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+{"file": "ukeme0401_NBK423961/s4.nxml", "text": "The EACH study was designed to evaluate the clinical effectiveness and cost-effectiveness of prenatal CMA compared with karyotyping within the UK\u2019s NHS. The study involved 8 of the 17 NHS regional genetics laboratories (representing six of the nine NHS Genetics Services in England) as well as the Welsh Genetics Service. All the participating laboratories had experience of running CMA on postnatal samples and, therefore, prenatal samples were integrated into existing postnatal laboratory array workflows. Through the NHS FASP all women in the UK are offered combined ultrasound and biochemical screening for common aneuploidies at 11\u201314 weeks\u2019 gestation and ultrasound screening for structural anomalies at 18\u201320 weeks of pregnancy.2 Women at a high risk of chromosomal anomalies are offered invasive testing with karyotyping of chorionic villus or amniotic fluid samples in QF-PCR-negative cases.2 Invasive testing and assessment of structural anomalies is undertaken by fetal medicine specialists working in large district hospitals or regional specialised fetal medicine centres. Cases for the EACH study were recruited from 10 of the largest specialised centres in England and Wales as well as 10 smaller units, reflecting current UK practice. The design of the EACH study was therefore predicated both by the existing national prenatal screening/diagnosis programmes and pathways but also by the published prenatal CMA data20 that confirmed that targeting fetuses with ultrasound anomalies would optimise the yield of clinically significant cryptic chromosomal anomalies.\nThe study has shown that, within this UK context, prenatal CMA in fetuses with a structural anomaly and a normal QF-PCR detects 3.5% more pathogenic CNVs than karyotyping. This rate is consistent with the early meta-analysis of Hillman et al.,36 which informed the EACH study, and their subsequent BAC array study conducted in Birmingham, which reported pathogenic CNVs in 4.4% of 243 fetuses referred with a structural anomaly on ultrasound.56 It is also consistent with the large National Institute of Child Health and Human Development (NICHD)-sponsored trial which performed oligonucleotide- or SNP-based arrays in 755 cases with an ultrasound anomaly; 21 (2.8%) were classified as having a pathogenic CNV.57 However, this is lower than the most recent systematic review of 18 studies (3359 cases), published in 2014, which reported a pooled prevalence of pathogenic CNVs in fetuses with one or more structural anomalies of 6.8% (95% CI 6.0% to 7.7%).58 This review was dominated by the experience from a single laboratory in the USA which reported pathogenic CNVs in 159 out of 2184 (7.3%) fetuses with ultrasound anomalies.59 Of note, a recent study from Belgium, which utilised 60,000 or 105,000 oligonucleotide arrays in 383 cases with undefined \u2018ultrasound abnormalities\u2019, identified submicroscopic \u2018causal\u2019 CNVs in 2.6% of cases, while CMA added valuable information over conventional karyotyping in another 3.9% of cases.60\nCurrently, the NHS FASP recommends the offer of invasive prenatal diagnosis when ultrasound screening identifies a NT of \u2265\u20093.5\u2009mm.4 The EACH study therefore sought to address the effectiveness of CMA in this specific group. Although CMA detected 4.5% more CNVs than karyotyping in fetuses with an increased NT, there was no difference in the rate of pathogenic variants. This finding was consistent when the NT cut-off point was increased to 5\u2009mm or 6\u2009mm. However, the rate of pathogenic CNVs on CMA was higher in fetuses with a NT of >\u20095\u2009mm than in those with a NT of 3.5\u20135\u2009mm. We are not aware of any other studies that have looked at the effectiveness of CMA at different NT cut-off points, although De Wit et al.58 found no difference in the rates of pathogenic submicroscopic CNVs in fetuses with a NT of >\u20093.5\u2009mm and those with a cystic hygroma. Grande et al.61 have recently published a meta-analysis of 17 studies (1696 cases) addressing the incremental yield of CMA over karyotyping in fetuses with an increased NT. When a NT cut-off point was defined, most authors used 3.5\u2009mm and included cystic hygroma, as in the current study. Overall, 5% more pathogenic CNVs were detected by CMA. In the subgroup of fetuses with isolated increased NT (1403 cases with no additional structural anomaly), the pathogenic CNV detection rate was 4% (95% CI 2.0% to 7.0%) higher with CMA. In the largest single study to date which reported CMA results in 215 fetuses with a NT of 3.5\u20139.0\u2009mm, CMA detected CNVs in 1.4% (95% CI 0.5% to 4.0%), but all three cases were classified as VOUS.62 These findings are also consistent with the NICHD trial, which included 187 cases with a NT of \u2265\u20093.5\u2009mm or a cystic hygroma or a NF of >\u20096\u2009mm; the rate of CNVs not detected by karyotyping (3.8%) was no different to that in the control group (for which CMA was performed for advanced maternal age in the absence of any detected fetal anomaly).63 Taken together, the results suggest that the added diagnostic yield of CMA over karyotyping is significantly lower in fetuses with an isolated increased NT than in fetuses with a structural anomaly (with or without an increased NT).\nThe results of the EACH study add to the growing evidence of the improved diagnostic accuracy of CMA compared with karyotyping for the detection of numerical and structural chromosomal imbalances in prenatal diagnosis. In a recent systematic review, using the combined rate of pathogenic imbalances detected by karyotyping and CMA as the reference standard, Saldarriaga et al.64 reported a higher sensitivity (0.942) for CMA than for karyotyping (0.673) with the same specificity (0.999). Negative and positive likelihood ratios were also improved (0.049 vs. 0.291 and 1340 vs. 860, respectively).\nThe two recent meta-analyses discussed above both showed statistically significant heterogeneity between studies.56,61 This is likely to reflect the inclusion of smaller cohorts with an artificially high detection rate.56 It is noteworthy that the results from the EACH study agree more closely with more recent, much larger, studies. However, differences in anomaly case selection, CNV calling strategy and array platform may also have contributed to the lower rates of pathogenic CNVs, than in the published meta-analyses, found in the current study.\nThe rate of pathogenic CNVs is known to be higher in fetuses with multiple sonographic anomalies. In De Wits\u2019 recent systematic review of studies in fetuses with structural anomalies and a normal karyotype,58 9.1% with multiple anomalies had a pathogenic CNV, compared with 5.6% of those with an isolated anomaly. In the EACH study, 11.1% of fetuses with multiple anomalies had a pathogenic CNV on CMA compared with 8.8% of those with one anomaly, although this difference was not statistically significant. A similar pattern was found in the NICHD study, in which CNVs (both pathogenic and VOUS) undetected by karyotype were more common in fetuses with multiple (n\u2009=\u2009206) than isolated (n\u2009=\u2009312) anomalies (13.6% vs. 6.7%).63 We also confirmed the specific benefit of CMA in fetuses with a cardiac anomaly.63,65,66 Other CMA studies have reported pathogenic CNVs in 2.0\u20136.6% of fetuses with a cardiac anomaly, normal karyotype and negative or no FISH results for 22q11.2 deletion syndrome.65,66 Yan et al.66 found the rate of pathogenic CNVs in this group was not affected by the presence of additional structural anomalies.\nConsiderable effort went into optimising the laboratory protocols before recruitment to the EACH study commenced. A detailed technical evaluation was performed by scientists at two of the participating laboratories (WRGL and NETRGL), aimed at developing an agreed technical workflow for performing CMA using minimal quantities of amniotic fluid (2\u20134\u2009ml), chorionic villi (2\u20135\u2009mg) and cultured cells (<\u2009150,000 cells). Guidance was provided about optimal methods of DNA extraction (using the iGENatalTM kit) and DNA quantitation (using Qubit\u00ae 2.0 Fluorometer and Quibit\u00ae dsDNA BR assay kit)48 to use with the agreed array design. As a result, 98.9% of arrays in the EACH study were performed on uncultured samples. This is a substantial improvement on the NICHD study in which the CMA result was derived from cultured cells in 21.5% of cases.57 The array design chosen was based on the ISCA Consortium\u2019s consensus49 and consisted of an 8-plex of 60,000 60-mer oligonucleotides with a backbone resolution of \u224875\u2009kb but with considerably higher coverage over telomeric and pericentromeric chromosomal regions, in known microdeletion/duplication syndromic regions and also for a number of developmental and haploinsufficent genes. Thus, the array design was felt to be optimal to detect known microdeletion and duplication syndrome regions as well as other chromosomal imbalances. The laboratories obtained their arrays from a number of commercial vendors and, in most cases, used the analytical software supplied with the arrays. With the described laboratory workflow, 3.3% of arrays failed (vs. 0.8% of karyotypes). This is higher than in other recent studies56,57 and, in part, may reflect the introduction of prenatal arrays in addition to karyotyping in busy NHS genetics laboratories but also may be due to the relatively small amounts of amniotic fluid and chorionic villi made available for DNA extraction (e.g. \u22485\u2009ml of amniotic fluid in EACH vs. 20\u2009ml exclusively available for DNA extraction in the NICHD study57). In fact, the preliminary work carried out by Callaway et al.48 was undertaken in anticipation of this problem and almost certainly contributed to the relatively high success rate with less then optimal volumes of starting fetal material for CMA.\nThe interpretation of CMAs and the strategy for calling CNVs is challenging. The identification of novel VOUS, and the perceived difficulties these cause for clinical management, is seen as the most significant disadvantage of prenatal CMA. To facilitate accurate and timely interpretation of prenatal CMA, the EACH study was designed so laboratories had ready access to parental DNA; blood samples were collected at the time of prenatal diagnostic procedure from 100% of mothers and 78% of fathers. Thus, for most identified CNVs, parental inheritance could be determined from a single parent versus parent array targeted at the area of imbalance. When necessary, FISH-ready clones were made rapidly available by WRGL. In-house interpretation of CNVs has been facilitated by the development and evolution of online international databases with results from normal individuals (e.g. Database of Genomic Variation67) and those with phenotypic anomalies (e.g. DECIPHER68). As laboratories have increased their experience of prenatal CMA many have also developed databases cataloguing CNVs. Through continuous updating of clinical CMA databases (e.g. ISCA Consortium database69), understanding of genotype/phenotype correlations and the clinical significance of novel CNVs is expanding rapidly with an emphasis on penetrance of key phenotypic traits associated with an increasing number of CNVs.70 To reflect current laboratory practice, local laboratory scientists used their standard in-house interpretation protocols including minimum size cut-off points for deletions and duplications. In this context, deletions as small as 5.5\u2009kb and 11\u2009kb were detected and included in the reported cases (see Tables 15 and 16). The exception to this was the agreed list of low-penetrance CNVs excluded from reporting. Furthermore, to provide additional support to local genetics services in making CNV calls and reporting decisions, an expert panel made up of five consultant clinical cytogeneticists and five consultant clinical geneticists, was available to offer advice when requested. This strategy was chosen to reflect how CMA may be introduced into the NHS. It differs markedly from the NICHD trial, for which all CMA results were interpreted at a single centre and all VOUS were referred to an independent advisory panel.\nVariants for which the possible genotype and phenotypic effects were uncertain (VOUS) were not reported in the EACH study. Overall, 38 (3.4%) cases were called as VOUS and the rate was similar in the two study groups. In the majority of cases (58%), the decision was made in-house by the local cytogenetics/clinical genetics team while, of the 32 cases referred to the EACH panel because of local uncertainty, half were not reported (i.e. regarded as a VOUS). The VOUS rate in the EACH study is very similar to the rate reported in the NICHD trial, in which 24 out of 755 (3.2%) arrays performed for an ultrasound anomaly were interpreted as \u2018potential for clinical significance\u2019. 57 These VOUS rates are higher than both the rates reported by Hillman et al.56 in their meta-analysis of studies for which the clinical indication for CMA was a structural anomaly (2.1%, 95% CI 1.3% to 3.3%) and the rates reported in the Leuven study (1.6%) of oligonucleotide arrays in fetuses with ultrasound anomalies.60 It is also higher than the rate reported from the meta-analysis of increased NT studies (0.8%, 95% CI 0.4% to 1.3%).61 This is likely to reflect the 60,000 oligonucleotide array design used in the EACH study in comparison with several studies included in both meta-analyses that used BAC arrays. VOUS rates are known to increase as the resolution of the array increases. Hillman et al.71 compared CNV detection rate in 62 fetal samples using two array platforms (1\u2009Mb targeted BAC array and 60,000 oligonucleotide array). The 60,000 array detected 4.8% (95% CI 1.6% to 13.3%) more pathogenic CNVs but 8% (95% CI 1.3% to 14.8%) more VOUS.71 Using information on CMA genome coverage (rather than a direct comparison in the same fetal samples), Shaffer et al.70 estimated that use of a 55,000 array would reduce VOUS rates by 32% compared with a higher resolution 135,000 array while still detecting all but one pathogenic variant (98%). Thus, we believe that the array design employed in the EACH study represents a sensible compromise between pathogenic and VOUS detection rates. It is expected that, as experience of prenatal CMA increases, the number of VOUS calls will reduce.61\nThe second part of the laboratory evaluation within the EACH study was to compare TATs. To reflect NHS laboratory practice, we adopted the definition used for CPA (which is one of the services within the UK Accreditation Service), that is, from the date of sample receipt to the date of the final report. Based on this definition, result times for karyotyping were consistent with the UK average (12 days) and times for CMA were consistent with previous research studies.72 CMA results took a median of 3 days longer than karyotyping (15 days vs. 12 days), although there was a large variation between laboratories. When further testing was required to interpret the array, median TATs were increased to 22\u201326 days, depending on the test; however, we acknowledge that this may not reflect TAT if CMA were to replace karyotyping. The TAT, as defined for CPA, is also predicated on whether or not rapid aneuploidy detection by QF-PCR is performed prior to setting up the array. We therefore also compared times from set-up to report; in this analysis CMA was 5 days quicker than karyotyping with a median time of 6 days. There remained substantial differences between laboratories (with median times ranging from 2 to 9 days) suggesting that, with greater clinical experience, a median TAT of 7 days is achievable.\nThere was complete concordance between the detection rate of cytogenetically visible euchromatic unbalanced chromosomal abnormalities and the results obtained by CMA. As expected, array CGH failed to detect 15 visible cytogenetic abnormalities, the majority of which were apparently balanced rearrangements and, therefore, no euchromatic gains or losses were observed on the matched array.73 The proportion of cases with an abnormal karyotype but normal CMA (1.3%) is comparable to the Hillman et al.56 series from the UK (2%) and the NICHD study (0.9%).57 Balanced translocations and inversions are reported in <\u20090.1% of prenatal diagnostic samples,74 suggesting that such changes are more common in prenatal series selected for fetal anomaly. Although an inherited rearrangement with a normal CMA would not explain ultrasound findings in the index pregnancy, the finding is of relevance to future reproductive counselling. Counselling parents with a de novo apparently balanced rearrangement is more challenging, especially when the indication for testing is an increased NT with no structural anomaly. In a large cytogenetic series, in which amniocentesis was performed primarily for maternal age, the risk of a serious anomaly in fetuses with an apparently balanced de novo rearrangement varied from 3.7% for Robertsonian translocations to 9.4% for inversions.75 Although many such cases will have an imbalance detected on CMA, either at one or more breakpoints or unrelated to the translocation/inversion,76 the residual risk in those with a normal CMA is unclear.\nConfined placental mosaicism, in which the karyotypes of the placental and fetal tissues are divergent, has been extensively reported and reviewed.77 It occurs in up to 2% of chorionic villus samples (compared with <\u20090.5% of amniotic fluid samples78). Overall, within the EACH cohort there were three cases with CPM (UCH312964, Birm3011 and SGH 86084) that were recognised prenatally and not reported. There were two further cases in which the CPM was only finally recognised postnatally. The first was a Xp22.31 steroid sulfatase enzyme deletion (UCH1285528) and was reported prenatally. Follow-up studies after delivery showed that the Xp22.31 deletion was confined to the placenta.79 The case prompted an observation by Kooper and Faas80 that similar false positives could be avoided if the source villi were dissociated into two separate cell suspensions of cytotrophoblast and mesenchymal cells using an adapted version of the protocol of Mann et al.81 This allows testing of DNA from both fractions whenever mosaicism is suspected. The second case (CAM62826) was reported prenatally to be a trisomy 7 mosaic on karyotyping and non-mosaic trisomy 7 using CMA. Postnatal karyotyping of peripheral blood found no evidence of trisomy 7, confirming CPM. These two cases, both with a normal outcome, represent the only false positives within the EACH study giving a frequency of 2 out of 1123 (0.18%). Aside from these CPM cases, there were three additional cases in the EACH study in which an apparent anomaly was identified by CMA (SGH 85534, QCH130141 and UCH1176628) but follow-up studies found to be cultural or technical artefacts. In addition, there was one case with a benign heterochromatic variant, t(Y;15) (NCL12275). All four cases were reported as normal.\nAn increasing number of prenatal studies are using SNP-based arrays. Srebniak et al.82 summarised the advantages of SNP-based arrays over non-SNP arrays in the prenatal setting. SNP arrays can detect triploidy and MCC, although this is less important in the current UK context, where rapid aneuploidy detection with QF-PCR is recommended. With male fetal samples interfering levels of MCC can be excluded when the sex chromosome plots are normal, but this is not possible with a female fetus and MCC testing is recommended to be confident that the results are reflective of the fetal DNA.83 However, depending on the array and software design, small CNVs can be detected with varying levels of MCC.81 SNP arrays are also reported to be able to detect levels of mosaicism as low as 5% depending on origin.82 However, recent studies have confirmed that oligoCGH arrays can detect mosaicism at a level as low as 10%.84,85 Finally, in contrast to oligoCGH arrays, SNP arrays also offer the advantage of detecting loss of heterozygosity; large regions of homozygosity throughout the genome can suggest consanguinity while regions of homozygosity involving chromosomes 6, 7, 11, 14, 15 and 20 can suggest clinically relevant UPD.82 In one of the 15 abnormal karyotype/normal CMA cases, additional molecular studies demonstrated paternal UPD(14) of the chromosome 14 homologues in association with mosaicism supernumerary heterochromatic marker 14 chromosome86 and represented the one case in the EACH study for which use of a SNP array would have provided a more comprehensive diagnostic utility.87,88\nThe health economic analysis indicated that the mean cost per test was higher for CMA than for karyotyping (\u00a3322 vs. \u00a3234\u2013272), as was the total cost of the pathway, including laboratory follow-up tests and clinical follow-up (\u00a3599 vs. \u00a3486). CMA detected more pathogenic CNVs than karyotyping in the structural anomaly group than the NT group (0.035 vs. 0.010 more per pregnancy) and, hence, the incremental cost per pathogenic CNV detected by CMA compared with karyotyping was lower in the structural anomaly group (\u00a33635 vs. \u00a39439). Results were sensitive to the number of pathogenic CNV results found by CMA, the number of abnormal karyotypes and the cost of CMA. If the NHS was willing to pay an extra \u00a310,000 to detect an extra pCNV, then CMA would be cost-effective on 87% of occasions. Few economic evaluations of CMA are available for comparison. Hillman et al.89 evaluated the cost-effectiveness of CMA compared with nine combinations of conventional techniques in the NHS using data from a cohort of women with a structural anomaly. Testing strategies including CMA detected the most CNVs. Using a base-case unit cost for a 1-Mb BAC array of \u00a3405, the authors calculated that, compared with QF-PCR followed by karyotype [if the polymerase chain reaction (PCR) was negative] followed by FISH for 22q11.2 deletion syndrome (if the karyotype was negative and the fetal anomaly was cardiac related), CMA cost an extra \u00a324,600 for every additional CNV. Compared with karyotyping alone, CMA cost an extra \u00a333,300 per pathogenic CNV detected, which fell to \u00a39768 if the unit cost of CMA was \u00a3360. The authors concluded that CMA was not cost-effective, although they acknowledged that it may become so in future if the costs of CMA fall; the EACH results support this (in the EACH study, the unit cost of CMA was \u00a3322). Hillman et al.89 included both pathogenic CNVs and VOUS in their definition of additional cases detected. When VOUS were treated as false positives, CMA was dominated by all other strategies (i.e. it was more costly and less effective). The authors noted that treating all VOUS or false positives would underestimate the specificity of CMA because some VOUS would in time be determined to be pathogenic. Definitive judgements about the cost-effectiveness of CMA are problematic because it is unclear what the NHS is willing to pay to detect an additional CNV.\nThe qualitative substudy aimed to inform the development of any policy product from the main study by providing an evaluation of the acceptability of CMA to major stakeholders. It brings together parents\u2019 perspectives with those of health-care providers (i.e. cytogeneticists, clinical geneticists, genetic counsellors, midwives, fetal medicine consultants and commissioners). No previous studies have evaluated CMA across this range of experiences. Two prior studies have reported parents\u2019 experience of prenatal CMA; Bernhardt et al.90 conducted 23 telephone interviews with women in the USA who received positive array results. Sixteen of the group had prenatal testing for maternal age or positive screening results. In the UK, Hillman et al.91 conducted 25 interviews with women after receiving the results of CMA, most of which were normal. In contrast, the women in our study were interviewed at least 3 months after the outcome of the pregnancy, allowing participants to comment on their experience of decision-making after a longer period of reflection. Participants were generally supportive of the process of prenatal diagnosis in general and the use of CMA in particular. However, in each of the participant groups, actual and potential problems were raised for discussion, which clustered around three key issues.\nThe first was around achieving integration of CMA into the existing care pathways. Accessing CMA involves referral to a fetal medicine centre. Health professional participants were concerned that delays in referral could reduce options for women in terms of termination of pregnancy for fetal anomaly at later gestations. The time between identification of a problem and referral to a specialist centre is a time of great stress for parents and a time when appropriate support is needed.92 Participants highlighted many advantages of CMA already reported in the literature, particularly the availability of more detailed genetic information.36 Furthermore, CMA was perceived to be a quicker test than karyotyping. Even when a novel variant was identified, the reporting time was felt to be no slower than karyotyping. Moreover, several health professionals acknowledged that, as more information became available about CNVs, reporting time for arrays was likely to reduce.25,93,94 The management of VOUS was seen by health professionals as the main challenge associated with implementation of CMA, which is consistent with previous literature.95\u201397 Professionals were primarily concerned with ensuring that parents who take up the offer of an array are well informed about the test, particularly the potential for a VOUS. Education programmes for health professionals and appropriate literature for parents were recommended, reinforcing recommendations from other studies of CMA in prenatal diagnosis.90,91 Commissioners provided a unique insight into the factors that influence commissioning decisions around new technologies. Evidence of clinical utility, improved diagnostic yield and the actual cost featured high on their list of priorities. However, they were also keen to ensure that there was equity of access to care and patients appeared central to their commissioning decisions in line with current policy.98 Finally, many participants highlighted the future impact of NIPT on care pathways, acknowledging the increased risk of miscarriage associated with invasive prenatal diagnosis. Although NIPT was seen as an appropriate part of the aneuploidy screening pathway,99 several professional participants commented that the higher diagnostic yield of CMA in cases with an ultrasound anomaly meant that this technology was more appropriate for informing pregnancy management decisions.\nThe second issue was around informed consent. Parent participants had variable understandings of the EACH study. Consistent with prior research,100 some parents participated in the research for altruistic reasons and there was some lack of recognition that the results could potentially have an impact on their own pregnancy. Many parents recalled being told about a concurrent study on NIPT and, although some parents\u2019 accounts demonstrated understanding of CMA as a genetic test, they mistakenly thought it would screen for all genetic abnormalities. These sorts of possible confusion related to CMA have been reported previously90,99 and were raised as concerns by health professionals. Even with apparent understanding at the time of consent, parents who opt for the test can still be surprised by an abnormal result and disappointed when health professionals are unable to interpret the results.90 Similarly, parents interviewed for the EACH study described feeling lured into a false sense of security by a normal karyotype result and, hence, were shocked by the array result.89 This highlights the difficulties of offering a test that is additional to standard care in a research study. Parents are unprepared for receiving such news92,101 and, when parents have not fully understood the implications of their participation in research, this can be more problematic.\nDespite many parents not fully understanding the array test, and some experiencing shock after receiving an abnormal array result, they tended to accept it on the basis that CMA offered additional information. The potential for uncertain information was either not an immediate concern for parents in the EACH study or was a risk they were prepared to take in seeking clarification/reassurance. Many described gaining a better understanding of potential problems which directly informed their decisions about pregnancy, which was experienced as empowering. The premise that \u2018knowledge is power\u2019 prevailed, which was consistent with previous studies, even though this knowledge may, in the end, be seen as \u2018toxic\u2019.90,91 Our findings suggest that informed consent for CMA is challenging because the information is just one facet of a complex scenario, which itself is a time of emotional distress for parents. Distress can have an impact on parents\u2019 recollection of what was said to them92,101 and this effect seems likely in this study.\nThe management of uncertain prognosis underpins much of the literature on prenatal diagnosis and it featured in discussions about test integration and informed consent. CMA does not necessarily offer certainty in terms of diagnosis or prognosis, raising questions about how much uncertainty can be tolerated given the potential decision to end a pregnancy. Professionals in particular were concerned about these issues; adopting an inclusive, interprofessional team approach to shared clinical decision-making was an important mechanism for managing this concern. This approach is associated with more collaborative team behaviour,102 leading to improved continuity of care and patient satisfaction.103 Our study suggests that clinicians, especially fetal medicine consultants, value this collaboration more when they are faced with uncertainty. Collaborative working was important to them in ensuring that parents received the best possible information. Other strategies for managing uncertainty were raised. A few health professionals who had faced dilemmas over VOUS saw the use of targeted arrays as a way to reconcile the ambivalence around uncertain results while acknowledging that this strategy did not remove all reporting uncertainty.85 The majority of the health professionals were keen to see CMA develop in the longer term and to gain more information to offer to parents. Developing collaborative databases of information and ensuring long-term follow-up were seen as fundamental requirements. The EACH study included a review panel to assist in decisions about reporting CNVs. Clinician experience of, and satisfaction with, the panel was variable and there was some ambiguity about the role of the panel in relation to decision-making. Although, in principle, the panel is an excellent resource for clinicians faced with uncertainty about the clinical significance of a CNV and whether or not to report it, clear guidance on what role the panel has in the final decision needs to be developed to increase the professional user experience.\nFinally, linked to the issue of managing uncertain prognosis, both parents and health professionals identified the importance of reporting of results. CMA results were reported only if it was felt that the CNV had a high probability of a phenotypic effect. This left some women with a \u2018normal\u2019 result unsure; they had understood that there would be longer-term results from the study, albeit that many seemed to have misunderstood the intricacies of the test. However, women who received prenatal testing often remained anxious about the health of their child104,105 and not reporting a result has the potential to add to the concerns of an already worried parent.\nThe geneticisation of health and disease has been identified as both dominant and problematic in how the social categories normal and abnormal are defined.106 Much of the literature on user experiences of CMA stems from its use in those with an established personhood status, for example children with developmental delay and/or congenital anomalies. In this context, the concept of informed consent is of critical importance to the understandings of the ethical and social acceptability of genetic testing as a medical practice. The issue of informed consent recurs in the literature on parent perspectives of prenatal diagnosis.88,91 The dominant biomedical narratives for prenatal diagnosis utilise notions of control, choice and reassurance, but these narratives have been criticised for being too simple.106 Factors such as strong social support and religious beliefs have influence regardless of social context while class and ethnicity are affected by social structures. The notion of choice in prenatal diagnosis has been problematised because, following the diagnosis of a serious fetal anomaly, parents have a time-limited decision about whether to continue or end the pregnancy. For some parents, this is problematic because it is conceptualised as an \u2018involuntary choice\u2019101 and, for others, the problem lies with the lack of true alternatives to termination. Decisions after prenatal diagnosis of fetal anomaly are, therefore, dependent on social context as well as individual views. Underpinning these debates is the idea that the social context in which western populations live make it difficult to say \u2018no\u2019 to new developments38,101 because the goal of a \u2018healthy baby\u2019 is perceived to be a self-evident good.107 As genetic technology has progressed, so the options for prenatal diagnosis have multiplied. Therefore, CMA is best conceptualised as a recent innovation in this dynamic sphere of activity. Many of the issues raised in the analysis of the EACH data are best understood as a part of a much bigger social and medical phenomenon. However, their occurrence in this novel situation provides opportunities to understand how those existing dilemmas have shaped the acceptability of the intervention in the study and how that may, in turn, shape policy in the future.\nThe EACH study has a number of strengths. It was designed to detect a clinically important difference in cryptic chromosomal imbalances in two distinct groups of fetuses with ultrasound anomalies after exclusion of a common aneuploidy by QF-PCR. This large cohort of women was recruited from 20 fetal medicine units with samples analysed in nine cytogenetic laboratories across England and Wales. Pilot work optimised laboratory workflows and a single oligoCGH array design was chosen, based on ISCA consensus, which optimised detection of clinically relevant imbalances while minimising VOUS rates. CNV calls and reporting decisions were made by local genetic teams but advice was available from an expert panel for challenging cases. The results are therefore likely to genuinely reflect the impact of replacing karyotyping with CMA in clinical practice. The study also included an economic analysis which investigated the cost-effectiveness of such a replacement strategy using main unit costs averaged from all nine participating laboratories. The EACH study also included an embedded qualitative study to determine the factors that influence parents\u2019 and health professionals\u2019 choice and decision-making about CMA in this context. Importantly, the sample included professionals making commissioning decisions about prenatal testing.\nThe study also has a number of limitations. Prenatal arrays were undertaken in addition to karyotyping on \u2018routine\u2019 clinical samples in busy NHS laboratories. As a result, limited amounts of fetal material were available for DNA extraction, which accounts for the relatively high CMA failure rate (3.4%) in the EACH study. Furthermore, TATs were calculated using the CPA standard for NHS laboratories even though arrays may not have been set up as soon as possible after sample receipt. Therefore, a more clinically meaningful time was calculated from the time of array set-up to final report. Unlike many prenatal array studies, we attempted to collect pregnancy outcome for all cases but this was not available in 32% of the cohort. However, we were able to get delivery details and some paediatric information from >\u200995% of the cases with an abnormal karyotype and/or CMA. Understandably, termination of pregnancy rates were very high but surprisingly, given the indications for testing, many parents opted not to have post-mortem examination, limiting the phenotypic information available for a significant number of infants. The main limitation of the health economic analysis was that cost-effectiveness was measured in terms of the incremental cost per pCNV detected. Although this is an appropriate measure for evaluating CMA, it means that it is difficult to make a firm judgement about whether or not CMA is cost-effective because it is unclear how much money the NHS is willing to pay to detect an additional pathogenic CNV. Further research to identify how much the NHS is willing to pay to detect an additional pCNV would be beneficial. In addition, we considered only costs during pregnancy, not following childbirth and, therefore, we did not capture all the potential costs and benefits associated with detected additional pCNVs. Moreover, the study was designed so that participating women underwent both karyotyping and CMA. This is an appropriate study design to investigate the diagnostic accuracy of CMA, but it meant that when the results of CMA and karyotyping were discordant it was difficult to attribute the subsequent laboratory follow-up tests and clinical follow-up to each option. It is acknowledged that, although the sample size for the qualitative study was sufficiently diverse for the purpose, few parents with a \u2018normal karyotype and abnormal CMA\u2019 were interviewed. The narratives of the two couples that did fall into this category offered a unique insight into the benefits of CMA as the test directly informed their decision to discontinue the pregnancy.\nThe EACH study began recruiting in May 2012. Since then there have been significant advances in prenatal testing for chromosomal anomalies which will have an impact on future implementation of CMA as well as future research. Numerous studies summarised in a recent meta-analysis,108 as well as the NHS RAPID project,47 have demonstrated the effectiveness of cell-free DNA in maternal blood to screen for trisomies 21, 18 and 13 using either sequencing (massively parallel or chromosome specific) or SNP-based methodology. As a result, in November 2016, the Department of Health announced the introduction of cell-free DNA as a second stage non-invasive screen for these trisomies.109 Benachi et al.110 studied the potential impact of cell-free DNA in 387 cases of structural anomaly (which included 193 cases with increased NT) and out of the 290 cases with a normal cell-free DNA analysis, 23 (7.9%) had additional pathogenic karyotypes. Unfortunately, CMA was not performed. The application of cell-free DNA is already being extended to sex chromosome aneuploidies111 and common microdeletion syndromes.112 Introduction of cell-free DNA testing is anticipated to dramatically reduce the number of women undergoing invasive testing for chromosomal anomalies. In 2012, the first report of whole-genome sequencing of amniocytes from a fetus with multiple anomalies was reported113 and, since then, further cases of prenatal exome sequencing have been reported, including a series of 30 prenatal and neonatal samples from cases with structural anomalies from Birmingham.114 Targeting exons has the advantage that only 1\u20132% of the genome is sequenced, but these key protein-coding regions contain up to 85% of mutations known to cause genetic disorders. In 2013, the Prenatal Assessment of Genome and Exomes (PAGE) project was funded with the aim of analysing 1000 exome sequences (with whole-genome sequencing in a smaller cohort) from a fetus undergoing invasive testing because of a structural anomaly.114 Samples will be collected from fetal medicine centres throughout the UK, building on the EACH collaboration, and PAGE will identify what additional information exome sequencing provides over CMA. Further studies will be needed to determine whether or not sequencing should replace CMA and, ultimately, if this technology can be applied to detect other chromosomal imbalances in cell-free fetal DNA in maternal blood.115\nConclusions\nThe EACH study was designed to provide guidance to health service providers on whether or not CMA should replace karyotyping in the prenatal diagnosis of fetal anomalies. The laboratory evaluation, conducted in nine cytogenetic laboratories across England and Wales, showed that, in the group of fetuses with a structural anomaly, array CGH detected 3.5% more clinically significant CNVs than karyotyping. Although CMA also detected more CNVs in fetuses with increased NT, the increase in pathogenic variants did not achieve statistical significance. Laboratory TATs were slightly longer for CMA than for karyotyping owing to the design of the study. However, analysis of actual set-up to reporting times indicated that, in clinical practice, CMA results would be available on average within 7 days. Cost calculations indicated that, per patient, CMA is on average \u00a3113 more costly than karyotyping. Whether or not CMA is cost-effective depends on how much money the NHS would be willing to pay to detect an additional pathogenic CNV. CMA is likely to be more cost-effective in cases with a structural anomaly than in those with increased NT. The qualitative evaluation of attitudes to CMA suggest that parents find the technology acceptable, despite the uncertainties it may introduce, and that, generally, it is acceptable to health professionals and commissioners. However, it will be important to ensure health professionals are able to better inform parents about CMA in order to enhance patient understanding of the implications of the test. The sharing of information on detected variants and the associated phenotypes and outcomes is seen as important to ensure consistent interpretation of CMA results.", "pairs": [], "interleaved": []}
+{"file": "ukeme0401_NBK423961/ack1.nxml", "text": "The authors would like to thank all the staff from each of the contributing fetal medicine units and cytogenetic laboratories. Special thanks go to the Trial Steering Committee (Natalie Teich, Sara Wynn, Katherine Payne and Alan Cameron), particularly Lorraine Gaunt (chairperson) and the Data Monitoring and Ethics Committee (Eddie Maher and Jenny Barrett), particularly Ruth Newbury-Ecob (chairperson). The authors would also like to thank the Newcastle Clinical Trials Unit, particularly Joanne Morrison (Trial Manager) and all the parents who participated in the study.\nContributions of authors\nThe first and final draft of the report was prepared by Stephen C Robson.\nThe report on the RAPID study was prepared by Lyn S Chitty.\nThe health economic methods, analysis and discussion were prepared by Stephen Morris and Talitha Verhoef.\nThe statistical analysis was prepared by Gareth Ambler.\nThe genetic methods, results and discussion were prepared by Diana G Wellesley and John A Crolla.\nThe qualitative substudy methods, analysis and discussion were prepared by Ruth Graham and Claire Leader.\nThe lay summary was prepared by Jane Fisher.\nPublications\nCallaway JLA, Shaffer LG, Chitty LS, Rosenfeld JA, Crolla JA. The clinical utility of microarray technologies applied to prenatal cytogenetics in the presence of a normal conventional karyotype: a review of the literature. Prenat Diagn 2013;33:1\u20135.\nKarampetsou E, Morrogh D, Ballard T, Waters JJ, Lench N, Chitty LS. Confined placental mosaicism: implications for fetal chromosomal analysis using microarray comparative genomic hybridization. Prenat Diagn 2014;34:98\u2013101.\nCallaway JLA, Huang S, Karampetsou E, Crolla JA. Perspective on the technical challenges involved in the implementation of array-CGH in prenatal diagnostic testing. Mol Biotechnol 2014;56:312\u201318.\nRobson SC, Crolla J, Chitty L, Wellesley D, Ambler G. Evaluation of array comparative genomic hybridisation in prenatal diagnosis of fetal anomalies (EACH study). BJOG 2015;122(Suppl. 2):3.\nData sharing statement\nAll available data can be obtained from the corresponding author.\nDisclaimers\nThis report presents independent research. The views and opinions expressed by authors in this publication are those of the authors and do not necessarily reflect those of the NHS, the NIHR, the MRC, NETSCC, the EME programme or the Department of Health. If there are verbatim quotations included in this publication the views and opinions expressed by the interviewees are those of the interviewees and do not necessarily reflect those of the authors, those of the NHS, the NIHR, NETSCC, the EME programme or the Department of Health.", "pairs": [], "interleaved": []}
+{"file": "ukeme0401_NBK423961/app6.nxml", "text": "Patients and public were involved throughout the EACH study, as follows.\nDesign: the application had a lay coapplicant (Jane Fisher, Antenatal Results and Choices, London, UK) who helped design the study and contributed to the study application and each version of the protocol.\nMethods: Jane Fisher, along with the two lay members of the Trial Steering Committee and two lay members of the Reproductive Health Research Group (Newcastle University, Newcastle, UK) contributed to the content of the patient information leaflet and the patient consent form. Jane Fisher also contributed to the interview guide used in the qualitative substudy.\nReport: Jane Fisher contributed to the content of final report and prepared the lay summary.", "pairs": [], "interleaved": []}
+{"file": "ukeme0401_NBK423961/app5.nxml", "text": "Provided by Professor Lyn Chitty (Institute of Child Health, University College London, 2015, personal communication).\nBackground\nThe presence in maternal plasma of cell-free fetal DNA, which is pregnancy specific and represents the whole of the fetal genome, offers enormous promise for the development of a range of prenatal diagnostic tests. Fetal sex can be determined by analysis of cell-free DNA and several other tests are available for definitive prenatal diagnosis of several monogenic disorders. However, it is the development of NIPT for aneuploidy that stands to have the greatest impact on maternity care. The first proof of principle studies using next-generation sequencing were published by academic units in 2008. Larger demonstration projects, led by industry, reporting high sensitivities and specificities (98% and 99%, respectively) for the detection of trisomy 21 in high-risk pregnancies were followed by the commercial launch of NIPT in Asia and the USA in 2011, and in the UK in 2012. It is clear that women and health professionals welcome NIPT but concerns have been raised that ease of access to NIPT may undermine informed consent as there is a risk of this test being seen as routine. These concerns may be overcome by careful pre- and post-test counselling, but this will require education of health professionals and adequate provision of counselling. In the UK, the limitation of NIPT services to private sector provision (at a cost of \u00a3400\u2013900) is resulting in inequality of access with increasing demands from women for implementation in public sector maternity care. However, this has significant implications for service provision. Should the test be developed in NHS laboratories or be outsourced to the commercial sector? How should we manage the 1\u20136% of pregnancies where NIPT fails? The current cost of NIPT is likely to prohibit implementation in the NHS as a replacement for traditional trisomy 21 screening: should we therefore offer it to high-risk women as an intermediate test?\nDevelopment of non-invasive prenatal testing for aneuploidy\nThe funding provided by Efficacy and Mechanism Evaluation assisted the RAPID team with the evaluation of NIPT for aneuploidy by providing consumables for the sequencing of cell-free DNA extracted from \u22481100 maternal blood samples, collected as part of the EACH study, and the development of the bioinformatics algorithm used for the massively parallel sequencing of maternal plasma. This has allowed the RAPID team to deliver an evaluation of NIPT for aneuploidy in the NHS through the NIHR Programme Grants for Applied Research RAPID programme (RP-PG-0707-10107) using sequencing performed at the local regional genetics laboratory at Great Ormond Street Hospital NHS Foundation Trust. The overall aim of this part of the RAPID programme was to deliver a report on all aspects of NIPT implementation (laboratory evaluation, health professional and patient education, and evaluation of the care pathway and a detailed health economic analysis) to the National Screening Committee to inform the decisions on if, how and when to introduce NIPT into the NHS maternity care pathway. The final report has been submitted to the national Screening Committee for comment in May 2015. In November 2016, the Department of Health announced the introduction of NIPT for new, non-invasive, prenatal Down, Edwards and Patau syndromes.109\nEvaluation of non-invasive prenatal testing for the detection of other chromosomal imbalances\nRecently, the scope of NIPT has been increased to include selected subchromosomal abnormalities, but the number of samples reported has been small, leading to considerable uncertainty in test sensitivity and specificity. As part of the RAPID programme, a novel calling pipeline based on a segmentation algorithm for the detection of fetal microdeletion and microduplication syndrome in maternal cell-free DNA has been developed. The algorithm has been tested on 31 samples with known subchromosomal abnormalities and many of those from whom these samples were taken were recruited as part of the EACH study.\nWith the same read depth used in our NIPT for aneuploidy pipeline, our algorithm detected 15 out of 18 samples with a CNV larger than 6\u2009Mb (sensitivity 83%) and three out of three samples with maternally derived abnormalities (sensitivity 100%). There were two false-positive calls in 534 samples with no known subchromosomal abnormalities (specificity 99.6%). Using funding from the Great Ormond Street Biomedical Research Centre, deeper sequencing has been performed. With the higher read depth, 26 out of the 28 samples with fetal subchromosomal abnormalities were detected.\nWe concluded that test sensitivity is a function of the fetal fraction, read depth and the fetal CNV size. Fetal fraction is a strong determinant of sensitivity and at least one of the two false negatives was due to low fetal fraction. The lack of an independent method for determining fetal fraction, especially for female fetuses, leads to uncertainty in the test sensitivity, which has implications for the future of this technique as a clinical diagnostic test.", "pairs": [], "interleaved": []}
+{"file": "ukeme0401_NBK423961/s1.nxml", "text": "Major congenital anomalies affect 1\u20131.5% of births and are a leading cause of neonatal death and disability, resulting in substantial emotional and economic burden for families and society.1 Chromosomal anomalies are responsible for 20\u201325% of major fetal anomalies.1 The national Fetal Anomaly Screening Programme (FASP) recommends ultrasound screening for congenital anomalies at 11\u201314 and 18\u201320 weeks of pregnancy.2 Early screening for chromosomal anomalies at 11\u201314 weeks incorporates ultrasound measurement of nuchal translucency (NT); 2\u20133% of pregnant women are identified as being at high risk of having a baby with the chromosomal anomaly after screening and are offered chorionic villus sampling (CVS) or amniocentesis.3 When the NT is \u2265\u20093.5\u2009mm (equivalent to a 99th centile) the risk of a chromosomal anomaly is >\u200920% and FASP recommends the offer of invasive prenatal diagnosis irrespective of other screening markers.4 Fetuses with a NT of \u2265\u20093.5\u2009mm are also at increased risk of structural anomalies and genetic syndromes.4\nScreening for structural anomalies is recommended at the 18+0 to 20+6 week ultrasound scan,5 although a recent systematic review has shown that approximately 50% of fetal structural anomalies can be detected by an ultrasound scan at 11\u201314 weeks of pregnancy.6 Approximately 15% of fetuses with a structural anomaly detected in the second trimester have an underlying chromosomal anomaly although rates vary with the specific structural anomaly and whether or not the anomaly is isolated or one of a number of ultrasound-detected defects;7,8 rates increase from 29%, when two or more structural anomalies are detected, to >\u200970% with six or more.7\nWhen women opt for invasive testing because of an increased fetal risk of chromosomal anomaly, laboratories perform quantitative fluorescent polymerase chain reaction (QF-PCR) for rapid aneuploidy detection. This test detects chromosome copy number by amplification of repeat sequences at polymorphic loci. It is efficient (one technician can examine up to 5000 samples per year) and detects trisomies 21, 18 and 13 as well as sex chromosome anomalies and triploidy within 2 days in 5\u201314% of cases.9 In addition, G-banded karyotyping of cultured cells detects other aneuploidies and large unbalanced chromosome rearrangements at a resolution of 5\u201310\u2009Mb in a further 5% of cases.10\u201312 However, karyotyping is slow (it can take 2 weeks to receive the results of the test) and labour intensive (one technician can examine \u2248250 samples/year).10 The majority of these other aneuploidies and rearrangements occur in fetuses with multiple anomalies or an increased NT.7,11,13,14 Furthermore, when a structural anomaly is suggestive of a well-defined microdeletion/duplication syndrome (e.g. del22q11), additional targeted fluorescence in situ hybridisation (FISH) or multiplex ligation-dependent probe amplification (MLPA) may be performed.12,15 When testing fails to identify a chromosomal anomaly in a fetus with a sonographic anomaly, counselling parents about aetiology and prognosis is difficult because some babies are later found to have other abnormalities and/or learning difficulties.\nArray comparative genomic hybridisation (CGH) or chromosomal microarray (CMA) involves the isolation and differential labelling of genomic deoxyribonucleic acid (DNA) from a patient and a reference (control) followed by co-hybridisation to a matrix of DNA fragments (probes) that can be short (25- to 60-mer) oligonucleotides or larger fragments (up to 250\u2009kb).16 The fluorescence ratio for each probe reflects the average copy number ratio between patient and control DNA. Use of probes with known genome positions allows identification of DNA copy number variants (CNVs).\nThe first CMA platforms used bacterial artificial chromosome (BAC) clones of 100\u2013150\u2009kb in length, generating an intense hybridisation signal with a high signal-to-noise ratio.17 BAC arrays have an average resolution of 0.5\u20131\u2009Mb and multiplexing is limited; therefore, they are suitable only for low throughput. Subsequent oligonucleotide comparative genomic hybridisation (oligoCGH) arrays use shorter probes (typically 50\u201360 base pairs), providing higher resolution (40\u2013400\u2009kb) but the shorter probes result in less specific hybridisation and lower signal-to-noise ratio. Single nucleotide polymorphism (SNP) arrays use probes that are either 25 or 50 base pairs long and, therefore, tend to have the lowest signal-to-noise ratio. The resolution of oligonucleotide-based arrays is dependent on the number and spacing (density) of the probes and the number of consecutive probes required for a CNV call. Resolution therefore varies with the array design and the calling parameters.17\nInterpretation of CMA results in a clinical setting can be challenging as copy number variation occurs in normal individuals; approximately 12% of the human genome exhibits such variation.18 Variants can be classified as pathogenic (i.e. causative of the phenotype), benign or novel variants of unknown significance (VOUS). More than 99% of benign CNVs are inherited and the vast majority of these are <\u2009500\u2009kb.19 Thus, in order to classify novel CNVs, follow-up studies (e.g. parental arrays sometimes followed by FISH, MLPA and QF-PCR on fetal/parental DNA) are often required. Regularly updated online databases that catalogue CMA results from normal individuals and those with phenotypic anomalies provide invaluable information to assist in array classification. One perceived disadvantage of the increased diagnostic yield of smaller CNVs with oligonucleotide-based (either CGH or SNP) arrays is the need for more parental follow-up tests and the increased rate of VOUS.17,20\nMicroarray technology has identified novel microdeletion/duplication syndromes, many of which are mediated by specific genomic motifs called segmental duplications. In postnatal life, these account for 25% of all recurring microdeletion/duplication cases. Array CGH has also been used to further characterise visible chromosomal rearrangements and, in some cases, unmask novel Mendelian disorders. When CMA has been applied to children with undiagnosed developmental delay and an apparently normal G-banded karyotype, 10\u201315% had pathogenic de novo CNVs of 150\u201315,000\u2009kb.21,22 The improved detection of pathogenic CNVs, together with reduced cost of arrays, led to the recommendation that CMA should be the first-line test for postnatal referrals with developmental delay and dysmorphism.23 UK guidelines, published in 2011,24 recommended postnatal CMA for the investigation of developmental delay and dysmorphism. This policy has been implemented in regional genetics laboratories across the country.\nInitial prenatal studies employed low-resolution BAC arrays on fetal DNA extracted after fetal loss25 and pathogenic CNVs were detected in \u224810% of cases. Subsequent studies confirmed that CMA could be applied to uncultured and cultured amniocytes and chorionic villi with detection of aneuploidies, known microdeletion syndromes and large unbalanced CNVs.26\u201328 Shaffer et al.29 and Coppinger et al.30 used BAC arrays in two studies of 151 and 182 prenatal samples with normal karyotypes collected for varying clinical indications. Pathogenic CNVs were detected in 1.3\u20132.7% of cases and benign CNVs in a further 8.8%. The highest yield of pathogenic CNVs (4.5%) was found in cases with an abnormal ultrasound scan.\nFollowing the introduction of higher resolution oligonucleotide arrays, Van den Veyver et al.31 used a targeted array in 300 prenatal samples from cases referred primarily for advanced maternal age. They detected 58 (19.3%) CNVs, of which 15 (5.0%) were deemed pathogenic and three (1.0%) of uncertain significance. Subsequent studies used CMA in fetuses with structural anomalies identified on ultrasound but a normal karyotype, and CNVs were reported in 2\u201315% of cases.32\u201335 In 2011, Hillman et al.36 reported a meta-analysis of eight prenatal studies which showed that overall array CGH detected 12% [95% confidence interval (CI) 8.8% to 16.4%] more chromosomal imbalances when karyotyping was normal. When the analysis was confined to fetuses with a structural anomaly on ultrasound (six studies, 359 cases) CMA detected 11.2% (95% CI 5.7% to 22.1%) more chromosomal imbalances overall. The rate fell to 5.2% (95% CI 1.9% to 13.9%) when benign CNVs were removed.36 This included 1.9% (95% CI 0.4% to 9.5%) for which the result reported was of unknown significance.36 The largest of these studies included only 151 cases. In the same year, Leung et al.37 reported the first study of CMA in a cohort of 48 fetuses with increased NT (>\u20093.5\u2009mm) at 11+2\u201314+1 weeks\u2019 gestation and a normal karyotype. CNVs were reported in six (12.5%) cases and considered pathogenic in four (8.3%) cases. Thus, at the time of commencing the Evaluation of Array Comparative genomic Hybridisation (EACH) study, the available literature suggested that CMA may detect more pathogenic CNVs in fetuses with ultrasound-identified structural anomalies and increased NT than karyotyping.\nPolicy development in NHS-based health care is complex. This is particularly apparent in the area of fetal anomaly, in which health care involves a significant degree of scientific uncertainty about prognoses, alongside difficult decision-making for parents about the care pathways and reproductive choices available to them following prenatal diagnosis. With respect to CMA, there is concern that reporting of VOUS may not only increase parental anxiety but lead parents to choose to terminate a fetus that is potentially at low risk of an adverse outcome.36 Although technological developments in fetal medicine can help reduce uncertainty, Williams38 noted that sometimes developments have been implemented without due consideration of the ethical and social issues that may arise from the technology for the parents and health professionals involved in care provision. Furthermore, implementation of health policy is influenced by a range of factors, including local level staff views about changes to service provision.39 Prior to commencing the EACH study, no work had been done on patient or professional views on prenatal CMA or on possible barriers to or facilitators of implementation. In their systematic review of prenatal CMA, Hillman et al.36 emphasised the need for qualitative research in this area.\nIn England and Wales, around 35,000 women undergo karyotyping per annum (at a cost of \u00a33.3M).10 Since CMA was introduced into prenatal diagnosis, when the cost per case was estimated at \u2248\u00a3600,40 array costs have decreased dramatically and higher-density oligonucleotide arrays are now available for <\u2009\u00a3150. However, given the reported increased detection rates of pathogenic variants and VOUS with CMA, the associated costs of additional tests, reviewing results and clinical follow-up to explain CMA results are expected to be higher than karyotyping. Thus, at the time of commencing the EACH study, there was an urgent need to assess the clinical and cost efficacy of CMA before introduction into NHS practice, as had already occurred in some US centres. Indeed, despite the limited evidence, an American College of Obstetrics and Gynecology Committee Opinion in 2009 stated that \u2018Targeted array CGH, in concert with genetic counselling, can be offered as an adjunct tool in prenatal cases with abnormal anatomic findings and a normal conventional karyotype\u2019.41\nIf CMA is shown to detect pathogenic genomic imbalances more effectively than karyotyping and at a lower cost, then array CGH should replace karyotyping, allowing the streamlining of genetic service delivery with a single diagnostic platform across prenatal and postnatal life. CMA could also dramatically further our understanding of fetal anomalies and, in so doing, enhance patient counselling and management. The rationale for the EACH study was, therefore, to evaluate the potential for CMA to replace karyotyping for the investigation of fetuses with ultrasound anomalies after rapid exclusion of common aneuploidies by QF-PCR. The specific research aim was to provide guidance to health service providers on whether CMA should replace karyotyping in the prenatal diagnosis of fetal anomalies by determining whether or not CMA is an acceptable, cost-effective and robust method to detect more clinically significant de novo chromosomal imbalances than conventional karyotyping in fetuses with (1) one or more structural anomalies identified by ultrasound and (2) an isolated NT of \u2265\u20093.5\u2009mm identified at the routine 11- to 14-week ultrasound scan.\nOver recent years, along with developments in CMA, there have also been considerable advances in the development of non-invasive prenatal testing (NIPT) based on cell-free fetal deoxyribonucleic acid (cffDNA) in maternal plasma. By 2011, when the EACH study was funded, several studies had demonstrated the potential for NIPT for common trisomies and even other large CNVs.42\u201345 The National Institute for Health Research (NIHR) had funded the Reliable Accurate Prenatal non-Invasive Diagnosis (RAPID) project to (1) develop standards for implementation of NIPT into routine NHS practice for fetal sex determination and single gene disorders, (2) evaluate the potential of NIPT for trisomy 21 and other major trisomies and (3) explore the potential for NIPT of significant unbalanced CNVs using next generating sequencing of cffDNA.46 As the EACH study would be recruiting a cohort enriched for both aneuploidy and other CNVs, combining studies would allow a large-scale evaluation of NIPT on the EACH cohort. Therefore, a secondary research aim was to contribute maternal blood samples to the RAPID project. The results of the RAPID project will be reported separately.46\nIn order to achieve these aims the specific research objectives of the EACH study were as follows.\nLaboratory evaluation\nTo compare detection rates of:\npathogenic CNVs and VOUS by karyotyping and CMA in two target populations of fetuses with ultrasound-detected anomalies after exclusion of major numerical sex and autosomal chromosomal anomalies by QF-PCR\ntrisomy 21 and other major trisomies by QF-PCR and NIPT (to be reported with the RAPID project).\nTo compare turnaround times (TATs) from receipt of sample to issuing of results for:\nkaryotyping and CMA\nQF-PCR and NIPT (to be reported with the RAPID project).\nCost issues\nTo calculate costs of karyotyping and CMA to NHS and Personal Social Services (including DNA extraction, quality metrics, labelling and follow-on tests on fetal/parental DNA).\nTo calculate the cost per additional pathogenic CNV detected by array CGH (and associated follow-on testing) relative to karyotyping.\nPublic and health professional attitudes\nTo evaluate parent and health professional attitudes to CMA.\nTo determine which factors influence parents\u2019 and health professionals\u2019 choices and decision-making about CMA (and the potential to replace karyotyping).\nAn economic evaluation of NIPT and an evaluation of public and health professional attitudes to NIPT was already included as part of the RAPID project.", "pairs": [], "interleaved": []}
+{"file": "ukeme0401_NBK423961/app3.nxml", "text": "\n(PDF download)\n", "pairs": [], "interleaved": []}
+{"file": "ukeme0401_NBK423961/app4.nxml", "text": "\n(PDF download)\n", "pairs": [], "interleaved": []}
+{"file": "ukeme0401_NBK423961/app2.nxml", "text": "\n(PDF download)\n", "pairs": [], "interleaved": []}
+{"file": "ukeme0401_NBK423961/s5.nxml", "text": "Summary of implications for practice\nThe evidence from the EACH study suggests that CMA should replace karyotyping in care pathways when the indication for fetal testing is one or more structural anomalies or an isolated NT of \u2265\u20093.5\u2009mm on ultrasound scan after a normal QF-PCR result. Replacement for both indications will ensure there is a consistent prenatal (and postnatal) diagnostic technology.\nIn order to achieve a detection rate greater than when using karyotyping, the experience from the EACH study suggests that a minimum array-CGH resolution of \u2248400\u2009kb throughout the genome is optimal.\nThe findings of the EACH study suggest that any variant that can be linked to a potential phenotype of the future child on the basis of the genes involved should be reported, regardless of the size of the imbalance. This should include high-penetrance neurosusceptibility loci. Other (incidental) variants should not be reported.\nThe evidence suggests that until more national (and international) information on prenatal variants has been collected, specifically linking clinical phenotypic and molecular data, there may be benefit in a national advisory group that can provide expert advice to local health-care professionals about variants of possible pathogenic significance. Such a group could also identify incidental findings not to be reported. The operating model of the EACH review panel could serve as a template for such a group.\nTo ensure that consistent, high-quality, information is provided to parents and to help support shared decision-making, consideration should be given to producing a national information sheet and consent form.\nSince completion of the EACH study and taking account of the findings, the Joint Committee on Genomics in Medicine published recommendations for the use of CMA in pregnancy in June 2015.116 The recommendations were endorsed by The Royal College of Pathologists in collaboration with The British Society for Genetic Medicine, The Royal College of Obstetricians and Gynaecologists and The British Maternal and Fetal Medicine Society. The implications for practice from the EACH study are consistent with the recommendations produced by the Joint Committee.\nImplications for future research\nThe evidence supporting the introduction of CMA in the prenatal diagnosis of fetal anomalies is robust. It is anticipated that NIPT for trisomies will be introduced into UK clinical practice very soon. Future research should focus on the role of next-generation sequencing not only on fetal DNA acquired by invasive testing, but also on cffDNA in maternal blood. A particular concern is the rate of false-positive results generated by high-depth sequencing. In addition, following on from the EACH health economic study and relevant to future decisions around new genetic technologies, further research is needed to decide how much money the NHS is willing to pay to detect an additional pathogenic CNV. Finally, following on from the qualitative substudy, and relevant to the introduction of next generation sequencing, further research is needed on methods of enhancing patient understanding and supporting collaborative decision-making when a CNV is detected.", "pairs": [], "interleaved": []}