Idiopathic hypogonadotropic hypogonadism (IHH) is a disorder defined as irreversible delayed puberty and infertility with low serum levels of steroid hormones and low gonadotropins. One subtype of IHH is Kallmann syndrome (KS), the combination of IHH and anosmia/hyposmia. IHH is due to a deficiency of GnRH and/or gonadotropin secretion. GnRH neurons migrate from the olfactory placode to the hypothalamus along with the olfactory neurons during the development of the embryo. The molecular basis for KS is not well understood. The purpose of this study was to identify gene mutations that cause IHH. The overall hypothesis was that genes that regulate GnRH or gonadotropins in the hypothalamic-pituitary (H-P) axis possess mutations in male and female IHH patients, but not in controls. Mutations in genes encoding proteins involved in this process can provide a potential molecular basis for KS. Methods. Genomic DNA custom microarrays provided an effective method to screen mutations in 6 candidate genes (GNRH1, FSHB, LHB, NELF, BRAN2, HESX1 ). Capillary based DNA sequencing, realtime RT-PCR, western blot analysis, immunocytochemistry, mutagenesis, flow cytometry (FACS), and migration analysis were performed to screen for mutations in candidate genes and analyze their functions in vitro. Results. Gene mutations were identified in KAL1, FGFR1 and NELF (nasal embryonic LHRH factor), each of which is involved in the GnRH migration pathway and known to have clinical relevance. Mutations in the KAL1 gene, encoding anosmin-1, were described in males with X-linked recessive KS. The prevalence for KAL1 mutations was 3.7% in 138 sporadic patients (males only), and 6.3% in the group of anosmic/hyposmic males. A nonsense mutation (R622X) mutation in FGFR1 was identified in an autosomal dominant family with normosmic IHH, which suggests FGFR1 mutations can cause fully penetrant, complete IHH only, without anosmia or other FGFR1-associated anomalies. No mutations were found in NELF, the third candidate gene suspected to be involved in the GnRH neuron migration pathway. However, studies on the biology of NELF suggest that NELF is present in both migratory and non-migratory GnRH neuronal cell lines, with high protein expression in migratory cells. NELF is a nuclear protein containing a nuclear localization signal and 2 putative zinc fingers. Different isoforms of NELF are present in migratory vs. non-migratory cell lines. Knock down of NELF via microRNA approaches decreases the number of migrating cells in migration analysis assay in vitro. Therefore NELF could play an indirect role in GnRH migration pathway as a nuclear transcription factor, which regulates downstream target genes that induce migration. Taken together, identification of gene mutations furthers our understanding of the molecular mechanism of IHH, and studies of NELF biology provide an inventory of components to significant to regulation of normal puberty and human reproduction.;Index words. Idiopathic hypogonadotropic hypogonadism, GnRH, Kallmann syndrome, gonadotropins, puberty, microarray, FGFR1, NELF.
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