Application Research Of HRM Technology In Detection Of FGFR1 And GNRHR Gene Mutations In Male IHH Patients

BACKGROUND:The complex components and regulation of the human hypothalamic pituitary gonadal (HPG) axis makes it susceptible to dysfunction in the face of a variety of genetic insults, leading to different degrees of hypogonadotrophic hypogonadism (HH). Although the genetic basis of some HH was recognised more than 60 years ago the first specific pathogenic defect, in the KAL1 gene, was only identified within the last 20 years. In the past decade, the rate of genetic discovery has dramatically accelerated, with defects in more than 10 genes(including KAL1, FGFR1, FGF8, PROKR2, PROK2, CHD7, NELF, GNRHR, GNRH1, TACR3, TAC3 and KISS1R)now associated with IHH. The genetic basis of IHH has gradually been uncovered.The association of some genes, including FGFR1, FGF8, PROK2 and PROKR2, both with IHH in association with hyposmia/anosmia (Kallmann syndrome), and with normosmic IHH, thus blurring the clinical distinction between ontogenic and purely functional defects in the HPG axis. Many examples of digenic inheritance of IHH have also been reported, sometimes producing variable reproductive and accessory phenotypes within a family with non-Mendelian inheritance patterns.Recent advances in fluorescent dyes, methods, instruments and software for DNA melting analysis have created versatile new tools for variant scanning and genotyping. High resolution melting analysis (HRM or HRMA) is faster, simpler, and less expensive than alternative approaches requiring separations or labeled probes. With the addition of a saturating dye before PCR followed by rapid melting analysis of the PCR products, the sensitivity of heterozygote scanning approaches 100%. Specificity can be increased by identifying common polymorphisms with small amplicon melting, unlabeled probes or snapback primers to decrease the sequencing burden.There is no application of HRM technology in detection of FGFR1 and GNRHR gene mutations in male IHH patients up to now.OBJECTIVE:There are more than 10 genes(including KAL1, FGFR1, FGF8, PROKR2, PROK2, CHD7, NELF, GNRHR, GNRH1, TACR3, TAC3 and KISS1R) now associated with IHH. High resolution melting analysis (HRM or HRMA) is faster, simpler, and less expensive than alternative approaches requiring separations or labeled probes and is a versatile new tool for variant scanning and genotyping. There is no application of HRM technology in genetic study of IHH up to now. In this paper, we mainly carried out research of HRM technology in detection of FGFR1 and GNRHR gene mutations in male IHH patients, studied the prevalance of FGFR1 and GNRHR gene mutation in Chinese male IHH patients and examined if there are cases of digenic inheritance of IHH in our cohort.METHODS:The cases of the study were the patients who came to The Clinical Hospital of Jilin University for abnormal puberty or infertility from January 2008 to May 2009. Among examining 60 cases, there are 20 patients with Kallmann syndrome (KS) and 40 patients with normosmic idiopathic hypogonadotropic hypogonadism (nIHH) .At the same time; 30 healthy volunteers was also collected as controls. All subjects are sporadic cases.The 20 patients with KS were diagnosed according to the clinical signs and symptoms of hypogonadism.The olfactory bulb structures of KS patients were examined by brain magnetic resonance imaging. Together 40 unrelated males with IHH were identified based on clinical signs and symptoms of hypogonadism, prepubertal testosterone (<1.6nmol L-1), low or inappropriately normal gonadotropin levels, normal baseline and reserve testing of other anterior pituitary hormones and normal radiological imaging of the hypothalamic–pituitary region. Anosmia/hyposmia was evaluated using the olfactory test described by Davidson and Murphy. Blood samples (5 mL taken into EDTA by venipuncture) were obtained from all subjects. Genomic DNA for PCR analysis was isolated from thawed whole blood using QuickGene DNA whole blood kit S (DB-S,Fuji Photo Film (China) Investment Co., Ltd. Hong Kong, P.R China). We used PCR-HRM analysis with exon-flanking primers and automated sequencing techniques with peripheral blood DNA samples. Sequencing of the coding regions of the FGFR1 gene (NM₀23110) was performed. Amplified products were sequenced in both directions by using the AmpliTaq Dye Terminator Cycle Sequencing kit and an ABI 3130 Genetic Analyzer (AppliedBiosystems). Subsequent sequence analysis was performed by using SeqScape (Version 2.5) and visual inspection. All sequence variations were found on both strands and confirmed in a separate PCR.RESULTS:1. The basic informationHypogonadotropic hypogonadism was documented in all patients. Basal testosterone concentrations were consistently in the prepubertal range (1.6 nmol/L).Meanwhile, gonadotropin levels were always below the normal adult male range (FSH: 3.0³0.0 IU/L; LH: 5.0²8.0 IU/L) and normal prepubertal male range(FSH: 1.0⁹.0 IU/L; LH: 3.0⁷.0 IU/L). All of them had uneventful pregnancies, and anosmia or hyposmia had been present since early childhood. In adolescence, there was an absence of subnormal pubertal development. On physical examination, certain clinical characteristics were found in all individuals: normal stature, infantile genitalia and scant pubic hair. The high-resolution G-banded karyotype was 46, XY, and computed axial tomography of the hypothalamic- pituitary region did not show any disorder in any of the patients.2. Molecular Analysis of FGFR1 GeneFour rare sequence variants of the FGFR1 gene were found in three unrelated KS patients(cases K51,K23 and K29) and one nIHH patient (case nIHH6) . A transversion c.709 G→A in exon 6 is predicted to substitute a glycine for serine at position 237 (G237S) in D2 within the extracellular region of FGFR1 (case K29). A transition c.2172 C→G in exon 16 leads to a substitution of asparagine for lysine at position 724 (N724K) in the TKD (case nIHH6). Finally a transversion c.346G→A in exon 3 leads to a substitution of valine for isoleucine at position 116 (V116I) in D1 (case K51) and a transition c.569G→C in exon 5 is predicted to substitute a tryptophan for serine at position 190 (W190S) in D2 within the extracellular region of FGFR1 (case K23).These changes were not detected in ethnically matched controls.The prevalence of FGFR1 mutation of KS and nIHH in our cohort are 15%(3/20) and 2.5%(1/40), respectively.3. Molecular Analysis of GNRHR GeneIn our cohort, the 40 nIHH patients were tested for GNRHR mutations using HRM combined sequencing and were negative. Only one SNP (rs4986942) c.2204C→T (S151S) was found in two unrelated patients.CONCLUSIONS:We examined the FGFR1 gene in 20 KS and 40 nIHH patients using HRM mutation scanning combined with sequencing. Four rare heterozygous FGFR1 mutations were found in three of 20 unrelated KS patients and one of 40 nIHH patients. (i) G237S in KS case K29; (ii) V116I in KS case K51; (iii) W190S in KS case K23 and (â…³) N724K in nIHH case nIHH6. The prevalence of FGFR1 mutation of KS and nIHH in our cohort were 15%(3/20) and 2.5%(1/40),respectively. No mutation of GNRHR gene was found. There was no case with digenic inheritance of FGFR1 and GNRHR gene mutation in our subjects.