Downstream Alternate Start Site Allows N-Terminal Nonsense Variants to Escape NMD and Results in Functional Recovery by Readthrough and Modulator Combination

Genetic variants that introduce premature termination codons (PTCs) have remained difficult to therapeutically target due to lack of protein product. Nonsense mediated mRNA decay (NMD) targets PTC-bearing transcripts to reduce the potentially damaging effects of truncated proteins. Readthrough compounds have been tested on PTC-generating variants in attempt to permit translation through a premature stop. However, readthrough compounds have not proved efficacious in a clinical setting due to lack of stable mRNA. Here, we investigate N-terminal variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which have been shown to escape NMD, potentially through a mechanism of alternative translation initiation at downstream AUG codons. We hypothesized that N-terminal variants in CFTR that evade NMD will produce stable transcript, allowing CFTR function to be restored by a combination of readthrough and protein modulator therapy. We investigate this using two cell line models expressing CFTR-expression minigenes (EMG; HEK293s and CFBEs) and primary human nasal epithelial (NE) cells, and we test readthrough compounds G418 and ELX-02 in combination with CFTR protein modulators. HEK293 cells expressing the variants E60X and L88X generate CFTR-specific core glycosylated products that are consistent with downstream translation initiation. Mutation of downstream methionines at codons 150 and 152 does not result in changes in CFTR protein processing in cells expressing L88X-CFTR-EMG. However, mutation of methionine at 265 results in loss of detectable CFTR protein in cells expressing E60X, L88X, and Y122X CFTR-EMGs, indicating that downstream translation initiation is occurring at the AUG codon at position M265. In HEK293 stable cells harboring L88X, treatment with readthrough compounds alone allows for formation of full-length, but misfolded CFTR protein. Upon addition of protein modulators in combination with readthrough, we observe formation of mature, complex-glycosylated CFTR. In CFBE and NE cells, addition of readthrough ELX-02 and modulator therapy results in substantial recovery of CFTR function. Our work indicates that N-terminal variants generate stable CFTR transcript due to translation initiation at a downstream AUG codon. Thus, individuals with CF bearing 5′ nonsense variants that evade NMD are ideal candidates for treatment with clinically safe readthrough compounds and modulator therapy

P523: The NeuroDev Study: Genetic characterization of neurodevelopmental disorders in African populations

Introduction: Genetic association studies have made significant contributions to our understanding of the etiology of neurodevelopmental disorders (NDDs). However, the vast majority of these studies have focused on populations of European ancestry, and few include individuals from the African continent, creating substantial gaps in our understanding of the expression and variability of such disorders. Further, individuals of African ancestry are less likely to receive accurate genetic diagnoses and benefit from advances in genomic science and medicine. The NeuroDev study aims to address this diversity gap through detailed phenotypic and genetic characterization of children with NDDs from South Africa and Kenya. Here, we present the genetic findings from the NeuroDev pilot study, which consists of the study’s first 99 parent-child trios. Methods: Exome sequencing and data processing were performed by the Genomics Platform at the Broad Institute of MIT and Harvard, and the resulting data was uploaded to the seqr platform for analysis. A standard analysis protocol, which involves both de novo/dominant and recessive searches, was deployed across all trios to identify potential causal variants. When evaluating variants in established disease genes, NeuroDev phenotype data was assessed for possible consistencies with known clinical presentations for that gene, bearing in mind potential deviations from expectation due to ancestry. Genes not yet associated with a well-established human disease were evaluated using a variety of sources of evidence, and candidate variants were shared with the Matchmaker Ex- change (MME) network to identify additional cases with similar genotypic and phenotypic profiles. Potential causal variants were subjected to rigorous evaluation of the evidence for pathogenicity following the ACMG/AMP guidelines, and variants meeting pathogenic or likely pathogenic (P/LP) criteria in South African probands were nominated for clinical validation and return to interested families. Results: Exome sequencing analysis identified P/LP variants in 22 of the 99 cases. A total of 13 (17%) of the 75 cases from South Africa were found to have P/ LP variants eligible for return, including 6 single nucleotide variants (SNVs) or indels and 7 copy number variants (CNVs) impacting established NDD genes or cytogenetic regions. Of the 24 Kenyan cases, 9 (38%) were solved with P/LP variants, including 6 SNVs and 3 CNVs. In addition to the solved cases, 14 cases were found to have suspicious variants of uncertain significance (VUS) in candidate disease genes, which were submitted to MME. Matches were made for 7 of the 14 submitted candidates, each of which will be included in collaborative case series reports on the genes of interest. These variants may be reclassified in the future after re-evaluation of the evidence collected for these emerging gene-disease associations. Conclusion: The NeuroDev study is making progress towards addressing the diversity gap in studies of the genetic etiology of neurodevelopmental disorders. The results of the first 99 trios demonstrate the potential for genetic characterization and rare disease discovery in an African context. Additionally, the NeuroDev trio pilot data is now the largest African NDD collection for which genetic and phenotypic data are publicly available to the research community, through the National Human Genome Research Institute Analysis Visualization and Informatics Lab-space (AnVIL) controlled access data repository. Over the course of the next few years, the NeuroDev study will, in total, perform exome sequencing and share data for approximately 2,000 children with NDDs, which will be of significant benefit to the global genetics community

Protospacer modification improves base editing of a canonical splice site variant and recovery of CFTR function in human airway epithelial cells

Canonical splice site variants affecting the 5′ GT and 3′ AG nucleotides of introns result in severe missplicing and account for about 10% of disease-causing genomic alterations. Treatment of such variants has proven challenging due to the unstable mRNA or protein isoforms that typically result from disruption of these sites. Here, we investigate CRISPR-Cas9-mediated adenine base editing for such variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We validate a CFTR expression minigene (EMG) system for testing base editing designs for two different targets. We then use the EMG system to test non-standard single-guide RNAs with either shortened or lengthened protospacers to correct the most common cystic fibrosis-causing variant in individuals of African descent (c.2988+1G>A). Varying the spacer region length allowed placement of the editing window in a more efficient context and enabled use of alternate protospacer adjacent motifs. Using these modifications, we restored clinically significant levels of CFTR function to human airway epithelial cells from two donors bearing the c.2988+1G>A variant

Synthesis and SAR of novel, non-MPEP chemotype mGluR5 NAMs identified by functional HTS

This Letter describes the discovery and SAR of three novel series of mGluR5 noncompetitive antagonists/negative allosteric modulators (NAMs) not based on manipulation of an MPEP/MTEP chemotype identified by a functional HTS approach. This work demonstrates fundamentally new mGluR5 NAM chemotypes with submicromolar potencies, and further examples of a mode of pharmacology `switch' to provide PAMs with a non-MPEP scaffold

Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies.

Elucidating the functional consequence of molecular defects underlying genetic diseases enables appropriate design of therapeutic options. Treatment of cystic fibrosis (CF) is an exemplar of this paradigm as the development of CFTR modulator therapies has allowed for targeted and effective treatment of individuals harboring specific genetic variants. However, the mechanism of these drugs limits effectiveness to particular classes of variants that allow production of CFTR protein. Thus, assessment of the molecular mechanism of individual variants is imperative for proper assignment of these precision therapies. This is particularly important when considering variants that affect pre-mRNA splicing, thus limiting success of the existing protein-targeted therapies. Variants affecting splicing can occur throughout exons and introns and the complexity of the process of splicing lends itself to a variety of outcomes, both at the RNA and protein levels, further complicating assessment of disease liability and modulator response. To investigate the scope of this challenge, we evaluated splicing and downstream effects of 52 naturally occurring CFTR variants (exonic = 15, intronic = 37). Expression of constructs containing select CFTR intronic sequences and complete CFTR exonic sequences in cell line models allowed for assessment of RNA and protein-level effects on an allele by allele basis. Characterization of primary nasal epithelial cells obtained from individuals harboring splice variants corroborated in vitro data. Notably, we identified exonic variants that result in complete missplicing and thus a lack of modulator response (e.g. c.2908G>A, c.523A>G), as well as intronic variants that respond to modulators due to the presence of residual normally spliced transcript (e.g. c.4242+2T>C, c.3717+40A>G). Overall, our data reveals diverse molecular outcomes amongst both exonic and intronic variants emphasizing the need to delineate RNA, protein, and functional effects of each variant in order to accurately assign precision therapies

Fiskeri-undervisningen 1966 - 1967

The NeuroDev study will deeply phenotype cognition, behavior, dysmorphias, and neuromedical traits on an expected cohort of 5,600 Africans (1,800 child cases, 1,800 child controls, and 1,900 parents) and will collect whole blood for exome sequencing and biobanking