Characteristic Analysis Of Kallmann Syndrome Patients With ANOS1 Gene Mutation And Spermatogenesis Efficacy Of Gonadotropin Therapy In Patients With Partial CHH

Part ?Characteristics of Kallmann syndrome patients with ANOS1 gene mutationBackgroundKallmann syndrome is a rare genetic disease characterized by hypogonadotropic hypogonadism and anosmia.ANOS1 gene is the first pathogenic gene found in Kallmann syndrome.It is located on the X chromosome and belongs to the X-linked genetic form,encoding the protein anosmin-1.ANOS1 gene mutation causes anosmin-1 protein dysfunction,which leads to the migration damage of GnRH neurons and olfactory neurons,resulting in Kallmann syndrome.In this study,we investigated the mutations of ANOS1 gene in the Chinese cohort of Kallmann syndrome with large sample size and single center,analyzed the relationship between clinical phenotype and gene mutation,and completed the biological function verification of new mutation sites.Objective1.To screen the mutations of ANOS1 gene in 212 Chinese male Kallmann syndrome patients.2.To determine whether the new ANOS1 gene mutation is pathogenic and its relationship with clinical phenotype.Objects and methodsParticipants:from January 2010 to October 2017,a total of 212 male patients with sporadic Kallmann syndrome were recruited from the Department of Endocrinology,Peking Union Medical College Hospital,and they agreed with pathogenic gene screening.Research methods1.Gene screening:31 genes related to Kallmann syndrome were screened by target sequence capture and next generation sequencing.Sanger sequencing was used to verify the ANOS1 mutation.2.Clinical study:collecting the clinical data of Kallmann syndrome patients,and sort out the clinical characteristics of ANOS1 gene mutation patients.3.Correlation between new mutation genotypes and phenotypes:functional prediction of new missense mutation sites of ANOS1 was made by using five biological prediction software:polyphen-2,sift,mutationtaster,mutationassessor and provean;functional prediction of new splice site mutationof ANOS1 was made by using two biological prediction software,namely Human splicing finder and Splice site score calculation4.In vitro function verification test:Transwell test5.To summarize the clinical characteristics of Kallmann syndrome caused by mutations at different sites and to explore the relationship between phenotype and genotype.Result1.In this study,212 patients with Kallmann syndrome were included,22 of whom had ANOS1 gene mutation.In these patients,the prevalence of cryptorchidism was as high as 36.4%(8/22),the probability of renal hypoplasia was as high as 18.2%(4/22).One patient had mirror movement of both hands,and 100%of the patients with olfactory MRI abnormality had olfactory abnormality2.In this study,19 mutations were found in ANOS1,including 5 nonsense mutations,8 missense mutations,3 frameshift mutations and 3 splice site mutations,of which 8 mutations have been previously reported(p.R257X?p.W258X?p.R262X?p.R423X?p.R424X?p.Y579fs?p.V560I?c.1843-1G>A).Two new mutations(p.P135fs?K512fs),seven missense mutations(p.A140G?p.C147Y?p.C157R?p.C164F?p.C164S?p.A264P?p.P504L)and two splice site mutations(c.1207+1G>c?c.1449+1G>A)were found.All the new missense mutations are considered to be pathogenic after being calculated by five biological softwares.The mutation sites of two new splice site mutations are located in the most classical human splice site(GT-AG sequence),and the two splice prediction software(human splicing finder and splice site score calculation)predict that the possibility of disease is high.3.Transwell experiment was used to verify the effect of ANOS1 gene mutation on the migration of NLT cells.The migration ability of seven ANOS1 missense mutations was significantly lower than that of the wild ANOS1 group.Conclusion1.Kallmann syndrome patients with ANOS1 gene mutation are more likely to have clinical phenotypes involving its non reproductive system.2.In this study,19 mutations of ANOS1 were found,8 of which were reported mutations were confirmed again,and 11 new mutations of ANOS1 were found,and the missense mutations were functional verified to confirm their pathogenicity.These mutations expanded the mutation spectrum of ANOS1,providing a basis for prenatal diagnosis and genetic consultation.Part ?Gonadotropin treatment for male partial congenital hypogonadotropic hypogonadism in Chinese patientsBackgroundCongenital hypogonadotropic hypogonadism(CHH)is a group of rare heterogeneous reproductive disorders in clinical and genetic phenotype,which is caused by the deficiency of GnRH secretion in hypothalamus or the deficiency of its function.According to the degree of puberty development,CHH patients can be divided into partial congenital hypogonadotropic hypogonadism(partial CHH,PCHH)and complete congenital hypogonadotropic hypogonadism(complete CHH,CCHH).partial CHH is caused by an insufficiency in,but not a complete lack of,gonadotropin secretion.This leads to reduced testosterone production,mild testicular enlargement,and partial pubertal development.No studies have shown the productivity of spermatogenesis in patients with partial CHH.We compared the outcomes of gonadotropin-induced spermatogenesis between patients with partial CHH and complete CHH.Previous studies on partial CHH were mainly limited to symptom description and mutation gene identification.At present,there is no report on gonadotropin treatment of partial CHH in Chinese.In this study,male CHH patients were used to analyze the efficacy of gonadotropin treatment of partial CHH and to investigate relevant genes mutations.Objective1.To analyze the efficacy of gonadotropin treatment between partial CHH patients and complete CHH patients.2.Partial CHH patients and complete CHH patients were screened for pathogenic gene mutations to determine the impact of different mutations on the clinical performance of patients.methods1.Subjects:From January 2008 to September 2016,122 male CHH patients were included.2.Retrospective study3.For all patients(122),human chorionic gonadotropin(HCG)and human menopausal gonadotropin(HMG)were intramuscular injected.Testicular size,serum luteinizing hormone levels,follicle-stimulating hormone levels,serum total testosterone levels,and sperm count were recorded at each visit.4.Gene screening:87 patients received next-generation sequencing,including 31 CHH related gene mutations,was conducted to identify the probable causative gene in partial CHH patients and complete CHH patients.Result1.After gonadotropin therapy,patients with partial CHH had a higher spermatogenesis rate(92.3%)than complete CHH patients(74.7%).During 24-month combined gonadotropin treatment,the partial CHH group took significantly less time to begin producing sperm compared with the complete CHH group(median time:11.7 vs 17.8 months,P<0.05).2.A total of 87 patients were screened for pathogenic genes.28 of them were screened for CHH related mutations by next-generation sequencing.The detection rate was about 32.2%(28/87).9 pathogenic genes were detected in 28 mutations,of which 7 were known pathogenic mutations in past report and 21 were new gene mutations.FGFR1,ANOSl,PROKR2 and CHD7 mutations were found in both partial and complete CHH groups,indicating that the same mutation may lead to different phenotypes and different prognosis.LHB and NELF mutations were found only in partial CHH group.GNRHR,FGF8 and PROK2 mutations were found in complete CHH group,and none of them were detected in partial CHH group.Conclusion1.We defined male patients with partial CHH as those with a testicular volume of>4 ml and complete CHH as those with a testicular volume of<4 ml.2.During the two-year regular gonadotropin treatment,the spermatogenesis rate of partial CHH patients is higher than complete CHH patients,and the first time of spermatogenesis is shorter.Under the same treatment time,the average density of spermatogenesis of partial CHH patients is much higher than complete CHH patients.3.FGFR1,PROKR2.CHD7,ANOS1 gene mutations can lead to partial CHH and complete CHH.According to the current research,no gene mutation that can only lead to partial CHH has been found.The results of gene detection show that the probability of gene mutation in partial CHH and complete CHH is the same.For patients who have been identified with pathogenic gene mutation,the two groups should be given intensive treatment.The rate of treatment failure was higher in the complete CHH group with pathogenic gene mutation,so we should pay attention to the results of gene detection in order to find the appropriate treatment.