Rapid Aneuploidy Testing Research Articles

Confined placental mosaicism is a diagnostic pitfall in dystrophinopathies: a clinical report.

Single-gene copy number variants (CNVs) limited to placenta although rarely identified may have clinical implications. We describe a pregnant woman referred for chorionic villus sampling due to increased fetal nuchal translucency. Incident intragenic deletion of Duchenne muscular dystrophy (DMD) gene, affecting exons 56 and 57, was identified in a male fetus in ~23-30% of placental cells by chromosomal microarray and confirmed using multiplex ligation-dependent probe amplification (MLPA). Rapid aneuploidy testing showed normal results and the deletion was not detected in the mother. Subsequent analyses on amniotic cells yielded a normal DMD gene result, corroborating the confined placental nature of the mosaicism. Hence, this report emphasizes the importance of conducting amniocentesis following detection of mosaicism for single gene CNVs on chorionic villi, in order to preclude confined placental mosaicism (CPM). As far as we know, this report marks only the second documented situation of CPM involving an intragenic DMD deletion.

Post-mortem rapid aneuploidy testing for holoprosencephaly.

Abortion and fetal death are common in fetuses with holoprosencephaly, so genetic examinations often have to be made in a post-mortem setting. The efficiency of the conventional karyotyping using cultured fibroblasts in these situations is limited due to frequent culture failure. In the current study, archived cases of holoprosencephaly, where post-mortem genetic evaluation was requested and sufficient frozen material was available, were reevaluated using the quantitative fluorescence polymerase chain reaction (QF-PCR) technique. Testing for aneuploidies of chromosomes 13, 15, 16, 18, 21, 22, X, and Y with the QF-PCR technique was carried out on DNA isolated from archived frozen chorionic villi in seven cases of holoprosencephaly. QF-PCR was successful in all seven cases. Two cases of trisomy 13, two cases of triploidy, and one case of trisomy 18 was found meaning a 71% diagnostic yield. The success rate of QF-PCR (100%, 7/7) was superior compared to conventional karyotyping (43%, 3/7). Rapid aneuploidy testing using the QF-PCR technique is a simple, reliable, time- and cost-effective method sufficient to conclude the etiologic investigation in the majority of holoprosencephaly cases post-mortem.

Chromosome microarray analysis should be offered to all invasive prenatal diagnostic testing following a normal rapid aneuploidy test result.

Chromosomal microarray analysis (CMA) has now replaced karyotyping in the analysis of prenatal cases with a fetal structural anomaly, whereas in those pregnancies undergoing invasive prenatal diagnosis with a normal fetal ultrasound, conventional karyotyping is still performed. The aims of this study were to establish the diagnostic yield of CMA in prenatal diagnosis, and to provide new data that might contribute to reconsider current practices. We reviewed 2905 prenatal samples with a normal rapid aneuploidy detection test referred for evaluation by CMA testing. Our study revealed pathogenic and reported susceptibility copy number variants associated with syndromic disorders in 4.8% (n = 138/2905) of cases, being 2.8% (n = 81/2905) the estimated added diagnostic value of CMA over karyotyping. Clinically significant CMA abnormality was detected in 5.4% (107/1975) of the fetuses with ultrasound anomalies and in 1.4% (5/345) of those considered as low-risk pregnancies. Our series shows that in prenatal samples, CMA increases 2-fold the diagnostic yield achieved by conventional karyotyping.

The prevalence of genetic diagnoses in fetuses with severe congenital heart defects

PurposeCongenital heart defects (CHD) are associated with geneticsyndromes. Rapid aneuploidy testing and chromosome microarray analysis (CMA) arestandard care in fetal CHD. Many genetic syndromes remain undetected with thesetests. This cohort study aims to estimate the frequency of causal geneticvariants, in particular structural chromosome abnormalities and sequencevariants, in fetuses with severe CHD at mid-gestation, to aid prenatalcounselling.MethodsFetuses with severe CHD were extracted from the PRECOR registry(2012–2016). We evaluated pre- and postnatal genetic testing resultsretrospectively to estimate the frequency of genetic diagnoses in general, aswell as for specific CHDs.Results919 fetuses with severe CHD were identified. After exclusion of 211cases with aneuploidy, a genetic diagnosis was found in 15.7% (111/708). Thesecomprised copy number variants in 9.9% (70/708). In 4.5% (41/708) sequencevariants were found that would have remained undetected with CMA. Interruptedaortic arch, pulmonary atresia with ventricular septal defect andatrioventricular septal defect were most commonly associated with a geneticdiagnosis.ConclusionIn case of normal CMA results, parents should be offered exomesequencing sequentially, if time allows for it, especially if the CHD isaccompanied by other structural malformations due to the large variety ingenetic syndromes.

Chromosomal Microarray Analysis versus Karyotyping in Fetuses with Increased Nuchal Translucency.

We have carried out a retrospective study of chromosome anomalies associated with increased nuchal translucency (NT) in order to compare yield rates of karyotype, chromosome microarray analysis (CMA), and non-invasive prenatal testing (NIPT) in this condition. Presenting with increased NT or cystic hygroma ≥3.5 mm as an isolated sign, 249 fetuses underwent karyotype and/or CMA from 11 to 18 gestational weeks. Karyotype and fluorescence in situ hybridization (FISH) analyses detected 103 chromosomal anomalies including 95 aneuploidies and eight chromosomal rearrangements or derivatives. Further, seven pathogenic copy number variants (CNV), five likely pathogenic CNVs, and 15 variants of unknown significance (VOUS) were detected by CMA in fetuses with normal karyotype. Genetic testing is now facing new challenges due to results with uncertain clinical impacts. Additional investigations will be necessary to interpret these findings. More than 15% of the anomalies that we have diagnosed with invasive techniques could not be detected by NIPT. It is therefore definitely not recommended in the case of ultrasound anomalies. These results, while corroborating the use of CMA in fetuses with increased NT as a second tier after rapid aneuploidy testing, do not suggest a dismissal of karyotype analysis.

Diagnostic yield of chromosomal microarray analysis in fetuses with isolated increased nuchal translucency: a French multicenter study.

To determine the frequency and nature of copy number variants (CNVs) identified by chromosomal microarray analysis (CMA) in a large cohort of fetuses with isolated increased nuchal translucency thickness (NT)≥ 3.5 mm. This was a retrospective, multicenter study, including 11 French hospitals, of data from the period between April 2012 and December 2015. In total, 720 fetuses were analyzed by rapid aneuploidy test and the fetuses identified as euploid underwent CMA. CNVs detected were evaluated for clinical significance and classified into five groups: pathogenic CNVs; benign CNVs; CNVs predisposing to neurodevelopmental disorders; variants of uncertain significance (VOUS); and CNVs not related to the phenotype (i.e. incidental findings). In 121 (16.8%) fetuses, an aneuploidy involving chromosome 13, 18 or 21 was detected by rapid aneuploidy test and the remaining 599 fetuses were euploid. Among these, 53 (8.8%) had a CNV detected by CMA: 16/599 (2.7%) were considered to be pathogenic, including 11/599 (1.8%) that were cryptic (not visible by karyotyping); 7/599 (1.2%) were CNVs predisposing to neurodevelopmental disorders; and 8/599 (1.3%) were VOUS. Additionally, there was one (0.2%) CNV that was unrelated to the reason for referral diagnosis (i.e. an incidental finding) and the remaining 21 were benign CNVs, without clinical consequence. Interestingly, we identified five genomic imbalances of the 1q21.1 or 15q11.2 regions known to be associated with congenital heart defects. Our study demonstrates the benefit of CMA in the etiological diagnosis of fetuses with isolated increased NT. It is worth noting that most (69%) of the detected pathogenic CNVs were cryptic. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Trophoblast CD200 expression in successful human pregancies and missed abortions

Presence of the CD200 immune check-point inhibitor at the feto-maternal interface is linked to prevention of spontaneous abortion in mice and humans. In human missed abortions (MA), absence of Th17-driven inflammation has been attributed to expression of villus trophoblast CD200 quantified using immunohistochemistry. While rapid aneuploidy (QF-PCR) testing linked low CD200 to pregnancy failure, data showing normal VT CD200 in first trimester normal pregnancy and in abortion of chromosomally abnormal embryos has not been demonstrated. The present report shows normal CD200 in a 7 week gestation termination with normal male QF-PCR and in a 10 week male trisomy 18 MA.

Segmental Duplication QF-PCR: A Simple and Alternative Method of Rapid Aneuploidy Testing for Developing Country Like India.

Aneuploidy screening is becoming an integral part of routine prenatal screening in developing countries like India, and the need for more cheaper and rapid aneuploidy testing methods are required to relive the anxiety and financial burden among the high-risk couples. Segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR) emerged as an alternative aneuploidy diagnostic method. This study was conducted to optimize and access the utility of SD-QF-PCR in routine prenatal diagnosis to complement existing short tandem repeats (STR) based QF-PCR. About 50 control samples, 50 Down's syndrome samples, and one each trisomy 18 and Klinefelter samples were studied to optimize the assay. Later, 100 amniotic fluid samples were also studied. The assay was able to successfully identify normal and aneuploidy samples with 100% sensitivity and specificity. The results of amniotic fluid analysis by SD-QF-PCR were in agreement with results of STR-QF-PCR. Observed results qualify SD-QF-PCR as a preliminary aneuploidy diagnosis method.

2086260 The Prenatal Diagnosis of Microdeletion/Microduplication Syndromes in Increased Nuchal Translucency with Normal Karyotype: Unselected Korean Populations

Though the presence of aneuploidy is ruled out, nuchal thickening is clinically associated with many problems. Some reports have examined the value of microdeletion testing in increased NT fetuses, normal karyotype and no structural anomalies, but the results have showed limited value. The purpose of this study was to assess the value of testing for microdeletion/micoduplication syndromes with NT greater or equal to 95th percentile according to crown-rump length and normal karyotype. During April 2010 to December 2013, a total of 3051 samples had a routine karyotyping and MLPA analyses performed for several indications. Among them a total of 974 pregnancies were collected the inclusion criteria of increased NT. All samples were processes for G-banding karyotyping in additional to rapid aneuploidy testing and Multiplex Ligation dependent-Probe Amplification (MLPA). The MLPA analysis for detection of microdeletion or microduplication syndrome were performed using commercial MLPA kits (SALSA P245,MRC- Holland, Netherland). Application of the screening detected 26 cases of microdeletion/microduplication syndromes in 3051 samples (0.85% incidence). Among the 974 in increased NT samples with a normal karyotype, we identified 3 cases of microdeletion syndromes; 1 cases of DiGeorge syndrome (22q11 deletion), 1 cases of Wolf- Hirschhorn syndrome (4p 16.3 deletion), 1 Sotos syndrome and 9 cases of microduplication syndromes(3q,7q,10p,15q,17q,22q). The diagnostic yield was 1.23%(12/974) in the thick NT, 0.73%(4/545) in advanced maternal age and 0.35%(3/843) in the high risk result following maternal serum biomarker screening. This is the largest single reported study of microdeletion/microduplication syndromes in increased NT fetuses with normal karyotype. The prevalence of microdeletion/microduplication syndromes with increased NT is obviously higher than that expected in the general population. For this reason, the additional MLPA analyses for selected microdeletion/microduplication syndrome are valuable and useful in counselling parents of pregnancies with increased NT.

Experience Using a Rapid Assay for Aneuploidy and Microdeletion/Microduplication Detection in over 2,900 Prenatal Specimens

Background: While microarray testing can identify chromosomal abnormalities missed by karyotyping, its prenatal use is often avoided in low-risk pregnancies due to the possible identification of variants of uncertain significance (VOUS). Methods: We tested 2,970 prenatal samples of all referral indications using a rapid BACs-on-Beads-based assay with probes for sex chromosomes, common autosomal aneuploidies, and 20 microdeletion/microduplication syndromes, designed as an alternative to microarray in low-risk pregnancies and an alternative to rapid aneuploidy testing in pregnancies also undergoing microarray analysis. Results: Interpretable results were obtained in 2,940 cases (99.0%), with 89% receiving results in 1 day. Aneuploidies were detected in 7.3% and partial chromosome abnormalities in 0.45% (n = 13), including 5 referred for maternal age, abnormal maternal serum screen, or isolated ultrasound markers. The added detection above karyotype was 1 in 745 in lower-risk cases with normal ultrasounds or isolated ultrasound markers/increased nuchal measurements and 1 in 165 for fetuses with structural/growth abnormalities. Neither false negatives nor false positives were found within test limitations. Female polyploidy could not be detected, while polyploidies with Y chromosomes were suspected and confirmed through additional analysis. Conclusion: When combined with karyotyping, this assay provides increased interrogation of specific chromosomal regions, while limiting VOUS identification.

BACs-on-beads: a new robust and rapid detection method for prenatal diagnosis

Karyotyping, the gold standard used for diagnosis of chromosomal abnormalities, is being progressively replaced by rapid aneuploidy testing (RAT) techniques such as quantitative fluorescence-PCR, FISH and multiplex ligation-dependent probe amplification for diagnosing the common aneuploidies or chromosomal microarray analysis for comprehensive genome-wide testing. However, due to technical limitations, current RATs are confined to the detection of common aneuploidies 13, 18, 21 and sex chromosomes. To overcome the limitations of RATs, a bacterial artificial chromosomes-on-beads (BoBs™) assay technology has been introduced for the detection of the common aneuploidies as well as specific microdeletion syndromes. The BoBs assay is a bead-based multiplex assay using polystyrene beads impregnated with two spectrally distinct infrared fluorochromes to create a liquid array of up to 100 unique spectral signatures that supports the analysis of that scale of simultaneous hybridization assays on a minute DNA sample. This review gives an overview on the collective experiences of BoBs applications in prenatal diagnosis.

Best diagnostic approach for the genetic evaluation of fetuses after intrauterine death in first, second or third trimester: QF-PCR, karyotyping and/or genome wide SNP array analysis.

BackgroundThe aim of this study was to evaluate the best diagnostic approach for the genetic analysis of samples from first, second and third trimester intrauterine fetal deaths (IUFDs). We examined a total of 417 IUFD samples from fetuses with and without congenital anomalies. On 414 samples, karyotyping (N = 46) and/or rapid aneuploidy testing by QF-PCR (N = 371) was performed). One hundred sixty eight samples with a normal test result were subsequently tested by genome wide Single Nucleotide Polymorphism (SNP) array analysis. Three samples were only analyzed by array.ResultsIn 50 (12.0%) samples an aneuploidy was detected by QF-PCR and/or karyotyping, representing 47.1% of first, 13.2% of second and 3.4% of third trimester pregnancies. Karyotyping and QF-PCR failed in 4 (8.7%) and 7 (1.9%) samples, respectively, concerning mostly contaminated amniotic fluid samples from third trimester pregnancies.Clinically relevant aberrations were identified in 4.2% (all fetuses with malformations) of the 168 samples tested by SNP array. Inherited copy number variants (CNVs) were detected in 5.4% and 8.9% showed CNVs of unknown clinical relevance as parental inheritance could not be studied yet. In a sample from a fetus suspect for Meckel-Grüber syndrome, the genotype information from the SNP array revealed various stretches of homozygosity, including one stretch encompassing the CEP290 gene. Subsequent CEP290 mutation analysis revealed a homozygous, pathogenic mutation in this gene.ConclusionsBased on our experience we recommend QF-PCR as the first-line test in IUFD samples of first and second trimester pregnancies to exclude aneuploidy before performing array analysis. The chance to detect aneuploidy in third trimester pregnancies is relatively low and therefore array analysis can be performed as a first-tier test. A tissue sample, instead of amniotic fluid, is preferred because of a higher success rate in testing.We emphasize the need for analysis of parental samples whenever a rare, unique CNV is detected to allow for better interpretation of such findings and to improve future pregnancy management. Furthermore, we illustrate the strength of SNP arrays for genotype analysis, even though we realize it is crucial to have detailed phenotypic information to make optimal use of the genotype data in finding candidate recessive genes that may be related to the fetal phenotype.

Completely discrepant results between prenatal QF‐PCR rapid aneuploidy testing and cultured cell karyotyping obtained from CVS: lessons from UK audit and re‐audit of 22,221 cases

Completely discrepant results between prenatal QF‐PCR rapid aneuploidy testing and cultured cell karyotyping obtained from CVS: lessons from UK audit and re‐audit of 22,221 cases

Locus‐specific dual color‐probe for the enumeration of chromosome 18 in rapid FISH aneuploidy testing on uncultured amniocytes

Locus‐specific dual color‐probe for the enumeration of chromosome 18 in rapid FISH aneuploidy testing on uncultured amniocytes

Defining fetal medicine practice in England 2009

IntroductionSubspecialty training in fetal medicine is nationally regulated, but provision of service has been developed regionally, leading to variation in how and where services are provided. As a result there...

Chinese women's preferences for prenatal diagnostic procedure and their willingness to trade between procedures

To assess Chinese women's preference for the choice of a prenatal diagnosis test, karyotyping or rapid aneuploidy, and its relationship to maternal psychological state. Three hundred consenting women completed a self administered structured questionnaire which documented their psychological state and their preferred choice of diagnostic test for chromosomal abnormality using a discrete choice experiment design. Diagnostic tests were categorised according to three attributes: completeness of chromosomal information, procedure-to-result time interval and cost. Participants indicated a preference towards the karyotype test irrespective of cost and procedure-to-result time interval. The value of obtaining the extra information provided by karyotyping above that of rapid aneuploidy was pound267.82 (95% CI 226.71-323.31). Women would only accept the rapid aneuploidy test if results where available 18 days sooner than if they were undergoing a karyotyping test. Among women who traded between diagnostic tests, the value of extra chromosomal information was reduced to pound153.83 (95% CI 125.81-192.19). Women with high trait anxiety scores (>40) did not preferentially select one test. Chinese women showed a strong preference towards performing a full karyotype test and having a full chromosomal assessment performed for their pregnancy.

Rapid aneuploidy testing versus traditional karyotyping in amniocentesis for certain referral indications

Objective. (1) To determine the suitability of replacing full karyotype analysis with quantitative fluorescent polymerase chain reaction (QF-PCR) for prenatal diagnosis in amniotic fluid samples obtained by amniocentesis. (2) To evaluate an indication-based classification of cases at risk of missing clinically relevant chromosomal disorders by QF-PCR.Methods. We reviewed all fetal karyotypes obtained by amniocentesis between January 2004 and December 2008. We compared the cytogenetic findings obtained through conventional karyotype with those that would have been theoretically obtained using QF-PCR.Results. Of the 4007 karyotypes obtained, 110 abnormal karyotypes were found (2.8%). Out of these, 30 (27%) were chromosomal abnormalities (CA) which would not have been detected by PCR alone. These included 16 cases (53%) predicted to confer no increased risk, 9 (30%) predicted to have a low risk, and 5 (17%) with an uncertain or high risk of fetal abnormality. A policy of QF-PCR alone would have identified 80 of 85 (94%) clinically significant CA. When QF-PCR shows a normal result, the overall residual risk is 0.75% for any CA and 0.12% for a clinical significant CA.Conclusion. In our population, a policy of QF-PCR alone would miss 0.12% clinically relevant CA. QF-PCR directed to common aneuploidies can be considered as an economically and clinically acceptable prenatal diagnosis policy, offering full karyotype only for specific indications.

Rapid aneuploidy test for positive Down screening: who is willing to pay for it?

Rapid aneuploidy test for positive Down screening: who is willing to pay for it?

A Prospective Study on the Effect of Rapid Aneuploidy Testing (Amnio-PCR) on Anxiety Levels and Quality of Life Measures in Women and Their Partners with Positive Down Screening Result

Objectives: To examine the effect of rapid aneuploidy testing by amnio-PCR on anxiety levels and quality of life measures in women and their partners with positive Down screening result. Methods: In the original design, screen-positive women were to be randomized to have amnio-PCR or not. Of the first 60 women approached to join the study between April 2004 and April 2005, 4 declined amniocentesis, 14 agreed to be randomized, while the other 42 (75%) chose to pay for the amnio-PCR themselves (3 excluded: 2 because of Down syndrome and 1 dropout). The study was thus performed as a prospective observational study on the remaining 39 women of the last group. The longitudinal profile of the state-anxiety and quality of life domain scores for this cohort were studied using the Spielberger State-Trait Anxiety Inventory and WHO Quality of Life Measure – abbreviated version (Hong Kong) Questionnaire at 5 time points: (1) before amniocentesis, (2) 2 days after amniocentesis when amnio-PCR report was disclosed, (3) 3 weeks after amniocentesis when karyotyping report was disclosed, (4) 30–32 weeks’ gestation, (5) 6 weeks after delivery. Results: In the final cohort of 39 women and 27 partners, a significant reduction in their state-anxiety scores was found when they received the normal amnio-PCR report. On the other hand, there was no significant change in their quality of life domain scores. Conclusions: There is a demand from women and their partners who had a positive Down screening result for rapid aneuploidy testing (amnio-PCR) which can effectively alleviate their anxiety. A rapid aneuploidy test should be made available to women in a Down screening programme.

Complete discrepancy between abnormal fetal karyotypes predicted by QF‐PCR rapid testing and karyotyped cultured cells in a first‐trimester CVS

A chorion villus sample (CVS) biopsied at 11 weeks' gestation for raised nuchal translucency, revealed monosomy X (presumptive 45,X karyotype) by QF-PCR for rapid aneuploidy testing for chromosomes 13, 18, 21, X and Y. Long-term culture gave the karyotype: 47,XY,+ 21[66]/49,XYY,+ 21,+ 21 [22]. This discrepancy prompted redigestion of the combined residual villus fragments from the original QF-PCR assay. The repeat QF-PCR assay identified the presence of trisomy 21 and a Y chromosome consistent with a 47,XY,+ 21 karyotype. A double non-disjunction event early in embryogenesis in a 47,XY,+ 21 conceptus with subsequent cell lineage compartmentalisation of the three observed cell lines (45,X; 47,XY,+ 21 and 49,XYY,+ 21,+ 21) would account for these results. This is the first reported case to describe complete discrepancy at diagnosis between abnormal karyotypes detected by QF-PCR rapid aneuploidy testing and a cultured karyotype in the same CVS.