Cases With Male Idiopathic Hypogonadotropic Hypogonadism: Clinical Data Analysis And Molecular Genetic Study

ObjectiveIdiopathic hypogonadotropic hypogonadism (IHH) refers to a group of patients who characterized by varying degrees of sexual development disruption, resulting in the failure to develop a mature reproductive system because of insufficient gonadotropin release for unknown reasons. In this study, we described the clinical features, genetic features, diagnosis, treatment methods and efficacy of all cases, and analyzed their genotype phenotype correlations. We also did a preliminary study for their molecular genetic pathogenesis.1. To clinically characterize all cases with IHH, include clinical manifestations, hormone levels, karyotype, thyroid function, adrenal, pituitary secretion, glucose and lipid metabolism, GnRH stimulation test stimulation, magnetic resonance imaging of the olfactory bulb and olfactory score features.2. To understand the molecular etiology of KS from a genome-wide perspective, we investigated the genome-wide profile of structural variation and linkage analysis in a consanguineous Han Chinese family using the Affymetrix Genome-Wide Human SNP Array 6.0 platform.3. To do direct DNA sequence analysis of the candidate gene KAL1, FGFR1, PROK2 and PROKR2 to reveal mutations in the IHH patients, and to perform single-strand conformation polymorphism (SSCP) to conform the mutations in the affected individuals and to exclude the possibility of polymorphism in a reference population.Methods1. Clinical study:1.1 To establish the follow-up files and disease histories of patients such as penile length, testicular volume, smell score, hormone levels, karyotype, thyroid function, adrenal cortex, pituitary secretion, glucose and lipid metabolism, GnRH stimulation test and other tests stimulation were recorded.1.2 For magnetic resonance imaging (MRI) of the hypothalamic and pituitary areas included olfactory bulb, olfactory tract and sulcus in patients and normal control subjects.1.3 To do the reproductive hormone therapy (HCG, or HCG plus Andriol therapy) in different groups and to follow-up regularly and assess the results of treatment.2. Molecular genetic study2.1 Genomic DNA:Genomic DNA was isolated from peripheral blood leukocytes of the patients and normal control subjects using TIANamp Blood DNA Kit (Tiangen Biotech, Beijin, China).2.2 Gene chip:To understand the molecular etiology of KS from a genome-wide perspective, we investigated the genome-wide profile of structural variation and linkage analysis in a consanguineous Han Chinese family using the Affymetrix Genome-Wide Human SNP Array 6.0 platform.2.3 Sequence Analysis:Direct nucleotide sequencing of the coding region of KALI, FGFR1, PROK2 and PROKR2 gene in DNA to reveal mutations in the IHH patients. PCR-SSCP was performed to reassess the DNA sequencing variation.Results1. Clinical study1.1 The mean age at evaluation was (21±3.2) years old and 52 patients were diagnosed as Kallmann syndrome (KS; IHH with anosmia/ hyposmia) while others were normosmic IHH (nIHH). Of them,47 cases showed some pubertal development,45 patients with no obvious puberty development,3 cases of gynecomastia,15 patients with unilateral cryptorchidism, and 5 cases of bilateral cryptorchidism. The karyotypes were 46,XY in all 92 cases. The serum thyroid gland, adrenal gland function and glucose and lipid metabolism levels were in normal and the levels of serum LH, FSH and T were low. 1.2 Magnetic resonance imaging examination showed that all KS patients had the absence or dysplasia of olfactory bulb, olfactory tract or sulcus. They all had normal radiological imaging of the hypothalamic and pituitary areas. There were found the unilateral olfactory bulb dysplasia in some nIHH patients with. In the normal control group there was not found the absence or dysplasia of the olfactory bulb or olfactory tract or sulcus.1.3 The level of serum LH, FSH and T as well as the clinical parameters (development of secondary sexual characteristics, sexual function, and serum testosterone levels.) were significant improvement after reproductive hormone therapy (T, HCG, or HMG therapy). The sexual function was normal in 3 married patients, and one of them had a child.2. Molecular genetic study2.1 While we investigated the genome-wide profile of structural variation and linkage analysis in a consanguineous Han Chinese family using the Affymetrix Genome-Wide Human SNP Array 6.0 platform, there was no significant linkage were found in polymorphic markers (LOD values were less than 1.0).But the results revealed that the three affected individuals had common copy number variants (microdeletions) on chromosomes 1p21.1,2q32.2,8q21.13,14q21.2 and Xp22.31. Moreover, the copy number variants on Xp22.31 were located in the intron of KALI, which causes X-linked KS. In addition, genomic microdeletions in this region were verified in one sporadic KS, but not in 100 unrelated Han Chinese normal controls.2.2 The present study reports on a male patient who presented with the KS phenotype with fertility. The patient had KS as a result of two missense mutations at exon 11 of KALI gene,1690 G>A and 1765 G>A, a G/A transition in codons 514 and 539, which resulted in the replacement of lysine by glutamic acid, respectively. One of them is a novel mutation. We also report a novel missense mutation of KALI at exon 12,1828G> A, which lead to a pVal/V610Ile/I substitution in the two brothers with KS and a heterozygous state in their mother. These identified molecular defects with the disease were not found in 100 unaffected individuals or the other patients. In addition, no mutations of FGFR1, PROKR2, or PROK2 were identified in these patients.Conclusion1. According to the clinical features and laboratory results, we can do diagnosis and differential diagnosis of IHH. Reproductive hormone therapy is the choice of treatment.2. The results about gene chip provide a novel insight into the relative contributions of certain copy number variants to KS's molecular etiology and generate a list of interesting candidate regions for further studies.3. The male patient who presented with the KS phenotype with fertility and two missense mutations at exon 11 of the KALI gene implies that this genotype may be more prevalent in reproductive KS. The results provided further support for the hypothesis that patients with KS can achieve normal sexual development and fertility after HCG treatment, and imply that this genotype is more prevalent in reproductive patients with KS than in the other patients with KS and incurable infertility. The report gives evidence that the Val610Ile mutation of KALI is associated with X-linked recessive KS with unilateral cryptorchidism or unilateral renal dysgenesis, which expand the spectrum of KALI mutations causing KS.