Clinical Indicators Of Bone Metabolism And Molecular Genetic Analysis In Idiopathic Hypogonadotropic Hypogonadism

Objectives1.A retrospective analysis of the clinical manifestations,sex hormone levels,bone metabolism indices and related complications in patients with idiopathic hypogonadotropic hypogonadism（IHH）,so as to improve clinicians’understanding,diagnosis and treatment of the disease.2.Using the WES method to screen the related pathogenic genes in IHH probs,and evaluate the detection rate of related genes,with the aim of finding new mutation genes and/or new mutation site,enrich the genetic spectrum of the disease,as well as explore the correlation between pathogenic genes and phenotypes,clarify the incidence of polygenic mutations and whether there are differences in the levels of sex hormones.Methods1.A total of 104 patients with idiopathic hypogonadotropic hypogonadism（IHH）who were treated in a hospital of Henan Provincial from May 2013 to October 2021 were collected,and their clinical case characteristics（including general information,physical examination,pituitary hormone axis and bone metabolism indexes,gonadotropin-releasing hormone（Gn RH）excitation test,pituitary MRI,bone mineral density,kidney and genital ultrasound and other inspection results）were analyzed retrospectively.2.The methods of Whole Exome Sequencing（WES）and Sanger sequencing was performed to screen for mutated genes in the probs and some family members,inquiried the mutation frequency of each mutated gene in the human database.The bioinformatics platforms such as SIFT,Poly Phen-2 and Mutation Taster were applied to carry out functional prediction of the mutant site,and evaluate the pathogenicity of the gene.3.Statistical software（SPSS 21.0）was applied to analyze the differences between hormone levels,non-reproductive phenotypes,and bone metabolism indexes in IHH patients,and the correlation between sex hormone and bone metabolic indexes was analyzed,the difference was considered statistically significant at P<0.05.Results1.A total of 104 patients（85 males and 19 females）with IHH were included in this study,and the age at first diagnosis ranged from 14 to 40 years,including 55 patients（47 males and 8 females）with Kalman syndrome（KS）and 49 patients（38 males and 11 females）with normosmic IHH（n IHH）.The main manifestations of males with micropenis and small testes,incomplete development of secondary sexual characteristics,mammary gland development（15.2%,13/85）,cryptorchidism（17.6%,15/85）,testicular microlithiasis（10.5%,9/85）,and short stature（2.3%,2/85）;the females were mainly presented with primary amenorrhea,infantile vulva,and mammary gland dysplasia（Tanner stage I-II）.In KS patients,the main non-reproductive phenotypes were obesity（10.9%,6/55）,elbow valgus（9.1%,5/55）,deviated nasal septum（3.5%,2/55）,and facial deformities（7.2%,4/55）;the n IHH group mainly had hearing impairment（8.1%,4/49）,history of fracture（8.1%,4/49）,elbow valgus（4.9%,2/49）,obesity（6.1%,3/49）,etc,there were no significant differences in the non-reproductive phenotypes.There were no statistical differences in FSH,LH,T and E2 levels between KS and n IHH groups,and the results of Gn RH excitation test in 84patients showed that the peak levels of FSH and LH were at 4.63（2.73～6.61）IU/L,2.64（1.22～6.16）IU/L,the peak time of LH was concentrated at 60 min（34.52%）and 120 min（36.90%）,64.2%（54/84）of the patients with non-activation of gonadal axis function and 27.3%（23/84）of patients with partially impaired of gonadal axis function.Spearman rank correlation coefficient was used to analyze the level of sex hormones and bone metabolism,there was a positive correlation between FSH,LH and 25（OH）D,r_s=0.491,0.453,and between E₂ and 25（OH）D,femoral neck BMD r_s=0.791,0.669;there was a negative correlation between LH and OC,β-Cross L,E₂andβ-Cross L,r_s=-0.287,-0.255,-0.418.There was a statistical significant in the levels of 25（OH）D and OC between the groups with LH≤0.7 U/L and LH>0.7 U/L（P<0.05）.In the analysis of complications associated with IHH patients,76.6%（46/61）of the patients had BMD below the expected range for the same age group;59.1%（29/49）had delayed bone age,mean age 21（19-24）years;17 patients had glucose tolerance test,2 patients had impaired glucose tolerance and 3 had diabetes mellitus;21.1%（22/104）of patients had a BMI>24 kg/m²,which exceeded the normal body fat index.2.96 patients with IHH were tested by WES and Sanger validation（KS 51,n IHH 45）,66 patients had mutated genes,with a total detection rate of 68.75%（66/96）,including 31 mutated genes and 82 mutated sites,most of which were missense heterozygous mutations（73/82）,and 42 pathogenic mutations were screened by bioinformatics analysis,most of them were novel mutations.The mutation rates were higher in PROKR2,CHD7,KAL1,FGFR1,IL17RD and AXL genes,accounting for 56.6%.In addition,the mutation rate of LEPR,RNF216,DMXL2,PNPLA6 and SEMA3A accounted for 17.1%,which could be considered as candidate genes.In addition,1 case of ATRX gene was detected,previously confirmed to be associated with alpha-thalassemia/mental retardation syndrome.Among the patients with genetic tested,single mutations accounted for 44.7%（43/96）and multiple mutations 23.9%（23/96）,of which 73.9%（17/23）were double mutations,with PROKR2 and KAL1 combined with other gene combinations were more common,and the three combinations of CHD7+DMXL2+PNPLA6,PROKR2+AXL,and PROKR2+FEZF1 were discovered as the combination pattern of pathogenic genes.There was no statistical difference in the levels of gonadotropin and sex hormone among the three groups of monogenic,polygenic and no gene mutations.3.The mutation spectrum of the disease was extended and found PROKR2（p.A103V,p.L218P,p.G229R,p.R270H,p.D42delins DED）,CHD7（p.D728N,p.S103T,p.G1048E,p.S1049C,p.Y1008C,p.H185Q,p.T730I,p.S8R,p.K2129E,p.R2319L,p.Q2073P）,FGFR1（p.185-189del,p.A526fs,p.E322fs）,AXL（p.W448L）,FGF17（p.P120L）,SEMA3A（p.R197Q）and other genes with novel mutation sites and their function predictions may have the potential harm to protein function.A total of four families had IHH symptoms,and mutations were detected in three families:KAL1:p.N155D pure mutation（family 1）,FGFR1:p.E322fs shift code mutation（family 2）,EIF2S3:3UTR locus（family 3）,which have not been reported in the literature and there are novel mutation sites,and no mutant gene was detected in family 4.4.The study found that KAL1 and AXL genes were more frequently associated with olfactory impairment;PROKR2 gene combined with obesity and deviated nasal septum were common;CHD7 gene could present with elbow ectropion,obesity,deviated nasal septum and visual/hearing impairment;FGFR1gene mutation was associated with congenital cleft lip/palate and congenital deafness.Conclusions1.The levels of sex hormones in IHH patients are significantly lower than the normal standard,and only hypoplasia of gonads and secondary sexual characteristics may occur,some with congenital cleft lip,elbow ectropion,facial midline abnormalities,cryptorchidism and other abnormal phenotypes.2.Both hypogonadotropic and hypogonadotropic hormones in IHH patients reduce the level of bone metabolism,and it is easy to combine with metabolic syndrome such as impaired glucose tolerance,obesity,osteoporosis,etc.so early prevention and treatment is essential.3.This study found several novel pathogenic mutations sites and polygenic combination patterns associated with IHH disease,enriching the gene spectrum of IHH disease.