Unexpected genotypes associated with severe paediatric conditions identified in a healthy population cohort.

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Saumya Jamuar, Yasmin Bylstra, Weng Khong Lim, Jing Xian Teo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6532096/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jan, 2026 Read the published version in European Journal of Human Genetics → Version 1 posted 11 You are reading this latest preprint version Abstract The expansion of genomics provides opportunity to screen individuals beyond clinical indication yet the classification of genomic variants and implications for health outcomes in this context is still emerging. We investigated this further by analysing clinically relevant variants and expected clinical implications in a population with no reported medical conditions. Whole genomes from 9637healthy unrelated research-consented participants in Singapore were analysed focusing on 1619 genes associated with severe paediatric disease. Association between causative variants and expected phenotype was assessed in correlation with participant characteristics and medical history where available. After considering protein impact, mode of inheritance and participant demographics for 110 unique variants, further analysis was performed for 44 variants occurring in 150 participants to understand clinical implications. Most carried variants associated with a mild phenotype (cystinuria), late onset (Fabry disease) or a potentially missed phenotype (Hajdu-Cheney syndrome). However, nine participants had variants associated with severe paediatric disease predicted to be symptomatic, such as limb-girdle muscular dystrophy and spastic paraplegia.Despite a cohort selected for absence of pre-existing health conditions, individuals were identified carrying variants associated with severe paediatric conditions. Further work is required to examine for subtle clinical symptoms. This study revealed the challenge of predicting clinical outcomes from genotype-derived screening and emphasises the importance of expanding phenotype characterisation which is highly relevant in population and reproductive screening settings. Trial registration: NCT02791152 Biological sciences/Genetics/Clinical genetics/Disease genetics Biological sciences/Genetics/Clinical genetics Population genomic screening genetic carrier screening genotype-phenotype correlation Figures Figure 1 Introduction The expansion of genomic applications has provided the opportunity to screen individuals beyond clinical indication. Initiating genomics as a screening approach can identify individuals with genetic variants leading to a diagnosis, an increased risk of developing, or a carrier of a genetic condition that may have otherwise been undetected. If implemented at a population level, genomics screening has the potential to transition from reactionary medical treatment to preventive care, as well as helping prospective parents to understand the chance of having a child with a genetic condition. However, the classification of genomic variants and their impact on health outcomes in absence of phenotype-driven testing is still being explored. Genomic screening, when applied to unselected cohorts, has identified individuals harbouring variants of clinical significance in absence of associated clinical features. 1 The American College of Medical Genetics and Genomics (ACMG) has emphasised that further ascertainment of genotype-phenotype correlation is necessary for the application of genomics screening to predict clinical outcomes in the general population. 2 This has generated focus on the variants associated with medically actionable genetic conditions proposed by ACMG 3 in large unselected cohort studies, and more recently in healthy newborns, refining prevalence, 4 – 9 revising penetrance 7 , 10 , 11 and evaluating the appropriateness of current genetic testing criteria. 12 , 13 Analysis beyond ACMG-recommended genes is less described; however, similar findings have been reported. Using a genotype-first approach, variants associated with severe paediatric conditions have been detected in individuals with no associated clinical features. 14 , 15 A targeted analysis of highly penetrant RASopathies and Marfan syndrome demonstrated only 14% (3/19) and 22% (9/41) respectively had received a clinical diagnosis and many did not exhibit associated clinical features. 16 Overall, these studies demonstrate that clinical indication can be either absent or highly variable, potentially resulting in missed diagnoses and uncertainties in predicting clinical outcomes. In this study we analysed the presence of variants associated with highly penetrant paediatric conditions amongst 9637 unrelated research participants from two Singaporean control study cohorts: SG10K_Health and the National Heart Centre Singapore biobank. 17 , 18 All participants reported no health issues at the time of recruitment. We focused on severe paediatric conditions given they are typically selected against in control populations and have clinical relevance for screening programs such as reproductive and newborn screening. We describe the process of assessing the clinical relevance of variants in parallel with participant characteristics, using phenotypic and medical history where available. Methods Study population and characteristics Whole genome sequencing data was obtained from 9637 participants of Chinese, Indian and Malay ancestry and inferred to be unrelated to the second degree. This study comprised 9051 participants from the SG10K_Health project, 19 who were recruited as a control study cohort with no reported pre-existing health conditions and 586 participants from the National Heart Centre Singapore biobank, 17 , 18 asymptomatic as ascertained by their health screen at recruitment. Exclusion criteria for each cohort is outlined in Supplementary Table 1. All studies contributing to the study cohort were approved by relevant institutional ethics review boards as previously outlined. Participant characteristics available included age, gender, ancestry, height, weight, low-density lipoprotein cholesterol and HbA1c glucose levels. Access to electronic medical records was available to a subset of 4750 (53%) participants and of these, recontact was consented for 1750 (19%) participants. Gene panel, variant curation and bioinformatics analysis Analysis focused on 1619 genes previously defined to be associated with severe paediatric disease and autosomal dominant, autosomal recessive and X-linked inheritance (Supplementary Table 2). 15 , 20 Genome sequencing, bioinformatic analysis and variant curation has been previously described in the SG10K Health project. 19 Briefly, variants were curated according to American College of Medical Genetics and Genomics–Association for Molecular Pathology (ACMG-AMP) guidelines, 21 and those occurring in the severe paediatric gene list were extracted for further review. For the additional 586 participants from the National Heart Centre Singapore biobank, the same bioinformatic pipeline and variants analysis processes previously described for the SG10K Health project were applied with focus on the severe paediatric gene list. Variants with a minimum genotype quality of 20, minimum depth of 6 and variant allele balance of 0.2–0.8 were included. Variants associated with severe early onset disease and low read counts were manually inspected using Integrative Genomics Viewer (IGV). To minimise the possibility of pathogenic variants present as a result of clonal hematopoiesis and not a germline event, given that somatic variant burden increases with age in healthy tissues such as blood 22 , variants occurring in genes subject to clonal hematopoiesis with a variant allele fraction of < 0.35 were removed. 23 Furthermore variants occurring in genes with homologous regions or pseudogenes 24 were also considered as sequencing can be technically challenging. Variant selection and clinical association Participants carrying pathogenic/likely pathogenic (P/LP) variants in genes associated with autosomal dominant disease, homozygous for autosomal recessive disease and hemizygous for X-linked disease reported to cause symptoms in childhood were selected for further review. Compound heterozygotes were not included in this analysis as phasing could not be confirmed. To analyse the clinical significance at a variant level, each P/LP variant was further interrogated to determine the associated genetic condition, inheritance pattern, protein impact, expected phenotype and severity using sources such as ClinVar, 25 ClinGen, 26 OMIM 27 and literature. The variant impact was reviewed by clinical genetics, genetic counsellors and bioinformaticians in correlation with participant characteristics (such as age and gender) and medical records where available, to assess clinical implications These details were discussed until consensus regarding potential variant impact was achieved. Variants were classified as highly penetrant if primarily associated with disease onset occurring before age 18 years. As a paediatric cohort was also included in this study, age of onset before 5 years was applied for these participants. Disease onset primarily after 18 years was considered as adult onset or variable if manifesting in both childhood and adulthood. Phenotype severity was assessed as either profound or severe based on classification proposed by Lazarin et al. 2014 28 which takes into consideration life span impact, intellectual disability, impaired mobility, physical malformation, sensory impairment and dysmorphic features. More recent gene panel reviews in the context of newborn screening were also referred to. 29 , 30 The phenotype was annotated as mild if clinical features could potentially be undiagnosed at time of recruitment. Results Participant characteristics The study cohort comprised of 9637 participants with no reported pre-existing health conditions and inferred to be unrelated to the second degree. The age of the cohort ranged from 6–88 years (median: 47), 57% were female and genetic ancestry was inferred to be Chinese (63%), Indian (20%), Malay (17%) and other (0.18%) (Table 1). Variant analysis overview Although the analysis included genes curated to be associated with severe paediatric disease, several variables were encountered when evaluating the validity and clinical significance at a variant level. The assessment required integration of published literature, participant demographics and medical history, where available (Table 2). Overall, there were 110 unique P/LP variants in genes reported to be associated with severe paediatric conditions detected in 244 participants as either homozygous, heterozygous or hemizygous. Further review resulted in the exclusion of 66 variants and selection of 44 variants for detailed analysis (Fig. 1 ). The 66 variants excluded from further consideration included five variants ambiguous after Integrative Genomics Viewer inspection (occurring in AMT, CDH7 , EHMT1, EP300 and LMX1B ). For example, the AMT missense variant has been reported homozygous in a patient with nonketotic hyperglycinemia since birth, 31 yet medical records indicated our male participant aged 70 years was unaffected. Inspection of IGV revealed the causative variant absent in one out of ten reads suggesting heterozygosity. Sanger sequencing, which has not yet been possible, would clarify this further. One variant potentially derived from a pseudogene ( CYP21A2 ), seven variants were inferred as a product of clonal hematopoiesis (occurring in genes ARL , ASXL1 , and CBL) , and one BTD hypomorphic variant were also excluded. Additionally, 22 variants were found in genes CLCN1 , CSF1R , MAT1A, SEC23B, SLC3A1 and WFS1 more likely to be associated with recessive disease although the gene can also be associated with dominant inheritance. There were 25 variants in females associated with X-linked inheritance where no or a mild phenotype was expected and five loss of function variants were found in genes where haploinsufficiency is not expected to disrupt protein function ( FBN2 , MAP2K1 , SMC3 , TMEM67 ) (Supplementary Table 3). The remaining 44 unique P/LP variants occurring in 184 participants were interrogated further for severity and penetrance of expected phenotype, as well as reported medical history where available. The age of onset and severity was determined at a variant level using literature and reviews. For novel missense variants, variant impact was evaluated by assessing previously reported variants in the same amino acid and for novel loss of function variants, the impact of downstream loss of function variants were reviewed (Table 3). Autosomal dominant and X-linked conditions There were 46 participants that harboured a heterozygous P/LP variant in a gene reported to be associated with an autosomal dominant paediatric condition. Medical records were available for 13 (28%) participants. The most frequent variants were either variable in severity and penetrance, for example GLMN (glomuvenous malformations, OMIM 138000) or associated with mild disease in heterozygous state, for example CAV3 (subclinical muscle weakness, OMIM 614321). Some participants could be at risk of developing a condition that they were not aware of. For example two participants had the same loss of function COL1A1 variant previously described in two patients with osteogenesis imperfecta (OMIM 166200) causing fractures; 32 , 33 one participant had a FLCN variant described previously in a patient with Birt-Hogg-Dubé (OMIM 135150); one participant with a MPZ variant reported previously with late-onset Charcot-Marie-Tooth type 2 (OMIM 607736); and one participant had a PTPN11 variant detected previously in patients with Noonan syndrome (OMIM 151100). Five participants carried variants associated with severe disease: a previously reported splice site variant COL2A1 (Stickler syndrome, OMIM 10830) in a female aged 54 years; a well characterised missense variant in SPAST (spastic paraplegia, OMIM 182601) associated with paediatric onset in a female aged 35 years; and a 12 year old male (asymptomatic according to medical records) was found to carry a missense variant in ATP1A3 ( ATP1A3 -related neurological disorders, OMIM 601338). Two novel loss of function variants were also identified, FZD4 (exudative vitreoretinopathy, OMIM 133780) in a male aged 27 years and KMT2A (Wiedemann-Steiner syndrome, OMIM 605130) in a male aged 55 years, who was asymptomatic according to medical records (Supplementary Table 4). Only two hemizygous carriers were identified, both with variants in GLA (Fabry disease, OMIM 301500) associated with late onset cardiac manifestations. Autosomal recessive conditions There were 102 participants who were homozygous for autosomal recessive conditions associated with severe paediatric disease and of these, medical history was available for 38 (37%). Overall, the homozygous variants were associated with a milder phenotype or later onset, for example, the majority (76%) carried GJB2 p.Val37Ile which is associated with incomplete penetrant hearing loss in the homozygous state. As expected, of the 35 participants where medical records were available, none had associated clinical manifestations. Other variants detected in multiple participants were CFTR p.Gln1352His (8 participants) and c.1210-11T > G (2 participants) associated with congenital bilateral absence of vas deferens and CFTR -related disorders. One participant (female, 62 years) was homozygous for CFTR p.Gln1352His and was reported to have an airway infection and mucus plug that could possibly be attributed to her genotype, yet she has not yet been evaluated for CFTR -related disorders (Table 3). Four participants were found to be homozygous for a variant associated with a severe phenotype. One participant (female, 30 years) was homozygous for a loss of function variant LAMA2 p.Arg362Ter expected to cause congenital muscular dystrophy, however, unfortunately her medical records cannot be accessed. The clinical manifestations of this variant in the homozygous state are unreported. The remaining participants had homozygous variants which were either reported pathogenic in ClinVar, for example SLC26A4 (Pendred syndrome, OMIM 274600) or loss of function associated with pathogenicity in GLRB (hereditary hyperekplexia, OMIM 614619) and TUBGCP6 (microcephaly and chorioretinopathy, OMIM 251270). Of these, two medical records were available, and no associated symptoms were reported (Sup Table 2). Discussion The genomic analysis of populations is increasingly being implemented in both research and clinical settings as it offers the opportunity to enhance health screening. Phenotype characterisation has typically been ascertained in patient cohorts or individuals with a relevant family history, thereby guiding relevant clinical management recommendations. However, predicting clinical implications of genomic variants derived from individuals with no known associated phenotype presents challenges. Despite applying stringent variant classification criteria, an analysis of variants occurring in 1619 genes associated with severe paediatric disease resulted in the exclusion of 60% (66/110) variants. These exclusions were based on potential sequencing artifacts, derivatives of clonal hematopoiesis, presence in homologous gene regions, insufficient evidence for pathogenicity in heterozygous state, haploinsufficiency not expected to disrupt protein function or hypomorphic in the homozygous state. Analysis of the remaining 44 variants revealed that whilst detected in genes associated with paediatric onset, variant level assessment supported their presence in this cohort. Several variants were reported to have milder presentations such as PTPN11 p.Tyr63Cys (Noonan syndrome). Others were variable in age of onset, such as FLCN c.1432 + 1G > T (Birt-Hogg Dube syndrome) and ABCA4 p.Gly1961Glu (Stargardt disease) or specifically adult onset, as observed with GLA c.640-801G > A (Fabry disease) and GALC p.Val681Met (Krabbe disease). Despite significant pre-existing conditions being excluded for this cohort, there were nine participants harbouring P/LP variants associated with severe paediatric disease. Unfortunately recontact of these participants for further phenotyping was not possible. Contrary to the search for causative variants in affected individuals, we speculated why these variants were observed in a cohort selected to be healthy. It is possible that the participants carrying variants associated with Stickler syndrome, Wiedemann-Steiner syndrome, Pendred syndrome, limb girdle muscular dystrophy and exudative vitreoretinopathy were undiagnosed at time of study recruitment, and we note that mild and adult onset phenotypes of these conditions, although rare, have been reported. 34 – 37 For example, although the mean age of FZDA -associated exudative vitreoretinopathy is 6 years, expressivity has been reported as variable with symptoms diagnosed at 49 years in one patient. 38 The participant harbouring this variant in our study was recruited at 27 years. The pathogenicity of ATP1A3 Gly358Arg associated with ATP1A3 -related neurological disorders is reported as two stars in ClinVar. Variants at the same position Gly358Cys, Gly358Ser, Gly358Val and Gly358Asp have been described in case reports associated with alternating hemiplegia of childhood 39 – 42 and the significance of this position has been demonstrated by functional data 40 , 41 . Although in silico predictions highly favour pathogenicity for ATP1A3 Gly358Arg, its consequence has not been reported in literature. However, three variants occurring in genes SPAST , GLRB and TUBGCP6 were strongly predicted to cause a severe phenotype after reviewing protein impact and literature. There are several reports of affected patients carrying the SPAST missense variant with symptoms such as hereditary spastic paraplegias, intellectual disability and loss of speech manifesting from infancy to six years of age. 43 While the GLRB loss of function variant is novel, there are at least six downstream loss of function variants described in patients with hyperekplexia since birth. 44 Similarly, the TUBGCP6 loss of function is novel and there are only a few TUBGCP6 pathogenic variants described in literature yet one patient homozygous for a loss of function variant downstream had retinal dystrophy and severe intellectual disability. These clinical presentations would preclude inclusion in a cohort designated as healthy. In a previous study of 874 genes associated with fully penetrant severe mendelian disease amongst a cohort of 589,306 participants, 13 were found to harbour pathogenic variants with no associated clinical manifestations, 14 thus, further questioning current understandings of disease penetrance. It is therefore unclear how current penetrance estimates and disease severity derived from patient cohorts translate to variants detected in unselected populations. There is increasing recognition that reproductive carrier screening should be offered to population-wide 45 and the clinical utility of proactive genomics screening at newborn and childhood has recently been explored. 8 , 9 The genes selected for screening are typically associated with severe disease such as those with life-limiting impacts or significant reductions in quality of life. Our findings highlight the complexities of predicting clinical impact from such genes which is particularly relevant in settings when counselling at-risk couples regarding disease onset and severity. While early identification of certain genetic conditions in otherwise healthy individuals may offer opportunities for prevention or early intervention the benefit of screening must be balanced with the potential psychological harm or anxiety, particularly for individuals who may never develop the condition. When applying this information for reproductive planning it is important to understand the likelihood of having an affected child to make a truly informed decision, which can be challenged when risk prediction is uncertain. These ethical complexities underscore the importance of informed consent, clear communication, and the need for careful consideration of the potential harms and benefits. Such considerations will need to be refined as further research regarding reduced penetrance, milder presentations, and late-onset variants in diverse populations expands. This study provides insights into the application of genomics screening at a population level, but it has several limitations due to the constraints of the available data. A major limitation was the inability to obtain detailed phenotypic information for all the participants carrying severe disease or highly penetrant clinically actionable variants. While no significant medical issues were documented at time of recruitment, it is possible that some individuals were taking medication which masked symptoms or may have developed symptoms after recruitment. Although medical record access is a common data source to document clinical outcomes, it is subject to misclassification or incomplete records presenting gaps in reliability. 10 , 46 The ability to capture relevant medical data emphasises the importance of recontact and follow-up to document prospective health outcomes and an important consideration at the time of study design and consent development. 14 , 15 Additionally, the significance of large cohort sizes is apparent when only a small minority of participants harbour variants warranting further investigation, highlighting the benefit of collaborative efforts to explore a range of variables potentially impacting clinical outcomes such as genetic modifiers and environmental factors. However, even in the absence of large cohort sizes, this study demonstrates that it is still possible to identify participants who carry unexpected genotypes. The genes analysed in this study were selected as they have been previously reported to be highly penetrant and associated with severe paediatric disease. However, our analysis of an apparently healthy cohort identified asymptomatic individuals, introducing significant implications for tailoring health screening advice in population screening programs and predicting clinical outcomes in reproductive carrier screening. Further work regarding characterisation of clinically significant variants generated from population sequencing in parallel with patient cohort studies will help refine existing understandings regarding the penetrance, phenotypic variability and clinical impact of genomic variants. This study revealed the challenges of understanding the clinical ramifications from genotype-derived screening and emphasises the importance of expanding phenotype characterisation which is highly relevant within the realms of population genomics and reproductive screening settings. Declarations Data availability Data is available in both Results and Supplementary Materials. Further data can be made available from the corresponding author by reasonable request. Ethical Approval Ethics approval and consent to participate from participants was obtained from six institutes: Growing Up in Singapore Towards healthy Outcomes birth cohort study (GUSTO), National University Hospital, Singapore CIRB/E/2019/2655 NCT01174875; Health for Life In Singapore Study (HELIOS), Nanyang Technology University, Singapore, IRB 2016-11-030; Singapore Multi-Ethnic Cohort Study (MEC), National University of Singapore, CIRB 13–512; SingHealth Duke-NUS Institute of Precision Medicine (PRISM), SingHealth Centralised Institutional Review Board, 2013/605/C NCT02791152; Singapore Epidemiology of Eye Diseases study (SEED), SingHealth Centralised Institutional Review Board, 2018/2717; Tan Tock Seng Hospital (TTSH ), National Health Group, TB-2020-001 & BTC-2020-001. All experiments were performed in accordance with relevant guidelines and regulations. This research conforms to the principles of the Helsinki Declaration. Competing Interests The authors declare that they have no competing interests. Funding statement This study made use of data generated as part of the Singapore National Precision Medicine program funded by the Industry Alignment Fund (Pre-Positioning) (IAF-PP: H17/01/a0/007). This study made use of data / samples collected in the following cohorts in Singapore: 1. The Health for Life in Singapore (HELIOS) study at the Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore (supported by grants from a Strategic Initiative at Lee Kong Chian School of Medicine, the Singapore Ministry of Health (MOH) under its Singapore Translational Research Investigator Award (NMRC/STaR/0028/2017) and the IAF-PP: H18/01/a0/016); 2. The Growing up in Singapore Towards Healthy Outcomes (GUSTO) study, which is jointly hosted by the National University Hospital (NUH), KK Women’s and Children’s Hospital (KKH), the National University of Singapore (NUS) and the Singapore Institute for Clinical Sciences (SICS), Agency for Science Technology and Research (A*STAR) (supported by the Singapore National Research Foundation under its Translational and Clinical Research (TCR) Flagship Programme and administered by the Singapore Ministry of Health’s National Medical Research Council (NMRC), Singapore - NMRC/TCR/004-NUS/2008; NMRC/TCR/012-NUHS/2014. Additional funding is provided by SICS and IAF-PP H17/01/a0/005); 3. The Singapore Epidemiology of Eye Diseases (SEED) cohort at Singapore Eye Research Institute (SERI) (supported by NMRC/CIRG/1417/2015; NMRC/CIRG/1488/2018; NMRC/OFLCG/004/2018); 4. The Multi-Ethnic Cohort (MEC) cohort (supported by NMRC grant 0838/2004; BMRC grant 03/1/27/18/216; 05/1/21/19/425; 11/1/21/19/678, Ministry of Health, Singapore, National University of Singapore and National University Health System, Singapore); 5. The SingHealth Duke-NUS Institute of Precision Medicine (PRISM) cohort was supported by core funding from SingHealth and Duke-NUS Institute of Precision Medicine (PRISM) and centre grant awarded to the National Heart Centre Singapore from the National Medical Research Council, Ministry of Health, Singapore (NMRC/CG/M006/2017_NHCS and MOH-000985) as well as NMRC/STaR/0011/2012, NMRC/STaR/ 0026/2015, Lee Foundation and Tanoto Foundation. 6. The TTSH Personalised Medicine Normal Controls (TTSH) cohort funded (supported by NMRC/CG12AUG17 and CGAug16M012). The National Precision Medicine Programme (NPM) PHASE II FUNDING (MOH-000588) supports W.K.L. The views expressed are those of the author(s) are not necessarily those of the National Precision Medicine investigators, or institutional partners. Author contributions Conceptualization: DA, SJ, MM, JH, YB, PT, KY. Data curation: WL, JT, YB. Formal analysis YB, DA, SJ, MM, JH. Funding acquisition: PT, KY, SJ, WK. Investigation: YB, SJ, DA. Methodology: YB, SJ, DA. Writing review and editing: SJ, DA. wrote the manuscript with support from DA and SJ. All authors read and approved the final manuscript. Acknowledgements We thank all investigators, staff members and study participants who made the National Precision Medicine Project possible. We also acknowledge the clinical research assistants for their roles in recruitment and participant contact as well as the study leads for their assistance in obtaining medical history. We would also like to thank the Lee Foundation for grant support to the SingHEART study conducted at the National Heart Centre Singapore. References Plon, S. and G. 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Clapcote, Genetically altered animal models for ATP1A3-related disorders . Dis Model Mech, 2021. 14(10). Paciorkowski, A.R., et al., Novel mutations in ATP1A3 associated with catastrophic early life epilepsy, episodic prolonged apnea, and postnatal microcephaly . Epilepsia, 2015. 56(3): p. 422–30. Pereira, P., et al., A Distinct Phenotype in a Novel ATP1A3 Mutation: Connecting the Two Ends of a Spectrum . Mov Disord Clin Pract, 2016. 3(4): p. 398–401. Nan, H., et al., A p. Arg499His mutation in SPAST is associated with infantile-onset complicated spastic paraplegia: a case report and review of the literature . BMC neurology, 2021. 21: p. 1–5. Chung, S.-K., et al., GLRB is the third major gene of effect in hyperekplexia . Human molecular genetics, 2013. 22(5): p. 927–940. Gregg, A.R., et al., Screening for autosomal recessive and X-linked conditions during pregnancy and preconception: a practice resource of the American College of Medical Genetics and Genomics (ACMG) . Genet Med, 2021. 23(10): p. 1793–1806. Buchanan, A.H., et al., Clinical outcomes of a genomic screening program for actionable genetic conditions . Genet Med, 2020. 22(11): p. 1874–1882. Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations There is no duality of interest Supplementary Files Table1ESHG.xlsx Table 1 Table2ESHG.xlsx Table 2 Table3ESHG.xlsx Table 3 Table4ESHG.xlsx Table 4 SupplementaryTable1EJHG.xlsx Supplementary 1 SupplementaryTable2EJHG.xlsx Supplementary 2 SupplementaryTable3EJHG.xlsx Supplementary 3 SupplementaryTable4EJHG.xlsx Supplementary 4 Cite Share Download PDF Status: Published Journal Publication published 10 Jan, 2026 Read the published version in European Journal of Human Genetics → Version 1 posted Editorial decision: revise 01 Oct, 2025 Review # 2 received at journal 07 Sep, 2025 Reviewer # 3 agreed at journal 21 Aug, 2025 Reviewer # 2 agreed at journal 19 Aug, 2025 Review # 1 received at journal 16 May, 2025 Reviewer # 1 agreed at journal 15 May, 2025 Reviewers invited by journal 09 May, 2025 Submission checks completed at journal 30 Apr, 2025 First submitted to journal 30 Apr, 2025 Unknown event 29 Apr, 2025 Editor assigned by journal 25 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6532096","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":454358647,"identity":"4895ff2d-8745-4098-a5c1-54b9b9dd17ac","order_by":0,"name":"Saumya 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School","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Tan","suffix":""},{"id":454358655,"identity":"06fce3f3-9674-47aa-93cb-37f70d3aef12","order_by":8,"name":"David Amor","email":"","orcid":"https://orcid.org/0000-0001-7191-8511","institution":"Murdoch Children's Research Institute","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Amor","suffix":""}],"badges":[],"createdAt":"2025-04-26 02:00:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6532096/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6532096/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41431-025-02009-2","type":"published","date":"2026-01-10T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82795844,"identity":"9e831b3c-8804-4f12-8c55-506b2db473fa","added_by":"auto","created_at":"2025-05-15 10:38:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94160,"visible":true,"origin":"","legend":"\u003cp\u003eFlow diagram showing the exclusion and inclusion of variants analysed according to severity, penetrance and participant characteristics\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6532096/v1/bb816bdee6b474621cfc5960.png"},{"id":99967804,"identity":"e75e2c05-cd05-49a0-987e-d389f383b039","added_by":"auto","created_at":"2026-01-11 08:05:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":695058,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6532096/v1/ad007194-c3f4-4893-8656-f474bf533831.pdf"},{"id":82794410,"identity":"d36f4ccc-f391-4cbe-b2aa-9cb5d818006a","added_by":"auto","created_at":"2025-05-15 10:30:01","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17567,"visible":true,"origin":"","legend":"\u003cp\u003eTable 1\u003c/p\u003e","description":"","filename":"Table1ESHG.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6532096/v1/885d604418e3a99da8586830.xlsx"},{"id":82797601,"identity":"eb59c060-9fc9-418a-b387-52fa9d66be83","added_by":"auto","created_at":"2025-05-15 10:46:01","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12433,"visible":true,"origin":"","legend":"Table 2","description":"","filename":"Table2ESHG.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6532096/v1/00720a3f226ad06d2b0d2599.xlsx"},{"id":82794412,"identity":"a8592391-d5d9-4a87-84e6-c6cd168a3cd9","added_by":"auto","created_at":"2025-05-15 10:30:01","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18145,"visible":true,"origin":"","legend":"\u003cp\u003eTable 3\u003c/p\u003e","description":"","filename":"Table3ESHG.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6532096/v1/ed06d42a096553c4bce83a97.xlsx"},{"id":82799204,"identity":"1fb34e2c-08d3-4b63-b3c6-2397bf0f11e3","added_by":"auto","created_at":"2025-05-15 10:54:01","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18133,"visible":true,"origin":"","legend":"Table 4","description":"","filename":"Table4ESHG.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6532096/v1/9c224290a0e53a4962c0cead.xlsx"},{"id":82797600,"identity":"7cc87759-4851-4422-9d3c-4e88fbc9c622","added_by":"auto","created_at":"2025-05-15 10:46:01","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":13607,"visible":true,"origin":"","legend":"Supplementary 1","description":"","filename":"SupplementaryTable1EJHG.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6532096/v1/96ba0ba86bb2eeae8c4bd1bd.xlsx"},{"id":82795847,"identity":"26392511-7a44-4185-914e-f1182068401e","added_by":"auto","created_at":"2025-05-15 10:38:01","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":63887,"visible":true,"origin":"","legend":"Supplementary 2","description":"","filename":"SupplementaryTable2EJHG.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6532096/v1/e88a80fd411a03eaab9289e2.xlsx"},{"id":82795850,"identity":"232f1bbd-95a1-4466-ad65-1c903b8cb85b","added_by":"auto","created_at":"2025-05-15 10:38:01","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":16285,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary 3\u003c/p\u003e","description":"","filename":"SupplementaryTable3EJHG.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6532096/v1/8b30a6caafbf963b4d62ad4c.xlsx"},{"id":82794419,"identity":"d609f6ea-1431-4e9d-aa4c-73a7401476d6","added_by":"auto","created_at":"2025-05-15 10:30:01","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":12922,"visible":true,"origin":"","legend":"Supplementary 4","description":"","filename":"SupplementaryTable4EJHG.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6532096/v1/4c88d5d133afdbe9e4452a31.xlsx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Unexpected genotypes associated with severe paediatric conditions identified in a healthy population cohort.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe expansion of genomic applications has provided the opportunity to screen individuals beyond clinical indication. Initiating genomics as a screening approach can identify individuals with genetic variants leading to a diagnosis, an increased risk of developing, or a carrier of a genetic condition that may have otherwise been undetected. If implemented at a population level, genomics screening has the potential to transition from reactionary medical treatment to preventive care, as well as helping prospective parents to understand the chance of having a child with a genetic condition. However, the classification of genomic variants and their impact on health outcomes in absence of phenotype-driven testing is still being explored.\u003c/p\u003e \u003cp\u003eGenomic screening, when applied to unselected cohorts, has identified individuals harbouring variants of clinical significance in absence of associated clinical features.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e The American College of Medical Genetics and Genomics (ACMG) has emphasised that further ascertainment of genotype-phenotype correlation is necessary for the application of genomics screening to predict clinical outcomes in the general population.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e This has generated focus on the variants associated with medically actionable genetic conditions proposed by ACMG\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e in large unselected cohort studies, and more recently in healthy newborns, refining prevalence,\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e revising penetrance\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and evaluating the appropriateness of current genetic testing criteria.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAnalysis beyond ACMG-recommended genes is less described; however, similar findings have been reported. Using a genotype-first approach, variants associated with severe paediatric conditions have been detected in individuals with no associated clinical features.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e A targeted analysis of highly penetrant RASopathies and Marfan syndrome demonstrated only 14% (3/19) and 22% (9/41) respectively had received a clinical diagnosis and many did not exhibit associated clinical features.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Overall, these studies demonstrate that clinical indication can be either absent or highly variable, potentially resulting in missed diagnoses and uncertainties in predicting clinical outcomes.\u003c/p\u003e \u003cp\u003eIn this study we analysed the presence of variants associated with highly penetrant paediatric conditions amongst 9637 unrelated research participants from two Singaporean control study cohorts: SG10K_Health and the National Heart Centre Singapore biobank.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e All participants reported no health issues at the time of recruitment. We focused on severe paediatric conditions given they are typically selected against in control populations and have clinical relevance for screening programs such as reproductive and newborn screening. We describe the process of assessing the clinical relevance of variants in parallel with participant characteristics, using phenotypic and medical history where available.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population and characteristics\u003c/h2\u003e \u003cp\u003eWhole genome sequencing data was obtained from 9637 participants of Chinese, Indian and Malay ancestry and inferred to be unrelated to the second degree. This study comprised 9051 participants from the SG10K_Health project,\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e who were recruited as a control study cohort with no reported pre-existing health conditions and 586 participants from the National Heart Centre Singapore biobank,\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e asymptomatic as ascertained by their health screen at recruitment. Exclusion criteria for each cohort is outlined in Supplementary Table\u0026nbsp;1. All studies contributing to the study cohort were approved by relevant institutional ethics review boards as previously outlined. Participant characteristics available included age, gender, ancestry, height, weight, low-density lipoprotein cholesterol and HbA1c glucose levels. Access to electronic medical records was available to a subset of 4750 (53%) participants and of these, recontact was consented for 1750 (19%) participants.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGene panel, variant curation and bioinformatics analysis\u003c/h3\u003e\n\u003cp\u003eAnalysis focused on 1619 genes previously defined to be associated with severe paediatric disease and autosomal dominant, autosomal recessive and X-linked inheritance (Supplementary Table\u0026nbsp;2).\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Genome sequencing, bioinformatic analysis and variant curation has been previously described in the SG10K Health project.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Briefly, variants were curated according to American College of Medical Genetics and Genomics\u0026ndash;Association for Molecular Pathology (ACMG-AMP) guidelines,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and those occurring in the severe paediatric gene list were extracted for further review. For the additional 586 participants from the National Heart Centre Singapore biobank, the same bioinformatic pipeline and variants analysis processes previously described for the SG10K Health project were applied with focus on the severe paediatric gene list.\u003c/p\u003e \u003cp\u003eVariants with a minimum genotype quality of 20, minimum depth of 6 and variant allele balance of 0.2\u0026ndash;0.8 were included. Variants associated with severe early onset disease and low read counts were manually inspected using Integrative Genomics Viewer (IGV). To minimise the possibility of pathogenic variants present as a result of clonal hematopoiesis and not a germline event, given that somatic variant burden increases with age in healthy tissues such as blood\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, variants occurring in genes subject to clonal hematopoiesis with a variant allele fraction of \u0026lt;\u0026thinsp;0.35 were removed.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Furthermore variants occurring in genes with homologous regions or pseudogenes\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e were also considered as sequencing can be technically challenging.\u003c/p\u003e\n\u003ch3\u003eVariant selection and clinical association\u003c/h3\u003e\n\u003cp\u003e Participants carrying pathogenic/likely pathogenic (P/LP) variants in genes associated with autosomal dominant disease, homozygous for autosomal recessive disease and hemizygous for X-linked disease reported to cause symptoms in childhood were selected for further review. Compound heterozygotes were not included in this analysis as phasing could not be confirmed.\u003c/p\u003e \u003cp\u003eTo analyse the clinical significance at a variant level, each P/LP variant was further interrogated to determine the associated genetic condition, inheritance pattern, protein impact, expected phenotype and severity using sources such as ClinVar,\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e ClinGen,\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e OMIM\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and literature. The variant impact was reviewed by clinical genetics, genetic counsellors and bioinformaticians in correlation with participant characteristics (such as age and gender) and medical records where available, to assess clinical implications These details were discussed until consensus regarding potential variant impact was achieved.\u003c/p\u003e \u003cp\u003eVariants were classified as highly penetrant if primarily associated with disease onset occurring before age 18 years. As a paediatric cohort was also included in this study, age of onset before 5 years was applied for these participants. Disease onset primarily after 18 years was considered as adult onset or variable if manifesting in both childhood and adulthood. Phenotype severity was assessed as either profound or severe based on classification proposed by Lazarin et al. 2014\u003csup\u003e28\u003c/sup\u003e which takes into consideration life span impact, intellectual disability, impaired mobility, physical malformation, sensory impairment and dysmorphic features. More recent gene panel reviews in the context of newborn screening were also referred to.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e The phenotype was annotated as mild if clinical features could potentially be undiagnosed at time of recruitment.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eParticipant characteristics\u003c/h2\u003e \u003cp\u003eThe study cohort comprised of 9637 participants with no reported pre-existing health conditions and inferred to be unrelated to the second degree. The age of the cohort ranged from 6\u0026ndash;88 years (median: 47), 57% were female and genetic ancestry was inferred to be Chinese (63%), Indian (20%), Malay (17%) and other (0.18%) (Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eVariant analysis overview\u003c/h2\u003e \u003cp\u003eAlthough the analysis included genes curated to be associated with severe paediatric disease, several variables were encountered when evaluating the validity and clinical significance at a variant level. The assessment required integration of published literature, participant demographics and medical history, where available (Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e Overall, there were 110 unique P/LP variants in genes reported to be associated with severe paediatric conditions detected in 244 participants as either homozygous, heterozygous or hemizygous. Further review resulted in the exclusion of 66 variants and selection of 44 variants for detailed analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe 66 variants excluded from further consideration included five variants ambiguous after Integrative Genomics Viewer inspection (occurring in \u003cem\u003eAMT, CDH7\u003c/em\u003e, \u003cem\u003eEHMT1, EP300\u003c/em\u003e and \u003cem\u003eLMX1B\u003c/em\u003e). For example, the \u003cem\u003eAMT\u003c/em\u003e missense variant has been reported homozygous in a patient with nonketotic hyperglycinemia since birth,\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e yet medical records indicated our male participant aged 70 years was unaffected. Inspection of IGV revealed the causative variant absent in one out of ten reads suggesting heterozygosity. Sanger sequencing, which has not yet been possible, would clarify this further. One variant potentially derived from a pseudogene (\u003cem\u003eCYP21A2\u003c/em\u003e), seven variants were inferred as a product of clonal hematopoiesis (occurring in genes \u003cem\u003eARL\u003c/em\u003e, \u003cem\u003eASXL1\u003c/em\u003e, and \u003cem\u003eCBL)\u003c/em\u003e, and one \u003cem\u003eBTD\u003c/em\u003e hypomorphic variant were also excluded. Additionally, 22 variants were found in genes \u003cem\u003eCLCN1\u003c/em\u003e, \u003cem\u003eCSF1R\u003c/em\u003e, \u003cem\u003eMAT1A, SEC23B, SLC3A1\u003c/em\u003e and \u003cem\u003eWFS1\u003c/em\u003e more likely to be associated with recessive disease although the gene can also be associated with dominant inheritance. There were 25 variants in females associated with X-linked inheritance where no or a mild phenotype was expected and five loss of function variants were found in genes where haploinsufficiency is not expected to disrupt protein function (\u003cem\u003eFBN2\u003c/em\u003e, \u003cem\u003eMAP2K1\u003c/em\u003e, \u003cem\u003eSMC3\u003c/em\u003e, \u003cem\u003eTMEM67\u003c/em\u003e) (Supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eThe remaining 44 unique P/LP variants occurring in 184 participants were interrogated further for severity and penetrance of expected phenotype, as well as reported medical history where available. The age of onset and severity was determined at a variant level using literature and reviews. For novel missense variants, variant impact was evaluated by assessing previously reported variants in the same amino acid and for novel loss of function variants, the impact of downstream loss of function variants were reviewed (Table\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAutosomal dominant and X-linked conditions\u003c/h3\u003e\n\u003cp\u003eThere were 46 participants that harboured a heterozygous P/LP variant in a gene reported to be associated with an autosomal dominant paediatric condition. Medical records were available for 13 (28%) participants. The most frequent variants were either variable in severity and penetrance, for example \u003cem\u003eGLMN\u003c/em\u003e (glomuvenous malformations, OMIM 138000) or associated with mild disease in heterozygous state, for example \u003cem\u003eCAV3\u003c/em\u003e (subclinical muscle weakness, OMIM 614321). Some participants could be at risk of developing a condition that they were not aware of. For example two participants had the same loss of function \u003cem\u003eCOL1A1\u003c/em\u003e variant previously described in two patients with osteogenesis imperfecta (OMIM 166200) causing fractures;\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e one participant had a \u003cem\u003eFLCN\u003c/em\u003e variant described previously in a patient with Birt-Hogg-Dub\u0026eacute; (OMIM 135150); one participant with a \u003cem\u003eMPZ\u003c/em\u003e variant reported previously with late-onset Charcot-Marie-Tooth type 2 (OMIM 607736); and one participant had a \u003cem\u003ePTPN11\u003c/em\u003e variant detected previously in patients with Noonan syndrome (OMIM 151100).\u003c/p\u003e \u003cp\u003eFive participants carried variants associated with severe disease: a previously reported splice site variant \u003cem\u003eCOL2A1\u003c/em\u003e (Stickler syndrome, OMIM 10830) in a female aged 54 years; a well characterised missense variant in \u003cem\u003eSPAST\u003c/em\u003e (spastic paraplegia, OMIM 182601) associated with paediatric onset in a female aged 35 years; and a 12 year old male (asymptomatic according to medical records) was found to carry a missense variant in \u003cem\u003eATP1A3\u003c/em\u003e (\u003cem\u003eATP1A3\u003c/em\u003e-related neurological disorders, OMIM 601338). Two novel loss of function variants were also identified, \u003cem\u003eFZD4\u003c/em\u003e (exudative vitreoretinopathy, OMIM 133780) in a male aged 27 years and \u003cem\u003eKMT2A\u003c/em\u003e (Wiedemann-Steiner syndrome, OMIM 605130) in a male aged 55 years, who was asymptomatic according to medical records (Supplementary Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eOnly two hemizygous carriers were identified, both with variants in \u003cem\u003eGLA\u003c/em\u003e (Fabry disease, OMIM 301500) associated with late onset cardiac manifestations.\u003c/p\u003e\n\u003ch3\u003eAutosomal recessive conditions\u003c/h3\u003e\n\u003cp\u003eThere were 102 participants who were homozygous for autosomal recessive conditions associated with severe paediatric disease and of these, medical history was available for 38 (37%). Overall, the homozygous variants were associated with a milder phenotype or later onset, for example, the majority (76%) carried \u003cem\u003eGJB2\u003c/em\u003e p.Val37Ile which is associated with incomplete penetrant hearing loss in the homozygous state. As expected, of the 35 participants where medical records were available, none had associated clinical manifestations. Other variants detected in multiple participants were \u003cem\u003eCFTR\u003c/em\u003e p.Gln1352His (8 participants) and c.1210-11T\u0026thinsp;\u0026gt;\u0026thinsp;G (2 participants) associated with congenital bilateral absence of vas deferens and \u003cem\u003eCFTR\u003c/em\u003e-related disorders. One participant (female, 62 years) was homozygous for \u003cem\u003eCFTR\u003c/em\u003e p.Gln1352His and was reported to have an airway infection and mucus plug that could possibly be attributed to her genotype, yet she has not yet been evaluated for \u003cem\u003eCFTR\u003c/em\u003e-related disorders (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eFour participants were found to be homozygous for a variant associated with a severe phenotype. One participant (female, 30 years) was homozygous for a loss of function variant \u003cem\u003eLAMA2\u003c/em\u003e p.Arg362Ter expected to cause congenital muscular dystrophy, however, unfortunately her medical records cannot be accessed. The clinical manifestations of this variant in the homozygous state are unreported. The remaining participants had homozygous variants which were either reported pathogenic in ClinVar, for example \u003cem\u003eSLC26A4\u003c/em\u003e (Pendred syndrome, OMIM 274600) or loss of function associated with pathogenicity in \u003cem\u003eGLRB\u003c/em\u003e (hereditary hyperekplexia, OMIM 614619) and \u003cem\u003eTUBGCP6\u003c/em\u003e (microcephaly and chorioretinopathy, OMIM 251270). Of these, two medical records were available, and no associated symptoms were reported (Sup Table\u0026nbsp;2).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe genomic analysis of populations is increasingly being implemented in both research and clinical settings as it offers the opportunity to enhance health screening. Phenotype characterisation has typically been ascertained in patient cohorts or individuals with a relevant family history, thereby guiding relevant clinical management recommendations. However, predicting clinical implications of genomic variants derived from individuals with no known associated phenotype presents challenges. Despite applying stringent variant classification criteria, an analysis of variants occurring in 1619 genes associated with severe paediatric disease resulted in the exclusion of 60% (66/110) variants. These exclusions were based on potential sequencing artifacts, derivatives of clonal hematopoiesis, presence in homologous gene regions, insufficient evidence for pathogenicity in heterozygous state, haploinsufficiency not expected to disrupt protein function or hypomorphic in the homozygous state.\u003c/p\u003e \u003cp\u003eAnalysis of the remaining 44 variants revealed that whilst detected in genes associated with paediatric onset, variant level assessment supported their presence in this cohort. Several variants were reported to have milder presentations such as \u003cem\u003ePTPN11\u003c/em\u003e p.Tyr63Cys (Noonan syndrome). Others were variable in age of onset, such as \u003cem\u003eFLCN\u003c/em\u003e c.1432\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;T (Birt-Hogg Dube syndrome) and \u003cem\u003eABCA4\u003c/em\u003e p.Gly1961Glu (Stargardt disease) or specifically adult onset, as observed with \u003cem\u003eGLA\u003c/em\u003e c.640-801G\u0026thinsp;\u0026gt;\u0026thinsp;A (Fabry disease) and \u003cem\u003eGALC\u003c/em\u003e p.Val681Met (Krabbe disease).\u003c/p\u003e \u003cp\u003eDespite significant pre-existing conditions being excluded for this cohort, there were nine participants harbouring P/LP variants associated with severe paediatric disease. Unfortunately recontact of these participants for further phenotyping was not possible. Contrary to the search for causative variants in affected individuals, we speculated why these variants were observed in a cohort selected to be healthy. It is possible that the participants carrying variants associated with Stickler syndrome, Wiedemann-Steiner syndrome, Pendred syndrome, limb girdle muscular dystrophy and exudative vitreoretinopathy were undiagnosed at time of study recruitment, and we note that mild and adult onset phenotypes of these conditions, although rare, have been reported.\u003csup\u003e\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e For example, although the mean age of \u003cem\u003eFZDA\u003c/em\u003e-associated exudative vitreoretinopathy is 6 years, expressivity has been reported as variable with symptoms diagnosed at 49 years in one patient.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e The participant harbouring this variant in our study was recruited at 27 years. The pathogenicity of \u003cem\u003eATP1A3\u003c/em\u003e Gly358Arg associated with \u003cem\u003eATP1A3\u003c/em\u003e-related neurological disorders is reported as two stars in ClinVar. Variants at the same position Gly358Cys, Gly358Ser, Gly358Val and Gly358Asp have been described in case reports associated with alternating hemiplegia of childhood\u003csup\u003e\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and the significance of this position has been demonstrated by functional data\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Although in silico predictions highly favour pathogenicity for \u003cem\u003eATP1A3\u003c/em\u003e Gly358Arg, its consequence has not been reported in literature.\u003c/p\u003e \u003cp\u003eHowever, three variants occurring in genes \u003cem\u003eSPAST\u003c/em\u003e, \u003cem\u003eGLRB\u003c/em\u003e and \u003cem\u003eTUBGCP6\u003c/em\u003e were strongly predicted to cause a severe phenotype after reviewing protein impact and literature. There are several reports of affected patients carrying the \u003cem\u003eSPAST\u003c/em\u003e missense variant with symptoms such as hereditary spastic paraplegias, intellectual disability and loss of speech manifesting from infancy to six years of age.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e While the \u003cem\u003eGLRB\u003c/em\u003e loss of function variant is novel, there are at least six downstream loss of function variants described in patients with hyperekplexia since birth.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Similarly, the \u003cem\u003eTUBGCP6\u003c/em\u003e loss of function is novel and there are only a few \u003cem\u003eTUBGCP6\u003c/em\u003e pathogenic variants described in literature yet one patient homozygous for a loss of function variant downstream had retinal dystrophy and severe intellectual disability. These clinical presentations would preclude inclusion in a cohort designated as healthy.\u003c/p\u003e \u003cp\u003eIn a previous study of 874 genes associated with fully penetrant severe mendelian disease amongst a cohort of 589,306 participants, 13 were found to harbour pathogenic variants with no associated clinical manifestations,\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e thus, further questioning current understandings of disease penetrance. It is therefore unclear how current penetrance estimates and disease severity derived from patient cohorts translate to variants detected in unselected populations. There is increasing recognition that reproductive carrier screening should be offered to population-wide\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e and the clinical utility of proactive genomics screening at newborn and childhood has recently been explored.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e The genes selected for screening are typically associated with severe disease such as those with life-limiting impacts or significant reductions in quality of life. Our findings highlight the complexities of predicting clinical impact from such genes which is particularly relevant in settings when counselling at-risk couples regarding disease onset and severity. While early identification of certain genetic conditions in otherwise healthy individuals may offer opportunities for prevention or early intervention the benefit of screening must be balanced with the potential psychological harm or anxiety, particularly for individuals who may never develop the condition. When applying this information for reproductive planning it is important to understand the likelihood of having an affected child to make a truly informed decision, which can be challenged when risk prediction is uncertain. These ethical complexities underscore the importance of informed consent, clear communication, and the need for careful consideration of the potential harms and benefits. Such considerations will need to be refined as further research regarding reduced penetrance, milder presentations, and late-onset variants in diverse populations expands.\u003c/p\u003e \u003cp\u003eThis study provides insights into the application of genomics screening at a population level, but it has several limitations due to the constraints of the available data. A major limitation was the inability to obtain detailed phenotypic information for all the participants carrying severe disease or highly penetrant clinically actionable variants. While no significant medical issues were documented at time of recruitment, it is possible that some individuals were taking medication which masked symptoms or may have developed symptoms after recruitment. Although medical record access is a common data source to document clinical outcomes, it is subject to misclassification or incomplete records presenting gaps in reliability.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e The ability to capture relevant medical data emphasises the importance of recontact and follow-up to document prospective health outcomes and an important consideration at the time of study design and consent development.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Additionally, the significance of large cohort sizes is apparent when only a small minority of participants harbour variants warranting further investigation, highlighting the benefit of collaborative efforts to explore a range of variables potentially impacting clinical outcomes such as genetic modifiers and environmental factors. However, even in the absence of large cohort sizes, this study demonstrates that it is still possible to identify participants who carry unexpected genotypes.\u003c/p\u003e \u003cp\u003eThe genes analysed in this study were selected as they have been previously reported to be highly penetrant and associated with severe paediatric disease. However, our analysis of an apparently healthy cohort identified asymptomatic individuals, introducing significant implications for tailoring health screening advice in population screening programs and predicting clinical outcomes in reproductive carrier screening. Further work regarding characterisation of clinically significant variants generated from population sequencing in parallel with patient cohort studies will help refine existing understandings regarding the penetrance, phenotypic variability and clinical impact of genomic variants. This study revealed the challenges of understanding the clinical ramifications from genotype-derived screening and emphasises the importance of expanding phenotype characterisation which is highly relevant within the realms of population genomics and reproductive screening settings.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData is available in both Results and Supplementary Materials. Further data can be made available from the corresponding author by reasonable request.\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eEthical Approval\u003c/h2\u003e \u003cp\u003eEthics approval and consent to participate from participants was obtained from six institutes: Growing Up in Singapore Towards healthy Outcomes birth cohort study (GUSTO), National University Hospital, Singapore CIRB/E/2019/2655 NCT01174875; Health for Life In Singapore Study (HELIOS), Nanyang Technology University, Singapore, IRB 2016-11-030; Singapore Multi-Ethnic Cohort Study (MEC), National University of Singapore, CIRB 13\u0026ndash;512; SingHealth Duke-NUS Institute of Precision Medicine (PRISM), SingHealth Centralised Institutional Review Board, 2013/605/C NCT02791152; Singapore Epidemiology of Eye Diseases study (SEED), SingHealth Centralised Institutional Review Board, 2018/2717; Tan Tock Seng Hospital (TTSH ), National Health Group, TB-2020-001 \u0026amp; BTC-2020-001. All experiments were performed in accordance with relevant guidelines and regulations. This research conforms to the principles of the Helsinki Declaration.\u003c/p\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003ch2\u003eFunding statement\u003c/h2\u003e \u003cp\u003eThis study made use of data generated as part of the Singapore National Precision Medicine program funded by the Industry Alignment Fund (Pre-Positioning) (IAF-PP: H17/01/a0/007). This study made use of data / samples collected in the following cohorts in Singapore: 1. The Health for Life in Singapore (HELIOS) study at the Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore (supported by grants from a Strategic Initiative at Lee Kong Chian School of Medicine, the Singapore Ministry of Health (MOH) under its Singapore Translational Research Investigator Award (NMRC/STaR/0028/2017) and the IAF-PP: H18/01/a0/016); 2. The Growing up in Singapore Towards Healthy Outcomes (GUSTO) study, which is jointly hosted by the National University Hospital (NUH), KK Women\u0026rsquo;s and Children\u0026rsquo;s Hospital (KKH), the National University of Singapore (NUS) and the Singapore Institute for Clinical Sciences (SICS), Agency for Science Technology and Research (A*STAR) (supported by the Singapore National Research Foundation under its Translational and Clinical Research (TCR) Flagship Programme and administered by the Singapore Ministry of Health\u0026rsquo;s National Medical Research Council (NMRC), Singapore - NMRC/TCR/004-NUS/2008; NMRC/TCR/012-NUHS/2014. Additional funding is provided by SICS and IAF-PP H17/01/a0/005); 3. The Singapore Epidemiology of Eye Diseases (SEED) cohort at Singapore Eye Research Institute (SERI) (supported by NMRC/CIRG/1417/2015; NMRC/CIRG/1488/2018; NMRC/OFLCG/004/2018); 4. The Multi-Ethnic Cohort (MEC) cohort (supported by NMRC grant 0838/2004; BMRC grant 03/1/27/18/216; 05/1/21/19/425; 11/1/21/19/678, Ministry of Health, Singapore, National University of Singapore and National University Health System, Singapore); 5. The SingHealth Duke-NUS Institute of Precision Medicine (PRISM) cohort was supported by core funding from SingHealth and Duke-NUS Institute of Precision Medicine (PRISM) and centre grant awarded to the National Heart Centre Singapore from the National Medical Research Council, Ministry of Health, Singapore (NMRC/CG/M006/2017_NHCS and MOH-000985) as well as NMRC/STaR/0011/2012, NMRC/STaR/ 0026/2015, Lee Foundation and Tanoto Foundation. 6. The TTSH Personalised Medicine Normal Controls (TTSH) cohort funded (supported by NMRC/CG12AUG17 and CGAug16M012). The National Precision Medicine Programme (NPM) PHASE II FUNDING (MOH-000588) supports W.K.L. The views expressed are those of the author(s) are not necessarily those of the National Precision Medicine investigators, or institutional partners.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eConceptualization: DA, SJ, MM, JH, YB, PT, KY. Data curation: WL, JT, YB. Formal analysis YB, DA, SJ, MM, JH. Funding acquisition: PT, KY, SJ, WK. Investigation: YB, SJ, DA. Methodology: YB, SJ, DA. Writing review and editing: SJ, DA. wrote the manuscript with support from DA and SJ. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003e We thank all investigators, staff members and study participants who made the National Precision Medicine Project possible. We also acknowledge the clinical research assistants for their roles in recruitment and participant contact as well as the study leads for their assistance in obtaining medical history. We would also like to thank the Lee Foundation for grant support to the SingHEART study conducted at the National Heart Centre Singapore.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePlon, S. and G. Jarvik, \u003cem\u003eTen Years of Incidental, Secondary, and Actionable Findings\u003c/em\u003e. N Engl J Med, 2023. 389(1741): p. 52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDirectors, A.B.o., \u003cem\u003eThe use of ACMG secondary findings recommendations for general population screening: a policy statement of the American College of Medical Genetics and Genomics (ACMG)\u003c/em\u003e. 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Hum Mol Genet, 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMandelker, D., et al., \u003cem\u003eNavigating highly homologous genes in a molecular diagnostic setting: a resource for clinical next-generation sequencing\u003c/em\u003e. Genet Med, 2016. 18(12): p. 1282\u0026ndash;1289.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLandrum, M.J., et al., \u003cem\u003eClinVar: improving access to variant interpretations and supporting evidence\u003c/em\u003e. Nucleic Acids Res, 2018. 46(D1): p. D1062-d1067.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRehm, H.L., et al., \u003cem\u003eClinGen\u0026ndash;the Clinical Genome Resource\u003c/em\u003e. N Engl J Med, 2015. 372(23): p. 2235\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmberger, J.S., et al., \u003cem\u003eOMIM.org: Online Mendelian Inheritance in Man (OMIM(R)), an online catalog of human genes and genetic disorders\u003c/em\u003e. Nucleic Acids Res, 2015. 43(Database issue): p. D789-98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLazarin, G.A., et al., \u003cem\u003eSystematic Classification of Disease Severity for Evaluation of Expanded Carrier Screening Panels\u003c/em\u003e. PLoS One, 2014. 9(12): p. e114391.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCeyhan-Birsoy, O., et al., \u003cem\u003eInterpretation of Genomic Sequencing Results in Healthy and Ill Newborns: Results from the BabySeq Project\u003c/em\u003e. Am J Hum Genet, 2019. 104(1): p. 76\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDownie, L., et al., \u003cem\u003eGene selection for genomic newborn screening: Moving toward consensus?\u003c/em\u003e Genet Med, 2024. 26(5): p. 101077.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKure, S., et al., \u003cem\u003eComprehensive mutation analysis of GLDC, AMT, and GCSH in nonketotic hyperglycinemia\u003c/em\u003e. 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Am J Med Genet A, 2023. 191(7): p. 1935\u0026ndash;1941.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteel, D., et al., \u003cem\u003eWhole exome sequencing reveals a MLL de novo mutation associated with mild developmental delay and without \u0026lsquo;hairy elbows\u0026rsquo;: expanding the phenotype of Wiedemann\u0026ndash;Steiner syndrome\u003c/em\u003e. Journal of genetics, 2015. 94: p. 755\u0026ndash;758.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith, N., U.K.-I. JM, and J. Karalliedde, \u003cem\u003eDelayed diagnosis of Pendred syndrome.\u003c/em\u003e BMJ Case Rep, 2016. 2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagri, F., et al., \u003cem\u003eLimb girdle muscular dystrophy due to LAMA2 gene mutations: new mutations expand the clinical spectrum of a still challenging diagnosis\u003c/em\u003e. Acta Myologica, 2020. 39(2): p. 67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRanchod, T.M., et al., \u003cem\u003eClinical presentation of familial exudative vitreoretinopathy\u003c/em\u003e. Ophthalmology, 2011. 118(10): p. 2070\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePanagiotakaki, E., et al., \u003cem\u003eClinical profile of patients with ATP1A3 mutations in Alternating Hemiplegia of Childhood-a study of 155 patients\u003c/em\u003e. Orphanet J Rare Dis, 2015. 10: p. 123.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNg, H.W.Y., J.A. Ogbeta, and S.J. Clapcote, \u003cem\u003eGenetically altered animal models for ATP1A3-related disorders\u003c/em\u003e. Dis Model Mech, 2021. 14(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaciorkowski, A.R., et al., \u003cem\u003eNovel mutations in ATP1A3 associated with catastrophic early life epilepsy, episodic prolonged apnea, and postnatal microcephaly\u003c/em\u003e. Epilepsia, 2015. 56(3): p. 422\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePereira, P., et al., \u003cem\u003eA Distinct Phenotype in a Novel ATP1A3 Mutation: Connecting the Two Ends of a Spectrum\u003c/em\u003e. Mov Disord Clin Pract, 2016. 3(4): p. 398\u0026ndash;401.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNan, H., et al., \u003cem\u003eA p. Arg499His mutation in SPAST is associated with infantile-onset complicated spastic paraplegia: a case report and review of the literature\u003c/em\u003e. BMC neurology, 2021. 21: p. 1\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChung, S.-K., et al., \u003cem\u003eGLRB is the third major gene of effect in hyperekplexia\u003c/em\u003e. Human molecular genetics, 2013. 22(5): p. 927\u0026ndash;940.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGregg, A.R., et al., \u003cem\u003eScreening for autosomal recessive and X-linked conditions during pregnancy and preconception: a practice resource of the American College of Medical Genetics and Genomics (ACMG)\u003c/em\u003e. Genet Med, 2021. 23(10): p. 1793\u0026ndash;1806.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuchanan, A.H., et al., \u003cem\u003eClinical outcomes of a genomic screening program for actionable genetic conditions\u003c/em\u003e. Genet Med, 2020. 22(11): p. 1874\u0026ndash;1882.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Population genomic screening, genetic carrier screening, genotype-phenotype correlation","lastPublishedDoi":"10.21203/rs.3.rs-6532096/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6532096/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe expansion of genomics provides opportunity to screen individuals beyond clinical indication yet the classification of genomic variants and implications for health outcomes in this context is still emerging. We investigated this further by analysing clinically relevant variants and expected clinical implications in a population with no reported medical conditions. Whole genomes from 9637healthy unrelated research-consented participants in Singapore were analysed focusing on 1619 genes associated with severe paediatric disease. Association between causative variants and expected phenotype was assessed in correlation with participant characteristics and medical history where available. After considering protein impact, mode of inheritance and participant demographics for 110 unique variants, further analysis was performed for 44 variants occurring in 150 participants to understand clinical implications. Most carried variants associated with a mild phenotype (cystinuria), late onset (Fabry disease) or a potentially missed phenotype (Hajdu-Cheney syndrome). However, nine participants had variants associated with severe paediatric disease predicted to be symptomatic, such as limb-girdle muscular dystrophy and spastic paraplegia.Despite a cohort selected for absence of pre-existing health conditions, individuals were identified carrying variants associated with severe paediatric conditions. Further work is required to examine for subtle clinical symptoms. This study revealed the challenge of predicting clinical outcomes from genotype-derived screening and emphasises the importance of expanding phenotype characterisation which is highly relevant in population and reproductive screening settings.\u003c/p\u003e\n\u003cp\u003eTrial registration: NCT02791152\u003c/p\u003e","manuscriptTitle":"Unexpected genotypes associated with severe paediatric conditions identified in a healthy population cohort.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 10:29:56","doi":"10.21203/rs.3.rs-6532096/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-10-01T20:56:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-08T03:42:45+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-21T16:36:36+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-20T02:07:43+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-05-16T14:37:28+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-15T15:26:59+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-05-09T13:38:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-30T18:17:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Human Genetics","date":"2025-04-30T05:39:18+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-04-29T10:30:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-26T01:59:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b2ef22d2-d234-4531-845d-895ba792e36c","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48315705,"name":"Biological sciences/Genetics/Clinical genetics/Disease genetics"},{"id":48315706,"name":"Biological sciences/Genetics/Clinical genetics"}],"tags":[],"updatedAt":"2026-01-11T08:05:28+00:00","versionOfRecord":{"articleIdentity":"rs-6532096","link":"https://doi.org/10.1038/s41431-025-02009-2","journal":{"identity":"european-journal-of-human-genetics","isVorOnly":false,"title":"European Journal of Human Genetics"},"publishedOn":"2026-01-10 05:00:00","publishedOnDateReadable":"January 10th, 2026"},"versionCreatedAt":"2025-05-15 10:29:56","video":"","vorDoi":"10.1038/s41431-025-02009-2","vorDoiUrl":"https://doi.org/10.1038/s41431-025-02009-2","workflowStages":[]},"version":"v1","identity":"rs-6532096","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6532096","identity":"rs-6532096","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}