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Fully understanding the genetic factors involved in Autism Spectrum Disorder (ASD) requires whole-genome sequencing (WGS), which theoretically allows the detection of all types of genetic variants. With the aim of generating an unprecedented resource for resolving the genomic architecture underlying ASD, we analyzed genome sequences and phenotypic data from 5,100 individuals with ASD and 6,212 additional parents and siblings (total n=11,312) in the Autism Speaks MSSNG Project, as well as additional individuals from other WGS cohorts. WGS data and autism phenotyping were based on high-quality short-read sequencing (>30x coverage) and clinically accepted diagnostic measures for ASD, respectively. For initial discovery of ASD-associated genes, we used exonic sequence-level variants from MSSNG as well as whole-exome sequencing-based ASD data from SPARK and the Autism Sequencing Consortium (>18,000 trios plus additional cases and controls), identifying 135 ASD-associated protein-coding genes with false discovery rate SCN2A and a nuclear mitochondrial insertion impacting SYNGAP1. Polygenic risk scores did not differ between children with ASD in multiplex families versus simplex, and rare, damaging recessive events were significantly depleted in multiplex families, collectively suggesting that rare, dominant variation plays a predominant role in multiplex ASD. Our study provides a guidebook for exploring genotype-phenotype correlations in the 15-20% of ASD families who carry ASD-associated rare variants, as well as an entry point to the larger and more diverse studies that will be required to dissect the etiology in the >80% of the ASD population that remains idiopathic. All data resulting from this study are available to the medical genomics research community in an open but protected manner.

Fully understanding autism spectrum disorder (ASD) genetics requires whole-genome sequencing (WGS). We present the latest release of the Autism Speaks MSSNG resource, which includes WGS data from 5,100 individuals with ASD and 6,212 non-ASD parents and siblings (total n = 11,312). Examining a wide variety of genetic variants in MSSNG and the Simons Simplex Collection (SSC; n = 9,205), we identified ASD-associated rare variants in 718/5,100 individuals with ASD from MSSNG (14.1%) and 350/2,419 from SSC (14.5%). Considering genomic architecture, 52% were nuclear sequence-level variants, 46% were nuclear structural variants (including copy-number variants, inversions, large insertions, uniparental isodisomies, and tandem repeat expansions), and 2% were mitochondrial variants. Our study provides a guidebook for exploring genotype-phenotype correlations in families who carry ASD-associated rare variants and serves as an entry point to the expanded studies required to dissect the etiology in the ∼85% of the ASD population that remain idiopathic.

Summary Induced pluripotent stem cells (iPSC) derived from healthy individuals are important controls for disease-modeling studies. Here we apply precision health to create a high-quality resource of control iPSCs. Footprint-free lines were reprogrammed from four volunteers of the Personal Genome Project Canada (PGPC). Multilineage-directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users demonstrated versatility by generating kidney organoids, T lymphocytes, and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole-genome sequencing-based annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harbored at least one pre-existing or acquired variant with cardiac, neurological, or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cells from six tissues for disease modeling, and variant-preferred healthy control lines were identified for specific disease settings., Graphical Abstract, Highlights • Precision health resource of high-quality control iPSC lines for disease modeling • Versatile differentiation into six functional cell types shown by resource users • CRISPR gene editing reveals expected phenotype of cardiomyopathy model • Variant annotation identified preferred lines for neurologic and cardiac disease, Ellis, Scherer, and colleagues apply precision health to upgrade iPSC quality for disease modeling. The resource provides control lines from four healthy individuals, clinical annotation of whole-genome variants, and identification of variant-preferred lines for neurologic and cardiac disease. Resource users demonstrated versatile differentiation into functional cells from six tissues, and CRISPR-edited cells phenocopied a cardiomyopathy model.

BackgroundWhole blood is currently the most common DNA source for whole-genome sequencing (WGS), but for studies requiring non-invasive collection, self-collection, greater sample stability or additional tissue references, saliva or buccal samples may be preferred. However, the relative quality of sequencing data and accuracy of genetic variant detection from blood-derived, saliva-derived and buccal-derived DNA need to be thoroughly investigated.MethodsMatched blood, saliva and buccal samples from four unrelated individuals were used to compare sequencing metrics and variant-detection accuracy among these DNA sources.ResultsWe observed significant differences among DNA sources for sequencing quality metrics such as percentage of reads aligned and mean read depth (pConclusionFor WGS, we recommend using DNA extracted from blood rather than saliva or buccal swabs; if saliva or buccal samples are used, we recommend against using methylation-based eukaryotic DNA enrichment. All data used in this study are available for further open-science investigation.

In this work, we explore whether raw genetic data generated during sequencing ought to be returned to a pediatric patient and/or their parents/guardians. We identify the principles used by various professional societies in their guidelines on the return of secondary findings and apply them to this new context. We conclude that since each situation is unique, decisions should be made on a case-by-case basis according to the best interests of the child.

Purpose Genetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use. Methods We prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing. Results WGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24% P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A. Conclusion WGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.

BACKGROUND: The Personal Genome Project Canada is a comprehensive public data resource that integrates whole genome sequencing data and health information. We describe genomic variation identified in the initial recruitment cohort of 56 volunteers. METHODS: Volunteers were screened for eligibility and provided informed consent for open data sharing. Using blood DNA, we performed whole genome sequencing and identified all possible classes of DNA variants. A genetic counsellor explained the implication of the results to each participant. RESULTS: Whole genome sequencing of the first 56 participants identified 207 662 805 sequence variants and 27 494 copy number variations. We analyzed a prioritized disease-associated data set (n = 1606 variants) according to standardized guidelines, and interpreted 19 variants in 14 participants (25%) as having obvious health implications. Six of these variants (e.g., in BRCA1 or mosaic loss of an X chromosome) were pathogenic or likely pathogenic. Seven were risk factors for cancer, cardiovascular or neurobehavioural conditions. Four other variants — associated with cancer, cardiac or neurodegenerative phenotypes — remained of uncertain significance because of discrepancies among databases. We also identified a large structural chromosome aberration and a likely pathogenic mitochondrial variant. There were 172 recessive disease alleles (e.g., 5 individuals carried mutations for cystic fibrosis). Pharmacogenomics analyses revealed another 3.9 potentially relevant genotypes per individual. INTERPRETATION: Our analyses identified a spectrum of genetic variants with potential health impact in 25% of participants. When also considering recessive alleles and variants with potential pharmacologic relevance, all 56 participants had medically relevant findings. Although access is mostly limited to research, whole genome sequencing can provide specific and novel information with the potential of major impact for health care.

SummaryInduced Pluripotent Stem Cells (iPSC) derived from healthy individuals are important controls for disease modeling studies. To create a resource of genetically annotated iPSCs, we reprogrammed footprint-free lines from four volunteers in the Personal Genome Project Canada (PGPC). Multilineage directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users further demonstrated line versatility by generating kidney organoids, T-lymphocytes and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole genome sequencing (WGS) based annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harboured at least one pre-existing or acquired variant with cardiac, neurological or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cell types found in six tissues for disease modeling, and clinical annotation highlighted variant-preferred lines for use as unaffected controls in specific disease settings.

Whole genome sequencing (WGS) is a transformative technology which promises improved diagnostic rates compared to conventional genetic testing strategies and tailored approaches to patient care. Due to the practical and ethical complexities associated with using WGS, particularly in the paediatric context, input from a broad spectrum of healthcare providers can guide implementation strategies. We recruited healthcare providers from the largest paediatric academic health science centre in Canada and conducted semi-structured qualitative interviews, exploring experiences with and perceptions of the opportunities and challenges associated with WGS. Interview transcripts were coded and analyzed thematically. Interviews were completed with 14 genetics professionals (geneticists and genetic counsellors) and 15 non-genetics professionals (physician sub-specialists and nurses). Genetics professionals ordered genetic tests more often and reported greater confidence on pre- and post-test genetic counselling compared to non-genetics professionals. Most healthcare providers endorsed WGS when a more specific test was either not available or not likely to yield a diagnosis. While genetics professionals raised concerns regarding the time demands associated with reviewing WGS variants, non-genetics professionals reflected concerns about knowledge and training. Providers' position on reporting secondary variants to parents drew upon but was not limited to the concept of best interests. Taken together, understanding practical and principled matters of WGS from healthcare providers' perspectives can guide ongoing efforts to implement WGS in paediatrics.

We are performing whole genome sequencing (WGS) of families with Autism Spectrum Disorder (ASD) to build a resource, named MSSNG, to enable the sub-categorization of phenotypes and underlying genetic factors involved. Here, we report WGS of 5,205 samples from families with ASD, accompanied by clinical information, creating a database accessible in a cloud platform, and through an internet portal with controlled access. We found an average of 73.8 de novo single nucleotide variants and 12.6 de novo insertion/deletions (indels) or copy number variations (CNVs) per ASD subject. We identified 18 new candidate ASD-risk genes such as MED13 and PHF3, and found that participants bearing mutations in susceptibility genes had significantly lower adaptive ability (p=6×10−4). In 294/2,620 (11.2%) of ASD cases, a molecular basis could be determined and 7.2% of these carried CNV/chromosomal abnormalities, emphasizing the importance of detecting all forms of genetic variation as diagnostic and therapeutic targets in ASD.

The publication of the ACMG recommendations has reignited the debate over predictive testing for adult-onset disorders in minors. Response has been polarized. With this in mind, we review and critically analyze this debate. First, we identify long-standing inconsistencies between consensus guidelines and clinical practice regarding risk assessment for adult-onset genetic disorders in children using family history and molecular analysis. Second, we discuss the disparate assumptions regarding the nature of whole genome and exome sequencing underlying arguments of both supporters and critics, and the role these assumptions play in the arguments for and against reporting. Third, we suggest that implicit differences regarding the definition of best interests of the child underlie disparate conclusions as to the best interests of children in this context. We conclude by calling for clarity and consensus concerning the central foci of this debate.

Objective To better understand the consequences of returning whole genome sequencing (WGS) results in paediatrics and facilitate its evidence-based clinical implementation, we studied parents9 experiences with WGS and their preferences for the return of adult-onset secondary variants (SVs)—medically actionable genomic variants unrelated to their child9s current medical condition that predict adult-onset disease. Methods We conducted qualitative interviews with parents whose children were undergoing WGS as part of the SickKids Genome Clinic, a research project that studies the impact of clinical WGS on patients, families, and the healthcare system. Interviews probed parents9 experience with and motivation for WGS as well as their preferences related to SVs. Interviews were analysed thematically. Results Of 83 invited, 23 parents from 18 families participated. These parents supported WGS as a diagnostic test, perceiving clear intrinsic and instrumental value. However, many parents were ambivalent about receiving SVs, conveying a sense of self-imposed obligation to take on the ‘weight’ of knowing their child9s SVs, however unpleasant. Some parents chose to learn about adult-onset SVs for their child but not for themselves. Conclusions Despite general enthusiasm for WGS as a diagnostic test, many parents felt a duty to learn adult-onset SVs. Analogous to ‘inflicted insight’, we call this phenomenon ‘inflicted ought’. Importantly, not all parents of children undergoing WGS view the best interests of their child in relational terms, thereby challenging an underlying justification for current ACMG guidelines for reporting incidental secondary findings from whole exome and WGS.

The American Academy of Pediatrics (AAP)/American College of Medical Genetics (ACMG) guidelines discourage predictive genetic testing of children at risk for adult-onset diseases, while the ACMG ex...

Background: In Ontario, Canada, patients who lack decision-making capacity and have no family or friends to act as substitute decision-makers currently rely on the Office of the Public Guardian and Trustee to consent to long-term care (nursing home) placement, but they have no legal representative for other placement decisions. Objectives: We highlight the current gap in legislation for difficult transition cases involving unrepresented patients and provide a novel framework for who ought to assist with making these decisions and how these decisions ought to be made. Research design: This paper considers models advanced by Volpe and Steinman with regard to who ought to make placement decisions for unrepresented patients, as well as current ethical models for analyzing how these decisions should be made. Participants and research context: We describe an anonymized healthcare transition case to illustrate the fact that there is no legally recognized decision-maker for placement destinations other than long-term care facilities and to show how this impacts all stakeholders. Ethical considerations: The case provided is an anonymized vignette representing a typical transition case involving an unrepresented patient. Findings: As a result of a gap in provincial legislation, healthcare providers usually determine the appropriate placement destination without a clear framework to guide the process and this can cause significant moral distress. Discussion: We argue for a team decision-making approach in the short term, and a legislative change in the long-term, to respect the patient voice, evaluate benefit and risk, enhance collaboration between healthcare providers and patients, and promote social justice. We believe that our approach, which draws upon the strengths of interprofessional teams, will be of interest to all who are concerned with the welfare and ethical treatment of the patients for whom they care. Conclusions: One of the main strengths of our recommendation is that it provides all members of the healthcare team (including nurses, social workers, therapists, and others) an increased opportunity to advocate on behalf of unrepresented patients.

▪ Abstract More than 80 genes associated with human photoreceptor degenerations have been identified. Attention must now turn toward defining the mechanisms that lead to photoreceptor death, which occurs years to decades after the birth of the cells. Consequently, this review focuses on topics that offer insights into such mechanisms, including the one-hit or constant risk model of photoreceptor death; topological patterns of photoreceptor degeneration; mutations in ubiquitously expressed splicing factor genes associated only with photoreceptor degeneration; disorders of the retinal pigment epithelium; modifier genes; and global gene expression analysis of the retina, which will greatly increase our understanding of the downstream events that occur in response to a mutation.

Background We report a consanguineous couple that has experienced three consecutive pregnancy losses following the foetal ultrasound finding of short limbs. Post-termination examination revealed no skeletal dysplasia, but some subtle proximal limb shortening in two foetuses, and a spectrum of mildly dysmorphic features. Karyotype was normal in all three foetuses (46, XX) and comparative genomic hybridization microarray analysis detected no pathogenic copy number variants. Results Whole-exome sequencing and genome-wide homozygosity mapping revealed a previously reported frameshift mutation in the OBSL1 gene (c.1273insA p.T425nfsX40), consistent with a diagnosis of 3-M Syndrome 2 (OMIM #612921), which had not been anticipated from the clinical findings. Conclusions Our study provides novel insight into the early clinical manifestations of this form of 3-M syndrome, and demonstrates the utility of whole exome sequencing as a tool for prenatal diagnosis in particular when there is a family history suggestive of a recurrent set of clinical symptoms.

Inherited photoreceptor degenerations (IPDs), a group of incurable progressive blinding diseases, are caused by mutations in more than 200 genes, but little is known about the molecular pathogenesis of photoreceptor (PR) death. Increased retinal expression of STAT3 has been observed in response to many retinal insults, including IPDs, but the role of this increase in PR death is unknown. Here, we show that the expression of Stat3 is increased in PRs of the Tg(RHO P347S) and Prph2(rds) (/+) mouse models of IPD and is activated by tyrosine phosphorylation. PR-specific deletion of Stat3 substantially accelerated PR degeneration in both mutant strains. In contrast, increased PR-specific expression of ROSA26 (R26) alleles encoding either WT STAT3 (Stat3(wt)) or the gain-of-function variant STAT3(C) (Stat3(C)) improved PR survival in both models. Moreover, PR signaling in Tg(RHO P347S) mice carrying either a R26-Stat3(wt) or R26-Stat3(C) allele demonstrated increased a-wave amplitude of the scotopic electroretinogram. Phosphorylation of STAT3 at tyrosine 705 was required for the prosurvival effect because an R26-Stat3(Y705F) allele was not protective. The prosurvival role of enhanced Stat3 activity was validated using recombinant adenoassociated virus (rAAV) vector-mediated PR Stat3 expression in Tg(RHO P347S) mice. Our findings (i) establish that the increase in endogenous PR Stat3 expression is a protective response in IPDs, (ii) suggest that therapeutic augmentation of PR Stat3 expression has potential as a common neuroprotective therapy for these disorders, and (iii) indicate that prosurvival molecules whose expression is increased in mutant PRs may have promise as novel therapies for IPDs.

The future clinical applications of whole genome sequencing come with speculation and enthusiasm but require careful consideration of the true system costs and health benefits of the clinical uses of this exciting technology., Summary The cost of whole genome sequencing is dropping rapidly. There has been a great deal of enthusiasm about the potential for this technological advance to transform clinical care. Given the interest and significant investment in genomics, this seems an ideal time to consider what the evidence tells us about potential benefits and harms, particularly in the context of health care policy. The scale and pace of adoption of this powerful new technology should be driven by clinical need, clinical evidence, and a commitment to put patients at the centre of health care policy.

In March of this year, G3: Genes | Genomes | Genetics published an early online version of a manuscript by Landry et al. documenting the genome sequence of a HeLa cell line ([Landry et al. 2013][1]). It prompted a commentary in the New York Times by Rebecca Skloot ([Skloot 2013][2]), who is well

Expression of the Endothelin-2 (Edn2) mRNA is greatly increased in the photoreceptors (PRs) of mouse models of inherited PR degeneration (IPD). To examine the role of Edn2 in mutant PR survival, we generated Edn2(-/-) mice carrying homozygous Pde6b(rd1) alleles or the Tg(RHO P347S) transgene. In the Edn2(-/-) background, PR survival increased 110% in Pde6b(rd1/rd1) mice at post-natal (PN) day 15, and 60% in Tg(RHO P347S) mice at PN40. In contrast, PR survival was not increased in retinal explants of Pde6b(rd1/rd1) ; Edn2(-/-) mice. This finding, together with systemic abnormalities in Edn2(-/-) mice, suggested that the increased survival of mutant PRs in the Edn2(-/-) background resulted at least partly from the systemic EDN2 loss of function. To examine directly the role of EDN2 in mutant PRs, we used a scAAV5-Edn2 cDNA vector to restore Edn2 expression in Pde6b(rd1/rd1) ; Edn2(-/-) PRs and observed an 18% increase in PR survival at PN14. Importantly, PR survival was also increased after injection of scAAV5-Edn2 into Pde6b(rd1/rd1) retinas, by 31% at PN15. Together, these findings suggest that increased Edn2 expression is protective to mutant PRs. To begin to elucidate Edn2-mediated mechanisms that contribute to PR survival, we used microarray analysis and identified a cohort of 20 genes with >4-fold increased expression in Tg(RHO P347S) retinas, including Fgf2. Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2(-/-) retinas. Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.

The standard of care for first-tier clinical investigation of the aetiology of congenital malformations and neurodevelopmental disorders is chromosome microarray analysis (CMA) for copy-number variations (CNVs), often followed by gene(s)-specific sequencing searching for smaller insertion–deletions (indels) and single-nucleotide variant (SNV) mutations. Whole-genome sequencing (WGS) has the potential to capture all classes of genetic variation in one experiment; however, the diagnostic yield for mutation detection of WGS compared to CMA, and other tests, needs to be established. In a prospective study we utilised WGS and comprehensive medical annotation to assess 100 patients referred to a paediatric genetics service and compared the diagnostic yield versus standard genetic testing. WGS identified genetic variants meeting clinical diagnostic criteria in 34% of cases, representing a fourfold increase in diagnostic rate over CMA (8%; P value=1.42E−05) alone and more than twofold increase in CMA plus targeted gene sequencing (13%; P value=0.0009). WGS identified all rare clinically significant CNVs that were detected by CMA. In 26 patients, WGS revealed indel and missense mutations presenting in a dominant (63%) or a recessive (37%) manner. We found four subjects with mutations in at least two genes associated with distinct genetic disorders, including two cases harbouring a pathogenic CNV and SNV. When considering medically actionable secondary findings in addition to primary WGS findings, 38% of patients would benefit from genetic counselling. Clinical implementation of WGS as a primary test will provide a higher diagnostic yield than conventional genetic testing and potentially reduce the time required to reach a genetic diagnosis.

The presenilin proteins (PS1 and PS2) with their partners (NCT, Aph1, and Pen2) are the major components of the high molecular weight gamma-secretase complex which facilitates the intramembraneous cleavage of various type 1 transmembrane proteins, including the amyloid-beta precursor protein (APP) and the Notch receptor. Additional gamma-secretase complex components may be involved in regulation of its activity and specificity. A recent investigation indicated that the Crumbs protein is a negative regulator of Notch signaling and may act by repressing gamma/epsilon-secretase activity in Drosophila [Herranz, H., Stamataki, E., Feiguin, F., and Milan, M. (2006) EMBO Rep. 7, 297-302]. To address this question, we investigated potential functional interactions between the human Crumbs homologues (CRB1, CRB2, and CRB3) and presenilin complexes which mediate gamma/epsilon-secretase cleavage of APP and Notch. We found no evidence for direct interaction between CRB1, CRB2, or CRB3 and presenilin complex components. Furthermore, overexpression of human CRB1 and related isoforms, CRB2 and CRB3, had no effect on the levels of presenilin complex components, on NCT maturation or on PS endoproteolysis, and did not alter Abeta AICD or NICD production. These results suggest that, in mammalian cells at least, Crumbs is unlikely to be a significant direct modulator of presenilin-dependent gamma/epsilon-secretase activity.

Objective The overall goal of the SickKids Genome Clinic is to pilot paediatric genomic medicine. To this end we have assessed parental interest in predictive secondary medically-actionable variants (MAVs) and used whole genome sequencing (WGS) to determine the frequency and nature of these MAVs in children. Design The Genome Clinic conducts diagnostic WGS for 150+ children/year who are undergoing genetic evaluations. With parents’ permission, we search children’s genomes for predictive MAVs in 2800+ disease genes listed in the NIH Clinical Genomic Database. Results Of 373 families approached to date, 56% agreed to participate. 58% of participants chose to learn their child’s secondary adult-onset MAVs. Among these parents, 79% decided to learn their own status for these variants. Bioinformatics analysis of the first 100 patient genomes yielded 2957 candidate variants in 1132 genes (~30 variants/genome). ~70% of candidates were listed in HGMD. However, subsequent manual assessment rejected >90% of variants for dominant diseases listed in HGMD as disease causing due to inadequate evidence of pathogenicity. After manual assessment, 33/100 children had at least one reportable predictive MAV. 9 MAVs occurred in a 2013 ACMG-guideline reportable gene. Expanding our search 50 fold to include 2800+ disease genes yielded 29 additional reportable predictive MAVs. Return of predictive MAVs and assessment of their penetrance is underway. Conclusions Parental opinions vary widely regarding return of predictive MAVs and comprehensive genomic analysis can yield predictive MAVs in 1/3 of children, with the number of reportable predictive MAVs constrained by disease prevalence and imperfect variant interpretation.

Background Chromosome microarray analysis (CMA) is the current first-tier clinical investigation for paediatric patients with congenital malformations and/or neurodevelopmental disorders. Whole genome sequencing (WGS) promises to capture all classes of genetic variation in a single experiment, but the diagnostic yield of WGS compared to CMA in the clinical setting has not been established. Objectives The purpose of the study was to compare the diagnostic yield of WGS with CMA and other targeted sequence analysis. Methods Through the SickKids genome clinic 100 consecutive patients undergoing CMA were recruited and WGS was performed to compare diagnostic yield. Results WGS identified variants meeting clinical diagnostic criteria in 32% of cases, representing a 4-fold increase in diagnostic rate over CMA (8%) alone and > 2-fold increase in CMA plus targeted sequence testing (15%). WGS identified all reportable rare CNVs that were detected by CMA. In an additional 27 patients, WGS revealed clinically significant SNV and indel mutations presenting in a dominant (72%) or a recessive (28%) manner. Four cases had variants in at least two genes involved in distinct genetic disorders, contributing to a more complex clinical phenotype. Conclusions Our data indicate that WGS is highly accurate and efficient, providing a diagnosis in 32% paediatric patients that meet clinical criteria for CMA. Clinical implementation of WGS as a single and primary molecular test will provide a higher diagnostic yield than conventional testing while decreasing the number of genetic tests and ultimately the time before reaching a genetic diagnosis.

DNA microarrays are a powerful technology that can provide a wealth of gene expression data for disease studies, drug development, and a wide scope of other investigations. Because of the large volume and inherent variability of DNA microarray data, many new statistical methods have been developed for evaluating the significance of the observed differences in gene expression. However, until now little attention has been given to the characterization of dispersion of DNA microarray data. Here we examine the expression data obtained from 682 Affymetrix GeneChips® with 22 different types and we demonstrate that the Gaussian (normal) frequency distribution is characteristic for the variability of gene expression values. However, typically 5 to 15% of the samples deviate from normality. Furthermore, it is shown that the frequency distributions of the difference of expression in subsets of ordered, consecutive pairs of genes (consecutive samples) in pair-wise comparisons of replicate experiments are also normal. We describe a consecutive sampling method, which is employed to calculate the characteristic function approximating standard deviation and show that the standard deviation derived from the consecutive samples is equivalent to the standard deviation obtained from individual genes. Finally, we determine the boundaries of probability intervals and demonstrate that the coefficients defining the intervals are independent of sample characteristics, variability of data, laboratory conditions and type of chips. These coefficients are very closely correlated with Student's t- distribution. In this study we ascertained that the non-systematic variations possess Gaussian distribution, determined the probability intervals and demonstrated that the K α coefficients defining these intervals are invariant; these coefficients offer a convenient universal measure of dispersion of data. The fact that the K α distributions are so close to t- distribution and independent of conditions and type of arrays suggests that the quantitative data provided by Affymetrix technology give "true" representation of physical processes, involved in measurement of RNA abundance. This article was reviewed by Yoav Gilad (nominated by Doron Lancet), Sach Mukherjee (nominated by Sandrine Dudoit) and Amir Niknejad and Shmuel Friedland (nominated by Neil Smalheiser).