Elevated ASCL1 activity creates de novo regulatory elements associated with neuronal differentiation.

BACKGROUND: The pro-neural transcription factor ASCL1 is a master regulator of neurogenesis and a key factor necessary for the reprogramming of permissive cell types to neurons. Endogenously, ASCL1 expression is often associated with neuroblast stem-ness. Moreover, ASCL1-mediated reprogramming of fibroblasts to differentiated neurons is commonly achieved using artificially high levels of ASCL1 protein, where ASCL1 acts as an "on-target" pioneer factor. However, the genome-wide effects of enhancing ASCL1 activity in a permissive neurogenic environment has not been thoroughly investigated. Here, we overexpressed ASCL1 in the neuronally-permissive context of neuroblastoma (NB) cells where modest endogenous ASCL1 supports the neuroblast programme. RESULTS: Increasing ASCL1 in neuroblastoma cells both enhances binding at existing ASCL1 sites and also leads to creation of numerous additional, lower affinity binding sites. These extensive genome-wide changes in ASCL1 binding result in significant reprogramming of the NB transcriptome, redirecting it from a proliferative neuroblastic state towards one favouring neuronal differentiation. Mechanistically, ASCL1-mediated cell cycle exit and differentiation can be increased further by preventing its multi-site phosphorylation, which is associated with additional changes in genome-wide binding and gene activation profiles. CONCLUSIONS: Our findings show that enhancing ASCL1 activity in a neurogenic environment both increases binding at endogenous ASCL1 sites and also results in additional binding to new low affinity sites that favours neuronal differentiation over the proliferating neuroblast programme supported by the endogenous protein. These findings have important implications for controlling processes of neurogenesis in cancer and cellular reprogramming.

Disease-specific ACMG/AMP guidelines improve sequence variant interpretation for hearing loss.

PURPOSE: The ClinGen Variant Curation Expert Panels (VCEPs) provide disease-specific rules for accurate variant interpretation. Using the hearing loss-specific American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines, the Hearing Loss VCEP (HL VCEP) illustrates the utility of expert specifications in variant interpretation. METHODS: A total of 157 variants across nine HL genes, previously submitted to ClinVar, were curated by the HL VCEP. The curation process involved collecting published and unpublished data for each variant by biocurators, followed by bimonthly meetings of an expert curation subgroup that reviewed all evidence and applied the HL-specific ACMG/AMP guidelines to reach a final classification. RESULTS: Before expert curation, 75% (117/157) of variants had single or multiple variants of uncertain significance (VUS) submissions (17/157) or had conflicting interpretations in ClinVar (100/157). After applying the HL-specific ACMG/AMP guidelines, 24% (4/17) of VUS and 69% (69/100) of discordant variants were resolved into benign (B), likely benign (LB), likely pathogenic (LP), or pathogenic (P). Overall, 70% (109/157) variants had unambiguous classifications (B, LB, LP, P). We quantify the contribution of the HL-specified ACMG/AMP codes to variant classification. CONCLUSION: Expert specification and application of the HL-specific ACMG/AMP guidelines effectively resolved discordant interpretations in ClinVar. This study highlights the utility of ClinGen VCEPs in supporting more consistent clinical variant interpretation.

ClinGen expert clinical validity curation of 164 hearing loss gene-disease pairs.

PURPOSE: Proper interpretation of genomic variants is critical to successful medical decision making based on genetic testing results. A fundamental prerequisite to accurate variant interpretation is the clear understanding of the clinical validity of gene-disease relationships. The Clinical Genome Resource (ClinGen) has developed a semiquantitative framework to assign clinical validity to gene-disease relationships. METHODS: The ClinGen Hearing Loss Gene Curation Expert Panel (HL GCEP) uses this framework to perform evidence-based curations of genes present on testing panels from 17 clinical laboratories in the Genetic Testing Registry. The HL GCEP curated and reviewed 142 genes and 164 gene-disease pairs, including 105 nonsyndromic and 59 syndromic forms of hearing loss. RESULTS: The final outcome included 82 Definitive (50%), 12 Strong (7%), 25 Moderate (15%), 32 Limited (20%), 10 Disputed (6%), and 3 Refuted (2%) classifications. The summary of each curation is date stamped with the HL GCEP approval, is live, and will be kept up-to-date on the ClinGen website ( https://search.clinicalgenome.org/kb/gene-validity ). CONCLUSION: This gene curation approach serves to optimize the clinical sensitivity of genetic testing while reducing the rate of uncertain or ambiguous test results caused by the interrogation of genes with insufficient evidence of a disease link.

Correction: ClinGen expert clinical validity curation of 164 hearing loss gene-disease pairs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeted Droplet-Digital PCR as a Tool for Novel Deletion Discovery at the DFNB1 Locus.

Pathogenic variants at the DFNB1 locus encompassing the GJB2 and GJB6 genes account for 50% of autosomal-recessive, congenital nonsyndromic hearing loss in the United States. Most cases are caused by sequence variants within the GJB2 gene, but a significant number of DFNB1 patients carry a large deletion (GJB6-D13S1830) in trans with a GJB2 variant. This deletion lies upstream of GJB2 and was shown to reduce GJB2 expression by disrupting unidentified regulatory elements. First-tier genetic testing for hearing loss includes GJB2 sequence and GJB6-D13S1830 deletion analysis; however, several other deletions in this locus, each with distinct breakpoints, have been reported in DFNB1 patients and are missed by current panels. Here, we report the development of a targeted droplet digital polymerase chain reaction-based assay for comprehensive copy-number analysis at the DFNB1 locus that detects all deletions reported to date. This assay increased detection rates in a multiethnic cohort of 87 hearing loss patients with only one identified pathogenic GJB2 variant. We identify two deletions, one of which is novel, in two patients (2/87 or 2.3%), suggesting that other pathogenic deletions at the DFNB1 locus may be missed. Mapping the assayed DFNB1 deletions also revealed a ∼ 95 kb critical region, which may harbor the GJB2 regulatory element(s).

Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction.

Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein-coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCbeta homolog. Here we show that the sphingolipid biosynthetic enzyme, ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, we show that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP(2)), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP(2) and its partitioning into ordered membrane domains. Thus ceramide kinase-mediated maintenance of ceramide level is important for the local regulation of PIP(2) and PLC during phototransduction.

Rapid and accurate interpretation of clinical exomes using Phenoxome: a computational phenotype-driven approach.

Clinical exome sequencing (CES) has become the preferred diagnostic platform for complex pediatric disorders with suspected monogenic etiologies. Despite rapid advancements, the major challenge still resides in identifying the casual variants among the thousands of variants detected during CES testing, and thus establishing a molecular diagnosis. To improve the clinical exome diagnostic efficiency, we developed Phenoxome, a robust phenotype-driven model that adopts a network-based approach to facilitate automated variant prioritization. Phenoxome dissects the phenotypic manifestation of a patient in concert with their genomic profile to filter and then prioritize variants that are likely to affect the function of the gene (potentially pathogenic variants). To validate our method, we have compiled a clinical cohort of 105 positive patient samples that represent a wide range of genetic heterogeneity. Phenoxome identifies the causative variants within the top 5, 10, or 25 candidates in more than 50%, 71%, or 88% of these exomes, respectively. Furthermore, we show that our method is optimized for clinical testing by outperforming the current state-of-art method. We have demonstrated the performance of Phenoxome using a clinical cohort and showed that it enables rapid and accurate interpretation of clinical exomes. Phenoxome is available at https://phenoxome.chop.edu/ .

PCDH19-related epilepsy in a male with Klinefelter syndrome: Additional evidence supporting PCDH19 cellular interference disease mechanism.

Heterozygous de novo or inherited pathogenic variants in the PCDH19 gene cause a spectrum of neurodevelopmental features including developmental delay and seizures. PCDH19 epilepsy was previously known as "epilepsy and mental retardation limited to females", since the condition almost exclusively affects females. It is hypothesized that the co-existence of two populations of neurons, some with and some without PCDH19 protein expression, results in pathologically abnormal interactions between these neurons, a mechanism also referred to as cellular interference. Consequently, PCDH19-related epilepsies are inherited in an atypical X-linked pattern, such that hemizygous, non-mosaic, 46,XY males are typically unaffected, while individuals with a disease-causing PCDH19 variant, mainly heterozygous females and mosaic males, are affected. As a corollary to this hypothesis, an individual with Klinefelter syndrome (KS) (47,XXY) who has a heterozygous disease-causing PCDH19 variant should develop PCDH19-related epilepsy. Here, we report such evidence: - a male child with KS and PCDH19-related epilepsy - supporting the PCDH19 cellular interference disease hypothesis.