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Angelman syndrome is a neurodevelopmental disorder caused by disrupted function of the maternal copy of the imprinted UBE3A gene; here, targeting a long non-coding RNA that is responsible for silencing the paternal copy of UBE3A with antisense oligonucleotides is shown to partially restore UBE3A expression in the central nervous system and correct some cognitive deficits in a mouse model of the disease. Therapy for Angelman syndrome Frank Rigo and colleagues report the development of the first gene-specific therapy for Angelman syndrome, a severe neurodevelopmental disorder caused by disrupted function of the maternal copy of the imprinted gene UBE3A. The paternal copy of UBE3A is intact but silenced by a long non-coding RNA antisense transcript, UBE3A-ATS . The authors show that by reducing Ube3a-ATS with antisense oligonucleotides (ASOs), the silencing of paternal Ube3a can be reversed in cultured mouse neurons and in vivo . Some phenotypes in an Angelman syndrome mouse model, including obesity and memory impairment can also be corrected. Angelman syndrome is a single-gene disorder characterized by intellectual disability, developmental delay, behavioural uniqueness, speech impairment, seizures and ataxia 1 , 2 . It is caused by maternal deficiency of the imprinted gene UBE3A , encoding an E3 ubiquitin ligase 3 , 4 , 5 . All patients carry at least one copy of paternal UBE3A , which is intact but silenced by a nuclear-localized long non-coding RNA, UBE3A antisense transcript ( UBE3A-ATS ) 6 , 7 , 8 . Murine Ube3a-ATS reduction by either transcription termination or topoisomerase I inhibition has been shown to increase paternal Ube3a expression 9 , 10 . Despite a clear understanding of the disease-causing event in Angelman syndrome and the potential to harness the intact paternal allele to correct the disease, no gene-specific treatment exists for patients. Here we developed a potential therapeutic intervention for Angelman syndrome by reducing Ube3a-ATS with antisense oligonucleotides (ASOs). ASO treatment achieved specific reduction of Ube3a-ATS and sustained unsilencing of paternal Ube3a in neurons in vitro and in vivo . Partial restoration of UBE3A protein in an Angelman syndrome mouse model ameliorated some cognitive deficits associated with the disease. Although additional studies of phenotypic correction are needed, we have developed a sequence-specific and clinically feasible method to activate expression of the paternal Ube3a allele.

Whole genome sequencing (WGS) shows promise as a first-tier diagnostic test for patients with rare genetic disorders. However, standards addressing the definition and deployment practice of a best-in-class test are lacking. To address these gaps, the Medical Genome Initiative, a consortium of leading health care and research organizations in the US and Canada, was formed to expand access to high quality clinical WGS by convening experts and publishing best practices. Here, we present best practice recommendations for the interpretation and reporting of clinical diagnostic WGS, including discussion of challenges and emerging approaches that will be critical to harness the full potential of this comprehensive test.

The effectiveness of a social skills training group for adolescents with Asperger syndrome and high-functioning autism (AS/HFA) was evaluated. Parents of six groups of adolescents (n = 46, 61% male, mean age 14.6) completed questionnaires immediately before and after the 12-week group. Parents and adolescents were surveyed regarding their experience with the group. Significant pre- to post-treatment gains were found on measures of both social competence and problem behaviors associated with AS/HFA. Effect sizes ranged from 0.34 to 0.72. Adolescents reported more perceived skill improvements than did parents. Parent-reported improvement suggests that social skills learned in group sessions generalize to settings outside the treatment group. Larger, controlled studies of social skills training groups would be valuable.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by maternal deficiency of the imprinted gene UBE3A. Individuals with AS suffer from intellectual disability, speech impairment, and motor dysfunction. Currently there is no cure for the disease. Here, we evaluated the phenotypic effect of activating the silenced paternal allele of Ube3a by depleting its antisense RNA Ube3a-ATS in mice. Premature termination of Ube3a-ATS by poly(A) cassette insertion activates expression of Ube3a from the paternal chromosome, and ameliorates many disease-related symptoms in the AS mouse model, including motor coordination defects, cognitive deficit, and impaired long-term potentiation. Studies on the imprinting mechanism of Ube3a revealed a pattern of biallelic transcription initiation with suppressed elongation of paternal Ube3a, implicating transcriptional collision between sense and antisense polymerases. These studies demonstrate the feasibility and utility of unsilencing the paternal copy of Ube3a via targeting Ube3a-ATS as a treatment for Angelman syndrome.

Background Exome sequencing is now being incorporated into clinical care for pediatric and adult populations, but its integration into prenatal diagnosis has been more limited. One reason for this is the paucity of information about the clinical utility of exome sequencing in the prenatal setting. Methods We retrospectively reviewed indications, results, time to results (turnaround time, TAT), and impact of exome results for 146 consecutive “fetal exomes” performed in a clinical diagnostic laboratory between March 2012 and November 2017. We define a fetal exome as one performed on a sample obtained from a fetus or a product of conception with at least one structural anomaly detected by prenatal imaging or autopsy. Statistical comparisons were performed using Fisher’s exact test. Results Prenatal exome yielded an overall molecular diagnostic rate of 32% ( n  = 46/146). Of the 46 molecular diagnoses, 50% were autosomal dominant disorders ( n  = 23/46), 41% were autosomal recessive disorders ( n  = 19/46), and 9% were X-linked disorders ( n  = 4/46). The molecular diagnostic rate was highest for fetuses with anomalies affecting multiple organ systems and for fetuses with craniofacial anomalies. Out of 146 cases, a prenatal trio exome option designed for ongoing pregnancies was performed on 62 fetal specimens, resulting in a diagnostic yield of 35% with an average TAT of 14 days for initial reporting (excluding tissue culture time). The molecular diagnoses led to refined recurrence risk estimates, altered medical management, and informed reproductive planning for families. Conclusion Exome sequencing is a useful diagnostic tool when fetal structural anomalies suggest a genetic etiology, but other standard prenatal genetic tests did not provide a diagnosis.

Improved resolution of molecular diagnostic technologies enabled detection of smaller sized exonic level copy-number variants (CNVs). The contribution of CNVs to autosomal recessive (AR) conditions may be better recognized using a large clinical cohort. We retrospectively investigated the CNVs’ contribution to AR conditions in cases subjected to chromosomal microarray analysis (CMA, N=~70,000) and/or clinical exome sequencing (ES, N=~12,000) at Baylor Genetics; most had pediatric onset neurodevelopmental disorders. CNVs contributed to biallelic variations in 87 cases, including 81 singletons and three affected sibling pairs. Seventy cases had CNVs affecting both alleles, and 17 had a CNV and a single-nucleotide variant (SNV)/indel in trans. In total, 94.3% of AR-CNVs affected one gene; among these 41.4% were single-exon and 35.0% were multiexon partial-gene events. Sixty-nine percent of homozygous AR-CNVs were embedded in homozygous genomic intervals. Five cases had large deletions unmasking an SNV/indel on the intact allele for a recessive condition, resulting in multiple molecular diagnoses. AR-CNVs are often smaller in size, transmitted through generations, and underrecognized due to limitations in clinical CNV detection methods. Our findings from a large clinical cohort emphasized integrated CNV and SNV/indel analyses for precise clinical and molecular diagnosis especially in the context of genomic disorders.

Purpose: To investigate pan-ethnic SMN1 copy-number and sequence variation by hybridization-based target enrichment coupled with massively parallel sequencing or next-generation sequencing (NGS). Methods: NGS reads aligned to SMN1 and SMN2 exon 7 were quantified to determine the total combined copy number of SMN1 and SMN2 . The ratio of SMN1 to SMN2 was calculated based on a single-nucleotide difference that distinguishes the two genes. SMN1 copy-number results were compared between the NGS and quantitative polymerase chain reaction and/or multiplex ligation-dependent probe amplification. The NGS data set was also queried for the g.27134T>G single-nucleotide polymorphism (SNP) and other SMN1 sequence pathogenic variants. Results: The sensitivity of the test to detect spinal muscular atrophy (SMA) carriers with one copy of SMN1 was 100% (95% confidence interval (CI): 95.9–100%; n = 90) and specificity was 99.6% (95% CI: 99.4–99.7%; n = 6,648). Detection of the g.27134T>G SNP by NGS was 100% concordant with an restriction fragment-length polymorphism method ( n = 493). Ten single-nucleotide variants in SMN1 were detectable by NGS and confirmed by gene-specific amplicon-based sequencing. This comprehensive approach yielded SMA carrier detection rates of 90.3–95.0% in five ethnic groups studied. Conclusion: We have developed a novel, comprehensive SMN1 copy-number and sequence variant analysis method by NGS that demonstrated improved SMA carrier detection rates across the entire population examined. Genet Med advance online publication 19 January 2017

Background Although mosaic variation has been known to cause disease for decades, high-throughput sequencing technologies with the analytical sensitivity to consistently detect variants at reduced allelic fractions have only recently emerged as routine clinical diagnostic tests. To date, few systematic analyses of mosaic variants detected by diagnostic exome sequencing for diverse clinical indications have been performed. Methods To investigate the frequency, type, allelic fraction, and phenotypic consequences of clinically relevant somatic mosaic single nucleotide variants (SNVs) and characteristics of the corresponding genes, we retrospectively queried reported mosaic variants from a cohort of ~ 12,000 samples submitted for clinical exome sequencing (ES) at Baylor Genetics. Results We found 120 mosaic variants involving 107 genes, including 80 mosaic SNVs in proband samples and 40 in parental/grandparental samples. Average mosaic alternate allele fraction (AAF) detected in autosomes and in X-linked disease genes in females was 18.2% compared with 34.8% in X-linked disease genes in males. Of these mosaic variants, 74 variants (61.7%) were classified as pathogenic or likely pathogenic and 46 (38.3%) as variants of uncertain significance. Mosaic variants occurred in disease genes associated with autosomal dominant (AD) or AD/autosomal recessive (AR) (67/120, 55.8%), X-linked (33/120, 27.5%), AD/somatic (10/120, 8.3%), and AR (8/120, 6.7%) inheritance. Of note, 1.7% (2/120) of variants were found in genes in which only somatic events have been described. Nine genes had recurrent mosaic events in unrelated individuals which accounted for 18.3% (22/120) of all detected mosaic variants in this study. The proband group was enriched for mosaicism affecting Ras signaling pathway genes. Conclusions In sum, an estimated 1.5% of all molecular diagnoses made in this cohort could be attributed to a mosaic variant detected in the proband, while parental mosaicism was identified in 0.3% of families analyzed. As ES design favors breadth over depth of coverage, this estimate of the prevalence of mosaic variants likely represents an underestimate of the total number of clinically relevant mosaic variants in our cohort.

Background Exome sequencing (ES) has been successfully applied in clinical detection of single nucleotide variants (SNVs) and small indels. However, identification of copy number variants (CNVs) using ES data remains challenging. The purpose of this study is to understand the contribution of CNVs and copy neutral runs of homozygosity (ROH) in molecular diagnosis of patients referred for ES. Methods In a cohort of 11,020 consecutive ES patients, an Illumina SNP array analysis interrogating mostly coding SNPs was performed as a quality control (QC) measurement and for CNV/ROH detection. Among these patients, clinical chromosomal microarray analysis (CMA) was performed at Baylor Genetics (BG) on 3229 patients, either before, concurrently, or after ES. We retrospectively analyzed the findings from CMA and the QC array. Results The QC array can detect ~ 70% of pathogenic/likely pathogenic CNVs (PCNVs) detectable by CMA. Out of the 11,020 ES cases, the QC array identified PCNVs in 327 patients and uniparental disomy (UPD) disorder-related ROH in 10 patients. The overall PCNV/UPD detection rate was 5.9% in the 3229 ES patients who also had CMA at BG; PCNV/UPD detection rate was higher in concurrent ES and CMA than in ES with prior CMA (7.2% vs 4.6%). The PCNVs/UPD contributed to the molecular diagnoses in 17.4% (189/1089) of molecularly diagnosed ES cases with CMA and were estimated to contribute in 10.6% of all molecularly diagnosed ES cases. Dual diagnoses with both PCNVs and SNVs were detected in 38 patients. PCNVs affecting single recessive disorder genes in a compound heterozygous state with SNVs were detected in 4 patients, and homozygous deletions (mostly exonic deletions) were detected in 17 patients. A higher PCNV detection rate was observed for patients with syndromic phenotypes and/or cardiovascular abnormalities. Conclusions Our clinical genomics study demonstrates that detection of PCNV/UPD through the QC array or CMA increases ES diagnostic rate, provides more precise molecular diagnosis for dominant as well as recessive traits, and enables more complete genetic diagnoses in patients with dual or multiple molecular diagnoses. Concurrent ES and CMA using an array with exonic coverage for disease genes enables most effective detection of both CNVs and SNVs and therefore is recommended especially in time-sensitive clinical situations.

Neurodevelopmental disorders (NDDs) affect brain function and development, with 90% lacking approved treatments. Understanding their pathogenic mechanisms is critical for developing precision gene therapies. An autosomal recessive NDD associated with variants in the Mediator complex subunit 27 (MED27) gene is previously identified. The Mediator complex is essential for transcription initiation by bridging transcription factors (TFs) at enhancers to RNA polymerase II at promoters. All patients with MED27 variants exhibit cerebellar hypoplasia or atrophy, underscoring the cerebellum's heightened vulnerability to MED27 dysfunction. To investigate the disease mechanisms, in vitro stem cells carrying patient‐specific MED27 variants and in vivo mouse models with Med27 loss‐of‐function (LoF) are generated. These preclinical models recapitulate key patient phenotypes, including progressive cerebellar atrophy and motor deficits. Molecular analyses reveal that mutant MED27 destabilizes the Mediator complex, impairing its chromatin occupancy and altering chromatin interactions. Comprehensive transcriptomic profiling, including single‐cell resolution spatial transcriptomics, identifies dysregulation of downstream targets regulated by MED27, such as critical master regulatory TFs involved in neurogenesis and cerebellar development. This study elucidates a partial LoF mechanism underlying MED27‐associated NDDs and establishes a prototype for investigating NDDs caused by pathogenic variants in Mediator subunits. MED27 is one of the 26 subunits in the human Mediator complex (MED). Neurodevelopmental disorder‐causing MED27 genetic variants induce instability of MED, leading to disrupted DNA occupancy, altered chromatin interaction, and subsequent transcriptional dysregulation of critical downstream genes, including master regulatory transcription factors essential for early neurogenesis and cerebellar development.