Mutations Analysis Of KAL1 And Fibroblast Growth Factor Receptor 1 Gene In Male Patients With Idiopathic Hypogonadotropic Hypogonadism

BACKGROUND : Idiopathic hypogonadotropic hypogonadism (IHH) is a congenital hypothalamic disease. Due to the hypothalamic secretion of gonadotropin-releasing hormone (GnRH) dysfunction, testicular can be dysfunctional, which could mainly results adolescents still have no secondary sexual characteristics, external genitalia is naive type and adults are infertile. If the IHH patients have hyposmia and anosmia, the Kallmann syndrome (KS) could be defined.The incidence rate of Kallmann syndrome is 1:10 000 in males and 1:50 000 in females. The cases are mainly sporadic.The patients could return to gonadal function and partially recover fertility by means of early detection using molecular biology, and then the hormone replacement therapy could be carried out.OBJECTIVES: A genetic diagnosis methods of IHH resulted from KAL1 and FGFR1 mutations should be established, which could provide important basis for clinicians to make the correct diagnosis, offer some help to detect and intervene this disease more earlier and lay the foundation for patients to access to fertility ability.METHODS:The objects in this article were 60 cases of male patients with IHH in the male urology outpatient clinic of Clinical Hospital of Jilin University between October 2008 and December 2009.Besides that, 50 cases of normal male were collected as the control group, who are normal male volunteers. The basic information of the object should be collected, including age, development of secondary sexual characteristics, olfactory sense condition, level of intelligence, family and inherited disease history and past history of drug and surgery and so on. The polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) technology combined with PCR product direct sequencing technique could be applied in the peripheral blood taken in the research objects in order to analysis the KAL1 and FGFR1 gene exon mutation of IHH patients.RESULTS:60 cases of the patients can be amplified 14 exons of KAL1 gene by PCR. After PCR amplification, the SSCP screening could reveal that the single-strand conformation bands of three samples, in which the sequencing analysis reveals there are three mutations, are significantly different from the normal controls. 50 cases of controls were not found the KAL1 gene single nucleotide polymorphisms.The patient 1 transforms C into T in the 1270 locus of the 9th exon coding sequences (c.1270C>T,p. R424X), resulting that the CGA encoding arginine (Arg) change into the stop codon TGA. The AG deletion was found in the 279₂80 locus in the 3rd coding sequences in patient 2(c.279₂80delAG,p.G94fs), leading to frameshift mutations. patient 3 was found that TT was deleted in c.1886₁887 locus in the 13th exon coding sequence (c.1886₁887delTT,p.L629fs), which could also leads to frameshift mutations. In addition, after seeking 1,2,3 patients' mother's consent, their mother's peripheral blood was extracted in order to carry out mutation screening of exon in KAL1 gene. By the means of gene sequencing analysis, there is a heterozygous point mutations c.C1270T (p.Arg424ter)in the 9th exon of KAL1 gene in the mother of patient 1; there is a heterozygous frameshift mutation c.279₂80delAG (p.G94fs) in the 3rd exon of KAL1 gene in the mother of patient 2; there is a heterozygous frameshift mutation 1886₁887delTT (p.L629fs) in the 13th exon of KAL1 gene in the mother of patient 3. 60 cases of the patients can be amplified 18 exons of FGFR1 gene by PCR. After PCR amplification, the SSCP screening could reveal that the single-strand conformation bands in three cases of samples, in which the sequencing analysis reveals there are three mutations, are significantly different from the normal controls. 50 cases of controls were not found the FGFR1 gene single nucleotide polymorphisms.The patient 4 transforms G into C in the 569 locus of the 9th exon coding sequences(c.569G>C,p.W190S), resulting that the TGG encoding Trp changes into Ser; the patient 5 transforms G into A in the 709 locus of the 5th exon coding sequences(c.709G>A,p.G237S), resulting that the GGC encoding Gly changes into Ser; the patient 6 transforms G into A in the 346 locus of the 3rd exon coding sequences(c.346G>A,p.V116I), resulting that the GTC encoding Val changes into Ile.CONCLUSIONS:1. This study detected the KAL1 gene 14 exons of the 60 patients with IHH, and then found one case of nonsense mutations and 2 cases of frameshift mutation. We detected the FGFR1 gene 18 exon of the 60 patients with IHH and found three cases of missense mutations.2. In the KAL1 gene mutation screening, we found two cases of novel mutations, (c.279₂80delAG,p.G94fs) and (c.1886₁887delTT,p.L629fs), respectively. In the FGFR1 gene mutation screening, we found two cases of novel mutations mutation, (c.569G>C,p.W190S) and (c.346G>A,p.V116I), respectively. The discovery of the new gene mutation not only enriches the human gene mutation database, but also provides new clues for the KS molecular studies, laying the foundation for future research.3. This study revealed that 3 KAL1 gene mutated patients'mother have the same mutations in the same place, who is the mutation carriers, suggesting that the mutation of the patients comes from their mothers.4. KAL1 gene mutations accounted for 5% of patients with IHH(3/60) and 15% of patients with KS(3/20); FGFR1 gene mutations accounted for 5% of patients with IHH(3/60) and 15% of patients with KS(3/20).5. This study established the gene diagnosis technology for KAL1 genes and FGFR1 gene in IHH patients, which provided a favorable basis for early diagnosis for IHH patients.