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BackgroundClinical exome sequencing typically achieves diagnostic yields of 30%–57.5% in individuals with monogenic rare diseases. Undiagnosed diseases programmes implement strategies to improve diagnostic outcomes for these individuals.AimWe share the lessons learnt from the first 3 years of the Undiagnosed Diseases Program-Victoria, an Australian programme embedded within a clinical genetics service in the state of Victoria with a focus on paediatric rare diseases.MethodsWe enrolled families who remained without a diagnosis after clinical genomic (panel, exome or genome) sequencing between 2016 and 2018. We used family-based exome sequencing (family ES), family-based genome sequencing (family GS), RNA sequencing (RNA-seq) and high-resolution chromosomal microarray (CMA) with research-based analysis.ResultsIn 150 families, we achieved a diagnosis or strong candidate in 64 (42.7%) (37 in known genes with a consistent phenotype, 3 in known genes with a novel phenotype and 24 in novel disease genes). Fifty-four diagnoses or strong candidates were made by family ES, six by family GS with RNA-seq, two by high-resolution CMA and two by data reanalysis.ConclusionWe share our lessons learnt from the programme. Flexible implementation of multiple strategies allowed for scalability and response to the availability of new technologies. Broad implementation of family ES with research-based analysis showed promising yields post a negative clinical singleton ES. RNA-seq offered multiple benefits in family ES-negative populations. International data sharing strategies were critical in facilitating collaborations to establish novel disease–gene associations. Finally, the integrated approach of a multiskilled, multidisciplinary team was fundamental to having diverse perspectives and strategic decision-making.

SHQ1 is essential for biogenesis of H/ACA ribonucleoproteins, a class of molecules important for processing ribosomal RNAs, modifying spliceosomal small nuclear RNAs and stabilizing telomerase. Components of the H/ACA ribonucleoprotein complex have been linked to neurological developmental defects. Here, we report two sibling pairs from unrelated families with compound heterozygous variants in SHQ1. Exome sequencing was used to detect disease causing variants, which were submitted to ‘matching’ platforms linked to MatchMaker Exchange. Phenotype comparisons supported these matches. The affected individuals present with early-onset dystonia, with individuals from one family displaying additional neurological phenotypes, including neurodegeneration. As a result of cerebrospinal fluid studies suggesting possible abnormal dopamine metabolism, a trial of levodopa replacement therapy was started but no clear response was noted. We show that fibroblasts from affected individuals have dramatic loss of SHQ1 protein. Variants from both families were expressed in Saccharomyces cerevisiae, resulting in a strong reduction in H/ACA snoRNA production and remarkable defects in rRNA processing and ribosome formation. Our study identifies SHQ1 as associated with neurological disease, including early-onset dystonia, and begins to delineate the molecular etiology of this novel condition.

Sharing genomic variant interpretations across laboratories promotes consistency in variant assertions. A landscape analysis of Australian clinical genetic-testing laboratories in 2017 identified that, despite the national-accreditation-body recommendations encouraging laboratories to submit genotypic data to clinical databases, fewer than 300 variants had been shared to the ClinVar public database. Consultations with Australian laboratories identified resource constraints limiting routine application of manual processes, consent issues, and differences in interpretation systems as barriers to sharing. This information was used to define key needs and solutions required to enable national sharing of variant interpretations. The Shariant platform, using both the GRCh37 and GRCh38 genome builds, was developed to enable ongoing sharing of variant interpretations and associated evidence between Australian clinical genetic-testing laboratories. Where possible, two-way automated sharing was implemented so that disruption to laboratory workflows would be minimized. Terms of use were developed through consultation and currently restrict access to Australian clinical genetic-testing laboratories. Shariant was designed to store and compare structured evidence, to promote and record resolution of inter-laboratory classification discrepancies, and to streamline the submission of variant assertions to ClinVar. As of December 2021, more than 14,000 largely prospectively curated variant records from 11 participating laboratories have been shared. Discrepant classifications have been identified for 11% (28/260) of variants submitted by more than one laboratory. We have demonstrated that co-design with clinical laboratories is vital to developing and implementing a national variant-interpretation sharing effort. This approach has improved inter-laboratory concordance and enabled opportunities to standardize interpretation practices.

PURPOSE: Somatic variants in tumor necrosis factor receptor-associated factor 7 (TRAF7) cause meningioma, while germline variants have recently been identified in seven patients with developmental delay and cardiac, facial, and digital anomalies. We aimed to define the clinical and mutational spectrum associated with TRAF7 germline variants in a large series of patients, and to determine the molecular effects of the variants through transcriptomic analysis of patient fibroblasts.METHODS: We performed exome, targeted capture, and Sanger sequencing of patients with undiagnosed developmental disorders, in multiple independent diagnostic or research centers. Phenotypic and mutational comparisons were facilitated through data exchange platforms. Whole-transcriptome sequencing was performed on RNA from patient- and control-derived fibroblasts.RESULTS: We identified heterozygous missense variants in TRAF7 as the cause of a developmental delay-malformation syndrome in 45 patients. Major features include a recognizable facial gestalt (characterized in particular by blepharophimosis), short neck, pectus carinatum, digital deviations, and patent ductus arteriosus. Almost all variants occur in the WD40 repeats and most are recurrent. Several differentially expressed genes were identified in patient fibroblasts.CONCLUSION: We provide the first large-scale analysis of the clinical and mutational spectrum associated with the TRAF7 developmental syndrome, and we shed light on its molecular etiology through transcriptome studies.

The evolutionarily conserved hedgehog (Hh) pathway is essential for organogenesis and plays critical roles in postnatal tissue maintenance and renewal. A unique feature of the vertebrate Hh pathway is that signal transduction requires the primary cilium (PC) where major pathway components are dynamically enriched. These factors include smoothened (SMO) and patched, which constitute the core reception system for sonic hedgehog (SHH) as well as GLI transcription factors, the key mediators of the pathway. Here, we report biallelic loss-of-function variations in SMO in seven individuals from five independent families; these variations cause a wide phenotypic spectrum of developmental anomalies affecting the brain (hypothalamic hamartoma and microcephaly), heart (atrioventricular septal defect), skeleton (postaxial polydactyly, narrow chest, and shortening of long bones), and enteric nervous system (aganglionosis). Cells derived from affected individuals showed normal ciliogenesis but severely altered Hh-signal transduction as a result of either altered PC trafficking or abnormal activation of the pathway downstream of SMO. In addition, Hh-independent GLI2 accumulation at the PC tip in cells from the affected individuals suggests a potential function of SMO in regulating basal ciliary trafficking of GLI2 when the pathway is off. Thus, loss of SMO function results in abnormal PC dynamics of key components of the Hh signaling pathway and leads to a large continuum of malformations in humans.

usc Refereed/Peer-reviewed 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.

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

The vast quantities of short-read sequencing data being generated are often exchanged and stored as aligned reads. However, aligned data becomes outdated as new reference genomes and alignment methods become available. Here we describe Bazam, a tool that efficiently extracts the original paired FASTQ from alignment files (BAM or CRAM format) in a format that directly allows efficient realignment. Bazam facilitates up to a 90% reduction in the time for realignment compared to standard methods. Bazam can support selective extraction of read pairs from focused genomic regions for applications such as targeted region analyses, quality control, structural variant calling, and alignment comparisons. Electronic supplementary material The online version of this article (10.1186/s13059-019-1688-1) contains supplementary material, which is available to authorized users.

The lipid phosphatase gene FIG4 is responsible for Yunis-Varon syndrome and Charcot-Marie-Tooth disease Type 4J, a peripheral neuropathy. We now describe four families with FIG4 variants and prominent abnormalities of central nervous system (CNS) white matter (leukoencephalopathy), with onset in early childhood, ranging from severe hypomyelination to mild undermyelination, in addition to peripheral neuropathy. Affected individuals inherited biallelic FIG4 variants from heterozygous parents. Cultured fibroblasts exhibit enlarged vacuoles characteristic of FIG4 dysfunction. Two unrelated families segregate the same G > A variant in the +1 position of intron 21 in the homozygous state in one family and compound heterozygous in the other. This mutation in the splice donor site of exon 21 results in read-through from exon 20 into intron 20 and truncation of the final 115 C-terminal amino acids of FIG4, with retention of partial function. The observed CNS white matter disorder in these families is consistent with the myelination defects in the FIG4 null mouse and the known role of FIG4 in oligodendrocyte maturation. The families described here the expanded clinical spectrum of FIG4 deficiency to include leukoencephalopathy.

The DNA damage-binding protein 1 (DDB1) is part of the CUL4-DDB1 ubiquitin E3 ligase complex (CRL4), which is essential for DNA repair, chromatin remodeling, DNA replication, and signal transduction. Loss-of-function variants in genes encoding the complex components CUL4 and PHIP have been reported to cause syndromic intellectual disability with hypotonia and obesity, but no phenotype has been reported in association with DDB1 variants. Here, we report eight unrelated individuals, identified through Matchmaker Exchange, with de novo monoallelic variants in DDB1, including one recurrent variant in four individuals. The affected individuals have a consistent phenotype of hypotonia, mild to moderate intellectual disability, and similar facies, including horizontal or slightly bowed eyebrows, deep-set eyes, full cheeks, a short nose, and large, fleshy and forward-facing earlobes, demonstrated in the composite face generated from the cohort. Digital anomalies, including brachydactyly and syndactyly, were common. Three older individuals have obesity. We show that cells derived from affected individuals have altered DDB1 function resulting in abnormal DNA damage signatures and histone methylation following UV-induced DNA damage. Overall, our study adds to the growing family of neurodevelopmental phenotypes mediated by disruption of the CRL4 ubiquitin ligase pathway and begins to delineate the phenotypic and molecular effects of DDB1 misregulation.

In about half of all patients with a suspected monogenic disease, genomic investigations fail to identify the diagnosis. A contributing factor is the difficulty with repetitive regions of the genome, such as those generated by segmental duplications. TheWhole exome, whole genome and long-read DNA sequencing techniques combined with studies of RNA and quantitative proteomics were used to investigate 17 subjects from 16 unrelated families with suspected mitochondrial disease.We report six differentAustralian NHMRC, US Department of Defense, Japanese AMED and JSPS agencies, Australian Genomics Health Alliance and Australian Mito Foundation.

Summary Background In about half of all patients with a suspected monogenic disease, genomic investigations fail to identify the diagnosis. A contributing factor is the difficulty with repetitive regions of the genome, such as those generated by segmental duplications. The ATAD3 locus is one such region in which recessive deletions and dominant duplications have recently been reported to cause lethal perinatal mitochondrial diseases characterized by pontocerebellar hypoplasia or cardiomyopathy, respectively. Methods Whole-exome, whole-genome, and long-read DNA sequencing techniques combined with studies of RNA and quantitative proteomics were used to investigate 17 subjects from 16 unrelated families with suspected mitochondrial disease. Findings We report 6 different de novo duplications in the ATAD3 gene locus causing a distinctive presentation, including lethal perinatal cardiomyopathy, persistent hyperlactacidemia, and frequently, corneal clouding or cataracts and encephalopathy. The recurrent 68-kb ATAD3 duplications are identifiable from genome and exome sequencing but usually missed by microarrays. The ATAD3 duplications result in the formation of identical chimeric ATAD3A/ATAD3C proteins, altered ATAD3 complexes, and a striking reduction in mitochondrial oxidative phosphorylation complex I and its activity in heart tissue. Conclusions ATAD3 duplications appear to act in a dominant-negative manner and the de novo inheritance infers a low recurrence risk for families, unlike most pediatric mitochondrial diseases. More than 350 genes underlie mitochondrial diseases. In our experience, the ATAD3 locus is now one of the five most common causes of nuclear-encoded pediatric mitochondrial disease, but the repetitive nature of the locus means ATAD3 diagnoses may be frequently missed by current genomic strategies. Funding Australian NHMRC, US Department of Defense, US National Institutes of Health, Japanese AMED and JSPS agencies, Australian Genomics Health Alliance, and Australian Mito Foundation.

Rapid identification of clinically significant variants is key to the successful application of next generation sequencing technologies in clinical practice. The Melbourne Genomics Health Alliance (MGHA) variant prioritization framework employs a gene prioritization index based on clinician-generated a priori gene lists, and a variant prioritization index (VPI) based on rarity, conservation and protein effect. We used data from 80 patients who underwent singleton whole exome sequencing (WES) to test the ability of the framework to rank causative variants highly, and compared it against the performance of other gene and variant prioritization tools. Causative variants were identified in 59 of the patients. Using the MGHA prioritization framework the average rank of the causative variant was 2.24, with 76% ranked as the top priority variant, and 90% ranked within the top five. Using clinician-generated gene lists resulted in ranking causative variants an average of 8.2 positions higher than prioritization based on variant properties alone. This clinically driven prioritization approach significantly outperformed purely computational tools, placing a greater proportion of causative variants top or in the top 5 (permutation P-value=0.001). Clinicians included 40 of the 49 WES diagnoses in their a priori list of differential diagnoses (81%). The lists generated by PhenoTips and Phenomizer contained 14 (29%) and 18 (37%) of these diagnoses respectively. These results highlight the benefits of clinically led variant prioritization in increasing the efficiency of singleton WES data analysis and have important implications for developing models for the funding and delivery of genomic services.

USP9X is an X-chromosome gene that escapes X-inactivation. Loss or compromised function of USP9X leads to neurodevelopmental disorders in males and females. While males are impacted primarily by hemizygous partial loss-of-function missense variants, in females de novo heterozygous complete loss-of-function mutations predominate, and give rise to the clinically recognisable USP9X-female syndrome. Here we provide evidence of the contribution of USP9X missense and small in-frame deletion variants in USP9X-female syndrome also. We scrutinise the pathogenicity of eleven such variants, ten of which were novel. Combined application of variant prediction algorithms, protein structure modelling, and assessment under clinically relevant guidelines universally support their pathogenicity. The core phenotype of this cohort overlapped with previous descriptions of USP9X-female syndrome, but exposed heightened variability. Aggregate phenotypic information of 35 currently known females with predicted pathogenic variation in USP9X reaffirms the clinically recognisable USP9X-female syndrome, and highlights major differences when compared to USP9X-male associated neurodevelopmental disorders.

The RNA editing enzyme ADAR2 is essential for the recoding of brain transcripts. Impaired ADAR2 editing leads to early-onset epilepsy and premature death in a mouse model. Here, we report bi-allelic variants in ADARB1, the gene encoding ADAR2, in four unrelated individuals with microcephaly, intellectual disability, and epilepsy. In one individual, a homozygous variant in one of the double-stranded RNA-binding domains (dsRBDs) was identified. In the others, variants were situated in or around the deaminase domain. To evaluate the effects of these variants on ADAR2 enzymatic activity, we performed in vitro assays with recombinant proteins in HEK293T cells and ex vivo assays with fibroblasts derived from one of the individuals. We demonstrate that these ADAR2 variants lead to reduced editing activity on a known ADAR2 substrate. We also demonstrate that one variant leads to changes in splicing of ADARB1 transcript isoforms. These findings reinforce the importance of RNA editing in brain development and introduce ADARB1 as a genetic etiology in individuals with intellectual disability, microcephaly, and epilepsy.

Multiple studies have shown that genomic testing has a high diagnostic yield and an impact on clinical management for patients with suspected genetic conditions. Therefore, there has been a push worldwide to apply rapid genomic sequencing in critically ill neonatal and pediatric patients. The goal of this study was to investigate the practicality of applying ultrarapid genomic testing for critically ill neonatal and pediatric patients with suspected monogenic conditions in Australia. The study recruited a total of 108 patients prospectively from March 2018 to February 2019, and data were collected until May 2019. Of the 12 hospitals that acted as collaborating sites, 5 were women's hospitals, 3 women's and children's hospitals, and 4 children's hospitals. Eligible patients included those who were admitted to a neonatal or pediatric intensive care unit (NICU or PICU) and were referred to clinical genetics for a possible monogenic condition. Chromosomal microarray was a requirement before enrollment if there was a suspected chromosomal condition. Chromosomal microarrays were performed concurrently with ultrarapid exome sequencing when the probability of a positive result on microarray was low. Additionally, rapid mitochondrial genome sequencing was performed alongside ultrarapid exome sequencing if a mitochondrial condition was suspected. When possible, ultrarapid exome sequencing was performed in both parents as well as the child (trio). The primary outcome of this study measured the time from the last sample received to the ultrarapid exome sequencing report finalized. Other outcomes measured were the diagnostic yield, change in clinical management after the report was finalized, number of reports returned before hospital discharge, and time from admission to the report being finalized. Of the 108 patients enrolled, the median age was 28 days, with a range of 0 days to 17 years. Overall, 34% were female, 57% were NICU admissions, 33% were PICU admissions, and 9% were from other hospital wards. The mean time it took from the last sample received to the report being issued was 3.3 days (95% confidence interval [CI], 3.2-3.5 days). The mean time from hospital admission to the sequencing report being finalized was 17.5 days (95% CI, 14.6-21.1 days). The mean time from hospital admission to clinical genetics referral was 8.1 days (95% CI, 6.0-10.8 days). Overall, 94% of reports were issued within the goal of 5 calendar days, and 56 molecular diagnoses were made for 55 patients (51%). Among the 62 patients in the NICU, 35 had a clear molecular diagnosis from ultrarapid exome sequencing (56%), whereas in the 36 PICU patients, 17 had a clear molecular diagnosis (47%), and in the 10 patients from other hospital wards, 3 had a clear molecular diagnosis (30%). After the ultrarapid exome sequencing report was finalized, clinical management changed for 48 of the 108 patients enrolled (44%). Overall, the ultrarapid sequencing reports that revealed a clear molecular diagnosis were regarded as “very useful” or “useful” by referring providers in 52 of 55 cases (95%) and “neutral” in 3 of 55 cases (5%). Negative reports were regarded as “very useful” or “useful” in 31 of 53 cases (58%), “neutral” in 20 of 53 cases, and “not useful” in 2 of 53 cases (4%). Altogether, this study demonstrates the utility of ultrarapid exome sequencing in critically ill pediatric patients with a suspected monogenic condition in Australia. Refereed/Peer-reviewed

Premature ovarian insufficiency involves amenorrhea and elevated follicle-stimulating hormone before age 40, and its genetic basis is poorly understood. Here, we study 13 premature ovarian insufficiency (POI) patients using whole-exome sequencing. We identify PREPL and TP63 causative variants, and variants in other potentially novel POI genes. PREPL deficiency is a known cause of syndromic POI, matching the patients' phenotype. A role for TP63 in ovarian biology has previously been proposed but variants have been described in multiorgan syndromes, and not isolated POI. One patient with isolated POI harbored a de novo nonsense TP63 variant in the terminal exon and an unrelated patient had a different nonsense variant in the same exon. These variants interfere with the repression domain while leaving the activation domain intact. We expand the phenotypic spectrum of TP63-related disorders, provide a new genotype:phenotype correlation for TP63 and identify a new genetic cause of isolated POI.

ObjectiveTo describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia.MethodsExome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for DMD premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle.ResultsSplice-altering intronic single nucleotide variants or structural rearrangements in DMD were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced DMD mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%–5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness.ConclusionsWhole-genome sequencing relied heavily on RNA studies to identify DMD splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMD mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant.

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 meantime 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. Refereed/Peer-reviewed

Diagnostic exome sequencing (ES) can be performed on the proband only (singleton; sES) or with additional samples, often including both biological parents with the proband (trio; tES). In this study we sought to compare the efficiencies of exome sequencing (ES) by trio (tES) versus singleton (sES) approach, determine costs, and identify factors to consider when deciding on optimal implementation strategies for the diagnosis of monogenic disorders. We undertook ES in 30 trios and analysed each proband's sES and tES data in parallel. Two teams were randomly allocated to either sES or tES analysis for each case and blinded to each other's work. Each task was timed and cost analyses were based on time taken and diagnostic yield. We modelled three scenarios to determine the factors to consider in the implementation of tES. sES diagnosed 11/30 (36.7%) cases and tES identified one additional diagnosis (12/30 (40.0%)). tES obviated the need for Sanger segregation, reduced the number of variants for curation, and had lower cost-per-diagnosis when considering analysis alone. When sequencing costs were included, tES nearly doubled the cost of sES. Reflexing to tES in those who remain undiagnosed after sES was cost-saving over tES in all as first-line. This approach requires a large differential in diagnostic yield between sES and tES for maximal benefit given current sequencing costs. tES may be preferable when scaling up laboratory throughput due to efficiency gains and opportunity cost considerations. Our findings are relevant to clinicians, laboratories and health services considering tES over sES.

BackgroundAs costs of high throughput sequencing have fallen, we are seeing vast quantities of short read genomic data being generated. Often, the data is exchanged and stored as aligned reads, which provides high compression and convenient access for many analyses. However, aligned data becomes outdated as new reference genomes and alignment methods become available. Moreover, some applications cannot utilise pre-aligned reads at all, necessitating conversion back to raw format (FASTQ) before they can be used. In both cases, the process of extraction and realignment is expensive and time consuming.FindingsWe describe Bazam, a tool that efficiently extracts the original paired FASTQ from reads stored in aligned form (BAM or CRAM format). Bazam extracts reads in a format that directly allows realignment with popular aligners with high concurrency. Through eliminating steps and increasing the accessible concurrency, Bazam facilitates up to a 90% reduction in the time required for realignment compared to standard methods. Bazam can support selective extraction of read pairs from focused genomic regions, further increasing efficiency for targeted analyses. Bazam is additionally suitable as a base for other applications that require efficient paired read information, such as quality control, structural variant calling and alignment comparison.ConclusionsBazam offers significant improvements for users needing to realign genomic data.

Purpose: RAC3 is an underexamined member of the Rho GTPase gene family that is expressed in the developing brain and linked to key cellular functions. De novo missense variants in the homolog RAC1 were recently associated with developmental disorders. In the RAC subfamily, transforming missense changes at certain shared residues have been observed in human cancers and previously characterized in experimental studies. The purpose of this study was to determine whether constitutional dysregulation of RAC3 is associated with human disease. Methods: We discovered a RAC3 variant in the index case using genome sequencing, and searched for additional variants using international data-sharing initiatives. Functional effects of the variants were assessed using a multifaceted approach generalizable to most clinical laboratory settings. Results: We rapidly identified five individuals with de novo monoallelic missense variants in RAC3, including one recurrent change. Every participant had severe intellectual disability and brain malformations. In silico protein modeling, and prior in vivo and in situ experiments, supported a transforming effect for each of the three different RAC3 variants. All variants were observed in databases of somatic variation in cancer. Conclusions: Missense variants in RAC3 cause a novel brain disorder, likely through a mechanism of constitutive protein activation.

Detection of copy number variation (CNVs) is a challenging but highly valuable application of exome and targeted high throughput sequencing (HTS) data. While there are dozens of CNV detection methods available, using these methods remains challenging due to variable accuracy both across different data sets and within the same data set with different methods. We propose that extracting good results from CNV detection on HTS data requires a systematic approach involving rigorous quality control, adjustment of method parameters and calibration of confidence measures for filtering results. We present Ximmer, a tool which supports an end to end process for applying these procedures including a simulation framework, CNV detection analysis pipeline, and a visualisation and curation tool which enables interactive exploration of CNV results. We apply Ximmer to perform a comprehensive evaluation of CNV detection on four data sets using four different detection methods, representing one of the most comprehensive evaluations to date. Ximmer is open source and freely available at http://ximmer.org (example results are viewable at http://example.ximmer.org).

Baker, Gordon et al. present the first international case series describing the neurodevelopmental disorder associated with Synaptotagmin 1 (SYT1) de novo missense mutations. Key features include movement abnormalities, severe intellectual disability, and hallmark EEG alterations. Expression of patients’ SYT1 mutations in mouse neurons disturbs presynaptic vesicle dynamics in a mutation-specific manner., Synaptotagmin 1 (SYT1) is a critical mediator of fast, synchronous, calcium-dependent neurotransmitter release and also modulates synaptic vesicle endocytosis. This paper describes 11 patients with de novo heterozygous missense mutations in SYT1. All mutations alter highly conserved residues, and cluster in two regions of the SYT1 C2B domain at positions Met303 (M303K), Asp304 (D304G), Asp366 (D366E), Ile368 (I368T) and Asn371 (N371K). Phenotypic features include infantile hypotonia, congenital ophthalmic abnormalities, childhood-onset hyperkinetic movement disorders, motor stereotypies, and developmental delay varying in severity from moderate to profound. Behavioural characteristics include sleep disturbance and episodic agitation. Absence of epileptic seizures and normal orbitofrontal head circumference are important negative features. Structural MRI is unremarkable but EEG disturbance is universal, characterized by intermittent low frequency high amplitude oscillations. The functional impact of these five de novo SYT1 mutations has been assessed by expressing rat SYT1 protein containing the equivalent human variants in wild-type mouse primary hippocampal cultures. All mutant forms of SYT1 were expressed at levels approximately equal to endogenous wild-type protein, and correctly localized to nerve terminals at rest, except for SYT1M303K, which was expressed at a lower level and failed to localize at nerve terminals. Following stimulation, SYT1I368T and SYT1N371K relocalized to nerve terminals at least as efficiently as wild-type SYT1. However, SYT1D304G and SYT1D366E failed to relocalize to nerve terminals following stimulation, indicative of impairments in endocytic retrieval and trafficking of SYT1. In addition, the presence of SYT1 variants at nerve terminals induced a slowing of exocytic rate following sustained action potential stimulation. The extent of disturbance to synaptic vesicle kinetics is mirrored by the severity of the affected individuals’ phenotypes, suggesting that the efficiency of SYT1-mediated neurotransmitter release is critical to cognitive development. In summary, de novo dominant SYT1 missense mutations are associated with a recognizable neurodevelopmental syndrome, and further cases can now be diagnosed based on clinical features, electrophysiological signature and mutation characteristics. Variation in phenotype severity may reflect mutation-specific impact on the diverse physiological functions of SYT1.

Highly conserved TREX-mediated mRNA export is emerging as a key pathway in neuronal development and differentiation. TREX subunit variants cause neurodevelopmental disorders (NDDs) by interfering with mRNA export from the cell nucleus to the cytoplasm. Previously we implicated four missense variants in the X-linked THOC2 gene in intellectual disability (ID). We now report an additional six affected individuals from five unrelated families with two de novo and three maternally inherited pathogenic or likely pathogenic variants in THOC2 extending the genotypic and phenotypic spectrum. These comprise three rare missense THOC2 variants that affect evolutionarily conserved amino acid residues and reduce protein stability and two with canonical splice-site THOC2 variants that result in C-terminally truncated THOC2 proteins. We present detailed clinical assessment and functional studies on a de novo variant in a female with an epileptic encephalopathy and discuss an additional four families with rare variants in THOC2 with supportive evidence for pathogenicity. Severe neurocognitive features, including movement and seizure disorders, were observed in this cohort. Taken together our data show that even subtle alterations to the canonical molecular pathways such as mRNA export, otherwise essential for cellular life, can be compatible with life, but lead to NDDs in humans. Refereed/Peer-reviewed

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 Melbourne Genomics 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 Measures The study examined the clinical utility of a molecular diagnosis and the cost-effectiveness of alternative diagnostic trajectories, depending on timing of WES. Results Of 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 incremental cost savings of A$9020 (US$6838) per additional diagnosis (95% CI, A$4304-A$15 404 [US$3263-US$11 678]) compared with the standard diagnostic pathway. Even if WES were performed at the first genetics appointment, there would be an incremental cost savings of A$5461 (US$4140) (95% CI, A$1433-A$10 557 [US$1086- US$8004]) per additional diagnosis compared with the standard diagnostic pathway. Conclusions and Relevance Singleton WES in children with suspected monogenic conditions has high diagnostic yield, and cost-effectiveness is maximized by early application in the diagnostic pathway. Pediatricians should consider early referral of children with undiagnosed syndromes to clinical geneticists.

Short tandem repeat (STR) expansions have been identified as the causal DNA mutation in dozens of Mendelian diseases. Historically, pathogenic STR expansions could only be detected by single locus techniques, such as PCR and electrophoresis. The ability to use short read sequencing data to screen for STR expansions has the potential to reduce both the time and cost to reaching diagnosis and enable the discovery of new causal STR loci. Most existing tools detect STR variation within the read length, and so are unable to detect the majority of pathogenic expansions. Those tools that can detect large expansions are limited to a set of known disease loci and as yet no new disease causing STR expansions have been identified with high-throughput sequencing technologies.Here we address this by presenting STRetch, a new genome-wide method to detect STR expansions at all loci across the human genome. We demonstrate the use of STRetch for detecting pathogenic STR expansions in short-read whole genome sequencing data with a very low false discovery rate. We further demonstrate the application of STRetch to solve cases of patients with undiagnosed disease and apply STRetch to the analysis of 97 whole genomes to reveal variation at STR loci. STRetch assesses expansions at all STR loci in the genome and allows screening for novel disease-causing STRs.STRetch is open source software, available fromgithub.com/Oshlack/STRetch.

Exome sequencing in the context of developmental disorders is a useful technique, but variants found need to be interpreted in the context of detailed phenotypic information. Whole gene deletions and loss-of-function-mutations in the HNRNPU gene have been associated with intellectual disability and seizures in some patients. However, a unifying syndromic phenotype has not been previously elucidated. Here, we report a total of seven patients (six patients identified through the Wellcome Trust Deciphering Developmental Disorders study, with one additional patient), who have heterozygous de novo mutations in HNRNPU. These were found via trio-based exome sequencing. All but one of the mutations is predicted to cause loss-of-function. These patients have dysmorphic features in common, including prominent eyebrows, long palpebral fissures, overhanging columella, and thin upper lip. All patients have developmental delay and intellectual disability (ID), ranging from moderate to severe. Seizures are common from early childhood. These initially occur in the context of febrile episodes. This series demonstrates common phenotypic features, including emerging dysmorphism, associated with heterozygous HNRNPU mutations. This allows us to define a novel neurodevelopmental syndrome, with a likely mechanism of haploinsufficiency.

PNPT1 (PNPase—polynucleotide phosphorylase) is involved in multiple RNA processing functions in the mitochondria. Bi-allelic pathogenic PNPT1 variants cause heterogeneous clinical phenotypes affecting multiple organs without any established genotype−phenotype correlations. Defects in PNPase can cause variable combined respiratory chain complex defects. Recently, it has been suggested that PNPase can lead to activation of an innate immune response. To better understand the clinical and molecular spectrum of patients with bi-allelic PNPT1 variants, we captured detailed clinical and molecular phenotypes of all 17 patients reported in the literature, plus seven new patients, including a 78-year-old male with the longest reported survival. A functional follow-up of genomic sequencing by cDNA studies confirmed a splicing defect in a novel, apparently synonymous, variant. Patient fibroblasts showed an accumulation of mitochondrial unprocessed PNPT1 transcripts, while blood showed an increased interferon response. Our findings suggest that functional analyses of the RNA processing function of PNPase are more sensitive than testing downstream defects in oxidative phosphorylation (OXPHPOS) enzyme activities. This research extends our knowledge of the clinical and functional consequences of bi-allelic pathogenic PNPT1 variants that may guide management and further efforts into understanding the pathophysiological mechanisms for therapeutic development.

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. 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. 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. This prospective study provides strong evidence for increased diagnostic and clinical utility of singleton WES as a first-tier 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. Genet Med 18 11, 1090–1096.

The benefits of implementing high throughput sequencing in the clinic are quickly becoming apparent. However, few freely available bioinformatics pipelines have been built from the ground up with clinical genomics in mind. Here we present Cpipe, a pipeline designed specifically for clinical genetic disease diagnostics. Cpipe was developed by the Melbourne Genomics Health Alliance, an Australian initiative to promote common approaches to genomics across healthcare institutions. As such, Cpipe has been designed to provide fast, effective and reproducible analysis, while also being highly flexible and customisable to meet the individual needs of diverse clinical settings. Cpipe is being shared with the clinical sequencing community as an open source project and is available at http://cpipeline.org. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0191-x) contains supplementary material, which is available to authorized users.

Systemic juvenile idiopathic arthritis (sJIA) shares clinical features with classic monogenic autoinflammatory diseases, characterised by fevers, arthritis and evanescent rashes. Disease exacerbations are associated with elevated serum cytokine levels including 1L-1β, IL-6, and IL-18; and clinical response to anakinra, canakinumab and tocilizumab suggests that cytokine dysregulation is a key pathophysiological mechanism. Macrophage activation syndrome (MAS) may complicate sJIA, rendering individuals clinically indistinguishable from their familial haemophagocytic lymphohistiocytosis (fHLH) counterparts; with NK and CD8+ cell dysfunction leading to sustained immune cell activation and cytokine storm. Whilst there have been HLA associations and polymorphisms noted on sJIA genome-wide association studies, in rare cases mutations have been found in genes encoding key components of the inflammatory response, which may contribute to disease pathogenesis.

Summary: Bpipe is a simple, dedicated programming language for defining and executing bioinformatics pipelines. It specializes in enabling users to turn existing pipelines based on shell scripts or command line tools into highly flexible, adaptable and maintainable workflows with a minimum of effort. Bpipe ensures that pipelines execute in a controlled and repeatable fashion and keeps audit trails and logs to ensure that experimental results are reproducible. Requiring only Java as a dependency, Bpipe is fully self-contained and cross-platform, making it very easy to adopt and deploy into existing environments. Availability and implementation: Bpipe is freely available from http://bpipe.org under a BSD License. Contact: simon.sadedin@mcri.edu.au Supplementary information: Supplementary data are available at Bioinformatics online.