Matching variants for functional characterization of genetic variants.

Rapid and low-cost sequencing, as well as computer analysis, have facilitated the diagnosis of many genetic diseases, resulting in a substantial rise in the number of disease-associated genes. However, genetic diagnosis of many disorders remains problematic due to the lack of interpretation for many genetic variants, especially missenses, the infeasibility of high-throughput experiments on mammals, and the shortcomings of computational prediction technologies. Additionally, the available mutant databases are not well-utilized. Toward this end, we used Caenorhabditis elegans mutant resources to delineate the functions of eight missense variants (V444I, V517D, E610K, L732F, E817K, H873P, R1105K, and G1205E) and two stop codons (W937stop and Q1434stop), including several matching variants (MatchVar) with human in ciliopathy associated IFT-140 (also called CHE-11)//IFT140 (intraflagellar transport protein 140). Moreover, MatchVars carrying C. elegans mutants, including IFT-140(G680S) and IFT-140(P702A) for the human (G704S) (dbSNP: rs150745099) and P726A (dbSNP: rs1057518064 and a conflicting variation) were created using CRISPR/Cas9. IFT140 is a key component of IFT complex A (IFT-A), which is involved in the retrograde transport of IFT along cilia and the entrance of G protein-coupled receptors into cilia. Functional analysis of all 10 variants revealed that P702A and W937stop, but not others phenocopied the ciliary phenotypes (short cilia, IFT accumulations, mislocalization of membrane proteins, and cilia entry of nonciliary proteins) of the IFT-140 null mutant, indicating that both P702A and W937stop are phenotypic in C. elegans. Our functional data offered experimental support for interpreting human variants, by using ready-to-use mutants carrying MatchVars and generating MatchVars with CRISPR/Cas9.

WDR31 displays functional redundancy with GTPase-activating proteins (GAPs) ELMOD and RP2 in regulating IFT complex and recruiting the BBSome to cilium.

The correct intraflagellar transport (IFT) assembly at the ciliary base and the IFT turnaround at the ciliary tip are key for the IFT to perform its function, but we still have poor understanding about how these processes are regulated. Here, we identify WDR31 as a new ciliary protein, and analysis from zebrafish and Caenorhabditis elegans reveals the role of WDR31 in regulating the cilia morphology. We find that loss of WDR-31 together with RP-2 and ELMD-1 (the sole ortholog ELMOD1-3) results in ciliary accumulations of IFT Complex B components and KIF17 kinesin, with fewer IFT/BBSome particles traveling along cilia in both anterograde and retrograde directions, suggesting that the IFT/BBSome entry into the cilia and exit from the cilia are impacted. Furthermore, anterograde IFT in the middle segment travels at increased speed in wdr-31;rpi-2;elmd-1 Remarkably, a non-ciliary protein leaks into the cilia of wdr-31;rpi-2;elmd-1, possibly because of IFT defects. This work reveals WDR31-RP-2-ELMD-1 as IFT and BBSome trafficking regulators.

ConVarT: Search Engine for Missense Variants Between Humans and Other Organisms.

ConVarT (https://convart.org/) is a search engine for searching for conjugate variants between humans and other species. The search engine is based on matching conjugate variants called MatchVars between species. Matching equivalent variants requires correct alignment of orthologous proteins with the use of multiple sequence alignments (MSA). Indeed, the ConVarT pipeline has performed over a million MSAs and integrated variants and variant-specific annotations (pathogenicity, phenotypic variants; etc.) into the corresponding positions on MSAs. When a clinically relevant variant is discovered whose functional relevance is unknown, ConVarT offers clinician scientists the possibility to search for a MatchVar in other species and to look for functional data on that variant. Fortunately, ConVarT enables users to paste a protein sequence in FASTA format to search for human orthologous proteins. A pairwise sequence alignment (PSA) is then performed between the provided protein sequence and the human orthologous protein, allowing users to visualize human variants on the PSA. Here, we describe the step-by-step usage of ConVarT. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Searching matching variants (MatchVar) with gene/protein identifiers. Basic Protocol 2: Searching with a FASTA sequence. Alternate Protocol: Search with gene name in multiple species. Basic Protocol 3: Search genes associated with a disease.

ConVarT: a search engine for matching human genetic variants with variants from non-human species.

The availability of genetic variants, together with phenotypic annotations from model organisms, facilitates comparing these variants with equivalent variants in humans. However, existing databases and search tools do not make it easy to scan for equivalent variants, namely 'matching variants' (MatchVars) between humans and other organisms. Therefore, we developed an integrated search engine called ConVarT (http://www.convart.org/) for matching variants between humans, mice, and Caenorhabditis elegans. ConVarT incorporates annotations (including phenotypic and pathogenic) into variants, and these previously unexploited phenotypic MatchVars from mice and C. elegans can give clues about the functional consequence of human genetic variants. Our analysis shows that many phenotypic variants in different genes from mice and C. elegans, so far, have no counterparts in humans, and thus, can be useful resources when evaluating a relationship between a new human mutation and a disease.