Proteome-Wide Assessment of Clustering of Missense Variants in Neurodevelopmental Disorders Versus Cancer.

Missense de novo variants (DNVs) and missense somatic variants contribute to neurodevelopmental disorders (NDDs) and cancer, respectively. Proteins with statistical enrichment based on analyses of these variants exhibit convergence in the differing NDD and cancer phenotypes. Herein, the question of why some of the same proteins are identified in both phenotypes is examined through investigation of clustering of missense variation at the protein level. Our hypothesis is that missense variation is present in different protein locations in the two phenotypes leading to the distinct phenotypic outcomes. We tested this hypothesis in 1D protein space using our software CLUMP. Furthermore, we newly developed 3D-CLUMP that uses 3D protein structures to spatially test clustering of missense variation for proteome-wide significance. We examined missense DNVs in 39,883 parent-child sequenced trios with NDDs and missense somatic variants from 10,543 sequenced tumors covering five TCGA cancer types and two COSMIC pan-cancer aggregates of tissue types. There were 57 proteins with proteome-wide significant missense variation clustering in NDDs when compared to cancers and 79 proteins with proteome-wide significant missense clustering in cancers compared to NDDs. While our main objective was to identify differences in patterns of missense variation, we also identified a novel NDD protein BLTP2. Overall, our study is innovative, provides new insights into differential missense variation in NDDs and cancer at the protein-level, and contributes necessary information toward building a framework for thinking about prognostic and therapeutic aspects of these proteins.

Quantitative determination of SLC2A1 variant functional effects in GLUT1 deficiency syndrome.

OBJECTIVE: The goal of this study is to demonstrate the utility of a growth assay to quantify the functional impact of single nucleotide variants (SNVs) in SLC2A1, the gene responsible for Glut1DS. METHODS: The functional impact of 40 SNVs in SLC2A1 was quantitatively determined in HAP1 cells in which SLC2A1 is required for growth. Donor libraries were introduced into the endogenous SLC2A1 gene in HAP1-Lig4KO cells using CRISPR/Cas9. Cell populations were harvested and sequenced to quantify the effect of variants on growth and generate a functional score. Quantitative functional scores were compared to 3-OMG uptake, SLC2A1 cell surface expression, CADD score, and clinical data, including CSF/blood glucose ratio. RESULTS: Nonsense variants (N = 3) were reduced in cell culture over time resulting in negative scores (mean score: -1.15 ± 0.17), whereas synonymous variants (N = 10) were not depleted (mean score: 0.25 ± 0.12) (P < 2e-16). Missense variants (N = 27) yielded a range of functional scores including slightly negative scores, supporting a partial function and intermediate phenotype. Several variants with normal results on either cell surface expression (p.N34S and p.W65R) or 3-OMG uptake (p.W65R) had negative functional scores. There is a moderate but significant correlation between our functional scores and CADD scores. INTERPRETATION: Cell growth is useful to quantitatively determine the functional effects of SLC2A1 variants. Nonsense variants were reliably distinguished from benign variants in this in vitro functional assay. For facilitating early diagnosis and therapeutic intervention, future work is needed to determine the functional effect of every possible variant in SLC2A1.

Germline mosaicism of a missense variant in KCNC2 in a multiplex family with autism and epilepsy characterized by long-read sequencing.

Currently, protein-coding de novo variants and large copy number variants have been identified as important for ~30% of individuals with autism. One approach to identify relevant variation in individuals who lack these types of events is by utilizing newer genomic technologies. In this study, highly accurate PacBio HiFi long-read sequencing was applied to a family with autism, epileptic encephalopathy, cognitive impairment, and mild dysmorphic features (two affected female siblings, unaffected parents, and one unaffected male sibling) with no known clinical variant. From our long-read sequencing data, a de novo missense variant in the KCNC2 gene (encodes Kv3.2) was identified in both affected children. This variant was phased to the paternal chromosome of origin and is likely a germline mosaic. In silico assessment revealed the variant was not in controls, highly conserved, and predicted damaging. This specific missense variant (Val473Ala) has been shown in both an ortholog and paralog of Kv3.2 to accelerate current decay, shift the voltage dependence of activation, and prevent the channel from entering a long-lasting open state. Seven additional missense variants have been identified in other individuals with neurodevelopmental disorders (p = 1.03 × 10-5 ). KCNC2 is most highly expressed in the brain; in particular, in the thalamus and is enriched in GABAergic neurons. Long-read sequencing was useful in discovering the relevant variant in this family with autism that had remained a mystery for several years and will potentially have great benefits in the clinic once it is widely available.

ACES: Analysis of Conservation with an Extensive list of Species.

MOTIVATION: An abundance of new reference genomes is becoming available through large-scale sequencing efforts. While the reference FASTA for each genome is available, there is currently no automated mechanism to query a specific sequence across all new reference genomes. RESULTS: We developed ACES (Analysis of Conservation with an Extensive list of Species) as a computational workflow to query specific sequences of interest (e.g. enhancers, promoters, exons) against reference genomes with an available reference FASTA. This automated workflow generates BLAST hits against each of the reference genomes, a multiple sequence alignment file, a graphical fragment assembly file and a phylogenetic tree file. These data files can then be used by the researcher in several ways to provide key insights into conservation of the query sequence. AVAILABILITY AND IMPLEMENTATION: ACES is available at https://github.com/TNTurnerLab/ACES. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Coding and noncoding variants in EBF3 are involved in HADDS and simplex autism.

BACKGROUND: Previous research in autism and other neurodevelopmental disorders (NDDs) has indicated an important contribution of protein-coding (coding) de novo variants (DNVs) within specific genes. The role of de novo noncoding variation has been observable as a general increase in genetic burden but has yet to be resolved to individual functional elements. In this study, we assessed whole-genome sequencing data in 2671 families with autism (discovery cohort of 516 families, replication cohort of 2155 families). We focused on DNVs in enhancers with characterized in vivo activity in the brain and identified an excess of DNVs in an enhancer named hs737. RESULTS: We adapted the fitDNM statistical model to work in noncoding regions and tested enhancers for excess of DNVs in families with autism. We found only one enhancer (hs737) with nominal significance in the discovery (p = 0.0172), replication (p = 2.5 × 10-3), and combined dataset (p = 1.1 × 10-4). Each individual with a DNV in hs737 had shared phenotypes including being male, intact cognitive function, and hypotonia or motor delay. Our in vitro assessment of the DNVs showed they all reduce enhancer activity in a neuronal cell line. By epigenomic analyses, we found that hs737 is brain-specific and targets the transcription factor gene EBF3 in human fetal brain. EBF3 is genome-wide significant for coding DNVs in NDDs (missense p = 8.12 × 10-35, loss-of-function p = 2.26 × 10-13) and is widely expressed in the body. Through characterization of promoters bound by EBF3 in neuronal cells, we saw enrichment for binding to NDD genes (p = 7.43 × 10-6, OR = 1.87) involved in gene regulation. Individuals with coding DNVs have greater phenotypic severity (hypotonia, ataxia, and delayed development syndrome [HADDS]) in comparison to individuals with noncoding DNVs that have autism and hypotonia. CONCLUSIONS: In this study, we identify DNVs in the hs737 enhancer in individuals with autism. Through multiple approaches, we find hs737 targets the gene EBF3 that is genome-wide significant in NDDs. By assessment of noncoding variation and the genes they affect, we are beginning to understand their impact on gene regulatory networks in NDDs.