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Background: Chromosome 22q11.2 deletion syndrome (22q11DS) is the most common microdeletion syndrome, typically presenting in neonates with congenital cardiac anomalies, hypocalcaemia and thymic hypoplasia. Some patients are diagnosed later in adolescence and adulthood, with less known about the clinical phenotype of these patients., Aim: To summarise key clinical features in cases of 22q11DS diagnosed during adolescence and adulthood., Methods: This is a retrospective cohort study of 22q11DS patients diagnosed after 13 years of age over 2010-2021, with a literature review of published cases highlighting other late diagnoses. The study was performed in a large multicentre tertiary health network in Melbourne, Australia. Patients diagnosed with 22q11DS after the age of 13 years were included in the study. Main outcome measures were key clinical features in cases of late diagnosis of 22q11DS., Results: A literature search yielded 53 published case reports and one cohort study for review (62 subjects). Additionally, 10 cases of late diagnosis of 22q11DS were identified through a retrospective electronic medical chart review. Findings suggest that intellectual disability and learning difficulties, hypocalcaemia with hypoparathyroidism and facial dysmorphism remain key features in patients with a late diagnosis of 22q11DS, with hypocalcaemia being the most common presentation leading to diagnosis. Patients diagnosed in adulthood may lack classical clinical features of congenital cardiac anomalies and thymic hypoplasia. Immunological consequences of 22q11DS are also an important late-onset consideration. Atypical features may include basal ganglia calcification., Conclusions: Chromosome 22q11DS has diverse clinical features and a highly variable phenotype, likely contributing to underdiagnosis and later diagnoses., (© 2024 The Author(s). Internal Medicine Journal published by John Wiley & Sons Australia, Ltd on behalf of Royal Australasian College of Physicians.)

Purpose: To characterize the diagnostic and clinical outcomes of a cohort of critically ill infants and children with suspected mitochondrial disorders (MD) undergoing ultra-rapid genomic testing as part of a national program., Methods: Ultra-rapid genomic sequencing was performed in 454 families (genome sequencing: n=290, exome sequencing +/- mitochondrial DNA sequencing: n=164). In 91 individuals, MD was considered, prompting analysis using an MD virtual gene panel. These individuals were reviewed retrospectively and scored according to modified Nijmegen Mitochondrial Disease Criteria., Results: A diagnosis was achieved in 47% (43/91) of individuals, 40% (17/43) of whom had an MD. Seven additional individuals in whom an MD was not suspected were diagnosed with an MD following broader analysis. Gene-agnostic analysis led to the discovery of two novel disease genes, with pathogenicity validated through targeted functional studies (CRLS1 and MRPL39). Functional studies enabled diagnosis in another four individuals. Of the 24 individuals ultimately diagnosed with an MD, 79% had a change in management, which included 53% whose care was redirected to palliation., Conclusion: Ultra-rapid genetic diagnosis of MD in acutely unwell infants and children is critical for guiding decisions about the need for additional investigations and clinical management., (Copyright © 2024. Published by Elsevier Inc.)

Background: The Cut Homeobox 1 (CUX1) gene has been implicated in a number of developmental processes and has recently emerged as an important cause of developmental delay and impaired intellectual development. Individuals with variants in CUX1 have been described with a variety of co-morbidities including variations in sex development (VSD) although these features have not been closely documented., Case Presentation: The proband is a 14-year-old male who presented with congenital complex hypospadias, neurodevelopmental differences, and subtle dysmorphism. A family history of neurodevelopmental differences and VSD was noted. Microarray testing and whole exome sequencing found the 46,XY proband had a large heterozygous in-frame deletion of exons 4-10 of the CUX1 gene., Conclusions: Our review of the literature has revealed that variants in CUX1 are associated with a range of VSD and suggest this gene should be considered in cases where a VSD is noted at birth, especially if there is a familial history of VSD and/or neurodevelopmental differences. Further work is required to fully investigate the role and regulation of CUX1 in sex development., (© 2024. The Author(s).)

Childhood conditions that feature developmental regression are poorly understood. Phenotype-genotype characterization and diagnostic yield data are needed to inform clinical decision-making. The aim of this study was to report the conditions featuring developmental regression and assess diagnostic yields of investigations. A retrospective chart review of children presenting with developmental regression to a tertiary pediatric genetic clinic between 2018 and 2021 was performed. Of 99 children, 30% (n = 30) had intellectual disability (ID), 21% (n = 21) were autistic, 39% (n = 39) were autistic with ID, and 9% (n = 9) did not have ID or autism. Thirty-two percent (n = 32) of children received a new diagnosis, including eight molecular findings not previously reported to feature developmental regression. Of the children investigated, exome sequencing (ES) provided the highest diagnostic yield (51.1%, n = 24/47), highest (63.6%, n = 14/22) for children with ID, 50% for autistic children with ID (n = 6/12) and children without autism or ID (n = 3/6), and 14.3% (n = 1/7) for autistic children without ID. We highlight the conditions that feature developmental regression and report on novel phenotypic expansions. The high diagnostic yield of ES, regardless of autism or ID diagnosis, indicates the presence of developmental regression as an opportunity to identify the cause, including for genetic differences not previously reported to include regression., (© 2024 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Purpose: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively., Methods: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID., Results: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis., Conclusion: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway - KYNU, HAAO, and NADSYN1 - have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain-specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/- mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1-/- mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Developmental regression describes when a child loses previously established skills, such as the ability to speak words and is most recognised in neurodevelopmental conditions including Autism; Developmental Epileptic Encephalopathies, such as Landau Kleffner syndrome, and genetic conditions such as Rett syndrome and Phelan McDermid syndrome. Although studies have reported developmental regression for over 100 years, there remain significant knowledge gaps within and between conditions that feature developmental regression. The certainty of evidence from earlier work has been limited by condition-specific studies, retrospective methodology, and inconsistency in the definitions and measures used for classification. Given prior limitations in the field, there is a paucity of knowledge about neurocognitive mechanisms, trajectories and outcomes for children with developmental regression, and their families. Here we provide a comprehensive overview, synthesise key definitions, clinical measures, and aetiological clues associated with developmental regression and discuss impacts on caregiver physical and mental health to clarify challenges and highlight future directions in the field., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Critically ill infants and children with rare diseases need equitable access to rapid and accurate diagnosis to direct clinical management. Over 2 years, the Acute Care Genomics program provided whole-genome sequencing to 290 families whose critically ill infants and children were admitted to hospitals throughout Australia with suspected genetic conditions. The average time to result was 2.9 d and diagnostic yield was 47%. We performed additional bioinformatic analyses and transcriptome sequencing in all patients who remained undiagnosed. Long-read sequencing and functional assays, ranging from clinically accredited enzyme analysis to bespoke quantitative proteomics, were deployed in selected cases. This resulted in an additional 19 diagnoses and an overall diagnostic yield of 54%. Diagnostic variants ranged from structural chromosomal abnormalities through to an intronic retrotransposon, disrupting splicing. Critical care management changed in 120 diagnosed patients (77%). This included major impacts, such as informing precision treatments, surgical and transplant decisions and palliation, in 94 patients (60%). Our results provide preliminary evidence of the clinical utility of integrating multi-omic approaches into mainstream diagnostic practice to fully realize the potential of rare disease genomic testing in a timely manner., (© 2023. The Author(s).)

Fragile X syndrome (FXS) is caused by hypermethylation of the FMR1 promoter due to the full mutation expansion (full mutation [FM]: CGG ≥ 200 repeats) and silencing of FMR1. Assessment of mosaicism for active-unmethylated alleles has prognostic utility. This study examined relationships between FMR1 methylation in different tissues with FMR1 messenger ribonucleic acid (mRNA) and intellectual functioning in 87 males with FXS (1.89-43.17 years of age). Methylation sensitive Southern blot (mSB) and Methylation Specific-Quantitative Melt Aanalysis (MS-QMA) were used to examine FMR1 methylation. FMR1 mRNA levels in blood showed strong relationships with FMR1 methylation assessed using MS-QMA in blood (n = 68; R 2  = 0.597; p = 1.4 × 10 -10 ) and buccal epithelial cells (BEC) (n = 62; R 2  = 0.24; p = 0.003), with these measures also showing relationships with intellectual functioning scores (p < 0.01). However, these relationships were not as strong for mSB, with ~40% of males with only FM alleles that were 100% methylated and non-mosaic by mSB, showing methylation mosaicism by MS-QMA. This was confirmed through presence of detectable levels of FMR1 mRNA in blood. In summary, FMR1 methylation levels in blood and BEC examined by MS-QMA were significantly associated with FMR1 mRNA levels and intellectual functioning in males with FXS. These relationships were not as strong for mSB, which underestimated prevalence of mosaicism., (© 2022 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Background: Despite the increasing number of clinical trials involving children with neurodevelopmental disorders, appropriate and objective outcome measures for behavioral symptoms are still required., Aim: This study assessed the agreement between parents' and clinical researchers' ratings of behavioral problem severity in children with fragile X syndrome (FXS) and chromosome 15 imprinting disorders., Methods and Procedures: The cohort comprised 123 children (64% males), aged 3-17 years, with FXS (n = 79), Prader-Willi (PWS; n = 19), Angelman (AS; n = 15), and Chromosome 15q duplication (n = 10) syndromes. Specific items from the Autism Diagnostic Observation Schedule-Second Edition and Aberrant Behavior Checklist-Community Edition mapping to corresponding behavioral domains were selected ad-hoc, to assess behavioral problems., Outcomes and Results: Inter-rater agreement for the cohort was slight for self-injury (Intraclass Correlation Coefficient (ICC) = 0.12), fair for tantrums/aggression (0.24) and mannerisms/stereotypies (0.25), and moderate for hyperactivity (0.48). When stratified by diagnosis, ICC ranged from poor (0; self-injury, AS and PWS) to substantial (0.48; hyperactivity, females with FXS)., Conclusions and Implications: The high level of inter-rater disagreement across most domains suggests that parents' and researchers' assessments led to discrepant appraisal of behavioral problem severity. These findings have implications for treatment targets and outcome measure selection in clinical trials, supporting a multi-informant approach., Competing Interests: Declarations of conflicting interests All authors declare no other competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Verheij syndrome (VRJS) is a rare craniofacial spliceosomopathy presenting with craniofacial dysmorphism, multiple congenital anomalies and variable neurodevelopmental delay. It is caused by single nucleotide variants (SNVs) in PUF60 or interstitial deletions of the 8q24.3 region. PUF60 encodes a splicing factor which forms part of the spliceosome. To date, 36 patients with a sole diagnosis of VRJS due to disease-causing PUF60 SNVs have been reported in peer-reviewed publications. Although the depth of their phenotyping has varied greatly, they exhibit marked phenotypic heterogeneity. We report 10 additional unrelated patients, including the first described patients of Khmer, Indian, and Vietnamese ethnicities, and the eldest patient to date, with 10 heterozygous PUF60 variants identified through exome sequencing, 8 previously unreported. All patients underwent deep phenotyping identifying variable dysmorphism, growth delay, neurodevelopmental delay, and multiple congenital anomalies, including several unique features. The eldest patient is the only reported individual with a germline variant and neither neurodevelopmental delay nor intellectual disability. In combining these detailed phenotypic data with that of previously reported patients (n = 46), we further refine the known frequencies of features associated with VRJS. These include neurodevelopmental delay/intellectual disability (98%), axial skeletal anomalies (74%), appendicular skeletal anomalies (73%), oral anomalies (68%), short stature (66%), cardiac anomalies (63%), brain malformations (48%), hearing loss (46%), microcephaly (41%), colobomata (38%), and other ocular anomalies (65%). This case series, incorporating three patients from previously unreported ethnic backgrounds, further delineates the broad pleiotropy and mutational spectrum of PUF60 pathogenic variants., (© 2022 Wiley Periodicals LLC.)

Purpose: To evaluate whether the additional cost of providing increasingly faster genomic results in pediatric critical care is outweighed by reductions in health care costs and increases in personal utility., Methods: Hospital costs and medical files from a cohort of 40 children were analyzed. The health economic impact of rapid and ultra-rapid genomic testing, with and without early initiation, relative to standard genomic testing was evaluated., Results: Shortening the time to results led to substantial economic and personal benefits. Early initiation of ultra-rapid genomic testing was the most cost-beneficial strategy, leading to a cost saving of AU$26,600 per child tested relative to standard genomic testing and a welfare gain of AU$12,000 per child tested. Implementation of early ultra-rapid testing of critically ill children is expected to lead to an annual cost saving of AU$7.3 million for the Australian health system and an aggregate welfare gain of AU$3.3 million, corresponding to a total net benefit of AU$10.6 million., Conclusion: Early initiation of ultra-rapid genomic testing can offer substantial economic and personal benefits. Future implementation of rapid genomic testing programs should focus not only on optimizing the laboratory workflow to achieve a fast turnaround time but also on changing clinical practice to expedite test initiation., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Purpose: Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy)., Methods: A total of 74 families with diverse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases., Results: Informative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with samples from ≥2 affected individuals or heterozygotes and 10 cases with ≥2 biospecimens. PCR amplicons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing., Conclusion: RNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long amplicons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data., Competing Interests: Conflict of Interest Sandra T. Cooper is director of Frontier Genomics Pty Ltd (Australia). Sandra T. Cooper currently receives no consultancy fees or other remuneration for this role. Frontier Genomics Pty Ltd (Australia) has no existing financial relationships that will benefit from publication of these data. Samuel P. Strom is an employee and shareholder of Fulgent Genetics. Michael F. Buckley is an employee and shareholder of Invitae. The remaining coauthors declare no conflicts of interest., (Copyright © 2021. Published by Elsevier Inc.)

Purpose: Cost-effectiveness evaluations of first-line genomic sequencing (GS) in the diagnosis of children with genetic conditions are limited by the lack of well-defined comparative cohorts. We sought to evaluate the cost-effectiveness of early GS in pediatric patients with complex monogenic conditions compared with a matched historical cohort., Methods: Data, including investigation costs, were collected in a prospective cohort of 92 pediatric patients undergoing singleton GS over an 18-month period (2016-2017) with two of the following: a condition with high mortality, multisystem disease involving three or more organs, or severe limitation of daily function. Comparative data were collected in a matched historical cohort who underwent traditional investigations in the years 2012-2013., Results: GS yielded a diagnosis in 42% while traditional investigations yielded a diagnosis in 23% (p = 0.003). A change in management was experienced by 74% of patients diagnosed following GS, compared with 32% diagnosed following traditional investigations. Singleton GS at a cost of AU$3100 resulted in a mean saving per person of AU$3602 (95% confidence interval [CI] AU$2520-4685). Cost savings occurred across all investigation subtypes and were only minimally offset by clinical management costs., Conclusion: GS in complex pediatric patients saves significant costs and doubles the diagnostic yield of traditional approaches.

Background: Our primary aim was to evaluate the systematic reanalysis of singleton exome sequencing (ES) data for unsolved cases referred for any indication. A secondary objective was to undertake a literature review of studies examining the reanalysis of genomic data from unsolved cases., Methods: We examined data from 58 unsolved cases referred between June 2016 and March 2017. First reanalysis at 4-13 months after the initial report considered genes newly associated with disease since the original analysis; second reanalysis at 9-18 months considered all disease-associated genes. At 25-34 months we reviewed all cases and the strategies which solved them., Results: Reanalysis of existing ES data alone at two timepoints did not yield new diagnoses. Over the same timeframe, 10 new diagnoses were obtained (17%) from additional strategies, such as microarray detection of copy number variation, repeat sequencing to improve coverage, and trio sequencing. Twenty-seven peer-reviewed articles were identified on the literature review, with a median new diagnosis rate via reanalysis of 15% and median reanalysis timeframe of 22 months., Conclusion: Our findings suggest that an interval of greater than 18 months from the original report may be optimal for reanalysis. We also recommend a multi-faceted strategy for cases remaining unsolved after singleton ES., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Rapid genomic diagnosis programs are transforming rare disease diagnosis in acute pediatrics. A ventilated newborn with cerebellar hypoplasia underwent rapid exome sequencing (75 h), identifying a novel homozygous ASNS splice-site variant (NM&#95;133436.3:c.1476+1G>A) of uncertain significance. Rapid ASNS splicing studies using blood-derived messenger RNA from the family trio confirmed a consistent pattern of abnormal splicing induced by the variant (cryptic 5' splice-site or exon 12 skipping) with absence of normal ASNS splicing in the proband. Splicing studies reported within 10 days led to reclassification of c.1476+1G>A as pathogenic at age 27 days. Intensive care was redirected toward palliation. Cost analyses for the neonate and his undiagnosed, similarly affected deceased sibling, demonstrate that early diagnosis reduced hospitalization costs by AU$100,828. We highlight the diagnostic benefits of adjunct RNA testing to confirm the pathogenicity of splicing variants identified via rapid genomic testing pipelines for precision and preventative medicine., (© 2020 Wiley Periodicals LLC.)

Fragile X syndrome (FXS) is a leading single-gene cause of intellectual disability (ID) with autism features. This study analysed diagnostic and prognostic utility of the Fragile X-Related Epigenetic Element 2 DNA methylation (FREE2m) assessed by Methylation Specific-Quantitative Melt Analysis and the EpiTYPER system, in retrospectively retrieved newborn blood spots (NBS) and newly created dried blood spots (DBS) from 65 children with FXS (~2-17 years). A further 168 NBS from infants from the general population were used to establish control reference ranges, in both sexes. FREE2m analysis showed sensitivity and specificity approaching 100%. In FXS males, NBS FREE2m strongly correlated with intellectual functioning and autism features, however associations were not as strong for FXS females. Fragile X mental retardation 1 gene ( FMR1 ) mRNA levels in blood were correlated with FREE2m in both NBS and DBS, for both sexes. In females, DNAm was significantly increased at birth with a decrease in childhood. The findings support the use of FREE2m analysis in newborns for screening, diagnostic and prognostic testing in FXS.

Fragile X syndrome (FXS) is caused by a hypermethylated full mutation (FM) expansion with ≥ 200 CGG repeats, and a decrease in FMR1 mRNA and its protein. However, incomplete silencing from FM alleles has been associated with more severe autism features in FXS males. This study compared scores on the Aberrant Behavior Checklist-Community-FXS version (ABC-C FX ) in 62 males affected with FXS (3 to 32 years) stratified based on presence or absence of mosaicism and/or FMR1 mRNA silencing. Associations between ABC-C FX subscales and FMR1 mRNA levels, assessed using real-time PCR relative standard curve method, were also examined. The FXS group mosaic for premutation (PM: 55-199 CGGs) and FM alleles had lower irritability (p = 0.014) and inappropriate speech (p < 0.001) scores compared to males with only FM alleles and complete loss of FMR1 mRNA. The PM/FM mosaic group also showed lower inappropriate speech scores compared to the incomplete silencing (p = 0.002) group. Increased FMR1 mRNA levels were associated with greater irritability (p < 0.001), and lower health-related quality of life scores (p = 0.004), but only in the incomplete silencing FM-only group. The findings suggest that stratification based on CGG sizing and FMR1 mRNA levels may be warranted in future research and clinical trials utilising ABC-C FX subscales as outcome measures.

Importance: Widespread adoption of rapid genomic testing in pediatric critical care requires robust clinical and laboratory pathways that provide equitable and consistent service across health care systems., Objective: To prospectively evaluate the performance of a multicenter network for ultra-rapid genomic diagnosis in a public health care system., Design, Setting, and Participants: Descriptive feasibility study of critically ill pediatric patients with suspected monogenic conditions treated at 12 Australian hospitals between March 2018 and February 2019, with data collected to May 2019. A formal implementation strategy emphasizing communication and feedback, standardized processes, coordination, distributed leadership, and collective learning was used to facilitate adoption., Exposures: Ultra-rapid exome sequencing., Main Outcomes and Measures: The primary outcome was time from sample receipt to ultra-rapid exome sequencing report. The secondary outcomes were the molecular diagnostic yield, the change in clinical management after the ultra-rapid exome sequencing report, the time from hospital admission to the laboratory report, and the proportion of laboratory reports returned prior to death or hospital discharge., Results: The study population included 108 patients with a median age of 28 days (range, 0 days to 17 years); 34% were female; and 57% were from neonatal intensive care units, 33% were from pediatric intensive care units, and 9% were from other hospital wards. The mean time from sample receipt to ultra-rapid exome sequencing report was 3.3 days (95% CI, 3.2-3.5 days) and the median time was 3 days (range, 2-7 days). The mean time from hospital admission to ultra-rapid exome sequencing report was 17.5 days (95% CI, 14.6-21.1 days) and 93 reports (86%) were issued prior to death or hospital discharge. A molecular diagnosis was established in 55 patients (51%). Eleven diagnoses (20%) resulted from using the following approaches to augment standard exome sequencing analysis: mitochondrial genome sequencing analysis, exome sequencing-based copy number analysis, use of international databases to identify novel gene-disease associations, and additional phenotyping and RNA analysis. In 42 of 55 patients (76%) with a molecular diagnosis and 6 of 53 patients (11%) without a molecular diagnosis, the ultra-rapid exome sequencing result was considered as having influenced clinical management. Targeted treatments were initiated in 12 patients (11%), treatment was redirected toward palliative care in 14 patients (13%), and surveillance for specific complications was initiated in 19 patients (18%)., Conclusions and Relevance: This study suggests feasibility of ultra-rapid genomic testing in critically ill pediatric patients with suspected monogenic conditions in the Australian public health care system. However, further research is needed to understand the clinical value of such testing, and the generalizability of the findings to other health care settings.

Congenital hearing impairment (HI) is the most common sensory impairment and can be isolated or part of a syndrome. Diagnosis through newborn hearing screening and management through early intervention, hearing aids and cochlear implantation is well established in the Australian setting; however understanding the genetic basis of congenital HI has been missing. This population-derived cohort comprised infants with moderate-profound bilateral HI born in the 2016-2017 calendar years, detected through newborn hearing screening. Participants were recruited through an integrated paediatric, otolaryngology and genetics HI clinic and offered whole exome sequencing (WES) on a HiSeq4000 or NextSeq500 (Illumina) platform with a targeted average sequencing depth of 100x and chromosome microarray on the Illumina Infinium core exome-24v1.2 platform. Of those approached, 68% (106/156) consented to participate. The rate of genetic diagnosis was 56% (59/106), significantly higher than standard of care (GJB2/6 sequencing only), 21% (22/106). There were clinical implications for the 106 participants: 36% required no further screening, 9% had tailored screening initiated, 2% were offered treatment and 4% had informed care for a complex neurodevelopmental syndrome. WES in this cohort demonstrates the range of diagnoses associated with congenital HI and confirms the genetic heterogeneity of congenital HI. The high diagnostic yield and clinical implications emphasises the need for genomic sequencing to become standard of care.

Context: The WNT/β-catenin pathway is central to the pathogenesis of various human diseases including those affecting bone development and tumor progression., Objective: To evaluate the role of a gain-of-function variant in CTNNB1 in a child with a sclerosing bone dysplasia and an adrenocortical adenoma., Design: Whole exome sequencing with corroborative biochemical analyses., Patients: We recruited a child with a sclerosing bone dysplasia and an adrenocortical adenoma together with her unaffected parents., Intervention: Whole exome sequencing and performance of immunoblotting and luciferase-based assays to assess the cellular consequences of a de novo variant in CTNNB1., Main Outcome Measure(s)/result: A de novo variant in CTNNB1 (c.131C>T; p.[Pro44Leu]) was identified in a patient with a sclerosing bone dysplasia and an adrenocortical adenoma. A luciferase-based transcriptional assay of WNT signaling activity verified that the activity of β-catenin was increased in the cells transfected with a CTNNB1p.Pro44Leu construct (P = 4.00 × 10-5). The β-catenin p.Pro44Leu variant was also associated with a decrease in phosphorylation at Ser45 and Ser33/Ser37/Thr41 in comparison to a wild-type (WT) CTNNB1 construct (P = 2.16 × 10-3, P = 9.34 × 10-8 respectively)., Conclusion: Increased β-catenin activity associated with a de novo gain-of-function CTNNB1 variant is associated with osteosclerotic phenotype and adrenocortical neoplasia., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: Clinical genetics is an evolving specialty impacted by the availability of increasingly sophisticated investigational technologies. Methods for monitoring the changes in workload and workflow are necessary to ensure adequate service resourcing., Methods: A literature search of known workload and workflow studies was completed, identifying metrics of value. A framework of metrics to allow consistent capture in clinical genetics practice was developed. This framework was then applied to local general genetics service data to evaluate recent changes in service delivery., Results: Literature regarding service delivery metrics in clinical genetics services is limited and inconsistent in application. The metric framework generated is a useful tool for consistent and ongoing evaluation of general genetics services. Through application of the framework, new service delivery trends and significant changes in workload were identified., Conclusion: Studies of clinical genetics service delivery suffer from the use of inconsistent metrics. This framework will allow for monitoring of changes to service delivery, caseload volume, caseload complexity, and workforce over time. Local data presented demonstrate the significant effect that implementing clinical genomic sequencing has had on clinical service delivery. Applying this framework produces a comprehensive service characterization, enabling funding bodies to justify resourcing that addresses the growing demand of clinical genetics.

Background: Fragile X syndrome (FXS) is a common cause of intellectual disability and autism spectrum disorder (ASD) usually associated with a CGG expansion, termed full mutation (FM: CGG ≥ 200), increased DNA methylation of the FMR1 promoter and silencing of the gene. Mosaicism for presence of cells with either methylated FM or smaller unmethylated pre-mutation (PM: CGG 55-199) alleles in the same individual have been associated with better cognitive functioning. This study compares age- and sex-matched FM-only and PM/FM mosaic individuals on intellectual functioning, ASD features and maladaptive behaviours., Methods: This study comprised a large international cohort of 126 male and female participants with FXS (aged 1.15 to 43.17 years) separated into FM-only and PM/FM mosaic groups (90 males, 77.8% FM-only; 36 females, 77.8% FM-only). Intellectual functioning was assessed with age appropriate developmental or intelligence tests. The Autism Diagnostic Observation Schedule-2nd Edition was used to examine ASD features while the Aberrant Behavior Checklist-Community assessed maladaptive behaviours., Results: Comparing males and females (FM-only + PM/FM mosaic), males had poorer intellectual functioning on all domains (p < 0.0001). Although females had less ASD features and less parent-reported maladaptive behaviours, these differences were no longer significant after controlling for intellectual functioning. Participants with PM/FM mosaicism, regardless of sex, presented with better intellectual functioning and less maladaptive behaviours compared with their age- and sex-matched FM-only counterparts (p < 0.05). ASD features were similar between FM-only and PM/FM mosaics within each sex, after controlling for overall intellectual functioning., Conclusions: Males with FXS had significantly lower intellectual functioning than females with FXS. However, there were no significant differences in ASD features and maladaptive behaviours, after controlling for intellectual functioning, independent of the presence or absence of mosaicism. This suggests that interventions that primarily target cognitive abilities may in turn reduce the severity of maladaptive behaviours including ASD features in FXS.

Rapid genomic testing is a valuable new diagnostic tool for acutely unwell infants, however exome sequencing does not deliver clinical-grade mitochondrial genome sequencing and may fail to diagnose mitochondrial disorders caused by mitochondrial DNA (mtDNA) variants. Rapid mitochondrial genome sequencing and analysis are not routinely available in rapid genomic diagnosis programmes. We present two critically ill neonates with transfusion-dependent anaemia and persistent lactic acidosis who underwent rapid mitochondrial genome sequencing in tandem with exome sequencing as part of an exome sequencing-based rapid genomic diagnosis programme. No diagnostic variants were identified on examination of the nuclear exome data for either infant. Mitochondrial genome sequencing identified a large mtDNA deletion in both infants, diagnosing Pearson syndrome within 74 and 55 h, respectively. Early diagnosis in the third week of life allowed the avoidance of a range of other investigations and appropriate treatment planning. Rapid mitochondrial genome analysis provides additional diagnostic and clinical utility and should be considered as an adjunct to exome sequencing in rapid genomic diagnosis programmes.

Germline mutations in fundamental epigenetic regulatory molecules including DNA methyltransferase 3 alpha ( DNMT3A ) are commonly associated with growth disorders, whereas somatic mutations are often associated with malignancy. We profiled genome-wide DNA methylation patterns in DNMT3A c.2312G > A; p.(Arg771Gln) carriers in a large Amish sibship with Tatton-Brown-Rahman syndrome (TBRS), their mosaic father, and 15 TBRS patients with distinct pathogenic de novo DNMT3A variants. This defined widespread DNA hypomethylation at specific genomic sites enriched at locations annotated as genes involved in morphogenesis, development, differentiation, and malignancy predisposition pathways. TBRS patients also displayed highly accelerated DNA methylation aging. These findings were most marked in a carrier of the AML-associated driver mutation p.Arg882Cys. Our studies additionally defined phenotype-related accelerated and decelerated epigenetic aging in two histone methyltransferase disorders: NSD1 Sotos syndrome overgrowth disorder and KMT2D Kabuki syndrome growth impairment. Together, our findings provide fundamental new insights into aberrant epigenetic mechanisms, the role of epigenetic machinery maintenance, and determinants of biological aging in these growth disorders., (© 2019 Jeffries et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Fragile X syndrome (FXS) is a common monogenic cause of intellectual disability with autism features. While it is caused by loss of the FMR 1 product (FMRP), mosaicism for active and inactive FMR1 alleles, including alleles termed premutation (PM: 55-199 CGGs), is not uncommon. Importantly, both PM and active full mutation (FM: ≥ 200 CGGs) alleles often express elevated levels of mRNA that are thought to be toxic. This study determined if complete FMR1 mRNA silencing from FM alleles and/or levels of FMR1 mRNA (if present) in blood are associated with intellectual functioning and autism features in FXS., Methods: The study cohort included 98 participants (70.4% male) with FXS (FM-only and PM/FM mosaic) aged 1-43 years. A control group of 14 females were used to establish control FMR1 mRNA reference range. Intellectual functioning and autism features were assessed using the Mullen Scales of Early Learning or an age-appropriate Wechsler Scale and the Autism Diagnostic Observation Schedule-2nd Edition (ADOS-2), respectively. FMR1 mRNA was analysed in venous blood collected at the time of assessments, using the real-time PCR relative standard curve method., Results: Females with FXS had significantly higher levels of FMR1 mRNA ( p  < 0.001) than males. FMR1 mRNA levels were positively associated with age ( p  < 0.001), but not with intellectual functioning and autistic features in females. FM-only males (aged < 19 years) expressing FM FMR1 mRNA had significantly higher ADOS calibrated severity scores compared to FM-only males with completely silenced FMR1 ( p  = 0.011). However, there were no significant differences between these subgroups on intellectual functioning. In contrast, decreased levels of FMR1 mRNA were associated with decreased intellectual functioning in FXS males ( p  = 0.029), but not autism features, when combined with the PM/FM mosaic group., Conclusion: Incomplete silencing of toxic FM RNA may be associated with autistic features, but not intellectual functioning in FXS males. While decreased levels of mRNA may be more predictive of intellectual functioning than autism features. If confirmed in future studies, these findings may have implications for patient stratification, outcome measure development, and design of clinical and pre-clinical trials in FXS., Competing Interests: All procedures were approved by The Royal Children’s Hospital and INTA Human Research Ethics Committees (HREC #33066 and #15, respectively). All parents/caregivers provided written informed consent and those who were deemed cognitively able also provided written informed consent.D. Godler is an inventor of the following patents: PCT/AU2010/001134; filing No. AU2010/903595; filing No. AU2011/902500; and filing No. 2013/900227, related to the technology described in this publication. All other authors have no conflicts of interest to declare.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Increased intragenic DNA methylation of the Fragile X Related Epigenetic Element 2 (FREE2) in blood has been correlated with lower intellectual functioning in females with fragile X syndrome (FXS). This study explored these relationships in a paediatric cohort of males with FXS using Buccal Epithelial Cells (BEC). BEC were collected from 25 males with FXS, aged 3 to 17 years and 19 age-matched male controls without FXS. Methylation of 9 CpG sites within the FREE2 region was examined using the EpiTYPER approach. Full Scale IQ (FSIQ) scores of males with FXS were corrected for floor effect using the Whitaker and Gordon (WG) extrapolation method. Compared to controls, children with FXS had significant higher methylation levels for all CpG sites examined (p < 3.3 × 10 -7 ), and within the FXS group, lower FSIQ (WG corrected) was associated with higher levels of DNA methylation, with the strongest relationship found for CpG sites within FMR1 intron 1 (p < 5.6 × 10 -5 ). Applying the WG method to the FXS cohort unmasked significant epi-genotype-phenotype relationships. These results extend previous evidence in blood to BEC and demonstrate FREE2 DNA methylation to be a sensitive epigenetic biomarker significantly associated with the variability in intellectual functioning in FXS.

Background: Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously., Results: We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management., Conclusions: Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.

Export of mRNA from the cell nucleus to the cytoplasm is essential for protein synthesis, a process vital to all living eukaryotic cells. mRNA export is highly conserved and ubiquitous. Mutations affecting mRNA and mRNA processing or export factors, which cause aberrant retention of mRNAs in the nucleus, are thus emerging as contributors to an important class of human genetic disorders. Here, we report that variants in THOC2, which encodes a subunit of the highly conserved TREX mRNA-export complex, cause syndromic intellectual disability (ID). Affected individuals presented with variable degrees of ID and commonly observed features included speech delay, elevated BMI, short stature, seizure disorders, gait disturbance, and tremors. X chromosome exome sequencing revealed four missense variants in THOC2 in four families, including family MRX12, first ascertained in 1971. We show that two variants lead to decreased stability of THOC2 and its TREX-complex partners in cells derived from the affected individuals. Protein structural modeling showed that the altered amino acids are located in the RNA-binding domains of two complex THOC2 structures, potentially representing two different intermediate RNA-binding states of THOC2 during RNA transport. Our results show that disturbance of the canonical molecular pathway of mRNA export is compatible with life but results in altered neuronal development with other comorbidities., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

α-Isopropylmalate synthase (IPMS) catalyses the reaction between α-ketoisovalerate and acetyl coenzyme A (AcCoA) in the first step of leucine biosynthesis. IPMS is closely related to homocitrate synthase, which catalyses the reaction between AcCoA and the unbranched α-ketoacid α-ketoglutarate. Analysis of these enzymes suggests that several differently conserved key residues are responsible for the different substrate selectivity. These residues were systematically substituted in the Mycobacterium tuberculosis IPMS, resulting in changes in substrate specificity. A variant of IPMS was constructed with a preference for the unbranched α-ketoacids α-ketobutyrate and pyruvate over the natural branched substrate α-ketoisovalerate., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Mutations in FOXP1, located at 3p13, have been reported in patients with global developmental delay (GDD), intellectual disability (ID), and speech defects. Mutations in FOXP2, located at 7q31, are well known to cause developmental speech and language disorders, particularly developmental verbal dyspraxia (DVD). FOXP2 has been shown to work co-operatively with FOXP1 in mouse development. An overlap in FOXP1 and FOXP2 expression, both in the songbird and human fetal brain, has suggested that FOXP1 may also have a role in speech and language disorders. We report on a male child with a 0.19 MB intragenic deletion that is predicted to result in haploinsufficiency of FOXP1. Review of our patient and others reported in the literature reveals an emerging phenotype of GDD/ID with moderate to severe speech delay where expressive speech is most severely affected. DVD appears not to be a distinct feature in this group. Facial features include a broad forehead, downslanting palpebral fissures, a short nose with broad tip, relative or true macrocephaly, a frontal hair upsweep and prominent digit pads. Autistic traits and other behavioral problems are likely to be associated with haploinsufficiency of FOXP1. Congenital malformations may be associated., (© 2013 Wiley Periodicals, Inc.)

Alpers syndrome is a rare autosomal recessive hepatocerebral degenerative disorder. Seventeen patients with Alpers syndrome or polymerase-γ gene mutations were identified. Case records of 12 patients with Alpers syndrome and polymerase-γ mutations in both alleles were reviewed. All patients manifested developmental delay or regression, refractory epilepsy, and biochemical liver dysfunction. Liver failure occurred in three patients previously treated with valproate. Other signs included ataxia, visual disturbance, motor paresis, and tremor. Myoclonic and focal motor seizures were common, often manifesting as status epilepticus. Electroencephalograms demonstrated absent/slow posterior dominant rhythms. Interictal discharges were common, usually involving the occipital lobes. Rhythmic high-amplitude delta with (poly)spikes was evident in four patients. Magnetic resonance imaging showed migratory, cortical, and subcortical T(2) hyperintensities in four children most often affected the parietal and occipital lobes. Developmental regression and refractory focal motor or myoclonic seizures are consistent clinical features of Alpers syndrome with polymerase-γ mutations. Liver dysfunction constitutes a late manifestation. Migratory T(2)/fluid attenuated inversion recovery signal abnormalities involving metabolically active occipital and sensorimotor cortical regions comprise characteristic imaging findings. Interictal and ictal electroencephalogram patterns are more variable than previously reported. Three common polymerase-γ mutations, in patients of European descent, can assist with rapid diagnosis, circumventing the need for liver biopsy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

alpha-Isopropylmalate synthase (alpha-IPMS) catalyses the first committed step in leucine biosynthesis in many pathogenic bacteria, including Neisseria meningitidis. This enzyme (NmeIPMS) has been purified, characterised, and compared to alpha-IPMS proteins from other bacteria. NmeIPMS is a homodimer which catalyses the condensation of alpha-ketoisovalerate (alpha-KIV) and acetyl coenzyme A (AcCoA), and is inhibited by leucine. NmeIPMS can use alternate alpha-ketoacids as substrates and, in contrast to alpha-IPMS from other sources, is activated by a range of metal ions including Cd(2+) and Zn(2+) that have previously been reported as inhibitory, since they suppress the dithiodipyridone assay system rather than the enzyme itself. Previous studies indicate that alpha-IPMS is a TIM barrel enzyme with an allosteric leucine-binding domain. To assess the importance of this domain, a truncated form of NmeIPMS was generated and characterised. Loss of the regulatory domain resulted in a loss of the ability to catalyse the aldol reaction, although the enzyme was still able to slowly hydrolyse AcCoA independently of alpha-KIV at a rate similar to that of the WT enzyme. This implies that the regulatory domain is not only required for control of enzymatic activity but may assist in the positioning of key residues in the catalytic TIM barrel. The importance of this domain to catalytic function may offer new strategies for inhibitor design., (Copyright 2010 Elsevier Inc. All rights reserved.)

Aim: To audit the identification and screening of graduates from a neonatal intensive care unit with risk factors for sensorineural hearing loss., Methods: Hospital medical records of newborn infants discharged from the neonatal intensive care unit, Christchurch Womens Hospital, between 1 July 1994 and 30 June 1995 (n=564), were examined to identify those at risk for sensorineural hearing loss according to the American Speech-Language Hearing Association risk criteria 1991. Auditory brainstem response test results were obtained from the Christchurch Hospital Audiology Department. Outcome measures were: presence of hearing loss risk factors, numbers tested with auditory brainstem response, age at test and presence and degree of hearing impairment., Results: Of 5,215 live births in Christchurch, 564 infants were discharged through the neonatal intensive care unit. Of these, 86 had risk factors for sensorineural hearing loss. There were 72 (84%) infants tested at audiology, with fifteen (17%) having abnormal test results. There were fourteen with risk factors who did not get audiology screening., Conclusion: A high proportion (84%) of high risk newborn infants had auditory brainstem response testing. Further improvement would require strict implementation of standard procedures. Auditory brainstem response screening is part of a wider population surveillance approach to identify hearing loss as early as possible.