Objectives: Congenital hypogonadotropic hypogonadism (CHH) is a rare congenital gonadal dysplasia caused by defects in the synthesis, secretion or signal transduction of hypothalamic gonadotropin releasing hormone. The main manifestations of CHH are delayed or lack puberty, low levels of sex hormones and gonadotropins, and may be accompanied with other clinical phenotypes. Some patients with CHH are also accompanied with anosmia or hyposmia, which is called Kalman syndrome (KS). ANOS1, located on X chromosome, is the first gene associated with CHH in an X-linked recessive manner. This study aims to provide a basis for the genetic diagnosis of CHH by analyzing the gene variant spectrum of ANOS1 in CHH and the relationship between clinical phenotype and genotype.
Methods: In this study, whole exome sequencing (WES) was used to screen rare sequencing variants (RSVs) of ANOS1 in a Chinese cohort of 165 male CHH patients. Four commonly used in silico tools were used to predict the function of the identified RSVs in coding region, including Polyphen2, Mutation Taster, SIFT, and Combined Annotation Dependent Depletion (CADD). Splice Site Prediction by Neural Network (NNSPLICE) was employed to predict possibilities of intronic RSVs to disrupt splicing. American College of Medical Genetics and Genomics (ACMG) guidelines was used to assess the pathogenicity of the detected RSVs. The ANOS1 genetic variant spectrum of CHH patients in Chinese population was established. The relationship between clinical phenotype and genotype was analyzed by collecting detailed clinical data.
Results: Through WES analysis for 165 CHH patients, ANOS1 RSVs were detected in 17 of them, with the frequency of 10.3%. A total of 13 RSVs were detected in the 17 probands, including 5 nonsense variants (p.T76X, p.R191X, p.W257X, p.R262X, and p.W589X), 2 splicing site variants (c.318+3A>C, c.1063-1G>C), and 6 missense variants (p.N402S, p.N155D, p.P504L, p.C157R, p.Q635P, and p.V560I). In these 17 CHH probands with ANOS1 RSVs, many were accompanied with other clinical phenotypes. The most common associated phenotype was cryptorchidism (10/17), followed by unilateral renal agenesis (3/17), dental agenesis (3/17), and synkinesia (3/17). Eight RSVs, including p.T76X, p.R191X, p.W257X, p.R262X, p.W589X, c.318+3A>C, c.1063-1G>C, and p.C157R, were predicted to be pathogenic or likely pathogenic ANOS1 RSVs by ACMG. Eight CHH patients with pathogenic or likely pathogenic ANOS1 variants had additional features. In contrast, only one out of nine CHH patients with non-pathogenic (likely benign or uncertain of significance) ANOS1 variants according to ACMG exhibited additional features. And function of the non-pathogenic ANOS1 variants accompanied with other CHH-associated RSVs.
Conclusions: The ANOS1 genetic spectrum of CHH patients in Chinese population is established. Some of the correlations between clinical phenotype and genotype are also established. Our study indicates that CHH patients with pathogenic or likely pathogenic ANOS1 RSVs tend to exhibit additional phenotypes. Although non-pathogenic ANOS1 variants only may not be sufficient to cause CHH, they may function together with other CHH-associated RSVs to cause the disease.
目的: 先天性低促性腺激素性性腺功能减退症(congenital hypogonadotropic hypogonadism，CHH)是一种罕见的先天性疾病，由于下丘脑促性腺激素释放激素的合成、分泌或信号转导缺陷引起先天性性腺发育不良。CHH以青春期发育延迟或缺乏、性激素及促性腺激素水平低下为主要表现，同时可能伴有其他临床表型。一部分CHH患者伴有嗅觉丧失或低下，被称为卡尔曼综合征(Kallmann syndrome，KS)。ANOS1基因是第一个被发现的CHH致病基因，位于X染色体上，其突变可导致X-连锁隐性遗传的CHH。本研究拟通过分析CHH患者中ANOS1的基因突变图谱以及临床表型和基因型的关系，为CHH的遗传学诊断奠定基础。方法: 利用全外显子组测序(whole exome sequencing，WES)的方法筛选来自中国的165名男性CHH患者中ANOS1基因的罕见变异(rare sequencing variants，RSVs)。利用Polyphen2、Mutation tastaster、SIFT和CADD(Combined Annotation Dependent Depletion)4种常见的生物信息学工具预测编码区变异的功能，基于神经网络的剪接位点预测(Splice Site Prediction by Neural Network，NNSPLICE)软件对检测到的内含子区的RSVs进行注释，并利用美国医学遗传学和基因组学学院(American College of Medical Genetics and Genomics，ACMG)遗传变异分类标准与指南判断ANOS1 RSVs是否具有致病性。初步建立中国人群CHH患者ANOS1的遗传突变谱，通过收集部分患者详细的临床资料，建立临床表型和基因型的相关性。结果: WES分析显示165名CHH患者中17例患者发生了ANOS1突变，突变频率为10.3%。在17名CHH患者中共检测到13个ANOS1 RSVs，包括5个无义突变(p.T76X、p.R191X、p.W257X、p.R262X和p.W589X)，2个剪切位点突变(c.318+3A>C和c.1063-1G>C)和6个错义突变(p.N402S、p.N155D、p.P504L、p.C157R、p.Q635P及p.V560I)。在17名携带ANOS1 RSVs的患者中，很多同时伴有其他临床表型，其中最常见的为隐睾(10/17)，其次是单侧肾脏发育不全(3/17)，牙齿发育不全(3/17)和联带运动(3/17)。利用ACMG对上述检测到的ANOS1 RSVs进行分析，8个RSVs包括p.T76X、p.R191X、p.W257X、p.R262X、p.W589X、c.318+3A>C、c.1063-1G>C和p.C157R被预测为致病性或可能致病性的罕见突变，且携带这些突变的患者经过临床检查全部伴随其他临床表型；然而，在携带其他被预测为非致病性(不确定性或可能良性)的ANOS1 RSVs患者中，只有1例患者同时伴随其他临床表型，且这些患者中大部分同时伴有其他CHH致病基因的RSVs。结论: 初步建立了中国人群CHH患者的ANOS1基因突变谱及基因型-表型相关性。携带致病性或可能致病性ANOS1 RSV的CHH患者往往伴随其他表型。单纯非致病性ANOS1 RSV可能不足以引起CHH，但它们可能与其他CHH致病基因突变一起发挥作用，导致该疾病的发生。.
目的: 先天性低促性腺激素性性腺功能减退症(congenital hypogonadotropic hypogonadism，CHH)是一种罕见的先天性疾病，由于下丘脑促性腺激素释放激素的合成、分泌或信号转导缺陷引起先天性性腺发育不良。CHH以青春期发育延迟或缺乏、性激素及促性腺激素水平低下为主要表现，同时可能伴有其他临床表型。一部分CHH患者伴有嗅觉丧失或低下，被称为卡尔曼综合征(Kallmann syndrome，KS)。 ANOS1基因是第一个被发现的CHH致病基因，位于X染色体上，其突变可导致X-连锁隐性遗传的CHH。本研究拟通过分析CHH患者中 ANOS1的基因突变图谱以及临床表型和基因型的关系，为CHH的遗传学诊断奠定基础。
方法: 利用全外显子组测序(whole exome sequencing，WES)的方法筛选来自中国的165名男性CHH患者中 ANOS1基因的罕见变异(rare sequencing variants，RSVs)。利用Polyphen2、Mutation tastaster、SIFT和CADD(Combined Annotation Dependent Depletion)4种常见的生物信息学工具预测编码区变异的功能，基于神经网络的剪接位点预测(Splice Site Prediction by Neural Network，NNSPLICE)软件对检测到的内含子区的RSVs进行注释，并利用美国医学遗传学和基因组学学院(American College of Medical Genetics and Genomics，ACMG)遗传变异分类标准与指南判断 ANOS1 RSVs是否具有致病性。初步建立中国人群CHH患者 ANOS1的遗传突变谱，通过收集部分患者详细的临床资料，建立临床表型和基因型的相关性。
结果: WES分析显示165名CHH患者中17例患者发生了 ANOS1突变，突变频率为10.3%。在17名CHH患者中共检测到13个 ANOS1 RSVs，包括5个无义突变(p.T76X、p.R191X、p.W257X、p.R262X和p.W589X)，2个剪切位点突变(c.318+3A>C和c.1063-1G>C)和6个错义突变(p.N402S、p.N155D、p.P504L、p.C157R、p.Q635P及p.V560I)。在17名携带 ANOS1 RSVs的患者中，很多同时伴有其他临床表型，其中最常见的为隐睾(10/17)，其次是单侧肾脏发育不全(3/17)，牙齿发育不全(3/17)和联带运动(3/17)。利用ACMG对上述检测到的 ANOS1 RSVs进行分析，8个RSVs包括p.T76X、p.R191X、p.W257X、p.R262X、p.W589X、c.318+3A>C、c.1063-1G>C和p.C157R被预测为致病性或可能致病性的罕见突变，且携带这些突变的患者经过临床检查全部伴随其他临床表型；然而，在携带其他被预测为非致病性(不确定性或可能良性)的 ANOS1 RSVs患者中，只有1例患者同时伴随其他临床表型，且这些患者中大部分同时伴有其他CHH致病基因的RSVs。
结论: 初步建立了中国人群CHH患者的 ANOS1基因突变谱及基因型-表型相关性。携带致病性或可能致病性 ANOS1 RSV的CHH患者往往伴随其他表型。单纯非致病性 ANOS1 RSV可能不足以引起CHH，但它们可能与其他CHH致病基因突变一起发挥作用，导致该疾病的发生。
Keywords: ANOS1; Kallmann syndrome; congenital hypogonadotropic hypogonadism; cryptorchidism.