Pathogenic Research Of A Novel Variant In SEMA3A Gene And Clinical Analysis Of Congenital Hypogonadotropic Hypogonadism

Part I Identification and pathogenic research of a novel variant in SEMA3A gene Background:Congenital hypogonadotropic hypogonadism(CHH)is a rare genetic disease characterized by delayed or absent pubertal development and/or infertility due to an isolated defect in gonadotropin-releasing hormone(GnRH)secretion or action.When CHH is accompanied by anosmia/hyposmia,it is named as Kallmann syndrome(KS),which accounts for about 40%to 60%of CHH cases;and when the sense of smell is normal,it is considered normosmic congenital hypogonadotropic hypogonadism(nCHH).With low incidence and complex genotype-phenotype relationship of CHH,it is difficult to make a clear diagnosis through simple clinical manifestations in some cases.Reproductive system defects such as"small penis,small testicles,and infertility" have brought heavy psychological burdens to patients and families.Therefore,genetic testing plays an increasingly important role in the early diagnosis and differential diagnosis of CHH.Currently,genetic defects account for about 50%of all CHH cases and about 50 genes can cause CHH,of which about 20 genes can lead to KS,including ANOS1,FGFR1,PROK2,CHD7,SEMA3A and so on.Among these genes,Semaphorin3A(SEMA3A)is a secreted protein encoded by the SEMA3A gene,which is mainly guided by axons and is essential for the development of olfactory organs and the migration of GnRH neurons.However,SEMA3A variants only account for around 6%of KS cases and the related molecular mechanism remain ambiguous,which needs further research.This section explored the pathogenicity of the novel SEMA3A variant in KS and its associated molecular mechanisms,which provided additional data for genetic counseling in patients with KS.Objectives:1.Identification of the novel variant in the SEMA3A gene in a family with KS.2.To clarify the pathogenicity of the novel variant in SEMA3A.3.To analyze the molecular pathogenic mechanism of KS caused by SEMA3A.Methods:1.Make a clinical data collection on the proband.2.Peripheral blood of the proband and his family members was collected for DNA extraction.Whole exome sequencing technology was used to screen the related pathogenic genes,which were verified by Sanger sequencing.3.Bioinformatics analysis of the novel variant in SEMA3A was carried out by Mutation Taster,PolyPhen-2 and PROVEAN.The homodimer model of SEMA3A protein was established by SWISS-MODEL and the regions involved in the variant were visualized by Deepview.4.The expression of SEMA3A mutant protein was detected by in vitro functional experiments such as Western blot(WB)and Enzyme linked immunosorbent assay(ELISA).5.The pathogenic molecular mechanism of SEMA3A leading to KS was analyzed by PubMed and all reported SEMA3A variants related to KS were summarized by Human gene mutation database(HGMD).Results:1.The proband was a 31-year-old male who had clinical manifestations of hyposmia and infertility.Laboratory tests showed low levels of sex hormones.GnRH stimulation test showed delayed LH peak.Semen routine showed that the number and the motility of sperm were low.Ultrasound showed small bilateral testes and gynecomastia.The proband’s father had been diagnosed with KS.Based on symptoms,signs auxiliary examinations and family history,,the proband was initially diagnosed as KS.2.A de novo(newborn)heterozygous variant(c.814G>T,p.D272Y)of SEMA3A was identified in the proband and his father,which had not been reported at home and abroad.The variant was located at the highly conserved Sema domain with important functions.3.Bioinformatics analysis suggested that it had pathogenic significance with a high damage score.The three-dimensional structure modeling of the protein showed that the overall spatial structure of the mutant protein was relatively looser than that of the wild type.In terms of amino acid properties,the original negatively charged acidic state had become the uncharged neutral polarity state,which was the carbonyl group became a benzene ring.Ultimately it altered the charge distribution of the entire protein and its stability.4.In vitro functional experiments showed that the novel variant significantly reduced the intracellular and extracellular expression of the secreted SEMA3A protein(P<0.05),which proved that the variant could cause the secretion defect of SEMA3A protein.5.The analysis of the pathogenic molecular mechanism suggested that mutant SEMA3A may cause the disease by failing to induce the phosphorylation of Focal adhesion kinase(FAK)in GnRH neurons or by affecting the formation of the SEMA3A-NRP-Plexin ligand-receptor complex.6.Through the HGMD database,there were 19 variants of SEMA3A related to KS(including the novel variant),including 16 missenses,1 splicing,1 small deletion and 1 gross deletion.68.4%of the variants were located at the Sema domain and 20.8%of the patients had additional mutations in two or more genes.Conclusions:1.A novel heterozygous variant(c.814G>T,p.D272Y)in SEMA3A was identified by WES in the patient and his father with KS.2.Bio informatics analysis and in vitro functional experiments elucidate the pathogenicity of de novo SEMA3A variant in KS.3.The novel variant in SEMA3A extend the genetic mutation spectrum of SEMA3A and help KS patients get additional data in genetic counselling.It also help identify the function of SEMA3A and reveal the underlying pathogenesis of KS.Part II Clinical analysis of congenital hypogonadotropic hypogonadism Background:Congenital hypogonadotropic hypogonadism(CHH)is a rare disease caused by defective GnRH neuron development or migration,resulting in insufficient secretion of pituitary gonadotropins,which in turn causes gonadal developmental disorders.According to the presence or absence of olfactory disorder,it can be divided into Kallmann syndrome(KS)with olfactory disorder and CHH with normal olfactory sense(nCHH).However,care should be taken when using these terms.Routine clinical consultations tend to underestimate the olfactory disturbance of patients with KS.KS can easily be misdiagnosed as nCHH without professional olfactory assessment or image examination.In addition,the boundary between KS and nCHH is sometimes blurred and the pathogenesis of both can be caused by the same pathogenic gene such as FGFR1.Therefore,we made a detailed difference analysis of the clinical manifestations and hormone levels of KS and nCHH,which provided an important reference for clinicians to better understand and identify these two disorders.In addition,CHH has obvious clinical and genetic heterogeneity and has different clinical manifestations at different ages.A small number of CHH patients also have congenital non-reproductive developmental abnormalities,such as cleft lip and palate,midline defects,hearing loss,visual impairment,mirror movements and so on,which led to a greater challenge for clinicians to diagnose and differentially diagnose CHH.This part retrospectively analyzed the clinical features of 90 male CHH patients which expanded the spectrum of clinical manifestations of CHH,improved clinicians’ awareness of the disease,and helped clinicians in diagnosis,differential diagnosis and treatment of CHH.Objectives:1.Collect male CHH cases and analyze the clinical features of CHH.2.To compare whether there are statistical differences between KS and nCHH patients in terms of age,height,weight,BMI,penis length,testicular volume,sex hormone levels and other indicators,which provide a reference for the diagnosis of CHH subtypes.Methods:1.A retrospective analysis of the clinical data of male CHH patients admitted to the Department of Endocrinology and Pediatrics of Shandong Provincial Hospital from October2011 to November 2021,including current illness history,birth history,family history,penis length,testicular volume,sex hormone levels,GnRH test,bone age detection,karyotype analysis and treatment plan,etc.2.SPSS 26.0 software was used for statistical analysis of CHH-related clinical data.3.Follow-up of CHH patients by clinic or telephone.Results:1.A total of 90 male CHH patients were included in this study,including 33(36.7%)KS patients and 57(63.3%)nCHH patients.Gynecomastia was detected by ultrasound in 49patients(54.4%).82 patients with CHH underwent bone age testing,of which 57(69.5%)patients showed a backward bone age,with an average of 4.1 years behind.2.There was no statistical difference between the KS and nCHH groups in terms of age,height,weight,BMI,penis,testosterone,estradiol,and prolactin.However,compared with nCHH,KS had more severe reproductive phenotypic defects manifested as lower gonadotropin levels,smaller testis and a higher incidence of cryptorchidism(p<0.05).3.In addition to the reproductive phenotypic abnormalities associated with GnRH deficiency alone,CHH can also be combined with non-reproductive phenotypic abnormalities such as hearing impairment,visual impairment,ptosis,and midline deviation.4.Different treatment regimens for CHH had advantages and disadvantages and there were great differences in the response of different patients to the same treatment regimen.CHH reversal may occur in a minority of patients.Conclusions:1.In addition to reproductive system defects,CHH also has non-reproductive phenotypes such as skeletal age retardation,hearing impairment,visual impairment andptosis.2.The more severe reproductive phenotypic defects of KS than nCJIH such as "lower gonadotropin level,smaller testis,and higher incidence of cryptorchidism" provide an important reference for clinicians to diagnose and differentially diagnose KS and nCHH.3.Through appropriate hormone replacement therapy,CHH patients can induce the development of sexual characteristics,maintain normal sex hormone levels and healthy sexual life and achieve fertility.