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.

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.

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.

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.

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.

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.

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.

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.

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.

Detection of mosaicism for genome imbalance in a cohort of 3,042 clinical cases using an oligonucleotide array CGH platform.

Mosaicism for chromosome imbalance has traditionally been detected by karyotype analysis. The introduction of array CGH into clinical diagnostic laboratories and routine clinical practice has raised concerns as to the ability of this new test to detect the presence of more than one cell line. We present our validation data on the detection of chromosome mosaicism by oligonucleotide array CGH, and the cases detected in a cohort of 3042 clinical referrals. Using an artificial mosaicism series, we found that oligonucleotide array CGH using specific analysis parameters could accurately measure levels of mosaicism down to 10% and that the degree of mosaicism could be predicted from fluorescence ratios. We detected 12 cases of mosaicism in our clinical cohort, in 9 of which there was no previous indication of mosaicism. In two cases, G-banded chromosome analysis had been carried out previously, and had failed to detect the abnormal cell line. Three cases had mosaicism for the X chromosome and 9 involved autosomes, of which 4 were mosaic for whole chromosome trisomies, one for whole chromosome monosomy, and four were mosaic for segmental imbalances. We conclude that oligonucleotide array CGH has the power to detect a range of mosaic abnormalities in clinical diagnostic samples.

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.

MLPA for confirmation of array CGH results and determination of inheritance.

BACKGROUND: Array CGH has recently been introduced into our laboratory in place of karyotype analysis for patients with suspected genomic imbalance. Results require confirmation to check sample identity, and analysis of parental samples to determine inheritance and thus assess the clinical significance of the abnormality. Here we describe an MLPA-based strategy for the follow-up of abnormal aCGH results. RESULTS: In the first 17 months of our MLPA-based aCGH follow-up service, 317 different custom MLPA probes for novel aCGH-detected abnormalities were developed for inheritance studies in 164 families. In addition, 110 samples were tested for confirmation of aCGH-detected abnormalities in common syndromic or subtelomeric regions using commercial MLPA kits. Overall, a total of 1215 samples have been tested by MLPA. A total of 72 de novo abnormalities were confirmed. CONCLUSIONS: Confirmation of aCGH-detected abnormalities and inheritance of these abnormalities are essential for accurate diagnosis and interpretation of aCGH results. The development of a new service utilising custom made MLPA probes and commercial MLPA kits for follow-up studies of array CGH results has been found to be efficient and flexible in our laboratory.

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.

Validation and implementation of array comparative genomic hybridisation as a first line test in place of postnatal karyotyping for genome imbalance.

BACKGROUND: Several studies have demonstrated that array comparative genomic hybridisation (CGH) for genome-wide imbalance provides a substantial increase in diagnostic yield for patients traditionally referred for karyotyping by G-banded chromosome analysis. The purpose of this study was to demonstrate the feasibility of and strategies for, the use of array CGH in place of karyotyping for genome imbalance, and to report on the results of the implementation of this approach. RESULTS: Following a validation period, an oligoarray platform was chosen. In order to minimise costs and increase efficiency, a patient/patient hybridisation strategy was used, and analysis criteria were set to optimise detection of pathogenic imbalance. A customised database application with direct links to a number of online resources was developed to allow efficient management and tracking of patient samples and facilitate interpretation of results. Following introduction into our routine diagnostic service for patients with suspected genome imbalance, array CGH as a follow-on test for patients with normal karyotypes (n = 1245) and as a first-line test (n = 1169) gave imbalance detection rates of 26% and 22% respectively (excluding common, benign variants). At least 89% of the abnormalities detected by first line testing would not have been detected by standard karyotype analysis. The average reporting time for first-line tests was 25 days from receipt of sample. CONCLUSIONS: Array CGH can be used in a diagnostic service setting in place of G-banded chromosome analysis, providing a more comprehensive and objective test for patients with suspected genome imbalance. The increase in consumable costs can be minimised by employing appropriate hybridisation strategies; the use of robotics and a customised database application to process multiple samples reduces staffing costs and streamlines analysis, interpretation and reporting of results. Array CGH provides a substantially higher diagnostic yield than G-banded chromosome analysis, thereby alleviating the burden of further clinical investigations.

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.

A novel deletion in proximal 22q associated with cardiac septal defects and microcephaly: a case report.

BACKGROUND: Proximal 22q is rich in low copy repeats (LCRs) which mediate non-allelic homologous recombination and give rise to deletions and duplications of varying size depending on which LCRs are involved. METHODS: A child with multiple septal defects and other congenital anomalies was investigated for genome imbalance using multiplex ligation-dependent probe amplification (MLPA) for subtelomeres and microdeletion loci, followed by array comparative genomic hybridization (CGH) using oligonucleotide arrays with 44,000 probes across the genome. RESULTS: MLPA identified a single probe deletion in the SNAP29 gene within band 22q11.21. Follow-up array CGH testing revealed a ~1.4-Mb deletion from 19,405,375 bp to 20,797,502 bp, encompassing 28 genes. CONCLUSION: This deletion is likely to be causally associated with the proband's congenital anomalies. Previous publications describing deletions in proximal 22q have reported deletions between LCRs 1 to 4, associated with 22q11 deletion syndrome; in addition, deletions between LCRs 4 and 6 have been described associated with "distal 22q11 deletion syndrome". To our knowledge, this is the first deletion which spans LCR4 and is not apparently mediated by LCRs. Comparison of the phenotypes found in conjunction with previously reported deletions, together with the function and expression patterns of genes in the deleted region reported here, suggests that haploinsufficiency for the Crk-like (CRKL) gene may be responsible for the reported cardiac abnormalities.

Submicroscopic chromosome imbalance in patients with developmental delay and/or dysmorphism referred specifically for Fragile X testing and karyotype analysis.

BACKGROUND: Microdeletion syndromes are generally identified because they usually give rise to specific phenotypic features; many of these deletions are mediated by duplicons or LCRs. The phenotypes associated with subtelomeric deletions are also becoming recognised. However, reciprocal duplication events at these loci are less easily recognised and identified, as they may give rise to milder phenotypic features, and the individuals carrying them may not therefore be referred for appropriate testing. 403 patients with developmental delay and/or dysmorphism, referred to our Genetics Centre for karyotyping and Fragile X expansion testing, were assessed for chromosome imbalance by Multiplex Ligation-dependent Probe Amplification (MLPA). Two MLPA kits were used, one containing probes for the subtelomere regions, and one containing probes for common microdeletion loci. 321 patients were tested with both kits, 75 with the subtelomere kit alone, and 7 with the microdeletion kit alone. RESULTS: 32 patients had abnormal results; the overall abnormality detection rate was 2.5% for karyotype analysis and 7.2% for MLPA testing; 5.5% of subtelomere tests and 2.1% of microdeletion tests gave abnormal results. Of the abnormal MLPA results, 5 were in cases with cytogenetically visible abnormalities; of the remaining, submicroscopic, changes, 3 results were established as de novo and 8 were inherited; parental samples were not available for the remaining cases. None of the patients was found to have a Fragile X expansion. CONCLUSION: Karyotype analysis in combination with MLPA assays for subtelomeres and microdeletion loci may be recommended for this patient group.