The evolving genetic etiology of conotruncal anomalies

To assess the diagnostic yield of genetic testing for antenatally detected conotruncal defects.

� Exome or genome sequencing significantly increases genetic diagnosis in fetuses with nonisolated conotruncal anomalies that is, fetuses that have other anomalies visible

| INTRODUCTION
Conotruncal heart malformations are a heterogenous group of abnormalities of the cardiac outflow tract, representing approximately 20% of all antenatally detected cardiac anomalies. 1Within this group of conotruncal anomalies, the rate of genetic diagnoses antenatally is 17%-26%. 12The detection rate varies according to the condition being studied. 3,4The commonest genetic findings in patients with conotruncal defects are trisomy 13, trisomy 18, trisomy 21 and 22q11.21deletion. 2,5The majority of studies assessing genetic conditions in antenatally detected conotruncal anomalies are over 10 years old and used karyotyping to establish a genetic diagnosis.
Currently in England prenatal exome testing is available within the NHS in certain situations, which include multiple abnormalities or raised nuchal translucency >6.5 mm plus another anomaly but not for isolated cardiac anomalies. 6In high-income countries, prenatal exome testing is currently offered variably from not being recommended outside a clinical trial (US) 7 to being considered in isolated anomalies (Canada). 8th the evolution of genetic testing and the move toward routine use of newer diagnostic methods such as microarray testing, exome sequencing (ES) and gemone sequencing (GS), it is possible that a greater proportion of fetuses with an antenatal conotruncal defect diagnosis will also have a concurrent genetic diagnosis than previously identified.Importantly, this may better help to correlate a genotype-phenotype relationship in major cardiac lesions, paving the way for both refined antenatal counseling about prognosis and providing an opportunity to develop future treatment avenues.Therefore, the aim of this project was to assess the diagnostic yield of genetic disorders in antenatally detected conotruncal defects since the clinical introduction of microarray and exome sequencing.Initially, genetic testing offered was QF-PCR for common aneuploidies and whole genome microarray; after 1 st January 2021, if no pathogenic findings were identified using these techniques, then exome sequencing was offered as part of a study.Antenatal exome sequencing was performed as previously described 9 with analysis limited to a targeted panel of 1205 genes where there is deemed sufficient evidence for a prenatal phenotype detectable by imaging. 10io sequencing was performed when possible, but these results are not shown in this study.Postnatal genetic testing offered included QF-PCR, microarray, and GS on a case-by-case basis if there were further clinical concerns.GS was not performed on all conotruncal cases after birth as this is not current clinical management and the study offering ES in cases of fetal cardiac anomalies did not extend to the postnatal period.It is worth noting that postnatal GS panels offer an analysis of different genes compared to the fetal anomaly panel.GS panels and numbers of genes tested for can be viewed at https:// www.england.nhs.uk/publication/national-genomic-test-directories/.

| Data collection
Data from identified patients were extracted and entered into a standardised Excel (Microsoft, Washington, USA) form.Maternal data collected included age, parity, BMI, consanguinity, medical history, toxin exposure, previous pregnancy abnormalities and parental cardiac history.Fetal data collected included gestational age at diagnosis, singleton/multiple pregnancy, cardiac diagnosis and details of this, additional anomalies, genetic testing performed (antenatal or postnatal, type, results), parental choice of management, survival, and details of cardiac surgery to date.If data were not documented (e.g.maternal medical history), it was assumed to be normal.Fetal growth restriction or small for gestational age was noted but not counted as an additional anomaly.

| Statistics
Data analysis was carried out using Excel and SPSS v27 (IBM, New York, US).

| Ethics
This study was discussed with the UCLH and GOSH information governance teams and registered as a clinical audit (GOSH Reg ID 3241).Trust guidance on data protection was followed.

| Cases and demographics
During the study period, 302 cases of conotruncal defects were identified antenatally.One of these was excluded from further analysis due to incomplete records, giving a final sample size of 301.
Sixteen of these fetuses (5.3%) were part of a multiple pregnancy.In six cases (2.0%) there was a history of congenital heart disease in a previous pregnancy and in one case (0.3%) there was a history of maternal or paternal congenital heart disease.

| Cases with both antenatal and postnatal genetic testing
There were 21 cases where genetic testing was performed antenatally (QF-PCR and microarray) and repeated postnatally, where genetic testing dependant on clinical circumstance.In three cases postnatal GS was performed; in two cases this showed genetic abnormalities (Noonans and Rhizomelic chondrodysplasia punctata) and in one case this was normal.The remaining 18 cases had further genetic testing, which was not GS.Of these, 17 were normal.One case had 22q11.21deletion detected antenatally, which was reconfirmed on postnatal testing at clinician request.

| Exclusion of trisomy 13/18
In modern practice, trisomy 13 and 18 are very commonly detected by a combination of first trimester screening and ultrasound scan.Removing these conditions from the results gives an overall genetic abnormality rate of 26.0% (47/181); of these 23.2% (42/181) were considered to be pathogenic or clinically significant.

| Types of cardiac anomalies
There were differences in both the rate of testing and the rate of genetic findings according to the exact type of cardiac anomaly, as seen in Table 3.For women with fetal TOF there was a 79.8% (99/124) rate of genetic testing with pathogenic findings in 25.3% (25/99) of those.shown in Table 3.

| Availability of exome sequencing
The availability of ES in addition to QF-PCR and whole genome

| Pregnancy outcomes
This was a retrospective consecutive cohort study of all antenatally detected cases of conotruncal anomalies seen in the Fetal Medicine Unit of University College London Hospital (UCLH), London, UK or in the Cardiology Department of Great Ormond Street Hospital (GOSH), London, UK from 1 st January 2018 to 31 st December 2021.Patients were referred to these tertiary clinics with a suspicion of cardiac abnormality from surrounding hospitals or within UCLH.A pre-existing database of all antenatal patients with cardiac diagnoses was searched to identify patients with the following antenatal diagnoses: tetralogy of Fallot (TOF), transposition of the great arteries (TGA), truncus arteriosus (TA), double outlet right ventricle (DORV), interrupted aortic arch (IAA) with VSD that is, typical type B IAA, common arterial trunk (CAT) and pulmonary atresia with ventricular septal defect (PA-VSD).Cases included women booked locally and referred from other units.Cardiac diagnoses were made by 2D ultrasonography performed by a consultant cardiologist.All patients were offered invasive testing to investigate genetic causes.

T A B L E 1 2
Genetic diagnoses of clinical significance in cases of antenatally detected conotruncal anomalies.Genetic diagnoses of no clinical significance in cases of antenatally detected conotruncal anomalies.
The most common was 22q11.21deletion.TOF can be subdivided depending on whether the aortic arch is left-sided (LAA) or right-sided (RAA); of the 124 cases in this study, 71 had LAA and 30 had RAA, with 23 unknown.Women with fetal TOF LAA PA-VSD, CAT, IAA and TA had smaller numbers in each group, as 13there are a significant number of genetic abnormalities found postnatally.13It is also possible that in other cases further testing was performed locally without the knowledge of the tertiary center.ES/GS was performed only in 16 of the 192 cases having genetic testing and so it is unclear what the true increased yield of ES/GS would be in this group.Additionally, WGS allows for the detection of variants in non-coding regions and increased sensitivity to detect structural variants so the application of WGS rather than ES has potential to increase diagnostic yield futher.However, in this study, all the pathogenic findings detected by genome sequencing would have been detected by exome sequencing.Due to the small cohort, the two methods have not been compared in this study.From these data, ES/GS seems to have a role in non-isolated conotruncal anomalies where no pathogenic findings were identified by standard QF-PCR and microarray testing but further work is needed in applying this technique consistently to patients with conotruncal anomalies.Due to the small sample size, further work is needed to assess the usefulness of ES/GS in isolated conotruncal anomalies.CONCLUSION Genetic abnormalities are present in approximately one quarter of cases of antenatally detected conotruncal anomalies.The commonest abnormality is 22q11.21 deetion.Exome sequencing orgenome sequencing leads to a significant increase in genetic diagnosis in nonisolated cases, but further work is needed to assess its usefulness in other groups.
5When looking at subgroups of different types of cardiac anomalies, 22q11.21deletionwas the commonest genetic abnormality in TOF and IAA, which is also in keeping with published literature.5In nn-isolated anomalies, and in TGA and DORV, aneuploidies such as trisomy 13, 18 and 21 were the most common genetic abnormality found.In TGA overall the rates of 5 |