尿道下裂患者MID1基因突变的研究

目的：探讨MID1基因在尿道下裂发病中的作用。方法：收集96例先天性尿道下裂患者外周静脉血，提取DNA，采用PCR扩增直接测序的方法检测MID1基因全部外显子序列。结果：在2个患者中发现2种突变，位于第一外显子的T64T同义突变和位于第九外显子的A620V错义突变，均没有报道过。结论：MID1基因在尿道下裂的发病中并不起主要作用。     

单纯尿道下裂患者Cxorf6基因突变分析

目的探讨Cxorf6基因突变与单纯性尿道下裂的相关性.方法采用聚合酶链反应(PCR)及基因测序技术,分别对70例单纯男性尿道下裂患者和40例健康正常对照的Cxorf6基因序列进行分析.结果其中1例尿道下裂患者CXorf6基因第3外显子802(T→C)致无意义突变;另1例尿道下裂患者CXorf6基因第3外显子第620位点缺失一个碱基G致移码突变;另1例尿道下裂患者CXorf6基因在第3外显子第663位插入一个碱基G致移码突变.在40例正常对照组中未发现CXorf6基因存在突变.结论 CXorf6基因突变可能也是中国人群单纯性尿道下裂患者的易感基因之一.     

2型常染色体显性遗传性多囊肾病致病基因突变研究

目的建立检测2型常染色体显性遗传性多囊肾病致病基因PKD2突变的方法,分析中国汉族人PKD2基因的突变。方法收集临床确诊的中国汉族人常染色体显性遗传性多囊肾病(ADPKD)患者48例,用试剂盒提取外周血白细胞DNA,利用聚合酶链反应-单链构象多态性分析(PCR-SSCP)技术进行突变分析,对异常条带的PCR产物进行核苷酸序列测定,明确突变位点和方式。结果从48例中成功检测到4种突变,包括1种无义突变、1种移码突变、2种错义突变。第1种为外显子5的1249C→T,417位编码氨基酸发生无义突变。第2种为外显子13的第2401位碱基A缺失,造成编码氨基酸移码突变。第3种突变为外显子1的568G→A,编码氨基酸改变为190Ala→Thr;第4种为外显子5的1168G→A,编码氨基酸改变为390Gly→Ser。结论PCR-SSCP技术可用于PKD2的直接基因诊断,并从本组患者中检测到4种突变,丰富了PKD2基因突变谱,为今后开展ADPKD的直接基因诊断、产前诊断和囊肿前诊断提供了一种有用方法。     

尿道下裂患者雄激素受体基因突变的研究

目的研究国人尿道下裂患者雄激素受体基因突变情况。方法采集92例各型尿道下裂患者外周抗凝血,使用酚氯仿变性法提取基因组DNA,通过PCR扩增DNA测序的方法,检测了雄激素受体2～7外显子全部序列。结果4例患者外周血提取的基因组DNA中具有雄激素受体基因外显子突变,第4外显子1例(664ATT→ACT);第7外显子3例(840CGT→CAT、855CGC→CAC、859CTC→CTA)。结论雄激素受体基因点突变可能是尿道下裂的发病原因之一,但是所占比例仅为43%。     

尿道下裂患者SOX9基因突变的研究

目的：探讨SOX9基因在尿道下裂发病中的作用。方法：收集96例先天性尿道下裂患者外周静脉血，提取DNA，采用PCR扩增直接测序的方法检测SOX9基因全部外显子序列。结果：在实验组尿道下裂患者和对照组的SOX9基因外显子片段中，我们未发现任何位点的突变。只是在外显子3的3’UTR发现了1个SNPA／C，经分析没有统计学意义。结论：SOX9基因在性别分化的初期起作用，其变异直接影响性剐取向，结果导致两性畸形或性反转，而非单纯性尿道下裂。     

性连锁Alport综合征COL4A5基因突变检测

目的 检测16个家系20例性连锁Alport综合征患者COL4A4基因突变。方法：采用PCR－变性凝胶梯度电泳（DGGE）－直接测序法检测患者COL4A5基因中30个外显子及其相邻内含子区域（外显子1－25，31，32，41，50，51），另选取100例政党人外周血DNA作为对照。结果 共发现4种突变，包括1种位于1号外显子的无义突变（266C→T谷氨酰胺22终止密码），1种位于31号外显子上的错义突变（2575G→T甘氨酸852缬氨酸），以及2种分别位于1，25号内含子区域的剪接突变（283＋1G→T，2150＋1G→T）。结论 COL4A5基因为性连锁Alport综合征的致病基因，突变类型多样，尚未发现热点突变。类似于外显子突变，内含子突变同样具有致病意义。患者临床症状典型，查阅基因库，此4种突变均为首次报道。     

瘢痕疙瘩组织p53基因突变的实验研究

目的：分析瘢痕疙瘩中p53基因第4—8外显子的突变及其意义。方法：取手术切除的瘢痕疙瘩和正常瘢痕各12例，并分别取同一患者正常皮肤对照，采用聚合酶链反应-单链构象多态性分析方法(PCR-SSCP)和基因测序，检测各组织p53基因的突变情况。结果：12例瘢痕疙瘩标本中有9例p53基因外显子4、5、6、7出现点突变和移码突变，正常瘢痕标本、正常皮肤标本均未检出突变。结论：p53基因突变是瘢痕疙瘩形成和发展的重要因素之一。     

Denys-Drash综合征及其致病基因突变鉴定二例报道

目的 研究中国Denys-Drash综合征(DDS)患者Wilms瘤基因1(WT1)的基因突变。 方法 应用PCR方法扩增出WT1基因全部10个外显子及其相邻内含子序列，经纯化后进行PCR产物直接测序。 结果 2例患者WT1基因分别存在1个杂合错义突变。例1的X外显子9第1180位碱基C→T突变，造成第394位精氨酸改变为色氨酸，即p.R394W(c.1180C>T)。例号2的S外显子9第1203位碱基C→A突变，造成第401位组氨酸改变为谷氨酰胺，即p.H401Q(c.1203C>A)。其中第1203位碱基C→A突变，p.H401Q (c.1203C>A)，在国内外文献及突变数据库中均未见报道，属新发现的突变。 结论 DDS综合征WT1基因中外显子9为突变热点，并发现一种新的WT1基因突变。     

雄激素不敏感症疑似病例AR基因全长测序位点突变筛查与结果分析

目的对临床疑似雄激素不敏症患者进行AR基因全长测序连锁分析位点突变。方法提取雄激素不敏感(AIS)疑似患者外周血DNA,做染色体核型分析,了解AR基因各外显子缺失或重复突变情况,进行基因全长测序连锁分析位点突变。A组外生殖器异常表型,表现为尿道下裂并阴茎体发育不良,B组无尿道下裂但为小阴茎。结果 A组12例患者中发现3例AR基因突变。第1例发生新的c.D841 CGTAGT(精氨酸丝氨酸)突变,第2例发生c.D172和c.D173插入了TGC三个碱基。第3例存在c.D946 TC,c.D 316络氨酸组氨酸突变。3例均确诊为雄激素不敏感,2例为完全型,1例部分型。B组未发现AR基因突变。结论 AR基因全长测序可从分子水平初步阐明雄激素不敏感症的病因,指导临床诊治。     

高通量基因测序调查尿道下裂患者雄激素作用相关基因甲基化研究

目的通过高通量基因测序方法,对我院尿道下裂患者包皮内板组织中雄激素作用相关的基因CpG岛甲基化情况进行深度定量检测,研究雄激素相关基因甲基化表观遗传学在我院尿道下裂患者疾病发病机制中的作用地位。方法收集自2012年到2015年于我院接受尿道下裂修补术患者96名,年龄从3.5岁～8岁。平均年龄(5±1.5)岁。根据术前尿道开口的位置,将尿道下裂患者分为轻中度和重度组。其中轻中度组患者55例,重度患者41例,术中取尿道下裂患者包皮内板组织。对照组为收集在我院行包皮环切的患者包皮内板27例,年龄从3.7岁至7.5岁,平均(5±0.8)y,术前均确定有正常的尿道开口,阴茎及尿道发育正常。使用Illumina Genome Analyzer IIx对雄激素作用相关基因进行定量甲基化深度检测。结果 AR:均值0.106±0.013 vs 0.112±0.051,P0.05. SRD5A2均值0.046±0.007 vs 0.043±0.009,P0.05。结论在我们的患者中雄激素相关基因AR及SRD5A2基因CpG岛甲基化抑制了雄激素发挥作用这条途径,可能并不是尿道下裂发病的主要机制。     

Compound heterozygous mutations in the SRD5A2 gene exon 4 in a male pseudohermaphrodite patient of Chinese origin

The goal of this study was to perform 5-alpha-reductase type 2 gene (SRD5A2) analysis in a male pseudohermaphrodite (MPH) patient with normal testosterone (T) production and normal androgen receptor (AR) gene coding sequences. A patient of Chinese origin with ambiguous genitalia at 14 months, a 46,XY karyotype, and normal T secretion under human chorionic gonadotropin (hCG) stimulation underwent a gonadectomy at 20 months. Exons 1-8 of the AR gene and exons 1-5 of the SRD5A2 gene were sequenced from peripheral blood DNA. AR gene coding sequences were normal. SRD5A2 gene analysis revealed 2 consecutive mutations in exon 4, each located in a different allele: 1) a T nucleotide deletion, which predicts a frameshift mutation from codon 219, and 2) a missense mutation at codon 227, where the substitution of guanine (CGA) by adenine (CAA) predicts a glutamine replacement of arginine (R227Q). Testes located in the inguinal canal showed a normal morphology for age. The patient was a compound heterozygote for SRD5A2 mutations, carrying 2 mutations in exon 4. The patient showed an R227Q mutation that has been described in an Asian population and MPH patients, along with a novel frameshift mutation, Tdel219. Testis morphology showed that, during early infancy, the 5-alpha-reductase enzyme deficiency may not have affected interstitial or tubular development.     

Molecular characterization of 6 unrelated Italian patients with 5alpha-reductase type 2 deficiency

Steroid 5alpha-reductase (5alphaR) deficiency (OMIM number #264600) is a rare 46,XY disorder of sex differentiation caused by mutations in the 5alphaR type 2 gene (SRD5A2) resulting in dihydrotestosterone deficiency during fetal development. We report on the analysis of the SRD5A2 gene in 6 unrelated 46,XY Italian patients with external genitalia morphology ranging from predominantly female to nearly completely male. Three subjects were seen and assessed at birth, 1 patient was referred to us before puberty, and 2 at postpubertal age. Six different causative mutations (5 missense and 1 nonsense) and a rare polymorphism were identified. Four patients presented homozygous single-base substitutions. These SRD5A2 mutations were located in exon 2 (variant Cys133Gly), exon 4 (Gly196Ser and Ala207Asp) and exon 5 (Tyr235Phe). A fifth subject was a compound heterozygote who carried a nonsense mutation in exon 1 (Trp53X) and a second SRD5A2 alteration in exon 5 (Tyr235Phe). The final patient presented a mutation in only 1 allele (Gly34Trp) together with the Ala49Thr variant. The molecular characterization of these patients made it possible to identify novel mutations and to confirm, before gender assignment or any surgical approach, the suspected 5alphaR deficiency in 2 newborns, 1 of whom had inconclusive hormonal data. 5alphaR deficiency in subjects without parental consanguinity and the presence of compound heterozygotic patients suggest that SRD5A2 mutations carrier frequency may be higher than previously thought.     

Isolated micropenis reveals partial androgen insensitivity syndrome confirmed by molecular analysis

Partial androgen insensitivity syndrome （PAIS） is the milder variant of androgen receptor （AR） defects. The subtle effects of AR mutations present in a patient with micropenis, peno-scrotal hypospadias, infertility, clitoromegaly and posterior labial fusion. We studied the association of isolated micropenis with the genetic defects resulting in androgen resistance, that is, AR gene defects and 5-α reductase type 2 （SRD5A2） deficiency. We describe two cases of isolated micropenis： one in a 14-year-old boy and the other in a 3-year-old boy who was followed until he was 10 years old. There were no findings of hypospadias, cryptorchidism or gynecomastia in either of these patients. Serum gonadotrophin and androgen levels were obtained and karyotyping was done. Human chorionic gonadotropin （hCG） stimulation testing assessed the functional capacity of the testes. DNA was extracted from peripheral leukocytes, and all exons of the SRD5A2 and AR genes were amplified by polymerase chain reaction and sequenced. In both patients, baseline testosterone （T） level was low and the values were elevated after hCG testing. The sequence of the SRD5A2 gene was normal in patient 1, and a heterozygous polymorphism, V89L, was found in patient 2. Two known mutations, P390S and A870V, were identified in patients 1 and 2, respectively. Mutations in the AR gene can be associated with isolated micropenis without other features of PAIS, such as hypospadias or gynecomastia. This underlines the importance of including AR gene analysis in the evaluation of isolated micropenis with normal plasma T to ensure proper management of the patient and appropriate genetic counseling for the family.     

Phenotype, hormonal profile and genotype of subjects with partial androgen insensitivity syndrome: report of a family with four adult males and one child with disorder of sexual differentiation

There is little information on the molecular basis of intrafamilial and inter-familial phenotypic heterogeneity with the same androgen receptor (AR) mutation in patients with partial androgen insensitivity syndrome. A genetic analysis was performed in a large kindred with ambiguous genitalia and the genotype–phenotype correlations were analysed. The index case was brought for sex assignment. Family history revealed four other affected members who had hypospadias and varying degrees of virilisation. All the affected males had hemizygous mutations in the third exon of the AR gene (A596T). One was also found to have a heterozygous mutation in the fourth exon of the 5 alpha reductase type 2 gene (G196S). This affected male with double mutations was better virilised compared with the other affected members with a single mutation. The degree of virilisation correlated with serum testosterone levels. Gynaecomastia was not present in any of these subjects. It is concluded that the subject with dual gene defects also had higher levels of testosterone and pubertal virilsation. Testosterone levels possibly govern the degree of pubertal virilisation in subjects with A596T gene defects. It is not clear whether the better pubertal virilsation and higher testosterone are in any way causally related to the SRD5A2 gene defect.     

Molecular investigation of mutations in androgen receptor and 5‐alpha‐reductase‐2 genes in 46,XY Disorders of Sex Development with normal testicular development

In this study, we aimed to determine androgen receptor (AR) and SRD5A2 gene mutations in 45 patients characterised by 46,XY Disorders of Sex Differentiation (DSD) signs with normal testicular development referred to the Children's Medical Center from February 2015 to September 2017. Karyotype and sex hormone analyses were performed. Cytogenetic investigation showed that seven patients were 46,XX DSD, six patients were chromosomal DSD and 32 patients were 46,XY DSD. Eight exons of the AR gene and five exons of the SRD5A2 gene were amplified. Two cases were affected with androgen insensitivity syndrome (AIS) (missense mutation on exon 7, position c.3637 G>A: p.R841H and position c.3610 G>A: p.R832Q), one case was affected with 5‐alpha‐reductase deficiency type 2 (missense mutation at c.578A>G: p.N193S on exon 4), and 22 cases (88%) did not demonstrate AIS or 5α‐RD2 gene abnormality. Due to the great impact of these disorders on human lifestyle, evaluation of genes involved can improve genetic counselling and therapeutic management. We focused on the AR and SRD5A2 genes in patients with 46,XY DSDs with normal testicular development referred to the Children's Medical Center from all over the country to eventually culminate in a reliable prenatal diagnosis protocol at this major referral centre giving service to a great number of families with consanguineous marriages.     

Heterozygous mutations in the LDL receptor-related protein 5 (LRP5) gene are associated with primary osteoporosis in children.

Three of 20 patients with juvenile osteoporosis were found to have a heterozygous mutation in the LRP5 gene. No mutations were found in the type I collagen genes. Mutations in the other family members with similar bone phenotype confirmed that LRP5 has a role in both juvenile and adult osteoporosis. INTRODUCTION: The gene encoding the low-density lipoprotein receptor-related protein 5 (LRP5) gene has recently been shown to affect bone mass accrual during growth and to be involved in osteoporosis-pseudoglioma syndrome and a high bone mass phenotype. Mutations in the type I collagen genes (COL1A1 and COL1A2) are known to cause osteogenesis imperfecta, characterized by increased bone fragility. MATERIALS AND METHODS: Here we analyzed COL1A1, COL1A2, and LRP5 for mutations in 20 pediatric patients with primary osteoporosis characterized by low BMD, recurrent fractures, and absent extraskeletal manifestations. RESULTS AND CONCLUSIONS: No mutations were detected in the type I collagen genes, but two missense mutations (A29T and R1036Q) and one frameshift mutation (C913fs) were found in the LRP5 gene in three of the patients. The frameshift mutation was also seen in the proband's father and brother, who both were found to have significant osteoporosis. R1036Q was observed in the proband's mother and two brothers, who all had osteoporosis. These results indicate that heterozygous mutations in the LRP5 gene can cause osteoporosis in both children and adults.     

Screening for mutations in candidate genes for hypospadias

Hypospadias, a condition with a frontally placed urethral orifice on the penis, is the most common malformation in males. During fetal development several components are necessary for normal male genital development. Testosterone and dihydrotestosterone act via the androgen receptor but a defective receptor function results in different degrees of genital malformations. Testosterone-5α-reductase converts testosterone to dihydrotestosterone, which is crucial for normal differentiation, and a total lack of this enzyme results, in syndromes with hypospadias. The Wilms' tumour 1 (WT1) gene is expressed in the fetal gonad and genital malformations can occur due to WT1 gene mutations. These genes are therefore strong candidate genes for hypospadias. We have analysed 35 boys with hypopadias and one girl diagnosed as with complete androgen insensitivity syndrome, using exon by exon polymerace chain reaction (PCR) amplification of the AR, WT1 and 5α-reductase genes and screened for point mutations and performed subsequent DNA sequencing. No mutations in any of these genes were found in the 26 patients with isolated hypospadias. Two patients with severe hypospadias with cryptorchidism were found to carry mutations in the androgen receptor gene. Also the girl with clinically diagnosed complete androgen insensitivity was found to be homozygous for a splice mutation in the 5α-reductase gene. In summary, mutations in the WT1, AR and 5α-reductase genes are not common causes of isolated hypospadias. Received: 1 October 1997 / Accepted: 4 May 1998     

Sixteen novel mutations identified in COL4A3, COL4A4, and COL4A5 genes in Slovenian families with Alport syndrome and benign familial hematuria

Alport syndrome (ATS) and benign familial hematuria (BFH) are type IV collagen inherited disorders. Mutations in COL4A5 are generally believed to cause X-linked ATS, whereas mutations in COL4A3 and COL4A4 genes can be associated with the autosomal-recessive and -dominant type of ATS or BFH. In view of the wide spectrum of phenotypes, an exact diagnosis is sometimes difficult to achieve. This study involved screening each exon with boundary intronic sequences of COL4A3, COL4A4, and COL4A5 genes by optimized polymerase chain reaction-single-stranded conformational polymorphism analysis in 17 families with ATS and in 40 families diagnosed as having BFH. Twelve different mutations were found in the COL4A5 gene in ATS patients, comprising nine missense mutations, a splice site mutation, a mutation causing frameshift, and a nonsense mutation. One of the missense mutations (p.G624D) was present not only in one family with ATS but also in five families with suspected BFH. Three heterozygous mutations in the COL4A3 gene (two missense and one frameshift) and four heterozygous mutations in COL4A4 (two splice site, one in-frame deletion, and one missense) were identified in patients with BFH. Sixteen mutations are to the best of our knowledge new and private.