【作者】 张世林；

【导师】 刘继红；

【作者基本信息】 华中科技大学 ， 外科学， 2011， 博士

【摘要】 目的收集男性特发性低促性腺激素性机能减退症(idiopathic hypogonadotropic hypogonadism,IHH)病例,研究其临床特点、诊断、治疗的方法及疗效,同时对其分子遗传学病因作初步的研究,探讨IHH的分子发病机制与临床表型间的关系。1.研究IHH病例的临床表现、性激素水平、染色体核型、甲状腺功能、肾上腺皮质功能、垂体分泌功能、糖脂代谢功能、GnRH刺激兴奋试验、磁共振嗅球部成像及嗅觉评分特征。2.基因定位：应用Affymetrix全基因组SNP 6.0基因芯片对一包含三个卡尔曼综合征子代患者的近亲婚配家庭进行连锁分析及基因组结构变化的分析。3.基因序列分析：采取直接DNA序列分析对所有患者进行KAL1.FGFR1.PROK2及PROKR2基因的突变检测,并用单链构象多态性分析(single-strand conformation polymorphism,SSCP)证实已发现的突变,同时在正常对照人群中进行检测排除是多态性的可能。方法1.临床研究1.1男性特发性低促性腺激素性机能减退症病例病史采集,建立随访治疗档案、分别进行阴茎长度、睾丸体积、嗅觉评分、性激素水平、染色体核型、甲状腺功能、肾上腺皮质功能、垂体分泌功能、糖脂代谢功能、GnRH刺激兴奋试验等检查。1.2对IHH患者及正常对照人群进行磁共振嗅球部成像检测,并测量嗅球容积。1.3分组分别行HCG肌注替代疗法或HCG肌注加雄激素(安特尔)口服治疗,定期随访评估治疗结果。2.分子遗传学研究2.1取患者及正常对照者外周静脉血4-8ml,采用TIANamp Blood DNA Kit血基因组DNA提取试剂盒提取基因组DNA。2.2应用Affymetrix全基因组SNP 6.0基因芯片对一包含三个卡尔曼综合征子代患者的近亲婚配家庭进行连锁分析及基因组结构变化的分析。2.3基因序列分析：采取直接DNA测序分析,对所有患者的KAL1、FGFR1、PROK2及PROKR2基因的全部外显子进行突变检测,并用SSCP证实已发现的突变,同时在正常对照人群中进行SSCP检测排除该突变是多态性的可能。结果1.临床研究1.1我们收治了105例男性IHH患者,其中92例患者诊治的临床及随访资料达到随访要求,纳入本次报告。92例IHH患者就诊年龄平均(21±3.2)岁,52例伴有嗅觉减退或缺失(卡尔曼综合征,KS),40例为嗅觉正常的IHH (nIHH);其中47例表现为部分青春期发育,45例无明显青春期发育,3例男性乳腺发育,15例单侧隐睾,5例双侧隐睾。92例染色体核型均为46,XY。检测血清甲状腺、肾上腺功能及糖脂代谢正常,黄体生成素(LH)、卵泡刺激素(FSH)及睾酮(T)基础值低,6例IHH患者行骨龄测定5例落后于实际年龄。1.2头颅磁共振成像检查显示所有KS患者均存在嗅球或嗅沟的缺失或发育不良,所有患者均无下丘脑-垂体区占位性器质性病变。部分nIHH患者存在单侧嗅球或嗅沟发育不良。在正常对照人群中未发现存在嗅球或嗅沟的缺失或发育不良。1.3经生殖激素替代治疗后,92例均有明显的第二性征发育,性功能改善,血清睾酮水平增高。3例结婚,性功能评价正常,一例患者的妻子生育后代。2.分子遗传学研究2.1应用Affymetrix全基因组SNP 6.0基因芯片对一包含三个卡尔曼综合征子代患者的近亲婚配家庭进行连锁分析时未发现多态性标记出现明显连锁,优势对数计分(LOD)值均小于1.0。进行基因组结构变化的分析时发现三个患者在染色体1p21.1,2q32.2,8q21.13,14q21.2和Xp22.31上存共同的染色体拷贝数变异(微缺失)。而发生于染色体Xp22.31的拷贝数变异位于KAL1基因的内含子区域,该基因已被证实可导致X-连锁的卡尔曼综合征。该微缺失还出现于一例散发病例中。100例正常对照者中均未发现该位点的微缺失。2.2测序发现在一例生育后代的患者中发现KAL1基因的11外显子存在双位点的错意突变(1690 G>A和1765 G>A,致密码子位置514和539均出现G>A的突变,致赖氨酸突变为谷氨酸),其中之一为该基因上新发现的突变,SSCP证实此突变与疾病表型共分离,家系中其他成员及所生育后代无此突变,100例正常人无此突变。在另一家庭的一对患病兄弟中发现存在KAL1基因外显12碱基1828G>A的错意突变,致缬氨酸突变为亮氨酸,其母亲为杂合子,俩患者一个存在单侧肾缺如,一个为单侧隐睾。家庭中无患病者及100例正常人无此突变。在本组患者中候选基因FGFR1、PROK2及PROKR2未检测出突变。结论1.根据第二性征发育异常患者合并的各种先天异常、病史、体格检查、染色体核型分析、性激素水平、头颅磁共振成像检查等可进行IHH的诊断与鉴别诊断。嗅球部磁共振成像可作为诊断KS的重要手段。HCG与雄激素替代疗法是治疗该疾病的有效方法。2.我们为卡尔曼综合征分子发病机制的研究提供了一个新的视野即某些相关的拷贝数变异可能参与其中；同时也为进一步研究提供了一些感兴趣的拷贝数变异候选区域。3. KAL1基因的突变可能参与卡尔曼综合征的发病机制：即使出现KAL1基因的突变,经过早期积极治疗仍有生育后代的可能。更多还原

【Abstract】 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.更多还原