Conclusion:Despite exciting advances, underlying biologic mechanisms will never allow 100% sensitivity or specificity.Genet Med 16 8, 620–624.Genetics in Medicine (2014); 16 8, 620–624. doi:10.1038/gim.2014.3

Prevalence of recurrent pathogenic microdeletions and microduplications in over 9500 pregnancies.

The implementation of chromosomal microarray analysis (CMA) in prenatal testing for all patients has not achieved a consensus. Technical alternatives such as Prenatal BACs‐on‐BeadsTM (PNBoBsTM) have thus been applied. The aim of this study was to provide the frequencies of the submicroscopic defects detectable by PNBoBsTM under different prenatal indications.

Confirmation of mosaicism and uniparental disomy in amniocytes, after detection of mosaic chromosome abnormalities in chorionic villi

Chromosome mosaicism is detected in about 1–2% of chorionic villi samples (CVS), and may be due to a postzygotic nondisjunction event generating a trisomic cell line in an initially normal conceptus (mitotic origin) or the postzygotic loss of one chromosome in an initially trisomic conceptus (meiotic origin and trisomy rescue). Depending on the distribution of the abnormal cell line, the mosaic can be confined to the placenta (CPM) or generalised to the fetus (TFM, true fetal mosaicism). Trisomy rescue could theoretically be associated with a 33.3% probability of uniparental disomy (UPD) in the fetus. The aim of this study was to determine the risk of fetal involvement in a cohort of numerical and structural chromosome mosaics revealed in chorionic villi by means of combined direct and long-term culture analyses; we also determined the incidence of UPD associated with mosaic aneuploidies and supernumerary markers involving imprinted chromosomes. A total of 273 of a consecutive series of 15 109 CVS evaluated during a period of 5 years showed a mosaic condition in direct preparations and/or long-term cultures; confirmatory amniocentesis was performed in 203 cases. The abnormal cell line was extended to the fetus in 12.8% cases in terms of structural and numerical abnormalities involving autosomes and sex chromosomes; the risk of TFM varied and depended on the placental tissue distribution of the abnormal cell line. One of the 51 cases in which the mosaic involved an imprinted chromosome showed UPD, thus indicating a risk of 1.96%.

Interpreting mosaicism in chorionic villi: results of a monocentric series of 1001 mosaics in chorionic villi with follow‐up amniocentesis

Chromosomal mosaicism in chorionic villi (CV) is detected in ~1–2% of cases. When a mosaic in CV is detected during prenatal diagnosis, a confirmatory karyotype should be performed on amniocytes to discriminate between a mosaic confined to the placenta [confined placental mosaicism (CPM)] and one generalized to the fetus [true fetal mosaicism (TFM)]. We determined the likelihood that any mosaic abnormalities identified through CV samples are confirmed in the fetus.

Chromosome Abnormalities Investigated by Non-Invasive Prenatal Testing Account for Approximately 50% of Fetal Unbalances Associated With Relevant Clinical Phenotypes

During the past 20 years non‐invasive screening tests have been increasingly utilized in prenatal diagnosis (PD) practice. Considerable effort has been exerted by multicenter consortia to evaluate the reliability of non‐invasive screening tests in detecting those women with an increased risk of having a pregnancy affected by trisomies 21, 18, and 13, monosomy X, and triploidies. To what extent this group of abnormal karyotypes accounts for the total number of phenotypically relevant fetal chromosome abnormalities has, however, never been investigated. The present report is an attempt aimed to quantify this proportion. A retrospective analysis of a homogeneous survey of 115,128 consecutive invasive prenatal tests was undertaken. All cases were classified in accordance with the indication given for the invasive testing. Cytogenetic results regarding 96,416 karyotype analyses performed because of advanced maternal age (≥35 years) or gestational anxiety (<35 years) were considered since these are the patients who usually undergo non‐invasive screening tests. We calculated the number of cases (T21, T18, T13, 45,X, and triploidy) that would have been detected by prenatal screening on the basis of the published detection rate of the combined‐2 test or the quadruple test. Our findings indicate that the chromosomal abnormalities investigated by screening tests represent <50% of the fetal chromosomal abnormalities associated with an abnormal outcome ranging from intermediate‐to‐severe in women <35 years (45.8% and 39.6% in the first and second trimesters, respectively), and sensitivity >50% in women ≥35 years (65.1% and 61.8%, respectively). To conclude, approximately 50% of the phenotypically relevant abnormal karyotypes cannot be detected by non‐invasive prenatal screening tests.

Prenatal BACs-on-Beads™: a new technology for rapid detection of aneuploidies and microdeletions in prenatal diagnosis†

Molecular cytogenetic techniques on uncultured prenatal samples are the sole tests applied in some countries in cases with advanced maternal age (AMA) or increased risk after prenatal screening. Moreover, there is a trend to perform invasive prenatal diagnosis (PD) during the first trimester before ultrasound manifestations, so new rapid and reliable assays are necessary to investigate microdeletions not detectable with the conventional karyotype. We report the validation study of the prenatal bacterial artificial chromosomes‐on‐Beads™ (BoBs™; CE‐IVD), a bead‐based multiplex assay detecting chromosomes 13, 18, 21, X/Y aneuploidies and nine microdeletion regions having an overall detection rate of 1/1700.

The type of feto‐placental aneuploidy detected by cfDNA testing may influence the choice of confirmatory diagnostic procedure

Cell‐free DNA (cfDNA) screening can provide false positive/negative results because the fetal fraction originates primarily from trophoblast. Consequently, invasive diagnostic testing is recommended to confirm a high‐risk result. Currently, there is debate about the most appropriate invasive method. We sought to estimate the frequency in which a chorionic villus sampling (CVS) performed after a high‐risk cfDNA result would require a follow‐up amniocentesis due to placental mosaicism.

Chromosome 11 segmental paternal isodisomy in amniocytes from two fetuses with omphalocoele: new highlights on phenotype–genotype correlations in Beckwith–Wiedemann syndrome

Background: The phenotypic variability in Beckwith–Wiedemann syndrome (BWS) reflects the genetic heterogeneity of the mechanism which by default leads to the deregulation of genes located at 11p15.5. Genotype–phenotype correlation studies have demonstrated an association between omphalocoele and CDKN1C/p57 mutations or hypermethylation. Paternal uniparental disomy 11 (pUPD11) has been described only in the mosaic condition with both uniparental and biparental cell lines, and no association with omphalocoele has been pointed out. Methods: Two cases are presented here, in which a paternal segmental UPD11 was detected by molecular investigation of amniotic fluid cell cultures after the presence of apparently isolated omphalocoele was revealed in the fetuses by ultrasound scan. Further studies were performed on additional autoptic feto-placental tissues to characterise the distribution of the uniparental cell line and to unmask any biparental lineage in order to document in more detail the as yet unreported association between omphalocoele and pUPD11. Results: Results on the UPD distribution profile showed that the abdominal organs have a predominant uniparental constitution. This condition could mimic the effect of CDKN1C/p57 inactivation, causing the omphalocoele. Conclusion: New genotype–phenotype correlations emerge from the investigated cases, suggesting that molecular analysis be extended to all cases with fetal omphalocoele in order to establish the incidence of pUPD11 in complete BWS and in monosymptomatic/mild forms.

Response to “QF-PCR as a substitute for karyotyping of cytotrophoblast for the analysis of chorionic villi: advantages and limitations from a cytogenetic retrospective audit of 44 727 first-trimester prenatal diagnoses”

We read with interest the comments on our paper from Dr Toutain and colleagues. Their letter focuses on their diagnostic strategy for CVS analysis. We know very well that a few laboratories have dismissed direct preparation and substituted it with alternative approaches, including QF-PCR, MLPA or interphase FISH analyses. The choice of the strategy depends on the history and expertise of each laboratory. We are still convinced that all diagnostic strategies on CVS should be targeted to obtain the highest detection rate of cytogenetic abnormalities. For this reason, we try to combine STC and LTC in all cases, and, whenever a mosaic condition is found, a follow-up amniocentesis is always recommended. We believe that our approach provides the maximum of the information for genetic counseling, and in addition, in our experience, we have never had feedback on discrepancies between the chorionic villi cytogenetic results and the true fetal karyotype. The aim of our paper is not to suggest our diagnostic strategy as model for other centers but to estimate the residual risk of chromosome abnormalities and epigenetic defects associated with imprinting syndromes if a genome wide approach by STC is substituted by a targeted assay. We would like to clarify that we do not believe that the 11 TFM type IV female cases are caused by massive MCC for different reasons: Multiple CV cultures were harvested and analyzed (>15mg). Chromosome heteromorphisms detected in STC were always compared with those of LTC in female fetus and no discrepancies were detected. Discrepancies among STC and LTC and the corresponding AF karyotypes were not detected. The incidence of MCC in the overall LTC in the present experience is 1.8%, in line with the literature and below the requested level by SIGUCERT (≤2%).

Application of a new molecular technique for the genetic evaluation of products of conception

Karyotyping is a well‐established method of investigating the genetic content of product of conceptions (POCs). Because of the high rate of culture failure and maternal cell contamination, failed results or 46,XX findings are often obtained. Different molecular approaches that are not culture dependent have been proposed to circumvent these limits. On the basis of the robust experience previously obtained with bacterial artificial chromosomes (BACs)‐on‐Beads™ (BoBs™), we evaluated the same technology that we had used for the analysis of prenatal samples on POCs.