Functional Analysis Of PROKR2 Gene,a Pathogenic Gene For Isolated GnRH-Deficiency

isolated GnRH deficiency is a rare reproductive dysfunction disease caused by abnormal secretion of gonadotropin-releasing hormone from hypothalamus.The typical clinical phenotypes of IGD patients are partial or complete absence of pubertal development,hypogonadism,and infertility.With early diagnosis and treatment,IGD patients can have normal adolescent development,maintain the corresponding secondary sex characteristics,and have normal fertility.Therefore,clarifying the pathogenesis of IGD and establishing comprehensive and accurate diagnostic methods are of very positive significance for the diagnosis,prevention and treatment of IGD.PROKR2 gene is one of the most frequently mutated pathogenic genes carried by IGD patients.PROKR2 has a variety of physiological functions,and can be involved in the migration of neural stem cells,circadian rhythm,feeding,pain perception,etc.The olfactory bulb atrophy and gonadal dysplasia of Prokr2 knockout mice showed typical IGD phenotype.There have been international reports on the mutation and function of PROKR2,but the molecular mechanism of its pathogenesis remains to be improved,especially the mechanism of PROKR2 mutation affecting its cell migration is still unclear.Objective: To clarify the signaling pathway of PROKR2 regulating cell migration,study the effect of IGD-related PROKR2 mutation on its signal transmission,and compare and analyze with prediction software to clarify the applicability of them,so as to provide data support for accurate diagnosis of IGD.Methods:1.Collected the DNA samples of IGD patients,conducted whole exome sequencing of the proband,screened the pathogenic gene mutations,including the PROKR2 gene to find the novel mutation sites of PROKR2;2.Combined with literature reports,PROKR2 mutations were summarized that were not detected for signaling pathway activity,mutant plasmids were constructed to improve the detection of all PROKR2 mutation activity related to IGD;3.A variety of G protein signaling pathway inhibitors were used to determine the effects of different G protein signaling pathways on PROKR2-mediated cell migration;4.Five common software were used to predict the harmfulness of all PROKR2 mutations,and compare with the experimental data to determine the pros and cons of the prediction software.Results:1.A database of 255 Chinese IGD patients was established,including the whole exome sequencing results and clinical phenotypes of the probands.35 IGD patients carried PROKR2 mutations,and a total of 39PROKR2 mutations were screened,among which 4 patients carried PROKR2 complex heterozygous mutations.Only one proband carried homozygous PROKR2 mutation,and the rest were heterozygous mutations.Among the IGD patients with PROKR2 mutation,14 patients carried only PROKR2 gene mutation,and 25 patients carried other IGD pathogenic gene mutations;2.The activity of 23 PROKR2 mutations in three G protein signal pathways was detected,and 3 frameshift mutations and 2 truncating mutations(R80fs*82,Y140 X,K162X,P256Lfs*51 and T330fs*5),R135 C,P302L and S324 R were inactive in three signal pathways;The three signaling pathways of E319 K and M239 T were damaged in different degrees.;Seven mutations(Y33H,R80 H,L91F,A103 V,A189S,V236 M,and H310Q)only showed normal ERK1/2 signal transduction,while Gαq and Gαs signal transduction were damaged;Gαs signal pathway of T11 I and Gαq signal pathway of V233 M were not significantly damaged,while the activities of the other two signaling pathways were reduced to varying degrees.;ERK1/2 signaling pathway of G57 C and Gαs signal pathway of R264 H,Gαq signaling pathway of L176 F and V180 M were damaged,while the other two signaling pathways were normal;3.When Gαq signal pathway was inhibited,PROKR2-mediated cell migration was inhibited;Inhibition of ERK signaling pathway had no effect on PROKR2-mediated cell migration;4.SIFT software can predict PROKR2 transmembrane mutations more accurately,and CADD software can predict PROKR2 mutations in intracellular and extracellular cells more accurately.Conclusion: Our study is the first to demonstrate that the Gαq signaling pathway is necessary for PROKR2-mediated cell migration,suggesting that PROKR2 mutations with impaired Gαq signaling may contribute to the development of IGD.For functional studies of newly discovered PROKR2 mutations,if PROKR2 mutation activity can be detected,we recommend that the Gαq signaling pathway activity be analyzed preferentially.In addition,SIFT software was recommended as a predictive tool for PROKR2 mutations located in transmembrane regions,and CADD software was recommended as a predictive tool for PROKR2 mutations located in both intracellular and extracellular regions in clinical diagnosis.