Author Correction: Scalable and robust SARS-CoV-2 testing in an academic center.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multiplex ligation-dependent probe amplification (MLPA): a reliable alternative for fetal chromosome analysis?

OBJECTIVE: To determine whether molecular karyotyping using multiple ligation probe amplification (MLPA) is a reliable alternative for quick and accurate diagnosis of fetal chromosomal abnormalities. METHODS: MLPA, using specialised probe sets designed to detect aneuploidy, major chromosomal rearrangements and recognised microdeletion syndromes, was used to analyse chorionic villi or amniocytes left after traditional karyotyping of 476 fetuses for clinical indications. RESULTS: An abnormal result was obtained in 190 cases, including 124 trisomies, 21 sex chromosome anomalies, 14 triploidies, and 31 rearrangements or mosaics. All trisomies were detected by all three techniques, but triploidies were only detected by karyotyping and QF-PCR. In 19 of the 31 cases of rearrangements or mosaicism there was an uncertain or high risk of adverse outcome. Traditional karyotyping detected 13 of the 19 pathogenic rearrangements, MLPA detected 18, and QF-PCR did not detect any. CONCLUSION: MLPA, using specialized probe sets, detects more chromosomal rearrangements, conferring significant risk of adverse outcome than karyotyping. A combination of qfPCR and MLPA could be a good, rapid alternative to current practice. In the future, used in conjunction with non-invasive prenatal diagnosis based on cell free fetal DNA it might provide a rapid and efficient approach to fetal karyotyping.

Incidence of placental mosaicism leading to discrepant results between QF-PCR and karyotyping in 22,825 chorionic villus samples.

OBJECTIVE: To review the frequency and analyse the origin of completely discrepant results observed between QF-PCR and karyotyping in chorionic villus samples (CVS) as a result of placental mosaicism. Also, to assess QF-PCR results for biallelic or triallelic patterns and determine their significance. METHODS: Between May 2002 and December 2009, 22 825 CVS were received at TDL Genetics for processing by QF-PCR and karyotype. The QF-PCR and karyotype data were compared to determine the incidence of discrepant results. RESULTS: Of the 22 825 samples received, 22 779 (99.8%) gave concordant results between the PCR and karyotype, and 46 samples (0.2%) gave discordant results. Of these discrepant cases, 5 displayed triallelic peaks and 41 displayed biallelic peaks. All discordant results are due to the presence of placental mosaicism, a known limitation of using this sample type for prenatal diagnosis. CONCLUSION: This retrospective study of placental mosaicism in CVS is the largest single centre study to date and provides a figure for the occurrence of completely discrepant results between QF-PCR and karyotype due to placental mosaicism. This study also demonstrates that the presence of triallelic peaks at QF-PCR is not sufficient to exclude the presence of placental mosaicism.

A novel method for extracting DNA from chorionic villus samples for use in CVS-PCR, which ensures complete villus dissociation.

OBJECTIVE: To demonstrate that glass disruption beads dissociate chorionic villus samples releasing DNA from mesenchymal and cytotrophoblast cells that is suitable for processing by CVS-PCR (rapid molecular aneuploidy testing). This method is quicker than conventional methods and may limit discrepancies between PCR and karyotype in certain types of placental mosaicism. METHOD: DNA was extracted from villus samples by mechanical disruption of the cells using glass beads. This method was compared to collagenase incubation followed by chelex extraction of the digested villus. PCR data generated were compared using standard criteria. RESULTS: DNA extracted by glass bead disruption generated data of equivalent quality to that obtained from DNA extracted using conventional collagenase and chelex-based extraction method. The case study demonstrates probable cytotrophoblast enrichment of a sample when processed by collagenase digestion and chelex incubation. Re-extraction of the digested sample by glass bead disruption resulted in cytotrophoblast and mesenchyme cells contributing to the supernatant. CONCLUSION: Glass bead disruption of chorionic villus samples is an effective, inexpensive and rapid DNA extraction method that dissociates villus ensuring that DNA from both cytotrophoblast and mesenchyme cells is represented in the supernatant. Extracted DNA produced is suitable for CVS-PCR and can be stored stably at - 20 degrees C.

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.

Use of real-time polymerase chain reaction for the detection of fetal aneuploidies.

With the advent of real-time polymerase chain reaction (PCR), it is now possible to measure nucleic acid concentrations with an accuracy that was not possible only a few years ago. Examples are the analysis of gene expression or gene duplications/losses, where twofold differences in nucleic acid concentration have routinely been determined with almost 100% accuracy. As our primary interest is in prenatal diagnosis, we have investigated whether real-time PCR could be used for the diagnosis of chromosomal anomalies, in particular the aneuploidies such as trisomy 21, where the difference in copy number is only 50%. The feasibility of such an approach was first tested in a pilot study, in which we were able to demonstrate that trisomy 21 samples could be detected with 100% specificity. We have recently modified this test in order to permit the simultaneous analysis of trisomies 18 and 21, and have in a large scale analysis demonstrated that our approach can be used for the highly reproducible and robust detection of only 1.5-fold differences in gene copy number.

Kinetic analysis of the interaction between the QutA and QutR transcription-regulating proteins.

The QutR protein is a multidomain repressor protein that interacts with the QutA activator protein. Both proteins are active in the signal transduction pathway that regulates transcription of the quinic acid utilization (qut) gene cluster of the microbial eukaryote Aspergillus nidulans. In the presence of quinate, production of mRNA from the eight genes of the qut pathway is stimulated by the QutA activator protein. The QutR protein plays a key role in signal recognition and transduction, and a deletion analysis has shown that the N-terminal 88 amino acids are sufficient to inactivate QutA function in vivo. Using surface plasmon resonance we show here that the N-terminal 88 amino acids of QutR are able to bind in vitro to a region of QutA that genetic analysis has previously implicated in transcription activation. We further show that increasing the concentration of a full-length (missense) mutant QutR protein in the original mutant strain can restore its repressing function. This is interpreted to mean that the qutR mutation in this strain increases the equilibrium dissociation constant for the interaction between QutA and QutR. We propose a model in which the QutA and QutR proteins are in dynamic equilibrium between bound (transcriptionally inactive) and unbound (transcriptionally active) states.

The QUTA activator and QUTR repressor proteins of Aspergillus nidulans interact to regulate transcription of the quinate utilization pathway genese.

Genetic evidence suggests that the activity of the native QUTA transcription activator protein is negated by the action of the QUTR transcription repressor protein. When Aspergillus nidulans was transformed with plasmids containing the wild-type qutA gene, transformants that constitutively expressed the quinate pathway enzymes were isolated. The constitutive phenotype of these transformants was associated with an increased copy number of the transforming qutA gene and elevated qutA mRNA levels. Conversely, when A. nidulans was transformed with plasmids containing the qutR gene under the control of the constitutive pgk promoter, transformants with a super-repressed phenotype (unable to utilize quinate as a carbon source) were isolated. The super-repressed phenotype of these transformants was associated with an increased copy number of the transforming qutR gene and elevated qutR mRNA levels. These copy-number-dependent phenotypes argue that the levels of the QUTA and QUTR proteins were elevated in the high-copy-number transformants. When diploid strains were formed by combining haploid strains that contained high copy numbers of either the qutA gene (constitutive phenotype) or the qutR gene (super-repressing; non-inducible phenotype), the resulting diploid phenotype was one of quinate-inducible production of the quinate pathway enzymes, in a manner similar to wild-type. The simplest interpretation of these observations is that the QUTR repressor protein mediates its repressing activity through a direct interaction with the QUTA activator protein. Other possible interpretations are discussed in the text. Experiments in which truncated versions of the QUTA protein were produced in the presence of a wild-type QUTA protein indicate that the QUTR repressor protein recognizes and binds to the C-terminal half of the QUTA activator protein.