IGSF10 mutations dysregulate gonadotropin‐releasing hormone neuronal migration resulting in delayed puberty

Abstract Early or late pubertal onset affects up to 5% of adolescents and is associated with adverse health and psychosocial outcomes. Self‐limited delayed puberty (DP) segregates predominantly in an autosomal dominant pattern, but the underlying genetic background is unknown. Using exome and candidate gene sequencing, we have identified rare mutations in IGSF10 in 6 unrelated families, which resulted in intracellular retention with failure in the secretion of mutant proteins. IGSF10 mRNA was strongly expressed in embryonic nasal mesenchyme, during gonadotropin‐releasing hormone (GnRH) neuronal migration to the hypothalamus. IGSF10 knockdown caused a reduced migration of immature GnRH neurons in vitro, and perturbed migration and extension of GnRH neurons in a gnrh3:EGFP zebrafish model. Additionally, loss‐of‐function mutations in IGSF10 were identified in hypothalamic amenorrhea patients. Our evidence strongly suggests that mutations in IGSF10 cause DP in humans, and points to a common genetic basis for conditions of functional hypogonadotropic hypogonadism (HH). While dysregulation of GnRH neuronal migration is known to cause permanent HH, this is the first time that this has been demonstrated as a causal mechanism in DP.‡

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I look forward to seeing a revised form of your manuscript as soon as possible. ***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System): The study presents novel findings that will help in the diagnosis of delayed puberty in patients and will contribute to our understanding of the mechanisms governing GnRH migration and development.

Referee #1 (Remarks):
In the present study, Howard and colleagues present a compelling series of human genetic, in vitro and in vivo studies that elegantly describe the novel role of IGSF10 in the migration of GnRH neurons to the hypothalamus during the embryonic period. Their data is supported by a large analysis of patients suffering hypogonadotropic hypogonadism and the function of this molecule postulated and assessed by in vitro models using GN11 cells, which showed reduced migration after Igsf10 knockdown, and reduced migration of GnRH neurons in zebra fish with a morpholino knockdown approach of this molecule. Overall, the study is innovative, informative, well designed and the results clearly stated. There are only a few comments regarding the proposed mechanism of action for Igsf10: -The authors tested the migration of GN11 cells after KD of Igsf10. This experiment assumes that GnRH neurons express Igsf10, which would be acting, perhaps, in an auto synaptic feedback loop in GnRH neurons. Still, the authors showed the expression of Igsf10 in other hypothalamic areas and, in the discussion, mentioned that this molecule probably participates in the creation of a gradient needed to direct GnRH neuronal migration. It is therefore not clear whether GnRH neurons may also express this molecule or whether GN11 cells, due to their immortalized nature, are not a faithful replication of GnRH neurons in vivo. It would be good if the authors clarified this by assessing the expression of Igsf10 in other GnRH cell lines and, if possible, through double label ISH with better resolution than the images presented in Figure 4.
-The authors nicely explain that the amount of mutations accumulated in a single individual may account for the different magnitudes in the HH phenotype observed, however, this does not explain the adult onset of HH discussed inlines 321+. If the role of Igsf10 is solely in the migration of GnRH neurons, as suggested by the disappearance of its expression in late embryonic phases, why would these mutations induce secondary amenorrhea after the HPG axis has been properly activated during puberty? Do they know whether this molecule has further developmental regulation? Would it be possible that its expression increases again at the time of puberty onset and/or is regulated by sex steroids in adulthood? Including a few samples from mice at critical developmental time points (e.g. infantile, juveline, peripubertal and adult) would address this question.
-Line 193: do they mean "presence" instead of absence? -Line 197: One of the mutations has less than 3 D and would be therefore "possibly damaging" according to their criteria. - Figure 4: The data depicting IGSF10 expression in the human tissue is too weak. Are they sure this is specific? Please, include controls using the sense probe in the supplementals. - Figure 4: Please, include a scale bar in each panel.
Referee #2 (Comments on Novelty/Model System): The authors demonstrate that IGSF10 regulates embrionic GnRH neuronal migration and mutations result in delayed puberty. This is, indeed, a new concept in Molecular Medicine. The manuscript has a high technical quality and the information is novel.

Referee #2 (Remarks):
Sasha et al, with the corresponding author being Prof. Dunkel, present an elegant multinational study where they have identified mutations in IGSF10 in 6 unrelated families, resulting in intracellular retention of this protein , thus with failure in the secretion of mutant proteins. Furthermore, the authors show that knock out of IGSF10 caused reduced migration of immature GnRH neurons in vitro and perturbed migration and extension of GnRH neurons in a gnrh3:EGFP zebrafish model. Furthermore, loss-of-function mutations in IGSF10 were identified in patients with hypothalamic amenorrhea. The authors conclude by saying that mutations in IGSF10 cause delayed puberty in humans with common genetic basis for functional hypogonadotropic hypogonadism. Indeed, this is the first time that this has been demonstrated as a casual mechanism in delayed puberty. This is a beautiful manuscript with important data to better understand patients with delayed puberty and hypogonatotropic hypogonadism. With the study, very elegant methodology was used. It is well written and easy to read. Comments: 1. The Introduction should be shortened. Background on IGSF10 should be included in this section.
2. After whole exome sequencing and targeted exome sequencing, the authors found 4 mutations (all of them are heterozygous missense variants predicted to be deleterious by {greater than or equal to}3/5 prediction tools) in IGSF10 (3 of them present in public databases). This is important information; however, with what certitude are these variants pathological?
3. To your knowledge, what kind of differences can be established between mutations in IGSF10 and IGSF1 genes in relation with delayed puberty? 4. Do the authors see any differences in the phenotype between patients with IGSF10 mutations and patients with mutations in KAL1 or PROKR2? 5. In table III the characteristics of Delayed Puberty Probands indicate that the sex is predominantly males (9 out of 10). Any specific comment about the only female subject? Regarding estradiol levels in males, did you measure them? 6. It looks like the induction of puberty was done only in 5 patients. Could the authors comment on the response and the degree of puberty obtained? 7. If would be of interest to include in Table IV   The study presents novel findings that will help in the diagnosis of delayed puberty in patients and will contribute to our understanding of the mechanisms governing GnRH migration and development.
Referee #1 (Remarks): In the present study, Howard and colleagues present a compelling series of human genetic, in vitro and in vivo studies that elegantly describe the novel role of IGSF10 in the migration of GnRH neurons to the hypothalamus during the embryonic period. Their data is supported by a large analysis of patients suffering hypogonadotropic hypogonadism and the function of this molecule postulated and assessed by in vitro models using GN11 cells, which showed reduced migration after Igsf10 knockdown, and reduced migration of GnRH neurons in zebra fish with a morpholino knockdown approach of this molecule. Overall, the study is innovative, informative, well designed and the results clearly stated. There are only a few comments regarding the proposed mechanism of action for Igsf10: -The authors tested the migration of GN11 cells after KD of Igsf10. This experiment assumes that GnRH neurons express Igsf10, which would be acting, perhaps, in an auto synaptic feedback loop in GnRH neurons. Still, the authors showed the expression of Igsf10 in other hypothalamic areas and, in the discussion, mentioned that this molecule probably participates in the creation of a gradient needed to direct GnRH neuronal migration. It is therefore not clear whether GnRH neurons may also express this molecule or whether GN11 cells, due to their immortalized nature, are not a faithful replication of GnRH neurons in vivo. It would be good if the authors clarified this by assessing the expression of Igsf10 in other GnRH cell lines and, if possible, through double label ISH with better resolution than the images presented in Figure 4.  5A).' -The authors nicely explain that the amount of mutations accumulated in a single individual may account for the different magnitudes in the HH phenotype observed, however, this does not explain the adult onset of HH discussed inlines 321+. If the role of Igsf10 is solely in the migration of GnRH neurons, as suggested by the disappearance of its expression in late embryonic phases, why would these mutations induce secondary amenorrhea after the HPG axis has been properly activated during puberty? Do they know whether this molecule has further developmental regulation? Would it be possible that its expression increases again at the time of puberty onset and/or is regulated by sex steroids in adulthood? Including a few samples from mice at critical developmental time points (e.g. infantile, juveline, peripubertal and adult) would address this question.
An overlapping phenotype between DP and HA has been seen in a previous study ( -Line 197: One of the mutations has less than 3 D and would be therefore "possibly damaging" according to their criteria. Damaging or possibly damaging were both assessed as 'deleterious' by our pipeline, but this clarification has been included in the revised manuscript -Line 184-6: 'All four IGSF10 variants are heterozygous missense variants predicted to be deleterious, damaging or possibly damaging by ≥3/5 prediction tools (Table 2).' - Figure 4: The data depicting IGSF10 expression in the human tissue is too weak. Are they sure this is specific? Please, include controls using the sense probe in the supplementals. Panel N is the expression image for the sense probe for human IGSF10, which shows no visible expression, as compared to the purple-colour nasal mesenchyme staining for IGSF10 seen in panels K, L and M.
- Figure 4: Please, include a scale bar in each panel.
Included in revised manuscript.

Referee #2 (Comments on Novelty/Model System):
The authors demonstrate that IGSF10 regulates embryonic GnRH neuronal migration and mutations result in delayed puberty. This is, indeed, a new concept in Molecular Medicine. The manuscript has a high technical quality and the information is novel.

Referee #2 (Remarks):
Sasha et al, with the corresponding author being Prof. Dunkel, present an elegant multinational study where they have identified mutations in IGSF10 in 6 unrelated families, resulting in intracellular retention of this protein, thus with failure in the secretion of mutant proteins. Furthermore, the authors show that knock out of IGSF10 caused reduced migration of immature GnRH neurons in vitro and perturbed migration and extension of GnRH neurons in a gnrh3:EGFP zebrafish model. Furthermore, loss-of-function mutations in IGSF10 were identified in patients with hypothalamic amenorrhea. The authors conclude by saying that mutations in IGSF10 cause delayed puberty in humans with common genetic basis for functional hypogonadotropic hypogonadism. Indeed, this is the first time that this has been demonstrated as a casual mechanism in delayed puberty. This is a beautiful manuscript with important data to better understand patients with delayed puberty and hypogonadotropic hypogonadism. With the study, very elegant methodology was used. It is well written and easy to read. Comments: 1. The Introduction should be shortened. Background on IGSF10 should be included in this section.
Please find the introduction shortened as requested. We do believe, however, that discussion of IGSF10 should not appear in the introduction, as this is the main discovery of the study and so details of this gene would logically follow in the results section. IGSF10 was not a candidate gene prior to the start of the study, and the study design was based on an unbiased analysis of WES data, which makes it difficult to highlight one gene in the introduction.
We feel that earlier disclosure would preempt this exciting discovery in the results section, and interrupt the flow of the argument. We are happy to take further guidance from the editor on this point.
2. After whole exome sequencing and targeted exome sequencing, the authors found 4 mutations (all of them are heterozygous missense variants predicted to be deleterious by {greater than or equal to}3/5 prediction tools) in IGSF10 (3 of them present in public databases). This is important information; however, with what certitude are these variants pathological?
Our in vitro data demonstrates failure of secretion of the two N-terminal mutations and retention within the intracellular compartment, which we believe shows clear evidence of their pathogenicity. These mutations were found in 6 out of the 10

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This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal's authorship guidelines in preparing your manuscript. PLEASE NOTE THAT THIS CHECKLIST WILL BE PUBLISHED ALONGSIDE YOUR PAPER The ethical consents gained from the study participants did not include permission to publish genetic data in public databases. Additionally, this whole exome sequencing data is the source of ongoing gene discovery projects that are central to the corresponding author's ongoing research portfolio. As such, although amendments to the original ethical permissions are being sought, this data cannot yet be deposited in a public repository.