Vincenzo Cirigliano

Prenatal detection of unbalanced chromosomal rearrangements by array CGH

Background: Karyotype analysis has been the standard method for prenatal cytogenetic diagnosis since the 1970s. Although highly reliable, the major limitation remains the requirement for cell culture, resulting in a delay of as much as 14 days to obtaining test results. Fluorescent in situ hybridisation (FISH) and quantitative fluorescent PCR (QF-PCR) rapidly detect common chromosomal abnormalities but do not provide a genome wide screen for unexpected imbalances. Array comparative genomic hybridisation (CGH) has the potential to combine the speed of DNA analysis with a large capacity to scan for genomic abnormalities. We have developed a genomic microarray of approximately 600 large insert clones designed to detect aneuploidy, known microdeletion syndromes, and large unbalanced chromosomal rearrangements. Methods: This array was tested alongside an array with an approximate resolution of 1 Mb in a blind study of 30 cultured prenatal and postnatal samples with microscopically confirmed unbalanced rearrangements. Results: At 1 Mb resolution, 22/30 rearrangements were identified, whereas 29/30 aberrations were detected using the custom designed array, owing to the inclusion of specifically chosen clones to give increased resolution at genomic loci clinically implicated in known microdeletion syndromes. Both arrays failed to identify a triploid karyotype. Thirty normal control samples produced no false positive results. Conclusions: Analysis of 30 uncultured prenatal samples showed that array CGH is capable of detecting aneuploidy in DNA isolated from as little as 1 ml of uncultured amniotic fluid; 29/30 samples were correctly diagnosed, the exception being another case of triploidy. These studies demonstrate the potential for array CGH to replace conventional cytogenetics in the great majority of prenatal diagnosis cases.

Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells

We have refined polymerase chain reaction (PCR) assays for the detection of sickle cell anaemia, the delta F 508 deletion causing cystic fibrosis, and the IVS1–110 mutation leading to beta‐thalassaemia, allowing them to be successfully performed upon single cells using fluorescent primers. We have also assessed the possibility of detecting aneuploidies of chromosomes 13, 18 and 21 using a quantitative fluorescent polymerase chain reaction (QF‐PCR) with primers flanking polymorphic short tandem repeat (STR) markers. Trisomies were readily diagnosed by the detection of tri‐allelic patterns. However some heterozygote normal and trisomic diallelic patterns did not produce the expected ratios of amplified PCR products due to preferential DNA sequence amplification. Total allelic drop out (ADO) did not occur with any of the cells tested. Multiplex QF‐PCR assays can be performed on a single cell in under 6 h and simultaneously provide diagnosis of single gene defects, sex determination and an indication of selected chromosome aneuploidy.

Rapid prenatal diagnosis of common chromosome aneuploidies by QF‐PCR, results of 9 years of clinical experience

Despite being deliberately targeted to common chromosome aneuploidies, the rapid quantitative fluorescent polymerase chain reaction (QF‐PCR) tests can detect the majority of chromosome abnormalities in prenatal diagnosis. The main advantages of this assay are low cost, speed and automation allowing large‐scale application.

Rapid detection of chromosomes X and Y aneuploidies by quantitative fluorescent PCR

Quantitative fluorescent polymerase chain reaction (QF‐PCR) assays and small tandem repeat (STR) markers have been successfully employed for the rapid detection of major numerical aneuploidies affecting human autosomes. So far, the analysis of chromosomes X and Y disorders has been hampered by the rarity of highly polymorphic markers which could distinguish normal female homozygous PCR patterns from those seen in patients with Turners syndrome. A new marker (X22) of the X/Y chromosomes has been identified which maps in the Xq/Yq pseudoautosomal region PAR2; used together with the HPRT it allows the rapid diagnosis of numerical aneuploidies of the sex chromosomes. Blood samples from normal male and female subjects and from patients with X and Y chromosome disorders (45,X and 47,XXY) have been tested by QF‐PCR with the X22 polymorphic pentanucleotide (12 alleles) together with the HPRT and P39 markers. The samples were also tested by multiplex QF‐PCR with STRs specific for chromosomes 21,18,13 and amelogenin (AMXY). Tested by QF‐PCR, all samples from normal females were heterozygous for either the X22 or the HPRT marker with fluorescent peak ratios near 1:1, thus allowing a correct, rapid diagnosis of their chromosome complement. Turners patients (45,X) showed only one X22 and one HPRT fluorescent peak, thus documenting the presence of a single X chromosome. Turners patients with mosaicism showed a major fluorescent peak for the X22 and HPRT markers and a minor peak revealing the presence of a second minor population of cells. Two 47,XXY cases could also be diagnosed. Multiplex analyses can be performed using simultaneously STR markers for chromosomes 21,18,13 X and Y. The diagnostic value of a third X‐linked marker (P39) was also investigated. These results suggest that rapid diagnosis of major numerical anomalies of the X and Y chromosomes can be performed using QF‐PCR with a new highly polymorphic X‐linked marker, X22, which maps in the Xq/Yq pseudoautosomal region PAR 2. Multiplex QF‐PCR tests—using the X22 STR in association with HPRT and, in rare cases, a third P39 marker—allow the rapid diagnosis of major aneuploidies affecting chromosomes 21, 18, 13, X and Y. The X22 marker can also be employed for the detection of fetal cells present in maternal peripheral blood or the endocervical canal. Copyright

Performance of screening for aneuploidies by cell‐free DNA analysis of maternal blood in twin pregnancies

To report clinical implementation of cell‐free DNA (cfDNA) analysis of maternal blood in screening for trisomies 21, 18 and 13 in twin pregnancies and examine variables that could influence the failure rate of the test.

Summary of the ISPD Preconference Day, June 3, 2012, Miami Beach

Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands Department of Obstetrics and Gynecology, Washington University School of Medicine, St Louis, MO, USA Molecular Genetics, Labco Quality Diagnostics, c/Londres 28, 08029 Barcelona and Biologia Cellular, Fisiologia y Immunologia, Universitat Autonoma de Barcelona, Barcelona, Spain Laboratory for Infectious Diseases and Screening, National Institute for Public Health and the Environment, Bilthoven, The Netherlands Children’s Law Group and DePaul University College of Law, Chicago, IL, USA University of North Carolina, North Carolina, USA Fetal Medicine Section, Leiden University Medical Center, Leiden, The Netherlands Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA *Correspondence to: Brigitte H. W. Faas. E-mail: b.faas@gen.umcn.nl Presented at the 16th International Conference on Prenatal Diagnosis and Therapy, Miami, Florida, June 3–6, 2012. Overall organizer preconference day. Current position: Verinata Health Inc., Redwood Cyto, CA, USA.

Rapid prenatal diagnosis by QF-PCR: evaluation of 30,000 consecutive clinical samples and future applications.

Abstract:  Rapid prenatal diagnoses of major chromosome abnormalities can be performed on a large scale using highly polymorphic short tandem repeats (STRs) amplified by the quantitative fluorescent polymerase chain reaction (QF‐PCR). The assay was introduced as a preliminary investigation to remove the anxiety of the parents waiting for the results by conventional cytogenetic analysis using amniotic fluid or chorionic cells. However, recent studies, on the basis of the analyses of several thousand samples, have shown that this rapid approach has a very high rate of success and could reduce the need for cytogenetic investigations. Its high efficiency, for example, allows early interruption of affected fetuses without the need of waiting for completion of fetal karyotype. The main advantages of the QF‐PCR are its accuracy, speed, automation, and low cost that allows very large number of samples to be analyzed by few operators. Here, we report the results of using QF‐PCR in a large series of consecutive clinical cases and discuss the possibility that, in a near future, it may even replace conventional cytogenetic analyses on selected samples.

Assessment of new markers for the rapid detection of aneuploidies by quantitative fluorescent PCR (QF–PCR)

Rapid prenatal diagnoses of major chromosome aneuploidies have been achieved successfully using quantitative fluoresent PCR (QF–PCR) assays and small tandem repeat (STR) markers. Here we report the results of evaluating the use of previously untested X‐linked STRs, (DXS6803) and (DXS6809), together with modified amelogenin (AMXY) sequences and the X22 marker that maps in the pseudoautosomal region PAR2 on the long arm of the X and Y chromosomes. These markers will allow prenatal diagnoses of sex chromosome aneuploidies such as 45,X (pure Turner Syndrome), 47,XXY and 47,XYY, while assessing the sex of the fetuses. Data are also presented concerning the difficulties associated with the evaluation of the frequencies of the various types of sub‐populations of cells in amniotic fluid samples collected from fetuses with sex chromosome mosaicism. The results of evaluating the use of new markers for the rapid diagnosis of aneuploidies affecting chromosomes 21,18 and 13 are also presented. Three chromosome 21 specific STRs have been found to produce trisomic triallelic or diallelic patterns from all amniotic samples retrieved from fetuses with Down Syndrome. Since all samples tested were amplified and no false positive or negative results were observed, the present results confirm the diagnostic value of QF–PCR for the prenatal detection of major numerical chromosome disorders.

Rapid methods for targeted prenatal diagnosis of common chromosome aneuploidies

Improvements in non-invasive screening methods for trisomy 21 (Down syndrome) and other aneuploidies during the first and second trimester of pregnancy have radically changed the indications for prenatal diagnosis over the last decade. Consequently, there was a need for rapid tests for the detection of common chromosome aneuploidies resulting in the development of molecular methods for the rapid, targeted detection of (an)euploidies of the chromosomes 13, 18, 21 and the sex chromosomes. The analysis of large series of prenatal samples has shown that such tests can detect the great majority of chromosome abnormalities in prenatal diagnosis. This resulted in lively discussions on whether conventional karyotyping should remain the standard method for the majority of prenatal cases or can be replaced by rapid tests only. This review gives an overview of different aspects of the three most common tests for rapid, targeted prenatal detection of (an)euploidies, i.e. interphase fluorescence in-situ hybridisation (iFISH), quantitative fluorescent polymerase chain reaction (QF-PCR) and multiplex ligation-dependent probe amplification (MLPA).

Counseling for non-invasive prenatal testing (NIPT): what pregnant women may want to know

Sequencing of cell-free fetal and maternal DNA fragments (cfDNA) in maternal plasma can be used to test for fetal chromosomal abnormalities. In particular, prediction of the presence or absence of fetal trisomy 21, the most common fetal chromosomal abnormality, has been proved to be highly accurate. The first studies, showing > 99% accuracy, were done in selected high-risk groups1,2. More recent studies in average-risk populations of pregnant women confirm, as was expected biologically, that the test works equally well in the general population3–7. Not surprisingly, this safe and accurate test, commonly referred to as non-invasive prenatal testing (NIPT), increasingly is being offered by clinicians and requested by pregnant women who want to be informed about the possibility of trisomy 21 in their unborn child. With the first studies suggesting very high accuracy of trisomy 21 detection, there was hope that after decades of searching for this ‘Holy Grail’, a safe blood test could replace chorionic villus sampling and amniocentesis, eliminating (fear of) procedure-related miscarriages. From larger follow-up studies, we now know that while an NIPT result positive for trisomy 21 often means that the fetus is affected, this is not always the case, and therefore confirmation using an invasive test remains necessary, at least when the woman is considering an irreversible decision. Furthermore, sensitivity for detection of trisomy 21 is > 99% in practically all studies, but some missed cases have been reported. Thus, although highly accurate, NIPT is not perfect. In the not-so-distant past, the use of maternal age alone to select women to undergo invasive testing was replaced by various forms of measuring maternal serum markers with or without nuchal translucency (NT) measurement. In countries in which this was implemented well, unnecessary invasive tests (and related miscarriages) significantly decreased, with concomitant improved detection, thus improving women’s reproductive choices8. Still, the vast majority of invasive tests following screening for trisomy reveal a normal result, while the screening test is falsely reassuring in at least one in 10 pregnancies with a trisomy 21 fetus. The use of NIPT enables us to further improve the quality of prenatal testing for fetal abnormalities. The aim of counseling a pregnant woman before she chooses to undergo any test which can have major consequences is to provide sufficient understanding of the test characteristics, limitations and risks for her to make what we call an ‘informed choice’ regarding whether she wants to undergo this test, another one or no test at all. The introduction of NIPT does not change this general principle. We have been counseling women of advanced maternal age on invasive testing for decades, and we are used to discussing serumand NT-based screening, which, when all aspects of the various tests are to be explained, is quite a complex task. Following the first publications on NIPT for trisomy 21, clinicians for a while were under the impression that pretest counseling would become an easier, if not almost superfluous, task. A simple message (‘If you want to know about trisomy 21, we take a tube of blood and let you know in a week or so whether your baby is affected.’) was thought, at least by some, to be capable of replacing the complex explanation involving serum markers, the meaning of this rim of fluid in the baby’s neck, an algorithm including the age of the mother and not so easy-to-understand reasons behind the cut-off between high and low risk. However, with the increasing use of NIPT in clinical practice, there is a rising awareness among professionals and policy makers that adequate pre-test counseling is still important, even for NIPT, in order to prevent misconceptions, disappointments and, in some cases, inappropriate selection of this test by women or doctors. In this Opinion paper, we describe what pregnant women may want to know about NIPT before consenting to undergo this test, and summarize useful aspects, which could be included in various forms of patient information (websites, guidelines, booklets or by personal contact in the clinic).