Purpose:To prospectively evaluate the diagnostic and clinical utility of singleton whole-exome sequencing (WES) as a first-tier test in infants with suspected monogenic disease. Methods:Singleton WES was performed as a first-tier sequencing test in infants recruited from a single pediatric tertiary center. This occurred in parallel with standard investigations, including single-or multigene panel sequencing when clinically indicated. The diagnosis rate, clinical utility, and impact on management of singleton WES were evaluated.Results: Of 80 enrolled infants, 46 received a molecular genetic diagnosis through singleton WES (57.5%) compared with 11 (13.75%) who underwent standard investigations in the same patient group. Clinical management changed following exome diagnosis in 15 of 46 diagnosed participants (32.6%). Twelve relatives received a genetic diagnosis following cascade testing, and 28 couples were identified as being at high risk of recurrence in future pregnancies. Conclusions:This prospective study provides strong evidence for increased diagnostic and clinical utility of singleton WES as a firsttier sequencing test for infants with a suspected monogenic disorder. Singleton WES outperformed standard care in terms of diagnosis rate and the benefits of a diagnosis, namely, impact on management of the child and clarification of reproductive risks for the extended family in a timely manner.

IMPORTANCE Optimal use of whole-exome sequencing (WES) in the pediatric setting requires an understanding of who should be considered for testing and when it should be performed to maximize clinical utility and cost-effectiveness.OBJECTIVES To investigate the impact of WES in sequencing-naive children suspected of having a monogenic disorder and evaluate its cost-effectiveness if WES had been available at different time points in their diagnostic trajectory. DESIGN, SETTING, AND PARTICIPANTS This prospective study was part of the MelbourneGenomics Health Alliance demonstration project. At the ambulatory outpatient clinics of the Victorian Clinical Genetics Services at the Royal Children's Hospital, Melbourne, Australia, children older than 2 years suspected of having a monogenic disorder were prospectively recruited from May 1 through November 30, 2015, by clinical geneticists after referral from general and subspecialist pediatricians. All children had nondiagnostic microarrays and no prior single-gene or panel sequencing.EXPOSURES All children underwent singleton WES with targeted phenotype-driven analysis. MAIN OUTCOMES AND MEASURESThe study examined the clinical utility of a molecular diagnosis and the cost-effectiveness of alternative diagnostic trajectories, depending on timing of WES. RESULTSOf 61 children originally assessed, 44 (21 [48%] male and 23 [52%] female) aged 2 to 18 years (mean age at initial presentation, 28 months; range, 0-121 months) were recruited, and a diagnosis was achieved in 23 (52%) by singleton WES. The diagnoses were unexpected in 8 of 23 (35%), and clinical management was altered in 6 of 23 (26%). The mean duration of the diagnostic odyssey was 6 years, with each child having a mean of 19 tests and 4 clinical genetics and 4 nongenetics specialist consultations, and 26 (59%) underwent a procedure while under general anesthetic for diagnostic purposes. Economic analyses of the diagnostic trajectory identified that WES performed at initial tertiary presentation resulted in an

Purpose: Mutations in KAT6A have recently been identified as a cause of syndromic developmental delay. Within 2 years, the number of patients identified with pathogenic KAT6A mutations has rapidly expanded and the full extent and variability of the clinical phenotype has not been reported. Methods: We obtained data for patients with KAT6A mutations through three sources: treating clinicians, an online family survey distributed through social media and a literature review. Results: We identified 52 unreported cases, bringing the total number of published cases to 76. Our results expand the genotypic spectrum of pathogenic mutations to include missense and splicing mutations. We functionally validated a pathogenic splice site mutation and identified a likely hot-spot location for de novo missense mutations. The majority of clinical features in KAT6A syndrome have highly variable penetrance. For core features such as intellectual disability, speech delay, microcephaly, cardiac anomalies and gastrointestinal complications, genotype-phenotype correlations show that late-truncating mutations (exons 16-17) aare significantly more prevalent. We highlight novel associations, including an increased risk of gastrointestinal obstruction. Conclusion: Our data expands the genotypic and phenotypic spectrum for individuals with genetic mutations in KAT6A and we outline appropriate clinical management.

Fibronectin is a master organizer of extracellular matrices (ECMs) and promotes the assembly of collagens, fibrillin-1, and other proteins. It is also known to play roles in skeletal tissues through its secretion by osteoblasts, chondrocytes, and mesenchymal cells. Spondylometaphyseal dysplasias (SMDs) comprise a diverse group of skeletal dysplasias and often manifest as short stature, growth-plate irregularities, and vertebral anomalies, such as scoliosis. By comparing the exomes of individuals with SMD with the radiographic appearance of ''corner fractures'' at metaphyses, we identified three individuals with fibronectin (FN1) variants affecting highly conserved residues. Furthermore, using matching tools and the SkelDys emailing list, we identified other individuals with de novo FN1 variants and a similar phenotype. The severe scoliosis in most individuals and rare developmental coxa vara distinguish individuals with FN1 mutations from those with classical Sutcliffe-type SMD. To study functional consequences of these FN1 mutations on the protein level, we introduced three disease-associated missense variants (p.Cys87Phe [c.260G>T], p.Tyr240Asp [c.718T>G], and p.Cys260Gly [c.778T>G]) into a recombinant secreted N-terminal 70 kDa fragment (rF70K) and the full-length fibronectin (rFN). The wild-type rF70K and rFN were secreted into the culture medium, whereas all mutant proteins were either not secreted or secreted at significantly lower amounts. Immunofluorescence analysis demonstrated increased intracellular retention of the mutant proteins. In summary, FN1 mutations that cause defective fibronectin secretion are found in SMD, and we thus provide additional evidence for a critical function of fibronectin in cartilage and bone.