RAPID NEWSLETTER JANUARY 2014

1. RAPID NEWSLETTER JANUARY 2014 Welcome to an update on the RAPID programme As I am sure most of you know, RAPID is a five year NIHR funded programme grant for applied research that aims to improve the quality of NHS prenatal diagnostic services by evaluating early non-invasive prenatal diagnosis or testing (NIPD/T) based on cell free (cf)DNA in maternal plasma. When we arrived at the acronym ‘RAPID’ we had no idea how apt it was going to be, and it is incredible to think how fast things have moved since we first got together to write the outline application in 2007. We now have UKGTN approved tests for fetal sex determination available in several regional genetics laboratories in the UK, our service at Great Ormond Street is now offering NIPD for some single gene disorders (more of this later) and NIPT for aneuploidy is widely available, albeit currently only in the private sector. Before going into detail we must thank all of you who have helped with the research, be it recruiting women undergoing invasive tests in your fetal medicine units, answering health professional questionnaires about various aspects of service delivery, or helping us to recruit women and their partners to answer questionnaires which will inform the development of educational packages and appropriate care pathways. We would certainly not be so far along the road towards delivering a comprehensive NIPD service if it were not for the >12,000 samples in the RAPID bank, samples that we and others have used/are using to develop NIPD for a variety of single gene disorders and that we are now using to develop the laboratory and bioinformatic standards to deliver NIPT for aneuploidy in our own regional laboratories. So please keep up the good work. Without your help we will not be able to continue to expand the range of NIPD we can offer for genetic disorders. We would very much like to ask you to focus on collecting samples from families at risk of the conditions listed in Table 1 on the next page as these are our priorities at the moment – Cystic fibrosis, congenital adrenal hyperplasia, Duchenne and Becker muscular dystrophies and skeletal dysplasias. For CAH and skeletal dysplasias we need maternal blood samples regardless of whether or not parents elect to undergo invasive testing as we can ascertain mutation status from the results of postnatal investigations if necessary. Just briefly, why NIPD or NIPT. Well we tend to use NIPD for those situations which are diagnostic – thus for the diagnosis of Achondroplasia or Thanatophoric dysplasia, we consider that cell free DNA testing is diagnostic and so have stuck with the term NIPD – Non-Invasive Prenatal Diagnosis. For aneuploidy the situation is different because of the risk of discordant results, as we will explain in more detail later, and confirmation of a positive NIPT – Non-Invasive Prenatal Testing - result via invasive testing is recommended. Maybe we should just stick to cell free DNA analysis; your thoughts on this would be welcomed. We hope you enjoy reading about the progress RAPID has made to date. If you have any queries or would like to help with our research please contact us at the RAPID email address – rapid@ucl.ac.uk Lyn Chitty Chief Investigator, RAPID Programme • RAPID Programme Highlights • The RAPID team have secured a funded extension to the RAPID programme to evaluate NIPT for aneuploidy in an NHS setting to provide information for the UK National Screening Committee (NSC) that will be used to inform decisions on the introduction of NIPT into the national Down Syndrome Screening (DSS) programme. • We now have over 12,000 samples in the RAPID bank to help us develop NIPD for a variety of single gene disorders, as well as developing the laboratory and bioinformatic standards required to develop NIPT for aneuploidy in our own regional laboratories. • Our website has been updated and now includes information on various aspects of NIPD/T for use by the public and health professionals. This information is readily accessible on computers, tablets, phones and other mobile devices. • We have developed a next generation sequencing panel that covers the majority of FGFR3 mutations and therefore expands the potential for accurate and speedy NIPD for families with unexpected findings in pregnancy. This panel has just been approved by the UKGTN . • The RAPID team have conducted several studies exploring patients’ and health professionals’ views regarding NIPT/D for single gene disorders and aneuploidies using interviews, focus groups and surveys. Information gathered has been used to inform development of educational packages. • We have just begun to gather the views of women who have been offered NIPT for Down syndrome and are conducting surveys and one-to-one interviews. • The RAPID team have an increasing list of well received publications, with 23 manuscripts now published and 4 submitted or in press. • The RAPID team have been successful at recent conferences including the International Society of Prenatal Diagnosis in Portugal, the American Society of Human Genetics, the British Society of Genomic Medicine and the British Maternal and Fetal Medicine Society. • How can you help? • If your service laboratory would like to develop NIPT for a specific single gene disorder not mentioned here, please contact us. If we have samples in the sample bank we would be keen to help you develop further new tests. • Finally, in order to continue to develop NIPD for a wider range of conditions please do keep recruiting women undergoing invasive tests for any single gene disorder. As we explained above we are particularly keen to have samples from women at risk of carrying a baby with CAH, CF or a skeletal dysplasia regardless of whether or not they have an invasive test. • Thank you! • We are grateful to the patients and health professionals who have given their time to attend focus groups and/or interviews to allow us to explore stakeholder opinions. • Thank you also to all those who are assisting with sample collection or recruitment to psychosocial studies. Funding Acknowledgement: This newsletter presents independent research funded by the National Institute for Health Research (NIHR) under the Programme Grants for Applied Research programme RP-PG-0707-10107 (the "RAPID" project). LSC is partly funded by the GOSH Children’s Charity and the NIHR BRC at GOSH. The views expressed here are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

2. Focus on NIPD for single gene disorders Fiona McKay, Principal Scientist, NIPD Section, NE Thames Regional Genetics Laboratory The development of NIPD for single gene disorders is being undertaken by the RAPID team and NE Thames Regional Genetics Service scientists at GOSH and a number of the regional genetics laboratories (Table 1) who are collaborating with RAPID and using samples from the RAPID sample bank. In 2012 gene dossiers for NIPD for Achondroplasia and Thanatophoric dysplasia (TD) were approved by the UKGTN. At this time the tests were based on a PCR-Restriction enzyme digest assay that we developed for four FGFR3 mutations, the most common one for Achondroplasia,1 one for TDII and the two most common mutations causing TDI.2 Whilst this approach was very effective we did have 4/53 inconclusive results in cases at risk of Achondroplasia and several cases at risk of TD where we could not detect the less common mutations. We have since developed a next generation sequencing panel for use on the MiSeq that covers the majority of FGFR3 mutations. This is proving very effective and has proved accurate in all cases tested, including the detection of one rare achondroplasia mutation and several less common TD ones. This panel has just been approved by the UKGTN and we are now beginning to see a shift from invasive to non-invasive testing for these conditions. We have offered NIPD to families at risk of several other conditions: Apertsyndrome (7), Crouzon syndrome (1), Torsion Dystonia (4), Frasers syndrome (2), Autosomal recessive polycystic kidney disease (1), Osteogenesisimperfecta (1) but usually only when we have plasma from an affected pregnancy in the sample bank. This year we are going to submit gene dossiers for NGS panels for the exclusion of the paternal CF allele and also for Apert syndrome (FGFR2) mutations. The main barrier to the development of NIPD for recessive conditions where parents carry the same mutant allele is identifying an accurate way to assess the fetal fraction. However, we are hopeful that taking the NGS panel approach to NIPD will facilitate estimation of fetal fraction. Progress in NIPD for monogenic disorders is slow, but so far as we are aware we are the only country offering definitive diagnostic testing for any single gene disorders. There is one other European centre focussing mainly on HD, but they always offer invasive testing to confirm the NIPD result, whereas, for the conditions discussed above, we do not think that is necessary. References 1. Chitty et al. 2011. Ultrasound ObstetGynecol 37: 283-9 2. Chitty et al. 2013. PrenatDiagn33:416-23 Table 1: Current status of the development of NIPD for single gene disorders in the RAPID programme in the UK • NIPT for aneuploidies • Lyn Chitty, RAPID CI , UCL Institute of Child Health, GOSH and UCLH • The development of NIPT for aneuploidies and sex chromosome anomalies has continued to move incredibly fast, and large scale studies consistently report detection rates of greater than 99% with false positive rates of 0.3-0.5%. The small false positive rate means NIPT for Down’s syndrome cannot be considered fully diagnostic and is thus being considered as an advanced screening test. Reasons for the occurrence of false positives include: • There is an increased rate of failure to report results in women with raised BMI, thought to be due to an increased rate of release of maternal cell free DNA (cfDNA) from maternal adipose tissue. • cffDNA is shed from the placenta, and so it would be reasonable to expect that NIPT might detect confined placental mosaicism. • The sequencing tests used analyse all cfDNA in the maternal blood, the majority of which emanates from the mother. Therefore maternal chromosomal rearrangements can be detected. Indeed, in one instance a ‘discordant’ result was found to be due to a maternal malignant tumour secreting aneuploidy cell lines. • We have moved rapidly from diagnosing the major trisomies with NIPT, to determining fetal sex and offering the potential to screen for sex chromosome abnormalities in early pregnancy. The ease of access of NIPT through the private sector has no precedent in prenatal diagnosis and raises significant ethical concerns. These are concerns that we must address as we consider implementation into routine public sector maternity care. Finally, despite the fact that it is only two years since the clinical introduction of NIPT for aneuploidy, we are already seeing publications on the detection of sub-chromosome rearrangements using this technology, and indeed some companies have already introduced NIPT for some microdeletion disorders such as Di George syndrome. Evaluation of NIPT for aneuploidy in the NHS Lyn Chitty, RAPID CI , UCL Institute of Child Health, GOSH and UCLH The rapid introduction into the private sector has been driven by commercial companies, but, whilst we should not procrastinate, introduction into the public sector requires further evaluation and development of the infrastructure required to deliver this test in a safe and appropriate way in routine NHS care. To that end the RAPID team have secured a funded extension to the RAPID programme to evaluate NIPT for aneuploidy in an NHS setting. This will provide information for the UK National Screening Committee (NSC) that will be used to inform decisions on the introduction of NIPT into the national Down’s Syndrome Screening (DSS) programme. In the study NIPT will be introduced into the current DSS and diagnosis pathway in five NHS maternity units in London and the South of England. These units have been chosen as they represent a variety of approaches to DSS and have a varied ethnic and social mix. We will formally evaluate the changes in uptake of DSS, NIPT and invasive testing, differences in false positive rates between DSS and NIPT, NHS and patient costs and sensitivity of NIPT in a medium-risk population as well as developing health professional and patient educational packages. We have developed an MPSS approach to NIPT in our laboratory at GOSH using a HiSeq 2500 which was provided by the GOSH Children’s Charity. As part of this project we are developing standard operating procedures and bioinformatics packages that can be rolled out to other Regional Genetics units when the study is completed and they are fully validated. We recruited the first patient to the NIPT arm of the study on 1st November 2013 and plan to end recruitment at the end of December 2014. We are working closely with the NSC and will be meeting them regularly to keep them updated.

3. Exploring women’s views on using NIPT for Down’s syndrome Celine Lewis, RAPID Research Psychologist, GOSH The successful implementation of non-invasive prenatal testing for Down’s syndrome requires us to understand the views of women (and their partner’s) who might be likely to use this new test in the future. We conducted qualitative interviews with 40 pregnant women to explore their views on NIPT including the perceived benefits and concerns around the test, and whether they would be likely to use the test if it were offered to them in a future pregnancy. In order to hear a variety of viewpoints, we spoke to women with differing experiences of prenatal testing. This included: 1) women who had declined Down’s syndrome screening; 2) women who had been identified as low risk through screening; 3) women who had been identified as high risk through screening and had declined invasive testing; and 4) women who had been identified as high risk through screening and had undergone invasive testing. Key findings The overwhelming majority of women viewed NIPT for Down’s syndrome as a positive advance in prenatal care. They highlighted numerous practical and psychological advantages of a safe test that was highly accurate and could be conducted early in pregnancy. Women also felt that a blood test specifically looking at the baby’s DNA was a much more satisfactory method compared to one which involved ultrasound measurements, maternal biomarkers and ultimately the skill of the health professional. Whilst the majority of comments women made related to the benefits of the test, a number of concerns did surface during interviews. The potential for NIPT to become routinised preventing women from making an informed decision was a concern raised, in particular because the test is simple to perform and will be one of many blood tests conducted during pregnancy. It also became evident during discussions that for many women, the availability of a medical test and the fact that it is offered by ‘trusted’ medical professionals creates a perceived need. We also identified that many women feel they should take advantage of new technologies and that by doing so, they are being responsible parents. Another concern raised by women related to the societal impact of the test. Many acknowledged the likely impact that fewer people would be born with Down’s syndrome and whether this might lead to stigma for those parents who do have children affected by the condition. Nevertheless, the vast majority of women (32 out of 40) said they would be likely to use NIPT if it were offered to them in a future pregnancy, including half of the women we spoke to who had previously declined screening. The successful introduction of NIPT into routine prenatal care will require guidelines and counselling strategies which ensure women are offered this test in a way which is appropriate, informative and safeguards informed consent. Our research exploring service users views on using NIPT for Down’s syndrome has now been published.1 References 1. Lewis et al. Public Health Genomics 2013; 16:223-232 A survey of service users’ views and likely uptake of NIPT for Down’s syndrome Celine Lewis, RAPID Research Psychologist, GOSH Results The vast majority of responders (96%) thought NIPT would be a positive development in prenatal care, and 88% of responders said they would accept the test if it were offered to them in a future pregnancy, indicating a high likely uptake. Concerning what factors were considered most important when deciding whether or not to accept prenatal tests, ‘not wanting to risk the safety of the baby’ was considered the most important factor followed by ‘the desire to have as much information as possible about the baby’. The least important factor was ‘religious beliefs’ followed by ‘the impact less people being born with Down's syndrome would have on the disabled community’. Interestingly, those women who said they would accept NIPT were significantly more likely to feel they should use all the medical tests available to them, an issue that was raised in our interview study. This highlights the importance of ensuring that it is made clear that NIPT is an optional test and is not accepted by women without sufficient knowledge about the purpose for which the test is being done. Of those responders who said they would accept NIPT, 31% said they would be likely to terminate an affected pregnancy and 37% said they would not be likely (the remainder were unsure). This suggests that there will be a significant number of women who use the test to prepare for the birth of an affected child rather than use the information to inform decisions about termination of pregnancy. Research into the type of information and support these women need during the remainder of their pregnancy will be essential to help parents prepare and minimise prenatal anxiety, but how this translates into practice when NIPT is introduced remains to be seen. Regarding service delivery, over half (51%) of responders thought NIPT should be routinely offered to all pregnant women and 26% thought it should only be offered to women found to be high risk through screening. However, until the cost of the test reduces, NIPT as a first line test is unlikely to be attractive to health care commissioners. The majority of responders didn’t mind where the test was conducted (antenatal clinic or GP) but they did want information about the test to be provided by their midwife. Most (67%) said they would be happy to make a decision about NIPT on the same day as pre-test counselling. Only 32% said they would want a few days to think about it. When we asked this same question in relation to invasive testing, 71% said they would want a few days to think about it. This may indicate that it is easier to make a decision about NIPT than invasive testing as there is no risk of miscarriage. But it could also indicate that women may not consider the implications of NIPT as carefully as they would for invasive testing, even though both are highly accurate. For that reason, it is important that appropriate pre-test counselling is provided to ensure women understand the implications of the test and make informed decisions in line with their views. References Lewis et al. BJOG 2013 (in press) Following on from our interview study, we conducted a large scale survey through four antenatal clinics, the website Mumsnet and viewers of the website and Facebook page of the support group Antenatal Results and Choices (ARC). In total, 1131 people completed the survey. Up until this point, there had been no large scale survey with potential service users conducted in the UK on the topic of NIPT. This research was therefore important in order to gauge expected uptake and generalise the findings from our qualitative work to a larger population. “The first huge benefit is the lack of invasive requirement... It takes out a level of concern. You become purely concerned about the result, not the process, which is very significant when you’re in that situation.” Carrie, high risk and had invasive testing The survey questions focused on 1) factors impacting people’s decision to accept or decline prenatal tests; 2) likely uptake of NIPT and 3) what decisions people would be likely to make if they received a positive NIPT result. “I think that’s much more useful than the current [screening] which bases itself on a number of markers including your age and all that. I think obviously it’s better if it’s looking at DNA.” Sara, high risk and had invasive testing “We live in a country where you’re able to have all sorts of tests and all sorts of care and it seems unwise not to I suppose. I mean, that’s the whole reason why they offer them so you can look after your baby as best as possible.”Elenor – low risk

4. Patient and professional views of NIPD for single gene disorders Melissa Hill, RAPID Coordinator, GOSH NIPD is set to change the options available for prenatal diagnosis for couples who are carriers of single gene disorders. The aim of this qualitative study was to determine service users’ opinions on NIPD for single gene disorders to inform implementation of these new tests. In total, we spoke with 47 health professionals (obstetricians and midwives who specialised in fetal medicine, clinical geneticists, genetic counsellors, haemoglobinopathyspecialists and screening coordinators, CF specialists and general practitioners) and 33 patients (carriers of sickle cell disease, thalassaemia and cystic fibrosis) to understand their views on this new technology. Participants were very positive about the introduction of NIPD for single gene disorders, describing benefits arising from no risk of miscarriage, earlier testing and a simple procedure. NIPD was also described as potentially being “emotionally easier” as it reduced the anxiety associated with decision-making. A number of participants spoke about the difficulty of terminating a pregnancy after a certain time point from a religious or moral standpoint, and hence an early test was preferable. Another reported advantage of NIPD cited by health professionals was that it would be easier to access than invasive testing as it is a blood test that women can have locally without needing to go to a specialist centre. Whilst discussion around NIPD was predominantly positive, concerns did emerge during interviews. A key concern was that the test had to be highly accurate and that thorough validation was essential. Health professionals emphasised that it would be important to clearly explain the limitations of the test to women. In addition to concerns about routinisation of testing and the potential for women to feel pressured into testing (due to the availability and ease of the testing procedure), there was also discussion around the potential misuse of the technology and how decisions would be made regarding which conditions were acceptable to test for. A number of health professionals raised the concern that as the technology advances it might become acceptable to test for more trivial issues. Service users and health professionals expressed some concern about testing for information only. Further work exploring views from families at risk of CF found that many would decline invasive testing for this condition because of the miscarriage risk, but would opt for NIPD to be forewarned of the baby’s condition. Health professionals’ hesitance about expanding testing options without a clear indication suggests there is a need for further debate in this area, as well as the need to establish regulations for testing and guidance from professional bodies. When discussing their preferred options for service delivery, most participants felt that NIPD should continue to be offered through specialist services such as genetics units or haemoglobinopathyscreening programmes so that current counselling and support pathways could be maintained. The findings from this work will be useful in the development of educational packages and implementation strategies for NIPD for single gene disorders. References Hill et al. Am J Med Genet A. 2013 Jul;161(7):1612-8 Hill et al. J Genet Couns. 2013 (in press) Ethical issues associated with NIPD for single gene disorders Heather Skirton, University of Plymouth Building on the previous work on NIPD for single gene disorders, we have conducted a study of the views of carriers of autosomal recessive conditions. The particular focus of this work is to ascertain potential users’ views on particular ethical issues, specifically informed consent, the father’s input into the request for the test and handling of situations where the father was not known to be a carrier and this is revealed by the fetal result. We are using qualitative methodology based on thematic analysis for this phase of the RAPID study. Purposive sampling has been used to obtain a maximum variation sample, and we have sought the views of potential fathers and as well as mothers. This far, we have collected in depth data from 26 participants (19 females and seven males) who have a risk of having a child with one of three conditions: cystic fibrosis, beta thalassaemia and spinal muscular atrophy (Type 1). We have tried strenuously to also recruit carriers of sickle cell disease (SCD) but have been unsuccessful. We are continuing our efforts in that regard as carriers of SCD may have a different approach to other parents, due to different ethnic backgrounds. While the participants view the possible use of NIPD to detect an affected fetus positively, in general they believe that use of a consent form to obtain a signed record of consent will encourage those accepting the test to think about the serious nature of the results and the possible impact on their decisions during the pregnancy. Time for consideration and discussion with a partner was also viewed by the majority as essential before consent could be given, although it was acknowledged that this was not necessary legally. Regarding the potential for incidentally determining paternal carrier status through fetal results, most respondents could not envisage a reason for a father not taking responsibility to have a carrier test himself. However, the view was expressed that, within different cultures, paternal responsibility differed according to tradition and therefore this might be an issue in some sections of the community. It was felt that where this was the case the fetus should still be tested at the mother’s request, and the father should be informed of the test and, if relevant, his inferred carrier status, with support from health professionals. In keeping with the results of other studies performed under the RAPID project, participants welcomed a test that did not carry a risk to the fetus: participants who had lost children were keen to avoid loss of a healthy fetus, although some indicated that this was a minor issue compared to losing their living child. The majority stressed that reduction of fetal risk was only a benefit providing the accuracy of the result could be assured. “The risk of miscarriage, albeit small, was something that I was really worried about initially and having something non-invasive that’s easy, straightforward, like a blood test, it does make life so much easier.”Avani, thalassaemia carrier “I didn’t like the idea of potentially having to abort at a late stage. So I think the earlier you can detect it, the better.” Jenny, CF carrier Information and education packages update Patient Information As part of the NIPT evaluation study we are piloting patient information on NIPT for Down’s Syndrome. The results of this study and an information leaflet will be published later this year. Using the data collected in the studies above, we have developed information for the public on a range of aspects of NIPD. These are on our website which has recently been updated. Information can be downloaded or viewed in static or video format on desk top computers, tablets and other mobile devices such as phones. Health Professional Education As above, using information collected in previous studies, we have developed information for health professionals on a variety of aspects of NIPD/T. These can all be found on the RAPID website (www.rapid.nhs.uk). As part of the NIPT evaluation study we are offering formal education to all midwives involved in discussing DSS and this is then being evaluated using established techniques. In this way, as part of the study we hope to produce validated health professional educational materials which can amended by the NSC if and when implementation is deemed appropriate. “If the couple is only coming for you to look for haemoglobinopathies, then it would be wrong to then say ok let’s look for everything else that there is. Where would you draw the line?” Leanne, haemoglobinopathyscreening coordinator