This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag051), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer 2：

The authors present METRIN-KG, a knowledge graph integrating plant metabolomic, trait, and biotic interaction datasets. The work should have substantial value for multiple plant sciences and ecology domains. The overall effort to harmonize disparate resources into an integrated, semantically coherent resource is impressive. The methodology includes a notable pipeline for ontology alignment across multiple sources. However, despite its technical strengths, several issues regarding data accessibility and manuscript structure and clarity should be addressed. The manuscript is highly technical throughout. While this level of precision is exemplary, it may alienate biologically oriented readers, and effort should be made so that the impact and manuscript is clear to a larger audience.

Major comments

1. The online deployment currently presents several problems that must be resolved before publication.

2. The existence of multiple SPARQL UIs (https://kg.earthmetabolome.org/metrin which redirects to https://qlever.earthmetabolome.org/metrin-kg/ and https://sib-swiss.github.io/sparql-editor/metrin-kg) is not explained, and the redundancy is potentially confusing.

3. At the time of review, https://qlever.earthmetabolome.org/metrin-kg/ showed expired SSL certificates, making it inaccessible for most users. Automatic certificate renewal (e.g., using certbot) should be implemented.

4. Attempting to use the SIB UI resulted in an error due to the redirect.

5. The certificate issue also prevented evaluation of ExpasyGPT querying against METRIN-KG.

6. Usability represents a significant barrier. Many potential users such as biologists without semantic-web or RDF experience are unlikely to be able to interpret the current figures or formulate SPARQL queries. The manuscript briefly mentions ExpasyGPT, which has strong potential to overcome this barrier by allowing natural language querying. This tool should be emphasized more prominently, potentially with a dedicated subsection and discussion of its role in broadening the resource's accessibility.

7. To fully demonstrate the relevance of METRIN-KG, the use-case section would benefit from quantitative summaries, visualizations (e.g., distributions, network visualizations), and biological interpretations of query results.

8. The overall manuscript organisation would benefit from restructuring to improve readability and better expose the impact of the work done.

9. The methodological content currently in "Mapping of TRY data" and "Mapping of GloBI data" should be moved to the Methods section, while the quantitative outputs (e.g., numbers of records) should be moved into a dedicated Results section.

10. The current "Data re-use and case studies" section could be reorganised into Results and Discussion sections,

11. Results include: description of outputs from the methodological steps (e.g. the ontology, the successfulness of the mapping process, the size of the final knowledge graph/number of triples, and other relevant metrics; the user interface, including being able to share and add example questions and write NL questions via ExpasyGPT; examples of SPARQL queries and case studies.

12. Discussion includes: Reuse potential; case-study interpretations or impact; future directions including planned expansion and enhancing of ontological structure, etc.

13. An overview and evaluation of the KG should be provided, for example the number of plant species, and the distributions of connections. Any gaps or any (potentially) biases due to the input data needs to be acknowledged. For example, it is very likely certain species may be under/overrepresented in either metabolome or interaction datasets, or possibly geographic skews could exist.

14. The ontology is variously referred to as the "Earth Metabolome Ontology", "EMI ontology", and "EMI". Consistent naming should be adopted throughout the manuscript and associated repositories. It is also unclear whether the ontology is a result of this work. As written, the "Ontology" section under "Methods" reads more like a result/description than a methodological step. Clarifying what components are original contributions and presenting them in Results would strengthen the manuscript. Additionally, the phrases "our proposed framework" and "our approach" are ambiguous, do these refer to the ontology itself, the metadata-mapping pipeline, or the overall integration process? Finally, referring to METRIN-KG as a "tutorial to build a knowledge graph" appears to be a bit out of place, given the topic of the manuscript.

15. Given its potential relevance beyond this project, the authors are strongly encouraged to publish the code for the metadata-mapping pipeline. In addition, the following details would strengthen the methodological rigor:

16. Were any acceptability criteria implemented in the automated step, e.g. a minimum Cosine similarity threshold?

17. Were the manual corrections systematically documented?

18. In how many cases were manual corrections needed?

19. Was any evaluation done on the embedding/model version or the source of errors?

Minor comments

1. The manuscript would benefit from proofreading for consistent use of Oxford commas (and an "&" instead of "and").

2. Reference 190 contains a typo "GiHub"

3. References should be checked (e.g. citation for [95] references both METRIN-KG Zenodo and GloBI Zenodo)

4. The METRIN-KG Zenodo link in the article is not to the latest version (Version 5)

5. Consider improving the figures, e.g. use of colour to better communicate the content and refining layouts.

6. The authors should deposit a snapshot of the GitHub repository to Zenodo.

The following are suggestions to the authors, to be followed by their own judgement:

1. Table 1, Figure 3, Figure 4 could be moved to supplementary material to make space for figures for the case studies.

2. The dense in-text list of ontologies in the "Metadata mapping" section could be replaced with a summarized table (e.g. by moving Supplementary Table 2, but including references to the main text).

3. The full SPARQL queries in "Taxonomy mapping" could be moved to supplementary materials, with a high-level description left in the main text.

This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag051), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer 1：

This study makes an important and timely contribution to plant ecology by combining multiple data sources of plant functional traits, metabolites, and interaction partners. Linking different aspects of a plants' phenotype (morphological and physiological traits, as well as metabolite profiles) with their potential ecological functions (biotic interactions) represents a much-needed step forward in both chemical and functional ecology. The effort the authors have put into compiling these datasets and establishing connections across sources is evident and deserves appreciation. The potential applications of this framework are manifold, and the examples provided on how the database can be used to explore research questions through knowledge paths convincingly demonstrate its value. The Introduction could be strengthened. At present, it feels somewhat long and diffuse in scope, which may make it harder for the reader to quickly grasp the importance of the contribution. Streamlining the text and sharpening the focus on the added value that linking different data sources provides would considerably improve clarity and help the reader more fully appreciate the strength of this novel approach. Minor comments: It would be helpful to specify more clearly what is meant by the "tremendous chemical diversity" that challenges metabolomic data analysis, and to clarify whether the "limited resources available to manage such complexity" refers to analytical limitations, data availability, or both. Similarly, the phrase "multi-level interaction data also lies locked in the guise of pairwise correlation metrics" could benefit from further explanation. As far as I am aware, the GLOBI database reports individual pairwise observations, which could already be seen as an effective way of documenting such interactions.

The Qid for descriptive standard / Erschließungsrichtlinie is (Q117460293), now with additional statements and linking items such as Encoded Archival Description (Q1340077), ISAD(G) (Q1654544), Rules for Archival Description (Q22908401) and Records in Contexts–Conceptual Model (Q30216914).

Where do you see this happening?

Why does that happen? Has this every happened to you?

普通人发布....

long-horizon environment learning

eLife Assessment

This is a valuable paper looking at nanoscale organization of the membrane associated periodic cytoskeleton in mouse sciatic nerve axons. Despite previous studies, the precise organisation of the structure remains unclear, especially in vivo, and this manuscript significantly adds to this knowledge with solid data. An unexpected observation is the presence of discrete nanoscale clusters, regularly distributed around sections of axons. However, the paper misses a description of these clusters along the longitudinal axis of the axons.

Reviewer #1 (Public review):

Summary:

The article "Nanoscale organization of beta-II spectrin within segments of the membrane-associated periodic skeleton in mouse sciatic nerve axons" by Gazal et al. looks into the organization of the spectrin scaffold in mouse sciatic nerves using super-resolution microscopy. It is now well established that axons, across species, contain a membrane-associated periodic scaffold mainly composed of circumferential actin filaments and longitudinally arranged spectrin tetramers. While super-resolution imaging of neurons in cell culture is relatively easy, exploring the ultrastructure of myelinated axons in intact nerve fibers is a daunting task. Nevertheless, the authors have attempted this by fixing and preparing cross-sections of sciatic nerves. They have then tried to quantify the fluorescence intensity patterns of specific components, especially that of labeled beta-II spectrin and have analysed its distribution.

One of the main findings is that spectrin is distributed along the axonal periphery and along the outer part of the myelin sheath. By labelling multiple cellular components and using intensity analysis, the authors show the sequence of structural organization of a few key components. They see that, unlike in the case of axons in culture, the axonal cross-sections within the sciatic nerve deviate significantly from a circular shape. They then use 3D-dSTORM to investigate the distribution of beta-II spectrin along the axonal circumference. They see that this distribution is very heterogeneous, both in the sizes of spectrin puncta and their arrangement along the periphery. The amount of spectrin scales linearly with axonal circumference.

Strengths:

Super-resolution imaging of axons of intact nerve fibers to investigate the organization of beta-II spectrin.

Weaknesses:

While most of the findings, like the spatial distribution of spectrin and related components, are reasonably well supported by data, I have concerns regarding the subsequent claims made in the article. The detection of axial periodicity based on the observation of a peak in the inter-tetramer spacing distribution is not very convincing, and a 3D representation (or a video of 3D reconstruction) would have been better. And so are the claims on characteristic spectrin spacing of 200 nm along the axonal circumference. A peak in the distribution does not imply a periodic arrangement.

Reviewer #2 (Public review):

Summary:

This is an interesting paper by the Unsain lab looking at the nanoscale organization of the membrane-associated periodic cytoskeleton in mouse sciatic nerve axons. The precise organization of the structure remains unclear, especially in vivo, and this manuscript significantly adds to our knowledge of this important structure. While some of the findings in the study are somewhat expected (though still valuable to see in an in vivo setting), an interesting observation is the presence of discrete nanoscale clusters that scale up with the size of the axon, which challenges previous assumptions.

Strengths:

Strong, convincing data; clever combination of imaging and analytical tools to make novel points; well written; excellent composition of figures.

Weaknesses:

(1) Figure 2A/3A: The large and small clusters of spectrin, as seen in cross sections, are unexpected and novel. The authors have done a clever job of combining imaging and analyses, but some things are still unclear. First, the authors should be consistent in their language when they talk about the spectrin clusters. Recommend precise language to define the small and large clusters when they first appear in the text, and then use the definitions consistently throughout the text. Second, based on the data shown, one does not get a clear idea of how the small and large clusters are organized along the longitudinal axis of the axon. In that context, are Figure 2B and C from imaging along the longitudinal axis? If not, it's unclear how the authors can conclude that the spectrin assemblies have a distance of ~170 nm along the linear axis. In general, a perceived limitation of this study is that while the authors have done a good job looking at cross sections, there is no information on the longitudinal distribution of spectrin in these axons. Looking at both cross- and longitudinal sections would also clarify details about the large spectrin clusters. For instance, are they small sausage-like structures, or long rods of spectrin running along the length of the axon? One assumes that all the analyses in Figures 3 and 4 are from the small clusters. Can the authors do a similar analyses of the large clusters? Finally, a schematic model showing both cross- and longitudinal- sections would make things clearer, but the authors would need to show the longitudinal data for that.

(2) It is interesting to think that the larger spectrin accumulations may be similar to the condensate-like structures seen by Boyer et al., as the authors mention in the discussion. In that context, it is possible that these focal accumulations are local reservoirs of spectrin that are also seen in mature axons (indeed, these accumulations were also seen in mature axons in the Boyer et al. paper, and they also speculated that these accumulations may be local reservoirs). Can the authors check if actin/adducin is also present in these larger spectrin accumulations?

(3) While talking about the nanoscale clusters, it is important to specify that the authors are talking about circumferential clusters. Though the writing is excellent, one still does not get the precise definition of "clusters" from just reading the abstract, and it would be good if the authors could work on that more (I recognize that this is not easy to do).

Reviewer #3 (Public review):

Summary:

In the presented work, the authors investigate spectral staining in axons of the sciatic nerve, where the MPS has been detected before using STED microscopy. They employ 3D-dSTORM in tissue sections and analyze the data, measuring localization of clusters on the axon perimeter and the relative distribution of those. From these data the conclude that large gaps in spectrum localizations exist and that clusters around the axon exist that are spaced at 200nm.

Major Comments:

(1) The presented data are at times overinterpreted, and the discussion lacks a critical view of the data. For example, the statement "...Unlike previous suggestions from qualitative evidence in cultured neurons (REfs), βII‑spectrin distribution in MPS segments of peripheral nerves is discontinuous, with extensive stretches of the perimeter lacking βII‑spectrin." is quite strong, given it is based on immunofluorescence staining and dSTORM microscopy in tissue. Absence of evidence of staining is not evidence of absence.

(2) The authors claim in the abstract that "The number of these clusters scales linearly with the axonal perimeter, maintaining a constant membrane occupancy of ~20% across varying axon diameters." Again, this is from a cut through an axon, while measuring the density of clusters on the perimeter. If they claim area occupancy, an area should be imaged, and the dots (clusters) should be measured in surface coverage in a 2D projection of the axonal surface.

(3) In general, this reviewer suggests being a bit more moderate in statements such as: "These findings challenge simplified models of the MPS based on cultured systems and demonstrate that the MPS in peripheral nerves is composed of discrete structural units." These statements are bold from the relatively few measurements in a single method and a single viewpoint. Especially when considering that techniques such as dSTORM depend extremely highly on labeling density, and apparent clustering of localization is highly prone to misinterpretation. If the authors desire to make such statements, working with endogenously labeled protein would be warranted. The authors should at least hedge such statements.

(4) If the authors want to make statements about general organization, why do they not compare adjacent cuts through the axon? If there are continuous spectrin filaments, the clusters should appear at the same site across repeated cuts through the axon.

Besides this, this reviewer welcomes the effort that has been made to establish dSTORM in tissue sections and to investigate the MPS in native tissue.

Author response:

We sincerely thank the editors and reviewers for their overall positive assessment and constructive feedback on our manuscript detailing the nanoscale organisation of βII-spectrin of the membrane-associated periodic skeleton (MPS) in mouse sciatic nerve axons. Their perspective and comments will help refining the manuscript.

A common comment by the reviewers relates to the description of the characteristic longitudinal periodicity of the MPS. We value these comments, which we believe are motivated by the fact that the longitudinal periodicity of the MPS is undoubtedly the most studied and prominent feature of the MPS in cultured neurons. However, the main goal of the present project was to describe how βII-spectrin is organised in the transverse axis of individual segments of the MPS in nerve tissue. This is why we utilised cross-sections of the sciatic nerve, hence achieving the best resolution possible in that plane, at the expense of the resolution in the axial axis. Furthermore, this study clearly shows that the transverse morphology of axons, and thus of the MPS, of neurons in the tissue is highly irregular, in comparison to cultured neurons. This imposes an extra challenge to observe correlated longitudinal structures when the observation length is limited, as in our studies. Nonetheless, to improve this aspect of the manuscript, we will revise our data and previous evidence, clarify the methodological trade-offs made, and make our interpretations more accurate.

Additionally, we will clarify several imaging- and definition-related inquiries, including tests for insufficient staining, the interpretation of βIII-tubulin staining, the assessment of axon–glia boundaries, and consistency in the use of terms like ‘clusters’ and ‘periodicity’, among others.

We believe these and other revisions will substantially strengthen the manuscript and comprehensively address the reviewers' feedback.

https://seed.bytedance.com/zh/edgebench

腾讯可怕的产品力

eLife Assessment

This fundamental manuscript describes a key role for the integrated stress response-regulated transcription factor CHOP in regulating liver biology in response to endoplasmic reticulum stress through both the downregulation of transcription factors involved in regulating hepatic identity and altering the capacity for integrated stress response and unfolded protein response signaling to induce protective signaling. The data supporting this model is convincing, but including some additional discussion on the mechanism and importance of the work in the context of the published literature would be helpful to better define the complex importance of CHOP signaling. This work will be of interest to a wide range of biologists interested in liver biology, stress-responsive signaling, and ER stress.

Reviewer #1 (Public review):

Summary:

The predominant view on CHOP's functions during ER stress is that it promotes cell death. This is in contrast to a handful of reports in the literature that claim that CHOP is a positive regulator of protein synthesis during chronic ER stress, and therefore is part of the adaptation program to ER stress. These previous studies were performed in tissue culture cells. Velarde and co-authors have used a mouse model of induction of mild ER stress to study the function of CHOP in hepatocytes.

Major strengths and weaknesses of the methods and results:

The authors use state-of-the-art mice to manipulate (i) CHOP and (ii) ATF6, a protective factor of ER proteostasis, and address the hepatocyte responses to mild ER stress in vivo and in cultures. Validated gene expression programs are well correlated to liver pathology in the mouse models. This is a very well-done study.

The authors clearly show that CHOP transitions hepatocytes under mild ER stress to a chronic ISR state, which is phenocopied by ATF6-depleted hepatocytes. So the conclusion that CHOP exacerbates ER stress in hepatocytes during mild ER stress is correct. It is also clear that CHOP targets negatively the transcription of hepatocyte identity genes, which opens a new direction of studies on the function of CHOP in secretory cells in general.

Conclusion:

This is a significant study that will benefit different research fields, and specifically studies on proteostasis, as was recently highlighted in Nat. Str. Mol. Biol. by experts in the field.

To this reviewer, the importance of the study is that it links the function of a transcription factor (CHOP) to stress intensity (mild versus severe) in a physiological experimental model (hepatocyte function and pathology).

Reviewer #2 (Public review):

The Unfolded protein response (UPR) and related integrated stress response (ISR) are critical signaling systems for cell survival in response to acute stresses. While the UPR directs critical adaptive gene expression, certain chronic stresses switch this pathway towards cell death and disease. An important question concerns the mechanisms by which the UPR switches from being adaptive to maladaptive. Prevailing models focus on the transcription factor CHOP (DDIT3 or GADD153), whose levels are enhanced via the UPR, and extended/amplified amounts of CHOP are suggested to boost death-related gene expression. However, the literature and this manuscript point out a number of observations that do not neatly fit with this model, suggesting that there are still unresolved processes by which CHOP adjusts cell outcomes via the UPR.

This manuscript features a nice hepatocyte-targeted knockout of CHOP to discern the contribution of CHOP in the transition between adaptive and maladaptive outcomes. The key ideas presented in this study are that CHOP-directed gene expression is focused on protein synthesis, metabolism, and hepatocyte identity. In the progression of the UPR, CHOP expression can lead to resumption of protein synthesis, which can assist in the translation of the UPR-directed transcriptome, which includes ATF6/XBP1-directed genes that aid the processing capacity of the endoplasmic reticulum (ER). However, enhanced nascent protein can further stress the ER. CHOP directs gene expression in both the first phase- acute and second phase-chronic in the UPR, and the pivotal decision lies in the transition between the phases.

Overall, the manuscript includes some new ideas as well as refinements of earlier ones for CHOP-determination of UPR-directed cell fate. The CHOP-hepatocyte knockout mouse model helps to delineate the different tissue functions of CHOP, which has been a problem for some earlier studies. The manuscript progression of experiments is solid, and experimental design and documentation are rigorous. The manuscript text is largely clear, but there are portions that would benefit from fuller explanations of ideas.

There are three points of concern. First, the manuscript model (Figure 7) lays out a timeline for the progression of the UPR between two phases. The study is not always clear about the times assayed, and there appears to be a single time point for measurements. Second, there is emphasis on protein synthesis changes in the model. It is true that the literature argues that resumption of protein synthesis concurrent with stress damage (i.e., GADD34-directed gene expression) is a key reason for the potentially debilitating effects of CHOP (e.g., Marciniak et al 2004, Han et al 2013). However, the manuscript does not feature protein synthesis measurements. Inclusion of bulk protein synthesis measurements in the context of this model system would strengthen the study and support for the model. Finally, for this reviewer, some of the most interesting ideas center on CHOP-directed transcription of genes that regulate hepatocyte identity. There is solid evidence for direct CHOP regulation of these genes, but the manuscript does not really develop and test the ramifications of these networks on cell fate during ER stress.

Reviewer Concerns:

(1) The abstract packs in a lot of information. The ideas would not be clear to a general reader. Furthermore, the UPR and ISR are referred to in the second-to-last sentence, but not defined earlier in the abstract.

(2) There are some typos/grammar concerns.

(3) ATF4 diminished with CHOP-depletion (Figure S2A). What is the mechanism here? Does this complicate the analysis of CHOP-directed gene expression? How does this fit with Figure 6J? The timelines for TM treatment are critical. The authors should more fully explain the time courses in the experiments.

(4) Figures 2 and 3: There is a discussion on enhanced protein synthesis with loss of CHOP (reduced GADD34 expression). What is the time point - 8 hours TM? Emphasize, explain, and justify time points of experiments here and in later panels. It would strengthen the model with direct measurements of protein synthesis. The authors could include GADD34 protein measurements in these panels. Figure 3 - panel D - some abbreviations are not standard.

(5) Figure 4: One of the most interesting in the manuscript is the transcription factors downstream of CHOP that are linked with hepatocyte differentiation and metabolism. The manuscript would be bolstered by developing some of these target genes into the Figure 7 transition model.

(6) Figure 6: The comparison of CHOP and ATF6 target genes is a highlight of the manuscript. The literature on this topic is complex, and there are some suggestions that CHOP can be downstream of ATF6. Furthermore, there were some earlier models by Walter and others about extended induction of Perk (death) vs induction of other UPR sensors (survival) (e.g. PMID: 17991856). It would be helpful in the Discussion to delineate between these models and their critical differences.

Reviewer #3 (Public review):

In this manuscript, the authors aim to understand the function of the transcription factor CHOP, which is known to promote cell death during severe stress in the ER. The authors note that CHOP is induced during less severe stress, but its functional output is not well understood in these cases. Here, they study the effects of conditional knockouts of CHOP in hepatocytes of mice challenged with chemical inducers of ER stress.

Tunicamycin (an ER stress inducer) injection leads to the upregulation of CHOP and lipid accumulation in the liver, but no significant cell death in the experiments outlined here. Conditional knockout of CHOP results in a number of differences in the way hepatocytes respond to stress, notably resulting in lower steatosis.

There are two main findings supported by the data presented here. First, the authors show that CHOP suppresses the expression of ONECUT, a master regulator of hepatocyte differentiation and metabolism, during ER stress. They show by ChIP-seq that CHOP binds to the promoter region of this gene, and by RNA-seq that ONECUT expression is suppressed by ER stress in a CHOP-dependent manner. Many predicted targets of ONECUT1 were also suppressed by ER stress in a CHOP-dependent manner, though they were not bound directly by CHOP. The data support a model where CHOP down-regulates hepatocyte metabolism and identity via regulation of ONECUT1. This is a new and interesting finding, perhaps explaining the steatosis phenotype of livers that accompanies ER stress, although this was not tested directly.

The second main finding of this paper is that CHOP deletion leads to an interesting assortment of effects on genes related to the ER stress response and integrated stress response (ISR). As expected, based on prior work, CHOP deletion led to more phosphorylation of eIF2alpha (CHOP is known to upregulate the phosphatase for this translation factor). However, unexpectedly, this did not cause increased expression of ATF4 (a transcription factor whose upregulation during stress is dependent on eIF2alpha phosphorylation) and its downstream targets; in fact, CHOP deletion had the opposite effect on these. In other words, CHOP seems to both turn off the initiating signal for the ISR (namely, eIF2alpha phosphorylation) and also promote the downstream signaling events that rely on this initiating signal. It makes sense that cells would do this, as restoring translation would be important for realizing the effects of the massive changes in gene expression initiated by ER stress, and yet this would exacerbate stress in the short term, so it would be counterproductive to also turn off the entire stress-regulated program. Having a factor (perhaps CHOP) that coordinates these two events makes sense. It will be interesting in future work to understand the mechanisms behind this regulation.

Finally, CHOP deletion led to less activity of other aspects of the ER stress response, notably IRE1 (determined through measurement of XBP1 splicing and RIDD of Bloc1s1). This is explained by the continued phosphorylation of eIF2alpha in these knockouts, as the continued attenuation of translation would lessen the burden of misfolded proteins in the ER. Somewhat confusingly, the same pattern is not seen in downstream targets of XBP1. Less splicing, coupled with perhaps less translation of the spliced mRNA, should result in less active transcription factor and lower expression of its target genes in the CHOP KO. This is not observed in Figure 2, although the more global gene expression analysis suggests that all stress-dependent gene expression changes were weaker in the CHOP KO livers.

The authors characterize the effects of CHOP, promoting restoration of protein synthesis and the accompanying exacerbation of stress while preserving the signaling that should relieve ER stress, as a switch from an acute to chronic phase of ER stress. This is mirrored in their analysis of ATF6 in a similar series of experiments. Although this is an interesting framework for thinking about the stress response, whether CHOP is the key factor or a supporting actor in regulating this transition will require a better understanding of the mechanisms involved.

┌─────────────────────────────────────┐ │ 模型库层：Transformers / Diffusers │ ← 用户写模型代码 ├─────────────────────────────────────┤ │ 🤗 Kernels（内核分发与加载层） │ ← 新增的这一层 │ - 从 Hub 拉取预编译内核 │ │ - 匹配当前硬件/OS/框架版本 │ │ - 安全验证（签名、可信发布者） │ ├─────────────────────────────────────┤ │ 框架层：PyTorch / JAX / CuPy │ ← 张量运算、autograd ├─────────────────────────────────────┤ │ 厂商运行时：CUDA / ROCm / Metal │ ← GPU 编程接口 ├─────────────────────────────────────┤ │ 硬件：NVIDIA / AMD / Apple GPU │ └─────────────────────────────────────┘

The stratification of privilege in the queer community is an important thing to remember. Folx who break away from heteronormative self expression experience more violence than others. The wheel of privilege helps us locate both ourselves and other identities to approximate the priveleges or unearned social benefits of people.

It seems so bizarre at first but then if you think about the social climate at the time, it makes perfect sense why folks did not feel safe about publishing at this time period.

I recently read Malcolm Gladwell's Revenge of the Tipping Point where he discusses the mid-90s resurgence of homophobia around the time that Bill Clinton signed the Defense of Marriage Act (DOMA) in 1996 after the don't ask don't tell policy in 1993 allowing members of military service to serve, so long as they conceal their identity. Gladwell goes on to discuss how the sitcom Will & Grace premiered in 1998 and the intentional characterization of Will as largely heteronormative with a deeply caring relationship with the other main character, Grace. Will's character was a counter-narrative to the pervasive homophobic societal expectation at the time that gay people were not capable of deep relationships in a same sex union. Gladwell's point in the book was that it took only a few years for a critical mass to shift (the tipping point he describes as a critical third) and we see that playing out in 2003 under George Bush, that the United States Supreme Court striking same-sex intimacy. While in 2004, Massachusetts became the first state to permit same-sex marriage. (American Queer History)

In 1992, Canada ended the prohibition on gay and lesbian service members, and in 1995 The Supreme Court recognized sexual orientation as a protected ground under the Canadian Charter of Rights and Freedoms. By 1996, sexual orientation was added to the Canadian Charter of Rights and Freedoms. By 2005, the Civil Marriage Act provided for the legalization of same-sex marriage across the whole of Canada.

I understand that this is a journal article and perhaps needed peer-reviewed articles for submission to this journal (???) but I think there can be merit to using non-peer-reviewed articles, especially for a topic like queerness. From a participant perspective, someone might feel more comfortable expressing themselves in a blog or book format, as opposed to a study, due to power dynamics.

Both author's have acknowledged their social positions as white and cis-gender. It seems like culture is largely absent from this literature review and only mentioned a few times. As we have been discussing intersectionality and our positionality this week, I wonder whether acknowledge of our social identities, and perhaps what we cannot personally speak to, warrants excluding articles from authors that can speak to that perspective?

I personally think that it is not difficult to find authors who have written about the perspectives we cannot personally speak to, but I'm curious to know other folks opinions!

I agree that academic leaders should focus on queer student, but also wonder whether there should also be a focus on queer staff? In my experience, queer students feel more comfortable and empowered when queer staff also feel comfortable to share their identity and experiences. Lee (2022) did a case study highlighting how "sign" and "symbols" of queerness were important in helping queer staff feel comfortable in the workplace, which then allowed them to be support for students. This study specifically mentioned that senior leadership visibility was important to staff empowerment.

There is actually quite a lot of racism, transphobia and sexism in white gay male spaces. White people and white culture are often treated as the default in queer spaces. Because of that, queer and trans people of color frequently feel excluded and have to push back against that logic in both personal interactions and institutions.

This is happening right now in North America. Withdrawing of support, cutting funding, silencing, looking the other way etc. Trans people in particular are under direct legal, social and cultural attack from the far right. This started in the US, and now is happening in our more conservative provinces.

Do crowds help us better understand ourselves?

Do you agree with this statement? Who is the main crowd / collective in your life? How are you impacted?

How is this different from the traditional notion of how a mob behaves? Do you agree with this view?

Traditional notion of mob mentality

0710

三种不同的行为

The author says that there doesn't have to be one answer to this question and that us readers should be able to have our say in it.

The other half of the argument says that all dialects and languages are just as good.

Treating a person poorly because they didn't use proper grammar is like treating a kid badly because they didn't get the right answer on a math equation.

Is losing local dialects worth it so everyone would use just one common one?

This means that knowing the standard language can actually help someone get a better job, not just speak and write more formally.

The argument about standard English and dialects has been around for a long time.

At fist I was surprised they said this, but it makes sense, because it is what they've been taught their whole lives.

The students think that the the classroom is only for standard english and other dialects are supposed to stay outside.

This essay is already starting to sound similar Young's argument! He also argued for keeping the english that people spoke at home.

As a vice principal, I have very little power within the board and even less power within the hiring process. I think it's important to address this issue of lack of diversity within our schools because in elementary schools especially, teachers are mostly homogenous group. However, I think when we talk about increasing diversity in school administration, we need to include senior administrations (superintendents) as well. They have more power within the board, and in our board, they make all the hiring decisions. I often find within the school boards, the further up the power chain we go, the more homogenous the group tends to be,

This has been my experience with the local public board. My colleague and I created East Asian Educator Affinity Network at my board to support and network with East Asian educators. The stories shared during our first meeting were heart-breaking. We also felt very invisible as a group within the board. To give an example, the board sends out monthly list of holidays and celebrations from many religious and ethnic groups to all the schools. Yet, they didn't include Chinese New Year, a very obvious but important East Asian holiday.

Given Regulation 274/12 was introduced in 2012, I am curious about how nepotism and favouritism were still allowed to continue when this report was conducted in 2020. When I worked for Peel, teacher hiring was strictly done through seniority list and principals no longer had any input. Even with the Regulation revoked, some boards are still continuing to use the seniority based hiring, not that I am suggesting that solves this issue of teacher diversity. I think lack of teacher diversity comes from a much wider systemic issues.

At least in TDSB and Peel, there were many BIPOC teachers. However, this was not the case for administrators. I always wondered why this was the case. I took my PQP online asynchronously and everyone in my class came from different parts of Ontario (and some outside of Ontario). Yet, the class was not very diverse. In comparison, I am really enjoying how diverse our cohort is for the PhD program where so many different perspectives are brought together because of the diversity. I just wonder what the difference is between these two programs.

At times, this EDI policies feel very surface level to me. At Greater Essex County District School Board, they spent an enormous amount of money on surveys and reports to address anti-black racism but beyond the surveys and reports, nothing more came out of it - similar to how so many projects were unaddressed in the Truth and Reconciliation Commission's 94 Calls to Action.

I am not familiar with this Aspiring Racialized Leaders Mentoring Project but we cannot put the onus of bringing more diverse teachers and administrators to BIPOC...simliar to how Gaudry and Lorenz talked about not requiring Indigenous people to bear the responsibility for change in Indigenous inclusion policy.

I think some of the teacher hiring processes were the results of political and economic climate and its responses from the union. For many years, there were very few teaching jobs which meant that teacher graduates who needed steady income to survive did not stay in teaching. It used to be the case that when teachers were hired at the board, they stayed in the profession for many decades. There was a teacher shortage at the beginning of 2000 when there was a change to retirement requirements (going from 90 factors to 85 factors). So a large number of today's teachers were hired around then where teacher diversity was not considered.

During my master's thesis I learned that I really knew nothing about education system in other provinces. In fact, being an elementary teacher, I really knew very little about what was happening in secondary schools, either. I think this is a disservice to teachers as it encourages teachers to hold a very narrowed view of education. As well, I've learned over the years that the comprehensive and cohesive educational strategies differ not just region to region but swings rather massively based on the political parties and time. For example, for many years, reading was taught through whole language approach and now we are moving back into phonics phase.

In fact, this often leads to Black, Indigenous, and other racialized students to being pushed into “special education” streams and disproportionately ignored, punished, disciplined, or expelled compared with white students. Yet many schools refused to confront the systemic racism driving these patterns, choosing instead to blame the students or their parents for the challenges they face (Gillborn, 2015; James, 2019).

The vast majority of teachers in Canada are white women. Gebhard (2020) discusses this as an issue by asserting that “the role of white women in education has historically been to reproduce rather than disrupt colonial epistemologies and discourses of white dominance as these intersect with white womanhood” (p.207). This continued system of ‘keeping the status quo’ may be partly due to ignorance and partly because thinking about or confronting systemic racism and white supremacy is deeply distressing to many people, particularly those in a position of power.

I am going to leave this short piece I wrote in 2023:

Black children are not scary. They are scared.

Over and over again, we see news about black children being arrested, beaten or murdered on our streets. There is no respite in the school system either, where black children are neglected, punished, and expelled disproportionally over white children. As Munroe (2021) says so powerfully, “within all of these conversations about racist practices, what often gets left out are stories about the ways school disciplinary practice and policies affect Black students’ emotional well-being and traumatizes them. Black students continue to be judged as inferior and dangerous. Even with ‘progressive discipline,’ Black students who violate rules are seen as offenders rather than teens in need of support” (para. 4).

It is very clear that black children are not scary. They are scared. Cole (2020) goes on further and very plainly outlines the emotional and systemic consequences of suspending and expelling black children from our schools: The gun violence issues in the city of Toronto have been one of the number one stories in this city for the past two years. Why does no one make the connection to poverty in the city of Toronto and to the expulsion of black students, which further alienates them. We've heard so many times the impact of not giving kids a place at school where they feel welcome or they feel like they have an environment to learn. We push kids out of the education system and we have this huge epidemic of violence and anti-social behaviour (para. 32). This means that keeping kids in school is of utmost importance.

In addition to keeping them in school, black children need support, care, love and respect. This means teaching black histories, contributions and perspectives and integrating them year-round, including black authors, resources, and contributions in the classroom (Facing History & Ourselves, 2021). This also involves celebrating and appreciating black excellence and cultural contributions in art, music and literature. Love (2014) says ignoring students' culture is an injustice and an oversight. Welcoming black joy into the classroom by allowing students to be themselves and share their culture will enable them to feel seen and appreciated. Emdin (2016) adds to that assertion and further emphasizes that “the classroom that reflects the culture of youth while highlighting how beautiful it could be challenges the ways that the teacher and students engage with each other even before teaching begins” (para. 17).

References

Cole, D. (2020, February 6). What’s it like to be black in Canada? Under policing, it’s hell [Interview]. In cbc.ca. https://www.cbc.ca/radio/thecurrent/the-current-for-feb-6-2020-1.5454037/what-s-it-like-to-be-black-in-canada-under-policing-it-s-hell-says-desmond-cole-1.5454039

Emdin, C. (2016, March 22). For White Folks Who Teach in the Hood (L. Ferlazzo, Interviewer) [Interview]. In Education Week. https://www.edweek.org/teaching-learning/opinion-for-white-folks-who-teach-in-the-hood-an-interview-with-chris-emdin/2016/03

Facing History & Ourselves. (2021). Black History Month Resources: Approaches, Identities, Histories, Legacies & Inclusion. Facingcanada.facinghistory.org. https://facingcanada.facinghistory.org/black-history-month-resources

Love, B. (2014). Hip hop, grit, and academic success [Video]. In YouTube. https://www.youtube.com/watch?v=tkZqPMzgvzg

Munroe, T. (2021, February 17). How to curb anti-Black racism in Canadian schools. The Conversation. https://theconversation.com/how-to-curb-anti-black-racism-in-canadian-schools-150489

Hiring processes, though presented as neutral, can reproduce racial bias through credentials, geography, and names. Also, there is a policy contradiction: diversification efforts without anti-racist structural change may reinforce inequality rather than reduce it. Administrator autonomy, then, demands critical self-reflection and accountability.

I found the emphasis on "Whiteness” and White privilege as a self-reinforcing system a significant idea. According to the article, educational systems validate Whiteness and White privilege and, as a result, promote policies and practices that further oppress BIPOC. This idea also promotes a discussion regarding how the dominant cultural norms that the benefactors are often unaware of, actively manifest obstacles and sustain inequities, rather than being a background that has no impact on the situation.

Canada is seen as standing for racially stratified society, where Whiteness is privileged. That's especially the case in resource distribution, for instance in employment and education. Canada is different from other countries as it lacks a national education framework. This means that the construction of educational policies and regulations is the responsibility of the province. This allows each province, including Ontario, to set its own policies in regard to education, teaching, and educational administrators which may lead to different levels of diversity.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

Reviewer #1:

Major comments:

1. Lines 103-116 (first paragraph of the results section) describe mainly published data that is more suitable for the introduction section. It is annoying to refer to different published articles in the Results section to strengthen the results instead of showing them. The same goes for paragraphs two and three. Why mention those data in the Results section if they are already published and known?

We have reorganized this material by moving some background information to the Introduction. Our intention was not to incorporate published data to strengthen our results, but rather to provide essential context for interpreting our findings. We have therefore left some of this foundational information in the results section to create a clear narrative flow, enabling readers to understand the basis for our experimental design and interpretations without needing to recall details from earlier paragraphs in the Introduction. For example, we considered it crucial to restate the earlier report of the BiP:sfGFP:HDEL phenotype in Atlastin mutants, since our results supporting luminal ER protein displacement contradict the previous fragmentation model.

The following concept was in line 103 in the Results section, and is now in the introduction in lines 82-92: "Conventional light microscopy, commonly used in studies of neuronal ER structure, lacks the resolution necessary to visualize individual ER tubules in small structures, such as presynaptic terminals. The ER is highly sensitive to fixation, and live imaging experiments in neurons in vivo have been conducted on upright microscopes using water dipping objectives with a typical axial resolution limit of >300 nm, which cannot distinguish the densely packed ER tubules at presynaptic terminals (3,8,21,28,39-42). Electron microscopy offers higher resolution, but cannot be used in live samples and has typically been limited to thin 2D sampling (in which it is difficult to distinguish ER cross-sections from synaptic vesicles) (8,20,22)."

Figure Legends-(in all Figures): The number of experimental repeats must be mentioned in the figure legends.

This information is provided in Supplementary Table 1, which contains detailed information about the genotype, statistical analysis, and number of larvae and NMJs analyzed. If the journal requires this information in figure legends, we can move it.

The way the figures are labeled is worrisome; supplementary figures are not ordered numerically.

We will be happy to rename supplementary figures according to journal guidelines.

The tubule extension in Figure 2D is not convincing. Is there a movie showing those changes? Better images are needed. It is essential to show which supplemental movie corresponds to which panel.

We have now included a corresponding video of the same neuron used as an example of tubule extension. We also added another frame to the figure to provide further information on the tubule event we captured. (Figure 2D, Movie S10)

This is unnecessary in the results section: "To investigate the relationship between ER structure and function at synapses, we examined mutants of Atlastin, a GTPase that regulates ER tubule fusion. Drosophila has a single homolog while mammals have three Atlastin homologs, with Atlastin-1 enriched in the brain (Rismanchi et al., 2008)."

This information was moved to the introduction.

"This reduction in ER membrane marker intensity has also been observed in other HSP mutants, suggesting this is a common feature of ER shaping mutants and could indicate changes in ER membrane composition, integrity, or tubule thickness (Perez-Moreno et al., 2023)." This comparison is important and should be shown in the same settings as for the Atlastin mutant rather than referring to published data.

We agree with the reviewer that it is important to determine whether other ER-shaping proteins, besides Atlastin, also show a decrease in tdTomato:Sec61b to support our claim that this could be a common feature among ER-shaping mutants. To do this, we examined mutants of another ER-shaping protein, Reticulon 1, which regulates membrane bending and stabilization in ER tubules. These loss-of-function mutants were a gift from Dr. Cahir O'Kane at the University of Cambridge and were used in his lab's Pérez-Moreno et al., 2023 publication. We found that in our hands tdTomato:Sec61b levels were reduced in Reticulon 1 mutants, consistent with the results reported by Pérez-Moreno et al. (2023). These results are in Figure 3E-F. We also examined the synaptic distribution of the luminal ER marker, BiP:sfGFP:HDEL, in Reticulon 1 mutants to see if it is displaced to the cytosol. Notably, it remained ER-associated, unlike in Atlastin mutants. These results are in Figure 6F-G, results lines 267-270, and discussion lines 542-545.

Does the distribution of the luminal ER marker in Figure 6F diffuse due to mislocalization or reflux after being localized to the ER and then refluxed to the cytosol as was previously shown for the ER to Cytosol signaling (ERCYS) mechanism? Could you assess other ER-luminal protein localization biochemically? It is highly recommended to look at another soluble ER-protein localization in the Atlastin mutant without overexpression, which can be an artifact.

ER stressors can induce ERCYS, in which some luminal proteins, including PDIA3, DNAJB11, ERp29, and an eroGFP reporter, reflux by 30-70% to the cytoplasm without subsequent degradation (unlike ERAD (ER-associated degradation). This phenomenon has only previously been observed in yeast and glioblastoma tumor cells from mice and human . We believe that our work provide the first suggestion that this may occur in neurons, and particularly in a neurological disease model.

We do not believe that the reflux phenotype for BiP:sfGFP:HDEL is due to its overexpression for two reasons: (1) we observe reflux in our neuronal Atlastin knockdown experiments, even when the levels of BiP:sfGFP:HDEL are significantly reduced artificially because of titration of the GAL4 between the RNAi and the reporter (Figure 7A), and (2) BiP:sfGFP:HDEL overexpression somewhat suppresses endogenous BiP upregulation ((Figure 10 and see Reviewer 1.10), arguing that the transgene does not induce ER stress). We included a new "limitations of the study" section to be transparent about the caveats of the BiP:sfGFP:HDEL reporter (lines 639-664).

Identifying potential endogenous neuronal ERCYS substrates in our in vivo preparation poses several challenges. First, biochemical approaches, such as fractionation, are not possible in our complex in vivo sample because neuronal ER proteins would mix with ER from other tissues upon homogenization. Second, detecting endogenous proteins with antibodies requires fixation and permeabilization, which notoriously disrupts ER structure and even causes our reporter BiP:sfGFP:HDEL to collapse from a smooth distribution, as visualized by live imaging and FRAP, to a punctate distribution. Third, using antibodies rather than neuronally restricted transgenes makes it challenging to determine whether the signal originates from the neuron or from dense ER structures in the surrounding muscle. Fourth, some ER luminal proteins can displace as little as 30% in the ERCYS examples cited above, and the sensitivity of our imaging assays may limit our ability to detect these small changes. Finally, the limited availability of tagged transgenes and antibodies specific to Drosophila luminal ER proteins (see next paragraph) poses additional challenges. These limitations highlight the need for future studies to develop novel tools and techniques to more definitively test whether we are indeed observing ERCYS. We have included a paragraph on these future challenges in our discussion in lines 639-664. Identifying endogenous targets of ERCYS in fly neurons is a worthwhile goal, but beyond the scope of the current study. These next steps will particularly benefit from identifying the machinery involved in the reflux of our BiP:sfGFP:HDEL reporter.

Tools we tested: We investigated several options: (1) a tagged PDI transgene (a gift from Karen Hibbard), which was not detectable at presynaptic terminals, (2) a tagged BiP (FlyORF; F000956) that did not localize to the ER, and (3) full-length endogenous BiP detected by antibody staining. We did not detect obvious reflux of endogenous BiP to the cytoplasm (Figure 9), with the caveat that in fixed samples, the BiP signal was not tightly co-localized with the ER marker even under control conditions. However, we did use this antibody to detect an increase in BiP in Atlastin mutant presynaptic terminals, indicating ER stress (see Reviewer 1.10).

Though we have not identified endogenous targets, we believe that our studies with the exogenous reporter will be of great interest to the field, as they clarify the previously reported Atlastin phenotype and provide the first report of a new defect in a human disease animal model.

In comparison to Summerville et al. (2016) in Figure 7, the experiment was not done in the same way. It is important to keep the same settings for comparison

In Figure 7D-E, we compare the distribution of BiP:sfGFP:HDEL in cell bodies, axons, and muscles between controls and Atlastin mutants. To clarify the experimental approach relative to Summerville et al. (2016): while both our studies examined the same cellular compartments (cell bodies, axons and nerve terminals) using the BiP:sfGFP:HDEL reporter, we employed super-resolution Airyscan microscopy. This enhanced resolution was critical for definitively demonstrating that this is a functional rather than a structural phenotype and that ER displacement is progressive, and repeating this experiment at lower resolution as previously reported does not provide any new information. We identified two distinct distribution phenotypes in Atlastin mutants expressing BiP:sfGFP:HDEL, which were not described in the Summerville et al., 2016 paper. From our manuscript (lines 249-251): "We identified two distinct ER network phenotypes in Atlastin mutants expressing BiP:sfGFP:HDEL: "Partial loss" NMJs retained both diffuse signal and identifiable ER network structures, while "Complete loss" NMJs showed no visible ER network structures. Note that the "Complete loss" phenotype in Atlastin mutants reflects the absence of detectable luminal marker signal in organized ER structures, but not the complete absence of ER membranes, as demonstrated by our ER membrane marker tdTomato:Sec61β results."

Does the Atlastin mutant induce the unfolded protein response and stress within the ER? It is necessary to look for UPR markers in those settings. It was shown previously that ER stress leads to protein reflux from the ER to the cytosol. Is there a difference in the ER stress markers in the presynaptic terminal?

The reviewer suggested that Atlastin mutant synapses may exhibit ER stress. To address this, we examined levels of the ER chaperone BiP, a well-established ER stress marker whose expression increases during UPR activation. We first validated that our BiP antibody can detect changes in ER stress by feeding control larvae with 50mM DTT for 24 hours. These results are in the new Figure 10A. Note that we were unable to test sensitivity to ER stress in this way in Atlastin mutant larvae because they did not consume the DTT-treated food, as assessed by blue food coloring in the larvae's guts.

Using this antibody, we measured baseline BiP levels at NMJs of Atlastin mutants on normal food, and found they were slightly increased compared to controls. We conclude from these experiments that Atlastin mutant synapses have mild ER stress. Notably however, Atlastin mutants co-expressing UAS-BiP:sfGFP:HDEL or UAS-tdTomato:Sec61b did not show significantly increased endogenous BiP levels, suggesting that transgene expression at least partly suppresses the mild ER stress response, even though there is extensive cytosolic displacement. These results argue (1) that the mild ER stress in Atl mutants does not strictly correlate with the reflux phenotype, and (2) that the reflux phenotype is not an artifact of overexpression-induced stress. These results are described on lines 430-436 in the results section and shown in Figure 10B-E, and their implications discussed on lines 585-598.

We also explored another strategy to detect ER stress by assessing eIF2α phosphorylation, a key event in the Unfolded Protein Response (UPR) pathway. We obtained a phospho-eIF2α antibody (Cell Signaling; #3597) that was reported to work in Drosophila. However, when we tested this antibody by Western blot, we were unable to detect a band at the expected molecular weight for phosphorylated eIF2α, even in positive-control samples treated with DTT to induce ER stress. We therefore concluded that this antibody is not suitable for reliably detecting ER stress in our experimental system. The failure of this antibody highlights the challenges of finding robust tools to measure ER stress in Drosophila.

It is important to add biochemical experiments to show that no fragmentation of the ER membrane occurred. It can be simply demonstrated by looking at the redox state of the ER, which would change if it were mixed with the reducing cytosol. Moreover, this can be shown by using an ER-targeted redox-sensitive fluorescent protein that is tethered to the ER membrane to follow changes in the redox state of the ER.

The reviewer asked us to test whether the redox state of the ER is disrupted, which could indicate exchange between the cytosol and ER due to membrane rupture. As noted above, biochemical approaches such as fractionation are not possible in this in vivo sample. We attempted to address this concern by creating a UAS-Sec61β:roGFP construct, using the roGFP sequence from Igbaria et al. (2019) to monitor the ER lumen redox environment in Atlastin mutants. Since Sec61β is membrane-tethered, it should remain in the ER and not undergo reflux, making it an ideal sensor for detecting any mixing between the reducing cytosolic environment and the oxidizing ER lumen that would occur if membrane fragmentation and/or ruptures were present. We tested this approach in wild-type Drosophila S2 cells and used the Gal4-UAS binary expression system to co-express Actin-Gal4 (to drive expression of UAS constructs), UAS-Sec61β:roGFP (redox sensor), and UAS-BiP:Halo:HDEL (as a control reporter insensitive to DTT treatment).

Our experiments showed no detectable changes in the fluorescent properties of UAS-Sec61β:roGFP following 30 min 10mM DTT treatment compared to DMSO vehicle control, including no increase in 405-nm excitation fluorescence or changes in 488nm/405nm excitation ratios. These results suggest that either the roGFP sensor requires further optimization for sensitivity in this cellular system or that additional controls and calibration steps are needed to establish the dynamic range of the assay. We believe this experiment falls beyond the scope of the current study, given the extensive optimization required. However, it represents an important future direction for testing membrane fragmentation as a mechanism underlying the phenotypes observed in Atlastin mutants. The possibility of ER integrity defects is mentioned in the discussion on lines 547-559.

Minor comments:

1. It is important to call figures by order. Figure 2C is called before 2A-B. Figure 2B is called before Figure 2A.

The revised manuscript has all figures in order of appearance in the text.

Figure legends (Figure 2): "The same control dataset used in E-G was used in Figure 5 and Figure 5_Supplement." Why is this relevant?

We wanted to be transparent about reusing the same control dataset across multiple figures to avoid any appearance of data duplication. This notation clarifies that, although the data appear in different contexts (Figures 2 and 5. This version does not contain a Figure 5_Supplement), it represents the same biological samples analyzed for different parameters, ensuring readers understand that these are not independent datasets.

Figure 4F is called before Figure-4D-E which are not called.

We revised our manuscript and reorganized Figure 4 to ensure that all figure panels are referenced in sequential order and that panels 4D-E, which were previously not cited in the text, are now properly referenced when discussing their corresponding results.

Figure 5B is called before the previous ones. Same for Figure 5A supplement.

We referenced Figure 5A in lines 211-212, which precedes our discussion of Figure 5B. To clarify the figure order, we removed the early references to Figures 2D-G and Movies 7-14, which were mentioned only to indicate that we were analyzing the same dataset in different ways.

The revised manuscript has all figures in order of appearance in the text.

Referees cross-commenting

I agree with the comments raised by reviewer2 and 3. Basically it is highly important to validate those data by genetic rescue. Moreover, it is essential to know the source of the displaced luminal marker to the cytosol. Is it mislocalization or it is a reflux of pre-existing protein to the cytosol after insertion to the ER. It is also recommended by me and the reviewers and me to test the endogenous protein rather than overexpression.

We have addressed these points in our responses to the following reviewer questions:

• Genetic rescue: Please see our responses to Reviewer 1/Question #10 and Reviewer 2/Question #1.
• Source of displaced luminal marker: We provide some evidence addressing this in our response to Reviewer 3/Question #1.
• Endogenous protein localization: We have examined this and detailed our findings in our responses to Reviewer 1/Question #7 and Reviewer 2/Question #6.

  Reviewer #1 (Significance (Required)):

General assessment: This interesting paper shows that proteins can escape the ER under special conditions. However, the authors need more evidence to show that and rely less on the overexpression system, especially of BIP-GFP, which can cause proteostasis stress within the ER. Advance: The results have been oversimplified in their explanations, and some points and complexities of the study need to be addressed further to make the most of them. These are often some of the more interesting concepts in the paper. I think many points can be addressed in the text by the authors being clear and concise with their reporting. At the same time, other experiments would turn this paper from an observational one into a very interesting mechanistic one. This paper is based on previously published articles from the group and other groups, and it is a nice progression. However, as mentioned, this paper depends primarily on published data, and the novelty is somehow lost between all the comparisons to other published data instead of emphasizing that. Without a substantial mechanistic improvement, the paper would remain observatory.

Audience: The microscopy tools can be great addition to researchers in the field to monitor protein trafficking especially Cell biologists (basic research)

My expertise: ER homeostasis, protein trafficking, cell biology

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

Summary The endoplasmic reticulum (ER) is a continuous organelle that extends throughout neurons to regulate fundamental processes. The analysis of ER dynamics at synaptic terminals is limited by the challenge of imaging these structures at high resolution. In this manuscript, the authors use super-resolution (~170 nm) live imaging and a combination of membrane and luminal ER markers at the Drosophila larval NMJ, an important model synapse, to investigate dynamic ER architecture in vivo. They report a detailed characterization of the presynaptic ER organization and dynamics at wild-type and GTPase Atlastin mutant NMJs. Their analysis using the ER membrane marker tdTomato:Sec61b reveals the presence of an intact ER network in Atlastin mutants. This contrasts with the apparent ER fragmentation phenotype previously reported and replicated here when using a luminal marker. Their findings instead point to the progressive displacement of luminal proteins to the cytosol in Atlastin mutants specifically at synapses. The authors propose that the disruption of ER protein dynamics at synapses is a compartment-specific ER stress response. The manuscript is well written, results are clearly presented, and experiments are technically rigorous.

Major comments

1. The baseline ER phenotypes in Atlastin mutants are mild with complete loss of ER network only observed in terminal boutons. This interesting and unexpected result should be further confirmed by genetic rescue. The authors can use a UAS rescue line previously reported in PMID: 19341724.

We tested the UAS-Atl-myc rescue line and unfortunately found that even in wild-type neurons, overexpression of Atlastin produced strong ER organization defects that precluded the rescue experiment. Instead, to confirm the cell autonomy of the phenotype and to test it wth an independent tool, we performed a presynaptic knockdown of Atlastin by RNAi and found that BiP:sfGFP:HDEL is displaced, as observed in the Atlastin null mutant. These results are in now shown in Figure 7A-C.

Lines 204-7: It's not clear how a greater coefficient of variation indicates that the marker is more concentrated in subsynaptic structures or what is meant by 'subsynaptic structures.'

We added the following text to explain, in lines 181-183: "A higher CoV indicates an uneven distribution of tdTomato:Sec61β within the presynaptic terminal, with some areas showing higher concentrations than others (in contrast to the uniform, diffuse signal expected from fragmentation)." To avoid confusion with postsynaptic structures called the subsynaptic reticulum, we have removed the term "subsynaptic". The intended meaning is distinct structures found within the presynaptic terminal.

There's a mistake in Figure 6C and the associated text. The summed percentage of the three phenotypic categories adds up to 110% for Atlastin mutants. *

The reviewer noted that the summed percentage of the three phenotypic categories in Figure 6C adds up to 110% for Atlastin mutants, which appears to be a mathematical error. However, this is not an error, but rather a reflection of our quantification methodology, in which a single bouton can exhibit more than one type of ER dynamics per movie recorded. Our quantification counts each phenotype independently, so boutons displaying multiple phenotypes contribute to more than one category. This approach provides a more comprehensive view of the range of ER dynamics present in Atlastin mutants, as restricting the analysis to mutually exclusive categories would underrepresent the complexity of the phenotypes observed. To make this point clear, we made the following change to the text in lines 257-259: "We note that the sum of these percentages exceeds 100% because one NMJ exhibited multiple phenotypes: one branch had a complete loss, while the other branch had no phenotype. These phenotypes were counted separately."

Figure 8: the ER looks fragmented in 1st instar controls and mutants. The authors should address this difference from more mature NMJs.

We would like to clarify that the bulk of experiments in this manuscript (including all ER dynamics, luminal marker redistribution, and membrane marker analyses discussed throughout the Results) were performed in 3rd instar larvae, which are more mature larval NMJ preparations standard in the field. Figure 8 was included specifically to test whether the Atlastin mutant phenotype we describe throughout the paper is also detectable at an earlier developmental stage, not to replace or reinterpret our primary findings.

Regarding the specific observation that the ER appears more fragmented in Figure 7F-H relative to the more mature NMJs shown elsewhere: this fragmentation, observed similarly in both control and Atlastin mutant 1st instar larvae, likely reflects technical challenges associated with dissecting these smaller, more delicate early-stage specimens rather than a genotype-specific effect. Because fragmentation occurred similarly in both genotypes, we could still reliably assess the redistribution of BiP:sfGFP:HDEL as our primary phenotypic readout in this experiment. We have added the following text (lines 306-309) to clarify this point: "Note that in 1st instar larvae, both normal networks in controls and residual networks in Atlastin mutants appeared more fragmented than in 3rd instar preparations, likely due to the technical challenges of dissecting these smaller, more delicate specimens. Since ER fragmentation occurred similarly in both genotypes, we could still reliably assess the redistribution of BiP:sfGFP:HDEL as our primary phenotypic readout.

The images in figure 9B do not seem representative of the quantification in Figure 9D. Specifically, the partial loss Atlastin NMJ appears to have recovered as fully as the complete loss Atlastin NMJ.

The images showed FRAP recovery across the entire bouton, but we photobleached only a small region within each bouton and quantified only this region. We have now added outlines to clearly delineate the specific FRAP regions that were analyzed in each image, which clarify that the partial loss Atlastin showed less recovery than the overall bouton. We have also reordered the figures to more clearly convey our message (Figure 9 is now Figure 8).

We also made a few changes to the paragraph on lines 347-350 to clarify our experimental reasoning: "We photobleached en passant boutons using a defined region of 6.8 x 7.8 microns (dashed box in Figure 8D) to ensure that BiP:sfGFP:HDEL could recover from the ER networks surrounding the FRAP region (Movies S20-S23)."

We also added this sentence to the figure legends of Figure 8: "The dashed boxes in (D) indicate areas that were photobleached and analyzed for recovery quantification in (E-F)."

Optional: An overexpressed luminal marker is displaced to the cytoplasm in Atlastin mutants. It would be interesting to know and increase the significance of the findings if the same is true of endogenous luminal proteins under biological stress conditions.

As noted in our response to Reviewer #1 suggested that Atlastin mutant synapses may exhibit ER stress. To address this, we examined levels of the ER chaperone BiP, a well-established ER stress marker whose expression increases during UPR activation. We first validated that our BiP antibody can detect changes in ER stress by feeding control larvae with 50mM DTT for 24 hours. We were unable to perform this experiment in Atlastin mutant larvae because they did not consume the DTT-treated food, as assessed by blue food coloring in the larvae's guts. These results are in Figure 10A. In the future, it will be of interest to establish a protocol to examine Atlastin mutants by feeding or treating larval fillets with DTT.

We measured BiP levels at NMJs of Atlastin mutants and found they were slightly increased compared to controls. Atlastin mutants co-expressing UAS-BiP:sfGFP:HDEL or UAS-tdTomato:Sec61b did not show significantly increased endogenous BiP levels, suggesting that transgene expression suppresses the mild ER stress response. We conclude from these experiments that Atlastin mutant synapses have mild ER stress. These results are in Figure 10B-E).

Optional: Applying this approach in stimulated conditions (high potassium, increased temperature) might reveal a greater activity-dependent role for Atlastin at synaptic terminals.

This is a very interesting idea, as we have only examined synapses at rest. However, this is beyond the scope of this paper.

Minor Comments

1. Line 16: Atlastin should be italicized.

Thank you for catching this typo. We have fixed it.

Figure 5A: Based on the relative intensities, it appears that control and mutant images are not contrast matched but this isn't stated.

Thank you for catching this omission. We added to the figure legend: "Control and Atlastin mutant images are not contrast matched."

Line 822: The number of static Atlastin mutant boutons used for analysis is missing.

Thank you for catching this omission. We have fixed this supplementary table.

Figure 9: The blue arrows are not annotated in the figure legend.

Thank you for catching this omission. We have fixed this figure legend.

Reviewer #2 (Significance (Required)):

Atlastin is linked to Hereditary Spastic Paraplegia (HSP) and this study changes our understanding of the compartment-specific impacts of its loss. This study reveals the importance of using both membrane and luminal ER markers to accurately interpret phenotypes as well as the importance of considering compartment-specific effects on ER. These findings represent significant mechanistic and conceptual advances. The lack of genetic rescue is a limitation and adding an investigation of an endogenous luminal protein under basal and stress conditions would add significantly to our understanding of Atlastin dysfunction in HSP. Notably, the in vivo imaging approach introduced here can be adapted broadly for live imaging of Drosophila larvae. Thus, this work will be of interest to both neuronal cell biologists and the wider Drosophila community. This review is based on our expertise in neuronal cell biology.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

In this manuscript, the authors investigate the structural dynamics of the endoplasmic reticulum (ER) in Drosophila neurons and examine the role of the ER-shaping protein Atlastin in ER morphology. Their discovery on the neuromuscular junction (NMJ)-specific contribution of Atlastin to ER integrity is intriguing and may provide valuable insights into the pathological mechanisms underlying Atlastin mutations associated with hereditary spastic paraplegia (HSP) and hereditary sensory neuropathy. The key observation on ER protein showing an aberrant cytoplasmic localisation in mutant cells appears convincing. Though this phenomenon's characterisation stays at the point of primary observation with its mechanics unclarified, establishing this new and unexpected functional rather than structural Atl effect is important and useful for the field. The observation that ER is structurally preserved in this mutant with absolute lack of Atl are also extremely useful.

It is unclear if the cytoplasmic localisation affects an exogenous overexpressed ER marker or endogenous protein would also appear in cytoplams, the authors should consider adding an immunostaining data to test that.

Authors offer speculations on potential reasons for the cyto localisation of the ER marker suggesting that relocation at the cell periphery specifically combined with slow clearance there is the most likely explanation (still unclear what stops the marker from spreading through the entire cell). They suggest that decrease in cotranslational translocation is unlikely as this would result in somatic accumulation of the marker. However, if the clearance in the periphery is less efficient than in soma, the accumulation there might reflect a compromised translocation. Any clarifying experiments, if practical, to directly demonstrate how ER proteins in relocates to the cytoplasm in atl mutant would help understanding better the phenomenon. For example, would proteasomal inhibition make the marker accumulate more across the cell? Authors also suggest links to ER stress. Would stress induction phenocopy the mutant?

Reviewer #3 asked whether defective proteasomal clearance underlies the cytosolic accumulation of BiP:sfGFP:HDEL in Atlastin mutants. We addressed this directly. First, proteasome function appears intact in the mutants: baseline ubiquitinated protein levels (FK1 antibody) were comparable between control and Atlastin mutants, and MG132 treatment produced a similar increase in ubiquitination in both genotypes, confirming both antibody specificity and normal proteasome activity. We then examined BiP:sfGFP:HDEL directly. In controls, MG132 caused the marker to accumulate at axons and presynaptic terminals, showing that it is normally cleared from these compartments by the proteasome. Critically, this accumulated marker remained associated with intact ER networks: MG132 did not induce diffuse cytosolic BiP:sfGFP:HDEL in any compartment (cell bodies, axons, or presynaptic terminals), even where levels rose substantially. Thus, blocking proteasomal clearance raises ER-localized marker but does not generate the cytosolic pool seen in Atlastin mutants, indicating that impaired clearance is not sufficient to cause the displacement phenotype. We separately noted that BiP:sfGFP:HDEL was already elevated in Atlastin mutant axons without MG132, paralleling the axonal tdTomato:Sec61β accumulation in Figure 4, consistent with reduced baseline clearance specifically in mutant axons, but this does not lead to cytosolic displacement. This experiment is now shown in Figure 11, described in Results (lines 445-475), and discussed in lines 576-581.

Minor comments:

Line 146:

"fast dynamics (Thank you for catching this mistake. We have corrected it.

Fig. 2D: The data representation of "Tubule displacement" image is unclear. The ER tubule indicated by the red arrow does not seem to show any changes over time (like static). time 0 in stamp appears behind the image.

Thank you for catching the typo. We have fixed it. Additionally, we added black arrows to highlight a tubule that is not moving, allowing the reader to compare it with the moving tubule. We also included a video of all types of ER tubule dynamics to ensure the reader can also look at the raw data (Movies S9-11).

• • Line 157-158 (and relevant method sections):

The definition of static and dynamic boutons is ambiguous. The author should describe in more detail this point including how long they observed the structure to define the changes in ER tubule dynamics.

We provide in the methods (lines 779-791) a detailed explanation of how we categorized boutons as dynamic or static. In addition, we added the following to explain in the results section how we defined static vs dynamic:

Old sentence: We qualitatively categorized boutons as "static" if we observed no change in ER network structure or "dynamic" if we observed at least one change.

New sentence in lines 143-147: "We imaged boutons for 40 sec at 0.92 sec intervals to capture ER dynamics over this observation period. Boutons were qualitatively categorized as "static" if we observed no detectable changes in ER network structure throughout the entire 40 sec imaging session, or "dynamic" if we observed at least one of the three defined dynamic events during this time window."

Fig. 2E: What n=75 and n=29 represent is unclear, are these the number of boutons in en passant and terminal subjected for qualitative analysis?

We removed these n values from the figure and added this information to the Supplementary Table 1, which contains detailed information about the genotype, statistical analysis, and number of larvae and NMJs analyzed.

Fig. 2: What the qualitative analysis represents is unclear, are the points pulled from different experiments?

The data in Fig. 2 E-F comes from movies acquired in the same experiment. The number of independent animals and NMJs imaged is described in Table 1.

* *Line 231: Regarding "...we found a small but significant reduction in dynamic boutons in Atlastin mutants (76%), ...", how do the authors assess significance. If proportion of static/dynamic ER in boutons was obtained from multiple experiments, it should be presented e.g. as in average {plus minus} standard deviation, or clarify that the proportion is representative of x independent experiments.

The videos used for this figure were acquired from a single experiment. We use a chi-square test to determine significance relative to the "expected" distribution of dynamics types from controls, as these are categorical rather than continuous data (see PMID 31145670). Information regarding genotype, statistical analysis and number of larvae and NMJs can also be found in Supplementary Table 1.

Line 267-269 and Fig. 6B: The author's conclusion that "Complete loss of ER network structure in NMJ of BiP:sfGFP:HDEL overexpressing Atl mutant" seem to be based on the lack of signal from luminal marker, which may be undetectable due to changes to tubular volume or marker loss to the cytoplasm, as suggested by the authors, while the membranous ER structure is intact. It would be useful to discuss this point and potentially add ER membrane-stained control.

We agree with the reviewer that Atlastin mutants categorized as 'complete loss mutants' do not actually lack ER at synapses. We think this is an important point so we added the following to the results in lines 251-254: "Note that the "Complete loss" phenotype in Atlastin mutants reflects the absence of detectable luminal marker signal in organized ER structures, not the complete absence of ER membranes, as demonstrated by our ER membrane marker tdTomato:Sec61β results."

We attempted to co-label the ER membrane and ER lumen, but these crosses yielded very few live larvae (in either controls or Atlastin mutants, and those that survived had severely deformed NMJs. We added Figure 6-Supplement showing the results of this experiment, and described them on lines 270-273.

Fig. 6C: In Atl mutant, why does the total of the proportion exceed 100% (10 + 45 + 55)?

The reviewer noted that the summed percentage of the three phenotypic categories in Figure 6C adds up to 110% for Atlastin mutants. This is not an error, but rather a reflection of our quantification methodology because a single bouton can exhibit more than one type of ER dynamics per movie recorded. Our quantification counts each phenotype independently, so boutons displaying multiple phenotypes contribute to more than one category. This approach provides a more comprehensive view of the range of ER dynamics present in Atlastin mutants, as restricting the analysis to mutually exclusive categories would underrepresent the complexity of the phenotypes observed. To make this point clear, we made the following change to the text in lines 257-259: "We note that the sum of these percentages exceeds 100% because one NMJ exhibited multiple phenotypes: one branch had a complete loss, while the other branch had no phenotype. These phenotypes were counted separately."

Fig. 9C, line 342-344: In FRAP experiment using CD8, it seems that the Partial loss Atl mutant shows slower recovery that control. There seems to be a mismatch in triangle symbols of Partial loss Atl mutant between legend and plot (one is filled and the other is empty). This should be clarified.

Thank you for catching this mistake. We have fixed the figure.

fig. 10 is a clever way to verify the cytoplasmic localization of the ER marker; however, its description and annotation can be improved, and it would be stronger if 4 curves in F for mutant and controls with the trap and normal were shown.

The reviewer suggested merging our graphs but we believe that keeping them separate is clearer.

Line 495: Drosophila have ReepA and ReepB, but not Reep1-4. If the authors discuss their speculation based on their observation (using Drosophila), the gene names should be unified in the same species, and explain the corresponding genes to mammalian cells.

We made the following changes to address the reviewer's concern about gene nomenclature consistency (lines 502-506): "These ER-derived vesicles are likely to involve ReepA and ReepB, the Drosophila orthologs of mammalian REEP1-4, which regulate ER vesicle formation in mammalian cells (67). Notably, while overexpression of Atlastin can regulate REEP vesicle fusion in mammalian systems (67), it is not essential for vesicle formation, suggesting similar regulatory relationships may exist between Atlastin and Reep genes in Drosophila."

Line 548; should UPR be Unfolded Protein Response?

Thank you for catching the typo. We have fixed it.

Reviewer #3 (Significance (Required)):

This study advances the understanding of how ER morphogens affect neuronal cells specifically, the lack of which limits researchers ability to comprehend the neuronal pathologies associated with ER structure-function. The observation on ER content aberrant localisation caused by the lack of key structural protein should be of a great interest for cell and neuronal biologists and researchers of the associated diseases and shows the field a new direction. Though, mechanistic details remain to be unraveled, it constitutes a fundamental, conceptual advance.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

In this manuscript, the authors investigate the structural dynamics of the endoplasmic reticulum (ER) in Drosophila neurons and examine the role of the ER-shaping protein Atlastin in ER morphology. Their discovery on the neuromuscular junction (NMJ)-specific contribution of Atlastin to ER integrity is intriguing and may provide valuable insights into the pathological mechanisms underlying Atlastin mutations associated with hereditary spastic paraplegia (HSP) and hereditary sensory neuropathy. The key observation on ER protein showing an aberrant cytoplasmic localisation in mutant cells appears convincing. Though this phenomenon's characterisation stays at the point of primary observation with its mechanics unclarified, establishing this new and unexpected functional rather than structural Atl effect is important and useful for the field. The observation that ER is structurally preserved in this mutant with absolute lack of Atl are also extremely useful.

It is unclear if the cytoplasmic localisation affects an exogenous overexpressed ER marker or endogenous protein would also appear in cytoplams, the authors should consider adding an immunostaining data to test that.

Authors offer speculations on potential reasons for the cyto localisation of the ER marker suggesting that relocation at the cell periphery specifically combined with slow clearance there is the most likely explanation (still unclear what stops the marker from spreading through the entire cell). They suggest that decrease in cotranslational translocation is unlikely as this would result in somatic accumulation of the marker. However, if the clearance in the periphery is less efficient than in soma, the accumulation there might reflect a compromised translocation. Any clarifying experiments, if practical, to directly demonstrate how ER proteins in relocates to the cytoplasm in atl mutant would help understanding better the phenomenon. For example, would proteasomal inhibition make the marker accumulate more across the cell? Authors also suggest links to ER stress. Would stress induction phenocopy the mutant?

Minor comments:

Line 146: "fast dynamics (<1 sec)" a velocity should be presented as distance/time

Fig. 2D: The data representation of "Tubule displacement" image is unclear. The ER tubule indicated by the red arrow does not seem to show any changes over time (like static). time 0 in stamp appears behind the image.

Line 157-158 (and relevant method sections): The definition of static and dynamic boutons is ambiguous. The author should describe in more detail this point including how long they observed the structure to define the changes in ER tubule dynamics.

Fig. 2E: What n=75 and n=29 represent is unclear, are these the number of boutons in en passant and terminal subjected for qualitative analysis?

Fig. 2: What the qualitative analysis represents is unclear, are the points pulled from different experiments?

Line 231: Regarding "...we found a small but significant reduction in dynamic boutons in Atlastin mutants (76%), ...", how do the authors assess significance. If proportion of static/dynamic ER in boutons was obtained from multiple experiments, it should be presented e.g. as in average {plus minus} standard deviation, or clarify that the proportion is representative of x independent experiments.

Line 267-269 and Fig. 6B: The author's conclusion that "Complete loss of ER network structure in NMJ of BiP:sfGFP:HDEL overexpressing Atl mutant" seem to be based on the lack of signal from luminal marker, which may be undetectable due to changes to tubular volume or marker loss to the cytoplasm, as suggested by the authors, while the membranous ER structure is intact. It would be useful to discuss this point and potentially add ER membrane-stained control.

Fig. 6C: In Atl mutant, why does the total of the proportion exceed 100% (10 + 45 + 55)?

Fig. 9C, line 342-344: In FRAP experiment using CD8, it seems that the Partial loss Atl mutant shows slower recovery that control. There seems to be a mismatch in triangle symbols of Partial loss Atl mutant between legend and plot (one is filled and the other is empty). This should be clarified

fig. 10 is a clever way to verify the cytoplasmic localisatoin of the ER marker, however its description and annotation can be improved, and it would be stronger if 4 curves in F for mutant and controls with the trap and normal were shown.

Line 495: Drosophila have ReepA and ReepB, but not Reep1-4. If the authors discuss their speculation based on their observation (using Drosophila), the gene names should be unified in the same species, and explain the corresponding genes to mammalian cells.

Line 548; should UPR be Unfolded Protein Response?

Significance

This study advances the understanding of how ER morphogens affect neuronal cells specifically, the lack of which limits researchers ability to comprehend the neuronal pathologies associated with ER structure-function. The observation on ER content aberrant localisation caused by the lack of key structural protein should be of a great interest for cell and neuronal biologists and researchers of the associated diseases and shows the field a new direction. Though, mechanistic details remain to be unraveled, it constitutes a fundamental, conceptual advance.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Summary

The endoplasmic reticulum (ER) is a continuous organelle that extends throughout neurons to regulate fundamental processes. The analysis of ER dynamics at synaptic terminals is limited by the challenge of imaging these structures at high resolution. In this manuscript, the authors use super-resolution (~170 nm) live imaging and a combination of membrane and luminal ER markers at the Drosophila larval NMJ, an important model synapse, to investigate dynamic ER architecture in vivo. They report a detailed characterization of the presynaptic ER organization and dynamics at wild-type and GTPase Atlastin mutant NMJs. Their analysis using the ER membrane marker tdTomato:Sec61b reveals the presence of an intact ER network in Atlastin mutants. This contrasts with the apparent ER fragmentation phenotype previously reported and replicated here when using a luminal marker. Their findings instead point to the progressive displacement of luminal proteins to the cytosol in Atlastin mutants specifically at synapses. The authors propose that the disruption of ER protein dynamics at synapses is a compartment-specific ER stress response. The manuscript is well written, results are clearly presented, and experiments are technically rigorous.

Major comments

1. The baseline ER phenotypes in Atlastin mutants are mild with complete loss of ER network only observed in terminal boutons. This interesting and unexpected result should be further confirmed by genetic rescue. The authors can use a UAS rescue line previously reported in PMID: 19341724.
2. Lines 204-7: It's not clear how a greater coefficient of variation indicates that the marker is more concentrated in subsynaptic structures or what is meant by 'subsynaptic structures.'
3. There's a mistake in Figure 6C and the associated text. The summed percentage of the three phenotypic categories adds up to 110% for Atlastin mutants.
4. Figure 8: the ER looks fragmented in 1st instar controls and mutants. The authors should address this difference from more mature NMJs.
5. The images in figure 9B do not seem representative of the quantification in Figure 9D. Specifically, the partial loss Atlastin NMJ appears to have recovered as fully as the complete loss Atlastin NMJ.
6. Optional: An overexpressed luminal marker is displaced to the cytoplasm in Atlastin mutants. It would be interesting to know and increase the significance of the findings if the same is true of endogenous luminal proteins under biological stress conditions.
7. Optional: Applying this approach in stimulated conditions (high potassium, increased temperature) might reveal a greater activity-dependent role for Atlastin at synaptic terminals.

Minor Comments

1. Line 16: Atlastin should be italicized.
2. Figure 5A: Based on the relative intensities, it appears that control and mutant images are not contrast matched but this isn't stated.
3. Line 822: The number of static Atlastin mutant boutons used for analysis is missing.
4. Figure 9: The blue arrows are not annotated in the figure legend.

Significance

Atlastin is linked to Hereditary Spastic Paraplegia (HSP) and this study changes our understanding of the compartment-specific impacts of its loss. This study reveals the importance of using both membrane and luminal ER markers to accurately interpret phenotypes as well as the importance of considering compartment-specific effects on ER. These findings represent significant mechanistic and conceptual advances. The lack of genetic rescue is a limitation and adding an investigation of an endogenous luminal protein under basal and stress conditions would add significantly to our understanding of Atlastin dysfunction in HSP. Notably, the in vivo imaging approach introduced here can be adapted broadly for live imaging of Drosophila larvae. Thus, this work will be of interest to both neuronal cell biologists and the wider Drosophila community. This review is based on our expertise in neuronal cell biology.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

In the present manuscript, the authors address an important question related to the ultrastructure and the dynamics of the ER in HSP. In contrast to previous studies, the authors here show (by using a membrane and luminal protein markers) that in the presynaptic terminals, the overexpressed BIP "mislocalizes" to the cytosol without affecting (or with minimal effect) the integrity of the ER membrane. Although they used an artificial system by overexpressing (overexpression) of BIP-sfGFP-HDEL (fused protein), the findings lack validation of the endogenous protein by biochemical and fluorescent tools.

Concerns:

I am worried about how the article is presented, mainly in the results section, as most of it refers to published data. The Results section is reserved for presenting new findings without external interpretation or comparison. The paper is written mainly as a comparison paper with other studies or relies on previous studies to strengthen their findings rather than coming up with novel findings. Up to figure 4, I missed the relevance of the new findings. The manuscript needs rewriting to emphasize its novelty and significance without comparing it to previous data. Moreover, the manuscript emphasizes the technology and the findings of the localization of the ER luminal proteins to the cytosol (which is not novel and was previously reported in other settings). Those two aspects were not given enough focus. Here are the main major and minor comments:

Major comments:

1. Lines 103-116 (first paragraph of the results section) describe mainly published data that is more suitable for the introduction section. It is annoying to refer to different published articles in the Results section to strengthen the results instead of showing them. The same goes for paragraphs two and three. Why mention those data in the Results section if they are already published and known?
2. Figure Legends-(in all Figures): The number of experimental repeats must be mentioned in the figure legends.
3. The way the figures are labeled is worrisome; supplementary figures are not ordered numerically.
4. The tubule extension in Figure 2D is not convincing. Is there a movie showing those changes? Better images are needed. It is essential to show which supplemental movie corresponds to which panel.
5. This is unnecessary in the results section: "To investigate the relationship between ER structure and function at synapses, we examined mutants of Atlastin, a GTPase that regulates ER tubule fusion. Drosophila has a single homolog while mammals have three Atlastin homologs, with Atlastin-1 enriched in the brain (Rismanchi et al., 2008)."
6. "This reduction in ER membrane marker intensity has also been observed in other HSP mutants, suggesting this is a common feature of ER shaping mutants and could indicate changes in ER membrane composition, integrity, or tubule thickness (P.rez-Moreno et al., 2023)." This comparison is important and should be shown in the same settings as for the Atlastin mutant rather than referring to published data.
7. Does the distribution of the luminal ER marker in Figure 6F diffuse due to mislocalization or reflux after being localized to the ER and then refluxed to the cytosol as was previously shown for the ER to Cytosol signaling (ERCYS) mechanism? Could you assess other ER-luminal protein localization biochemically? It is highly recommended to look at another soluble ER-protein localization in the Atlastin mutant without overexpression, which can be an artifact
8. "(data not shown)" in line 288. This affects the process of judging those data.
9. In comparison to Summerville et al. (2016) in Figure 7, the experiment was not done in the same way. It is important to keep the same settings for comparison
10. Does the Atlastin mutant induce the unfolded protein response and stress within the ER? It is necessary to look for UPR markers in those settings. It was shown previously that ER stress leads to protein reflux from the ER to the cytosol. Is there a difference in the ER stress markers in the presynaptic terminal?
11. It is important to add biochemical experiments to show that no fragmentation of the ER membrane occurred. It can be simply demonstrated by looking at the redox state of the ER, which would change if it were mixed with the reducing cytosol. Moreover, this can be shown by using an ER-targeted redox-sensitive fluorescent protein that is tethered to the ER membrane to follow changes in the redox state of the ER.

Minor comments:

1. It is important to call figures by order. Figure 2C is called before 2A-B. Figure 2B is called before Figure 2A.
2. Figure legends (Figure 2): "The same control dataset used in E-G was used in Figure 5 and Figure 5_Supplement." Why is this relevant?
3. Figure 4F is called before Figure-4D-E which are not called.
4. Figure 5B is called before the previous ones. Same for Figure 5A supplement.

Referees cross-commenting

I agree with the comments raised by reviewer2 and 3. Basically it is highly important to validate those data by genetic rescue. Moreover, it is essential to know the source of the displaced luminal marker to the cytosol. Is it mislocalization or it is a reflux of pre-existing protein to the cytosol after insertion to the ER. It is also recommended by me and the reviewers to test the endogenous protein rather than overexpression.

Significance

General assessment: This interesting paper shows that proteins can escape the ER under special conditions. However, the authors need more evidence to show that and rely less on the overexpression system, especially of BIP-GFP, which can cause proteostasis stress within the ER.

Advance: The results have been oversimplified in their explanations, and some points and complexities of the study need to be addressed further to make the most of them. These are often some of the more interesting concepts in the paper. I think many points can be addressed in the text by the authors being clear and concise with their reporting. At the same time, other experiments would turn this paper from an observational one into a very interesting mechanistic one. This paper is based on previously published articles from the group and other groups, and it is a nice progression. However, as mentioned, this paper depends primarily on published data, and the novelty is somehow lost between all the comparisons to other published data instead of emphasizing that. Without a substantial mechanistic improvement, the paper would remain observatory.

Audience: The microscopy tools can be great addition to researchers in the field to monitor protein trafficking especially Cell biologists (basic research)

My expertise: ER homeostasis, protein trafficking, cell biology

I always found it very interesting to hear from or about someone who stepped up in a crisis or high stress scenario. Especially if that person is not regularly in a leadership role. In a high-pressure group setting finding the nerve to not only take initiative but also direct others is impressive to me. Its also interesting that the others who didn't step up are usually willing and receptive to the directions of someone else even if they are a stranger. It seems the higher the stakes the quicker everyone falls in line and moves once someone has stood up as a leader even if it's a self-appointed role.

For all figures:

I think the font size for the axis labels adn legends are too small.

I also wonder if it would be more intuitive to show plots of proportions over time rather than cumulative coounts. So on top you'd have proportion uninfects vs infected with susceptible strain vs resistant strain. On the bottom you'd have proportion in the NI-T, NI-NT, I-T, I-NT categories. (I know that wouldn't let you visualize the inter-quartile spread though .. hmm).

The long and short is that I think you want to decide what the key message is that you want readers to understand from each figure and then plot the results in a way that is as easy as possible to see that result. Also state that key result in the legend. As it stands, I'm not sure that cummulative doses is germane to your key message(s).

I'd mention how the leaky balloon model parameters for these antibiotics compare. Are they the same?

What other attributes can help with decision making? I'm getting a little lost ...

Explain why this is this more realistic - is the idea that there is no update to environmental conditions until 10 patients are simulated?

It's unclear what aspects of these features are / aren't observed

Not sure what this means. I thought the ground truth is simply which patients are vs aren't infected?

For all three scenarios: I'd be more explicit about what this means. I.e. does it mean the agents know if people are high vs low risk, and that high risk ahs mu of 0.84 while low risk has mu of 0.56?

Can agents tell if patients are in the high vs low risk groups? If so - I'd remind readers of that. If not, then how else can the ground-truth stratification be exploited?

If a Gaussian distributio nis used, it seems that the probability of infection could fall outside of [0,1]?

'recent'

maybe 'antibiograms and clinical practice guidelines'

just 'homongeneous'

In the classic sense, 'Experiments' test hypotheses. So I might call these 'Simulation Sets' instead

can just say 'constant'

I would also comment on the chosen reward function. What values were used and why?

Typo

Maybe 'patient information and variably-updated clinical practice guidelines, but do not update behavior based on the most current data on clinical performance.'

Maybe add ('as shown in the results section')

I might explain that a bit more. Is this a penalty for how much the 'optimized' parameters vary across seeds?

It is unclear what the output is

they are flower organ

inside of the a carpel, The receptacle will become the red culinary “fruit,”

it wil probably dies, because its cut of the xylem and phloem supply connections

The most exterior layer of cells in a five-year-old woody perennial plant stem is called the cork (or phellem)

Annual rings originate from the seasonal variation in the activity of the vascular cambium, which alters the size and density of the secondary xylem cells it produces throughout the yea

Trace the boundary between the first, second, and third-year woodIdentify the vascular rays that transport water radiallyLocate the crushed remnants of the primary phloem

it yes get divided and other sister cells become derivative cell

look for the external structure and tissue arrangement root apical has a root cap and shoot apiece meristem has the leaf permordia

De ahi saca Gillespie el nùmero.

The attempt to assimilate African American students into using only standard English?

💻/thinkpad/🧊/me/📓/2026/7/0/9/do/search/metadesign/

search-metadesign@hypothesis

This is the main idea of the article. Multilingualism should be seen as a strength rather than a weakness. I can use this in my research about language and identity.

This connects to my own experience learning English while continuing to speak Spanish. Supporting children's home languages creates better learning environments.

messages IndyLab - 1

• 2
• 3

lifelong

kind of Life Box

Seeding the Indy Learning Commons

It will be better to add some NOTE that this works only when Object Storage is configured as Direct Mode in VBR side.

It doesn't increase restore speed, rather increase time when agent tries to find out which VBR Endpoint is accessible and mounts the backup to local machine.

I noticed that step indeed is called "Package", but Windows User Page page names it as "Option". Better merge it.

Better we align with Windows text

This variant was found in Patient 074752 in cis with a nonsense (c.6088C>T) and in trans with an intronic variant (c.4253+43G>A); neither of which has been curated yet by the ABCA4 VCEP

Not sure if this variant is c.1988G>A or c.1989G>A.

This variant is listed for Stargardt DNAID#067322 in trans with c.5603A>T p.(Asn1868Ile). No phenotype information provided.

Case#: Female, family #5, Patient #20

DiseaseAssertion: STGD

FamilyInfo: Proband's sister presented with same clinical prognosis. Sister diagnosed with pattern dystrophy and photoaversion at age 57, with difficulty seeing at night. Sister has nuclear sclerotic and cortical cataracts in both eyes.

CasePresentingHPOs: HP:0000662, HP:0000603, HP:0000603, HP:0000493

CaseHPOFreeText: Proband presented with localized blur at age 62, (late onset) in her left eye. BVCA 20/20-3 and 20/20-2 at age 70. Also has macular lesions with stage 2 fundus flecks.

CaseNotHPOs: n/a

CaseNotHPOFreeText: n/a

Genotyping Method: Genotyping performed at Columbia University, sequencing technology used is not disclosed.

PreviouslyPublished: n/a

Variant: p.N18681, IVS36:c.5196+1G>A

ClinVar: M2) 99067, M6) 99351

CAID: N/A

SupplementalData: Fig 1: Pedigree illustrating ABCA4 variants and the associated Stargardt phenotype for 5 families. Proband Labeled w/ white arrow for each family. Fig 2: retinal scan measuring melanin in 4 patients of family 2. Panel shows bull's-eye ring of RPE atropy. Fig 3: Macular SD-OCT line profile from b-scans. Reflectivity plotted against function of retinal depth. Table 1: table shows patients with p.N18681 variant, type of mutation, and pathogenicity class. Table 2: Patients, age on-set and first symptom

Case#: Patient 225, Female, age of onset 7 y.o, Poland

DiseaseAssertion: STGD-1

FamilyInfo: no given family information.

CasePresentingHPOs: HP:0007722, HP:0000608, HP:0025158

CaseHPOFreeText: RPE atrophy, macular degeneration, central hyper-autofluorescence in fundus autofluorescence

CaseNotHPOs: n/a

CaseNotHPOFreeText: n/a, non-proband identified HPO's mentioned, but not assignable to individual proband.

Genotyping Method: DNA isolated from peripheral blood from patients and relatives via MagNA Pure 24, samples screened with MIPs targeting 108 genes involved in pathogensis of IRD's. PCR completed on library, analysed with NGS fragment analysis kit.

PreviouslyPublished: yes

Variant: c.[1622T>C;3113C>T]

ClinVar: 99067, 7894

CAID: n/a

gnomeAD 0.0001266 allele frequency

SupplementalData: Fig1: List of families displaying pseudo-dominant inheritance. Fig2: Number of alleles for most common variants.

It is known that patients harbouring p.(Gly1961Glu) or p.(Asn1868Ile) allele share some common clinical characteristics and present with a milder disease phenotype than patients carrying other alleles. A typical feature of patients with p.(Gly1961Glu) is BEM, which is otherwise present in around 20% of all STGD1 patients.

It is known that patients harbouring p.(Gly1961Glu) or p.(Asn1868Ile) allele share some common clinical characteristics and present with a milder disease phenotype than patients carrying other alleles. A typical feature of patients with p.(Gly1961Glu) is BEM, which is otherwise present in around 20% of all STGD1 patients.

ABCA4 variant revealed but not target of research and doesn't seem to amount to anything Variantc.3113C>T p.(Ala1038Val)

ABCA4 variant revealed but not target of research and doesn't seem to amount to anything Variantc.3113C>T p.(Ala1038Val)

PatientID: 10

KindredID: 10

Case: M, 22Y0M, Caucasian

DiseaseAssertion: VHL

FamilyInfo: Positive family history.

CohortInfo: Case series of 14 patients with definite or presumed von Hippel-Lindau disease and retinal vascular proliferation.

CasePresentingHPOs: HP:0002011, HP:0001732, HP:0007850, HP:0012210, HP:0009711 (Morphological central nervous system abnormality, abnormality of the pancreas, retinal vascular proliferation, abnormal renal morphology, retinal capillary hemangioma)

CaseHPOFreeText: At age 12, his left eye had a large juxtapapillary vascular complex extending from the nerve along the superior arcade that was associated with dense fibrovascular tissue. During the next 29 months, this fibrovascular complex enlarged, extending into the center of the macula and causing a decrease in visual acuity to 20/80. The patient underwent pars plana vitrectomy and membrane peel. When the patient was reexamined 7 years after his surgery, there was no recurrence of the lesion. In the patient's contralateral right eye, 3 typical peripheral RCHs were observed during this 7-year follow-up. At age 14 years, he was also noted to have a small patch of superficial retinal vessels in the inferior retinal quadrant near the equator of the right eye that resembled an arteriovenous anastomosis.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: N/A

Variant: Partial Deletion

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: N/A

CAID: N/A

gnomAD: N/A

VariantEvidence: N/A

MutationType: deletion

CivicName:Null (Partial deletion)

MultipleGeneVariants: N/A

GroupID/ KindredID: 28

PatientID: II

Case: 24Y0M, Sex unknown, Polish

DiseaseAssertion: VHL

FamilyInfo: Family 28 consisted of 4 members; one relative (age 27Y) was found with multiple cerebellar HABs at 25Y, and multiple spinal HABs, another (40Y) diagnosed at 30Y with multiple cerebellar HABs, and multiple spinal HABs, and a final relative (62Y), diagnosed with a single HAB.

CasePresentingHPOs: HP:0006880 (cerebellar hemangioblastoma)

CaseHPOFreeText: Patient was diagnosed with a single cerebellar HAB at 24Y

GroupNotHPOs: HP:0009713; HP:0009711; HP:0005584 (spinal hemangioblastoma, retinal capillary hemangioma; renal cell carcinoma)

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: N/A

Variant: complete deletion of VHL gene

CaseProblemVariantFreeText: N/A

LegacyVariant: N/A

ClinVarID: N/A

CAID: N/A

gnomAD: N/A

VariantEvidence: Haplotype analysis information in Table 3

MutationType: deletion

CivicName: deletion

MultipleGeneVariants: N/A

GroupID/ KindredID: 22

Case: Age unknown, Sex unknown, Chinese

DiseaseAssertion: hemangioblastoma

FamilyInfo: No family history. Genetic testing of their parents confirmed a de novo mutation.

CasePresentingHPOs: HP:0010797 (hemangioblastoma)

CaseHPOFreeText: N/A

CaseNotHPOs: HP:0002666; HP:0005584; HP:0009711; HP:0001732 (pheochromocytoma; renal cell carcinoma; retinal capillary hemangioma; pancreatic lesion)

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: N/A

Variant: Exon 1 deletion

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVar: N/A

CAID: N/A

gnomAD: N/A

VariantEvidence:N/A

MutationType: exon_loss_variant

CivicName: Exon 1 Deletion

MultipleGeneVariants: N/A

PatientID: unknown (brother)

KindredID: A

Case: M (deceased), Age unknown, Turkish

DiseaseAssertion: assumed VHL

FamilyInfo: 5 family members are grouped with this deletion (ages 16-37Y; mean 31Y). VHL was clinically diagnosed in, at minimum, the proband. See Fig. 2 for family pedigree. This patient and his one brother were not tested for the DNA deletion however it is assumed by the authors due to the segregation observed.

CasePresentingHPOs: HP:0010797 (hemangioblastoma)

CaseHPOFreeText: N/A

CaseNotHPOs: HP:0009711; HP:0002666; HP:0005584 (retinal capillary hemangioma; pheochromocytoma; renal cell carcinoma)

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: N/A

Variant: Deletion of exons 1 and 2

CaseProblemVariantFreeText: N/A

LegacyVariant: N/A

ClinVarID: N/A

CAID: N/A

gnomAD: N/A

VariantEvidence: not tested for the DNA deletion however it is assumed by the authors due to the segregation observed.

MutationType: exon_loss_variant

CivicName: exon 1-2 deletion

MultipleGeneVariants: N/A

GroupID/ KindredID: F28

Case: Sex unknown, 23Y0M, Spanish

DiseaseAssertion: assumed VHL

FamilyInfo: familial antecedents found; unknown of any additional features of index case relatives

CasePresentingHPOs: HP:0000107; HP:0009711; HP:0006880; HP:0006770; HP:0002666 (renal cysts, retinal capillary hemangioma, cerebellar hemangioblastoma, clear cell renal cell carcinoma, pheochromocytoma)

CaseHPOFreeText: unilateral pheo, bilateral ccRCC and multiple lesions were found with the patient’s renal cysts. Age of onset is 23Y0M.

CaseNotHPOs: HP:0001737 (pancreatic cysts)

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: N/A

Variant: rearrangement (unspecified)

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: N/A

CAID: N/A

gnomAD: N/A

VariantEvidence: Southern blot positive

MutationType: rearrangement_region

CivicName: Rearrangement

MultipleGeneVariants: N/A

PatientID: 246

KindredID: 246

Case: Sex Unknown, 26Y0M, Ethnicity Unknown

DiseaseAssertion: VHL

FamilyInfo: N/A

CasePresentingHPOs: HP:0010797; HP:0009711; HP:0006770 (Hemangioblastoma, Retinal capillary hemangioma, Clear cell renal cell carcinoma)

CaseHPOFreeText: CNS and retinal hemangioblastoma and clear cell renal cell carcinoma.

CaseNotHPOs: HP:0002666; HP:0000107; HP:0001732 (Pheochromocytoma, Renal cyst, Abnormality of the pancreas)

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished:N/A

SupplementalData: Table S1, S2 and S3

Variant: Partial Deletion (0.7kb)

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: N/A

CAID: N/A

gnomAD: N/A

VariantEvidence: N/A

MutationType: deletion

CivicName: Partial deletion of 0.7kb

MultipleGeneVariants: N/A

PatientID: IX-I

KindredID: 9

Case: M, 68Y0M, Ethnicity Unknown

DiseaseAssertion: VHL

FamilyInfo: No family history reported.

CasePresentingHPOs: HP:0006748; HP:0002668; HP:0030405; HP:0001737; HP:0006880 (adrenal pheochromocytoma, paraganglioma, pancreatic endocrine tumor, pancreatic cyst, cerebellar hemangioblastoma)

CaseHPOFreeText: Patient presented with adrenal pheochromocytoma, paraganglioma, pancreatic endocrine tumor, pancreatic cyst, and cerebellar hemangioblastoma at age 55Y0M and was diagnosed with VHL type 2A (see table 2).

CaseNotHPOs: HP:0000107; HP:0005584 (renal cyst; renal cell carcinoma)

CaseNotHPOFreeText: Patient negative for renal cell carcinoma and renal cysts.

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: N/A

Variant: complete deletion

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: N/A

CAID: N/A

gnomAD: N/A

VariantEvidence: N/A

MutationType: deletion

CivicName: NULL (deletion)

MultipleGeneVariants: N/A

Performed a meta-analysis of all data published on the rs2710102 CNTNAP2 variant up to 2015, and included Anney et al, 2012 (PMID: 20663923), Toma et al., 2013 (PMID: 23277129), Sampath et al, 2013 (PMID: 24147096), Poot et al., 2014 (PMID: 25337070 ), plus there current cohort.

No significant increase in cases vs controls for CNTNAP2 rs2710102

While this data does help support Contradictory evidence for CNTNAP2 involvement in autism, the lack of reporting of the total number of controls, as well as the number of cases and controls with the SNP prevents me from curating this information within the ClinGen gene curation interface.

Performed a meta-analysis of all data published on the rs7794745 CNTNAP2 variant up to 2015, and included Arking et al., 2008 (PMID:18179894), Li et al 2010 (PMID: 20414140), Anney et al, 2012 (PMID: 20663923), Toma et al., 2013 (PMID: 23277129), Sampath et al, 2013 (PMID: 24147096), plus there current cohort.

No significant increase in cases vs controls for CNTNAP2 rs7794745

Genotyped for the two common SNPs in CNTNAP2 rs2710102 rs7794745

CaseControlLabel: Toma 2013 CNTNAP2 rs7794745 analysis

CaseCohortLabel: 322 Autism patients

ControlCohortLabel: 524 controls

CaseCohortDisease: MONDO:0005260 (Autism)

CaseCohortPhenotype:

CaseCohortPhenotypesFreeText: Authors indicate that all individuals fulfilled DSM-IV criteria for autism or Asperger disorder. Additionally, some cases included where diagnosed with pervasive developmental disorder they may or may not have been characterized based on ADI-R and ADOS-G.

CaseControlNOTPhenotype:

CaseControlNOTPhenotypeFreeText:

CaseDemographics: 269 men, 53 women, Average age= = 17y.o. of Spanish and/or Caucasian descent.

ControlDemographics: controls were noted to be sex-matched and unrelated, however no numbers or ages were indicated. Sample obtained from the Blood and Tissues Bank of Hospital Universitari Vali d'Hebron.

CaseGenotypingMethod: One SNP, rs7794745, was genotyped using PCR-RFLP, from DNA obtained from peripheral blood lymphocytes (salting out method).

ControlGenotypingMethod: One SNP, rs7794745, was genotyped using PCR-RFLP, from DNA obtained from peripheral blood lymphocytes (salting out method).

CasePower: 312/322

ControlPower: 505/524

CaseAddInfo: Other genetic variation is not noted.

ControlAddInfo: Other genetic variation is not noted.

CaseControlStudyType: Single Variant Analysis

CaseControlDetectionMethod: Cases and controls genotyped for single variant

CaseControlStats: p-value= 0.87

CaseControlBias: Cases and controls are ethinically matched, and noted to be sex-matched.

CaseControlComments:

GWAS identified rs2799573 as being associated with five disorders (autism spectrum disorder, attention deficit-hyperactivity disorder, bipolar disorder, major depressive disorder, and schizophrenia)

This paper is flagged in Mastermind as containing this variant. The rs12720449 ID is mentioned in the supplemental info under the controls tab, but the specific variant is not listed

Case: Multiple individuals with the same GRIN2B variant (the mean age was 76 ± 11 SD years (Range: 38–101), the mean age at PD onset was 67 ± 12 SD years (Range: 32–94), and 421 patients (63%) were male).

Disease Assertion: Parkinson's disease

FamilyInfo: None

CasePresentingHPOs: HP:0001300

CaseHPOFreeText: Parkinson's disease

CaseNotHPOs: HP:0001250

CasePreviousTesting: Polymorphism genotyping on Sequenom iPLEX ® platform

PreviouslyPublished: No

Variant: rs7301328

ClinVarID: 129205

Case#:Not assigned

DiseaseAssertion:MPS type I with evidence of cognitive impairment defined as a score of at least 1 SD below mean on IQ testing or in one domain of neuropsychological function; attenuated MPS I

FamilyInfo:No information

CasePresentingHPOs:HP:0100543

CaseHPOFreeText:

CaseNotHPOs:HP:0032557

CaseNotHPOFreeText:Subjects were excluded if they had undergone hematopoietic stem cell transplantation, were unable to comply with study procedures, had significant lumbar pathology precluding access to the intrathecal space via lumbar puncture, or significantly impaired spinal CSF flow as detected on a nuclear medicine flow study as part of screening.

CaseEnzymeAssay:No information

CaseUrineGAGs:No information

CaseERT:Yes

CaseBMT:No (exclusion criterion)

Variant1:L238Q

Variant1ClinVarID:265418

Variant1CAID:

Variant2:W402X

Variant2ClinVarID:

Variant2CAID:

AdditionalVariants:N/A

ParentalGenotype:Not specified

PreviouslyPublished

Case#:Not assigned

DiseaseAssertion:MPS type I with evidence of cognitive impairment defined as a score of at least 1 SD below mean on IQ testing or in one domain of neuropsychological function; attenuated MPS I

FamilyInfo:No information

CasePresentingHPOs:HP:0100543

CaseHPOFreeText:

CaseNotHPOs:HP:0032557

CaseNotHPOFreeText:Subjects were excluded if they had undergone hematopoietic stem cell transplantation, were unable to comply with study procedures, had significant lumbar pathology precluding access to the intrathecal space via lumbar puncture, or significantly impaired spinal CSF flow as detected on a nuclear medicine flow study as part of screening.

CaseEnzymeAssay:No information

CaseUrineGAGs:No information

CaseERT:Yes

CaseBMT:No (exclusion criterion)

Variant1:L238Q

Variant1ClinVarID:265418

Variant1CAID:

Variant2:63delC

Variant2ClinVarID:

Variant2CAID:

AdditionalVariants:N/A

ParentalGenotype:Not specified

PreviouslyPublished

Case#:Not assigned

DiseaseAssertion:MPS type I with evidence of cognitive impairment defined as a score of at least 1 SD below mean on IQ testing or in one domain of neuropsychological function; attenuated MPS I

FamilyInfo:No information

CasePresentingHPOs:HP:0100543

CaseHPOFreeText:

CaseNotHPOs:HP:0032557

CaseNotHPOFreeText:Subjects were excluded if they had undergone hematopoietic stem cell transplantation, were unable to comply with study procedures, had significant lumbar pathology precluding access to the intrathecal space via lumbar puncture, or significantly impaired spinal CSF flow as detected on a nuclear medicine flow study as part of screening.

CaseEnzymeAssay:No information

CaseUrineGAGs:No information

CaseERT:Yes

CaseBMT:No (exclusion criterion)

Variant1:65delC

Variant1ClinVarID:

Variant1CAID:

Variant2:H240R

Variant2ClinVarID:947028

Variant2CAID:

AdditionalVariants:N/A

ParentalGenotype:Not specified

PreviouslyPublished

PMID:34884460

GeneName:GRIN1

ClinVarID:209159

ClinVarID:397504

ClinVarID:1076783

ClinVarID:658023

ClinVarID:430264

ClinVarID:487503

ClinVarID:691274

Case:Patient 1, Male, 4 y/o, German and Thai

DiseaseAssertion:FOXG1 syndrome

FamilyInfo: Non-consanguineous parents. De novo. Patient 1 has maternally inherited duplications at 15q21.12(47292250-47309868)x3 and Xp22.31(6451676-8115124)x3 of unknown clinical significance.

ParentalGenotype:Not provided

CasePresentingHPOs:HP:0002197, HP:0005484, HP:0011344, HP:0001252, HP:0020045,HP:0000540, HP:0010808, HP:0002019, HP:0003781, HP:0012741, HP:0002421, HP:0000748, HP:0003763, HP:5200017, HP:0025112, HP:0000957

CaseHPOFreeText: At 19 months, could not sit independently and no speech development. Stereotypic hand and head movements. High pain tolerance. X-ray at age 2 showed broad ribs. Improved head control by age 4.

CaseNotHPO:HP:0410263, HP:0002376

CaseNotHPOFreeText:Dysmorphic features. breathing abnormalities

CasePreviousTesting:No previous testing

PreviouslyPublished:Not previously published

GenotypingMethod:Mate-pair sequencing, Sanger sequencing

Gene:FOXG1

Variant:46,XY,t(9;14)(q22.3;q11.2)dn

HGVS:Not provided

ClinVarID:426096

gnomAD:Not provided

Case:Patient 3, Male, 1 y/o, Brazilian

DiseaseAssertion: FOXG1 syndrome

FamilyInfo: Non-consanguineous parents

ParentalGenotype: No family testing mentioned.

CasePresentingHPOs: HP:0000252, HP:0001252, HP:0000486

CaseHPOFreeText: Developed microcephaly within 1 year. Developmental delay, unable to sit without assistance at 1 year old. Central Nervous System (CNS) MRI showed a volumetric reduction of the cerebral parenchyma, hypomyelination, and hypoplasia of corpus callosum mainly involving the anterior part and prominent lateral ventricles

CaseNotHPO: HP:0030917

CaseNotHPOFreeText: Normal length, weight, HC at birth. No retinal abnormalities.

CasePreviousTesting: Not asserted.

PreviouslyPublished: Not previously published.

GenotypingMethod: Sanger sequencing, mate-pair sequencing

Gene: FOXG1

Variant: 46,XY,t(2;14)(q37;q11.2)dn

HGVS: Not provided.

ClinVarID:426094

gnomAD:Not provided

Case:Patient 2, Female, 5 y/o, Brazilian

DiseaseAssertion:quadriplegic cerebral palsy with axial hypotonia and distal spasticity

FamilyInfo: Non-consanguineous parents. Parents had normal karyotypes. Older sister was clinically normal.

ParentalGenotype: Normal karyotypes for both parents.

CasePresentingHPOs: HP:0002098, HP:0010959, HP:0008755, HP:0002090, HP:0032661, HP:0001274, HP:0011344, HP:0001249, HP:0001344, HP:0000748, HP:0034435, HP:0000486, HP:0000483, HP:0200136

CaseHPOFreeText: Dilated superficial blood vessels seen on brain MRI.

CaseNotHPO:N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: Not asserted

PreviouslyPublished: Not previously published

GenotypingMethod:Mate-pair sequencing, Sanger sequencing, chromosome analysis

Gene: FOXG1

Variant: 46,XX,t(4;14)(q27;q13)dn

HGVS: Not provided

ClinVarID: 426095

gnomAD: Not provided

EXTL3, HGNC:3518

Neuro-immuno-skeletal Dysplasia Syndrome

OMIM: 617425

Selection A- Gene with same function implicated in disease

ExperimentName: Moye et al. 2018

GOterm: GO:0051179 (localization)

FunctionalEvidence: It was discovered that without ARL2BP, the photoreceptor OS does not develop normally or have correct DMT formation, axoneme extension, and disk alignment.

HGNC: HGNC:10295 (RPGR), HGNC:10263 (RP1), HGNC:6816 (MAK)

GeneEvidence: ARL2BP, RPGR, RP1 and MAK are all localized within the ciliary basal body and rootlet.

SharedGeneImplication: RPGR was classified as having a "definitive" relationship with RP 15 by the Retina GCEP. Refer to the curation for further details. RP1 was classified as having a "definitive" relationship with RP1 by the Retina GCEP. Refer to the curation for further details. MAK was classified as having a "definitive" relationship with MAK-related retinopathy by the Retina GCEP. Refer to the curation for further details.

Selection A- Gene with same function implicated in disease

ExperimentName: Aprea et al. 2023

GOterm: GO:0001535 (radial spoke head)

FunctionalEvidence: Taken together, our results indicate that the ruler proteins CCDC39 and CCDC40, the radial spoke head proteins RSPH1 and RSPH9 as well as the central pair associated apparatus proteins SPEF2 and HYDIN have a similar functional role in motile cilia and sperm flagella, as hypothesized in Figure 1.

HGNC: HGNC:12371

GeneEvidence: PMID: 24518672 documents how RSPH1 and RSPH9, which encode homologs of components of the ‘head’ structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. They both also have a relationship with PCD.

SharedGeneImplication: The gene-disease relationship between RSPH1 and PCD has been established and published in multiple papers. See Motile Ciliopathy GCEP curations of RSPH1 for more information.

Selection A- Gene with same function implicated in disease

ExperimentName: Wilken et al. 2024

GOterm: GO:0036156 (inner dynein arm).

FunctionalEvidence: Both DNAH6 and DNAH1 are both IDA components and localized to the same region.

HGNC: HGNC:2940 (DNAH1).

GeneEvidence: Both DNAH6 and DNAH1 are both IDA components and localized to the same region.

SharedGeneImplication: DNAH1 was classified as having a "limited" relationship with PCD 37 and a “defintive” relationship with spermatogenic failure 18 by the Motile Ciliopathy GCEP. Refer to the curation for further details.

Selection A - Gene with same function implicated in disease

ExperimentName: Shapiro 2007 et al.

GOterm: GO:0051179 (localization)

FunctionalEvidence: RSPH3 and RSPH9 are both localized to radial spoke heads and have a similar phenotype profile.

HGNC: HGNC:21057.

GeneEvidence: PMID: 20301301 documents how RSPH9 and RSPH3, which encode homologs of components of the ‘head’ structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. They both also have a relationship with PCD.

SharedGeneImplication: The gene-disease relationship between RSPH9 and PCD has been established and published in multiple papers. See Motile Ciliopathy GCEP curations of RSPH9 for more information.

PMID: 8541851

GeneName: GARS1

HGNC: HGNC:4162

Disease: CMT2D

MonDO ID:

Inheritance: Autosomal Dominant

The source focuses on the clinical, immunological, and molecular features of Severe Combined Immune Deficiency (SCID) based on a multi-institutional experience from India. The specific gene studied in this case is IL2RG. The HGNCID for this gene is HGNC:6016. The sources provide information about a 6-month-old male patient with SCID. The patient's family history suggests a possible X-linked inheritance pattern. The sources note the absence of autoimmunity and malignancies in the patient. Information regarding the patient's variant, previous testing, and publication history is not available in the sources.

Case#:Patient 52, female, 3 years old

DiseaseAssertion:Neonatal/Infantile Epileptic Encephalopathy (NIEE)

FamilyInfo:DeNovo. The family is Chinese

ParentalGenotype:The authors only conducted singleton and not trio-based exome sequencing so the parents' exomes were not sequenced.

CasePresentingHPOs:HP:0011344, HP:0002069, HP:0007359, HP:0011097, HP:0100704, HP:0001332, HP:0002072, HP:0012171.

CaseHPOFreeText:Patient 52 presents with severe global developmental delay and epilepsy.

Patient 52 has generalized tonic/clonic/tonic-clonic seizures, focal seizures and spasms. Patient 52's seizure onset occurred at 3 months old.

Patient 52 has cortical visual impairment (CVI), dystonia, chorea, and hand-washing sterotypies.

Patient History

@ 3 months - Patient 52 had generalized tonic/clonic/tonic-clonic seizures.

Patient 52 was on 3 antiepileptic drugs at most recent follow-up visit which reduced seizure frequency by >50%.

CaseNotHPOs:Not provided

CaseNotHPOFreeText:Not provided

CasePreviousTesting:The authors selected a cohort of 31 patients with seizure cryptogenic Neonatal/Infantile Epileptic Encephalopathy (NIEE) and seizure onset before 24 months.

Exclusion criteria included: (1) Patients with a definite history of brain insult, malformation of cortical development, neurocutaneous and syndromal disorders, and confirmed or highly suspected neurometabolic disorders based on clinical and biochemical markers. (2) Patients with Dravet syndrome and epilepsy at infancy with migrating focal seizure were also excluded because the majority of variants are detected in the SCN1A (>85%) and KCNT1 (approximately 50%) genes.

Formal neuropsychological testing or best clinical assessment was used to classify patient development or intelligence.

PreviouslyPublished:Not previously published

GenotypingMethod:Whole Exome Sequencing (WES) variant results were filtered in a panel of 430 epilepsy-associated genes. After selection of variants from the 430-gene panel, the synonymous variants, variants with variant frequency <10%, and variants with allele frequency >1% were removed.

Gene:CDKL5

Variant:NM_003159.2 c. 1849delC (p. Arg617Valfs*4)

The authors state that the variant is a heterozygous frameshift deletion.

The authors state that this is a novel variant and is pathogenic.

HGVS:Not provided

ClinVarID:Not found

CAID:Not found.

gnomAD:Not found

MultipleGeneVariants:Not provided

Case#:Patient 7, male, 5 years old

DiseaseAssertion:Neonatal/Infantile Epileptic Encephalopathy (NIEE)

FamilyInfo:The family is French/Chinese

ParentalGenotype:The variant was inherited from Patient 7's asymptomatic mother.

CasePresentingHPOs:HP:0010864, HP:0012758, HP:0000729, HP:0007359, HP:0002069, HP:0032794, HP:0001250, HP:0000252.

CaseHPOFreeText:Patient 7 presents with severe intellectual disability, developmental slowdown, and various seizure types. Patient 7 has Autistic Spectrum Disorder (ASD) and microcephaly.

Patient History

@ 12 months - Patient 7 presented with seizures.

Patient 7 developed additional seizure types including: focal seizures with/without generalization, generalized tonic/clonic/tonic-clonic seizures, myoclonic seizures, and hypomotor seizures.

Patient 7 was on two antiepileptic drugs at most recent followup visit which reduced seizure frequency by >50%.

CaseNotHPOs:Not provided

CaseNotHPOFreeText:Not provided

CasePreviousTesting:The authors selected a cohort of 31 patients with seizure cryptogenic Neonatal/Infantile Epileptic Encephalopathy (NIEE) and seizure onset before 24 months.

Exclusion criteria included: (1) Patients with a definite history of brain insult, malformation of cortical development, neurocutaneous and syndromal disorders, and confirmed or highly suspected neurometabolic disorders based on clinical and biochemical markers. (2) Patients with Dravet syndrome and epilepsy at infancy with migrating focal seizure were also excluded because the majority of variants are detected in the SCN1A (>85%) and KCNT1 (approximately 50%) genes.

Formal neuropsychological testing or best clinical assessment was used to classify patient development or intelligence.

PreviouslyPublished:Not previously published

GenotypingMethod:Whole Exome Sequencing (WES) variant results were filtered in a panel of 430 epilepsy-associated genes. After selection of variants from the 430-gene panel, the synonymous variants, variants with variant frequency <10%, and variants with allele frequency >1% were removed.

Gene:SLC9A6

Variant:Hemizygous splice site NM_001042537.1 c. 794-2A>G was assessed by the authors to be likely pathogenic.

HGVS:Not provided

ClinVarID:Not found

CAID:CA414750320

gnomAD:Not found

MultipleGeneVariants:Not provided

Case: Patient 1, female, 2 years old

DiseaseAssertion: Rett Syndrome

FamilyInfo: DeNovo. Patient 1 was the second child of a nonconsanguineous Chinese couple. Patient 1 was born at full term with a birth weight of 3.83 kg.

ParentalGenotype(s):Not provided

CasePresentingHPOs:HP:0011344, HP:0001250, HP:0001252, HP:0000252, HP:0000748, HP:0003763, HP:0005469, HP:0000486, HP:0012171.

CaseHPOFreeRext:Patient 1 presents with severe global developmental delay, epilepsy, and hypotonia. The Patient History contains all of Patient 1's phenotypes along with the progression of her condition.

Patient History

@ 3 months - Patient 1 had microcephaly (head circumference was less than 3rd percentile with a body weight and body height at 75th percentile). Patient 1 had hypotonia.

Patient 1 was given a metabolic screening, a muscle enzyme, and a brain tomography with normal results.

@ 6 months - Patient 1 had microcephaly, flat occiput, right divergent squint, and hypotonia. No syndromal diagnosis could be ascertained at that time.

@ 2 years - Patient 1 had epilepsy.

Patient 1 had severe global development delay.

Patient 1 was given an EEG which showed nonspecific background slowing, but no epileptiform abnormalities. Patient 1 was also given a brain MRI which showed mild thinning of corpus callosum without major structural defect. Patient 1 had no developmental regression but she developed stereotypical hand movements, bruxism, and occasional outburst of laughter.

Based on these phenotypes, Angelman/Rett Syndrome was suspected.

CaseNOTHPOs: HP:0002353.

CaseNOTHPOFreeText: Patient 1 was given an EEG which detected no epileptic abnormalites.

CasePreviousTesting: Genetic investigations (including methylation-specific multiplex ligation-dependent probe amplification (MS-MLLPA)) was conducted for Angelman Syndrome, UBE3A gene, MECP gene, and array CGH. These studies were negative. A FOXG1 related disease was suspected

PreviouslyPublished:Not previously published

GenotypingMethod: A FOXG1 gene test showed a de novo frameshift pathogenic mutation FOXG1 {NM_005249.3} c. 396_397ins26; FOXG1{NP_005240.3} :(p. Gly133TRPfs*68) which confirmed the diagnosis of a FOXG1 related congenital variant of Rett Syndrome.

Gene:FOXG1

Variant: (NM_005249.3) c. 396_397ins26 (p. Gly133Trpfs*68)

HGVS:Not provided

ClinVarID:Not found

CAID:Not found

gnomAD:Not found

Case:Patient 2, female, Chinese

DiseaseAssertion:Global delay

FamilyInfo:Patient 2 was the first child of nonconsanguineous Chinese couple born at 38-week gestation. The mother had gestational diabetes mellitus that required insulin therapy.

ParentalGenotype:Not provided

CasePresentingHPOs:HP:0000365, HP:0020049, HP:0000252, HP:0001263, HP:0012171, HP:0000154, HP:0000708, HP:0003763, HP:0002376, HP:0012433.

CaseHPOFreeText:

Patient History

@ birth - Patient 2 presented with mild grade bilateral hearing impairment and left divergent squint diagnosed at birth.

@ Follow-up visit - Patient 2 had microbrachycephaly and global developmental delay. Brain MRI, metabolic screening and array CGH were normal.

@ 1 year - Patient 2 had stereotypical handwashing movement.

There was no clinical or electrical seizure.

Patient 2 has craniofacial features like microbrachycephaly, wide mouth, divergent squint, and behavioral phenotype.

@ 1.5 years - Patient 2 had bruxism and developmental regression. Patient 2 also had loss of some motor and social skills.

CaseNotHPOs:HP:0001250

CaseNotHPOFreeText:Patient 2 has no seizures.

CasePreviousTesting:Not provided

PreviouslyPublished:Not previously published

GenotypingMethod:Not provided

Gene:MECP2 (MN_004992.3) (NP_004983.1)

Variant:c. 808C>T (p. Arg270*)

HGVS:Not provided

ClinVarID:Not found

CAID:CA172577

gnomAD:Not found

MultipleGeneVariants:NA

Case:Patient 1, female, 11 years old

DiseaseAssertion:Rett Syndrome - Atypical Variant

FamilyInfo:De Novo. Patient 1's parents were healthy. When Patient 1 was clinically examined, the head circumferences of her mother and father were 53 cm (P25) and 59 cm (P90), respectively. The parents had normal weight.

ParentalGenotype:Both parents were sequenced for Patient 1's mutation, and the deletion was not detected.

CasePresentingHPOs:HP:0001249, HP:0001513, HP:0001626, HP:0000256, HP:0010465, HP:0012758, HP:0000750, HP:0012433, HP:0002591, HP:0001250, HP:0020174, HP:0000316, HP:0000336, HP:0000431, HP:0000377, HP:0000470, HP:0001156, HP:0001500.

CaseHPOFreeText:

Patient history

@ birth - Patient 1 was born at 38 weeks of gestation after an uncomplicated pregnancy. Her weight was 3,390 g (P69), height 51 cm (P60), and her head circumference was 36 cm (P90).

@ 6 months - Patient 1 experienced developmental delay.

@ 9 months - Patient 1 sat without support.

@ 2 years - Patient 1 began to walk.

@ later on - Patient 1 presented with speech delay and behavioral disturbances. The behavioral disturbances were reduced by the drug risperidone.

@ early childhood - Patient 1 has been obese and appeared to display hyperphagia.

@ 8 years - Patient 1 developed precocious puberty.

@ 10 years - Patient 1 had her first epileptic seizure. Treatment with lamotrigine prevented further seizures. The seizures later became refractory to this treatment.

@ 10 years - Patient 1 presented with a height of 160 cm (P>97) and a head circumference of 59 cm (P>97). She had hypertelorism and prominent eyebrows. Her nasal bridge was broad and auricles were fleshy. In addition, Patient 1's neck was short and the fingers were short and wide.

Patient 1 has an intellectual disability, metabolic syndrome, and macrocephaly.

CaseNotHPOs:HP:0002540.

CaseNotHPOFreeText:Patient 1 sat without support at 9 months old. She walked at 2 years.

CasePreviousTesting:Patient 1 was given a conventional cytogenetic analysis. The chromosomal analyses (46,XX) and array CGH results (BlueGnome CytoChip ISCA 4×180K v1.0; Agilent Human Genome CGH Microarray 180K) were normal. Patient 1 was also given a methylation-specific MLPA to exclude a Temple syndrome, which is also characterized by weight gain and precocious puberty. In addition, Prader-Willi syndrome was ruled out by methylation testing. This syndrome is another imprinting disease causing obesity and intellectual disability.

PreviouslyPublished:Not previously published

GenotypingMethod:Whole-exome sequencing analysis of the entire exome was conducted for Patient 1. Whole-genome sequencing showed heterozygosity in Patient 1, which was confirmed by Sanger sequencing. Macrocephalic syndrome genes including PTEN, NSD1, NFIX, SETBP1, RAI1, and PHF6 were analyzed, and no additional variants of interest (pathogenic, likely pathogenic, or variants of uncertain significance) were observed.

Gene:MECP2

Variant:c. 1162_1172del (p. Pro388*), heterozygosity, frameshift.

HGVS:NM_004992.3

ClinVarID:Not found

CAID:CA1139667881

gnomAD:Not found

MultipleGeneVariants:Not provided

Case:Patient 2, female, 2.5 years old

DiseaseAssertion:CDKL5 Disorder

FamilyInfo:De Novo with an unremarkable family history.

ParentalGenotype:Not provided

CasePresentingHPOs:HP:0032792, HP:0007359, HP:0011154, HP:0002194, HP:0010862, HP:0000750, HP:0012434, HP:0000710, HP:0100023, HP:0000252, HP:0009062, HP:0010845, HP:0020174, HP:0010841.

CaseHPOFreeText:

Patient history

@ 2 months to 2.5 years - Patient 2 experienced tonic and focal seizures with autonomic symptoms.

@ 2 years - Patient 2 was diagnosed with CDKL5 disorder.

Patient 2 had severe delayed gross motor development, severe delayed fine motor development, severe delayed language development and delayed social development. Patient 2 had hyperoral and hand flapping stereotypies. She also had microcephaly (< 2 SD) and axial hypotonia.

Patient 2 was given a brain EEG which detected Delta slowing of the background followed by generalized attenuation during seizures and multifocal interictal epileptiform abnormalities.

@ 2.5 years - Patient 2 experienced seizures in clusters.

Treatments

Antiepleptic treatments included:Phenobarbital, Topirimate, Clobazam, Valproate, Keppra, Vitamin B6, Phenytoin, Lamotrigine, Nitrazepam, Oxcarbazepine, Mirtazipine, KCI, Levetiracetam, ant ketogenic diet.

Patient 2 was also treated with Carnitine. Patient 2's seizures seem to have changed in type over time but continued.

Other testing

Tests were conducted on Patient 2 to obtain data in the following areas: NBS, lactate, lipoprotein profile, plasma and urine amino acids, urine organic acids, acylcarnitine profile, total and free serum carnitine levels, plasma ammonia, total plasma homocysteine, serum CK, liver enzymes, urine alpha-AASA, creatine, biotinidase, VLCFA, Batten disease screen, CSF analysis (amino acids, lactate, glucose, protein, cell count, neurotransmitters), MRI-brain with spectroscopy, and karyotype.

CaseNotHPOs:Not provided

CaseNotHPOFreeText:Not provided

CasePreviousTesting:Patient 2 was given a gene sequence test for the genes SCNIA and MECP2. No gene mutation was found for these two genes.

PreviouslyPublished:Not previously published

GenotypingMethod:Patient 2 was given a CDKL5 gene sequence test. The test detected a mutation in the CDKL5 gene.

Gene:CDKL5

Variant:c. 2480_2486dupCAGATCT. frameshift

The authors state that CDKL5 gene sequencing detected a de novo duplication in exon 17, c. 2480_2486dupCAGATCT, resulting in a frameshift (Boston University School of Medicine, Center for Human Genetics, Boston, MA). This is a novel change that has not been reported before in ExAC. Only pathogenic point mutations in exon 17 have previously been reported. In a patient with a previously reported frameshift mutation in exon 18, a truncated CDKL5 transcript was detected. A truncated protein would lack the C-terminus and would not localize correctly in the cell, as demonstrated in vitro. Accordingly, any reading frame altering mutations proximal to exon 18 are null-variants. Therefore, this mutation is classified as pathogenic according to ACMG criteria.

HGVS:Not provided

ClinVarID:547188

NM_001323289.2 (CDKL5): c. 2480_2486dup (p. Gln830fs)

Allele ID: 538303

CAID:Not found

gnomAD:Not found

MultipleGeneVariants:Not provided

Case: Patient, Male, 4 months old <br /> DiseaseAssertion: SCID <br /> FamilyInfo: third child of non-consanguineous parents; has twin brother that is asymptomatic with no immune defects; no family history of primary immunodeficiencies <br /> CasePresentingHPOs: HP:0002014, HP:0020099, HP:0030148 <br /> CaseHPOFreeText: diarrhea, norovirus infection, heart murmur <br /> CaseNotHPOs: increased CRP <br /> CasePreviousTesting: N/A <br /> Gene: IL2RG <br /> Variant: NM_000206(IL2RG): <br /> ClinVar: <br /> CAID: <br /> gnomAD: <br /> SupplementalData:

PMID: 26986123 Gene: von Willebrand factor HGNCID: 12726 Note: reported for group analysis with no associated probands/patient

PMID: 21362127 Gene: von Willebrand factor HGNCID: 12726

PMID: 23311757 Gene: von Willebrand factor HGNCID: 12726

PMID: 18421900

Gene: SGCA

HGNCID: 10805