雄激素不敏感综合征家系遗传学分析与产前诊断北大核心CSCD

目的探讨雄激素不敏感综合征(AIS)患者的雄激素受体(AR)基因突变情况以及相应的遗传咨询和产前诊断。方法应用染色体G显带核型分析技术、基因测序技术检测2013年云南省第一人民医院遗传诊断中心产前诊断的1例AIS患者及家系部分成员的AR基因,然后进行遗传学分析。结果核型分析显示先证者和胎儿的染色体核型为46,XY,先证者检出AR基因第4外显子2069-2071del ACG,母亲为AR基因缺失的携带者。该突变造成AR基因的691位氨基酸(天冬氨酸)的缺失,最后编码了918个氨基酸的截短AR蛋白。正常人群中未发现该突变。结论通过对家系成员的临床遗传学分析确定了1种AR基因病理性新突变,AR基因缺失691位氨基酸(天冬氨酸)的截短突变可引起完全型雄激素不敏感综合征。通过遗传咨询和基因检测,可对该家系做出准确的产前诊断。     

7例完全型雄激素不敏感综合征患者的临床及遗传学分析

目的:探讨完全型雄激素不敏感综合征(CAIS)患者的临床和分子遗传特征。方法:收集7例CAIS患者及家族相关成员的临床资料,提取外周血全基因组DNA,用特异性引物-聚合酶链反应(PCR)扩增AR基因的8个外显子,扩增产物Sanger测序后与基因库中正常人的序列进行比对,查找可能存在的致病突变。应用Mutation taster、PolyPhen-2等生物信息学软件预测突变对蛋白功能的影响。结果:7例CAIS患者的AR基因均检测到突变,并且在家系中共分离,其中有4个突变未报道过:p.Y764N、p.Q876X、p.A356Efs*123、p.S704R。3例行腹腔镜探查术及性腺活检,病理检查结果证实性腺为睾丸。结论:CAIS患者常因原发闭经就诊而被发现,多数存在异常性激素水平,家系中常有多例患者,呈X连锁隐性遗传模式。AR基因的检测有助于CAIS患者的确诊并有助于CAIS家庭的生殖咨询。     

应用Ion Torrent半导体测序检测马凡综合征患者FBN1基因致病突变

目的:对3个马凡综合征(MFS)家系及1例MFS患者的原纤维蛋白1基因(FBN1)进行基因检测,以明确致病突变,为患者提供遗传咨询及产前诊断。方法:应用高通量Ion Torrent半导体测序技术对3个MFS家系的先证者及1例MFS患者的FBN1基因进行检测,筛选致病突变位点,并用Sanger测序法验证。对1例胎儿FBN1基因相应的位点进行检测,以明确其受累情况。结果:患者P1 FBN1基因检测到c.7125_7126 del TG杂合性缺失突变,为可疑致病位点;家系1患者FBN1基因检测到IVS 27-1G>C(c.3464-1G>C)杂合性突变,为致病突变;家系2患者FBN1基因检测到c.4981G>C(p.Gly1661Arg)杂合性错义突变,为致病突变,胎儿携带同样突变;家系3患者FBN1基因检测到c.1546C>T(p.Arg516Term)杂合性无义突变,为致病突变。结论:应用高通量Ion Torrent半导体测序技术检测到3个MFS家系及1个MFS患者的致病基因突变,为临床诊断及遗传咨询提供分子遗传学依据,并对1例胎儿进行了产前诊断。     

全外显子测序检测6个囊性肾病胎儿及其核心家系的致病突变

目的全外显子测序技术检测6个囊性肾病胎儿及其核心家系,寻找致病变异,为遗传咨询、产前诊断或植入前诊断提供遗传学数据支持。方法应用Illumina Hiseq2500测序平台,对超声检查提示为囊性肾病、染色体微阵列检测(CMA)未见异常的胎儿标本进行家系全外显子检测,参考数据库Human Genome 19(hg19/GRCh37),根据美国医学遗传学与基因组学学会指南(ACMG,2015)及指南应用建议评估变异致病性,对评级可疑致病位点应用Sanger测序验证。结果家系WES检出3个家系存在异常突变,其中1个家系的突变为遗传性,2个家系胎儿的突变为新发突变;余3个家系未检出明确致病突变。胎儿1 PKHD1基因c.5869G>A(父源)和c.9455delA复合杂合突变(母源),均为可能致病的变异。胎儿5 HNF1B基因c.826C>T杂合突变,为致病性新发突变。胎儿6 HNF1B基因c.1318G>T杂合突变,为可能致病性新发突变。Sanger测序验证结果与全外显子检测结果一致。结论对囊性肾病可运用家系核心成员全外显子检测,筛选出候选变异后进行Sanger测序验证,并对家系进行遗传分析,有助于遗传咨询、再次妊娠的产前诊断或辅助生殖的植入前诊断。本研究检出3个未见文献报道的可能致病突变位点,1个致病性突变位点已在个别文献报道但是未在国际公认数据库明确为致病突变位点,本研究拓宽了囊性肾病的致病基因位点谱。     

应用变性高效液相色谱技术检测结节性硬化症TSC1基因外显子15基因突变的研究

摘要：目的　探讨结节性硬化症（TSC）TSC1基因外显子15基因突变的特点。方法　1996—2006年采用多聚酶链扩增和变性高效液相色谱（DHPLC）技术,对21个家系59名研究对象进行了TSC1基因外显子15检测,对DHPLC检测异常者再进行多聚酶链扩增产物直接测序方法证实其突变类型。结果　对21个家系TSC1 基因外显子15共检测出4个家系的3种突变形式,其中c.1708～1709delAG（p.Arg570GlyfsX17）与c.1888～1891delAAAG（p.Lys630GlnfsX22） 两种突变为国内尚未报道的小的缺失突变,c.1460C > G（p.Ser487Cys）突变为1种罕见的错义突变。TSC1 基因外显子15突变基因检出频率为4/21（19%）。在检出突变的4个家系中1个为家系突变,其余3个为散发突变。结论　TSC1基因外显子15的c.1460C > G（p.Ser487Cys）,c.1708～1709delAG（p.Arg570GlyfsX17）与c.1888～1891delAAAG （p.Lys630GlnfsX22） 突变为目前国内首报突变。     

女性表型46,XY性发育障碍的临床、病理特征及遗传学检测

目的:总结女性表型的46,XY性发育障碍患者的临床及病理学特点,对其进行鉴别诊断及遗传学检测,为类似病例的诊断和鉴别诊断提供借鉴资料。方法:回顾分析2010年至2015年在深圳市妇幼保健院行妇科手术的3例46,XY性发育障碍患者的临床资料。将切除的性腺组织进行病理学诊断;提取患者及家属基因组DNA,应用Sanger测序、二代测序方法、MLPA、染色体基因组芯片分析等方法进行遗传学检测以寻找致病基因变异。结果:1例患者为完全型雄激素不敏感综合征(CAIS),病理结果证实一侧隐睾见精原细胞瘤,其AR基因第7外显子检测到移码突变c.2546_2547 insA(p.N849K,fs X32),此突变为已报道导致CAIS的突变方式;1例患者临床诊断为单纯性腺发育不良,性腺病理结果为不成熟的卵巢组织,患者SRY基因的HMG区域检测到c.206TC(p.V69A)突变,此突变未见报道;1例患者临床诊断为单纯性腺发育不良,病理结果为双侧性腺母细胞瘤伴无性细胞瘤,性发育相关基因未检测到明确的致病突变。结论:综合利用多种检测方法对女性表型46,XY性发育障碍患者进行致病基因检测,其中2例患者分别由AR基因、SRY基因突变引起,其中SRY基因c.206TC(p.V69A)为新发现的突变。     

假性软骨发育不全一家系COMP基因突变分析

对一假性软骨发育不全（PSACH） 家系进行软骨寡聚基质蛋白（COMP）基因突变分析。方法　2008年10月于北京协和医院儿科遗传门诊采集该家系中患者及正常人血样,提取DNA,用聚合酶链反应扩增COMP基因外显子8-19,并对扩增产物进行直接测序,确定突变位点。结果　家系中患病父子都存在COMP基因外显子8中c.815T > C突变,为已知突变。结论　该家系中患者发病是由COMP基因突变所致。     

雄激素不敏感综合征家系遗传学分析与产前诊断

目的探讨雄激素不敏感综合征(AIS)患者的雄激素受体(AR)基因突变情况以及相应的遗传咨询和产前诊断。方法应用染色体G显带核型分析技术、基因测序技术检测2013年云南省第一人民医院遗传诊断中心产前诊断的1例AIS患者及家系部分成员的AR基因,然后进行遗传学分析。结果核型分析显示先证者和胎儿的染色体核型为46,XY,先证者检出AR基因第4外显子2069-2071del ACG,母亲为AR基因缺失的携带者。该突变造成AR基因的691位氨基酸(天冬氨酸)的缺失,最后编码了918个氨基酸的截短AR蛋白。正常人群中未发现该突变。结论通过对家系成员的临床遗传学分析确定了1种AR基因病理性新突变,AR基因缺失691位氨基酸(天冬氨酸)的截短突变可引起完全型雄激素不敏感综合征。通过遗传咨询和基因检测,可对该家系做出准确的产前诊断。     

CYP1A1基因MspⅠ位点和SULT1A1基因Arg213His位点多态性与子宫肌瘤的关联性研究

目的 探讨细胞色素P450(cytochrome P450,CYP)1A1基因MspⅠ位点和硫酸氨基转移酶(sulfotransferase,SULT)1A1基因Arg213His位点多态性与鲁北地区汉族女性子宫肌瘤的关系.方法 采用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)方法检测123例子宫肌瘤患者和123例健康对照组的CYP1A1基因MspⅠ位点的基因型和SULT1A1基因Arg213His位点的基因型,分析基因多态性与子宫肌瘤的关系.结果 子宫肌瘤组CYP1A1基因MspⅠ位点的基因型与对照组中的分布比较,差异无统计学意义(P=0.927);而子宫肌瘤组SULT1A1基因Arg213His位点的基因型与对照组中的分布比较,差异有统计学意义(P=0.011).CYP1A1基因MspⅠ位点和SULT1A1基因Arg213His位点多态性在子宫肌瘤的发生过程中的交互作用比较,差异有统计学意义(P=0.024).结论 CYP1A1基因MspⅠ位点多态性与鲁北地区汉族女性子宫肌瘤的易感性无显著相关;SULT1A1基因Arg213His位点多态性与鲁北地区汉族女性子宫肌瘤的发生有关,并增加了子宫肌瘤的患病风险;CYP1A1基因MspⅠ位点和SULT1A1基因Arg213His位点多态性在子宫肌瘤的发生过程中具有交互作用.     

甲型血友病家系的F8基因检测及产前基因诊断

目的:探讨甲型血友病患者及其家属进行基因诊断与产前诊断的临床价值。方法:应用DHPLC、琼脂糖凝胶电泳及Sanger测序法分别对14个血友病家系进行F8致病基因检测,在明确致病突变基础上,对其中6例疑似致病基因携带者进行产前诊断。结果:14例甲型血友病家系中,检出F8基因内含子22倒位6例,F8基因外显子14缺失1例,F8基因编码区移码突变3例,F8基因编码区错义突变4例。通过羊水穿刺产前基因诊断检出异常胎儿4例。结论:通过基因测序的方法对先证者或高度疑似携带者进行基因诊断,明确突变类型及突变位点后对其进行优生优育指导和产前基因诊断,能有效降低甲型血友病的发病率。     

A double nucleotide insertion-induced frame-shift mutation of the androgen receptor gene in a familial complete androgen insensitivity syndrome

Objective

A wide spectrum of androgen receptor (AR) gene mutations has been reported in complete androgen insensitivity syndrome (CAIS). The molecular basis of androgen resistance was investigated in a female with familial CAIS.

Study design

AR gene and protein were investigated by PCR and direct sequencing and Western immunoblotting, respectively.

Results

Sequencing analysis of DNA of the patient identified a double nucleotide insertion in exon 4 that results in the frame-shift leading to premature terminal signal in the beginning of exon 6. This mutation predicted the synthesis of a truncated AR that lacks the entire ligand-binding molecules. Immunoblotting analysis of the gonad removed from the patient detected the mutated AR protein of 94 kDa. Positive control revealed the normal apparent molecular mass of 110 kDa. DNA sequencing of her mother demonstrated the presence of both canonical and mutated sequences in the exon 4 through 8.

Conclusion

These findings suggested that the previously undescribed insertion mutation in the AR gene is the cause of CAIS in this family.     

Identification of a mutant allele of the androgen receptor gene in a family with androgen insensitivity syndrome: detection of carriers and prenatal diagnosis

We report the results of a molecular study of a large family segregating the complete form of the Androgen Insensitivity Syndrome (CAIS) in several family members from three generations. We identified the mutant allele by polymerase chain reaction (PCR) amplification of the short tandem repeat (CAG)n, highly polymorphic in the population, present in the first exon of the androgen receptor (AR) gene. In this family four different alleles were detected and one of these showed a perfect segregation with the disease.This study enabled us to identify the heterozygous females in this family. We think that this simple, indirect test, is also suitable for prenatal diagnosis of Morris' syndrome when the mother is heterozygous for the size of the short tandem repeat and one affected subject in the family may be studied.     

Identification of a mutant allele of the androgen receptor gene in a family with androgen insensitivity syndrome: detection of carriers and prenatal diagnosis

We report the results of a molecular study of a large family segregating the complete form of the Androgen Insensitivity Syndrome (CAIS) in several members from three generations. We identified the mutant allele by Polymerase Chain Reaction (PCR) amplification of the short tandem repeat (CAG)n, highly polymorphic in the population, present in the first exon of the androgen receptor (AR) gene. In this family four different alleles were detected and one of these showed a perfect segregation with the disease. This study enabled us to identify the heterozygous females in this family. We think that this simple, indirect test, is also suitable for prenatal diagnosis of Morris' syndrome when the mother is heterozygous for the size of the short tandem repeat and one affected subject in the family may be studied.     

Primary amenorrhea in a young Polish woman with complete androgen insensitivity syndrome and Sertoli-Leydig cell tumor: identification of a new androgen receptor gene mutation and evidence of aromatase hyperactivity and apoptosis dysregulation within the tumor.

Primary amenorrhea in 46,XY females can be due to complete androgen insensitivity syndrome (CAIS), pure gonadal dysgenesis, 17-hydroxysteroid dehydrogenase deficiency, or mixed gonadal dysgenesis. The present paper describes a new de novo non-sense mutation in exon 1 (K141Z) of the androgen receptor gene (AR) and the expression in CAIS testis of aromatase, estrogen receptors, as well as proliferation- and apoptosis-associated proteins. CAIS is a rare disease characterized by absent virilization in 46,XY individuals and the development of a female phenotype despite normal or even elevated androgen levels. CAIS is usually caused by a mutation in AR, which leads to organ resistance to androgens. Testicular tumors such as Sertoli-Leydig cell tumor often develop in patients with CAIS. The immunohistochemical findings in the testes of our CAIS patient suggest that the high expression of aromatase and other molecular changes in the testis may be responsible for pubertal breast development and the increased risk of testicular tumor.     

Androgen receptor gene mutation identified by PCR-SSCP and sequencing in 4 patients with complete androgen insensitivity syndrome

To study the genetic defect of the human androgen receptor (hAR) gene in the complete androgen insensitivity syndrome (CAIS), we amplified each of the eight exons by PCR in genomic DNA extracted from the paraffin blocks of the resected gonads. We analyzed using SSCP, and directly sequenced the abnormally shifted bands. Mutations were found in 4 cases of CAIS. Patient 1 carried a point mutation; a G to A transition in exon 7 resulted in a change from arginine to glutamine at codon 831. Patient 2 carried a point mutation; a C to T transition in exon 7 resulted in a change from arginine to stop at codon 831. Patient 3 carried a point mutation and deletion in exon 7. A point mutation was an A to G transition that caused a glutamine to be substituted for the asparagine present at codon 819. A deletion of a G at codon 820 resulted in a frameshift and consequently in the introduction of a premature stop at codon 821. Patient 4 carried a mutation in 5’ splice donor site of intron 7; a G to T transition might have caused an abnormal splicing of the exon 7. All of the mutations were found in exon 7. These mutations of hAR gene might be related to the pathogenesis of CAIS. Received: May 1999 / Accepted: 17 August 1999     

完全型雄激素不敏感综合征患者的骨密度研究

目的探讨完全型雄激素不敏感综合征(CAIS)患者的骨密度特点及性腺切除前后骨密度的变化。方法回顾性分析北京协和医院28年收治的14例CAIS患者性腺切除术前后的骨密度特征,采用双能X线骨密度仪测定患者的腰椎和股骨颈骨密度,并与中国正常男性和女性的骨密度进行比较和分析。结果术前10例行骨密度检查的患者中,6例腰椎2~4骨密度为(0.92±0.08)g/cm2,正常男性为(1.17±0.15)g/cm2,正常女性为(1.17±0.10)g/cm2,与正常男、女性分别比较,差异均有统计学意义(P<0.01);5例股骨颈骨密度为(0.89±0.12)g/cm2,正常男性为(1.06±0.17)g/cm2,正常女性为(0.97±0.14)g/cm2,与正常男、女性分别比较,差异均有统计学意义(P<0.05)。术后7例进行过12次骨密度检查,腰椎2~4骨密度为(0.95±0.06)g/cm2,与正常男、女性分别比较,差异均有统计学意义(P<0.01);股骨颈骨密度为(0.91±0.08)g/cm2,与正常男、女性分别比较,差异均有统计学意义(P<0.01,P<0.05)。结论CAIS患者的骨密度均有不同程度的下降,尤其是腰椎,提示雌激素和雄激素在骨量的获得和维持中起重要作用。     

Androgen receptor gene mutation associated with complete androgen insensitivity syndrome and Sertoli cell adenoma.

We report a case of Sertoli cell adenoma in complete androgen insensitivity syndrome (CAIS) in a 22-year-old woman. Polymerase chain reaction-single strand conformation polymorphism and DNA sequencing revealed a single nucleotide substitution on exon 7 of the human androgen receptor (hAR) gene, resulting in a change of CGA (arginine) to CAA (glutamine) in codon 831.     

Long-term perspectives for 46,XY patients affected by complete androgen insensitivity syndrome or congenital micropenis

Controversy concerning optimal treatment for individuals affected by syndromes of abnormal sex differentiation can best be resolved with knowledge about long-term medical, surgical, and psychosexual outcomes of patients. Follow-up information has recently been gathered on older cohorts of the following patient groups: (1) those affected by complete androgen insensitivity syndrome (CAIS) raised female and (2) those affected by congenital micropenis raised male or female. As a group, women with CAIS were satisfied with their female gender and sexual function. However, a need for better patient education was identified for this specific population. Most patients with congenital micropenis, whether raised male or female, were satisfied with their gender. Regardless of sex of rearing, dissatisfaction with the appearance and function of the genitalia as judged by both physicians and subjects was evident. For patients with congenital micropenis, male sex of rearing was concluded to be optimal because genital reconstructive surgery is not required with this choice.     

A unique point mutation in the androgen receptor gene in a family with complete androgen insensitivity syndrome.

OBJECTIVE: To further delineate the diversity of genetic alterations in the gene coding for the androgen receptor in individuals with the androgen insensitivity syndrome and to increase our understanding of the disease at the molecular level. DESIGN: This was a prospective study in which genomic deoxyribonucleic acid (DNA) from individuals with androgen insensitivity were examined through the polymerase chain reaction and DNA sequencing analysis. PATIENTS: Eleven complete and four individuals with partial androgen insensitivity syndrome were examined. RESULTS: Exons two through eight were grossly intact in all study subjects. Nucleotide sequence analysis revealed that three of three related family members with complete androgen insensitivity had the same guanine to adenine base substitution in exon five of the steroid-binding domain. CONCLUSION: The subsequent alanine to threonine amino acid conversion may have resulted in a configurational change of the androgen receptor protein leading to complete androgen insensitivity. This precise alteration has not been previously identified in the human androgen receptor gene in patients with the androgen insensitivity syndrome.