Research On Isolated FSH Deficiency Caused By Arg97X Mutation In FSHβ Subunit

PartⅠClinical features and therapeutic process of Isolated FSH deficiencyObjective:To confirm the diagnostic criteria and the treatment principle of isolated FSH deficiency, to reveal the physiological function and the essential role in the process of follicle dominance, we have researched on the patient’s clinical features and the natural model of low levels’ FSH.Methods:The patient was asked to stop any endocrine therapy for at least three months. She came to our hospital at early morning to accept following examinations:ACTH, TSH, GH and GnRH stimulation experiments to evaluate pituitary function. Sex hormones, type-B ultrasonic inspection to assess ovarian function. Pituitary MRI and adrenal ultrasound to differentiate diagnosis. Sex hormones were detected by chemiluminescence method. The detection process has been carried out by Siemens instrument automatically. After acquire the patient’s agreement, we use the type-B ultrasonic inspection and sex hormone to monitor the follicular development dynamically, in case to adjust the dosage of drug and to understand the trend of follicular development and hormone changes.Result:1. Clinical features:1.1Clinical symptoms:primary amenorrhea, primary infertility1.2Physical examination:eunuchoidal proportion, breast development was Tanner IIIGynecological examination:uterine hypoplasia, normal development of the labia majora, poorly development of the labia minora, there is no abnormalities in two annexes.1.3Family history:Her parents are third cousins. There is no family history of menstrual disorders.1.4Laboratory examination:1.4.1Ovarian hormones:FSH0.08-0.47mIU/ml, LH27.11-64.53mIU/ml, E216.62pg/ml, T37.44ng/dl, PRL20.67ng/ml, P0.07ng/ml, AMH3.05ng/ml, INB1.23↓)pg/ml.1.4.2Adrenal hormones:DHEA2150↓nmol/1, AN6.06↑nmol/1.1.4.3Pituitary hormones:ACTH, TSH, GH are all normal.1.4.4GnRH stimulation examination:After30minutes of Gonadorelin injection, LH peak was appeared which was increased4-fold compared to baseline value of LH. While there was no fluctuation of values of FSH before and after Gonadorelin injection.1.4.5Chromosome:46,XX1.5Imaging examination:pituitary MRI, ultrasonography of adrenal glands were all normal.Type-B ultrasonic inspection indicated that uterine hypoplasia, and the numbers of antral follicles whose diameter≤3mm were6.2. Treatment outcomes: After we used marvelon(1#qd×21days) to reduce endogenous LH level and urofollitropin (75IU/d) to induce ovulation, the maximum follicle diameter was up to1.9cm, but there was no successfully pregnancy.Conclusion:1. According to patient’s clinical features, the disease was confirmed to be isolated FSH deficiency.2. Despite of lower FSH, higher LH is sufficient enough to suggest that hypothalamus is not the origin of IFD.3. GnRH stimulation test indicates that the ability of glycoprotein hormones’production and secretion of pituitary is normal. We suppose that isolated FSH deficiency is a kind of encoding gene defects.4. There were antral follicles in the patient’s ovary indicated that early antral follicles may also be non-hormone dependent growth of follicles, just like preantral follicles. Part twowhole-exon sequencing analysis of the proband and her familiesObjective:To determine whether there’s mutation in the FSHp encoding gene, we performed whole-exon sequencing analysis in the proband and her families.Methods:Blood samples of the patient and her families were placed into EDTA anticoagulant tubes. Using sanger sequencing technology-dideoxy termination method to perform the whole-exon sequencing analysis.Results:1. The proband and her families’sequencing results of whole exons of FSHP subunit:1.1Nonsense mutation:There is a C-T mutations occurred at No.184nucleotide site, which result in Arginine being replaced by termination codon (Arg97X). This nonsense mutation have truncated the expression of amino acids97-111. The patient is Arg97X homozygous mutant genotype, while her grandmother, parents, sister are Arg97X heterozygous mutant genotype. The Arg97X FSHβ subunit mutation has not been occured to her grandpa.1.2Single nucleotide polymorphisms:There is a C-T homozygous mutation at the No.69nucleotide site of exon3in all of the proband and her families, which is synonymous mutation and the occurrence probability in Chinese population is69.5percent.Conclusion:1. Considering the proband’s clinical features and her parents’ ill history of pregnancy, we can infer that Arg97X are a more serious subtype in gene mutations of isolated FSH deficiency. Even small amounts of homozygous mutant progeny can survive, they will be destined because of ther infertility.2. The disclosed crystal structure of FSHβ has inferred that Arg97X mutation would destroy the stability of FSHβ, inhibit the formation of α-β heterodimer, and affect the immunological and biological activity. Part threeIn vitro validation experiments of wild-type and mutant Arg97X FSHβ subunitObjective:To lay the foundation for studying the relationship between the mutation and immunological and biological activity of FSH, and to provide the basis for revealing pictures of FSH physiological functions and spatial structure, we have created the cell model which can express mutant Arg97X FSH by using the technology of gene amplification, vector construction and cell transfection. Methods:1. Primers designation:According to the sequencing results of second part, two pairs primers of wild-type and mutant gene’s fragments were designed. To distinguish the process of transcription and expression of target genes carried by plasmid with the process of translation and expression of CHO endogenous gene, we decided to insert HIS tag to the3’end. In that case, wild-type and mutant FSHβ had the same forward primer, while their reverse primer was different.2. Gene amplification:RNA was extracted from human’s uterine fibroid tissue. As a template, DNA was obtained and purified to gain wild-type, mutant FSHP gene fragment.3. Vector construction:the wild-type FSHβ, mutant FSHβ fragment and pcDNA3.1(+) vector were double digested by HindⅢ, EcoRI, respectively. After that, wild type FSHβ fragment, mutant FSHβ fragment were combined with the pcDNA3.1(+) corresponding to the restriction sites by using T4ligase to obtain pcDNA3.1(+)-FSHβ (WT) and pcDNA3.1(+)-FSHβ (MU) vector.4. Cell transfection, PCR, western-blot, chemiluminescence method:The three plasmids: pcDNA3.1(+)-FSHβ (WT), pcDNA3.1(+)-FSHβ (MU), pcDNA3.1(+) empty (KO) vector were transiently transfected into Chinese hamster ovary cells, after72hours of incubation, cell lysates were extracted for PCR and western-blot detection, cell culture supernatants were collected for ELISA.Result:1. PCR results:The wild-type mRNA was detected in WT group, suggesting that the wild-type FSHβ was successfully transcribed in CHO;The mutant mRNA was detected in MU group, suggesting that the mutant FSHβ was successfully transcribed in CHO;Neither the wild type, nor the mutant mRNA was detected in CK group, suggesting that there was no transcription occured. 2. western blot results:The protein of wild-type FSHP was detected in WT group, suggesting that wild-type FSHP can be successfully translated in CHO;The protein of mutant FSHp was detected in MU group, suggesting that mutant FSHP can be successfully translated in CHO;No His-tagged protein was detected in CK group, suggesting that there was no translation occurred in CHO.3. Chemiluminescence results:FSH(between1.92-2.12mIU/ml) could be detected in all of the supernatant of four groups (WT, MU, CK, media), and the difference was not statistically significant. In other words, the immunological activity of FSH in supernatant of WT and MU group had not been detected.Conclusion:1. These results confirm that, pcDNA3.1(+)-FSHp (WT) vector and pcDNA3.1(+)-FSHp (MU) vector were successfully constructed. Wild-type and mutant FSHP gene can be successful transcripted and expressed in CHO, but can not be secreted to extracellular fluid.2. Wild-type FSHp subunit can not be secreted, suggesting that FSH, even all of the glycoprotein hormone can be secreted extracellularly and play immunological and biological function only in the situation that α and β conbine together.3. Arg97X FSHP subunit mutant can not be secreted by the pituitary gland to peripheral blood circulation, even in the presence of normal amount of a-subunit (patients with normal TSH level) in circumstance, suggesting that the presence of the mutation could destroy the formation of α-β heterodimer and affect the immunological and biological activities.