Re-evaluation of whole exome sequencing, including intronic region, in combination with genetic intolerance score for detecting foetal structural anomalies in X-linked disorders

Background Whole-exome sequencing (WES) is a strong diagnostic tool for foetal structural anomalies, but the causative gene for more than half the anomalies have not been identified. Therefore, improving the diagnostic yield based on WES data is essential. Methods First, 138 foetuses with structural anomalies were assessed using conventional WES and copy number variation (CNV) analyses. For undiagnosed cases, we employed a three-step approach for diagnosis. We re-evaluated 1) candidate variants using a loss-of-function observed/expected upper bound fraction (LOEUF) score, 2) all variants of disease-causing genes for clinically diagnosed cases using spliceAI, and 3) the rare variants in all low LOEUF scored genes (< 0.35) using spliceAI. Results We identified molecular diagnoses in 53 of 138 cases (38.4%) using conventional WES and CNV. For undiagnosed cases, for the first step, we diagnosed two X-linked recessive diseases. For the second step, we diagnosed Meckel-Gruber syndrome by detecting likely pathogenic intron variant in TMEM67 . In the third step, we identified a de novo hemizygous pathogenic variant in one severe hydrops fetalis male, which caused aberrant splicing in CASK . We found a novel phenotype, hydrops fetalis, in CASK-related X-linked dominant disorder. Moreover, we revealed that the LOEUF score of X-linked disease-causing genes was significantly lower than that of autosomal genes among all OMIM-registered genes. Conclusion We showed that the evaluation of variants, including introns of WES data, in combination with the LOEUF score, could improve the WES diagnostic yield and be useful for evaluation of variants, especially on chromosome X. What is already known on this topic? Molecular genetic diagnosis of foetal structural anomalies using WES is being increasingly implemented. However, more than half of the cases cannot be diagnosed. There is signigicant potential to increase the diagnostic yield by re-analysing WES data. What this study adds In the present study, we focused on loss-of-function observed/expected upper bound fraction (LOEUF) scores to quantify genetic intolerance, and additional intron analysis for undiagnosed cases using conventional WES data. These approaches enabled the appropriate evaluation of candidate variants and detected overlooked candidate variants on intron. We diagnosed two X-linked recessive disorder cases (Hardikar syndrome and Ritscher-Schinzel syndrome), by re-evaluating candidate variants using the LOEUF score. We also diagnosed one Meckel-Gruber syndrome case caused by an intronic pathogenic variant, that had been overlooked by the conventional method. Moreover, evaluating all variants, including introns with low LOEUF score genes (2,971 genes) that could cause haploinsufficiency helped us find a pathogenic intronic variant on CASK in one hydrops fetalis case, which revealed that CASK-related X-linked dominant disorder could cause hydrops fetalis as severe phenotypes. Finally, the LOEUF score of X-linked genes was significantly lower than that of autosomal genes among all OMIM-registered genes, which meant that gene evaluation using the LOEUF score was helpful, for genetic diagnosis, especially for genes on chromosome X. How this study might affect research, practice, or policy The evaluation of variants, including introns, in combination with the LOEUF score is expected to contribute to the improvement of the diagnostic yield in WES. These approaches are easy and convenient to implement. The LOEUF score might be useful for evaluation of variants, especially in chromosome X. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by AMED under Grant Number JP21ek0109489, JSPS KAKENHI under Grant number 21H02887, JST COI-NEXT under Grant Number JPMJPF2017, and National Center for Child Health and Development under Grant Number NCCHD 2019A-4. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Institutional Review Board of the National Centre for Child Health and Development gave ethical approval for this work and written informed consent was provided by each participant (Institutional Review Board approval number: 236, 926). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.