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describe their experience with a pilot project that used a next-generation sequencing panel of 4813 \"clinically relevant\" genes to enable a specific genetic diagnosis in 8 of 20 neonates in whom a genetic or metabolic condition was suspected.2 The authors' aim was not to provide a rapid genetic result; however, the medical or surgical management of two patients was substantially altered because of an accurate genetic diagnosis. The complex presenting features of neonates in this cohort ranged from acute renal failure to multiple congenital anomalies with early-onset seizures, exemplifying the nonspecific phenotypes that neonatologists and their consulting specialist colleagues regularly face. No longer should a clinician expect to send a DNA sample to the molecular genetics laboratory with minimal clinical information, since accurate interpretation of the thousands of genetic variants in any large-scale sequencing experiment is an iterative process. The clinicians caring for the patient are uniquely placed to integrate their knowledge of the patient's phenotype with the genetic variants or mutations detected, with the ultimate aim of matching the phenotype to the genotype and helping to prevent false-positive or falsenegative results.4 In some instances, a definitive molecular diagnosis can be a relatively straightforward outcome of genomic sequencing, since the uniqueness of the patient's phenotype and the pathogenicity of the mutations will leave the molecular geneticist and the clinician in no doubt. However, some cases are solved only with further phenotyping of the patient according to a suggested or \"differential\" molecular diagnosis, a process sometimes termed \"reverse phenotyping.\" In these instances, the importance of a direct communication channel between members of the interpretation team cannot be overstated. This principle already holds true for all clinicians who order next-generation sequencing genetic test panels, but will become more important as the number of genes tested (and hence the number of variants detected) increases.

Background The clinical utility of whole genome sequencing (WGS) in paediatric cancer has been demonstrated in recent years. WGS has been routinely available in the National Health Service (NHS) England for all children with cancer in England since 2021, but its uptake has been variable geographically. To explore the underlying barriers to routine use of WGS in this population across England and more widely in the United Kingdom (UK) and the Republic of Ireland (ROI), a one-day workshop was held in Cambridge, United Kingdom in October 2022. Methods Following a series of talks, delegates participated in open, round-table discussions to outline local and broader challenges limiting routine WGS for diagnostic work-up for children with cancer in their Principal Treatment Centres (PTCs) and Genomic Laboratory Hubs (GLHs). Within smaller groups, delegates answered structured questions regarding clinical capability, education and training needs, and workforce competence and requirements. Data was recorded, centrally collated, and analysed following the event using thematic analysis. Results Sixty participants attended the workshop with broad representation from the 20 PTCs across the UK and ROI and the seven GLHs in England. All healthcare professionals involved in the WGS pathway were represented, including paediatric oncologists, clinical geneticists, clinical scientists, and histopathologists. The main themes highlighted by the group in ensuring equitable access to WGS identified were: lack of knowledge equity between NHS trusts, with a perception of WGS being for research only; and perception of lack of financial support for the clinical process surrounding WGS, including lack of time to take informed consent from patients. The latter also included limited trained staff available for data interpretation, affecting the turnaround time for reporting. Finally, the need for an integrated digital pathway to order, track, and return data to clinicians was highlighted. Conclusion At the workshop, the general motivation for including WGS in the diagnostic work up for children with cancer was high throughout the UK, however a perceived lack of resources and education opportunities limit the widespread use of this commissioned assay. This workshop has led to some recommendations to increase access to WGS in this population in England and more widely in the devolved national of the UK and the ROI.

Beckwith–Wiedemann Syndrome (BWS) results from mutations or epigenetic events involving imprinted genes at 11p15.5. Most BWS cases are sporadic and uniparental disomy (UPD) or putative imprinting errors predominate in this group. Sporadic cases with putative imprinting defects may be subdivided into (a) those with loss of imprinting (LOI) of IGF2 and H19 hypermethylation and silencing due to a defect in a distal 11p15.5 imprinting control element (IC1) and (b) those with loss of methylation at KvDMR1, LOI of KCNQ1OT1 (LIT1 ) and variable LOI of IGF2 in whom there is a defect at a more proximal imprinting control element (IC2). We investigated genotype/epigenotype–phenotype correlations in 200 cases with a confirmed molecular genetic diagnosis of BWS (16 with CDKN1C mutations, 116 with imprinting centre 2 defects, 14 with imprinting centre 1 defects and 54 with UPD). Hemihypertrophy was strongly associated with UPD ( P <0.0001) and exomphalos was associated with an IC2 defect or CDKN1C mutation but not UPD or IC1 defect ( P <0.0001). When comparing birth weight centile, IC1 defect cases were significantly heavier than the patients with CDKN1C mutations or IC2 defect ( P =0.018). The risk of neoplasia was significantly higher in UPD and IC1 defect cases than in IC2 defect and CDKN1C mutation cases. Kaplan–Meier analysis revealed a risk of neoplasia for all patients of 9% at age 5 years, but 24% in the UPD subgroup. The risk of Wilms’ tumour in the IC2 defect subgroup appears to be minimal and intensive screening for Wilms’ tumour appears not to be indicated. In UPD patients, UPD extending to WT1 was associated with renal neoplasia ( P =0.054). These findings demonstrate that BWS represents a spectrum of disorders. Identification of the molecular subtype allows more accurate prognostic predictions and enhances the management and surveillance of BWS children such that screening for Wilms’ tumour and hepatoblastoma can be focused on those at highest risk.

Despite advances in genome sequencing, many individuals with rare genetic disorders remain undiagnosed. Transcriptional profiling via RNA-seq can reveal functional impacts of DNA variants and improve diagnosis. We assessed blood-derived RNA-seq in the largely undiagnosed SickKids Genome Clinic cohort ( n  = 134), which has been subjected to multiple analyses benchmarking the utility of genome sequencing. Our RNA-centric analysis identifies gene expression outliers, aberrant splicing, and allele-specific expression. In one-third of diagnosed individuals (20/61), RNA-seq reinforced DNA-based findings. In 2/61 cases, RNA-seq revised diagnoses ( EPG5 to LZTR1 in an individual with a Noonan syndrome-like disorder) and discovered an additional relevant gene ( CEP120 in addition to SON in an individual with ZTTK syndrome). Additionally, ~7% (5/73) of undiagnosed cases had at least one plausible candidate gene identified. This study highlights both the benefits and limitations of whole-blood RNA profiling in refining genetic diagnoses and uncovering novel disease mechanisms.

Whole-genome sequencing and whole-exome sequencing have proven valuable for diagnosing inherited diseases, particularly in children. However, usage of sequencing data as a pharmacogenetic screening tool to ensure medication safety and effectiveness remains to be explored. Sixty-seven variants in 19 genes with known effects on drug response were compared between genome sequencing and targeted genotyping data for coverage and concordance in 98 pediatric patients. We used targeted genotyping data as a benchmark to assess accuracy of variant calling, and to identify copy number variations of the CYP2D6 gene. We then predicted clinical impact of these variants on drug therapy. We find genotype concordance across those panels to be > 97%. Concordance of CYP2D6 predicted phenotype between estimates of whole-genome sequencing and targeted genotyping panel were 90%; a result from a lower coverage depth or variant calling difficulties in our whole-genome sequencing data when copy number variation and/or the CYP2D6*4 haplotype were present. Importantly, 95 children had at least one clinically actionable pharmacogenetic variant. Diagnostic genomic sequencing data can be used for pre-emptive pharmacogenetic screening. However, concordance between genome-wide sequencing and target genotyping needs to be characterized for each of the pharmacologically important genes. Pediatric medicine: Diagnostic sequencing offers useful pharmacogenetic data, too Genome sequencing, in addition to helping diagnose disease, can inform medication decisions and improve drug safety for children. Ronald Cohn, Shinya Ito and colleagues at the Hospital for Sick Children in Toronto, Canada, studied a cohort of 98 pediatric patients who had undergone whole genome sequencing to help diagnose their unexplained congenital malformations or neurodevelopmental disorders. The researchers looked for 67 DNA variants found in 19 genes with known effects on drug responses. They used targeted genotyping to assess the accuracy of the sequence data. Sequencing proved to be more than 99% accurate for all but one of the pharmacologically important genes, showing the power of diagnostic genomic sequencing to identify DNA variants in children that affect medication safety and effectiveness. However, the accuracy of the method may need to be validated for each relevant gene.

Background Signalling by fibroblast growth factor receptor type 2 (FGFR2) normally involves a tissue-specific alternative splice choice between two exons (IIIb and IIIc), which generates two receptor isoforms (FGFR2b and FGFR2c respectively) with differing repertoires of FGF-binding specificity. Here we describe a unique chimeric IIIb/c exon in a patient with Apert syndrome, generated by a non-allelic homologous recombination event. Case Presentation We present a child with Apert syndrome in whom routine genetic testing had excluded the FGFR2 missense mutations commonly associated with this disorder. The patient was found to harbour a heterozygous 1372 bp deletion between FGFR2 exons IIIb and IIIc, apparently originating from recombination between 13 bp of identical DNA sequence present in both exons. The rearrangement was not present in the unaffected parents. Conclusions Based on the known pathogenesis of Apert syndrome, the chimeric FGFR2 protein is predicted to act in a dominant gain-of-function manner. This is likely to result from its expression in mesenchymal tissues, where retention of most of the residues essential for FGFR2b binding activity would result in autocrine activation. This report adds to the repertoire of rare cases of Apert syndrome for which a pathogenesis based on atypical FGFR2 rearrangements can be demonstrated.

Loeys–Dietz syndrome is a connective tissue disorder predisposing individuals to aortic and arterial aneurysms. Presenting with a wide spectrum of multisystem involvement, medical management for some individuals is complex. This review of literature and expert opinion aims to provide medical guidelines for care of individuals with Loeys–Dietz syndrome. Genet Med 16 8, 576–587.

Whole-genome sequencing (WGS) as a first-tier diagnostic test could transform medical genetic assessments, but there are limited data regarding its clinical use. We previously showed that WGS could feasibly be deployed as a single molecular test capable of a higher diagnostic rate than current practices, in a prospectively recruited cohort of 100 children meeting criteria for chromosomal microarray analysis. In this study, we report on the added diagnostic yield with re-annotation and reanalysis of these WGS data ~2 years later. Explanatory variants have been discovered in seven (10.9%) of 64 previously undiagnosed cases, in emerging disease genes like HMGA2. No new genetic diagnoses were made by any other method in the interval period as part of ongoing clinical care. The results increase the cumulative diagnostic yield of WGS in the study cohort to 41%. This represents a greater than 5-fold increase over the chromosomal microarrays, and a greater than 3-fold increase over all the clinical genetic testing ordered in practice. These findings highlight periodic reanalysis as yet another advantage of genomic sequencing in heterogeneous disorders. We recommend reanalysis of an individual’s genome-wide sequencing data every 1–2 years until diagnosis, or sooner if their phenotype evolves.

Clinical whole-genome sequencing (WGS) has been shown to deliver potential benefits to children with cancer and to alter treatment in high-risk patient groups. It remains unknown whether offering WGS to every child with suspected cancer can change patient management. We collected WGS variant calls and clinical and diagnostic information from 281 children (282 tumors) across two English units ( n  = 152 from a hematology center, n  = 130 from a solid tumor center) where WGS had become a routine test. Our key finding was that variants uniquely attributable to WGS changed the management in ~7% (20 out of 282) of cases while providing additional disease-relevant findings, beyond standard-of-care molecular tests, in 108 instances for 83 (29%) cases. Furthermore, WGS faithfully reproduced every standard-of-care molecular test ( n  = 738) and revealed several previously unknown genomic features of childhood tumors. We show that WGS can be delivered as part of routine clinical care to children with suspected cancer and can change clinical management by delivering unexpected genomic insights. Our experience portrays WGS as a clinically impactful assay for routine practice, providing opportunities for assay consolidation and for delivery of molecularly informed patient care. In a cohort of 281 children with diagnosed or suspected cancer presenting to the NHS, implementing routine whole-genome sequencing provided clinical benefit in 29% of cases and led to change in management in 7% of patients.