Performance of conventional cytogenetic analysis on chorionic villi when only one cell layer, cytotrophoblast or mesenchyme alone, is analyzed.

OBJECTIVE: To provide an estimation of the probability of error when chorionic villi (CV) cytogenetic analysis is limited to a single placental layer; either a direct preparation (Dir) or long-term culture (LTC). METHODS: We retrospectively reviewed cytogenetic studies on 81,593 consecutive CV samples in which both Dir and LTC were analyzed. All mosaic cases received amniocentesis. The false omission and false discovery rates were calculated by assessing the results that would have been reported when analysis was limited to either Dir or LTC. RESULTS: For all abnormalities combined, the proportion of normal Dir or LTC only reports that would have been inconsistent with a subsequent amniocentesis was 0.09% and 0.03%, respectively (false omissions). Among abnormal reports based on Dir or LTC alone, 8.01% and 3.17%, respectively, would be inconsistent with a subsequent amniocentesis result (false discoveries). Differences are present for individual abnormalities. CONCLUSIONS: From the perspective of identifying all abnormalities of potential clinical significance, the analysis of both placental layers is optimal. LTC alone is the preferred approach if only one layer of placenta is to be analyzed. Although rare, it is important to acknowledge that one cell layer analysis alone can cause misdiagnosis due to undetected mosaicism.

Implications of fetoplacental mosaicism on cell-free DNA testing for sex chromosome aneuploidies.

OBJECTIVE: The unique biological behavior of sex chromosomes has implications for cell-free DNA (cfDNA) testing. Our purpose is to predict the (1) false positive/negative rates of cfDNA testing consequent to fetoplacental mosaicism for any sex chromosome aneuploidies (SCA) and (2) positive predictive value (PPV) and negative predictive values of a high-risk and low-risk cfDNA result for any SCA. METHOD: This is a retrospective analysis of 67 030 chorionic villus sampling karyotypes, including fetoplacental mosaicism cases. RESULTS: Non-mosaic 45, X is associated with cystic hygroma/increased nuchal translucency and fetal anomalies. The false positive rate consequent to confined placental mosaicism is predicted to be 0.05%. The estimated false negative rate is in the range of 0% to 5.7% for all non-mosaic SCAs; it is 70% for mosaic 45, X with normal ultrasound. The predicted PPV on amniocytes is very high for most SCAs (94.4-99.4%). However, the stratified analysis shows that the PPV is much lower for 45, X without ultrasound anomalies compared with 45, X with abnormal scan (51% or 71%, vs 99%, respectively). CONCLUSION: Mosaicism is a major issue for SCA cfDNA testing, and prenatal confirmation, preferentially with amniocentesis if there are no ultrasound anomalies, remains important in counseling. As PPV varies on the basis of the presence of an ultrasound anomaly, skilled evaluation is critical. © 2017 John Wiley & Sons, Ltd.

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

OBJECTIVES: 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. METHODS: Over a period of 14 years, the laboratory analyzed both the cytotrophoblast and the mesenchyme of 60 347 CV samples. Cytogenetic results from CV samples showing mosaicism with follow-up amniocentesis were considered. The incidence of CPM and TFM and the risk of confirmation in the amniotic fluid (AF) were calculated. Uniparental disomy (UPD) was tested on ~300 cases at risk due to involvement of an imprinted chromosome. RESULTS: Overall, 1317 mosaic CV cases (2.18%) were detected, of which 1001 were subsequently investigated by amniocentesis. The overall risk of TFM was 13% and UPD incidence was 2.1%. CONCLUSIONS: The very large presented sample set and consistency in cytogenetic methodology, especially the analysis of both placental layers performed on all CV samples will enable genetic counselors to determine the risk of fetal involvement and the clinical relevance of an identified mosaic condition.

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

OBJECTIVES: 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. METHODS: Analyses of the frequencies of the different types of mosaicism involving cytotrophoblasts, for trisomies 21 (T21), 18 (T18), 13 (T13) and monosomy X (MX) among 52,673 CVS karyotypes obtained from cytotrophoblast, mesenchyme and confirmatory amniocentesis. RESULTS: After a high-risk cfDNA result for T21, 18, 13 and MX, the likelihood of finding CVS mosaicism and need for amniocentesis is, respectively, 2%, 4%, 22% and 59%. When mosaicism is detected by CVS, the likelihood of fetal confirmation by amniocentesis is, respectively, 44%, 14%, 4% and 26%. CONCLUSIONS: In cases of high-risk cfDNA results for T21/T18, CVS (combining cytotrophoblast and mesenchyme analysis) can be considered, but with the caveat of 2-4% risk of an inconclusive result requiring further testing. In high-risk results for MX/T13, amniocentesis would appear to be the most appropriate follow-up diagnostic test, especially in the absence of sonographic findings.

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.

De novo small supernumerary marker chromosomes detected on 143,000 consecutive prenatal diagnoses: chromosomal distribution, frequencies, and characterization combining molecular cytogenetics approaches.

OBJECTIVE: The risk of clinical consequences in prenatal cases with de novo small supernumerary marker chromosomes (sSMC), often in mosaic conditions, is not easy to predict, which results in difficulties in genetic counseling. METHOD: In this study, we evaluated the frequency, the chromosomal origin, and the clinical indication of 104 de novo sSMC detected in a monocenter survey on the basis of 143,000 consecutive prenatal diagnoses, and we assessed the reliability of molecular cytogenetics technologies for sSMC characterization. RESULTS: We detected a de novo sSMC frequency of 0.072%. Its incidence in advanced maternal age group is statistically different from that found in maternal anxiety indication (<35 years old). A higher prevalence of mosaicism in chorionic villi sampling (CVS) than in amniotic fluids was also revealed related to confined placental mosaicisms. The risk of confirmation in amniotic fluids of mosaics previously revealed at CVS was 33.3%. No uniparental disomy conditions were found when imprinted chromosomes were involved in the occurrence of de novo sSMC. The majority of de novo sSMC were acrocentric derived-chromosomes, and a neocentromere formation was observed in one pregnancy. CONCLUSION: Our data support that array comparative genomic hybridization has improved sSMC characterization and demonstrate its utility in supporting genetic counseling. We propose a workflow for de novo sSMC characterization.

Recombinant chromosome 4 from a familial pericentric inversion: prenatal and adulthood wolf-hirschhorn phenotypes.

Pericentric inversion of chromosome 4 can give rise to recombinant chromosomes by duplication or deletion of 4p. We report on a familial case of Wolf-Hirschhorn Syndrome characterized by GTG-banding karyotypes, FISH, and array CGH analysis, caused by a recombinant chromosome 4 with terminal 4p16.3 deletion and terminal 4q35.2 duplication. This is an aneusomy due to a recombination which occurred during the meiosis of heterozygote carrier of cryptic pericentric inversion. We also describe the adulthood and prenatal phenotypes associated with the recombinant chromosome 4.

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.

OBJECTIVES: Karyotyping on chorionic villous samples (CVS) includes the analysis of both cytotrophoblast (STC) and mesenchyme (LTC). This approach requires complex laboratory organization and trained technicians. The introduction of quantitative fluorescent polymerase chain reaction (QF-PCR) instead of conventional karyotyping in low-risk pregnancies opened its application in CVS analysis. Discordant QF-PCR and CVS cytogenetic results were reported, and strategies for CVS analysis were introduced to minimize this risk. The possibility to substitute the STC with QF-PCR was reported. The aim of this study is to evaluate benefits and limitations of the approach QF-PCR + LTC compared with the traditional method STC + LTC and to quantify the associated risks of false results. METHOD: This study is based on a retrospective cytogenetic audit of CVS results (n = 44 727) generated by the STC + LTC analytic approach. False-negative risks related to true fetal mosaicism type IV, imprinting syndromes and maternal contamination in LTC were calculated. RESULTS: Compared with STC + LTC, QF-PCR + LTC approach is associated with a cumulative false-negative risk of ~1/3100-1/4400. Costs and reporting time of STC in a high-throughput cytogenetic lab are similar to a CE-IVD marked QF-PCR analysis. CONCLUSIONS: These results should be clearly highlighted in the pre-test counseling and extensively discussed with the couple prior to testing for informed consent.

Prenatal BACs-on-Beads™: the prospective experience of five prenatal diagnosis laboratories.

OBJECTIVE: We previously reported on the validation of Prenatal BACs-on-Beads™ on retrospectively selected and prospective prenatal samples. This bead-based multiplex assay detects chromosome 13, 18, 21 and X/Y aneuploidies and the nine most frequent microdeletion syndromes. We demonstrated that Prenatal BACs-on-Beads(TM) is a new-generation, prenatal screening tool. Here, we describe the experience of five European prenatal diagnosis laboratories concerning the ongoing use of Prenatal BACs-on-Beads™ . METHODS: Some 1653 samples were analyzed. All results were confirmed by conventional karyotyping or another appropriate technique. All indications for invasive prenatal diagnosis were included. Amniotic fluid and chorionic villus samples were analyzed in equivalent proportions. RESULTS: The failure rate was 3.3% and the overall abnormality detection rate was ~1/10. Eighty-five percent of the detected abnormalities were common aneuploidies. Eleven microdeletions and duplications were identified, thus giving an overall yield for microdeletion and microduplication detection of 1/145. Compared with QF-PCR, Prenatal BACs-on-Beads™ provides an additional detection rate of ~1/250 for low-risk pregnancies. The false positive and negative rates were both <1%. CONCLUSION: When associated with conventional karyotyping, the Prenatal BACs-on-Beads™ assay combines a short turnaround time (typical of rapid aneuploidy detection tests) with valuable detection of the most frequent microdeletion syndromes that cannot be detected in cytogenetic analyses.

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 (>or=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 >or=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.

Pure monosomy and pure trisomy of 13q21.2-31.1 consequent to a familial insertional translocation: exclusion of PCDH9 as the responsible gene for autosomal dominant auditory neuropathy (AUNA1).

Insertional translocations (IT) are rare structural rearrangements. Offspring of IT balanced carriers are at high risk to have either pure partial trisomy or monosomy for the inserted segment as manifested by "pure" phenotypes. We describe an IT between chromosomes 3 and 13 segregating in a three-generation pedigree. Short tandem repeat (STR) segregation analysis and array-comparative genomic hybridization were used to define the IT as a 25.1 Mb segment spanning 13q21.2-q31.1. The phenotype of pure monosomy included deafness, duodenal stenosis, developmental and growth delay, vertebral anomalies, and facial dysmorphisms; the trisomy was manifested by only minor dysmorphisms. As the AUNA1 deafness locus on 13q14-21 overlaps the IT in the PCDH9 (protocadherin-9) gene region, PCDH9 was investigated as a candidate gene for deafness in both families. Genotyping of STRs and single nucleotide polymorphisms defined the AUNA1 breakpoint as 35 kb 5' to PCDH9, with a 2.4 Mb area of overlap with the IT. DNA sequencing of coding regions in the AUNA1 family and in the retained homologue chromosome in the monosomic patient revealed no mutations. We conclude that AUNA1 deafness does not share a common etiology with deafness associated with monosomy 13q21.2-q31.3; deafness may result from monosomy of PCHD9 or another gene in the IT, as has been demonstrated in contiguous gene deletion syndromes. Precise characterization of the breakpoints of the translocated region is useful to identify which genes may be contributing to the phenotype, either through haploinsufficiency or extra dosage effects, in order to define genotype-phenotype correlations.

Prenatal detection by subtelomeric FISH and MLPA of unbalanced meiotic recombinants resulting from parental pericentric inversions.

OBJECTIVE: Pericentric inversion carriers are predisposed to produce unbalanced gametes that result in conceptuses having either a partial trisomy for one distal segment and a partial monosomy for the other or vice versa. Larger inversions result in smaller unbalanced distal segments and a higher likelihood of a viable fetus. In these cases the structure of the recombinant chromosome is similar to the original balanced inverted or normal ones despite the (unbalanced) genetic content. Such cases may not be detected prenatally by conventional cytogenetic analysis. METHODS: In all prenatal samples from the pericentric inversion carriers we applied subtelomeric FISH probes specific for the chromosome involved in order to detect parental meiotic recombinants resulting from a single cross-over event. Confirmatory MLPA was also applied in unbalanced fetuses. RESULTS: The occurrence of a duplication deficiency unbalance from pericentric inversion carriers was successfully detected in all three fetuses by FISH. MLPA assays applied in two cases confirmed these results. CONCLUSIONS: The application of commercial FISH subtelomeric probes is a reliable method that could be routinely applied for the detection of single cross-over meiotic recombinants. MLPA is a sound alternative technique.

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%.

Prenatal search for UPD 14 and UPD 15 in 83 cases of familial and de novo heterologous Robertsonian translocations.

OBJECTIVES: The presence in the conceptus of a Robertsonian translocation predisposes to UPD formation, mainly by post-zygotic events of chromosome abnormality rescue. This is due to the increased risk of generating aneuploid zygotes because the rearranged chromosome and the respective homologues are prone to non-disjunction errors. Given this, carriers and karyotypically normal individuals conceived from a parent with a Robertsonian translocation are at risk for UPD. Abnormal phenotypes due to an imprinting effect have been found to be associated with UPD 14 and 15. The aim of the study was to refine, at the time of prenatal diagnosis, the risk for UPD 14 and 15 in a population with Robertsonian translocations involving these chromosomes. METHODS: Sixty-five cases of familial and de novo heterologous Robertsonian translocations involving chromosomes 14 and 15 and 18 fetuses with a normal karyotype, but conceived by a Robertsonian translocation carrier were prenatally studied to investigate the presence of UPD for chromosomes 14 and 15. RESULTS: Of the 65 Robertsonian translocation carriers, one fetus with a de novo der(14;21) showed maternal UPD 14. None of the 18 fetuses with a normal karyotype had UPD. CONCLUSION: Our data, combined with other previous prenatal investigations provide a general risk estimate for UPD 14 and 15 of 0.6%. Nevertheless, combining our data and those previously reported, all three fetuses with UPD had a de novo Robertsonian translocation, thus suggesting a risk of UPD formation of about 3% for this specific group of translocation carriers.