Severe Early-Onset Fetal Growth Restriction: The Yield of Antenatal and Postnatal Genetic Testing.

P381: Rare mechanisms causing UPD(14)pat and UPD(14)mat

eP388: Optical genome mapping capability expanded to enable detection of absence of heterozygosity

81. Maternal cardiovascular changes secondary to sildenafil intake in pregnancies complicated by severe fetal growth restriction: STRIDER trial

Background&Hypothesis: Single ventricle disease (SVD) is a subtype of congenital heart disease that frequently requires early staged surgical intervention for survival. It is well known that syndromic causes of SVD are associated with worse outcomes. However, the overall effect of variants of uncertain significance (VUSs) found on chromosomal microarray (CMA) has not been explored. We hypothesized that VUSs impart no significant effect on clinical outcomes for patients with SVD when compared to normal chromosomal findings while pathogenic variants have a negative impact on mortality and heart failure. Objective: To explore the impact of CMA VUSs on outcomes in patients with SVD. Methods: We conducted a review of consecutive patients with SVD born between 1985-2023 with a CMA and treated at our institution. Primary outcomes included heart failure, heart transplantation, cardiac arrest, and mortality. Secondary outcomes included gastrostomy tube (G-tube) insertion, tracheostomy, and extracorporeal membrane oxygenation (ECMO). We controlled for possible confounding factors such as gestational age, birth weight, and age at diagnosis. Rates of primary and secondary outcomes were compared using Fisher’s exact test. Freedom from event curves were generated using a log-rank test for comparison. Results: The cohort included 322 consecutive single ventricle patients. The CMA results showed 248 (77.02%) patients having normal CMAs, 42 (13.04%) with VUSs, and 32 (9.94%) with abnormal/pathogenic results. Of those with abnormal CMAs, 22 (68.75%) had a chromosomal abnormality associated with a known genetic syndrome. Analysis using Fisher’s exact test showed lower rates of cardiac arrest and G-tube insertion in VUSs compared to abnormal CMAs (p < 0.05). The one-year survival curve showed higher survival in the normal CMA group compared to the abnormal CMA group (p < 0.05). The cardiac arrest freedom from event curve showed lower event rates in the VUS and normal CMA group compared to the abnormal CMA group (p < 0.05). There was no difference in outcomes between VUSs and normal CMAs. Conclusions: Individuals with SVD and a VUS on CMA have survival rates comparable to those with normal CMAs. In contrast, abnormal CMAs are associated with reduced survival. These findings suggest that VUSs and normal CMAs may be less susceptible to cardiac arrest which is a critical complication of SVD.

BackgroundThe absence of heterozygosity (AOH) is a kind of genomic change characterized by a long contiguous region of homozygous alleles in a chromosome, which may cause human genetic disorders. However, no method of low-pass whole genome sequencing (LP-WGS) has been reported for the detection of AOH in a low-pass setting of less than onefold. We developed a method, termed CNVseq-AOH, for predicting the absence of heterozygosity using LP-WGS with ultra-low sequencing data, which overcomes the sparse nature of typical LP-WGS data by combing population-based haplotype information, adjustable sliding windows, and recurrent neural network (RNN). We tested the feasibility of CNVseq-AOH for the detection of AOH in 409 cases (11 AOH regions for model training and 863 AOH regions for validation) from the 1000 Genomes Project (1KGP). AOH detection using CNVseq-AOH was also performed on 6 clinical cases with previously ascertained AOHs by whole exome sequencing (WES).ResultsUsing SNP-based microarray results as reference (AOHs detected by CNVseq-AOH with at least a 50% overlap with the AOHs detected by chromosomal microarray analysis), 409 samples (863 AOH regions) in the 1KGP were used for concordant analysis. For 784 AOHs on autosomes and 79 AOHs on the X chromosome, CNVseq-AOH can predict AOHs with a concordant rate of 96.23% and 59.49% respectively based on the analysis of 0.1-fold LP-WGS data, which is far lower than the current standard in the field. Using 0.1-fold LP-WGS data, CNVseq-AOH revealed 5 additional AOHs (larger than 10 Mb in size) in the 409 samples. We further analyzed AOHs larger than 10 Mb, which is recommended for reporting the possibility of UPD. For the 291 AOH regions larger than 10 Mb, CNVseq-AOH can predict AOHs with a concordant rate of 99.66% with only 0.1-fold LP-WGS data. In the 6 clinical cases, CNVseq-AOH revealed all 15 known AOH regions.ConclusionsHere we reported a method for analyzing LP-WGS data to accurately identify regions of AOH, which possesses great potential to improve genetic testing of AOH.

Maternal sildenafil for severe fetal growth restriction (STRIDER): a multicentre, randomised, placebo-controlled, double-blind trial

Dear Editor, We conducted a comprehensive clinical assessment of our newly developed method, CNVseq-AOH, for the detection of absence of heterozygosity (AOH) using low-pass genome sequencing (LP GS) with ultra-low sequencing data in this multi-centre study. Although AOH in chromosomes does not necessarily have clinical consequences, the detection of AOH is clinically important when it is related to imprinting effects or autosomal recessive disease mechanisms.1-4 LP GS (also known as CNVseq) has enabled the detection of copy-number variants (CNVs) for its superiority in sensitivity and specificity.2, 5, 6 In China, it has been recommended as a first-line diagnostic method for foetuses displaying structural abnormalities.7 However, the investigation on the use of LP GS for the detection of AOH is limited, and furthermore, LP GS has never been reported for the detection of AOH in a low-pass setting of less than 1-fold. To establish a comprehensive clinical assessment of our newly developed method (https://github.com/helplessness/CNVseq-AOH), CNVseq-AOH, for the detection of AOH, and to investigate the optimal sequencing depth, we gathered 409 samples (by far the largest clinical samples) of amniotic fluid from three hospitals. Our data showed that CNVseq-AOH in large-scale clinical practice maintained high sensitivity (100%) and specificity (100%), providing good evidence for its clinical application. This multi-centre study demonstrated the feasibility of CNVseq-AOH for the detection of AOH in real clinical settings. First, using samples with positive AOH regions from the 1000 Genomes Project (1KGP), we performed concordant analysis for CNVseq-AOH and chromosomal microarray analysis (CMA). CNVseq-AOH was aimed to predict AOH using LP GS with ultra-low sequencing data. Compared to CMA, CNVseq-AOH showed high sensitivity and great potential to improve the genetic testing of AOH. Second, to assess and comprehensively study the performance of CNVseq-AOH in a real clinical environment, a total of 409 archived DNA samples of amniotic fluid (209 with positive AOH results (Table S1) and 200 samples with negative AOH results by CMA (Table S3)) from Women's Hospital, Zhejiang University School of Medicine, Jiaxing Maternity and Child Health Care Hospital and Anhui Province Maternity & Child Health Hospital from April 2017 to March 2023, were recruited. Among the 409 samples, CMA identified 209 cases with positive AOHs (506 AOH regions) (Figure 1). LP GS for the 409 samples were conducted on the MGISEQ-2000 platform for single-end (35 bp) sequencing as previously described.8 For these samples, an average of 71.48 M uniquely aligned high quality reads (UAHRs) were obtained, approximately 0.83-fold for each sample. After sequencing, AOH detection was performed for each sample using CNVseq-AOH. The results of CMA were blinded to individuals who were analysing using CNVseq-AOH. When using the CMA results as a reference, the diagnostic yield of CNVseq-AOH was found to be equivalent to that of CMA (Table S1). Specifically, CNVseq-AOH demonstrated a sensitivity of 100% (209/209) and a specificity of 100% (200/200) in our cohort. The overlap for the 506 AOH regions detected by CMA and CNVseq-AOH was further analysed (Table S1). It showed that ∼99.60% (504/506) of the AOHs detected by CNVseq-AOH had a reciprocal overlap of more than 50% with the AOHs detected by CMA (Table S2). Compared with CMA, two AOHs were detected to be with an overlap of less than 50% in case PS201 (Figure 2) and case PS44 (Figure 3). Case PS201 included 1 positive AOH across the whole chromosome 9 by CMA. In the CNVseq-AOH detection results, the region was divided into multiple subregions (Figure 2C), with a total overlap of less than 50% between the two methods. Further analysis revealed low-level mosaicism (∼6.7%) of the whole chromosome 9 (Figure 2E). The presence of this mosaicism can affect the performance of CNVseq-AOH, leading to the discrepancy observed between the two methods. For the ∼10.4 Mb AOH in case PS44, abnormal signals were detected in the reported regions of CMA (Figure 3C), with some regions showing relatively dispersed signals from allele difference analysis (Figure 3F). These differences may be due to the differences in detection principles between the two methods. Third, to examine how the detection sensitivity of CNVseq-AOH is affected by sequencing depth, depth evaluation was performed (Figure 1B). In total, the UAHRs for 504 AOHs were utilized to create downsampling samples (14 different sequencing depths for each sample). As a result, the performance of CNVseq-AOH in downsampling samples varied depending on the size of the AOH (Figure 4 and Table S2). The detection sensitivity of 4 AOHs (cases PS1-4) with sizes less than 5 Mb was greatly influenced by UAHR. The detection sensitivity of AOHs with sizes between 5 and 10 Mb was also influenced by UAHR (Figure 4) and reached a plateau at 20 M UAHR. For AOHs larger than 10 Mb and at the chromosome level, the impact of sequencing depth becomes less pronounced (Figure 4 and Table S2), reaching 99.74% and 99.89% at 10 M UAHR. Overall, the detection sensitivity tended to increase as the number of UAHRs increased, and it reached a plateau at 15 M UAHRs for all the 504 AOHs (Figure 4). When using 15 M UAHRs, the overall detection sensitivity is over 99.66%. Therefore, 15 M UAHRs were considered optimal for detecting AOHs using CNVseq-AOH based on our cohort, approximately 23 times (342.86 M reads) less than the existing method.9 What is more, CNVseq-AOH addressed the long-standing challenge of standard LP GS in detecting AOH. Typically, the cost of CMA for one sample is more than $600.10 In our laboratory, the overall cost of LP GS for a single case was approximately $248.8 Combining CNVseq-AOH, LP GS could achieve the same level of performance as CMA for AOH detection (> 10 Mb) at a very low sequencing depth. In summary, we developed a method for predicting AOH using LP GS data and tested its performance in this multi-centre study. CNVseq-AOH can identify regions of AOH accurately in the clinical setting and possesses great potential to improve the genetic testing of AOH. According to these findings, CNVseq-AOH has demonstrated high precision in detecting AOHs, indicating its promising application in clinical settings. Suping Li, Lijie Song, Jiansheng Zhu, Minyue Dong, Yeqing Qian, Jianjun Zhu, Zhiguo Tang and Yan Sun contributed to the conception and design of the study. Yan Sun wrote the first draft of the article. Yun Yang, Linlin Fan, Yixi Sun, Bei Liu, Min Chen, Yuqin Luo, Junjie Hu and Kai Yan designed and performed the experiments. Yan Sun, Zhonghua Wang, Fei Tang, Jianfen Man, Lina Wang, Cangcang Jia, Ping Tang, Xinyi Zhu, Chaohong Wang, Junxiang Tang, Yuanyuan Xia, Xueqin Guo, Kang Zhang and Xiaoli Wang performed data analysis. Yeqing Qian, Lijie Song, Minyue Dong and Yan Sun contributed to revising the manuscript. All authors reviewed the manuscript and approved the submitted version. We thank the patients for participating in this study. The authors declare no conflict of interest. This study was supported by the National Key R&D Program of China (2023YFC2705600). This work was also supported by the 4+X Clinical Research Project of Women's Hospital, Zhejiang University School of Medicine (ZDFY2023-4XPY201), Technology Bureau of Jiaxing, Zhejiang Province (2023AY31030), Anhui Key Research and Development Program (2022e07020031), and Zhejiang Provincial Natural Science Foundation of China (LY22H110004). These projects are non-profit research projects by the government and had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This study was approved by the Institutional Review Board of Women's Hospital, Zhejiang University School of Medicine (NO. IRB-20230313-R), Jiaxing Maternity and Child Health Care Hospital (NO. 2023−047), Anhui Province Maternity & Child Health Hospital (NO. 2023-005-01) and the Institutional Review Board of BGI (NO. BGI-IRB 23140). The data generated and analysed during the current study is not publicly available as they are patient samples and sharing them could compromise research participant privacy. The data that support the findings of this study are available on request from the corresponding author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Objective To investigate the clinical value of chromosomal microarray analysis (CMA) in identifying the genetic etiology of fetal growth restriction (FGR). Methods Eighty-five FGR cases were recruited from Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School from January 2014 to October 2016. Samples of amniotic fluid (n=74), skin tissues from aborted fetuses (n=9), umbilical cord blood (n=1) and peripheral blood from a premature infant (n=1) were collected. Affymetrix CytoScan 750K Array was used to detect copy number variation (CNV) in fetal samples. Microarray analysis or fluorescence quantitative polymerase chain reaction was further recommended for the parents if fetal CMA result was variants of unknown significance (VOUS). Karyotype analysis of umbilical cord blood was further recommended if fetal CMA result was chromosome mosaicism. Chromosome analysis of peripheral blood was further recommended for the parents if fetal CMA result of a fetus was submicroscopic CNVs. Adjusted Chi-square test was used as the statistical method. Results CMA was successful in all samples in identifying chromosomal abnormalities. Among the 36 isolated FGR cases (42.4%, 36/85), CMA identified in four cases of chromosome imbalance recombination and four cases of VOUS, and the rest 28 cases were normal. Besides, no CNV was detected. Among the other 49 FGR cases (57.6%, 49/85) with ultrasound abnormalities, there were five cases of VOUS, and five cases of chromosome imbalance recombination and nine cases of CNVs. No significance difference in the detection rate of chromosome imbalance recombination was observed between the isolated and non-isolated FGR groups [11% (4/36) vs 10% (5/49), adjusted χ2=0.000, P>0.999]. Parents of six cases of VOUS were further examined and the same variants was found in either one. One case of sex chromosome mosaicism was validated by cord blood karyotyping. One case of chromosome imbalance recombination was due to the paternal balanced translocation. Conclusions CMA is helpful in detecting the chromosome imbalance recombination in FGR cases. Key words: Fetal growth retardation; Microarray analysis; Chromosomes, human; DNA copy number variations

516: The effect of pravastatin administration on fetal and utero-placental blood hemodynamics in pregnancies complicated by severe early-onset fetal growth restriction

880: Variants of uncertain significance in prenatal microarrays

Prenatal diagnosis of Neu-Laxova syndrome with compound heterozygous variants in PHGDH in a fetus presenting increased nuchal translucency and severe early-onset fetal growth restriction in a dichorionic diamniotic twin pregnancy.

Chromosomal microarray (CMA) testing of children with intellectual disability (ID), developmental delay (DD), autism spectrum disorders (ASDs) and multiple congenital anomalies (MCAs) has been increasingly utilised in Australasia since 2010. CMA provides 10–100 times higher resolution over standard karyotyping with higher diagnostic yields.1 Palmer et al. have described the benefits and limitations of CMA testing in this journal, including detection of variants of uncertain significance (VOUS) and incidental findings (IFs).1 As these may have future health implications, informed consent is essential. With most data on CMA utility published by tertiary centres, we reviewed the practice of CMA testing at a secondary paediatric service in New Zealand. We retrospectively reviewed all 114 CMA laboratory requests for children 16 years and under in the 18-month period ending December 2012, following the introduction of CMA at Tauranga Hospital. Children frequently had more than one test indication with 75% showing DD/ID, 30% ASD and 25% MCA/dysmorphology. Current recommendations support CMA as first-line genetic testing for patients with MCA, DD/ID and ASD.1 While most requests followed these criteria, 14% did not. Recent research supports testing for other conditions, such as undiagnosed neurological disorders.2 Figure 1 illustrates that 6% of CMA tests showed a clinically significant abnormality and 19% VOUS, with the remaining 75% normal. No IFs were found. Referral rates to clinical geneticists were high with 86% of children with VOUS and 85% with clinically significant abnormalities referred. Surveys have shown paediatricians are uncomfortable explaining VOUS results to families with many desiring further education.3, 4 High referral rates may create difficulties where access to clinical geneticists is limited. Chromosomal microarray results and documented evidence of informed consent. We found 81% of children had no documented evidence of informed consent for CMA testing. This is concerning given the important ethical and health implications. It may reflect limited understanding of this new and complex diagnostic tool. The absence of specific paperwork and clinic time constraints are recognised barriers to adequate consent processes.3, 4 CMA laboratory costs were approximately $55 000 annually and $12 500 per clinically significant abnormality detected. While CMA is currently 60% more expensive than standard karyotyping, most abnormalities would have been missed by the latter. As a result of our review, we recommend improving general paediatricians' education and providing written information to staff and families to support the informed consent process. The implications for access to clinical genetics services also require consideration. The authors would like to thank Dr Alexa Kidd, Clinical Geneticist, Canterbury Health Laboratories and Tauranga Hospital Paediatric and the IT departments for their assistance with this review.

Severe early onset fetal growth restriction caused by placental dysfunction leads to high rates of perinatal mortality and neonatal morbidity. The phosphodiesterase 5 inhibitor, sildenafil, inhibits cyclic guanosine monophosphate hydrolysis, thereby activating the effects of nitric oxide, and might improve uteroplacental function and subsequent perinatal outcomes. To determine whether sildenafil reduces perinatal mortality or major morbidity. This placebo-controlled randomized clinical trial was conducted at 10 tertiary referral centers and 1 general hospital in the Netherlands from January 20, 2015, to July 16, 2018. Participants included pregnant women between 20 and 30 weeks of gestation with severe fetal growth restriction, defined as fetal abdominal circumference below the third percentile or estimated fetal weight below the fifth percentile combined with Dopplers measurements outside reference ranges or a maternal hypertensive disorder. The trial was stopped early owing to safety concerns on July 19, 2018, whereas benefit on the primary outcome was unlikely. Data were analyzed from January 20, 2015, to January 18, 2019. The prespecified primary analysis was an intention-to-treat analysis including all randomized participants. Participants were randomized to sildenafil, 25 mg, 3 times a day vs placebo. The primary outcome was a composite of perinatal mortality or major neonatal morbidity until hospital discharge. Out of 360 planned participants, a total of 216 pregnant women were included, with 108 women randomized to sildenafil (median gestational age at randomization, 24 weeks 5 days [interquartile range, 23 weeks 3 days to 25 weeks 5 days]; mean [SD] estimated fetal weight, 458 [160] g) and 108 women randomized to placebo (median gestational age, 25 weeks 0 days [interquartile range, 22 weeks 5 days to 26 weeks 3 days]; mean [SD] estimated fetal weight, 464 [186] g). In July 2018, the trial was halted owing to concerns that sildenafil may cause neonatal pulmonary hypertension, whereas benefit on the primary outcome was unlikely. The primary outcome, perinatal mortality or major neonatal morbidity, occurred in the offspring of 65 participants (60.2%) allocated to sildenafil vs 58 participants (54.2%) allocated to placebo (relative risk, 1.11; 95% CI, 0.88-1.40; P = .38). Pulmonary hypertension, a predefined outcome important for monitoring safety, occurred in 16 neonates (18.8%) in the sildenafil group vs 4 neonates (5.1%) in the placebo group (relative risk, 3.67; 95% CI, 1.28-10.51; P = .008). These findings suggest that antenatal maternal sildenafil administration for severe early onset fetal growth restriction did not reduce the risk of perinatal mortality or major neonatal morbidity. The results suggest that sildenafil may increase the risk of neonatal pulmonary hypertension. ClinicalTrials.gov Identifier: NCT02277132.

Reinterpretation of Chromosomal Microarrays with Detailed Medical History

BackgroundThe EVERREST Prospective Study is a multicentre observational cohort study of pregnancies affected by severe early-onset fetal growth restriction. The study recruits women with singleton pregnancies where the estimated fetal weight is less than the 3rd centile and below 600 g, between 20 + 0 and 26 + 6 weeks of pregnancy, in the absence of a known chromosomal, structural or infective cause.MethodThe reported study was retrospective descriptive qualitative interview study of women who had participated in the EVERREST Prospective Study. The aim of this study was to explore the experiences and perceptions of pregnant women taking part in research during a pregnancy affected by severe early-onset fetal growth restriction. Audio-recorded semi-structured telephone interviews were conducted with a purposive sample of 12 women, at least 1 year after delivery of their baby. Two of these pregnancies had ended in stillbirth and one in neonatal death, reflecting the outcomes seen in the EVERREST Prospective Study. Participants gave informed consent, were 16 years or older and were interviewed in English. A topic guide was used to ensure a consistent approach. Questions focused on pregnancy experiences, involvement with the EVERREST study and potential involvement in future research. Recordings were transcribed verbatim for thematic analysis using NVivo10.ResultsFour broad themes were identified; ‘before joining the EVERREST Prospective Study’, ‘participating in research’, ‘information and support’ and ‘looking back and looking forwards’. Each broad theme incorporated several subthemes. All participants recalled their reaction to being told their baby was smaller than expected. The way this news was given had a lasting impact. A range of benefits of participation in the EVERREST Prospective Study were described and the participants were positive about the way it was conducted. As a consequence, they were receptive to participating in future research. However, the findings suggest that research teams should be sensitive when approaching families at a difficult time or when they are already participating in other research.ConclusionsThis study highlights the willingness of pregnant women to participate in research and identifies strategies for researchers to engage participants.