An ESHG-ESHRE survey on the current practice of expanded carrier screening in medically assisted reproduction.

STUDY QUESTION: What is the current practice and views on (expanded) carrier screening ((E)CS) among healthcare professionals in medically assisted reproductive (MAR) practices in Europe? SUMMARY ANSWER: The findings show a limited support for ECS with less than half of the respondents affiliated to centres offering ECS, and substantial variation in practice between centres in Europe. WHAT IS KNOWN ALREADY: The availability of next-generation sequencing, which enables testing for large groups of genes simultaneously, has facilitated the introduction and expansion of ECS strategies, currently offered particularly in the private sector in the context of assisted reproduction. STUDY DESIGN, SIZE, DURATION: A cross-sectional survey evaluating practice and current views among professionals working in MAR practice in different European countries was designed using the online SurveyMonkey tool. The web-based questionnaire included questions on general information regarding the current practice of (E)CS in MAR and questions on what is offered, to whom the test is offered, and how it is offered. It consisted mostly of multiple-choice questions with comment boxes, but also included open questions on the respondents' attitudes/concerns relevant to (E)CS practice, and room to upload requested files (e.g. guidelines and gene panels). In total, 338 responses were collected from 8 February 2022 to 11 April 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: The online survey was launched with an invitation email from the ESHRE central office (n = 4889 emails delivered) and the European Society of Human Genetics (ESHG) central office (n = 1790 emails delivered) sent to the ESHRE and ESHG members, and by social media posts. The survey was addressed to European MAR centres or gamete banks and to centres located in non-European countries participating in the European IVF-monitoring Consortium. Two reminder emails were sent. After exclusion of 39 incomplete responses received (e.g. only background information), 299 respondents from 40 different countries were included for analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 42.5% (127/299) of respondents were affiliated to centres offering ECS. The perceived responsibility to enable prospective parents to make informed reproductive decisions and preventing suffering/burden for parents were the main reasons to offer ECS. A single ECS panel is offered by nearly 45% (39/87 received answers) of the centres offering ECS, 25.3% (22/87) of those centres offer a selection of ECS panels, and 29.9% (26/87) offer whole exome sequencing and a large in silico panel. Different ranges of panel sizes and conditions were included in the ECS panel(s) offered. Most of the respondents (81.8%; 72/88 received answers) indicated that the panels they offer are universal and target the entire population. Pathogenic variants (89.7%; 70/78 received answers), and to a lesser extent, likely pathogenic variants (64.1%%; 50/78 received answers), were included in the ECS report for individuals and couples undergoing MAR with their own gametes. According to 87.9% (80/91 received answers) of the respondents, patients have to pay to undergo an ECS test. Most respondents (76.2%; 61/80 received answers) reported that counselling is provided before and after the ECS test. Preimplantation genetic testing, the use of donor gametes, and prenatal diagnostic testing were the three main reproductive options discussed with identified carrier couples. The main reason, according to the respondents, for not offering ECS in their centre, was the lack of professional recommendations supporting ECS (52.5%; 73/139 received answers) and the high cost for couples or reimbursement not being available (49.6%; 69/139). The challenges and moral dilemmas encountered by the respondents revolved mainly around the content of the offer, including the variants classification and the heterogeneity of the panels, the counselling, and the cost of the test. LIMITATIONS, REASONS FOR CAUTION: Although the total number of respondents was acceptable, the completion rate of the survey was suboptimal. In addition, the heterogeneity of answers to open-ended questions and the ambiguity of some of the answers, along with incomplete responses, posed a challenge in interpreting survey results. It is also plausible that some questions were not easily understood by the respondents. For this reason, response and non-response bias are acknowledged as further limitations of the survey. WIDER IMPLICATIONS OF THE FINDINGS: The results of this survey could aid in identifying potential challenges or areas for improvement in the current practice of ECS in the MAR field and contribute to the discussion on how to address them. The results underline the need to stimulate a more knowledge-based debate on the complexity and the pros and cons of a possible implementation of ECS in MAR. STUDY FUNDING/COMPETING INTEREST(S): All costs relating to the development process were covered from European Society of Human Reproduction and Embryology and European Society of Human Genetics funds. There was no external funding of the development process or manuscript production. A.C. is full-time employee of Juno Genetics. L.H. declared receiving a research grant during the past 36 months from the Netherlands Organisation for Health Research and Development. She has also participated in a Health Council report of the Netherlands on preconception carrier screening and collaborated with the VSOP Dutch Genetic Alliance (patient umbrella organization on rare and genetic disorders). L.H. and C.v.E. are affiliated with Amsterdam University Medical Centre, a hospital that offers ECS in a non-commercial setting. R.V. received honoraria for presentations from Merck Academy and is unpaid board member of the executive committee of the Spanish Fertility Society. The other authors had nothing to disclose. TRIAL REGISTRATION NUMBER: N/A.

The impact of inversions across 33,924 families with rare disease from a national genome sequencing project.

Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.

PSMC3 proteasome subunit variants are associated with neurodevelopmental delay and type I interferon production.

A critical step in preserving protein homeostasis is the recognition, binding, unfolding, and translocation of protein substrates by six AAA-ATPase proteasome subunits (ATPase-associated with various cellular activities) termed PSMC1-6, which are required for degradation of proteins by 26S proteasomes. Here, we identified 15 de novo missense variants in the PSMC3 gene encoding the AAA-ATPase proteasome subunit PSMC3/Rpt5 in 23 unrelated heterozygous patients with an autosomal dominant form of neurodevelopmental delay and intellectual disability. Expression of PSMC3 variants in mouse neuronal cultures led to altered dendrite development, and deletion of the PSMC3 fly ortholog Rpt5 impaired reversal learning capabilities in fruit flies. Structural modeling as well as proteomic and transcriptomic analyses of T cells derived from patients with PSMC3 variants implicated the PSMC3 variants in proteasome dysfunction through disruption of substrate translocation, induction of proteotoxic stress, and alterations in proteins controlling developmental and innate immune programs. The proteostatic perturbations in T cells from patients with PSMC3 variants correlated with a dysregulation in type I interferon (IFN) signaling in these T cells, which could be blocked by inhibition of the intracellular stress sensor protein kinase R (PKR). These results suggest that proteotoxic stress activated PKR in patient-derived T cells, resulting in a type I IFN response. The potential relationship among proteosome dysfunction, type I IFN production, and neurodevelopment suggests new directions in our understanding of pathogenesis in some neurodevelopmental disorders.

Profiling PI3K-AKT-MTOR variants in focal brain malformations reveals new insights for diagnostic care.

Focal malformations of cortical development including focal cortical dysplasia, hemimegalencephaly and megalencephaly, are a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism and intellectual disability. Importantly, focal cortical dysplasia is the most common cause of focal intractable paediatric epilepsy. Gain and loss of function variants in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we performed deep molecular profiling of common PI3K-AKT-MTOR pathway variants in surgically resected tissues using droplet digital polymerase chain reaction (ddPCR), combined with analysis of key phenotype data. A total of 159 samples, including 124 brain tissue samples, were collected from 58 children with focal malformations of cortical development. We designed an ultra-sensitive and highly targeted molecular diagnostic panel using ddPCR for six mutational hotspots in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key clinical, neuroimaging and histopathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of 29.31%. Variant allele fractions ranged from 0.14 to 22.67% across all mutation-positive samples. Our data show that pathogenic MTOR variants are mostly associated with focal cortical dysplasia, whereas pathogenic PIK3CA variants are more frequent in hemimegalencephaly. Further, the presence of one of these hotspot mutations correlated with earlier onset of epilepsy. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or histopathology. Importantly, we could not identify these mutational hotspots in other types of surgically resected epileptic lesions (e.g. polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the focal cortical dysplasia-hemimegalencephaly spectrum. Finally, our data suggest that ultra-sensitive molecular profiling of the most common PI3K-AKT-MTOR mutations by targeted sequencing droplet digital polymerase chain reaction is an effective molecular approach for these disorders with a good diagnostic yield when paired with neuroimaging and histopathology.

Copy number variation analysis implicates novel pathways in patients with oculo-auriculo-vertebral-spectrum and congenital heart defects.

Oculo-auriculo-vertebral spectrum (OAVS) is a developmental disorder of craniofacial morphogenesis. Its etiology is unclear, but assumed to be complex and heterogeneous, with contribution of both genetic and environmental factors. We assessed the occurrence of copy number variants (CNVs) in a cohort of 19 unrelated OAVS individuals with congenital heart defect. Chromosomal microarray analysis identified pathogenic CNVs in 2/19 (10.5%) individuals, and CNVs classified as variants of uncertain significance in 7/19 (36.9%) individuals. Remarkably, two subjects had small intragenic CNVs involving DACH1 and DACH2, two paralogs coding for key components of the PAX-SIX-EYA-DACH network, a transcriptional regulatory pathway controlling developmental processes relevant to OAVS and causally associated with syndromes characterized by craniofacial involvement. Moreover, a third patient showed a large duplication encompassing DMBX1/OTX3, encoding a transcriptional repressor of OTX2, another transcription factor functionally connected to the DACH-EYA-PAX network. Among the other relevant CNVs, a deletion encompassing HSD17B6, a gene connected with the retinoic acid signaling pathway, whose dysregulation has been implicated in craniofacial malformations, was also identified. Our findings suggest that CNVs affecting gene dosage likely contribute to the genetic heterogeneity of OAVS, and implicate the PAX-SIX-EYA-DACH network as novel pathway involved in the etiology of this developmental trait.

8p23.2-pter Microdeletions: Seven New Cases Narrowing the Candidate Region and Review of the Literature.

To date only five patients with 8p23.2-pter microdeletions manifesting a mild-to-moderate cognitive impairment and/or developmental delay, dysmorphisms and neurobehavioral issues were reported. The smallest microdeletion described by Wu in 2010 suggested a critical region (CR) of 2.1 Mb including several genes, out of which FBXO25, DLGAP2, CLN8, ARHGEF10 and MYOM2 are the main candidates. Here we present seven additional patients with 8p23.2-pter microdeletions, ranging from 71.79 kb to 4.55 Mb. The review of five previously reported and nine Decipher patients confirmed the association of the CR with a variable clinical phenotype characterized by intellectual disability/developmental delay, including language and speech delay and/or motor impairment, behavioral anomalies, autism spectrum disorder, dysmorphisms, microcephaly, fingers/toes anomalies and epilepsy. Genotype analysis allowed to narrow down the 8p23.3 candidate region which includes only DLGAP2, CLN8 and ARHGEF10 genes, accounting for the main signs of the broad clinical phenotype associated to 8p23.2-pter microdeletions. This region is more restricted compared to the previously proposed CR. Overall, our data favor the hypothesis that DLGAP2 is the actual strongest candidate for neurodevelopmental/behavioral phenotypes. Additional patients will be necessary to validate the pathogenic role of DLGAP2 and better define how the two contiguous genes, ARHGEF10 and CLN8, might contribute to the clinical phenotype.

Genome-Wide DNA Methylation Analysis of a Cohort of 41 Patients Affected by Oculo-Auriculo-Vertebral Spectrum (OAVS).

Oculo-auriculo-vertebral-spectrum (OAVS; OMIM 164210) is a rare disorder originating from abnormal development of the first and second branchial arch. The clinical phenotype is extremely heterogeneous with ear anomalies, hemifacial microsomia, ocular defects, and vertebral malformations being the main features. MYT1, AMIGO2, and ZYG11B gene variants were reported in a few OAVS patients, but the etiology remains largely unknown. A multifactorial origin has been proposed, including the involvement of environmental and epigenetic mechanisms. To identify the epigenetic mechanisms contributing to OAVS, we evaluated the DNA-methylation profiles of 41 OAVS unrelated affected individuals by using a genome-wide microarray-based methylation approach. The analysis was first carried out comparing OAVS patients with controls at the group level. It revealed a moderate epigenetic variation in a large number of genes implicated in basic chromatin dynamics such as DNA packaging and protein-DNA organization. The alternative analysis in individual profiles based on the searching for Stochastic Epigenetic Variants (SEV) identified an increased number of SEVs in OAVS patients compared to controls. Although no recurrent deregulated enriched regions were found, isolated patients harboring suggestive epigenetic deregulations were identified. The recognition of a different DNA methylation pattern in the OAVS cohort and the identification of isolated patients with suggestive epigenetic variations provide consistent evidence for the contribution of epigenetic mechanisms to the etiology of this complex and heterogeneous disorder.

Opportunistic genomic screening. Recommendations of the European Society of Human Genetics.

If genome sequencing is performed in health care, in theory the opportunity arises to take a further look at the data: opportunistic genomic screening (OGS). The European Society of Human Genetics (ESHG) in 2013 recommended that genome analysis should be restricted to the original health problem at least for the time being. Other organizations have argued that 'actionable' genetic variants should or could be reported (including American College of Medical Genetics and Genomics, French Society of Predictive and Personalized Medicine, Genomics England). They argue that the opportunity should be used to routinely and systematically look for secondary findings-so-called opportunistic screening. From a normative perspective, the distinguishing characteristic of screening is not so much its context (whether public health or health care), but the lack of an indication for having this specific test or investigation in those to whom screening is offered. Screening entails a more precarious benefits-to-risks balance. The ESHG continues to recommend a cautious approach to opportunistic screening. Proportionality and autonomy must be guaranteed, and in collectively funded health-care systems the potential benefits must be balanced against health care expenditures. With regard to genome sequencing in pediatrics, ESHG argues that it is premature to look for later-onset conditions in children. Counseling should be offered and informed consent is and should be a central ethical norm. Depending on developing evidence on penetrance, actionability, and available resources, OGS pilots may be justified to generate data for a future, informed, comparative analysis of OGS and its main alternatives, such as cascade testing.

P5CS expression study in a new family with ALDH18A1-associated hereditary spastic paraplegia SPG9.

In 2015-2016, we and others reported ALDH18A1 mutations causing dominant (SPG9A) or recessive (SPG9B) spastic paraplegia. In vitro production of the ALDH18A1 product, Δ1 -pyrroline-5-carboxylate synthetase (P5CS), appeared necessary for cracking SPG9 disease-causing mechanisms. We now describe a baculovirus-insect cell system that yields mgs of pure human P5CS and that has proven highly valuable with two novel P5CS mutations reported here in new SPG9B patients. We conclude that both mutations are disease-causing, that SPG9B associates with partial P5CS deficiency and that it is clinically more severe than SPG9A, as reflected in onset age, disability, cognitive status, growth, and dysmorphic traits.