2018 ISPD Conference

Clinical application of targeted next-generation sequencing on fetuses with congenital heart defects
Fengchang Qiao1, Ping Hu2, Wang Yan3, Zhengfeng Xu3

1Department of Prenatal Diagnosis, the Affiliated Obstetrics and Gynecology Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, China
2Department of Prental Diagnosis, The Affiliated Obstetrics and Gynecology Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital., Nanjing, China
3Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing, China
Objectives
This study aimed to determine the diagnostic yield of targeted next-generation sequencing (NGS) in prenatal diagnosis of congenital heart defects (CHDs) and for investigating the possible genetic etiology of prenatal CHD cases.
Methods
Forty-four fetuses with CHDs and normal molecular karyotypes underwent targeted NGS using DNA obtained via amniocentesis, were recruited in this study. Fetal genomic DNA was directly extracted from amniotic fluid cells in each prenatal case. A customized targeted NGS panel containing 77 CHD-associated genes was designed to detect variants in the coding regions and the splicing sites of these genes. The detected variants were then interpreted following the guidelines recommended by American College of Medical Genetics and Genomics.
Results
In the 44 fetuses, the detection rates of pathogenic and likely pathogenic variations were 13.6% (6/44) and 2.27% (1/44), respectively. The 6 pathogenic variations were identified on genes of CHD7 (associated with CHARGE syndrome), CITED2 (associated with Tetralogy of Fallot, Ventricular Septal Defect and Atrial Septal Defect), ZFPM2 (associated with Tetralogy of Fallot), MYH6 (associated with Atrial Septal Defect, Familial Isolated Dilated Cardiomyopathy), KMT2D (associated with Kabuki syndrome). One likely pathogenic variation was on JAG1, associated with Tetralogy of Fallot and Alagille syndrome.
Conclusions
Targeted NGS of fetuses with isolated and non-isolated CHDs achieved a high diagnostic yield in our cohort, with an acceptable turnaround time for the prenatal setting. Our results have important implications for clinical management and genetic counseling.

Implementing fast whole exome sequencing (WES) as diagnostic test for fetal multiple congenital anomalies on ultrasound
Nicole Corsten-Janssen1, K. Bouman2, Joke Verheij1, Julia El Mecky1, Helga Westers1, Rianne Kinds1, Ron Suijkerbuijk3, Beike Leegte1, Arjen Scheper1, Birgit Sikkema-Raddatz4, Richard Sinke4, Irene Van Langen5, Rolf Sijmons1, Cleo van Diemen1

1University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, Netherlands
2University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands, Groningen, Groningen, Netherlands
3Department of Genetics, University Medical Centre Groningen, University of Groningen, 9700 RB, Groningen, Netherlands
4Department of Genetics, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands
5University Medical Center Groningen, University of Groningen, Groningen, Netherlands
Objectives
Identifying the cause of fetal anomalies seen on ultrasound is paramount to improve reproductive choice and/or perinatal management. The conventional test (chromosomal microarray) leads to a diagnosis in  approximately 25% of fetuses with multiple congenital anomalies (MCA) on ultrasound. WES  is a promising technique to improve diagnostic yield. However, implementing WES in prenatal setting is challenging due to uncertainties around fetal phenotyping, required short turnaround times, and technical and ethical handling of incidental findings and variant interpretation. Here, we discuss the implementation of WES as a routine fast test for fetuses with MCA on ultrasound in our center.
Methods
Phase 1 (concluded): Blind retrospective WES analysis of six fetuses with known postnatal genetic diagnosis to test if this diagnosis could be made on the fetal phenotype only. Variants were filtered using human phenotype ontology (HPO) or using our  custom virtual prenatal gene panel (all known disease genes, excluding late-onset diseases).
Phase 2 (Starting March 5th): Prospective study of rapid trio WES analysis in addition to conventional genetic tests for fetuses with two or more congenital malformations on ultrasound. Measures: diagnostic yield; turnaround times; clinical consequences; differences in prenatal and postnatal genotype interpretation; and impact on couples and caregivers.
Results
Phase 1: Five of six known diagnosis could be confirmed by WES using our prenatal gene panel. HPO was not helpful in filtering variants. One causal pathogenic PTPN11 mutation was missed due to generally low capture efficiency and thus coverage in WES data. Modeling the WES pipeline shows a theoretical turn-around time of 8 working days  after the invasive procedure, which is sufficient in most cases.
Phase 2: The preliminary results of the prospective study will be available during the conference. Approximately 20 rapid trio WES analysis are expected.
Conclusions
Retrospective analysis and modeling of our pipeline show that implementing WES as a routine test in the prenatal setting is technically feasible  in our center.  With our implementation study we aim to investigate the feasibility, pro’s and con’s of prenatal WES in daily practice.

The ethics of clinical applications of germline genome modification: A systematic review of reasons
Ivy Van Dijke1, Lance Bosch2, Annelien Bredenoord3, Martina Cornel4, Sjoerd Repping2, Saskia Hendriks2

1PhD student at VU University Medical Center Amsterdam (VUMC) and Academic Medical Center University of Amsterdam (AMC), Amsterdam, Netherlands
2Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands
3UMC Utrecht, Utrecht, Netherlands
4VU University Medical Center, Amsterdam, Netherlands
Objectives
Germline genome modification is still unsafe and insufficiently effective for clinical purposes. However, the recent progress made using CRISPR-Cas has led scientists to expect to overcome the technical hurdles in the foreseeable future. This has invited a fierce debate on the socio-ethical and legal implications of germline genome modification. A systematic overview of the reasons presented in the literature in favour or against germline genome modification is missing. We aimed to identify all reasons that have been presented for or against the future clinical application of germline genome modification.
Methods
The database ‘Medline/Pubmed’ was systematically searched for articles published between January 2011 and June 2016 Articles were selected based on eligibility, and the reference lists of eligible studies were hand searched. All types of articles (e.g. reviews, opinion articles), except for original biological research were eligible. Articles covering reasons for or against clinical application of intentional modification of the nuclear DNA of the germline (i.e. embryo, zygote, gametes or precursor cells of gametes) were included.
Results
A total of 169 reasons were identified: 90 reasons for, and 79 reasons against clinical application of germline genome modification. None of the included articles mentioned more than 60/169 reasons. The reasons could be categorised into: (i) quality of life of affected individuals; (ii) safety; (iii) effectiveness; (iv) existence of a clinical need or alternative; (v) costs; (vi) effects on homo sapiens as a species; (vii) social justice; (viii) potential for misuse; (ix) special interests exercising influence; (x) parental rights and duties; (xi) comparability to acceptable processes; (xii) rights of the unborn child; (xiii) human life and dignity.
Conclusions
Clinical introduction of germline genome modification should only be considered based on reassuring outcomes of appropriate preclinical effectiveness and safety studies. In the meantime, there is an evident need for a proper pre-implementation process which should address all reasons provided. Such a pre-implantation process is essential for a responsible introduction of germline genome modification or any other new medical technique with potential large scale socio-ethical and legal implications. The provided overview of all reasons will aid in allowing for a systematic and thorough debate on the introduction of germline genome editing.

Prenatal diagnosis for single gene disorders in Victoria, Australia, 1977-2015
Alice Poulton1, Jane Halliday1, Sharon Lewis2, David Amor3, Lisa Hui4

1Murdoch Childrens Research Institute, Parkville, VIC, Australia
2Murdoch Childrens Research Institute, Parkville, Victoria, Australia
3Murdoch Children's Research Institute, Parkville, Australia
4Murdoch Childrens Research Institute, Heidelberg, VIC, Australia
Objectives
This study aimed to examine the historical and contemporary use of prenatal diagnosis (PnDx) for single gene disorders in the Australian state of Victoria (~70,000 annual births), over a 39 year period.  The type and scope of disorders for which PnDx has been done were described to reflect how such testing had changed with advances in genetic technology and knowledge of the human genome. The study also aimed to examine information on preimplantation genetic diagnosis (PGD) for single gene disorders. 
Methods
This population-based study included data held in a register of all women in Victoria who had PnDx from 1977-2015. Single gene disorders were categorised using a systematic hierarchical approach designed to reflect potential distinctive aspects of the PnDx decision-making process e.g. type of potential disability (physical or neurodevelopmental), severity, and age of onset of the disorder. Data on PGD for single gene disorders from the two clinics undertaking these tests in Victoria were categorised in the same way for comparison. Trend data were analysed using chi squared tests.
Results
There was an initial increase in PnDx for single gene disorders (χ2=19.18, p<0.001), steadying at ≈115 each year since the late 1990s. The scope of disorders has doubled (n=22 in 1993, n=45 in 2015). Most tests (68%) were for disorders that primarily impair physical ability, while disorders impairing cognitive ability comprised 21%. Adult onset conditions (3%) and disorders lethal in infancy (2%) were proportionately low. 5% were unable to be categorised. PGD for single gene disorders has seen rapid growth with approximately 25% done for adult onset conditions.
Conclusions
PnDx for single gene disorders is performed in about 1 in 500 pregnancies, with no observable changes since PGD and carrier screening became available during the past decade. Testing for adult onset disorders is not common during pregnancy, but appears to be more acceptable in the assisted reproduction setting. Advancing technologies in fetal genomic sequencing will lead to further shifts in testing for single gene disorders, however the direction of change is unknown. Understanding and reporting trends and changes can contribute to planning future service delivery, providing an overview of interest in and scope of single gene testing to date.