Dealing with uncertain results from chromosomal microarray and exome sequencing in the prenatal setting: An international cross‐sectional study with healthcare professionals

Abstract Objectives To conduct qualitative interviews with healthcare providers working in different countries to understand their experiences of dealing with uncertain results from prenatal chromosome microarray analysis (CMA) and exome sequencing (ES). Methods Semi‐structured interviews with 31 healthcare providers who report or return prenatal CMA and/or ES results (clinicians, genetic counsellors and clinical scientists) in six countries with differing healthcare systems; Australia (4), Denmark (5), Netherlands (6), Singapore (4), Sweden (6) and United Kingdom (6). The topic guide explored the main sources of uncertainty and their management. Results There was variation in reporting practices both between and across countries for variants of uncertain significance, however, there was broad agreement on reporting practices for incidental findings. There was also variation in who decides what results are reported (clinical scientists or clinicians). Technical limitations and lack of knowledge (to classify variants and of prenatal phenotypes) were significant challenges, as were turnaround times and lack of guidelines. Conclusion Health professionals around the globe are dealing with similar sources of uncertainty, but managing them in different ways, Continued dialogue with international colleagues on ways of managing uncertain results is important to compare and contrast the benefits and limitations of the different approaches.


| INTRODUCTION
Routine fetal ultrasound identifies structural abnormalities in around 3% of pregnancies. 1 Traditionally, investigations to identify the underlying aetiology have relied on cytogenetic analysis, including karyotyping. Over the last 10 years, chromosome microarray analysis (CMA) has been widely adopted as the first-line test to detect submicroscopic pathogenic copy number variations, and has been shown to increase diagnostic yield over traditional karyotyping. 2 Genome sequencing (primarily through whole exome sequencing [ES] and targeted panels) is now increasingly being used in the prenatal setting, and has increased the frequency with which genetic causes are detected with karyotyping or CMA alone, 3,4 impacting clinical management through informing decisions around continuation of pregnancy. 5 A key counselling and ethical challenge associated with CMA and ES is the potential to detect variants of uncertain significance (VUS) or incidental findings (IFs) (findings additional to the original reason for testing which are not actively searched for) which may have implications for both the fetus and the parent (s). 6 Although uncertainty in the prenatal setting is not new, the scale and types of uncertainty that may occur is increasing because of the more detailed comprehensive analysis of the fetal genome. For example, detection of VUS has been reported in around 2%-6% of prenatal cases through CMA 2,7-9 and around 4%-20% of cases through ES, 3,10,11 although the proportion is likely to decrease as new knowledge is gained and VUS are reanalysed. 11 A body of evidence has been published over recent years looking at healthcare professionals (HPs) experiences and attitudes towards returning uncertain results following prenatal CMA, and more recently ES. These studies have been conducted in countries including the Netherlands, 12 United States of America (USA), [13][14][15] United Kingdom (UK), [16][17][18] Hong Kong 19 and Australia. 20,21 The findings have highlighted differences in opinions and practice both across and within countries, regarding the management of uncertain results. 22 This is not surprising given the lack of consensus from some of the most notable professional bodies in terms of specific guidance around the reporting of uncertain results. [23][24][25][26][27][28][29] Here we report the findings from an international cross-sectional study of multidisciplinary HPs involved in prenatal diagnosis. The aims of the study were to (1) identify the different sources of uncertainties that HPs are regularly experiencing as a result of prenatal CMA and/or ES, and (2) describe how these are being managed.

| Ethical approval
Local ethical approval was gained from each participating research team (see Supporting Information Ethical Approval). LEWIS ET AL. -721

| Study design
This was a qualitative study using purposive sampling (the identification and selection of participants who are especially knowledgeable about or experienced with the phenomenon of interest) and semi-structured interviews with (1) clinical scientists-sometimes referred to as laboratory scientists-who analyse and report prenatal CMA and/or ES results, and (2) clinicians who conduct posttest counselling around prenatal CMA and/or ES (e.g., geneticists, genetic counsellors, obstetricians, maternal fetal medicine specialists). We focused on clinicians who conduct posttest counselling as they are most likely to have an in-depth understanding of what types of uncertain results get reported and how such results are managed.
However, some also had experience of conducting pretest counselling.

| Study setting
Participants from six countries took part in interviews using the same topic guides: Australia, Denmark, The Netherlands, Singapore, Sweden, and the UK. These high-income countries were chosen as they have established prenatal screening and diagnosis programs within a range of healthcare systems, and previous research has also highlighted that health professionals from some of these countries hold differing attitudes towards prenatal testing. 30 At the time of the interviews, prenatal CMA and ES (where it was available clinically) were provided as part of a national health service or in an insurance-based system in Denmark, the Netherlands, Sweden. In the UK, CMA was available as part of prenatal testing in the National Health Service, but ES was only available in research settings pending national implementation. In Australia, CMA was covered by the national health service or an insurance-based system, but ES was self-funded. In Singapore, CMA was subsidised in public hospitals but self-funded in private hospitals, and ES was entirely self-funded.

| Theoretical framework
The overarching theoretical framework that guided the research was Han's taxonomy of uncertainty whereby uncertainty in healthcare is characterised according to its fundamental sources (cause of uncertainty), issues (implications of uncertainty) and locus (with whom the uncertainty lies). 31 In particular, we referred to a taxonomy of medical uncertainties that related to clinical next-generation sequencing. 32 This was developed by Han et al. to facilitate recognition of the uncertainties inherent in each step of genomic testing and help researchers, clinicians, patients and relatives establish realistic expectations of its processes and outcomes. Whilst Han's taxonomy informed the development of the topic guides and our analysis, the aim of this research was not to develop a definitive list of uncertainties in prenatal genomics (this has largely been addressed already 32 ), but rather to gain a 'snapshot' of which uncertainties are frequently encountered by clinical scientists and clinicians in their day-to-day practice (i.e., to explore sources and locus), the associated challenges, and how they are managed (issues) across different countries.

| Recruitment
Potential participants who were experts in the field and encompassed either laboratory diagnostics or face-to-face clinical care were identified and invited to take part in an interview via email, telephone or face-to-face. This approach was taken due to the small sample of experts working in this area, most of whom are known to the research team in each participating country. The recruitment target in each country was approximately five interviews including a mix of clinical scientists and clinicians to ensure that both the clinical and laboratory viewpoints were included. Participants could be recruited from one or multiple sites. Informed consent was obtained from all participants.

| Interviews
The development of the topic guide was informed by our theoretical framework, the literature 14,15,20 and was revised following feedback from the authors. The topic guide explored the main sources of uncertainty from CMA/ES and how these uncertainties are managed.

| Data analysis
Data were analysed using thematic analysis 33 using a 'codebook approach' 34 whereby questions in the topic guide initially informed 722potential codes of interest but where additional codes were identified inductively from the data. Data were coded by C. Lewis and J.
Hammond and multiple iterations of the codebook were developed.
Codes were grouped into subthemes, for example, eligibility for testing, and themes for example, managing uncertainty. These were informed through the theoretical framework, but also through discussion with the authors about how the themes and subthemes logically fitted together to tell a story about uncertainty in prenatal genomics. NVivo version 12 (QSR International, Pty Ltd) was used to facilitate the initial coding and data analysis. We also employed a framework approach, whereby data was copied into an excel spreadsheet in order to conduct structured comparisons between countries for certain questions, for example, to compare approaches to returning VUS. 35

| FINDINGS
In total, 31 participants from 14 hospital sites agreed to take part (79% recruitment rate) between January 2019 and March 2020.
Twenty interviews were conducted face-to-face, ten were over telephone (range: 22-82 min; mean = 47 min) and one was via email.
Whilst this particular interview was conducted in a different manner to the other 20, it was included on the basis that it was complementary to the verbal interviews and yielded information of relevance to the research question. Participants from Australia were recruited through two sites, the Melbourne Academic Centre for Health Women's and Newborn Health Network, and are representative of Australian practitioners working in publicly funded metropolitan health services. Participants from Denmark were recruited from all the three genetic centres where prenatal samples are analysed. There is a publicly funded national screening program in Denmark resulting in relatively uniform services being provided, however new methods (CMA or ES) have been implemented prenatally at different timepoints between the three centres. Participants from the Netherlands were recruited from one of the eight academic hospitals in the Netherlands that provides prenatal genetic testing. Participants from Singapore were recruited from two out of six sites across the country where prenatal CMA and ES are performed, and are representative of Singaporean practitioners working in government funded health services. Participants from Sweden were recruited from four out of six sites across the country where prenatal CMA and ES are performed and where there is both a genetic and a specialist obstetrician taking care of the patients. Participants from the UK were recruited from one regional genetics service in London (of which there are 21). Participants in the UK were recruited from one site which is a regional genetics centre. In England, the NHS fetal anomaly screening programme ensures that there is equal access to uniform and quality-assured screening for all pregnant women.

| Participant characteristics
sources of uncertainty explored during the interview, highlighting the commonality of those uncertainties in the prenatal setting ( 'Recently, I saw a couple with a baby with a 15q11.2 microdeletion, and I mean we know that-and that was just incidental. It was incidentally picked up when they were being evaluated for risk of Turner syndrome. And there was no family history of that' Singapore 2, paediatrician 'I had a case in which we found a de novo variant that caused a fatal disease. It was a foetus that was dead intrauterine and had malformations… But I find out that the foetus is also deaf. It has two variants in the 'deaf gene' and both parents are carriers. And then I find out that the mother also has a variant in a large 'heart gene', so she may also be carrier of a heart disease'.

| Limited knowledge of prenatal phenotypes
Participants noted that our current lack of knowledge around natural history in the prenatal setting was a significant challenge for them.
'A lot of our data from gene function and gene pa-  However, one participant commented that ES 'has the potential to give an answer faster than a microarray…sort of breaking that two week boundary'. (Australia 3, clinical geneticist).

| What results are reported?
The criteria for which uncertain results are reported varied both within and across countries. For some types of uncertain results, this was reflective of national guidelines. For other uncertain results, it reflected local practices or was determined on a case-by-case basis.

T A B L E 3
Managing uncertainty in pre-and posttest counselling

Explain the limitations of the technology 'One of the things we talk about is the limitations of the test which is that negative result doesn't mean it's not genetic. It could still be genetic it just means the technology has a limitation and we haven't tested everything that we need to test'. Singapore 3, clinical geneticist
Explain that there is the potential to receive uncertain results including VUS and/or IFs An important finding was the role that HPs play in influencing the management of uncertainty by how they process and discuss it with patients. Parents have expressed that the way uncertain results are communicated has a significant impact on their experience and ability to cope with uncertainty. 40 Going forwards, it will be important to ensure adequate training for HPs returning CMA and ES results, so that only the more complex cases need to be referred to clinical genetics. Those providing pre-and posttest counselling should also have training to support parents in managing uncertainty and make decisions about their pregnancy. 41 Some attention has been given to how HPs can help manage parental uncertainty in the prenatal setting, [41][42][43] for example, through emphasising what is known and certain, and through pointing out was is structurally normal. 43 Further research in this area from the perspective of parents would be valuable. This is particularly important given that there is the potential that uncertain results may impact long-term maternal psychological outcomes 44 as well as on the parent/child relationship. 45

| Strengths and limitations
Many of the uncertainties identified in this study mapped onto the Han et al. 32 taxonomy of uncertainty in genomic sequencing. For example, both studies identified sources of uncertainty including pathogenicity, gene-phenotype associations, and test limitations as causes of uncertainty. Here, we have validated the taxonomy for the prenatal setting. This is useful in that it supports the use of the taxonomy for education and training purposes amongst health professionals working in prenatal genomics, in particular in thinking about how to ascertain where uncertainties lie and how to evaluate them from a medical standpoint. A limitation of this study was that only a small number of interviews were conducted in each country in a limited number of sites including only one site in the Netherlands and the UK.
However, the aim of the study was not to provide a comprehensive overview, but rather provide a snapshot of current practices. In- interviews conducted; however, we tried to mitigate against this by using a standardised topic guide, and interviews were co-coded.
Finally, due to resource constraints, translations were not backtranslated which is often conducted to check accuracy and quality. 46 However, the interviewers/translators were all bilingual researchers working in this area and therefore have language and content knowledge to provide technically accurate translations.

| CONCLUSION
Our study highlights the different approaches taken to manage prenatal uncertainty in genomics across differing healthcare settings.
A key question raised is whether uniformity in practices is desirable, or indeed, necessary? One value of different healthcare systems adopting differing approaches is the opportunity to compare and contrast the benefits and limitations of the various approaches through both research and meaningful discussion with international colleagues. Further research with parents in different countries to compare their preferences towards and experiences of receiving uncertain findings would also add to our understanding in this area.
Whilst uncertainty in prenatal genomics clearly raises a myriad of challenges, we should not be too disheartened; these new genomic technologies are transforming healthcare and offering new opportunities for prospective parents to make important choices about their pregnancy.