Autism-linked NLGN3 is a key regulator of gonadotropin-releasing hormone deficiency

  1. Roberto Oleari
  2. Antonella Lettieri
  3. Stefano Manzini
  4. Alyssa Paganoni
  5. Valentina André
  6. Paolo Grazioli
  7. Marco Busnelli
  8. Paolo Duminuco
  9. Antonio Vitobello
  10. Christophe Philippe
  11. Varoona Bizaoui
  12. Helen L. Storr
  13. Federica Amoruso
  14. Fani Memi
  15. Valeria Vezzoli
  16. Valentina Massa
  17. Peter Scheiffele
  18. Sasha R. Howard
  19. Anna Cariboni

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Abstract

Gonadotropin-releasing hormone (GnRH) deficiency (GD) is a disorder characterized by absent or delayed puberty, with largely unknown genetic causes. The purpose of this study was to obtain and exploit gene expression profiles of GnRH neurons during development to unveil novel biological mechanisms and genetic determinants underlying GD. Here, we combined bioinformatic analyses of immortalized and primary embryonic GnRH neuron transcriptomes with exome sequencing from GD patients to identify candidate genes implicated in the pathogenesis of GD. Among differentially expressed and filtered transcripts, we found loss-of-function (LoF) variants of the autism-linked neuroligin 3 (NLGN3) gene in two unrelated patients co-presenting with GD and neurodevelopmental traits. We demonstrated that NLGN3 is upregulated in maturing GnRH neurons and that NLGN3 wild-type, but not mutant, protein promotes neuritogenesis when overexpressed in developing GnRH cells. Our data represent proof of principle that this complementary approach can identify new candidate GD genes and demonstrate that LoF NLGN3 variants can contribute to GD. This novel genotype–phenotype correlation implies common genetic mechanisms underlying neurodevelopmental disorders, such as GD and autistic spectrum disorder.

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  1. Version published to 10.1242/dmm.049996
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    Reply to the reviewers

    Manuscript number: RC-2022-01490

    Corresponding author(s): Cariboni, Anna; Howard, Sasha R

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

    The current manuscript in question is well written and of general interest to the reproductive neuroendocrinology field. Overall it is a well written and substantiated.

    Reply: We thank the reviewer for his/her positive and supportive comments on our manuscript.

    The primary problem with the paper is the data derived from the microarray. While the experimental design included replicates (n = 3), although weak, the actual microarray data was based on a single data point. A major weakness. This experiment should be repeated using more up-to-date approaches such as RNA-seq or left out of the manuscript, because this data set is compromised due to the data collection procedure.

    __Reply: __ We thank the Reviewer for raising these points, which we wish to clarify. We respectfully disagree that the microarray data generated in this study is not valuable. The transcriptomic analysis of immortalized cells was performed on 3 biological replicates (specifically, RNA was extracted from n=3 samples, obtained from each cell line at 3 different passages) and run as 3 independent samples (for a total of 6, 3 for GN11 cells and 3 for GT1-7 cells). For the primary embryonic GFP-GnRH neurons, given the difficulty of isolating with FACS a sufficient number of GFP+ cells from each embryo due their very small number (around 1000 GnRH neurons/head), we had to pool sorted cells from 2-3 embryos for each time-point. Thus, although the primary cell microarrays were run on one sample for each time point, the RNA was not derived from one embryo only, but from at least 2/3 embryos.

    Nevertheless, to overcome the issue of low number of replicates for the primary embryonic cells, we revised our manuscript by re-running our analyses, using as the starting dataset the analyses obtained from immortalized cells, which were based on a ‘true’ n=3 of biological replicates. In this context, we filtered DEGs from this microarray using logFC>2 and adj. p-value1) found in primary GFP-GnRH neurons. We believe that this revised analysis is statistically more powerful, as the core bioinformatic analyses were performed on triplicate samples, with a second filtering step to take advantage of biologically relevant data obtained from n=1 primary GFP-GnRH neurons to confirm in vivo the expression of selected genes. Whilst RNAseq offers wider coverage of the genome and has advantages over microarray, we do not believe that this renders unimportant the data generated from these unique experiments and the novel genomic discoveries it facilitated.

    In line with this, our work may be considered as a proof-of-principle that transcriptomic profiles from rodent GnRH neurons can be exploited at different levels, including the possibility to identify novel GD candidate genes. Overall, our work also highlights the existence of similarities between two immortalized GnRH neuron cell lines with primary GnRH neurons, which was so far demonstrated by several functional studies, but not at molecular level.

    The manuscript has been now edited as per the above amendments (see first and second paragraph of Results section, lines 86-135).

    __CROSS-CONSULTATION COMMENTS __Notwithstanding the importance of neuroligin 3 during glutaminergic synaptogenesis, I agree with the reviewers on both points. Further screenings of the patient's family members should be done and the microarray data should be removed or potentially moved to a supplementary status.

    Reply: we thank the reviewer for their comments and, accordingly with their suggestion, we revised the filtering strategy starting from immortalized cells microarray and therefore moved a substantial part of the microarray data from primary GFP+ neurons as supplementary data. We also unsuccessfully tried to collect information of the brother from case 2 and investigated datasets from both the DECIPHER and 100,000 genome projects, but have been limited to two cases for which we have familial consent to publish.

    __Reviewer #1 (Significance (Required)): __ The paper is of significance based on the neuroligin 3 data, which is indicative of abnormal synaptogenesis. However, these defects seem to only have a limited effect on the functionality of GnRH neuron system and do not seem to cause elimination of GnRH neurons themselves. Nevertheless these data do open end a new direction that may help explain some dysfunctions in reproductive health.

    Reply: we thank the reviewer for their comments and agree that our findings have the potential to facilitate new avenues for the investigation of reproductive disorders.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ Oleari et al performed comparative transcriptome analysis on the different developmental stages of GnRH neurons, as well as two immortalized GnRH neuronal cells GT1-7 and GN11 which represent mature and immature GnRH neurons. As a results, they identified a panel of differentially expressed genes (DEG). They further used top DEGs as candidate disease-related genes for GnRH-deficiency (GD), a disorder characterized with absent of delayed puberty and infertility. To this end, they found two loss-of-function mutations in NLGN3 in patients with GD combined with autism. This study provide a resource for the identification of novel GD-associated genes, and suggest an intrinsic connection between GD and other neurodevelopmental diseases, such as autism. I only have some minor concerns.

    According to the pedigree, both probands (case 1 and 2) inherited their NLGN3 mutations from their unaffected mother, consistent with an X-linked recessive inheritance. However, only "parent" was used in the manuscript, therefore, it is not clear if this "parent" is the probands' mother or father. __Reply: __Thank you for this comment. We were limited to the use of non-gendered terminology due to medRxiv policies. We have now amended the text and changed ‘parent’ to ‘mother’, lines 161, 173, 179, 185 and 730. We also integrated this sentence highlighting the X-linked pattern of inheritance: “Sanger sequencing of the probands’ mothers confirmed them to be the heterozygous carrier in each family, consistent with an X-linked recessive inheritance pattern.”, lines 185-186.

    It is suggested to integrate Figure 2 as a panel in Figure 1.

    __Reply: __We thank the reviewer for this suggestion. Due to our revision of first two Results paragraphs, we have now edited the Figures and the filtering flowchart has been added in Figure 2.

    What is the meaning of Peak LH and Peak FSH, and how are they measured in Table 2?

    Reply: This refers to peak value obtained after standard protocol GnRH stimulation testing with 100mcg GnRH (Gonadorelin) as an IV bolus and measurement of serum LH and FSH at 0, 20 and 60 minutes intervals. (e.g. Harrington et al., 2012, doi:10.1210/jc.2012-1598). This clarification has been added to the text in Table 2 legend (lines 681-683).

    A genotyping for the elder brother of Case 2 will be a strong evidence to support NLGN3 as a GD-associated gene.

    __Reply: __We thank the reviewer for this important point. In view of this issue, we have strived to collect DNA from this individual. Unfortunately, despite trying repeatedly to contact the family of proband 2, it has not been practically possible to collect these extra data from this family.

    We also identified a third case via a public database with central hypogonadism who carried a stop-gain variant in NLGN3, but unfortunately the family did not release their consent for publishing this case.

    The authors claimed neither probands carried deleterious variants in known GD genes. It is suggested to indicate the exclusion criteria (which genes? How do they define a variant is deleterious?)

    Reply: We thank this reviewer for raising this important point of clarification. Inclusion criteria for variants in known GD genes (updated gene list available in Supplemental Table 3) were as per Saengkaew et al., 2021 (doi: 10.1530/EJE-21-0387): “Only variants that met the ACMG criteria for pathogenicity, likely pathogenicity, or variants of uncertain significance (VUS) were retained in the analysis”. We have added this sentence in the manuscript, lines 150-151.

    Please also include a sequence chromatogram for proband 2.

    Reply: We thank the reviewer for their comment. We added the chromatograms for proband 2 and his heterozygous mother in revised Figure 3.

    CROSS-CONSULTATION COMMENTS I agree with Reviewer 3, the genetics is not very strong, as NLGN3 mutations were only found in one GD case from their cohort and one pre-pubertal case from the literature. It will be nice to analyze the genotype and phenotype of Case 2's older brother. Further, it is important to screen NLGN3 rare sequencing variants in larger GD cohorts.

    Reply: We thank the reviewer for their comment, but respectfully disagree with this assertion. The second case is not from the literature, but is a second case found thanks to GeneMatcher, an international tool that allows researchers to collaborate on novel gene discovery. We have also explored other cohorts that were available to us, including the DECIPHER and 100,000 genome project, but have been limited to two cases for which we have familial consent to publish. We anticipate that further international patient cohorts will be screened following the publication of this manuscript (added in Discussion section, lines 306-308). As described above, despite trying repeatedly to contact the family of proband 2, it has not been practically possible to collect these extra data from this family.

    __Reviewer #2 (Significance (Required)): __ This study provides a resource for the identification of novel GD-associated genes, and suggest an intrinsic connection between GD and other neurodevelopmental diseases, such as autism. It may welcome by researchers and clinicians in the filed of neurodevelopment.

    Reply: We thank the reviewer for their positive and supportive comments.

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

    __Summary: Oleari et al used murine GnRH1, and immortalized GnRH cell lines (GT1-7, Gn11) to define genes of interest in GnRH development and used this list to filter exome sequencing data from patients with some evidence for GnRH Deficiency.

    Title: I am concerned that the title of the paper overstates the results and conclusions.

    Intro: use of "candidate causative genes" overstates the evidence presented.

    __Reply: __We thank the reviewer for their comment and have revised the title to reflect the findings of the study. We have also edited the sentence in the abstract reporting "candidate causative genes" as follows: “Here, we combined bioinformatic analyses of primary embryonic and immortalized GnRH neuron transcriptomes with exome sequencing from GD patients to identify candidate genes implicated in GD pathogenesis”, lines 40-43.

    Results: The transcriptomic profile of the developing human GnRH neuron has been published via in vitro differentiation protocols twice (Lund et al 2020, and Keen et al 2021). Gene set data is publicly available. This should be explicitly compared in results not relegated to discussion -- two or three examples it not enough to say mouse can be used instead of human.

    __Reply: __We thank the reviewer for this comment. We apologize if our sentence in the Discussion was misleading, as we did not intend to make a conclusion on the similarities of the two datasets/cell types, neither to suggest the use of rodent instead of human.

    Although we are aware that differences among species might exist, mouse/rodent models including immortalized cells have been instrumental to understand the molecular mechanisms of GnRH neuron development and to predict candidate genes. Indeed, our aim was to demonstrate that transcriptomic profiles of rodent GnRH neurons could be integrated with exome sequencing data from human patients to reveal novel candidate genes.

    Therefore, the aim of our study was different to that of the Lund and Keen publications. Further, caution should be exercised in any deeper comparative analyses with our transcriptomes, for following reasons: first, the GnRH neurons generated from human iPSC and cultured for 20 and 27 days cannot be objectively defined for their ‘age’ in order to be then compared to immortalized or primary embryonic GnRH neurons; second, in these datasets a different and more extensive transcriptomic technique has been used (RNAseq vs microarrays).

    There was no intention to relegate to the discussion the possible similarities with other transcriptomic datasets, but we felt that these comparative analyses were beyond the scope of our work.

    However, following the Reviewer’s suggestion, we have tried to make comparative analyses with the publicly available datasets from Lund et al 2020 and Keen et al 2021, and with a paper just published (Wang et al 2022), as follows.

    In Lund et al. paper, GnRH-like neurons were obtained from human iPSCs by dual SMAD inhibition and FGF8 treatment. We selected data obtained from cells treated with FGF8 and cultured for 20 days and 27 days for comparison with our early and late genes, respectively.

    Because the authors of this paper did not publish the full list of differentially expressed genes (DEGs) from this specific comparison (20 vs 27days) and we were not able to retrieve it upon request, we used the normalized counts of these samples (available at ArrayExpress repository) to compare the two experimental groups with DESeq (Bioconductor release 3.15). To increase stringency of our analysis, we considered as differentially expressed those genes which displayed both an adjusted p-value of less than 0.05 and an absolute fold change of >2. The number of DEGs obtained was different and greater (5981) than from the published data, and this large number of genes may, by chance alone, contain a large fraction of any gene dataset (including the genes that we found with our analysis). For this reason, this particular comparison in this dataset cannot be informative or useful.

    Next, we considered the dataset from Keen et al. In this paper, the authors have tested different differentiation protocols to obtain GnRH-like neurons from human wild-type or mCherry embryonic stem cells (hESC). They transcriptomically profiled hESC-mCherry-derived GnRH neurons at 8,15 and 25 days of culture.

    Again, although we cannot precisely define the matching embryonic stage of cells cultured for 8, 15 or 25 days, we compared the lists of DEGs from immortalized GnRH neurons (GN11vsGT1-7) with the transcriptomic profiles of mCh-hESC at day 15 vs day 8 and mCh-hESC at day 25 vs day 15, respectively. We considered as differentially expressed the genes that displayed both an adjusted p-value of less than 0.05 and logFC>2. We found that the majority of the genes that were differentially expressed in one dataset were not in the other. However, the few genes that were differentially expressed in both datasets demonstrated a good correlation, i.e. the same expression trend. Although this latter approach was more fruitful, by suggesting a partial similarity between primary GFP-GnRH neurons and hESCs-derived GnRH neurons at day 25 vs day 15 time-point, we do not feel that we could draw significant and reliable conclusions.

    Further, if we compare these two datasets obtained by RNAseq from hiPSC and hESC, even by taking into account the large amount of DEGs found in our re-analysis of Lund et al., 2021 raw data, a relatively small number of common DEGs were found. These data also suggest that there is transcriptomic heterogeneity even among human-derived GnRH neurons.

    In addition to these two datasets, while our manuscript was under revision, a new paper was published, in which the authors dissected iPSC-derived GnRH neuron transcriptome with RNA-seq at single cell level (Wang et al., 2022, doi:10.1093/stmcls/sxac069). Again, although the same concerns may apply in comparing this dataset with ours and raw data of DEGs were not publicly available in this case, we compared the expression trends of our 29 candidates with gene expression trajectories identified in this work. As a result, 24/29 candidate genes, including NLGN3, were found to have an expression trend consistent with our dataset. The few remaining genes exhibited an opposite trend (2/29) or were not found in available data from this work (3/29). As this is a purely qualitative analysis, we do not feel it would be appropriate to include it in the Results section, but have included commentary on these comparative dataset analyses in the Discussion section (lines 247-257). A future study could be designed to mine the raw data from all the available transcriptomic profiles of developing GnRH neurons, but this is beyond the scope of our current manuscript.

    The authors need to comment on other GnRH1 expression in the brain of developing rodent and if they think the GnRH1 sorted neurons are just "GnRH Neurons" associated with reproduction (Parhar et al 2005) due to microdissection.

    __Reply: __We thanks the reviewer for raising this point of clarification. We have carefully selected by microdissection nasal areas from E14, nasal and basal forebrain areas from E17 and basal forebrain from E20 rat embryos (see revised Methods, lines 325-327). We are therefore confident that what we have obtained is RNA from ‘reproductive’ GnRH neurons only.

    Questions about Cases/Missing Phenotypic Information:

    1. Case 1: the patient underwent increased testicular volume on testosterone therapy -- testosterone therapy does not increase testicular volume. Has this patient undergone or been assessed for reversal of his hypogonadism?

    __Reply: __We thank the reviewer for their comment. The patient had minimal testicular development on testosterone (from 10ml to 12ml) but did not increase testes volume beyond 12mls, consistent with a partial HH phenotype. He has had two trial periods of 3-4 months off testosterone treatment and during these periods had both low serum testosterone concentrations and symptoms of hypogonadism (tiredness, low energy and reduced muscle strength).

    1. Case 2: Is too young to be classified as having a pubertal defect. Microphallus is mentioned but what size, was this diagnosed at birth and treated? I think the case for GD is overstated in the results and discussion (especially with the discussion of small testes).

    Reply: We thank the reviewer for requesting these clarifications. The patient has not received any treatment for his microphallus (2.5 cm length in mid-childhood). We agree that this case is too young to be classified as having a pubertal defect, but the presence of microphallus and small testes volume in infancy and early childhood, in association with low gonadotrophins and absent erections, are well recognized as red flag signs for hypogonadotropic hypogonadism (Swee & Quinton, 2019, doi:10.3389/fendo.2019.00097). We added this information to the Results section, lines 175-177.

    Genetic Information: Since this was a candidate gene search -- what other candidate genes were uncovered in these probands?

    Reply: The revised list of 29 candidate genes were screened in the two probands from our study using the whole exome sequencing datasets for these individuals, and only the variants of interest in NLGN3 described in the manuscript were found.

    By searching for mutations of the revised list of candidate genes in our GD cohort, we identified nonsense variants only in NLGN3 and no splice variants. We also found few rare and predicted damaging missense variants in this gene list identified. Indeed, two rare (MAF 25) missense variants were identified in the genes PLXNC1 and CLSTN2 in two further probands (now summarized in Supplemental table 4). We have not identified further probands with PLXNC1 or CLSTN2 variants of interest from additional cohorts and thus at present we have not yet taken these gene variants further for molecular characterization, but we will examine the relevance of this gene variant in future work.

    Do the probands have a clear explanation for their developmental disability other than the gene noted?

    __Reply: __We thank the reviewer for raising this point. Proband exomes were also screened for genes related to developmental delay and no other causal gene variant were identified. We added this information in the text, lines 183-185.

    I would encourage the authors to update Table 3: they are missing IHH/KS genes such as GLI3, SEMA7A, SOX2, STUB1, TCF12. I suggest they update the Table and analyses.

    Reply: we thank the reviewer for highlighting this point. Since we performed a new analysis, we also performed a new candidate gene prioritization using a more up-to-date gene list to instruct ToppGene (please see revised Supplemental table 3).

    CROSS-CONSULTATION COMMENTS Dear Reviewer #2, I am concerned that the paper presents only a single case of GD to support the scientific work. What do you think?

    __Reply: __We would like to highlight that, as we describe above, GD can be diagnosed prior to pubertal age in individuals with red flag phenotypic signs and biochemical evidence of hypogonadism.

    Dear Reviewer #1: In addition to the weakness in the microarray data, what do you think about the authors using publicly available data from human GnRH neuron transcriptomics for analysis?

    __Reply: __please see the above discussion on the comparison with publicly available datasets.

    Reviewer #3 (Significance (Required)):

    There is not high significance to this paper: This is not the first article with GnRH transcriptomes. I would argue the human data is more relevant. Developmental disability has been previously linked the GnRH deficiency (as even cited in this paper) The article presents one case of GnRH deficiency, and one pre-pubertal case -- providing some modest evidence for a candidate gene, NLGN3.

    __Reply: __We would like to rebuff this assessment of the paper’s significance. To our knowledge, this is the first report of transcriptomes from primary GnRH neurons isolated at key embryonic developmental time points. Other published reports refer to iPSC-derived or adult GnRH neurons (Keen et al., 2021; Lund et al., 2020; Wang et al., 2022; Vastagh et al., 2016 and 2020).

    Similarly, the association of central hypogonadism with developmental disabilities have been reported in registry-based studies, but few causative genes have been identified, nor patient variants functionally validated in order to investigate the molecular biology underpinning this association. In the Discussion, in the light of a recent paper (Manfredi-Lozano et al., 2022, doi: 10.1126/science.abq4515), we also postulate that NLGN3 might be required for neuritogenesis of extra-hypothalamic projections of GnRH neurons thus contributing to the pathogenesis of NDD (lines 294-300).

    Regarding to human data, we would like to acknowledge that we had a third case that we were not able to publish due to family consent. NLGN3 deficiency is likely to be a rare disorder, but that should not obviate the impact of investigating the molecular etiology – indeed, many insights into human biology have come from private mutations in rare disease.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Oleari et al used murine GnRH1, and immortalized GnRH cell lines (GT1-7, Gn11) to define genes of interest in GnRH development and used this list to filter exome sequencing data from patients with some evidence for GnRH Deficiency.

    Title: I am concerned that the title of the paper overstates the results and conclusions.

    Intro: use of "candidate causative genes" overstates the evidence presented.

    Results:

    The transcriptomic profile of the developing human GnRH neuron has been published via in vitro differentiation protocols twice (Lund et al 2020, and Keen et al 2021). Gene set data is publicly available. This should be explicitly compared in results not relegated to discussion -- two or three examples it not enough to say mouse can be used instead of human.

    The authors need to comment on other GnRH1 expression in the brain of developing rodent and if they think the GnRH1 sorted neurons are just "GnRH Neurons" associated with reproduction (Parhar et al 2005) due to microdissection.

    Questions about Cases/Missing Phenotypic Information:

    1. Case 1: the patient underwent increased testicular volume on testosterone therapy -- testosterone therapy does not increase testicular volume. Has this patient undergone or been assessed for reversal of his hypogonadism?
    2. Case 2: Is too young to be classified as having a pubertal defect. Microphallus is mentioned but what size, was this diagnosed at birth and treated? I think the case for GD is overstated in the results and discussion (especially with the discussion of small testes).

    Genetic Information:

    Since this was a candidate gene search -- what other candidate genes were uncovered in these probands? Do the probands have a clear explanation for their developmental disability other than the gene noted?

    I would encourage the authors to update Table 3: they are missing IHH/KS genes such as GLI3, SEMA7A, SOX2, STUB1, TCF12. I suggest they update the Table and analyses.

    Referees cross-commenting

    Dear Reviewer #2, I am concerned that the paper presents only a single case of GD to support the scientific work. What do you think?

    Dear Reviewer #1: In addition to the weakness in the microarray data, what do you think about the authors using publicly available data from human GnRH neuron transcriptomics for analysis?

    Significance

    There is not high significance to this paper: This is not the first article with GnRH transcriptomes. I would argue the human data is more relevant. Developmental disability has been previously linked the GnRH deficiency (as even cited in this paper) The article presents one case of GnRH deficiency, and one pre-pubertal case -- providing some modest evidence for a candidate gene, NLGN3.

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    Referee #2

    Evidence, reproducibility and clarity

    Oleari et al performed comparative transcriptome analysis on the different developmental stages of GnRH neurons, as well as two immortalized GnRH neuronal cells GT1-7 and GN11 which represent mature and immature GnRH neurons. As a results, they identified a panel of differentially expressed genes (DEG). They further used top DEGs as candidate disease-related genes for GnRH-deficiency (GD), a disorder characterized with absent of delayed puberty and infertility. To this end, they found two loss-of-function mutations in NLGN3 in patients with GD combined with autism. This study provide a resource for the identification of novel GD-associated genes, and suggest an intrinsic connection between GD and other neurodevelopmental diseases, such as autism. I only have some minor concerns.

    1. According to the pedigree, both probands (case 1 and 2) inherited their NLGN3 mutations from their unaffected mother, consistent with an X-linked recessive inheritance. However, only "parent" was used in the manuscript, therefore, it is not clear if this "parent" is the probands' mother or father.
    2. It is suggested to integrate Figure 2 as a panel in Figure 1.
    3. What is the meaning of Peak LH and Peak FSH, and how are they measured in Table 2?
    4. A genotyping for the elder brother of Case 2 will be a strong evidence to support NLGN3 as a GD-associated gene.
    5. The authors claimed neither probands carried deleterious variants in known GD genes. It is suggested to indicate the exclusion criteria (which genes? How do they define a variants is deleterious?)
    6. Please also include a sequence chromatogram for proband 2.

    Referees cross-commenting

    I agree with Reviewer 3, the genetics is not very strong, as NLGN3 mutations were only found in one GD case from their cohort and one pre-pubertal case from the literature. It will be nice to analyze the genotype and phenotype of Case 2's older brother. Further, it is important to screen NLGN3 rare sequencing variants in larger GD cohorts.

    Significance

    This study provide a resource for the identification of novel GD-associated genes, and suggest an intrinsic connection between GD and other neurodevelopmental diseases, such as autism. It may welcome by researchers and clinicians in the filed of neurodevelopment.

  5. Review Commons

    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

    The current manuscript in question is well written and of general interest to the reproductive neuroendocrinology field. Overall it is a well written and substantiated.

    The primary problem with the paper is the data derived from the microarray. While the experimental design included replicates (n = 3), although weak, the actual microarray data was based on a single data point. A major weakness. This experiment should be repeated using more up-to-date approaches such as RNA-seq or left out of the manuscript, because this data set is compromised due to the data collection procedure.

    Referees cross-commenting

    Notwithstanding the importance of neuroligin 3 during glutaminergic synaptogenesis, I agree with the reviewers on both points. Further screenings of the patient's family members should be done and the microarray data should be removed or potentially moved to a supplementary status.

    Significance

    The paper is of significance based on the neuroligin 3 data, which is indicative of abnormal synaptogenesis. However, these defects seem to only have a limited effect on the functionality of GnRH neuron system and do not seem to cause elimination of GnRH neurons themselves. Nevertheless these data do open end a new direction that may help explain some dysfunctions in reproductive health.

  6. Version published to 10.1101/2022.05.24.22275221v1 on medRxiv