Clinical Outcomes Analysis Of 110 Male Patients With IHH And Preliminary Study On The Function Of CHL1 And PAX6 Gene

Objective: To study the clinical characteristics of 110 Chinese male patients with idiopathic hypogonadotropic hypogonadism(IHH),analyze the association between genotype and outcome,and use in vitro functional assays to explore the role of CHL1 and PAX6 gene and verify whether these genes are new causal genes in IHH.Methods: Combined with the panel sequencing results of the previous study(Zhou C,et al.Fertil Steril.2018)and the clinical outcome of 110 male IHH patients,we analyzed the outcome of patients with different mutations using statistical methods.We used high-throughput sequencing and bioinformatic analysis to screen for likely pathogenic IHH genes,and further constructed CHL1 and PAX6-related si RNA or vectors.GN11 cell is a good Gn RH neuron cell migration model.After the above gene products were transfected into cells,we determined GN11 cell viability and migration,as well as changes in downstream signal pathways.Results: After 110 male IHH patients received human chorionic gonadotropin(hCG)treatment,94.55% of the patients restored normal serum testosterone levels,and 54.55%achieved seminal sperm appearance(SSA).Patients who remained abnormal testosterone levels harbored rare sequencing variants(RSVs),including PLXNB1,ROBO3,LHB,NRP2,CHD7,PLXNA1 RSVs.The present study found that 30% of patients carried pathogenic/likely pathogenic(P/LP)RSVs,and these patients with P/LP RSVs took a longer time to achieve SSA than non-carriers.The RSV with most significant correlation was PROKR2 RSVs.In a family with Kallman syndrome,three affected individuals harbored a same CHL1 RSV(p.G517E)detected by whole exome sequencing.Data of in vitro functional assays suggested that overexpression of CHL1 protein promoted Gn11 cell activity and migration.The involved mechanisms included regulation of ERK1/2 activation,calcium loading and RIPK3/MLKL transcription.On the contrary,the CHL1 RSV disrupted the original positive regulatory function of CHL1 protein.In addition,among the three sporadic male IHH patients,we found that these patients carried PAX6 RSVs using target gene panel sequencing,and the patient with the nonsense mutation was accompanied by iris hypoplasia and nystagmus.In vitro experimental results proved that overexpression of PAX6 protein could promote GN11 cell migration.PAX6 RSVs not only affected the PAX6 expression and destroyed the normal nuclear localization of the protein,but also inhibited GN11 cell migration by down-regulating the FOSB/SEMA3A/ERK1/2 signaling pathway.Conclusions: This study confirmed the correlation between multiple RSVs and poor outcome during the h CG treatment,and suggested that male patients with P/LP PROKR2 RSVs had poor recovery of spermatogenesis.Male patients with this genotype might be better to choose gonadotropin combination therapy or pulsatile Gn RH administration(subcutaneous injection)before treatments start.In addition,this study expands the genetic profile of Chinese IHH candidate genes,reveals the role of CHL1 and PAX6 genes in the pathogenesis of IHH,and further clarifies the regulatory mechanism of the Gn RH neuronal network.We provide the strong reference evidence for genetic diagnosis as well as individualized treatment in IHH.