A novel non-invasive prenatal sickle cell disease test for all at-risk pregnancies.

Sickle cell disease (SCD) is the most common genetic haematological disorder. The availability of non-invasive prenatal diagnosis (NIPD) is predicted to increase uptake of prenatal diagnosis for SCD, as it has no perceived procedure-related miscarriage risk. We report the development of a targeted massively parallel sequencing (MPS) assay for the NIPD of fetal SCD using fetal cell-free (cf)DNA from maternal plasma, with no requirement for paternal or proband samples. In all, 64 plasma samples from pregnant women were analysed: 42 from SCD carriers, 15 from women with homozygous (Hb SS) SCD and seven from women with compound heterozygous (Hb SC) SCD. Our assay incorporated a relative mutation dosage assay for maternal carriers and a wild type allele detection assay for affected women (Hb SS/Hb SC). Selective analysis of only smaller cfDNA fragments and modifications to DNA fragment hybridisation capture improved diagnostic accuracy. Clinical sensitivity was 100% and clinical specificity was 100%. One sample with a fetal fraction of <4% was correctly called as 'unaffected', but with a discordant genotype (Hb AA rather than Hb AS). Six samples gave inconclusive results, of which two had a fetal fraction of <4%. This study demonstrates that NIPD for SCD is approaching clinical utility.

Cytogenomic results following high-chance non-invasive prenatal testing: a UK national audit.

OBJECTIVE: Non-invasive prenatal testing (NIPT) is increasingly being adopted as a screening test in the UK and is currently accessed through certain National Health Service healthcare systems or by private provision. This audit aims to describe reasons for and results of cytogenomic investigations carried out within UK genetic laboratories following an NIPT result indicating increased chance of cytogenomic abnormality ('high-chance NIPT result'). METHOD: A questionnaire was sent out to 24 genetics laboratories in the UK and completed by 18/24 (75%). RESULTS: Data were returned representing 1831 singleton pregnancies. A total of 1329 (73%) invasive samples were taken following NIPT results showing a high chance of trisomy 21; this was confirmed in 1305 (98%) of these by invasive sampling. Trisomy 21 was confirmed in >99% of patients who also had high-screen risk results or abnormal scan findings. Amongst invasive samples taken due to NIPT results indicating a high chance of trisomy 18, 84% yielded a compatible result, and this number dropped to 49% for trisomy 13 and 51% for sex chromosomes. CONCLUSION: In the UK, the majority of patients having invasive sampling for high-chance NIPT results are doing so following an NIPT result indicating an increased chance of common trisomies (92%). In this population, NIPT performs particularly well for trisomy 21, but less well for other indications.

Recommended practice for laboratory reporting of non-invasive prenatal testing of trisomies 13, 18 and 21: a consensus opinion.

OBJECTIVE: Non-invasive prenatal testing (NIPT) for trisomies 13, 18 and 21 is used worldwide. Laboratory reports should provide clear, concise results with test limitations indicated, yet no national or local guidelines are currently available. Here, we aim to present minimum best practice guidelines. METHODS: All laboratories registered in the three European quality assurance schemes for molecular and cytogenetics were invited to complete an online survey focused on services provided for NIPT and non-invasive prenatal diagnosis. Laboratories delivering NIPT for aneuploidy were asked to submit two example reports; one high and one low risk result. Reports were reviewed for content and discussed at a meeting of laboratory providers and clinicians held at the ISPD 2016 conference in Berlin. RESULTS: Of the 122 laboratories that responded, 50 issued reports for NIPT and 43 of these submitted sample reports. Responses and reports were discussed by 72 attendees at the meeting. Consensus opinion was determined in several areas and used to develop best practice guidelines for reporting of NIPT results. CONCLUSIONS: Across Europe, there is considerable variation in reporting NIPT results. Here, we describe minimum best practice guidelines, which will be distributed to European laboratories, and reports audited in subsequent external quality assurance cycles. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Gestation related karyotype, QF-PCR and CGH-array failure rates in diagnostic amniocentesis.

BACKGROUND: Few data exist describing laboratory related failure rates in prenatal diagnosis. The aim of this study is to assess the laboratory associated failure rate for karyotype, QF-PCR and CGH-array following amniocentesis in relation to gestation. METHODS: Retrospective database study of amniocenteses performed 2004-2014 comparing laboratory failure rate for karyotype, QF-PCR and CGH-array between 16 + 0 and 40 + 0 weeks' gestation. RESULTS: A total of 10 484 amniotic fluid test results were collected in three databases. Karyotype failed in 41/1797 (2.3%) tests; failure rate was significantly greater with advancing gestation reaching 43% at 36-40 weeks. QF-PCR failed in 132/5715 tests (2.3%) and was significantly greater with advancing gestation reaching 7% at 36-40 weeks. For CGH-array, 10/298 tests (3.4%) failed analysis. In one case, no result was obtainable by any technique. CONCLUSIONS: These data provide gestation specific laboratory failure rates for amniocentesis enabling informed decisions about the timing and laboratory technique most applicable to the clinical situation. Before 20 weeks, karyotype is least likely to fail of the three techniques. However, in the late third trimester, QF-PCR and, in particular, karyotyping are more likely to fail than CGH-array. Although there is some overlap between the three different tests, they may be preferentially offered in different clinical scenarios. © 2016 John Wiley & Sons, Ltd.

QF-PCR: application, overview and review of the literature.

Quantitative fluorescent polymerase chain reaction has been in diagnostic use in the UK for over 10 years and has proved to be a cost-effective, robust and accurate rapid prenatal test for common aneuploidies. Specific advantages include detection of triploidy, mosaicism and maternal cell contamination. Its application at our centre is described, with developments including stand-alone testing and improvements in strategies for the preparation and testing of chorionic villus biopsies.

Quantitative fluorescence PCR analysis of >40,000 prenatal samples for the rapid diagnosis of trisomies 13, 18 and 21 and monosomy X.

OBJECTIVE: To present the results of 10 years of quantitative fluorescence PCR (QF-PCR) analysis of prenatal samples for the rapid diagnosis of the common aneuploidies. This represents the largest QF-PCR data set from a single testing centre. METHODS: QF-PCR analysis using a single assay containing 17 microsatellite markers was applied to all prenatal samples for the identification of trisomies 13, 18 and 21 and triploidy. A separate assay containing 14 sex chromosome markers was targeted to prenatal samples at increased risk of monosomy X. RESULTS: Results from 40,624 prenatal samples comprising 14,144 chorionic villus and 26,480 amniotic fluid samples are summarised. A QF-PCR result was not possible for 2.24% amniotic fluid and 0.25% chorionic villus samples because of the presence of an additional genotype consistent with maternal cell contamination. Just 0.08% samples were uninformative for one or more chromosomes and 0.05% of samples failed to produce a genotype. Ninety-eight percent of samples were reported the following working day from sample receipt. Consumable costs were £ 5/sample. CONCLUSION: QF-PCR analysis is proven to be an accurate, robust and efficient method for the rapid diagnosis of common aneuploidies in prenatal samples. It has the advantage of detecting triploidy and mosaicism and benefits from considerable economy of scale.

Combined QF-PCR and MLPA molecular analysis of miscarriage products: an efficient and robust alternative to karyotype analysis.

OBJECTIVES: To replace G-banded chromosome analysis for miscarriage products with a combined molecular approach: QF-PCR and MLPA, to increase efficiency, reduce costs, and improve the diagnostic success rate for these samples. METHODS: A review of 10 years of karyotype results for miscarriages products indicated that 2.7% of nonmosaic chromosome imbalance would not be detected by the molecular approach. The molecular approach was validated on 117 samples in parallel with karyotype analysis; no discrepancies were detected. The molecular approach was implemented in September 2007, and in the first 18 months 500 samples were processed. RESULTS: In 500 samples, 117 samples (23%) were abnormal. Of these abnormalities, 64% were trisomies, 12% triploid, 11% monosomy X and 13% other abnormalities. When compared to karyotype analysis, the success rate was higher (95% cf 70%) and the reporting time was lower (88% within 28 days cf 79%). In addition, efficiency was higher as labour-intensive cell culture and karyotyping were replaced by batch testing and automated analysis. CONCLUSIONS: This molecular approach is less labour-intensive, allows a higher sample throughput and has a higher success rate than karyotype analysis; it is therefore an efficient and cost-effective diagnostic testing strategy for miscarriage products.

QF-PCR as a stand-alone test for prenatal samples: the first 2 years' experience in the London region.

OBJECTIVE: To analyse the results of the first 2 years of a QF-PCR stand-alone testing strategy for the prenatal diagnosis of aneuploidy in the London region and to determine the advantages and disadvantages of this policy. METHODS: A review of the results of 9737 prenatal samples received for exclusion of chromosome abnormalities. All samples were subjected to QF-PCR testing for common aneuploidies but only samples fulfilling specific criteria subsequently had a full karyotype analysis. RESULTS: Of the 9737 samples received, 10.3% had a chromosome abnormality detected by QF-PCR testing. Of the 7284 samples received with no indication for karyotype analysis, 25 (0.3%) received a normal QF-PCR result but subsequently had an abnormal karyotype detected either prenatally as a privately funded test or postnatally. Of these samples, without subsequent abnormal ultrasound findings, five had a chromosome abnormality associated with a poor prognosis, representing 0.069% of samples referred for Down syndrome testing. CONCLUSION: While back-up karyotyping is required for some samples, using QF-PCR as a stand-alone prenatal test for pregnancies without ultrasound abnormalities reduces costs, provides rapid delivery of results, and avoids ambiguous and uncertain karyotype results, reducing parental anxiety.

Prenatal Detection of Chromosome Aneuploidy by Quantitative Fluorescence PCR.

Autosomal chromosome aneuploid pregnancies that survive to term, namely, trisomies 13, 18, and 21, account for 89% of chromosome abnormalities with a severe phenotype identified in prenatal samples. They are traditionally detected by full karyotype analysis of cultured cells. The average reporting time for a prenatal karyotype analysis is approximately 14 days, and in recent years, there has been increasing demand for more rapid prenatal results with respect to the common chromosome aneuploidies, to relieve maternal anxiety and facilitate options in pregnancy. The rapid tests that have been developed negate the requirement for cultured cells, instead directly testing cells from the amniotic fluid or chorionic villus sample, with the aim of generating results within 48 h of sample receipt. Interphase fluorescence in situ hybridization is the method of choice in some genetic laboratories, usually because the expertise and equipment are readily available. However, a quantitative fluorescence (QF)-PCR-based approach is now widely used and reported as a clinical diagnostic service in many studies. It may be used as a stand-alone test or as an adjunct test to full karyotype or array CGH analysis, which scan for other chromosome abnormalities not detected by the QF-PCR assay.

European guidelines for constitutional cytogenomic analysis.

With advancing technology and the consequent shift towards an increasing application of molecular genetic techniques (e.g., microarrays, next-generation sequencing) with the potential for higher resolution in specific contexts, as well as the application of combined testing strategies for the diagnosis of chromosomal disorders, it is crucial that cytogenetic/cytogenomic services keep up to date with technology and have documents that provide guidance in this constantly evolving scenario. These new guidelines therefore aim to provide an updated, practical and easily available document that will enable genetic laboratories to operate within acceptable standards and to maintain a quality service.

Prenatal detection of chromosome aneuploidy by quantitative fluorescence PCR.

Autosomal chromosome aneuploid pregnancies that survive to term, namely, trisomies 13, 18, and 21, account for 89% of chromosome abnormalities with a severe phenotype. They are normally detected by full karyotype analysis of cultured cells. The average reporting time for a prenatal karyotype analysis is approximately 14 days, and in recent years, there has been increasing demand for more rapid prenatal results with respect to the common chromosome aneuploidies, to relieve maternal anxiety and facilitate options in pregnancy. The rapid tests that have been developed negate the requirement for cultured cells, instead directly testing cells from the amniotic fluid or chorionic villus sample, with the aim of generating results within 48 h of sample receipt. Interphase fluorescence in situ hybridization is the method of choice in some genetic laboratories, usually because the expertise and equipment are readily available. However, a quantitative fluorescence (QF)-PCR-based approach is more suited to a high-throughput diagnostic service. This approach has been investigated in a small number of pilot studies and reported as a clinical diagnostic service in many studies. It may be used as a stand-alone test or as an adjunct test to full karyotype analysis, which subsequently confirms the rapid result and scans for other chromosome abnormalities not detected by the QF-PCR assay.

Detection of subtelomere imbalance using MLPA: validation, development of an analysis protocol, and application in a diagnostic centre.

BACKGROUND: Commercial MLPA kits (MRC-Holland) are available for detecting imbalance at the subtelomere regions of chromosomes; each kit consists of one probe for each subtelomere. METHODS: For validation of the kits, 208 patients were tested, of which 128 were known to be abnormal, corresponding to 8528 genomic regions overall. Validation samples included those with trisomy 13, 18 and 21, microscopically visible terminal deletions and duplications, sex chromosome abnormalities and submicroscopic abnormalities identified by multiprobe FISH. A robust and sensitive analysis system was developed to allow accurate interpretation of single probe results, which is essential as breakpoints may occur between MLPA probes. RESULTS: The validation results showed that MLPA is a highly efficient technique for medium-throughput screening for subtelomere imbalance, with 95% confidence intervals for positive and negative predictive accuracies of 0.951-0.996 and 0.9996-1 respectively. A diagnostic testing strategy was established for subtelomere MLPA and any subsequent follow-up tests that may be required. The efficacy of this approach was demonstrated during 15 months of diagnostic testing when 455 patients were tested and 27 (5.9%) abnormal cases were detected. CONCLUSION: The development of a robust, medium-throughput analysis system for the interpretation of results from subtelomere assays will be of benefit to other Centres wishing to implement such an MLPA-based service.

Efficient and cost-effective genetic analysis of products of conception and fetal tissues using a QF-PCR/array CGH strategy; five years of data.

BACKGROUND: Traditional testing of miscarriage products involved culture of tissue followed by G-banded chromosome analysis; this approach has a high failure rate, is labour intensive and has a resolution of around 10 Mb. G-banded chromosome analysis has been replaced by molecular techniques in some laboratories; we previously introduced a QF-PCR/MLPA testing strategy in 2007. To improve diagnostic yield and efficiency we have now updated our testing strategy to a more comprehensive QF-PCR assay followed by array CGH. Here we describe the results from the last 5 years of service. METHODS: Fetal tissue samples and products of conception were tested using QF-PCR which will detect aneuploidy for chromosomes 13, 14, 15, 16, 18, 21, 22, X and Y. Samples that were normal were then tested by aCGH and all imbalance >1Mb and fully penetrant clinically significant imbalance <1Mb was reported. RESULTS: QF-PCR analysis identified aneuploidy/triploidy in 25.6% of samples. aCGH analysis detected imbalance in a further 9.6% of samples; this included 1.8% with submicroscopic imbalance and 0.5% of uncertain clinical significance. This approach has a failure rate of 1.4%, compared to 30% for G-banded chromosome analysis. CONCLUSIONS: This efficient QF-PCR/aCGH strategy has a lower failure rate and higher diagnostic yield than karyotype or MLPA strategies; both findings are welcome developments for couples with recurrent miscarriage.

Rapid prenatal diagnosis of aneuploidy using quantitative fluorescence-PCR (QF-PCR).

Molecular cytogenetic aneuploidy testing for pregnant women at increased risk of chromosome abnormality leads to rapid reassurance for those with normal results and earlier decisions on pregnancy management in the case of abnormality. We tested 9080 prenatal samples using a one-tube QF-PCR test for trisomies 13, 18, and 21; the abnormality rate was 5.9%. There were no misdiagnoses for non-mosaic trisomy. A sex chromosome multiplex was developed that detects structural sex chromosome abnormalities as well as aneuploidies. The sex chromosome test was targeted at pregnancies (272) with specific abnormalities suggestive of Turner syndrome; 13.2% showed 45,X, confirmed by follow-up analysis.

Strategies for the rapid prenatal diagnosis of chromosome aneuploidy.

Rapid diagnosis of common chromosome aneuploidies in raised risk pregnancies, usually prior to full karyotype analysis, is now carried out in a number of European genetic centres; several techniques for detecting genomic copy number changes have been described. Prenatal diagnosis of genetic disease requires accurate and robust assays; the invasive procedures are associated with a risk of pregnancy loss and an abnormal result may lead to termination of the pregnancy. The testing of prenatal material (amniotic fluid, chorionic villi or, more rarely, fetal blood) is associated with specific problems, including the quality and quantity of the tissue and difficulties of interpretation due to phenomena such as maternal cell contamination and mosaicism. In addition, there are 24-h, high-throughput demands on centres offering such a service. The extent to which existing and proposed strategies, including different PCR-based assays, a multiplex ligation-dependent probe amplification approach, and microarrays, fulfil the requirements of rapid prenatal testing is discussed. In the past 3 years, we have tested 7720 prenatal samples for trisomies 13, 18 and 21 using a quantitative fluorescence-PCR (QF-PCR) approach. The abnormality rate was 5.7%. There were no misdiagnoses for nonmosaic trisomy, the amplification failure rate was 0.09% of samples, and 97% of samples received a report on the working day following sample receipt. Maternal cell contamination and mosaicism were also detected. Our data recommend a QF-PCR approach as the current method of choice for rapid aneuploidy testing.

Multiplex ligation-dependent probe amplification using a completely synthetic probe set.

The recent development of multiplex ligation-dependent probe amplification (MLPA) has provided an efficient and reliable assay for dosage screening of multiple loci in a single reaction. However, a drawback to this method is the time-consuming process of generating a probe set by cloning in single-stranded bacteriophage vectors. We have developed a synthetic probe set to screen for deletions in a region spanning 18.5 Mb within chromosome 3q. In a pilot study, we tested 15 synthetic probes on 4 control samples and on 2 patients previously found to possess a heterozygous deletion in the region 3q26-q28. These synthetic probes detected deletions at all previously known deleted loci. Furthermore, using synthetic probes, the variability of results within samples was similar to that reported for commercially available M13-derived probes. Our results demonstrate that this novel approach to MLPA provides a generic solution to the difficulties of probe development by cloning; such synthetically generated probes may be used to screen a large number of loci in a single reaction. We conclude that the use of synthetic probes for MLPA is a rapid, robust, and efficient alternative for research (and potentially diagnostic) deletion and duplication screening of multiple genomic loci.

Array CGH as a first line diagnostic test in place of karyotyping for postnatal referrals - results from four years' clinical application for over 8,700 patients.

BACKGROUND: Array CGH is widely used in cytogenetics centres for postnatal constitutional genome analysis, and is now recommended as a first line test in place of G-banded chromosome analysis. At our centre, first line testing by oligonucleotide array CGH for all constitutional referrals for genome imbalance has been in place since June 2008, using a patient vs patient hybridisation strategy to minimise costs. FINDINGS: Out of a total of 13,412 patients tested with array CGH, 8,794 (66%) had array CGH as the first line test. Referral indications for this first line group ranged from neonatal congenital anomalies through to adult neurodisabilities; 25% of these patients had CNVs either in known pathogenic regions or in other regions where imbalances have not been reported in the normal population. Of these CNVs, 46% were deletions or nullisomy, 53% were duplications or triplications, and mosaic imbalances made up the remainder; 87% were <5Mb and would likely not be detected by G-banded chromosome analysis. For cases with completed inheritance studies, 20% of imbalances were de novo. CONCLUSIONS: Array CGH is a robust and cost-effective alternative to traditional cytogenetic methodology; it provides a higher diagnostic detection rate than G-banded chromosome analysis, and adds to the sum of information and understanding of the role of genomic imbalance in disease. Use of novel hybridisation strategies can reduce costs, allowing more widespread testing.

Prenatal detection of chromosome aneuploidy by quantitative-fluorescence PCR.

QF-PCR refers to the amplification of chromosome-specific polymorphic microsatellite markers using fluorescence-labelled primers, followed by semi-quantitative analysis of the products on a genetic analyser to determine copy number and/or imbalances of specific chromosomal material. This approach is now widely used for rapid prenatal diagnosis of the common trisomies. In addition, it can successfully detect maternal cell contamination and mosaicism in prenatal material.