interactive case study for the search for a mutated gene

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Interactive Case Study for The Search for a Mutated Gene

This video case study explores the approaches scientists used to identify a mutation that causes retinitis pigmentosa (RP), a progressive disease that leads to blindness.

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Research Article

A machine learning enhanced EMS mutagenesis probability map for efficient identification of causal mutations in Caenorhabditis elegans

Roles Conceptualization, Data curation, Formal analysis, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Affiliation Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, McGovern Institute for Brain Research, State Key Laboratory of Membrane Biology, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China

Roles Investigation

Roles Formal analysis, Visualization

Roles Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

• Zhengyang Guo, 
• Shimin Wang, 
• Yang Wang, 
• Zi Wang, 
• Guangshuo Ou

• Published: August 26, 2024
• https://doi.org/10.1371/journal.pgen.1011377
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Chemical mutagenesis-driven forward genetic screens are pivotal in unveiling gene functions, yet identifying causal mutations behind phenotypes remains laborious, hindering their high-throughput application. Here, we reveal a non-uniform mutation rate caused by Ethyl Methane Sulfonate (EMS) mutagenesis in the C . elegans genome, indicating that mutation frequency is influenced by proximate sequence context and chromatin status. Leveraging these factors, we developed a machine learning enhanced pipeline to create a comprehensive EMS mutagenesis probability map for the C . elegans genome. This map operates on the principle that causative mutations are enriched in genetic screens targeting specific phenotypes among random mutations. Applying this map to Whole Genome Sequencing (WGS) data of genetic suppressors that rescue a C . elegans ciliary kinesin mutant, we successfully pinpointed causal mutations without generating recombinant inbred lines. This method can be adapted in other species, offering a scalable approach for identifying causal genes and revitalizing the effectiveness of forward genetic screens.

Author summary

Exploring gene functions through chemical mutagenesis-driven genetic screens is pivotal, yet the cumbersome task of identifying causative mutations remains a bottleneck, limiting their high-throughput potential. In this investigation, we uncovered a non-uniform mutation pattern induced by Ethyl Methane Sulfonate (EMS) mutagenesis in the C . elegans genome, highlighting the influence of proximate sequence context and chromatin status on mutation frequency. Leveraging these insights, we engineered a machine learning enhanced pipeline to construct a comprehensive EMS mutagenesis probability map for the C . elegans genome. This map operates on the principle that causative mutations are selectively enriched in genetic screens targeting specific phenotypes amid the backdrop of random mutations.

Applying this mapping tool to Whole Genome Sequencing (WGS) data derived from genetic suppressors rescuing a C . elegans ciliary kinesin mutant, we achieved precise identification of causal mutations without resorting to the conventional generation of recombinant inbred lines. Our work not only advances understanding of mutation dynamics but also revitalizes the efficacy of forward genetic screens, contributing to the refinement of genetic exploration methodologies with implications for various organisms.

Citation: Guo Z, Wang S, Wang Y, Wang Z, Ou G (2024) A machine learning enhanced EMS mutagenesis probability map for efficient identification of causal mutations in Caenorhabditis elegans . PLoS Genet 20(8): e1011377. https://doi.org/10.1371/journal.pgen.1011377

Editor: X. Z. Shawn Xu, University of Michigan, UNITED STATES OF AMERICA

Received: April 12, 2024; Accepted: July 27, 2024; Published: August 26, 2024

Copyright: © 2024 Guo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Mutation data of MMP dataset were directly downloaded from the MMP homepage ( http://genome.sfu.ca/mmp/mmp_mut_strains_data_Mar14.txt ).Python scripts and the model are accessible on GihHub at https://github.com/young55775/Genetorch-developing . C. elegans CHIP-chip and CHIP-seq data can be obtained from the modENCODE project ( https://compbio.med.harvard.edu/modencode/webpage/Chromatin.v0.6.html ) and visualized in Wormbase Genome Browser (JBrowse ( wormbase.org )). ModEncode data can also be downloaded from GEO Accession Viewer ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi ) with the accession number listed in S1 Table. Code availability: https://github.com/young55775/EMSForest-A-Machine-Learning-Enhanced-EMS-Mutagenesis-Probability-Map .

Funding: This work was supported by the National Natural Science Foundation of China Grants to GO (31991190, 31730052, 31861143042, 31525015, 31561130153) and National Key R&D Program of China Grants (2017YFA0503501, 2019YFA0508401). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Forward genetic screens have been instrumental in demystifying the molecular mechanisms underpinning myriad biological processes across a diverse array of organisms. The procedure commences with the creation of a genetically varied population, often achieved via the application of chemical mutagens such as Ethyl Methane Sulfonate (EMS) [ 1 – 3 ] or through irradiation [ 4 , 5 ], thereby seeding the genome with a variety of mutations. This random mutagenesis generates a pool of organisms each harboring a distinct set of genetic aberrations. Subsequent stages involve diligent phenotypic screening, identifying responsible genes, and decoding biological significance. This strategy has been pivotal in unveiling gene functions across multiple biological spectra, spanning developmental processes [ 6 , 7 ], signal transduction [ 8 ], and disease mechanisms [ 9 ]. Genes unearthed through these screens often illuminate deeper molecular mechanisms and are frequently employed to glean insights into analogous processes in more complex organisms, thereby contributing profoundly to our understanding of biological systems and phenomena.

Once a desirable mutant phenotype is identified through chemical mutagenesis, a key challenge lies in determining which among potentially thousands of induced mutations is responsible for the observed phenotype [ 10 ]. Identifying the causal mutation necessitates a substantial investment of labor and time, often demanding rigorous mapping and validation work to confirm the genetic change responsible for the observed phenotype [ 11 – 13 ]. These limitations, especially when contrasted with RNAi [ 14 , 15 ] or CRISPR-Cas9-based reverse genetics [ 16 ] methodologies, have led to the gradual obsolescence of EMS chemical mutagenesis and screens in the model organisms. RNAi, with its ability to knock down genes transiently and its amenability to high-throughput formats [ 17 ], enables the systematic analysis of gene function across the genome, offering a powerful tool for functional genomics [ 18 ]. With CRISPR-Cas9, not only can specific genes be targeted, but specific types of mutations (e.g., point mutations, deletions, insertions) can be introduced [ 19 ], offering a level of precision and control that is simply unattainable with chemical mutagenesis [ 20 , 21 ].

On the other hand, chemical mutagenesis has unique advantages in the generation of a broad spectrum of alleles [ 22 ], including hypomorphic, hypermorphic, and neomorphic alleles, providing a rich reservoir for investigating gene function in a depth unattainable through complete gene knockdown or knockout strategies like RNAi or CRISPR-Cas9. Thus, forward genetic screens facilitate the exploration of nuanced gene functions [ 23 ], interactions [ 24 ] and pathway dynamics [ 25 ], often unveiling surprising connections and uncharted biological landscapes that are not immediately evident through targeted gene perturbation approaches [ 26 ]. Furthermore, this strategy can illuminate the functionality of genes of unknown function or open reading frames that might be overlooked with hypothesis-driven reverse genetics [ 27 ]. This stochastic, phenotype-driven approach allows for the serendipitous discovery of novel genetic interactions and pathways without preconceived notions about gene function, offering the potential to uncover entirely new facets of biology.

Whole-genome sequencing (WGS) has emerged as a formidable instrument for pinpointing causal mutations derived from genetic screens [ 28 ]. In the context of Caenorhabditis elegans ( C . elegans ), several strategies have been devised to reduce the number of candidate causal mutations. One prevalent method involves mating mutants, which are in the N2 background, with the polymorphic Hawaiian strain (CB4856), while alternative strategies exploit DNA variants inherent to the initial background or introduced through mutagenesis [ 29 ]. For the identification of suppressor mutations, the Sibling Subtraction Method (SSM) has been developed, which, by excluding genetic variants present in both mutants and their non-mutant siblings, markedly diminishes the roster of candidates [ 10 ]. However, these strategies, given their reliance on genetic crosses, do not lend themselves to high-throughput applications. Given that the recent advancement of AlphaMissense has predicted millions of pathogenic variants [ 30 ], the incorporation of homologous ones into model organisms for genetic suppressor studies may enhance our comprehension of rescue mechanisms and forge paths towards the proposal of innovative therapeutic strategies. Consequently, there is a pressing demand for the development of scalable methods for identifying causal mutations.

Machine learning, widely used as a statistical method, is a powerful tool for detecting hidden relationships between variables, fitting predictive models to data, and identifying informative groupings within data [ 31 ]. It proves particularly valuable when datasets are too complex for straightforward human analysis, enabling the capture of underlying relationships between variables. With rapid advancements in this field, successful attempts have been made to link phenotypes to potential causal variants based on sequencing data [ 32 ] or structural information [ 30 ]. Most existing tools predict the impact of a variant based on sequence conservation or clinical datasets, such as SIFT [ 33 ] or SnpEff [ 34 ]. However, when dealing with genetic screening involving highly effective mutagens, pathogenic alleles are not always the causal ones, and the genotype/phenotype relationship can be complicated by different genetic backgrounds. Furthermore, there are many genes whose functions are not fully understood and are not necessarily disease-causing but still worthy of study. A new model for genetic screening is needed to detect causal mutations independent of their known functions to predict the significance of mutated genes in chemical screening experiments.

In this study, we systematically analyzed C . elegans WGS data derived from 737 EMS-mutagenized worms, furnished by the Million Mutation Project (MMP) [ 22 ], alongside chromatin immunoprecipitation sequencing (ChIP-seq) datasets of DNA-binding proteins from the modENCODE project [ 35 ]. Contrary to expectations, we discovered that EMS-induced mutations were not uniformly distributed across the C . elegans genome. Instead, a genome-wide EMS mutagenesis bias was uncovered, which correlates with both adjacent sequence context and chromatin structure, facilitating the development of a machine learning based model for predicting EMS-induced mutagenesis probabilities. This model enabled the generation of a genome-wide EMS mutagenesis probability map, which predicts the expected frequency of random mutations for each nucleotide within the C . elegans genome. Consequently, our method enhances our capability to discern causative mutations—enriched through genetic screening for a specific phenotype—from random mutations. By employing this pipeline to analyze WGS data of genetic suppressors that rescue a ciliary kinesin harboring a missense mutation in C . elegans , we identified the causal mutations without the requisite establishment of recombinant inbred lines, thus enhancing the efficiency of high-throughput forward genetics.

A non-uniform pattern of EMS-induced mutations across the C . elegans genome

We examined the distribution of a cumulative 265,169 Single Nucleotide Variants (SNVs) and insertion/deletions (InDels) across the 737 EMS-mutagenized genomes from the Million Mutation Project ( Fig 1A ). A conspicuous non-uniform pattern emerged, characterized by ‘hot spots’—regions exhibiting a higher-than-average SNV density—and ‘cold spots,’ or regions manifesting a lower density. Given that EMS predominantly introduces an alkyl group at N7-guanine and O6-guanine, consequently inducing transitions from ‘G/C’ to ‘A/T,’ we postulated that mutation frequency might be modulated by the genomic concentration of ‘G/C’ nucleotides. Nonetheless, our correlation analysis revealed that the ‘G/C’ pair distribution only partially elucidated the mutation frequency ( Fig 1A and 1B ). Moreover, we discerned variations in mutation frequency amongst different chromosomes ( Fig 1C ), with chromosome X exhibiting an elevated mutation frequency and chromosome IV manifesting a diminished frequency, neither of which could be attributed to the ‘G/C’ distribution. These observations parallel the findings from two additional WGS datasets procured from our recent Artificial Evolution Summer School, in which undergraduates perform forward genetic screens for animals exhibiting dumpy (Dpy) or uncoordinated movement (Unc) phenotypes ( S1A–S1F Fig ). These results imply that EMS-induced variations are not uniformly distributed across the genome, potentially due to disparate local chromosomal features.

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A) Scatter and line plots representing mutations in the MMP dataset. The Scatter plot shows the mutation number on each chromosome. Each dot represents the number of mutated bases within every 100,000 bp counted from the first base pair of each chromosome. The Line plot represents the number of ‘C/G’ base pairs in each 100,000 bp region. B) Scatter plot representing the relationship between ‘C/G’ base pair contents and the number of mutations from the MMP data of each chromosome. Each dot represents the number of mutated bases within every 100,000 bp counted from the first base pair of each chromosome. Pearson correlation was used to evaluate the association between them. C) Kernel density estimation plot of the number of mutations on each chromosome.

https://doi.org/10.1371/journal.pgen.1011377.g001

The adjacent sequence context influences the probability of EMS mutagenesis

A) The distribution patterns of flanking sequences of each kind of base (A, T, C, G) in the genome DNA. The number with ‘+’ or ‘-’ represents the base left (5’)/right (3’) to the position 0. These distributions were set as expectation values that if the mutagen selects randomly on a genome, it will cause mutations with the same distribution in flanking sequences. B) The distribution patterns of flanking sequences for each kind of mutated base in MMP dataset. Note that these distribution patterns are different from the corresponding distributions in (A). Chi-square test: * p<0.05, ** p<0.01, ***p<0.001, ****p<0.000. C) Heatmap recording the mutation rate of each 5-base pattern. X-axis was sorted by the first 3 bases and Y-axis containing the next two. D) Bar plots showing the variation of C mutagenesis rate when -1 and +1 positions are fixed, as shown around the inner circle. E) Bar plots showing the variation of G mutagenesis rate when -1 and +1 positions are fixed, as shown around the inner circle.

https://doi.org/10.1371/journal.pgen.1011377.g002

Multiple DNA-binding protein patterns show correlation with the mutation events

Employing the adjacent sequence context, we crafted a graphical representation that elucidates the frequency of EMS-induced mutations. To exemplify the method, we showcase the map in relation to mutation events across a broad range on chromosome V, spanning from position 1,800,000 to 3,450,000 ( Fig 3A and 3B ). We incorporated a representative gene, ttn-1 , situated on chromosome V between positions 6,120,909 and 6,202,632 ( S2A and S2B Fig ). In both scenarios, the maps reveal expansive genomic segments characterized by a notable diminution in EMS-induced mutations, the ‘silent region’ of which belies the predictions formulated on adjacent sequence context.

Take the MMP data and DNA-binding protein features on Chr V:1800000~3450000 as an example. A) Line plot showing the prediction made from the Flanking sequence preferences of EMS mutagenesis ( P 0 ). B) The mutations observed in the MMP dataset. C) Raw ChIP-chip signal data of UNC-62 binding in young adult worms. D) Raw ChIP-chip signal data of UNC-39 binding in worm embryo. E) Raw ChIP-seq signal data of histone H3 in L3 larval worms. F) Raw ChIP-seq signal data of histone H4 in L3 larval worms. G) Line plot showing the prediction made by a Random Forest regressor trained by DNA-binding protein data.

https://doi.org/10.1371/journal.pgen.1011377.g003

We posited that a unique chromatin state might elucidate the emergence of these ‘silent regions’. Tightly supercoiled DNA, potentially having circumscribed interactions with extraneous substances [ 36 ], along with particular DNA-binding proteins, might confer protection to their target sequences, rendering them less vulnerable to chemicals that engage with DNA [ 37 ]. Consequently, we probed the C . elegans ChIP-seq datasets of DNA-binding proteins and histone modifications from the modENCODE project (Figs 3C–3F and S2C–S2F ). On chromosome V, a silent region extends over 200kb in length ( Fig 3B ). Within this expanse, the binding activity of the transcription factor UNC-62 and UNC-39 manifests irregularly, as evidenced by the ChIP-seq signal ( Fig 3C and 3D ). The absence of ChIP-seq signals in this area might denote a distinctive chromatin state that is less amenable to external substances. Analogously, the ‘silent region’ of the gene ttn-1 also undergoes a comparable albeit less marked depletion of transcription factor binding ( S2C and S2D Fig ). Nonetheless, a distinct histone binding pattern is evident in this region ( S2E and S2F Fig ). While histone binding shows a slight increase across the preceding 200 kb ‘silent region’ on chromosome V ( Fig 3E and 3F ), particularly in H3, the profile of transcription factor binding provides a clearer boundary of this area. This suggests that transcription factor binding may be a more effective feature for capturing the relationship between abnormal mutation frequency and DNA-binding proteins in this case. Probing mutations and the attributes of DNA-binding proteins suggests that regions with elevated or reduced mutation probabilities may originate from direct causative factors or mere coincidence. Nevertheless, it seems judicious to elucidate the binding patterns of these proteins and explore their association with mutation probability.

Using ChIP-seq dataset collections from the WormBase, we next examined the potential correlation between DNA-binding protein features and mutagenesis probability with machine learning techniques.

Nevertheless, it seems judicious to elucidate the binding patterns of these proteins and explore their association with mutation probability. Employing ChIP-seq dataset compilations from WormBase, we subsequently scrutinized the potential correlation between DNA-binding protein characteristics and mutagenesis probability through the lens of machine learning methodologies.

Random Forest regressor (Rfr) modeling to predict the mutation frequency

Employing ChIP-seq dataset compilations from modENCODE and WormBase [ 38 ], we examined the potential correlation between DNA-binding protein characteristics and mutagenesis probability through machine learning methods, which can identify complex chromatin feature patterns and learn their relationship with mutation events

A decision tree is a simple yet powerful prediction method [ 39 ]. It splits data into subsets in order to create groups with similar values of the target feature. Therefore, the predictor can make predictions from new observations with the relationship it learned from the already exist data. Random Forests are a combination of tree predictors [ 40 ]. Each tree is trained on a randomly selected subset of the data and features, capturing slightly different information, and reducing the risk of overfitting, thus improving overall performance.

We developed a regression model using Random Forest regressor. The underlying hypothesis is that, despite the sparsity of mutation events in the Million Mutation Project (200–350 mutations per 100,000 bp, Fig 1A ), the mutation probability for base pairs with the same ’property’ is well-represented in the dataset. The random forest algorithm’s task is to categorize base pairs into different groups based on DNA binding protein patterns and flanking sequences, enabling accurate prediction of each group’s average mutation expectation ( Fig 4A ). Nucleotides with similar flanking sequences and DNA-binding protein attributes are grouped into the same output node of each tree. The mean mutagenesis frequency for that node then serves as the predicted mutagenesis frequency for any nucleotide with similar characteristics assigned to that node during predictions.

A) Schematic diagram of the Random Forest regressor (RFr) model. 600 trees were modeled and each tree randomly used at most 70% percent of the 24 features (≤16 features). The smallest leaf size is set to be 30000 to avoid overfitting. In this model, each decision tree in the forest will put bases with similar properties (flanking sequences and DNA-binding protein patterns) together into an output node, and use the average mutation rate in each node as output. Consequently, the forest will take the average output of every 600 trees as the final output, with which the mutation rate of this kind of base can be predicted. B) Prediction made only by P 0 . The worm genome was divided into 300,000 bp blocks and each dot represents the expectation of mutation number made by P 0 and the actual mutation counts in MMP dataset. C) The performance of RFr on the test set, which contains 20% of the whole dataset. The test set was sorted by the prediction and was divided into 100,000 bp blocks. Each dot represents the expectation of mutation number made by RFr and versus actual mutations counts in MMP dataset in each 100,000 bp long blocks. D) Scatter plot of the overall performance of RFr on the whole genome. The genome was divided into 300,000 bp blocks. Each dot represents the expectation of mutation number made by RFr and the actual mutations counts in MMP dataset in each 300,000 bp long blocks. E) Scatter plot of the overall performance of RFr on 19639 genes of C . elegans . Each dot represents the expectation of mutation number made by RFr and the actual mutations counts in MMP dataset of each gene. F) Representative EMS mutagenesis map of gene atm-1 . Followed by the mutation map in the MMP dataset and dumpy screening. G) Representative EMS mutagenesis map of gene hum-7 . Followed by the mutation map in the MMP dataset and dumpy screening.

https://doi.org/10.1371/journal.pgen.1011377.g004

To create our model, we used a dataset containing 24 DNA-binding protein datasets from modENCODE, which includes information about histone binding, modifications, transcription factor interactions, and other epigenetic modifications ( S1 Table ). We constructed 600 decision trees, each based on the mentioned method, with each tree trained on a randomly chosen subset of up to 16 protein features. To create our model, we used a dataset containing 24 DNA-binding protein datasets from modENCODE, which includes information about histone binding, modifications, transcription factor interactions, and other epigenetic modifications ( S1 Table ). each trained on a randomly chosen subset of up to 16 protein features, based on the method described above.

To validate the model’s performance, our initial validation assessed predictions grounded solely on P 0 , demonstrating congruent patterns across every 300 kb genomic block. These patterns were juxtaposed with the discerned fluctuations in mutation occurrences within the MMP dataset ( Fig 4B ). Upon training utilizing a randomly selected 80% of the data, the model’s performance was appraised against the test set. The test set was categorized based on prediction results and subsequently partitioned into 100kb blocks. Anticipated mutation tallies were then contrasted against actual mutation instances documented in the MMP dataset ( Fig 4C ). Further, a thorough evaluation juxtaposed the anticipated mutation tallies within 300kb genomic blocks ( Fig 4D ) and individual genes, as per the WBcel235 annotation ( Fig 4E ), against actual mutation tallies from the MMP dataset. Collectively, these comparisons refine the rudimentary P 0 map, facilitating a meticulous prediction of EMS mutagenesis probability at individual nucleotides (Figs 3G , 4F , 4G and S2G ).

Subsequently, we selected two representative genes and created EMS mutagenesis maps, highlighting variations in mutagenesis frequency across the entire gene. These maps were then compared to mutations observed in the MMP dataset and sequencing data from the dumpy screening mentioned earlier ( Fig 4F and 4G ). In regions identified as ‘silent region’ based on our predictions, neither screening approach revealed mutations. Conversely, due to the limited number of SNVs in the MMP dataset, numerous nucleotides remained unmutated in this dataset. Notably, the continuous absence of observed mutations on the actual mutation occurrence map did not diminish the model’s ability to predict the potential for mutagenesis: In the dumpy screening, we observed mutations in regions where the MMP dataset displayed minimal mutations ( Fig 4F and 4G ). This suggests that our model recognized the specific characteristics of nucleotides in these regions and transferred the knowledge that similar nucleotides were prone to mutation in other genomic regions in order to make accurate predictions.

The EMS mutagenesis probability map facilitates the identification of causal mutations

We employed the EMS mutagenesis probability map to discern the causal mutation within a genetic suppressor screen, amending defects instigated by a missense mutation in the ciliary kinesin OSM-3. This kinesin drives intraflagellar transport, a process pivotal for the construction of olfactory cilia in C . elegans sensory neurons. These segments harbor an abundance of G protein-coupled receptors [ 41 ], enabling the organism to perceive environmental stimuli, inclusive of various odorant molecules. A functional impairment of the OSM-3 kinesin culminates in a specific loss of distal ciliary segments [ 42 ]; concomitantly, the delocalization of GPCRs or the prevention of odorant-receptor interaction causes animal behavioral defects, such as an incapacitation in executing osmotic avoidance (Osm).

Our preceding genetic screens isolated the E251K missense mutation within the motor domain of OSM-3 kinesin. This mutation parallels a pathogenic variant, E253K, identified in the KIF1A kinesin [ 43 ], mutations within which give rise to a spectrum of neurological disorders collectively recognized as KIF1A-Associated Neurological Disorder (KAND). The E253K mutation in KIF1A is hypothesized to disturb the flexibility of switch I in the motor domain, thereby suppressing γ-phosphate release and subsequent ATP binding and hydrolysis within the motor domain [ 44 , 45 ]. An in vitro single-molecule motility assay revealed that E253K induces a stringent binding to the microtubule (MT) yet precludes the engagement in processive motion of KIF1A. Consistently, C . elegans harboring the E251K mutation in OSM-3 disrupts their distal ciliary segments with full penetrance. Whereas wild-type animals exploit their distal ciliary segments to uptake the fluorescent dye DiI from the culture medium [ 46 ], all examined OSM-3 E251K mutant animals failed to do so (N > 500), manifesting a dye filling defect (Dyf) phenotype unable to take up dye from the environment, which is detectable under a fluorescence stereoscope.

Utilizing the Dyf phenotype as an efficacious readout probing ciliary defects, we executed genetic suppressor screens aimed at restoring dye-filling capacity and recuperating ciliary distal segments. Our screens isolated 38 independent suppressors. We conducted whole-genome sequencing of all suppressors and employed the EMS mutagenesis probability map to analyze the WGS data. Initially, background mutations, defined as those shared across a wide range of samples, were identified, and removed. Subsequently, we evaluated the effectiveness of EMS mutagenesis ( Materials and Methods ). The remaining mutations were compared to the adjusted mutagenesis map ( Fig 5A ). To assess the enrichment effect on each gene by the genetic screening, we calculated the fold change and p-value for each gene showing mutations in the background-removed mutation pool ( Fig 5A ).

A) Schematic diagram of the analysis pipeline. After genetic screening of the EMS mutated worms, those with a target phenotype were sequenced and mutation information was pooled. Then it could be compared with the EMS mutagenesis map based on the RFr (see Materials and Methods ). Finally, candidate genes’ mutation profiles were analyzed by the loss of function impact of InDel, stop-gain, splicing, missense mutations. B) Volcano plot showing the result of an OSM-3 (E251K, cas22599, n = 38) suppressor screening comparing to the EMS mutagenesis map. There were 5 genes showing significant difference between mutation expectation and actual mutation number. C) Bar plot showing the mutation properties of these five genes. D) Taking all the mutations inducing a high impact on protein into consideration, each of the 38 samples has one high-impact mutation in one of the three genes. E) Ciliary defects in the OSM-3 (E251K, cas22599) mutant animals rescued by a K04F10.2 mutated allele (Arg506*, cas23441). K04F10.2 gDNA tagged with Scarlet overexpression in cas23441 exhibits shorter cilia. Arrowheads indicate the ciliary base and transition zone, and arrows indicate junctions between the middle and distal segments. F) Cilium length (mean ± SD) in each group, n = 30 to 50. *** P<0.001 one-way ANOVA. G) Volcano plot showing the result of a dumpy screening (N = 240) from EMS mutated N2 wildtype strain comparing to the EMS mutagenesis map. Dumpy-related or body wall related genes are labeled. H) Bar plot showing the mutation properties of genes labeled in (G).

https://doi.org/10.1371/journal.pgen.1011377.g005

Our analysis unveiled the top five candidate genes, which demonstrated a markedly biased EMS mutagenesis rates in our suppressor screen but did not exhibit any enriched mutation frequency in our Dpy or Unc screens ( Fig 5B ). Among these candidates, two ciliary kinases, DYF-5 and DYF-18, have been recognized for their efficacy in rescuing ciliary defects in osm-3 mutant animals. We obtained 10 and 16 mutant alleles for dyf-5 and dyf-18 , respectively. The majority of these mutant alleles induce missense mutations, introduce stop codons, or cause splicing mutations within the coding region. These results underscore that the EMS mutagenesis probability map facilitates the identification of causal mutations in genes known to participate in this process. The remaining three genes are not well characterized, and it is unclear which one or ones might be implicated in the regulatory mechanisms of OSM-3.

Through categorizing the mutational characteristics of three genes, we observed that two of them harbored mutations—other than missense, introduced stop, or splicing mutations—which are unlikely to disrupt the function of the gene products. In stark contrast, 12 genetic suppressors encompass various loss-of-function mutations within the coding region of the K04F10.2 gene. Noteworthily, given the substantial size of the initial group subjected to screening, the concurrence of two suppressor alleles within the same strain emerges as exceptionally rare. Notably, the mutations with a significant impact did not coincide across the 38 suppressor strains ( Fig 5D ), suggesting that the 38 genetic suppressors induce defects in three genes: dyf-5 , dyf-18 , and K04F10.2. Given that we have already procured 12 distinct alleles of K04F10.2, and generating additional mutants of this gene may not yield further insight, we endeavored to conduct transgenic rescue experiments to further determine whether K04F10.2 operates as the suppressor gene. To this end, we introduced the genomic DNA of K04F10.2, tagged with the red fluorescent protein Scarlet and controlled by its 2kb endogenous promoter, into OSM-3 E251K; K04F10.2 double mutant animals ( Fig 5E and 5F ). The IFT-dynein heavy chain CHE-3, which traverses the entire length of cilia, was marked with green fluorescence, and used as a ciliary marker to ascertain ciliary length ( Fig 5E ) [ 41 ]. As anticipated, GFP fluorescence was observed along cilia measuring 8.09±0.86 μm. In mutants harboring the OSM-3 E251K mutation, the distal ciliary segment was absent, resulting in cilia of the shorter length (4.40±0.93 μm). While OSM-3 E251K;K04F10.2 double mutants restored ciliary length to 7.54±0.60 μm, introducing Scarlet-tagged wild-type genomic DNA of K04F10.2 into the double mutant reduced ciliary length to 6.22±0.81 μm, similar to that of the OSM-3 E251K single mutant. These results indicate that K04F10.2 mutations act as suppressors for OSM-3 E251K. Henceforth, we designate this gene as Joubert syndrome homolog 26 ( jbts-26 ) due to its inferred role in primary cilia. These results demonstrated that the EMS mutagenesis probability map is instrumental in isolating causal mutations in genes previously unlinked to specific processes. 

In conclusion, this research delineates the non-uniform mutation rate instigated by EMS mutagenesis across the C . elegans genome, underscoring that both proximate sequence context and chromatin status are ostensibly correlated with mutation frequency. Leveraging these factors, we deployed a Machine Learning assisted pipeline to sculpt a genome-wide EMS mutagenesis probability map. Operating under the premise that causative mutations will achieve enrichment through genetic screens pertinent to a specific phenotype amidst random mutations, we utilized the map to scrutinize Whole Genome Sequencing (WGS) data derived from C . elegans forward genetic screens. The findings illuminate that the map expediently facilitates the discernment of genes known for their instrumental roles in these processes. Notably, the map also prognosticated a novel gene entwined in each regulation, a prediction substantiated through transgenic rescue experiments. This holistic approach to causal gene identification eschews labor intensive genetic crossing and demonstrates that bioinformatic analysis of WGS data via the EMS mutagenesis probability map can present a potent conduit for directly interfacing phenotype with genotype. Our model performs well when multiple alleles per gene are obtained but is less effective when screens yield only one or two alleles per gene. For challenging forward genetic screens such as manual fluorescence-based screens under high-power microscopy, we recommend using the sibling subtraction method. Machine learning approaches rely on statistical effects to achieve desired accuracy, assuming a sufficient number of candidates are screened—typically, we recommend 30 or more samples per experiment. Our tool was originally designed for large-scale causal gene identification. For small datasets, sibling subtraction remains the most reliable method due to its manageable workload when dealing with only a few alleles.

Given the affordability of WGS, WGS data are usually available before sibling subtraction experiments. Analyzing one hundred WGS datasets using the EMS probability map takes less than one minute, and only several seconds for 10 to 20 WGS datasets. Our model significantly enhances the likelihood of identifying potential causal mutations for genes that have low baseline mutation probabilities, irrespective of the number of isolated mutant alleles. Therefore, investing an additional minute in computation could potentially expedite mutant cloning efforts.

While the EMS probability map cannot be universally applied, especially in challenging screens that yield a limited number of mutant alleles, it proves valuable in many forward genetic screens capable of efficiently generating dozens of mutants with manageable effort. Nonetheless, it’s crucial to employ multiple complementary approaches to enhance forward genetic screens. Their strength lies in their capacity to uncover novel mutations that might otherwise go unnoticed, and ability to unveil unknown aspects of biology. Thus, the EMS probability map can be a useful addition to the forward genetic toolkit.

The Random Forest model elucidated the relationship between various DNA-binding protein patterns and mutation frequency. DNA-binding proteins such as histones and transcription factors are commonly employed to assess the accessibility of genomic DNA, from which the topological arrangement of nucleosomes can be inferred [ 47 ]. However, this relationship becomes more intricate due to the challenge of precisely uncovering how DNA-binding protein patterns affect EMS effectiveness. For example, the occupancy of transcription factors on chromatin may reduce accessibility to EMS, while regions lacking ChIP-seq signals likely indicate a tightly coiled DNA topology with nucleosomes, making them less accessible to other molecules. The machine learning model established the relationship between EMS mutation frequency and these features by leveraging these correlations, resulting in a comprehensive model capable of handling diverse patterns. Further experiments are necessary to decipher these patterns and gain a deeper understanding of the actual topology and conditions of these regions.

We summarize the mutation counts from various datasets: In the MMP project, 737 WGS datasets of EMS mutagenized worms revealed a total of 265,169 mutations, averaging 360 mutations per worm, or approximately 0.36 mutations per 100,000 base pairs (bp). The DPY dataset, consisting of 240 worms, showed 65,171 mutations, averaging 272 mutations per worm, or approximately 0.27 mutations per 100,000 bp. Lastly, the UNC dataset, with 118 worms, yielded 34,730 mutations, averaging 293 mutations per worm, or approximately 0.30 mutations per 100,000 bp. While the MMP project generates a slightly higher mutation rate (360 mutations per worm) compared to our DPY (272 mutations per worm) or UNC (293 mutations per worm) screens, all mutation rates fall within a similar range.

The elucidation of jbts-26 as a suppressor gene mitigating ciliary defects induced by the OSM-3 E251K mutation inaugurates avenues for mechanistic investigations. Previous research has illuminated that the abrogation of ciliary kinases DYF-5 or DYF-18 permits an alternative ciliary heterotrimeric kinesin-2 to ectopically enter the ciliary distal region [ 48 ], thereby substituting the functionality absent in OSM-3. In essence, these two ciliary kinases may not exert their influence directly upon OSM-3. Rather, they curtail a concurrent ciliary transport pathway, and their loss enables heterotrimeric kinesin-2 to compensate for the OSM-3 deficit. Consequently, in the singular mutants of dyf-5 or dyf-18 , the organisms exhibit aberrantly elongated cilia [ 48 ]. In contrast, jbts-26 might orchestrate its function through a mechanism disparate from these two ciliary kinases. We and others have not been able to discern any overt ciliary defects in the single mutant defective in jbts-26 . A pioneering systematic exploration of ciliary genes has annotated jbts-26 as a conserved putative binding associate of a microtubule-severing protein, namely katanin, hinting at a role in microtubule regulation [ 49 ]. Nonetheless, the underlying mechanisms remain mysterious. Considering that the E251K mutation in OSM-3 is analogous to the E253K mutation in KIF1A, future studies will ascertain whether the inhibition of jbts-26 can ameliorate neuronal defects induced by KIF1A E253K, potentially unveiling a novel therapeutic target for intervening in KIF1A E253K-associated neurological disorders.

Recent endeavors to conduct genetic suppressor screens for various missense mutations within OSM-3 have been undertaken, inclusive of published suppressors of a motor hinge mutation, OSM-3 G444E [ 44 , 45 , 50 ]. Nonetheless, no mutations of jbts-26 were unveiled as suppressors for the ciliary defects attributed to OSM-3 G444E, positing that jbts-26 might exert its suppressive effects on the E251K mutation in OSM-3 in a residue-specific modality. This observation accentuates the imperative of functional residuomics: each residue may exhibit intrinsic uniqueness, and mutations at each individual residue may be ameliorated via divergent, distinct mechanisms.

Given the ubiquitous chemical principle underpinning EMS’s capacity to induce genetic mutations across diverse species, it is conceivable that the adjacent sequence context and chromatin status might also engender a non-uniform distribution of EMS-induced alterations throughout all genomes. Congruent with our findings, the efficacy of EMS mutagenesis in rice ( Huanghuazhan ) is correlated with its flanking sequence and chromatin status [ 51 ]. Consequently, it may not be startling to observe that systematic analyses of published WGS data across species divulge a pervasive bias in EMS mutation rates at genome-wide levels. Identifying EMS-induced causal mutations in species characterized by complexities in genome size, ploidy number, and life cycle surpassing those of C . elegans poses a substantially more formidable challenge. Therefore, formulating analogous EMS mutagenesis probability maps for these species could markedly expedite forward genetics endeavors therein.

Materials and methods

Wgs data from the million mutation project.

WGS data from the Million Mutation project can be accessed via the official webpage of Simon Fraser University ( http://genome.sfu.ca/mmp/ ). For our study, we utilized mutation data from a total of 737 strains isolated after EMS mutagenesis was used to train and test the model. Raw sequencing data in fastq format are available from Home—SRA—NCBI (nih.gov). The average sequencing depth of these data were about 15x fold and 265169 variations were found in this EMS mutation dataset.

DNA-binding protein data

C . elegans ChIP-chip and ChIP-seq data can be obtained from the modENCODE project ( data.modencode.org ) and visualized in WormBase Genome Browser (JBrowse ( wormbase.org )).

P 0 calculation from the MMP dataset

C ′ represents the count of a specific five-base pattern underwent mutation in the MMP dataset and C 0 is the number of the same pattern in the reference genome.

Preparation of DNA-binding protein data and Random Forest regressor modeling

The raw ChIP-chip and ChIP-seq data were preprocessed to better reflect the actual distribution of DNA-binding protein on the genome. A python script was employed to apply a moving average with the window size of 100 bp to smooth the data across each chromosome. The smoothed DNA-binding protein dataset was then utilized to train and validate a random forest regressor after randomly spilt the data into a 80% training set and a 20% test set.

The random forest comprised a total of 600 decision trees, each trained on the provided training dataset. To prevent overfitting, we implemented feature selection by randomly considering up to 70% of the available features for each tree. Additionally, to maintain model integrity, we set a minimum leaf size of 30,000 instances. To produce the final prediction, the output from each individual tree was aggregated. This ensemble approach allowed us to generate a comprehensive mutation probability assessment for each type of nucleotide.

This approach allowed us to assess the model’s predictive accuracy across different genomic regions.

This formula is applicable within a population of n strains, and it allows us to estimate the anticipated number of mutations across the sequence under investigation.

Model evaluation and feature importance

Where feature j undergoes n shuffling iterations.

Worm culture

C . elegans were maintained under a consistent temperature of 20°C according to the standard method. Nematode growth medium (NGM) with Escherichia Coli OP50 seeded on it was used to cultivate these worms. C . elegans strains used in this study are listed in S2 Table .

EMS mutagenesis

Worms synchronized at the late L4 stage were carefully collected with 4 mL M9 buffer ( S1 Table ). Subsequently, these collected worms were placed in 50 mM EMS buffer at room temperature with continuous rotation for a duration of 4 hours. Following this treatment, the worms underwent a thorough washing process with M9 buffer and were then cultured under standard conditions. Approximately 20 hours later, the adult worms were subjected to a bleaching procedure to isolate their eggs (referred to as F1). These eggs were subsequently distributed across approximately 100 separate 9 cm NGM plates, with an average of 50 to 100 eggs placed on each plate. Any adult worms displaying the desired phenotype were meticulously collected and placed in individual culture settings. After a careful examination of their offspring, these individuals were subjected to sequencing using an Illumina next-generation sequencer.

WGS data analyze

Mutation data of MMP dataset were directly downloaded from the MMP homepage ( http://genome.sfu.ca/mmp/mmp_mut_strains_data_Mar14.txt ). Raw reads obtained from the next-generation WGS were assessed for duplication and quality with FastQC and were trimmed using Trim_galore (version 0.4.4) to remove the adaptor sequence and low-quality reads. After that, clean reads were aligned to the reference genome (WBcel235) using BWA-MEM2 (version 2.2) with default parameters. In all these sequencing results, > 20x average coverage is ensured. Variations were detected using freebayes (version 1.3.6) and annotated using SnpEff. Finally, a filter was set to ensure the variation quality and limit false positive rate: only sequence depth >5 and allele frequency > 0.8 variations were taken into further analyze.

Strain construction

To perform the rescue experiment, we synthesized the pK04F10.2::K04F10.2::Scarlet DNA fragment using SOEing PCR [ 47 ] and subsequently created the transgenic strain through microinjection.

Supporting information

S1 fig. uneven distribution of ems-induced snv in screening of dumpy and uncoordinated phenotype..

A) Scatter and line plots representing mutations in the Dumpy screening dataset (n = 240). The Scatter plot shows the mutation number on each chromosome. Each dot represents the number of mutated bases within every 100,000 bp counted from the first base pair of each chromosome. The Line plot represents the number of ‘C/G’ base pairs in each 100,000 bp regions. B) Scatter plot representing the relationship between ‘C/G’ base pair contents and the number of mutations from the Uncoordinated screening data of each chromosome. Each dot represents the number of mutated bases within every 100,000 bp counted from the first base pair of each chromosome. Pearson correlation was used to evaluate the association between them. C) Kernel density estimation plot of the number of mutations on each chromosome in uncoordinated screening dataset. D) Scatter and line plots representing mutations in the Uncoordinated screening dataset (n = 118). The Scatter plot shows the mutation number on each chromosome. Each dot represents the number of mutated bases within every 100,000 bp counted from the first base pair of each chromosome. The Line plot represents the number of ‘C/G’ base pairs in each 100,000 bp regions. E) Scatter plot representing the relationship between ‘C/G’ base pair contents and the number of mutations from the dumpy screening data of each chromosome. Each dot represents the number of mutated bases within every 100,000 bp counted from the first base pair of each chromosome. Pearson correlation was used to evaluate the association between them. F) Kernel density estimation plot of the number of mutations on each chromosome in dumpy screening dataset.

https://doi.org/10.1371/journal.pgen.1011377.s001

S2 Fig. Mutation numbers in the MMP data shown association with the DNA-binding protein features.

Take the MMP data and DNA-binding protein features on Chr V:6120909~6202632 ( ttn-1 ) as an example. A) Line plots showing the prediction made from the Flanking sequence preferences of EMS mutagenesis ( P 0 ). B) The mutations observed in the MMP dataset. C) Raw ChIP-chip signal data of UNC-62 binding in young adult worms. D) Raw ChIP-chip signal data of UNC-39 binding in worm embryo. E) Raw ChIP-seq signal data of histone H3 in L3 larval worms. F) Raw ChIP-seq signal data of histone H4 in L3 larval worms. G) Line plots showing the prediction made by a Random Forest regressor trained by DNA-binding protein data.

https://doi.org/10.1371/journal.pgen.1011377.s002

S3 Fig. Feature importance analysis of the Random Forest regressor.

A-F) Permutation importance (see Materials and Methods ) of each feature used to model the Random Forest regressor. The importance is shown in the heatmap alongside the chromosome. Mutation rate is normalized to 0~1 and is shown in the bar plot attaching to the right side of the heatmap.

https://doi.org/10.1371/journal.pgen.1011377.s003

S1 Table. Features for Random Forest training.

https://doi.org/10.1371/journal.pgen.1011377.s004

S2 Table. C . elegans Strains in this study.

https://doi.org/10.1371/journal.pgen.1011377.s005

S3 Table. PCR products for C . elegans transgenesis.

https://doi.org/10.1371/journal.pgen.1011377.s006

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Inheritance and Mutations in a Single-Gene Disorder

• Biotechnology
• Patterns of Inheritance
• Genetic Disease

Resource Type

Description.

This activity builds on information presented in the short film Genes as Medicine . Students interpret actual pedigrees to determine the inheritance pattern of Leber congenital amaurosis (LCA), an inherited form of blindness. They also examine protein sequence data to explore mutations that can cause LCA.

LCA is a disease that primarily affects the retina. Scientists have identified several types of LCA with different genetic causes and patterns of vision loss. In this three-part activity, students examine a form of LCA caused by mutations in the gene RPE65. 

The activity reinforces the connection between genotype and phenotype and illustrates that genes can have multiple alleles, including multiple disease-causing alleles. In the first part of the activity, students analyze several pedigrees of families with members who have LCA. The pedigrees were obtained from a published scientific study. In the second part, students explore mutations in the RPE65 protein in individuals with LCA and discover that some individuals (known as compound heterozygotes) carry two different disease-causing alleles. In the last part of the activity, students reflect on how LCA can be treated using gene therapy.

The “Resource Google Folder” link directs to a Google Drive folder of resource documents in the Google Docs format. Not all downloadable documents for the resource may be available in this format. The Google Drive folder is set as “View Only”; to save a copy of a document in this folder to your Google Drive, open that document, then select File → “Make a copy.” These documents can be copied, modified, and distributed online following the Terms of Use listed in the “Details” section below, including crediting BioInteractive.

Student Learning Targets

• Describe the relationship between genotype and phenotype.
• Use a pedigree to infer genotypes and determine the most likely inheritance pattern of a single-gene trait.
• Analyze amino acid sequence data to identify mutations.  

Estimated Time

allele, compound heterozygote, gene sequencing, gene therapy, genotype, Leber congenital amaurosis (LCA), pedigree chart, phenotype

Primary Literature

Morimura, Hiroyuki, Gerald A. Fishman, Sandeep A. Grover, Anne B. Fulton, Eliot L. Berson, and Thaddeus P. Dryja. “Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or Leber congenital amaurosis.” Proceedings of the National Academy of Sciences 95, 6 (1998): 3088–3093. https://doi.org/10.1073/pnas.95.6.3088. 

Li, Yumei, Hui Wang, Jianlan Peng, Richard A. Gibbs, Richard Alan Lewis, James R. Lupski, Graeme Mardon, and Rui Chen. “Mutation survey of known LCA genes and loci in the Saudi Arabian population.” Investigative Ophthalmology and Visual Science 50, 3 (2008): 1336–1343. https://doi.org/10.1167/iovs.08-2589. 

Russell, Stephen, Jean Bennett, Jennifer A. Wellman, Daniel C. Chung, Zi-Fan Yu, Amy Tillman, Janet Wittes, et al. “Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65 -mediated inherited retinal dystrophy: A randomised, controlled, open-label, phase 3 trial.” The Lancet 390, 10097 (2017): 849–860. https://doi.org/10.1016/s0140-6736(17)31868-8.  

Terms of Use

The resource is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International license . No rights are granted to use HHMI’s or BioInteractive’s names or logos independent from this Resource or in any derivative works.

Accessibility Level (WCAG compliance)

Version history, curriculum connections, ngss (2013).

HS-LS1-1, HS-LS3-1; SEP2, SEP6

AP Biology (2019)

IST-1.I, IST-1.N, IST-1.P, IST-2.E, IST-4.A, SYI-3.C; SP1, SP2, SP6

IB Biology (2016)

3.1, 3.4, B.4

Common Core (2010)

ELA.RST.9–12.4, ELA.RST.9-12.7 Math.S-IC.2; MP2, MP4  

Vision and Change (2009)

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