| ContextOlfaction is one of the most important sensations of human beings,which impacts individuals’ social skills,relationships,well-being,and quality of life.According to the olfactory function,congenital hypogonadotropic hypogonadism(CHH)is divided into normosmic CHH(nCHH)and Kallmann syndrome(KS),which is characterized by anosmia or hyposmia.The self-reported smell and MRI of the olfactory apparatus(MRI-OA)are the most commonly used methods to evaluate olfactory function in clinical practice.However,the results of self-reported smell are not always reliable and the MRI is not fully concordant with sense of smell.Psychophysical evaluation of the olfactory function has been widely used in clinical and research settings.However,those international used tools may not be suitable for the Chinese due to differences in culture and environment.In 2019,Wen Zhou et al.developed the Chinese smell identification test(CSIT),which provides an effective tool to assess the olfactory function in the Chinese population.The efficacy of CSIT has been verified in Parkinson disease and schizophrenia,but it has not been validated in CHH patients so far.ObjectiveTo evaluate the olfactory function of CHH patients by the Chinese olfactory function test(COFT).To compare and analyze the diagnostic accuracy of the Self-reported Olfactory Scale(SROS)and MRI-OA for KS and to analyze the influence factors of the olfactory function in CHH patients.To compare the therapeutic effect and genetic background between nCHH and KS patients.MethodsPatients with CHH were prospectively recruited at Peking Union Medical College Hospital between November 2020 and June 2021.The clinical data of the patients were retrospectively analyzed.Targeted next-generation sequencing was used to investigate the pathogenic gene variants.The COFT and SROS were adopted as subjective tools,and MRI-OA was adopted as the objective tool to evaluate olfactory function in CHH patients.SROS used a 5-point scale and was grouped into five categories,namely "good","above average","average","below average",and "poor".Patients in "good","above average" and "average" categories were assigned as normal olfactory function,while patients in "below average" and "poor" categories were assigned as abnormal olfactory function.The COFT includes three tests,the Chinese Smell Threshold Test(CSTT),the Chinese Smell Discrimination Test(CSDT),and CSIT.According to the total score of the three tests(TDI),patients were grouped into normal olfactory function(TDI≥29.25)and abnormal olfactory function(TDI<29.25).The olfactory bulb volume(OBV)and the olfactory sulcus depth(OSD)were quantitively evaluated on MRI.The olfactory function of nCHH patients and KS patients were compared,and linear regression models were built to analyze the determinants of olfactory function in CHH patients.Results1 Clinical characteristic:A total of 71 CHH patients were evaluated,including 69 male patients and 2 female patients.The average age of these patients was 23.6±5.8 years and the body mass index(BMI)was 25.1±4.2 kg/m2.The average serum LH and FSH level was 1.15±2.26 U/L and 2.33±3.65 U/L,respectively.The total testosterone(T)level was 1.55± 1.52 ng/ml and the estradiol(E2)level was 20.31± 11.66 pg/ml.The percentage of patients with smoking,drinking,and chronic rhinitis was 25%,28%,and 31%.2 Comparison of Olfactory function between nCHH and KS patients:Based on the results of COFT,36 patients were assigned as nCHH,and the other 35 patients were KS.In nCHH group,based on SROS,patients in "good","above-average","average" and"below-average" categories were 3,12,20 and 1,respectively.While in KS group,patients assigned to "above-average","average","below-average" and "poor" categories were 1,4,2 and 28,respectively.For MRI-OA,67%of nCHH patients showed normal olfactory apparatus,while 22%of nCHH patients showed unilateral olfactory bulb aplasia/hypoplasia[UOA(H)],and 11%of nCHH patients showed bilateral olfactory bulbs aplasia/hypoplasia[BOA(H)].Ninety-six percent of KS patients had BOA(H),while 4%of KS patients showed normal olfactory apparatus.The nCHH patients had larger OBV and OSD than KS patients(37.58 ± 20.64 VS 2.40± 10.72 mm3,p=0.000;6.82 ± 2.31 VS 2.64± 3.07 mm,p=0.000).3 Influence factors for olfactory function:In linear analysis,the TDI score was used as the dependent variable and other factors,including age,BMI,follow-up time,T,E2,smoking,drinking,rhinitis,SROS,OBV,OSD,were used as independent variables.It showed the larger OBV and OSD were associated with the higher TDI.4 Comparison of the accuracy of SROS and MRI-OA in diagnosing KS:Based on COFT,the accuracy of SROS in the diagnosis of nCHH and KS was 91.5%with a sensitivity of 85.7%,a specificity of 97.2%,a positive predictive value of 96.8%and a negative predictive value of 87.5%.The MRI-OA predicted the nCHH and KS patients with an accuracy of 92.7%,a sensitivity of 96.4%,a specificity of 88.9%,a positive predictive value of 90.0%and a negative predictive value of 96.0%.5 The analysis of therapeutic effect:A total of 26 patients were included.Nineteen patients had gonadotropin therapy and 7 patients had pulsatile gonadotropin-releasing hormone therapy.The average follow-up time was 49.4±29.5 months and the median age was 20.5(17.8,26.0)years.The basal testicular volume of these patients was 1.25(1.0,2.0)ml.The basal level of serum LH,FSH and total testosterone(T)was 0.32(0.20,0.89)U/L,0.57(0.30,1.80)U/L and 0.37(0.29,0.61)ng/ml.After treatment,the testicular volume was increased by 8.8±4.7 ml and the T level was increased by 2.6±1.6 ng/ml.Fourteen(67%)patients achieved spermatogenesis and the median period of spermatogenesis was 21.1 ±12.8 months.There was no difference in the therapeutic ending between nCHH and KS.6 The analysis of gene variants:A total of 47 patients went on gene sequencing tests,while 24(51.0%)patients were identified rare gene variants.Eleven variants were assigned as pathogenic or likely pathogenic variants.The genes detected in nCHH patients included CHD7,FGFR1,DUSP6,FGF8,HESX1,PROKR2,PROP1 and TACR3,while the genes detected in KS patients included ANOS1,CHD7,FGFR1,FGF17,IGSF10,SEMA3A.Conclusion1 The Chinese olfactory function test is valuable for differential diagnosis of CHH patients and can be used as an olfactory test tool for clinical and scientific research.2 The SROS is more reliable for self-reported "abnormal" olfaction sense.3 The structure of the olfactory apparatus does not fully match the olfactory function in CHH patients.The olfactory function of CHH patients with UOA(H)is normal.Some patients with BOA(H)also have the normal olfactory function,which is known as"Normal olfaction without apparent olfactory bulbs".4 In our cohort,there was no difference in the prognosis between nCHH and KS patients.5.In our cohort,the incidence of rare variants of CHH patients was 51%,and the incidence of pathogenic variants was 23.4%.Several genes associated with GnRH neuronal migration have been identified in nCHH patients,suggesting that genetic testing cannot distinguish KS from nCHH patients.ContextCongenital hypogonadotropic hypogonadism(CHH)is a genetic disease characterized by incomplete or absent puberty and infertility.At present,more than 40 pathogenic genes have been identified of the disease.However,only about 20%-50%of CHH patients had a molecular diagnosis,suggesting that some pathogenic genes of CHH have not been identified.In 2020,Pitteloud et al.reported 4 CHH patients with rare variants of the neuron-derived neurotrophic factor(NDNF)gene,which was detected among 240 CHH patients.And they confirmed that these variants affect the migration of GnRH neurons using in vitro functional studies,indicating that the NDNF gene is the pathogenic gene of CHH.However,in 2021,another study only detected 9 synonymous variants of the NDNF gene in 61 HH patients.All of these were predicted to be benign by the in silico tools and they indicated that NDNF gene variants are rare in CHH patients.Thus,more studies are needed to confirm whether the NDNF gene is the pathogenic gene of CHH.ObjectiveThis study aims to identify the incidence of NDNF gene pathogenic variants in a large sample of CHH patients,and analyze the clinical phenotype of patients with NDNF gene variants and verify the pathogenicity of the variants in vitro.MethodsPatients with CHH admitted to Peking Union Medical College Hospital were recruited between 2016.01 and 2021.12.The clinical data of the patients were retrospectively analyzed.Whole exome sequencing was used to investigate the pathogenic gene variants,and Sanger sequencing was used to verify the NDNF gene variants.The pathogenicity of the variants was analyzed using bioinformatics methods.The wild-type and mutant NDNF eukaryotic expression vectors were constructed,and in vitro functional studies were used to analyze the pathogenicity of the variants.Results(1)A total of 7 patients with rare heterozygous variants of NDNF gene were detected in 523 CHH patients.The incidence of NDNF gene rare variants was 1.3%.Among the 7 patients,4 were Kallmann syndrome patients,and 3 were normosmic CHH.The mean age of these patients was 20.9±4.9 years,and the mean basal testicular volume was 2.6± 1.2 ml.The mean serum LH level was 0.38±0.42 U/L,FSH level was 1.07±0.61 U/L,and total testosterone level was 0.78±1.17 ng/ml.(2)These variants include 3 missense variants c.31C>T(p.L11F),c.769T>G(p.F257V)and c.1430A>G(p.K477R),2 frameshift variants c.1656dupG(p.H553Afs*8)and c.1669dupT(p.Y557Lfs*4)and 2 nonsense variants c.930C>G(p.Y310*),c.1483A>T(p.K495*).These variants were located in exon 4 of NDNF gene except c.31C>T.According to ACMG guideline,p.L11F was likely benign variant.p.F257V,p.K477R,p.H553Afs*8 and p.Y557Lfs*4 were uncertain significance variants.p.Y310*and p.K495*were likely pathogenic variants.(3)The results of Western Blot showed that all mutants were expressed in whole cell extracts.L11F,F257V and K477R mutants were detected in the conditioned medium.ConclusionThe loss of function variants of NDNF gene was the pathogenic mechanism of CHH.In our study,the incidence of CHH patients with NDNF gene rare variants is about 1.3%,while the incidence of likely pathogenic variants was 0.4%.Exon 4 is a hot spot of NDNF gene variants.Patients with NDNF gene variants can present with congenital hypogonadotropic hypogonadism with or without olfactory dysfunction. |
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