Functional Profiling of 2,193 ASS1 Missense Variants: Insights into Variant Pathogenicity and Epistatic Interactions in Citrullinemia Type I

  1. Russell S. Lo
  2. Gareth A. Cromie
  3. Michelle Tang
  4. Amy Sirr
  5. Ljubica Caldovic
  6. Hiroki Morizono
  7. Nicholas Ah Mew
  8. Andrea Gropman
  9. Aimée M. Dudley

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Abstract

Sequence variants in the urea cycle gene argininosuccinate synthase ( ASS1 ) cause Citrullinemia type 1 (CTLN1), a rare autosomal recessive disease. Mechanistically, reduction in argininosuccinate synthetase (ASS) enzyme activity impairs the urea cycle, leading to an accumulation of citrulline and neurotoxic ammonia. Disease severity varies according to the degree of enzyme impairment, ranging from severe neonatal forms (classic citrullinemia) to milder, late-onset forms that may manifest in childhood or adulthood. We established a high-throughput yeast functional assay of human ASS and individually measured the impact of 2193 amino acid substitutions, representing 90% of all single nucleotide variant (SNV)-accessible substitutions. When benchmarked against existing clinical variant annotation, our assay distinguishes known benign variants from strong loss of function pathogenic variants, enabling identification of a functional score threshold below which variants show clinically relevant impairment of ASS activity. Using the ACMG OddsPath framework, our assay meets PS3_supporting criteria for pathogenicity classification and achieves full PS3-level strength when variants observed as homozygotes in other primates are used as benign proxies for calibration. These results provide direct functional evidence to inform reclassification of ASS1 missense variants, supporting their clinical interpretation and diagnostic utility. Mapping functional scores onto the protein structure, we confirmed that residues involved in catalysis are highly sensitive to substitution. In addition, we identified residues from adjacent subunits of the ASS homotetramer that form compound active sites. Assaying these positions revealed a capacity for intragenic complementation consistent with a variant sequestration model: a form of positive epistasis in which deleterious variants from different subunits are sequestered into only a subset of active sites, restoring function in the remaining variant-free sites. The discovery of intragenic complementation in ASS reveals a novel mode of functional interaction with clinical implications for interpreting variant combinations in heterozygous individuals.

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    Reply to the reviewers

    We thank the reviewers and editors for their careful evaluation of our manuscript and their positive comments on the importance and rigor of the work. Below you will find our point-by-point response to each reviewer's suggestions. We believe that we have addressed (in the response and the revised manuscript) all of the concerns. Please note that in some cases, we have numbered a reviewer's comments for clarity, however beyond this, we have not altered any of the reviewers' text.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Lo et al., report a high-throughput functional profiling study on the gene encoding for argininosuccinate synthase (ASS1), done in a yeast experimental system. The study design is robust (see lines 141-143, main text, Methods), whereby "approximately three to four independent transformants of each variant would be isolated and assayed." (lines 140 - 141, main text, Methods). Such a manner of analysis will allow for uncertainty of the functional readout for the tested variants to be accounted for.

    This is an outstanding study providing insights on the functional landscape of ASS1. Functionally impaired ASS1 may cause citrullinemia type I, and disease severity varies according to the degree of enzyme impairment (line 30, main text; Abstract). Data from this study forms a valuable resource in allowing for functional interpretation of protein-altering ASS1 variants that could be newly identified from large-scale whole-genome sequencing efforts done in biobanks or national precision medicine programs. I have some suggestions for the Authors to consider:

    1. The specific function of ASS1 is to condense L-citrulline and L-aspartate to form argininosuccinate. Instead of measuring either depletion of substrate or formation of product, the Authors elected to study 'growth' of the yeast cells. This is a broader phenotype which could be determined by other factors outside of ASS1. Whereas i agree that the experiments were beautifully done, the selection of an indirect phenotype such as ability of the yeast cells to grow could be more vigorously discussed.

    We appreciate the reviewer's point regarding the indirect nature of growth as a functional readout. In our system, yeast growth is tightly and specifically coupled to ASS enzymatic activity. The strains used are isogenic and lack the native yeast argininosuccinate synthetase, such that arginine biosynthesis, and therefore yeast replication on minimal medium lacking arginine, depends exclusively on the activity of human ASS1. Under these defined and limiting conditions, growth provides a quantitative proxy for ASS1 function. However, we acknowledge that this assay does not resolve specific molecular mechanisms underlying reduced function, such as altered catalytic activity versus effects on protein stability. We have updated the text to clarify these points.

    "While growth is an indirect phenotype relative to direct measurement of substrate turnover or product formation, it is tightly coupled to ASS enzymatic activity in this system and is expected to be impaired by amino acid substitutions that reduce catalytic activity or protein stability. Therefore, growth on minimal medium lacking arginine is a quantitative measure of ASS enzyme function, allowing the impact of ASS1 missense variants to be assessed at scale through a high-throughput growth assay, in a single isogenic strain background, under controlled, defined conditions that limit confounding factors unrelated to ASS1 activity. We expect that the assay will detect reductions in both catalytic activity and protein stability but will not distinguish between these mechanisms."

    1. One of the key reasons why studies such as this one are valuable is due to the limitations of current variant classification methods that rely on 'conservation' status of amino acid residues to predict which variants might be 'pathogenic' and which variants might be 'likely benign'. However, there are serious limitations, and Figures 2 and 6 in the main text shows this clearly. Specifically, there is an appreciable number of variants that, despite being classified as "ClinVar Pathogenic", were shown by the assay to unlikely be functionally impaired. This should be discussed vigorously. Could these inconsistencies be potentially due to the read out (growth instead of a more direct evaluation of ASS1 function)?

    We interpret this discrepancy as reflecting a sensitivity limitation of the growth-based readout rather than a fundamental disagreement between functional effect and clinical annotation. Specifically, we believe that our assay is unable to resolve the very mildest hypomorphic variants from true wild type, i.e., the residual activity of these variants is sufficient to fully support yeast growth under the conditions used. On this basis, we have chosen not to treat wild-type-like growth in our assay as informative for benignity; conversely, reduced growth provides evidence supporting pathogenicity (all clinically validated variants examined in this range are pathogenic).

    We have revised the manuscript to clarify this point explicitly and to frame these variants as lying outside the effective resolution limit of the assay rather than representing true false positives. Additional discussion of this limitation and its implications is provided in our responses to Reviewer 2 (points 1 and 4) along with specific changes made to the text.

    1. Figure 3 is very interesting, showing a continuum of functional readout ranging from 'wild-type' to 'null'. It is very interesting that the Authors used a threshold of less than 0.85 as functionally hypomorphic. What does this mean? It would be very nice if they have data from patients carrying two hypomorphic ASS1 alleles, and correlate their functional readout with severity of clinical presentation. The reader might be curious as to the clinical presentation of individuals carrying, for example, two ASS1 alleles with normalized growth of 0.7 to 0.8.

    I hope you will find these suggestions helpful.

    We thank the reviewer for this thoughtful comment. Figure 3 indeed illustrates a continuum of functional effects, and we agree that careful interpretation of the thresholds used is important. To clarify the rationale for the hypomorphic threshold, the interpretation of intermediate growth values, and to emphasize that these labels reflect only behavior in the functional assay, we have rewritten the relevant section of the Results:

    "The normalized growth scores of the 2,193 variants tested in our functional assay form a clear bimodal distribution (Figure 3), with two distinct peaks corresponding to functional extremes, as is commonly reported in large-scale functional assays of protein function [9, 10]. The smaller peak, centered around the null control (normalized growth = 0), represents variants that fail to support growth in the assay (growth 0.85). Variants with growth values falling between these two peak-based thresholds display partial functional impairment and are classified as functionally hypomorphic (n = 323). Crucially, these classifications are entirely derived from the observed peaks in the distribution of growth values and reflect differences in functional activity under the assay conditions. They do not provide direct evidence for clinical pathogenicity or benignity and should not be used for clinical variant interpretation without proper benchmarking against clinical reference datasets, as implemented below within an OddsPath framework."

    We agree with the reviewer that correlating functional measurements with clinical severity in individuals carrying two hypomorphic ASS1 alleles would be highly informative, particularly given that ASS1 deficiency is an autosomal recessive disorder. While mild hypomorphic variants (for example, variants with normalized growth values of 0.7-0.8 in our assay) could plausibly contribute to disease when paired with a complete loss-of-function allele, systematic analysis of combinatorial genotype effects and genotype-phenotype correlations is beyond the scope of the present study, which focuses on the functional effects of individual variants. We view this as an important direction for future work.

    Reviewer #1 (Significance (Required)):

    This is an outstanding study providing insights on the functional landscape of ASS1. Functionally impaired ASS1 may cause citrullinemia type I, and disease severity varies according to the degree of enzyme impairment (line 30, main text; Abstract). Data from this study forms a valuable resource in allowing for functional interpretation of protein-altering ASS1 variants that could be newly identified from large-scale whole-genome sequencing efforts done in biobanks or national precision medicine programs.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, Lo et al characterize the phenotypic effect of ~90% of all possible ASS1 missense mutations using an elegant yeast-based system, and use this dataset to aid the interpretation of clinical ASS1 variants. Overall, the manuscript is well-written and the experimental data are interpretated rigorously. Of particular interest is the identification of pairs of deleterious alleles that rescue ASS1 activity in trans. My comments mainly pertain to the relevance of using a yeast screening methodology to infer functional effects of human ASS1 mutations.

    1. Since human ASS1 is heterologously expressed in yeast for this mutational screen, direct comparison of native expression levels between human cells and yeast is not possible. Could the expression level of human ASS1 (driven by the pARG1 promoter) in yeast alter the measured fitness defect of each variant? For instance, if ASS1 expression in yeast is sufficiently high to mask modest reductions in catalytic activity, such variants may be misclassified as hypomorphic rather than amorphic. Conversely, if expression is intrinsically low, even mild catalytic impairments could appear deleterious. While it is helpful that the authors used non-human primate SNV data to calibrate their assay, experiments could be performed to directly address this possibility.

    The nature of the relationship between yeast growth and availability of functional ASS1 could also influence the interpretation of results from the yeast-based screen. Does yeast growth scale proportionately with ASS1 enzymatic activity?

    We completely agree that the expression level of human ASS1 in yeast could influence the measured fitness effects of individual variants. We expect the rank ordering of variants in our growth assay to reflect their relative enzymatic activity (i.e. a monotonic relationship) but acknowledge that the precise mapping between activity and growth is unknown and may include ceiling and floor effects that limit the assay's dynamic range. As the reviewer notes, under high expression conditions moderate loss-of-function variants could appear indistinguishable from wild type (ceiling effect), whereas under lower expression the same variants could behave closer to the null control (floor effect).

    In our system, ASS1 is expressed from the pARG1 promoter, chosen under the assumption that the native expression level of ARG1 (the yeast ASS1 ortholog) is appropriately tuned for yeast growth. Crucially, rather than assuming a fixed mapping from assay growth to clinical pathogenicity (given potential nonlinearities in the relationship between ASS function and growth) we benchmark the assay against external data, including known pathogenic and benign variants and non-human primate SNVs, to calibrate thresholds and guide interpretation within an OddsPath framework. This benchmarking indicates that ceiling effects are likely present, with some mild loss-of-function pathogenic variants appearing indistinguishable from wild type in the growth assay. We explicitly account for this by not using high-growth scores as evidence toward benignity. We have made the following changes the manuscript:

    "A subset of clinically pathogenic ASS1 variants exhibit near-wild-type growth in our yeast assay. In general, we expect a monotonic relationship between ASS function and yeast growth, but with the potential for floor and ceiling effects that constrain the assay's dynamic range. In this context, we interpret high-growth pathogenic variants as likely causing mild loss of function that cannot be distinguished from wild type in our assay"

    "Based on these findings and given that 22/56 pathogenic variants show >85% growth, we conclude that growth above this threshold should not be used as evidence toward benignity."

    1. It would be helpful to add an additional diagram to Figure 1A explaining how the screen was performed, in particular: when genotype and phenotype were measured, relative to plating on selective vs non-selective media? This is described in "Variant library sequence confirmation" and "Measuring the growth of individual isolates" of the Methods section but could also be distilled into a diagram.

    We thank the reviewer for this helpful suggestion. We have updated Figure 1 by adding a new schematic panel (Figure 1C) that distills the experimental workflow into a visual overview. This diagram is intended to complement the detailed descriptions in the Methods and improve clarity for the reader.

    1. The authors rationalize the biochemical consequences of ASS1 mutations in the context of ASS1 per se - for example, mutations in the active site pocket impair substrate binding and therefore catalytic activity, which is expected. Does ASS1 physically interact with other proteins in human cells, and could these interactions be altered in the presence of specific ASS1 mutations? Such effects may not be captured by performing mutational scanning in yeast.

    We are not aware of any specific protein-protein interactions involving ASS that are required for its enzymatic function. However, we agree that ASS could engage in non-essential interactions with other human proteins that might be altered by specific missense variants and that such interactions would not necessarily be captured in a yeast-based assay.

    Importantly, our complementation system depends on human ASS providing the essential enzymatic activity required for arginine biosynthesis in yeast. If ASS1 required obligate human-specific protein interactions to function, even the wild-type enzyme would fail to support yeast growth, which is clearly not the case. We therefore conclude that the assay robustly reports on the intrinsic enzymatic activity of ASS, while acknowledging that non-essential human-specific interactions may not be assessed. We have updated the manuscript to reflect this point.

    "Importantly, successful functional complementation indicates that ASS enzymatic activity does not depend on any obligate human-specific protein interactions."

    1. The authors note that only a small number (2/11) of mutations at the ASS1 monomer-monomer interface lead to growth defects in yeast. It would be helpful for the authors to discuss this further.

    As discussed in response to the reviewer's comments on the relationship between ASS activity and yeast growth (point 1 above), we expect growth to be a monotonic but nonlinear function of enzymatic activity, with potential ceiling effects at high activity. Under this model, variants causing weak or moderate loss of function may remain indistinguishable from wild type when residual activity is sufficient to support normal growth. We favor this explanation for the observation that only 2/11 interface variants show reduced growth, as many pathogenic interface substitutions are associated with milder disease presentations, consistent with higher residual enzyme function. Consistent with this interpretation, variants affecting the active site, where substitutions are expected to cause large reductions in catalytic activity, are readily detected by the assay.

    Although we cannot exclude partial buffering of dimerization defects in yeast, we interpret the reduced sensitivity to interface variants primarily as a general limitation of growth-based assays. Accordingly, our decision not to use growth >85% as evidence toward benignity is conservative relative to approaches that would classify high-growth variants as benign except at the monomer-monomer interface, avoiding reliance on structural subclassification and minimizing the risk of false benign interpretation. Reduced growth, by contrast, provides strong evidence of loss of ASS1 function and pathogenicity, validated under the OddsPath framework.

    We have updated the Results and Discussion sections to clarify these points (also see response to the reviewer's point 1).

    "A subset of clinically pathogenic ASS1 variants exhibit near-wild-type growth in our yeast assay. In general, we expect a monotonic relationship between ASS function and yeast growth, but with the potential for floor and ceiling effects that constrain the assay's dynamic range. In this context, we interpret high-growth pathogenic variants as likely causing mild loss of function that cannot be distinguished from wild type in our assay. Consistent with this view, many pathogenic variants with high assay growth are located at the monomer-monomer interface rather than the active site, and are associated with milder or later-onset clinical presentations, suggesting partial enzymatic impairment that is clinically relevant in humans but not resolved by the yeast assay."

    "Based on these findings and given that 22/56 pathogenic variants show >85% growth, we conclude that growth above this threshold should not be used as evidence toward benignity. Notably, this approach is conservative relative to treating high-growth variants as benign except at the monomer-monomer interface, avoiding reliance on structural subclassification and minimizing the risk of false benign interpretation arising from assay ceiling effects. Conversely, the variants with

    Reviewer #2 (Significance (Required)):

    This study presents the first comprehensive mutational profiling of human ASS1 and would be of broad interest to clinical geneticists as well as those seeking biochemical insights into the enzymology of ASS1. The authors' use of a yeast system to profile human mutations would be particularly useful for researchers performing deep mutational scans, given that it provides functional insights in a rapid and inexpensive manner.

    __Reviewer #3 __(Evidence, reproducibility and clarity (Required)):

    Section 1 - Evidence, reproducibility, and clarity Summary This manuscript presents a comprehensive functional profiling of 2,193 ASS1 missense variants using a yeast complementation assay, providing valuable data for variant interpretation in the rare disease citrullinemia type I. The dataset is extensive, technically sound, and clinically relevant. The demonstration of intragenic complementation in ASS1 is novel and conceptually important. Overall, the study represents a substantial contribution to functional genomics and rare disease variant interpretation.

    Major comments

    1. This is an exciting paper as it can provide support to clinicians to make actionable decisions when diagnosing infants. I have a few major comments, but I want to emphasize the label of "functionally unimpaired" variants to be misleading. The authors explain that there are several pathogenic ClinVar variants that fall into this category (above the >.85 growth threshold) but I think this category needs a more specific name and I would ask the authors to reiterate the shortcomings of the assay again in the Discussion section.

    We thank the reviewer for raising this important point. We agree that the label "functionally unimpaired" could be misleading if interpreted as implying clinical benignity rather than assay behavior. We have therefore clarified that this designation refers strictly to variant behavior in the yeast growth assay and does not imply absence of pathogenicity.

    In addition, we have expanded the Discussion to explicitly address the existence of clinically pathogenic variants with high growth scores (>0.85), emphasizing that these likely reflect a ceiling effect of the assay and represent a key limitation for interpretation. This clarification reiterates that high-growth scores should not be used as evidence toward benignity, while reduced growth provides strong functional evidence of pathogenicity. Relevant revisions are described in our responses to Reviewers 1 and 2.

    1. I think there's an important discussion to be had here, is the assay detecting variants that alter the function of ASS or is it detecting a complete ablation of enzymatic activity? The results might be strengthened with a follow-up experiment that identifies stably expressed ASS1 variants.

    We agree with the review that distinguishing between stability and enzyme activity would be valuable information. Unfortunately, we do not currently have the resources to perform this type of large-scale study. We have acknowledged in the text that our assay does not distinguish between enzyme activity and protein stability:

    "We expect that the assay will detect reductions in both catalytic activity and protein stability, but will not distinguish between these mechanisms."

    At the very least, it would be great to see the authors replicate some of their interesting results from the high-throughput screen by down-selecting to ~12 variants of uncertain significance that could be newly considered pathogenic.

    We have included new analysis of all 25 VUS variants falling in the pathogenic range of our assay (Supplemental Table S7). Reclassification under current guidelines (in the absence of our data) shifts six variants to Pathogenic/Likely Pathogenic and 11 more are reclassified to Likely Pathogenic with the application of our functional data as PS3_Supporting. The remaining eight VUS are all reclassified to Likely Pathogenic when inclusion of homozygous PrimateAI-benign variants allows the assay to satisfy full PS3 criteria.

    1. I would ask the authors to provide more citations of the literature in the introduction of the manuscript. I would be especially interested in knowing more about human ASS being identified as a homolog of yeast ARG1, as they share little sequence similarity (27.5%) at the protein level. That said, I find the yeast complementation assay exciting.

    We thank the reviewer for this suggestion. Human ASS and yeast Arg1 catalyze the same biochemical reaction and share approximately 49% amino acid sequence identity. We have revised the Introduction to clarify this relationship and to note explicitly that the Saccharomyces Genome Database (SGD) identifies the human gene encoding argininosuccinate synthase (ASS1) as the ortholog of yeast ARG1. An appropriate citation has been added to support this statement. The protein alignments have been provided as File S2.

    "This assay is based on the ability of human ASS to functionally replace (complement) its yeast ortholog (Arg1) in S. cerevisiae (Saccharomyces Genome Database, 2026). Importantly, successful functional complementation indicates that ASS enzymatic activity does not depend on any obligate human-specific protein interactions. At the protein level, human ASS and yeast Arg1 display 49% sequence identity (File S2) and share identical enzymatic roles in converting citrulline and aspartate into argininisuccinate."

    1. I appreciate the efforts made by the authors to share their work and make this study more reproducible, such as sharing the hASS1 and yASS1 plasmids being shared on NCBI Genbank (Line 121) and publishing the ONT reads on SRA (Line 154). I made a requests for additional data to be shared, such as the custom method/code for codon optimization and a table of Twist variant cassettes that were ordered. I would also love to see these results shared on MaveDB.org.

    We thank the reviewer for these suggestions regarding data sharing and reproducibility. As requested, we have provided the custom codon optimization script as File S1 and the amino acid alignment used to perform codon harmonization as File S2. The sequence of the underlying variant cassette is included in the corresponding GenBank entry, and we have clarified this point in the legend of Figure 1. For each amino acid substitution, Twist Bioscience used a yeast-specific codon scheme with a single consistent codon per amino acid; accordingly, the sequence of each variant cassette can be inferred from the base construct and the specified amino acid change. A complete list of variant amino acid substitutions used in this study is provided in Table S3.

    1. I find this manuscript very exciting as the authors have a compelling assay that identifies pathogenic variants, but I was generally disappointed by the quality and organization of the figures. For example, Figure 4 provides very little insight, but could be dramatically improved with an overlay of the normalized growth score data or highlighting variants surrounding the substrate or ATP interfaces. There are some very interesting aspects of this manuscript that could be shine through with some polished figures.

    We thank the reviewer for this feedback and agree that clear and well-organized figures are essential for conveying the key results of the study. In response, we have substantially revised Figure 4 by adding colored overlays showing residue conservation and median normalized growth scores (new panels Figure 4C and 4D), which more directly link structural context to functional outcomes and highlight patterns surrounding the active site and substrate interfaces.

    I would also encourage the authors to generate a heatmap of the data represented in Figure 2 (see Fowler and Fields 2014 PMID 25075907, Figure 2), this would be more helpful reference to the readers.

    The reviewer also suggested that a heatmap representation, similar to that used in Fowler and Fields (2014), might aid interpretation of the data shown in Figure 2. Because our dataset consists of sparse single-amino acid substitutions rather than a complete mutational scan, such heatmaps are inherently less dense and less effective at conveying patterns than in saturation mutagenesis studies. Nevertheless, to aid readers who may find this visualization useful, we have generated and included a single-nucleotide variant heatmap as Supplemental Figure S1.

    My major comments are as follows:

    1. Citations needed - especially in the introduction and for establishing that hASS is a homolog of yARG1

    We have added the requested citations and clarified the ASS1-ARG1 orthology in the Introduction, as described in our response to point 3 above.

    1. Generally, the authors do a nice job distinguishing the ASS1 gene from the ASS enzyme, though I found some ambiguities (Line 685). Please double-check the use of each throughout the manuscript.

    We have edited the manuscript to ensure consistent and unambiguous use of gene and enzyme nomenclature throughout.

    1. Generally, I'm confused about what strain was used for integrating all these variants, was is the arg1 knock-out strain from the yeast knockout collection or was it FY4? I think FY4 was used for the preliminary experiments, then the KO collection strain was used for making the variant library but I think this could be made more clear in the text and figures. Lines 226-229 describes introducing the hASS1 and yASS1 sequences into the native ARG1 locus in strain FY4, but the Fig1A image depicts the ASS1 variants going into arg1 KO locus. Fig1A should be moved to Fig2.

    We agree that the strain construction steps were not described as clearly as they could have been. We have therefore clarified the strain construction workflow in the Materials & Methods and Results sections, as well as in the Figure 1 legend, to explicitly distinguish preliminary experiments performed in strain FY4 from construction of the variant library in the arg1 knockout background.

    As we have also added an additional panel to Figure 1 that schematically explains how the screen was performed (per Reviewer #2's request), we believe that Figure 1A is appropriately placed and should remain in Figure 1.

    1. Line 303 - "We classify these variants as 'functionally unimpaired'", this is not an accurate description of these variants as Figure 2 highlights 24 pathogenic ClinVar variants that would fall into this category of "functionally unimpaired". The yeast growth assay appears to capture pathogenic variants, but there is likely some nuance of human ASS functionality that is not being assessed here. I would make the language more specific, e.g. "complementary to Arg1" or "growth-compatible".

    We agree that the label "functionally unimpaired" could be misinterpreted if read as implying clinical benignity. We have therefore clarified within the manuscript that this designation refers strictly to variant behavior in the yeast growth assay (i.e., wild-type-like growth under assay conditions) and does not imply absence of pathogenicity. We also expanded the Discussion to explicitly address the subset of clinically pathogenic variants with high growth scores (>0.85), consistent with a ceiling effect of the assay and a key limitation for interpretation. See response to reviewer #3 point 1. Relevant revisions are also discussed in our responses to Reviewers #1 and #2.

    1. Lines 345-355 - It is interesting that there are variants that appear functional at the substrate interfacing sites. Is there anything common across these variants? Are they maintaining the polarity or hydrophobicity of the WT residue? Are any of these variants included in ClinVar or gnomAD? Are pathogenic variants found at any of these sites

    Yes. For highly sensitive active-site residues that have few permissible variants, the vast majority of amino acid substitutions that do retain activity preserve key physicochemical properties of the wild-type residue, such as hydrophobicity or charge. We have added this important observation to the manuscript:

    "Any variants at these sensitive residues that are permissive for activity in our assay retain hydrophobicity or charged states relative to the original amino acid side chain (Figure 5A & Table S5)."

    None of these variants are present in ClinVar. Only L15V and E191D are present in gnomAD (Table S4).

    1. Lines 423-430 - The OddsPath calculation would seem to rely heavily on the thresholds of .85 for normalized growth. The OddsPath calculation could be bolstered with some additional analysis that emphasizes the robustness to alternative thresholds.

    We agree that the sensitivity of the OddsPath calculation to the choice of growth thresholds is an important consideration. In our assay, benign ClinVar variants and non-human primate variants are observed exclusively within the peak centered on wild-type growth, whereas clinically annotated variants falling below this peak are exclusively pathogenic. On this basis, we defined the upper boundary of the assay range interpreted as supporting pathogenicity as the lower boundary of the wild-type-centered peak in the growth distribution (as defined in Figure 3), rather than selecting a cutoff by direct optimization of the OddsPath. This choice reflects the observed concordance, in our dataset, between the onset of measurable functional impairment in the assay and clinical pathogenic annotation. Importantly, in practice the OddsPath value is locally robust to the precise placement of this boundary, remaining invariant across the range 0.82-0.88. Supporting our chosen threshold of 0.85, the lowest-growth benign or primate variant observed has a normalized growth value of 0.88, while the lowest growth observed among variants present as homozygotes in gnomAD was 0.86. We have clarified this rationale and analysis in the revised manuscript.

    "Notably, the "Among all nine of the human ASS1 missense variants observed as homozygotes in gnomAD which were tested as amino acid substitutions in our assay, the lowest observed growth value was 0.86 (Ala258Val) consistent with the lower boundary of the PrimateAI variants which was a growth value of 0.87 (Ala81Thr) (Figure 6) and with our use of a 0.85 classification threshold."

    "If we treat PrimateAI variants as benign (solely for OddsPath calculation purposes), the OddsPath for growth

    1. Lines 432-441 - This is an interesting idea to use variants observed in primates, has ACMG weighed in on this? I understand that CTLN1 is an autosomal recessive disorder but I'd still be interested in seeing how the observed ASS1 missense variants in gnomAD perform in your growth assay, possibly a supplemental figure?

    To our knowledge, the ACMG/AMP guidelines do not currently address the use of homozygous missense variants observed in non-human primates. We are currently in discussion with two ClinGen working groups to discuss the possibility of formalizing the use of this data source.

    We agree that comparison with human population data is also important. Accordingly, total gnomAD allele counts and homozygous counts for all applicable ASS1 missense variants are provided in Table S4, and the growth behavior of ASS1 missense variants observed in the homozygous state in gnomAD is shown in Figure 6. These homozygous variants uniformly exhibit high growth in our assay, consistent with the absence of strong loss-of-function effects. We have updated the manuscript text to clarify these points.

    Minor comments

    1. Lines 53-59 - This paragraph needs to cite the literature, especially lines 56, 57, and 59
    2. Line 61 - no need to repeat "citrullinemia type I", just use the abbreviation as it was introduced in the paragraph above
    3. Lines 61-71 - again, this paragraph needs more literature citations
    4. Line 62 - change to "results"

    The changes suggested in points 1-4 have all been implemented in the revised manuscript.

    1. Line 74-75 - "RUSP" acronym not needed as it's never used in the manuscript, the same goes for "HHS"

    We agree that the acronyms "RUSP" and "HHS" are not reused elsewhere in the manuscript. We have nevertheless retained them at first mention, alongside the expanded names, because these acronyms are commonly used in newborn screening and public health policy contexts and may be more familiar to some readers than the expanded terms. We would be happy to remove the acronyms if preferred.

    1. Line 86 - "ASS1" I think is referring to the enzyme and should just be "ASS"? If referring to the gene then italicize to "ASS1"
    2. Lines 91-93 - It would be helpful to mention this is a functional screen in yeast
    3. Line 101 - It would be helpful to the readers to define SD before using the acronym, consider changing to "minimal synthetic defined (SD) medium" and afterwards can refer to as "SD medium"
    4. 109-114 - It would be great if you could share your method for designing the codon-harmonized yASS1 gene, consider sharing as a supplemental script or creating a GitHub repository linked to a Zenodo DOI for publication.

    The changes suggested in points 6-9 have all been implemented in the revised manuscript. The codon harmonization script has been provided as File S1.

    1. Lines 135-137 - I think it's helpful to provide a full table of the cassettes ordered from Twist as well as the primers used to amplify them, consider a supplemental table.

    Details of Twist cassette and the primer sequences used for amplification have been added to the Materials & Methods.

    1. Line 138 - "standard methods" is a bit vague, I'm guessing this is a Geitz and Schiestl 2007 LiAc/ssDNA protocol (PMID 17401334)? Also, was ClonNAT used to select for natMX colonies?

    The reviewer is correct about which protocol was used, and we have added the citation. We have also clarified that selection was carried out based on resistance to nourseothricin.

    1. Line 150 - change to "sequence the entire open reading frame, as previously described [4]."
    2. Line 222-223 - remove "replace" and just use "complement" (and remove the parenthesis)
    3. Line 249 - It would be great to see a supplemental alignment of the hASS1 and yASS1 sequences.
    4. Line 261 - spelling "citrullemia" should be corrected to "citrullinemia"
    5. Line 280 - "using Oxford Nanopore sequencing" is a bit vague, I suggest specifying the equipment used (e.g. Oxford Nanopore Technologies MinION platform) or simplify to "via long-read sequencing (see Materials & Methods)"

    The changes suggested in points 12-16 have all been implemented in the revised manuscript. An alignment of the ASS and Arg1 protein sequences has been provided as File S2.

    1. Line 287-289 - It would be great to see the average number of isolates per variant, as well as a plot of the variant growth estimate vs individual isolate growth.

    We agree with the reviewer that conveying measurement precision is important. The number of isolates assayed per variant is provided in Table S4, and we have added explicit mention of this in the text. Because variants were assayed with a mixture of 1, 2, or {greater than or equal to}3 independent isolates, a scatterplot of variant-level growth estimates versus individual isolate measurements would be difficult to interpret and potentially misleading. Instead, we report standard error estimates for each variant in Table S4, derived from the linear model used to estimate growth effects, which more appropriately summarizes measurement uncertainty given the experimental design.

    1. Lines 324-25 - consider removing the last sentence of this paragraph, it is redundant as the following paragraph starts with the same statement.

    We have removed this sentence.

    1. Lines 327-335 - This is interesting and would benefit from its own subpanel or plot in which the normalized growth score is plotted against variants that are at conserved or diverse residues in human ASS, and see if there's a statistical difference in score between the two groupings.

    As suggested by the reviewer, we have added Supplemental Figure 2 (Figure S2) in which the normalized growth score of each variant is plotted against the conservation of the corresponding residue, as measured by ConSurf. The manuscript already includes a statistical analysis of the relationship between residue conservation and functional impact, showing that amorphic variants occur significantly more frequently at highly conserved residues than unimpaired variants do (one-sided Fisher's exact test). We now refer to this new supplemental figure in the relevant Results section.

    1. Lines 339-341 - As written, it is unclear if aspartate interacts with all of the same residues as citrulline or just Asn123 and Thr119.
    2. Lines 345-355 - As with my above comment, I find this interesting and would
    3. Line 353 - add a period to "al" in "Diez-Fernandex et al."

    The issues raised in points 20 and 22 have all addressed. Point 21 appears to be truncated.

    1. Figure 1 a. Remove "Figure" from the subpanels and show just "A" and "B" (as you do for Figure 4) and combine the two images into a single image. Also make this correction to Figure 5 and Figure 8. b. Panel A - I thought the hASS1 and yASS1 were dropped into FY4, not the arg1 KO strain. This needs clarification. c. Panel A - I'm assuming the natMX cassette contains its own promoter, you could use a right-angled arrow to indicate where the promotors are in your construct. d. Panel B - I'm not sure the bar graph is necessary, it would be more helpful to see calculations of the colony size (or growth curves for each strain) and plot the raw values (maybe pixel counts?) for each replicate rather than normalizing to yeast ARG1. I would be great to have a supplemental figure showing all the replicates side-by-side. e. Panel B - Would be helpful to denote the pathogenic and benign ClinVar variants with an icon or colored text.

    f. Figure 1 Caption - make "A)" and "B)" bold.

    We have implemented the requested changes in Figure 1 with the following exceptions. We have retained panels A and B as separate subfigures because they illustrate distinct experimental concepts. In addition, we respectfully disagree with point (d). The bar graph is intended to provide a clear, high-level comparison of functional complementation by hASS1 versus yASS1 and to illustrate the gross differences in growth between benign and pathogenic proof-of-principle variants. As the bar graph includes error bars for standard deviations, presenting raw colony size measurements or growth curves for individual replicates would substantially complicate the figure without materially improving interpretability for this purpose.

    1. Figure 2 a. "Shown in magenta are amino acid substitutions corresponding to ClinVar pathogenic, pathogenic/likely pathogenic, and likely pathogenic variants" is repeated in the figure caption. b. "Shown in green are amino acid substitutions corresponding to ClinVar benign and likely benign variants." I don't see any green points. c. Identify the colors used for ASS1 substrate binding residues. d. This plot would benefit from a depiction of the human ASS secondary structure and any protein domains (nucleotide-binding domain, synthase domain, and C-terminal helix from Fig4B)

    e. Line 685 675 - "ASS1" is being used in reference to the enzyme, is this correct or should it be "ASS"?

    We have made the requested changes to Figure 2. The repeated caption text has been removed, and references to green points have been corrected to orange points to match the figure. The colors used to indicate ASS substrate-binding residues are explicitly described in the figure key. Secondary structure annotations have been added. References to the enzyme have been corrected to "ASS" rather than "ASS1" where appropriate.

    1. Figure 3 a. Rename the "unimpaired" category as there are several pathogenic ClinVar variants that fall into this category.

    To address this point, we have clarified the labeling by adding "in our yeast assay" to the figure legend, making explicit that the "unimpaired" category refers only to wild-type-like behavior under assay conditions and does not imply clinical benignity. See also response to Reviewer #3, Major Comment 1.

    1. Figure 4 a. List the PDB or AlphaFold accession used for this structure b. Panel A - state which colors are used for to depict each monomer. It is confusing to see several shades of pink/purple used to depict a single monomer in Panel A. c. It is very difficult to make out the aspartate and citrulline substrates in the catalytic binding activity, consider making an inset zooming-in on this domain and displaying a ribbon diagram of the structure rather than the surface. d. Generally, it would be more helpful here to label any particular residues that were identified as pathogenic from your screen, or to overlay average grow scores per residue data onto the structure

    We have implemented the requested changes to Figure 4. The relevant PDB/AlphaFold accession is now listed, and the colors used to depict each monomer in Panel A are clarified in the figure legend. An inset focusing on the active site has been added to improve visualization of the citrulline and aspartate substrates. In addition, we have added new panels (Figure 4C and 4D) overlaying pathogenic residues and average growth scores onto the structure to more directly link structural context with functional data.

    1. Figure 5 a. Line 716 - Insert a page break to place Figure 5 on its own page b. I suggest using a heatmap for this type of plot, as it is very difficult to track which color corresponds to which residue.

    c. Fig5A - This plot could be improved by identifying which residue positions interface with which substrate.

    We have placed Figure 5 on its own page and added information to the legend identifying which residue positions interface with each substrate. We have retained the active-site variant strip charts raised in point (b), as we believe they effectively illustrate how the distribution of variant effects differs between residues. In addition, we have provided a supplemental heatmap showing variant growth across the entire protein (Figure S1), and individual variant scores for all residues are provided in Table S4.

    1. Figure 7 a. Line 735 - Insert page break to place figure on a new page

    List the PDB accession used for these images. c. For clarity I would mention "human ASS" in the figure title d. State the colors of the substrates e. Panels A and B could be combined into a single panel, making it easier to distinguish the active site and dimerization variants.

    f. Could be interesting to get SASA scores for the ClinVar structural variants to determine if they are surface-accessible

    We have implemented the requested changes in Figure 7 with the following exceptions. For point (e), there is no single orientation of the structure that allows a clear simultaneous view of both active-site and dimerization variants; accordingly, we have retained panels A and B as separate subfigures to preserve clarity. With respect to point (f), we agree that solvent accessibility analysis could be informative in other contexts. However, such an analysis does not integrate naturally with the functional and assay-based framework of the present study and was therefore not included.

    1. Figure 8 a. Panel B - overlay a square frame in the larger protein structure that depicts where the below inset is focused, and frame inset image as well.

    We have framed the inset image as requested. We did not add a corresponding frame to the full protein structure, as doing so obscured structural details in the region of interest.

    Reviewer #3 (Significance (Required)):

    Section 2 - Significance This study represents a substantial technical, functional, and translational advance in the interpretation of missense variation in ASS1, a gene of high clinical relevance for the rare disease citrullinemia type I. Its principal strength lies in the generation of an experimentally validated functional atlas of ASS1 missense variants that covers ~90% of all SNV-accessible substitutions. The scale, internal reproducibility, and careful benchmarking of the yeast complementation assay against known pathogenic and benign variants provide a robust foundation for identifying pathogenic ASS1 variants. Particularly strong aspects include the rigorous quality control of variant identities, the quantitative nature of the functional readout, and the thoughtful integration of results into the ACMG/AMP OddsPath framework. The discovery of intragenic complementation between variants affecting distinct structural regions of the enzyme is a notable conceptual and mechanistic contribution. Limitations include the assay's reduced sensitivity to variants impacting oligomerization or subtle folding defects, and the use of yeast as a heterologous system, which may mask disease-relevant mechanisms as several pathogenic ClinVar variants were found to be "functionally unimpaired". Future work extending functional testing to additional cellular contexts or expanding genotype-level combinatorial analyses would further enhance clinical applicability. Relative to prior studies, which have relied on small numbers of patient-derived variants or low-throughput biochemical assays, this work extends the field decisively by delivering a comprehensive, variant-resolved functional map for ASS1. To the best of my current knowledge, this is the first systematic functional screen of ASS1 at this scale and the first direct experimental demonstration that ASS active sites span multiple subunits, enabling intragenic complementation consistent with Crick and Orgel's classic variant sequestration model. As such, the advance is simultaneously technical (high-throughput functional genomics), mechanistic (defining structural contributors to catalysis and epistasis), and clinical (enabling evidence-based reclassification of VUS). I find the use of homozygous non-human primate variants as an orthogonal benign calibration set both creative and controversial, my hope would be that this manuscript will prompt a productive discussion.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    This manuscript presents a comprehensive functional profiling of 2,193 ASS1 missense variants using a yeast complementation assay, providing valuable data for variant interpretation in the rare disease citrullinemia type I. The dataset is extensive, technically sound, and clinically relevant. The demonstration of intragenic complementation in ASS1 is novel and conceptually important. Overall, the study represents a substantial contribution to functional genomics and rare disease variant interpretation.

    Major comments

    This is an exciting paper as it can provide support to clinicians to make actionable decisions when diagnosing infants. I have a few major comments, but I want to emphasize the label of "functionally unimpaired" variants to be misleading. The authors explain that there are several pathogenic ClinVar variants that fall into this category (above the >.85 growth threshold) but I think this category needs a more specific name and I would ask the authors to reiterate the shortcomings of the assay again in the Discussion section. I think there's an important discussion to be had here, is the assay detecting variants that alter the function of ASS or is it detecting a complete ablation of enzymatic activity? The results might be strengthened with a follow-up experiment that identifies stably expressed ASS1 variants. At the very least, it would be great to see the authors replicate some of their interesting results from the high-throughput screen by down-selecting to ~12 variants of uncertain significance that could be newly considered pathogenic. I would ask the authors to provide more citations of the literature in the introduction of the manuscript. I would be especially interested in knowing more about human ASS being identified as a homolog of yeast ARG1, as they share little sequence similarity (27.5%) at the protein level. That said, I find the yeast complementation assay exciting. I appreciate the efforts made by the authors to share their work and make this study more reproducible, such as sharing the hASS1 and yASS1 plasmids being shared on NCBI Genbank (Line 121) and publishing the ONT reads on SRA (Line 154). I made a requests for additional data to be shared, such as the custom method/code for codon optimization and a table of Twist variant cassettes that were ordered. I would also love to see these results shared on MaveDB.org. I find this manuscript very exciting as the authors have a compelling assay that identifies pathogenic variants, but I was generally disappointed by the quality and organization of the figures. For example, Figure 4 provides very little insight, but could be dramatically improved with an overlay of the normalized growth score data or highlighting variants surrounding the substrate or ATP interfaces. There are some very interesting aspects of this manuscript that could be shine through with some polished figures. I would also encourage the authors to generate a heatmap of the data represented in Figure 2 (see Fowler and Fields 2014 PMID 25075907, Figure 2), this would be more helpful reference to the readers.

    My major comments are as follows:

    1. Citations needed - especially in the introduction and for establishing that hASS is a homolog of yARG1
    2. Generally, the authors do a nice job distinguishing the ASS1 gene from the ASS enzyme, though I found some ambiguities (Line 685). Please double-check the use of each throughout the manuscript
    3. Generally, I'm confused about what strain was used for integrating all these variants, was is the arg1 knock-out strain from the yeast knockout collection or was it FY4? I think FY4 was used for the preliminary experiments, then the KO collection strain was used for making the variant library but I think this could be made more clear in the text and figures. Lines 226-229 describes introducing the hASS1 and yASS1 sequences into the native ARG1 locus in strain FY4, but the Fig1A image depicts the ASS1 variants going into arg1 KO locus. Fig1A should be moved to Fig2.
    4. Line 303 - "We classify these variants as 'functionally unimpaired'", this is not an accurate description of these variants as Figure 2 highlights 24 pathogenic ClinVar variants that would fall into this category of "functionally unimpaired". The yeast growth assay appears to capture pathogenic variants, but there is likely some nuance of human ASS functionality that is not being assessed here. I would make the language more specific, e.g. "complementary to Arg1" or "growth-compatible".
    5. Lines 345-355 - It is interesting that there are variants that appear functional at the substrate interfacing sites. Is there anything common across these variants? Are they maintaining the polarity or hydrophobicity of the WT residue? Are any of these variants included in ClinVar or gnomAD? Are pathogenic variants found at any of these sites
    6. Lines 423-430 - The OddsPath calculation would seem to rely heavily on the thresholds of <.05 and >.85 for normalized growth. The OddsPath calculation could be bolstered with some additional analysis that emphasizes the robustness to alternative thresholds.
    7. Lines 432-441 - This is an interesting idea to use variants observed in primates, has ACMG weighed in on this? I understand that CTLN1 is an autosomal recessive disorder but I'd still be interested in seeing how the observed ASS1 missense variants in gnomAD perform in your growth assay, possibly a supplemental figure?

    Minor comments

    1. Lines 53-59 - This paragraph needs to cite the literature, especially lines 56, 57, and 59
    2. Line 61 - no need to repeat "citrullinemia type I", just use the abbreviation as it was introduced in the paragraph above
    3. Lines 61-71 - again, this paragraph needs more literature citations
    4. Line 62 - change to "results"
    5. Line 74-75 - "RUSP" acronym not needed as it's never used in the manuscript, the same goes for "HHS"
    6. Line 86 - "ASS1" I think is referring to the enzyme and should just be "ASS"? If referring to the gene then italicize to "ASS1"
    7. Lines 91-93 - It would be helpful to mention this is a functional screen in yeast
    8. Line 101 - It would be helpful to the readers to define SD before using the acronym, consider changing to "minimal synthetic defined (SD) medium" and afterwards can refer to as "SD medium"
    9. 109-114 - It would be great if you could share your method for designing the codon-harmonized yASS1 gene, consider sharing as a supplemental script or creating a GitHub repository linked to a Zenodo DOI for publication.
    10. Lines 135-137 - I think it's helpful to provide a full table of the cassettes ordered from Twist as well as the primers used to amplify them, consider a supplemental table
    11. Line 138 - "standard methods" is a bit vague, I'm guessing this is a Geitz and Schiestl 2007 LiAc/ssDNA protocol (PMID 17401334)? Also, was ClonNAT used to select for natMX colonies?
    12. Line 150 - change to "sequence the entire open reading frame, as previously described [4]."
    13. Line 222-223 - remove "replace" and just use "complement" (and remove the parenthesis)
    14. Line 249 - It would be great to see a supplemental alignment of the hASS1 and yASS1 sequences
    15. Line 261 - spelling "citrullemia" should be corrected to "citrullinemia"
    16. Line 280 - "using Oxford Nanopore sequencing" is a bit vague, I suggest specifying the equipment used (e.g. Oxford Nanopore Technologies MinION platform) or simplify to "via long-read sequencing (see Materials & Methods)"
    17. Line 287-289 - It would be great to see the average number of isolates per variant, as well as a plot of the variant growth estimate vs individual isolate growth
    18. Lines 324-25 - consider removing the last sentence of this paragraph, it is redundant as the following paragraph starts with the same statement
    19. Lines 327-335 - This is interesting and would benefit from its own subpanel or plot in which the normalized growth score is plotted against variants that are at conserved or diverse residues in human ASS, and see if there's a statistical difference in score between the two groupings
    20. Lines 339-341 - As written, it is unclear if aspartate interacts with all of the same residues as citrulline or just Asn123 and Thr119.
    21. Lines 345-355 - As with my above comment, I find this interesting and would
    22. Line 353 - add a period to "al" in "Diez-Fernandex et al."
    23. Figure 1

    a. Remove "Figure" from the subpanels and show just "A" and "B" (as you do for Figure 4) and combine the two images into a single image. Also make this correction to Figure 5 and Figure 8

    b. Panel A - I thought the hASS1 and yASS1 were dropped into FY4, not the arg1 KO strain. This needs clarification

    c. Panel A - I'm assuming the natMX cassette contains its own promoter, you could use a right-angled arrow to indicate where the promotors are in your construct

    d. Panel B - I'm not sure the bar graph is necessary, it would be more helpful to see calculations of the colony size (or growth curves for each strain) and plot the raw values (maybe pixel counts?) for each replicate rather than normalizing to yeast ARG1. I would be great to have a supplemental figure showing all the replicates side-by-side

    e. Panel B - Would be helpful to denote the pathogenic and benign ClinVar variants with an icon or colored text

    f. Figure 1 Caption - make "A)" and "B)" bold

    1. Figure 2

    a. "Shown in magenta are amino acid substitutions corresponding to ClinVar pathogenic, pathogenic/likely pathogenic, and likely pathogenic variants" is repeated in the figure caption

    b. "Shown in green are amino acid substitutions corresponding to ClinVar benign and likely benign variants." I don't see any green points

    c. Identify the colors used for ASS1 substrate binding residues

    d. This plot would benefit from a depiction of the human ASS secondary structure and any protein domains (nucleotide-binding domain, synthase domain, and C-terminal helix from Fig4B)

    e. Line 685 - "ASS1" is being used in reference to the enzyme, is this correct or should it be "ASS"?

    1. Figure 3

    a. Rename the "unimpaired" category as there are several pathogenic ClinVar variants that fall into this category

    1. Figure 4

    a. List the PDB or AlphaFold accession used for this structure

    b. Panel A - state which colors are used for to depict each monomer. It is confusing to see several shades of pink/purple used to depict a single monomer in Panel A

    c. It is very difficult to make out the aspartate and citrulline substrates in the catalytic binding activity, consider making an inset zooming-in on this domain and displaying a ribbon diagram of the structure rather than the surface.

    d. Generally, it would be more helpful here to label any particular residues that were identified as pathogenic from your screen, or to overlay average grow scores per residue data onto the structure

    1. Figure 5

    a. Line 716 - Insert a page break to place Figure 5 on its own page

    b. I suggest using a heatmap for this type of plot, as it is very difficult to track which color corresponds to which residue

    c. Fig5A - This plot could be improved by identifying which residue positions interface with which substrate

    1. Figure 7

    a. Line 735 - Insert page break to place figure on a new page

    b. List the PDB accession used for these images

    c. For clarity I would mention "human ASS" in the figure title

    d. State the colors of the substrates

    e. Panels A and B could be combined into a single panel, making it easier to distinguish the active site and dimerization variants

    f. Could be interesting to get SASA scores for the ClinVar structural variants to determine if they are surface-accessible

    1. Figure 8

    a. Panel B - overlay a square frame in the larger protein structure that depicts where the below inset is focused, and frame inset image as well.

    Significance

    This study represents a substantial technical, functional, and translational advance in the interpretation of missense variation in ASS1, a gene of high clinical relevance for the rare disease citrullinemia type I. Its principal strength lies in the generation of an experimentally validated functional atlas of ASS1 missense variants that covers ~90% of all SNV-accessible substitutions. The scale, internal reproducibility, and careful benchmarking of the yeast complementation assay against known pathogenic and benign variants provide a robust foundation for identifying pathogenic ASS1 variants. Particularly strong aspects include the rigorous quality control of variant identities, the quantitative nature of the functional readout, and the thoughtful integration of results into the ACMG/AMP OddsPath framework. The discovery of intragenic complementation between variants affecting distinct structural regions of the enzyme is a notable conceptual and mechanistic contribution. Limitations include the assay's reduced sensitivity to variants impacting oligomerization or subtle folding defects, and the use of yeast as a heterologous system, which may mask disease-relevant mechanisms as several pathogenic ClinVar variants were found to be "functionally unimpaired". Future work extending functional testing to additional cellular contexts or expanding genotype-level combinatorial analyses would further enhance clinical applicability.

    Relative to prior studies, which have relied on small numbers of patient-derived variants or low-throughput biochemical assays, this work extends the field decisively by delivering a comprehensive, variant-resolved functional map for ASS1. To the best of my current knowledge, this is the first systematic functional screen of ASS1 at this scale and the first direct experimental demonstration that ASS active sites span multiple subunits, enabling intragenic complementation consistent with Crick and Orgel's classic variant sequestration model. As such, the advance is simultaneously technical (high-throughput functional genomics), mechanistic (defining structural contributors to catalysis and epistasis), and clinical (enabling evidence-based reclassification of VUS). I find the use of homozygous non-human primate variants as an orthogonal benign calibration set both creative and controversial, my hope would be that this manuscript will prompt a productive discussion.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Lo et al characterize the phenotypic effect of ~90% of all possible ASS1 missense mutations using an elegant yeast-based system, and use this dataset to aid the interpretation of clinical ASS1 variants. Overall, the manuscript is well-written and the experimental data are interpretated rigorously. Of particular interest is the identification of pairs of deleterious alleles that rescue ASS1 activity in trans. My comments mainly pertain to the relevance of using a yeast screening methodology to infer functional effects of human ASS1 mutations.

    • Since human ASS1 is heterologously expressed in yeast for this mutational screen, direct comparison of native expression levels between human cells and yeast is not possible. Could the expression level of human ASS1 (driven by the pARG1 promoter) in yeast alter the measured fitness defect of each variant? For instance, if ASS1 expression in yeast is sufficiently high to mask modest reductions in catalytic activity, such variants may be misclassified as hypomorphic rather than amorphic. Conversely, if expression is intrinsically low, even mild catalytic impairments could appear deleterious. While it is helpful that the authors used non-human primate SNV data to calibrate their assay, experiments could be performed to directly address this possibility.
    • The nature of the relationship between yeast growth and availability of functional ASS1 could also influence the interpretation of results from the yeast-based screen. Does yeast growth scale proportionately with ASS1 enzymatic activity?
    • It would be helpful to add an additional diagram to Figure 1A explaining how the screen was performed, in particular: when genotype and phenotype were measured, relative to plating on selective vs non-selective media? This is described in "Variant library sequence confirmation" and "Measuring the growth of individual isolates" of the Methods section but could also be distilled into a diagram.
    • The authors rationalize the biochemical consequences of ASS1 mutations in the context of ASS1 per se - for example, mutations in the active site pocket impair substrate binding and therefore catalytic activity, which is expected. Does ASS1 physically interact with other proteins in human cells, and could these interactions be altered in the presence of specific ASS1 mutations? Such effects may not be captured by performing mutational scanning in yeast.
    • The authors note that only a small number (2/11) of mutations at the ASS1 monomer-monomer interface lead to growth defects in yeast. It would be helpful for the authors to discuss this further.

    Significance

    This study presents the first comprehensive mutational profiling of human ASS1 and would be of broad interest to clinical geneticists as well as those seeking biochemical insights into the enzymology of ASS1. The authors' use of a yeast system to profile human mutations would be particularly useful for researchers performing deep mutational scans, given that it provides functional insights in a rapid and inexpensive manner.

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    Referee #1

    Evidence, reproducibility and clarity

    Lo et al., report a high-throughput functional profiling study on the gene encoding for argininosuccinate synthase (ASS1), done in a yeast experimental system. The study design is robust (see lines 141-143, main text, Methods), whereby "approximately three to four independent transformants of each variant would be isolated and assayed." (lines 140 - 141, main text, Methods). Such a manner of analysis will allow for uncertainty of the functional readout for the tested variants to be accounted for.

    This is an outstanding study providing insights on the functional landscape of ASS1. Functionally impaired ASS1 may cause citrullinemia type I, and disease severity varies according to the degree of enzyme impairment (line 30, main text; Abstract). Data from this study forms a valuable resource in allowing for functional interpretation of protein-altering ASS1 variants that could be newly identified from large-scale whole-genome sequencing efforts done in biobanks or national precision medicine programs. I have some suggestions for the Authors to consider:

    1. The specific function of ASS1 is to condense L-citrulline and L-aspartate to form argininosuccinate. Instead of measuring either depletion of substrate or formation of product, the Authors elected to study 'growth' of the yeast cells. This is a broader phenotype which could be determined by other factors outside of ASS1. Whereas i agree that the experiments were beautifully done, the selection of an indirect phenotype such as ability of the yeast cells to grow could be more vigorously discussed.
    2. One of the key reasons why studies such as this one are valuable is due to the limitations of current variant classification methods that rely on 'conservation' status of amino acid residues to predict which variants might be 'pathogenic' and which variants might be 'likely benign'. However, there are serious limitations, and Figures 2 and 6 in the main text shows this clearly. Specifically, there is an appreciable number of variants that, despite being classified as "ClinVar Pathogenic", were shown by the assay to unlikely be functionally impaired. This should be discussed vigorously. Could these inconsistencies be potentially due to the read out (growth instead of a more direct evaluation of ASS1 function)?
    3. Figure 3 is very interesting, showing a continuum of functional readout ranging from 'wild-type' to 'null'. It is very interesting that the Authors used a threshold of less than 0.85 as functionally hypomorphic. What does this mean? It would be very nice if they have data from patients carrying two hypomorphic ASS1 alleles, and correlate their functional readout with severity of clinical presentation. The reader might be curious as to the clinical presentation of individuals carrying, for example, two ASS1 alleles with normalized growth of 0.7 to 0.8.

    I hope you will find these suggestions helpful.

    Significance

    This is an outstanding study providing insights on the functional landscape of ASS1. Functionally impaired ASS1 may cause citrullinemia type I, and disease severity varies according to the degree of enzyme impairment (line 30, main text; Abstract). Data from this study forms a valuable resource in allowing for functional interpretation of protein-altering ASS1 variants that could be newly identified from large-scale whole-genome sequencing efforts done in biobanks or national precision medicine programs.

  5. Version published to 10.1101/2025.09.17.676623 on bioRxiv