De novo variants in the RNU4-2 snRNA cause a frequent neurodevelopmental syndrome.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals where it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologs. Using RNA-sequencing, we show how 5' splice site usage is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 bp region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide.

The Rocker Technique for Atlantoaxial Dislocation With or Without Basilar Invagination: A Prospective Observational Study.

BACKGROUND AND OBJECTIVES: Atlantoaxial dislocation (AAD) poses a complex surgical challenge. Surgical approaches vary for reducible and irreducible cases. Challenges persist in reducing the atlantodental interval, especially in cases with oblique or vertical C1-C2 joints. The Rocker instrument (MJ Surgical), a less-explored technique, seeks to simplify instrumentation, reduce complexity, and enhance translation and retroflection reduction of AAD. METHODS: This prospective observational study was conducted from January 2022 to July 2023 at a tertiary neurosurgical center. Inclusion criteria covered all age groups with AAD, with or without basilar invagination. Exclusions included medically unstable patients and severe osteoporotic spine conditions. Preoperative assessments included dynamic X-rays, magnetic resonance imaging, and computed tomography scans. The Rocker technique was used, and patients were followed up for 6 to 12 months. RESULTS: Fifty-five patients (30 males, 25 females) underwent surgery. The mean age was 40.41 ± 15.01 years. Successful Rocker technique application was observed in 53 cases. Functional outcomes, assessed using Modified Ranawat grading, showed improvement postoperatively. Radiological outcomes revealed a significant reduction in the anterior atlantodental interval (7.21 ± 0.94 to 2.98 ± 0.78). Basilar invagination was reduced in all cases, whenever present. The technique exhibited versatility, applicability in various joint orientations, and cost-effectiveness. CONCLUSION: The Rocker technique is a safe and effective alternative for managing both reducible and irreducible AADs, with or without basilar invagination. It simplifies the reduction process, offering advantages over established techniques. Further trials, especially in rotational deformities, are warranted for validation.

Stroke volume index (SVI) predicts all-cause mortality following transcatheter aortic valve replacement (TAVR).

BACKGROUND: Left ventricular systolic dysfunction in patients with severe aortic stenosis (AS) may result in low transvalvular gradients and underestimation of AS severity. A low-flow state may occur with reduced LVEF. Little is known about the implications of low compared to normal flow in patients with reduced LVEF undergoing transcatheter aortic valve replacement (TAVR). OBJECTIVES: We compared survival rates with degree of flow across stenosed aortic valves and left ventricular dysfunction. We hypothesized that the stroke volume index (SVI) offers essential information regarding survival following TAVR. METHODS: We retrospectively reviewed patients with LVEF <50 % undergoing TAVR at the Gates Vascular Institute in Buffalo, New York, from 2012 to 2017. We performed Receiver Operator Characteristics to examine the value of SVI in predicting the postoperative outcome of patients. Kaplan-Meier and Cox regression analyses were used to investigate the effect of a low-flow state on five-year survival in patients with systolic dysfunction undergoing TAVR. RESULTS: Five-year survival following TAVR was decreased in patients with low-flow AS (SVI <35 mL/m2) compared to patients with normal flow. Seventy-four percent (n = 50) of patients with low-flow compared to 43 % (n = 22) of patients with normal flow were deceased five years post-TAVR (p ≤0.001). ROC curve indicated SVI to be a clinical predictor of five year survival (AUC 0.732, 95 % CI: 0.641-0.823, p < 0.001). CONCLUSION: Patients with systolic dysfunction and low transvalvular flow AS had increased mortality five years following TAVR. These findings highlight a better prognosis in patients with normal flow and LV systolic dysfunction. CONDENSED ABSTRACT: Low-flow aortic stenosis can occur with reduced left ventricular function. We compared survival rates of patients with known reduced left ventricular function in low-flow and normal flow aortic stenosis. This retrospective single-center study examined mortality rates following transcatheter aortic valve replacement. The mean gradient was not a predictor of mortality. This study shows patients with low-flow aortic stenosis have decreased five-year survival following valve replacement.

Rapid Assessment of the Need for Evidence: Applying the Principles of Value of Information to Research Prioritisation.

We propose a short-cut heuristic approach to rapidly estimate value of information (VOI) using information commonly reported in a research funding application to make a case for the need for further evaluative research. We develop a "Rapid VOI" approach, which focuses on uncertainty in the primary outcome of clinical effectiveness and uses this to explore the health consequences of decision uncertainty. We develop a freely accessible online tool, Rapid Assessment of the Need for Evidence (RANE), to allow for the efficient computation of the value of research. As a case study, the method was applied to a proposal for research on shoulder pain rehabilitation. The analysis was included as part of a successful application for research funding to the UK National Institute for Health and Care Research. Our approach enables research funders and applicants to rapidly estimate the value of proposed research. Rapid VOI relies on information that is readily available and reported in research funding applications. Rapid VOI supports research prioritisation and commissioning decisions where there is insufficient time and resources available to develop and validate complex decision-analytic models. The method provides a practical means for implementing VOI in practice, thus providing a starting point for deliberation and contributing to the transparency and accountability of research prioritisation decisions.

Development of phenyl-urea-based small molecules that target penicillin-binding protein 4.

Staphylococcus aureus has the ability to invade cortical bone osteocyte lacuno-canalicular networks (OLCNs) and cause osteomyelitis. It was recently established that the cell wall transpeptidase, penicillin-binding protein 4 (PBP4), is crucial for this function, with pbp4 deletion strains unable to invade OLCNs and cause bone pathogenesis in a murine model of S. aureus osteomyelitis. Moreover, PBP4 has recently been found to modulate S. aureus resistance to ß-lactam antibiotics. As such, small molecule inhibitors of S. aureus PBP4 may represent dual functional antimicrobial agents that limit osteomyelitis and/or reverse antibiotic resistance. A high throughput screen recently revealed that the phenyl-urea 1 targets PBP4. Herein, we describe a structure-activity relationship (SAR) study on 1. Leveraging in silico docking and modeling, a set of analogs was synthesized and assessed for PBP4 inhibitory activities. Results revealed a preliminary SAR and identified lead compounds with enhanced binding to PBP4, more potent antibiotic resistance reversal, and diminished PBP4 cell wall transpeptidase activity in comparison to 1.

A World Update of Progress in Lower Extremity Transplantation: What's Hot and What's Not.

ABSTRACT: The field of vascularized composite allotransplantation (VCA) is the new frontier of solid organ transplantation (SOT). VCA spans life-enhancing/life-changing procedures such as upper extremity, craniofacial (including eye), laryngeal, tracheal, abdominal wall, penis, and lower extremity transplants. VCAs such as uterus transplants are life giving unlike any other SOT. Of all VCAs that have shown successful intermediate- to long-term graft survival with functional and immunologic outcomes, lower extremity VCAs have remained largely underexplored. Lower extremity transplantation (LET) can offer patients with improved function compared to the use of conventional prostheses, reducing concerns of phantom limb pain and stump complications, and offer an option for eligible amputees that either fail prosthetic rehabilitation or do not adapt to prosthetics. Nevertheless, these benefits must be carefully weighed against the risks of VCA, which are not trivial, including the adverse effects of lifelong immunosuppression, extremely challenging perioperative care, and delayed nerve regeneration. There have been 5 lower extremity transplants to date, ranging from unilateral or bilateral to quadrimembral, progressively increasing in risk that resulted in fatalities in 3 of the 5 cases, emphasizing the inherent risks. The advantages of LET over prosthetics must be carefully weighed, demanding rigorous candidate selection for optimal outcomes.

Targeting mTOR and survivin concurrently potentiates radiation therapy in renal cell carcinoma by suppressing DNA damage repair and amplifying mitotic catastrophe.

BACKGROUND: Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation. EXPERIMENTAL DESIGN: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization. RESULTS: EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy. CONCLUSION: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.

De novo TLK1 and MDM1 mutations in a patient with a neurodevelopmental disorder and immunodeficiency.

The Tousled-like kinases 1 and 2 (TLK1/TLK2) regulate DNA replication, repair and chromatin maintenance. TLK2 variants underlie the neurodevelopmental disorder (NDD) 'Intellectual Disability, Autosomal Dominant 57' (MRD57), characterized by intellectual disability and microcephaly. Several TLK1 variants have been reported in NDDs but their functional significance is unknown. A male patient presenting with ID, seizures, global developmental delay, hypothyroidism, and primary immunodeficiency was determined to have a heterozygous TLK1 variant (c.1435C>G, p.Q479E), as well as a mutation in MDM1 (c.1197dupT, p.K400∗). Cells expressing TLK1 p.Q479E exhibited reduced cytokine responses and elevated DNA damage, but not increased radiation sensitivity or DNA repair defects. The TLK1 p.Q479E variant impaired kinase activity but not proximal protein interactions. Our study provides the first functional characterization of NDD-associated TLK1 variants and suggests that, such as TLK2, TLK1 variants may impact development in multiple tissues and should be considered in the diagnosis of rare NDDs.

Exercise-Induced QRS Prolongation in Brugada Syndrome: Implications for Improving Disease Phenotyping and Diagnosis.

BACKGROUND: Abnormal ventricular activation at rest is reported in Brugada syndrome (BrS). OBJECTIVES: The aim of this study was to evaluate the usefulness of dynamic changes in ventricular activation during exercise to improve disease phenotyping and diagnosis of BrS. METHODS: Digital 12-lead electrocardiograms during stress testing were analyzed retrospectively at baseline, peak exercise, and recovery in 53 patients with BrS and 52 controls. Biventricular activation was assessed from QRS duration (QRSd), whereas right ventricular activation was assessed from S wave duration in the lateral leads (I and V6) and terminal R wave duration in aVR. Exercise-induced changes in QRS parameters to predict a positive procainamide response were assessed in separate test and validation cohorts with suspected BrS. RESULTS: Baseline electrocardiogram parameters were similar between BrS and controls. QRSd shortened with exercise in all controls but prolonged in all BrS (-6.1 ± 6.0 ms vs 7.1 ± 6.5 ms [P < 0.001] in V6). QRSd in recovery was longer in BrS compared with controls (90 ± 12 ms vs 82 ± 11 ms in V6; P = 0.002). Both groups demonstrated exercise-induced S duration prolongation in V6, with greater prolongation in BrS (8.2 ± 14.3 ms vs 1.2 ± 12.4 ms; P < 0.001). Any exercise-induced QRSd prolongation in V6 differentiated those with a positive vs negative procainamide response with 100% sensitivity and 95% specificity in the test cohort, and 87% sensitivity and 93% specificity in the validation cohort. CONCLUSIONS: Exercise-induced QRSd prolongation is ubiquitous in BrS primarily owing to delayed right ventricular activation. This electrocardiogram phenotype predicts a positive procainamide response and may provide a noninvasive screening tool to aid in the diagnosis of BrS before drug challenge.

Molecular cascade reveals sequential milestones underlying hippocampal neural stem cell development into an adult state.

Quiescent adult neural stem cells (NSCs) in the mammalian brain arise from proliferating NSCs during development. Beyond acquisition of quiescence, an adult NSC hallmark, little is known about the process, milestones, and mechanisms underlying the transition of developmental NSCs to an adult NSC state. Here, we performed targeted single-cell RNA-seq analysis to reveal the molecular cascade underlying NSC development in the early postnatal mouse dentate gyrus. We identified two sequential steps, first a transition to quiescence followed by further maturation, each of which involved distinct changes in metabolic gene expression. Direct metabolic analysis uncovered distinct milestones, including an autophagy burst before NSC quiescence acquisition and cellular reactive oxygen species level elevation along NSC maturation. Functionally, autophagy is important for the NSC transition to quiescence during early postnatal development. Together, our study reveals a multi-step process with defined milestones underlying establishment of the adult NSC pool in the mammalian brain.

Expert Panel Curation of 31 Genes in Relation to Limb Girdle Muscular Dystrophy.

Introduction: Limb girdle muscular dystrophies (LGMDs) are a group of genetically heterogeneous autosomal conditions with some degree of phenotypic homogeneity. LGMD is defined as having onset >2 years of age with progressive proximal weakness, elevated serum creatine kinase levels and dystrophic features on muscle biopsy. Advances in massively parallel sequencing have led to a surge in genes linked to LGMD. Methods: The ClinGen Muscular Dystrophies and Myopathies gene curation expert panel (MDM GCEP, formerly Limb Girdle Muscular Dystrophy GCEP) convened to evaluate the strength of evidence supporting gene-disease relationships (GDR) using the ClinGen gene-disease clinical validity framework to evaluate 31 genes implicated in LGMD. Results: The GDR was exclusively LGMD for 17 genes, whereas an additional 14 genes were related to a broader phenotype encompassing congenital weakness. Four genes (CAPN3, COL6A1, COL6A2, COL6A3) were split into two separate disease entities, based on each displaying both dominant and recessive inheritance patterns, resulting in curation of 35 GDRs. Of these, 30 (86%) were classified as Definitive, 4 (11%) as Moderate and 1 (3%) as Limited. Two genes, POMGNT1 and DAG1, though definitively related to myopathy, currently have insufficient evidence to support a relationship specifically with LGMD. Conclusions: The expert-reviewed assertions on the clinical validity of genes implicated in LGMDs form an invaluable resource for clinicians and molecular geneticists. We encourage the global neuromuscular community to publish case-level data that help clarify disputed or novel LGMD associations.

Critical assessment of variant prioritization methods for rare disease diagnosis within the rare genomes project.

BACKGROUND: A major obstacle faced by families with rare diseases is obtaining a genetic diagnosis. The average "diagnostic odyssey" lasts over five years and causal variants are identified in under 50%, even when capturing variants genome-wide. To aid in the interpretation and prioritization of the vast number of variants detected, computational methods are proliferating. Knowing which tools are most effective remains unclear. To evaluate the performance of computational methods, and to encourage innovation in method development, we designed a Critical Assessment of Genome Interpretation (CAGI) community challenge to place variant prioritization models head-to-head in a real-life clinical diagnostic setting. METHODS: We utilized genome sequencing (GS) data from families sequenced in the Rare Genomes Project (RGP), a direct-to-participant research study on the utility of GS for rare disease diagnosis and gene discovery. Challenge predictors were provided with a dataset of variant calls and phenotype terms from 175 RGP individuals (65 families), including 35 solved training set families with causal variants specified, and 30 unlabeled test set families (14 solved, 16 unsolved). We tasked teams to identify causal variants in as many families as possible. Predictors submitted variant predictions with estimated probability of causal relationship (EPCR) values. Model performance was determined by two metrics, a weighted score based on the rank position of causal variants, and the maximum F-measure, based on precision and recall of causal variants across all EPCR values. RESULTS: Sixteen teams submitted predictions from 52 models, some with manual review incorporated. Top performers recalled causal variants in up to 13 of 14 solved families within the top 5 ranked variants. Newly discovered diagnostic variants were returned to two previously unsolved families following confirmatory RNA sequencing, and two novel disease gene candidates were entered into Matchmaker Exchange. In one example, RNA sequencing demonstrated aberrant splicing due to a deep intronic indel in ASNS, identified in trans with a frameshift variant in an unsolved proband with phenotypes consistent with asparagine synthetase deficiency. CONCLUSIONS: Model methodology and performance was highly variable. Models weighing call quality, allele frequency, predicted deleteriousness, segregation, and phenotype were effective in identifying causal variants, and models open to phenotype expansion and non-coding variants were able to capture more difficult diagnoses and discover new diagnoses. Overall, computational models can significantly aid variant prioritization. For use in diagnostics, detailed review and conservative assessment of prioritized variants against established criteria is needed.

GPR1 and CMKLR1 control lipid metabolism to support development of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC), the most common type of kidney cancer, is largely incurable in the metastatic setting. ccRCC is characterized by excessive lipid accumulation that protects cells from stress and promotes tumor growth, suggesting that the underlying regulators of lipid storage could represent potential therapeutic targets. Here, we evaluated the regulatory roles of GPR1 and CMKLR1, two G-protein coupled receptors of the pro-tumorigenic adipokine chemerin that is involved in ccRCC lipid metabolism. Both genetic and pharmacological suppression of either receptor suppressed lipid formation and induced multiple forms of cell death, including apoptosis, ferroptosis and autophagy, significantly impeding ccRCC growth in cell lines and patient derived xenograft (PDX) models. Comprehensive lipidomic and transcriptomic profiling of receptor competent and depleted cells revealed overlapping and unique signaling of the receptors granting control over triglyceride synthesis, ceramide production, and fatty acid saturation and class production. Mechanistically, the receptors both enforced suppression of the triglyceride lipase ATGL but also demonstrated distinct functions, such as the unique ability of CMKLR1 to control lipid uptake through regulation of SREBP1c and the CD36 scavenger receptor. Treating PDX models with the CMKLR1-targeting small molecule α-NETA led to a dramatic reduction of tumor growth, lipid storage, and clear cell morphology. Together, these findings provide mechanistic insight into lipid regulation in ccRCC and identify a targetable axis at the core of the histological definition of this tumor that could be exploited therapeutically.

Exome copy number variant detection, analysis, and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

Liposomal Phenylephrine Nanoparticles Enhance the Antitumor Activity of Intratumoral Chemotherapy in a Preclinical Model of Melanoma.

Intratumoral injection of anticancer agents has limited efficacy and is not routinely used for most cancers. In this study, we aimed to improve the efficacy of intratumoral chemotherapy using a novel approach comprising peri-tumoral injection of sustained-release liposomal nanoparticles containing phenylephrine, which is a potent vasoconstrictor. Using a preclinical model of melanoma, we have previously shown that systemically administered (intravenous) phenylephrine could transiently shunt blood flow to the tumor at the time of drug delivery, which in turn improved antitumor responses. This approach was called dynamic control of tumor-associated vessels. Herein, we used liposomal phenylephrine nanoparticles as a "local" dynamic control strategy for the B16 melanoma. Local dynamic control was shown to increase the retention and exposure time of tumors to intratumorally injected chemotherapy (melphalan). C57BL/6 mice bearing B16 tumors were treated with intratumoral melphalan and peri-tumoral injection of sustained-release liposomal phenylephrine nanoparticles (i.e., the local dynamic control protocol). These mice had statistically significantly improved antitumor responses compared to melphalan alone (p = 0.0011), whereby 58.3% obtained long-term complete clinical response. Our novel approach of local dynamic control demonstrated significantly enhanced antitumor efficacy and is the subject of future clinical trials being designed by our group.

De novo variants in the non-coding spliceosomal snRNA gene RNU4-2 are a frequent cause of syndromic neurodevelopmental disorders.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Increasingly, large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a novel syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 119 individuals with NDD. The vast majority of individuals (77.3%) have the same highly recurrent single base-pair insertion (n.64_65insT). We estimate that variants in this region explain 0.41% of individuals with NDD. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to its contiguous counterpart RNU4-1 and other U4 homologs, supporting RNU4-2's role as the primary U4 transcript in the brain. Overall, this work underscores the importance of non-coding genes in rare disorders. It will provide a diagnosis to thousands of individuals with NDD worldwide and pave the way for the development of effective treatments for these individuals.

Genome and RNA sequencing boost neuromuscular diagnoses to 62% from 34% with exome sequencing alone.

OBJECTIVE: Most families with heritable neuromuscular disorders do not receive a molecular diagnosis. Here we evaluate diagnostic utility of exome, genome, RNA sequencing, and protein studies and provide evidence-based recommendations for their integration into practice. METHODS: In total, 247 families with suspected monogenic neuromuscular disorders who remained without a genetic diagnosis after standard diagnostic investigations underwent research-led massively parallel sequencing: neuromuscular disorder gene panel, exome, genome, and/or RNA sequencing to identify causal variants. Protein and RNA studies were also deployed when required. RESULTS: Integration of exome sequencing and auxiliary genome, RNA and/or protein studies identified causal or likely causal variants in 62% (152 out of 247) of families. Exome sequencing alone informed 55% (83 out of 152) of diagnoses, with remaining diagnoses (45%; 69 out of 152) requiring genome sequencing, RNA and/or protein studies to identify variants and/or support pathogenicity. Arrestingly, novel disease genes accounted for <4% (6 out of 152) of diagnoses while 36.2% of solved families (55 out of 152) harbored at least one splice-altering or structural variant in a known neuromuscular disorder gene. We posit that contemporary neuromuscular disorder gene-panel sequencing could likely provide 66% (100 out of 152) of our diagnoses today. INTERPRETATION: Our results emphasize thorough clinical phenotyping to enable deep scrutiny of all rare genetic variation in phenotypically consistent genes. Post-exome auxiliary investigations extended our diagnostic yield by 81% overall (34-62%). We present a diagnostic algorithm that details deployment of genomic and auxiliary investigations to obtain these diagnoses today most effectively. We hope this provides a practical guide for clinicians as they gain greater access to clinical genome and transcriptome sequencing.

Novel syndromic neurodevelopmental disorder caused by de novo deletion of CHASERR, a long noncoding RNA.

Genes encoding long non-coding RNAs (lncRNAs) comprise a large fraction of the human genome, yet haploinsufficiency of a lncRNA has not been shown to cause a Mendelian disease. CHASERR is a highly conserved human lncRNA adjacent to CHD2-a coding gene in which de novo loss-of-function variants cause developmental and epileptic encephalopathy. Here we report three unrelated individuals each harboring an ultra-rare heterozygous de novo deletion in the CHASERR locus. We report similarities in severe developmental delay, facial dysmorphisms, and cerebral dysmyelination in these individuals, distinguishing them from the phenotypic spectrum of CHD2 haploinsufficiency. We demonstrate reduced CHASERR mRNA expression and corresponding increased CHD2 mRNA and protein in whole blood and patient-derived cell lines-specifically increased expression of the CHD2 allele in cis with the CHASERR deletion, as predicted from a prior mouse model of Chaserr haploinsufficiency. We show for the first time that de novo structural variants facilitated by Alu-mediated non-allelic homologous recombination led to deletion of a non-coding element (the lncRNA CHASERR) to cause a rare syndromic neurodevelopmental disorder. We also demonstrate that CHD2 has bidirectional dosage sensitivity in human disease. This work highlights the need to carefully evaluate other lncRNAs, particularly those upstream of genes associated with Mendelian disorders.

Clinical-Genomic Analysis of 1261 Patients with Ehlers-Danlos Syndrome Outlines an Articulo-Autonomic Gene Network (Entome).

Systematic evaluation of 80 history and 40 history findings diagnosed 1261 patients with Ehlers-Danlos syndrome (EDS) by direct or online interaction, and 60 key findings were selected for their relation to clinical mechanisms and/or management. Genomic testing results in 566 of these patients supported EDS relevance by their differences from those in 82 developmental disability patients and by their association with general rather than type-specific EDS findings. The 437 nuclear and 79 mitochondrial DNA changes included 71 impacting joint matrix (49 COL5), 39 bone (30 COL1/2/9/11), 22 vessel (12 COL3/8VWF), 43 vessel-heart (17FBN1/11TGFB/BR), 59 muscle (28 COL6/12), 56 neural (16 SCN9A/10A/11A), and 74 autonomic (13 POLG/25porphyria related). These genes were distributed over all chromosomes but the Y, a network analogized to an 'entome' where DNA change disrupts truncal mechanisms (skin constraint, neuromuscular support, joint vessel flexibility) and produces a mirroring cascade of articular and autonomic symptoms. The implied sequences of genes from nodal proteins to hypermobility to branching tissue laxity or dysautonomia symptoms would be ideal for large language/artificial intelligence analyses.