A homozygous R148W mutation in Semaphorin 7A causes progressive familial intrahepatic cholestasis

Abstract Semaphorin 7A (SEMA7A) is a membrane‐bound protein that involves axon growth and other biological processes. SEMA7A mutations are associated with vertebral fracture and Kallmann syndrome. Here, we report a case with a mutation in SEMA7A that displays familial cholestasis. WGS reveals a SEMA7A R148W homozygous mutation in a female child with elevated levels of serum ALT, AST, and total bile acid (TBA) of unknown etiology. This patient also carried a SLC10A1 S267F allele, but Slc10a1 S267F homozygous mice exhibited normal liver function. Similar to the child, Sema7a R145W homozygous mice displayed elevated levels of serum ALT, AST, and TBA. Remarkably, liver histology and LC‐MS/MS analyses exhibited hepatocyte hydropic degeneration and increased liver bile acid (BA) levels in Sema7a R145W homozygous mice. Further mechanistic studies demonstrated that Sema7a R145W mutation reduced the expression of canalicular membrane BA transporters, bile salt export pump (Bsep), and multidrug resistance‐associated protein‐2 (Mrp2), causing intrahepatic cholestasis in mice. Administration with ursodeoxycholic acid and a dietary supplement glutathione improved liver function in the child. Therefore, Sema7a R145W homozygous mutation causes intrahepatic cholestasis by reducing hepatic Bsep and Mrp2 expression.


Minor comments
The liver phenotype of the Sema7a_R145W mice is different from the patients with respect to bilirubin levels. This is somewhat remarkable as hyperbilirubinemia is described for Slc10a1 deficiency (which is present in the patient). Are similar differences due to species differences and also observed with other PFIC genes?
The PFIC phenotype is different from PFIC1/2 in the sense that pruritus was absent. This should be discussed.
Recent data that Sema7A is crucial for resolution of severe inflammation (PNAS 2021; PMID: 33637648) should be discussed as inflammation plays a role in cholestatic liver injury..

Referee #2 (Comments on Novelty/Model System for Author):
In general the study is of interest but lack a mechanistic explanation ofm the findings.

Referee #2 (Remarks for Author):
This study reports on the characterization of a Semaphorin 7a mutation in a female infant showing features of liver injury and cholestasis at 2 months of age. The clinical syndrome is marked by increased levels of AST and ALT and total bile acids (TBA) in the blood. Both the father and the mother had normal AST and ALT and TBA. Liver histopathology also revealed a robust hydropic degeneration in hepatocytes and increased accumulation of conjugated BA in the liver, while immunoshistochemistry in mice demonstrated an internalization of canalicular membrane BSEP and MRP2 . Gene sequencing revealed a mutation in the semaphoring 7a gene. The newly identified mutation is a homozygous p.R148W mutation in SEMA7A, and SEMA7A R145W homozygous mice, displayed similar histopathology features than the patients, including a hydropic degeneration of hepatocytes and increased accumulation of intrahepatic BA. The data suggest that SEMA7AR148W homozygous mutation is a new genetic determinant of cholestatic liver injury. The data reported are of interest. My comments are the detailed here: 1. The first sentence of Discussion is an over-interpretation of the results: "Here, we first reported a case of familial cholestasis caused by a homozygous p.R148W mutation in SEMA7A, and provided evidence of its pathophysiologic mechanism. This new genetic cholestatic disorder is characterized by (1) elevated levels of serum ALT, AST, and TBA ( Fig.2A-C and Tables.3&S13); (2) striking hydropic degeneration in hepatocytes and increased accumulation of conjugated BA in the liver ( Fig.2D&E and Table.4); and (3) internalization of canalicular membrane BSEP and MRP2 (Figs.3D&E". The authors have no data on the histopathology of the patient described in this report, therefore there is no information on point 2 and 3. This need to be amended and the same should be done in the abstract and throughout the manuscript. 2. Also the fact that the patient has a homozygous p.S267F mutation in SLC10A1, which leads to very close clinical pattern, does not allow a clear separation between the two syndromes. This suggest a cautionary approach on the title and abstract. The abstract should inform the readers that the patients had both mutations. 3. Page 16, the human mutation SEMA7AR148W is described as a "gain of function" mutation. The patient harboring this mutation had cholestasis and therefore the mutation should lead to some degree of loss of function. Particularly if the SEMA7AR148W prevents insertion of BSEP and MRP2 in the canalicular membrane of hepatocytes. Please clarify. 4. A previous study has shown that lack of semaforin7a is protective again cholestasis in mice, which is in contrast with the data shown in this paper. Refence made to this paper in page 15 is wrong since the paper is cited in ref. 7 (not 6). Furthermore, this paper has been criticized because extensive image duplications: https://pubpeer.com/publications/089AD44F94ECA4B33A4BF1718F644E. I will suggest to remove it from reference. 5. One limitation of the study is the lack of a mechanism explaining how SEM7A regulates BSEP insertion at the canalicular membrane. The structure shown in Figure 1D-F is poorly informative. 6. Table 4, shows a disproportionate increase of TCDCA over TMCA, suggesting an impaired conversion of CDCA to MCA. Does the authors have data on Cyp2c70? 7. How the authors explain differential regulation of Ost and Mrp4 in figure 2 Referee #3 (Comments on Novelty/Model System for Author): We constantly learn about novel mutations. Many of these are variants of unknown significance. The locus the authors describe is interesting but they cannot convincingly correlate the in vitro and in silico findings to a human clinical phenotype.

Referee #3 (Remarks for Author):
This is potentially an interesting finding however I see several drawbacks that should be addressed before publication: 1. There is no convincing correlation of the in vitro and in silico findings to the clinical phenotype in the child. Histopathology and longitudinal clinical data is missing in particular as previous publication on this gene suggested alleviation of cholestasis rather than a PFIC phenotype. 2. Suggest you follow the reporting guidelines with (equator network) 3. The language needs revision.

Responses to the Editor:
As you will see from the reports below, the referees acknowledge the interest of the study but also raise serious and partially overlapping concerns that should be addressed in a major revision. Furthermore, I would like you to consider publishing your manuscript as a scientific report (3 figures, ~22000 characters), for more information please check our "Author Guidelines".
Response: Thank you very much for the suggestion, we now have revised the manuscript and submit it as a scientific report.

Referee #1 (Comments on Novelty/Model System for Author):
Some differences between patient and animal model.
Conclusions on BSEP/MRP2 trafficking that would require cellular models as 'internalization' is a dynamic process, while only static data is provided.
Impact is unclear as no data is provided how often Sema7A mutations could lead to PFIC. This is in itself already a rare disease, so this issue is difficult to answer and not required for a first paper identifying a novel disease-causing gene variant I think. Highly novel and important though The molecular explanation for cholestasis is somewhat preliminary and if not better addressed, I would recommend this paper for a short report instead.
Response: Thank you for your positive comments and constructive suggestions. Now, the manuscript has been revised as a short report.

Referee #1 (Remarks for Author):
The   (Table.1 and Table.EV16) and the activated PKCδ/ε signaling (Fig.EV4A) in Sema7a R145W homozygous mice, implying that Sema7a R145W homozygous mutation might activate the PKCδ/ε signaling. This hypothesis was verified in Sema7a R145W homozygous mouse primary hepatocytes and human HepG2 cells transfected with SEMA7A_R148W expression construct (Fig.EV4B&C). Therefore, we propose that Sema7a R145W mutation reduces canalicular membrane Bsep and Mrp2 expression by activating the PKCδ/ε signaling in hepatocytes, although the details remain to be revealed. We have added these new data in the revised manuscript.

References:
Chai Response: We thank you for your critical comments and professional suggestions, which allowed us to improve the manuscript. We have now performed additional experiments to further test our hypothesis according to your suggestions. First, over-expression of SEMA7A_R148W or SEMA7A_WT protein remarkably reduced canalicular membrane Bsep and Mrp2 expression in a dose dependent manner in primary mouse hepatocyte collagen sandwich cultures (Fig.2G). Second, in the same dose transfection, the levels of canalicular membrane Bsep and Mrp2 protein expression were substantially lower in primary hepatocytes in sandwich cultures transfected with SEMA7A_R148W construct than that of with SEMA7A_WT construct (Fig.2G). Third, Sema7a R145W (human R148W) homozygous mutation activated the PKCδ/ε signaling in mouse primary hepatocytes and human HepG2 cells (Fig.EV4B&C). Therefore, we speculate that the Sema7a R145W mutation is a gain-of-function mutation. Furthermore, Sema7a R145W homozygous mice displayed an intrahepatic cholestatic phenotype (Table.1 and EV16). Conversely, Sema7a deficiency in mice would protect against cholestatic liver injury, in agreement with observations seen in the Sema7a knockout mice. Regarding the issue of recessive inheritance, we need to collect and analyze additional patients in the future. We have added these new data and made the corresponding changes in the revised manuscript.

Minor comments
1. The liver phenotype of the Sema7a_R145W mice is different from the patients with respect to bilirubin levels. This is somewhat remarkable as hyperbilirubinemia is described for Slc10a1 deficiency (which is present in the patient). Are similar differences due to species differences and also observed with other PFIC genes?
Response: We agree that there is a difference in serum bilirubin levels between Sema7a R145W mice and the child patient. Slc10a1 deficiency displayed hypercholanemia but not hyperbilirubinemia or liver injury . Similar to other PFIC phenotype (e.g. PFIC3), Sema7a R145W homozygous mice also displayed the elevated levels of serum total BA and bilirubin, as well as serum ALT and AST. However, the child patient with this new PFIC exhibited the elevated levels of serum total BA, ALT and AST, but not bilirubin.
The differences may stem from species differences or the early stage of the new PFIC in this patient.

Referee #2 (Comments on Novelty/Model System for Author):
In general the study is of interest but lack a mechanistic explanation of the findings.
Response: We thank you for your positive and critical comments. We have performed additional experiments and tried our best to provide a mechanistic explanation based on our data. Also, we modified the manuscript into a short report, because of the lack of detail mechanisms.

Also the fact that the patient has a homozygous p.S267F mutation in SLC10A1, which leads to very close clinical pattern, does not allow a clear separation between the two syndromes. This suggest a cautionary approach on the title and abstract. The abstract should inform the readers that the patients had both mutations.
Response: We appreciate your professional advice. We have made changed the Abstract accordingly.

Page 16, the human mutation SEMA7A R148W is described as a "gain of function" mutation. The patient harboring this mutation had cholestasis and therefore the mutation should lead to some degree of loss of function. Particularly if the SEMA7A R148W prevents insertion of BSEP and MRP2 in the canalicular membrane of hepatocytes. Please clarify.
Response: We have studied your thoughtful comments carefully. After performing additional experiments, our data supported that SEMA7A R148W may be a "gain of function" mutation as addressed the question 2 from Reviewer #1. Briefly, over-expression of SEMA7A_R148W or SEMA7A_WT protein markedly decreased canalicular Bsep and Mrp2 proteins in primary mouse hepatocytes in sandwich cultures in a dose-dependent manner, particularly after over-expression of the mutant (Fig.2G). Cell culture experiments for Sema7a R145W primary mouse hepatocytes and human HepG2 cells transfected with SEMA7A_R148W construct revealed that this mutation also increased PKCδ/ε activities (Fig.EV4B&C). Furthermore, Sema7a R145W homozygous mice displayed an intrahepatic cholestatic phenotype with reduction in the levels of canalicular membrane Bsep and Mrp2 expression (Fig.2D, Table.1, and Table EV16). Therefore, the gain-of-function mutation of Sema7a R145W may cause cholestasis by repressing of hepatic Bsep and Mrp2 expression. We have added these new data and discussed these points in the revised manuscript.

A previous study has shown that lack of semaforin7a is protective again cholestasis in mice, which is in contrast with the data shown in this paper. Refence made to this paper in page 15 is wrong since the paper is cited in ref. 7 (not 6).
Response: Thank you for pointing out the wrong citations, which has been corrected in the revised manuscript. As described above, we speculate that Sema7a R145W homozygous mutation is a gain-of-function mutation and causes intrahepatic cholestasis in mice. This would be consistent with that Sema7a deficiency protects mice against cholestatic liver injury.

Furthermore, this paper has been criticized because extensive image duplications:
https://pubpeer.com/publications/089AD44F94ECA4B33A4BF1718F644E. I will suggest to remove it from reference.

Response:
We appreciate your advice. Now, we have removed this reference in the revised manuscript.

One limitation of the study is the lack of a mechanism explaining how SEMA7A regulates BSEP
insertion at the canalicular membrane. The structure shown in Figure 1D-F is poorly informative.

Response:
We have performed additional experiments to explore the preliminary mechanisms. We found that Sema7a R145W mutation markedly reduced the levels of canalicular membrane Bsep and Mrp2 expression in primary mouse hepatocytes in sandwich cultures (Fig.2F), and increased PKCδ/ε activities in mouse and human hepatocytes (Fig.EV4B&C) homozygous mutation may reduce canalicular membrane Bsep and Mrp2 expression by increasing PKCδ/ε activities in hepatocytes. Accordingly, we have added these new data in the revised manuscript.
Also, we modified the manuscript into a short report, because of the lack of detail mechanisms.

References:
Chai  Response: Thank you for the constructive suggestion. We have now determined Cyp2c70 mRNA expression in mouse livers and found a significant decrease in in homozygous mice, when compared to WT and heterozygous mice (Fig.2C). We have added this new data in the revised manuscript.

How the authors explain differential regulation of Ost and Mrp4 in figure 2
Response: After reading the question, we carefully examined the original data about hepatic bile acid transporters. We noticed that there was an outlier in the heterozygous group that had affected the qPCR data of Ostβ and Mrp4 in the former figure 2. To address this issue, we repeated these experiments and corrected these results in the revised manuscript.

Referee #3 (Comments on Novelty/Model System for Author):
We constantly learn about novel mutations. Many of these are variants of unknown significance. The locus the authors describe is interesting but they cannot convincingly correlate the in vitro and in silico findings to a human clinical phenotype.
Response: Thank you for your insightful and critical comments. We have added new clinical data as below and discussed this issue in the revised manuscript.

Referee #3 (Remarks for Author):
This is potentially an interesting finding however I see several drawbacks that should be addressed before publication: 1. There is no convincing correlation of the in vitro and in silico findings to the clinical phenotype in the child. Histopathology and longitudinal clinical data is missing in particular as previous publication on this gene suggested alleviation of cholestasis rather than a PFIC phenotype.

Response:
We greatly appreciate your critical comments and helpful suggestions. To address the concern, we performed additional experiments and collected new clinical data. Firstly, the child patient carried with a SEMA7A R148W homozygous mutation and displayed the elevated levels of serum ALT, AST and TBA (Table EV1). Secondly, Sema7a R145W homozygous mice also exhibited the elevated levels of serum ALT, AST and TBA (Table.1 and Fig.EV3) and intrahepatic cholestasis (Table.1), which recapitulated her clinical phenotypes. Thirdly, over-expression of SEMA7A_R148W protein in primary mouse hepatocytes in sandwich cultures remarkably reduced the levels of canalicular membrane Bsep and Mrp2 expression in a dose-dependent manner (Fig.2G), compared to that of with SEMA7A_WT over-expression (Fig.2G). Furthermore, Sema7a R145W mutation increased PKCδ/ε activities in mouse and human hepatocytes (Fig.EV4B&C), supporting that the Sema7a R145W mutation is a gain-of-function mutation. Collectively, the Sema7a R145W mutation is gain-of-function mutation and can cause intrahepatic cholestasis in mice. Conversely, Sema7a deficiency in mice should attenuate cholestasis.
We did not have histopathological data of the child patient because the patient health condition did not meet the requirements for liver biopsy. However, longitudinal clinical data were collected and added in the revised manuscript (Fig.3). After finding abnormal liver function in the child, the child was treated with a therapeutic drug for cholestasis, UDCA (13 mg/kg/d) and a dietary supplement GSH (40 mg/kg/d).
Interestingly, treatment with both UDCA and GSH for 2 weeks significantly corrected the abnormal levels of serum ALT and AST and reduced the levels of serum TBA in the patient (Fig.3). However, when the treatment was ceased, the levels of serum ALT, AST and TBA increased abnormally in the patient (Fig.3). These new clinical data supported that Sema7a R148W mutation caused intrahepatic cholestasis in the child patient. Accordingly, we have added these new data and discussed them in the revised manuscript.

Suggest you follow the reporting guidelines with (equator network)
Response: We appreciate your advice. We have revised the manuscript following the CARE case report guidelines. The checking list was attached to the revised manuscript.

The language needs revision.
Response: We appreciate your advice. Now, we have carefully checked each sentence to eliminate/reduce any potential syntax. The manuscript has been proof-read by two native English biologists. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) Please address all the points raised by the referees. Particular attention should be given to the western blots in Figure 2D that appear not to be form the same gel although single GAPDH is presented as a loading control as pointed out by the referee #2. Western blots from different gels should be presented with a corresponding loading control blot. 2) In the main manuscript file, please do the following: -Correct/answer the track changes suggested by our data editors by working from the attached document.
-Reduce keywords to max. 5. -Merge "Grants support" with "Acknowledgements". -Add full URL to your deposited data and please be aware that all deposited data have to be freely available before publication of the manuscript. Please use the following format to report the accession number of your data: The datasets produced in this study are available in the following databases: Please check "Author Guidelines" for more information. https://www.embopress.org/page/journal/17574684/authorguide#availabilityofpublishedmaterial 3) EV Tables: Please move all the EV Tables to "Appendix", rename them to "Appendix Table S1" etc., and update their callouts in the main text. 4) Source data: Please upload one file per figure for the main Figures (zipp were appropriate) and zipp source data for all EV Figures as one file. 5) CARE Checklist is not required and can be removed. 6) As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication. Please note that the Authors checklist will be published at the end of the RPF. The aut hors have adapt ed the manuscript to a short report . This bet ter reflect s the level of mechanist ical insight linking Sem7A variant to cholest asis. Furt hermore, they did a nice job in assesssing the direct effect s of SEM7A overexpression (WT and pat ient variant ) on BSEP/MRP2 abundance/localiz at ion. I would have prefered if the experiment s wit h SEMA7A overexpression were performed and analyzed in a blinded (and more quant it at ive) manner but don't consider this essent ial for this manuscript at this st age. The text is also improved and at present accurat ely reflect s the dat a. I have no furt her comment s Referee #2 (Remarks for Aut hor): The aut hors have provided convincing replies to my comment s. I have a technical comment relat ed to the qualit y of WB that is very low and should be increased. Furt her on, I'm concerned over figure  3D, since many prot eins are present ed wit h a single Gapdh as a cont rol. This will imply that the same gell has been used mult iple times, but the morphoplogy of the various bands clearly indicat e that is not the case. As such, relat ive cont rol for each prot ein or groups of prot ein should be provided.

A-Figures 1. Data
The data shown in figures should satisfy the following conditions: the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way. graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation.

Captions
Each figure caption should contain the following information, for each panel where they are relevant: a specification of the experimental system investigated (eg cell line, species name). the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner. the exact sample size (n) for each experimental group/condition, given as a number, not a range; a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.). a statement of how many times the experiment shown was independently replicated in the laboratory. definitions of statistical methods and measures: Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects.

B-Statistics and general methods
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) Sample sizes were carefully determined according to previously published of ours and other groups.
The sample sizes are similar to previous reports in the same field.
No data were excluded from the analyses.
All samples and animals used in this study were randomized into experimental groups.
All samples and animals used in this study were randomized into experimental groups. In vivo studies, all mice were age-and sex-matched and randomly assigned into control and treatment groups to ensure similar body weight.
In immunohistochemistry and multiplex immunofluorescence , images were acquired using the same laser power and sensitivity, and image processing was the same across the experimental groups.
The statistical methods are indicated in the figure legends. Yes.