PCR-HRM分析筛查成骨不全一家系患儿基因突变

【摘要】目的 采用PCR-高分辨率熔解曲线（HRM）分析筛查成骨不全（OI）一家系患儿（先证者）COL1A1基因突变位点,探讨其基因型与临床表型的联系。方法对先证者进行家族史及临床资料的调查,采集先证者、家属及 50名正常对照者血液标本,应用PCR-HRM分析筛查先证者及正常对照者COL1A1基因突变,基因测序确证突变位点。结果先证者COL1A1基因17外显子筛查结果异常,其熔解温度（Tm）值比正常对照者Tm值低约0.4℃。先证者与正常对照者的标准熔解曲线及差异熔解曲线均有明显差异。测序结果为c.1138G>A,突变导致380位氨基酸由甘氨酸（Gly）变成丝氨酸（Ser）：p.（Gly380Ser）,为错义突变。先证者父亲、祖母均具有相同突变位点。先证者母亲及正常对照者基因测序结果无此突变。该突变在中国人群中未见报道。该家系遗传特征为常染色体显性遗传,先证者临床诊断为Ⅳ型OI,临床表型较严重。结论PCR-HRM分析是有效的OI基因筛查新方法。COL1A1基因 c.1138G>A突变在中国人群中为新发现的突变位点。α螺旋结构域Gly被替换可能导致较严重的临床表型。 COL1A1     

甲状腺激素不敏感综合征三例报道及部分家系TRβ基因位点分析并文献复习

[目的] 甲状腺不敏感综合征是由于甲状腺激素受体缺陷导致甲状腺激素作用障碍,使机体组织器官对甲状腺激素敏感性降低的一种罕见疾病。发病与甲状腺激素受体β（thyroid hormone receptor β, TRβ）基因突变相关。 报道我院临床明确诊断的三例甲状腺激素不敏感综合征病例,并对部分患儿及家系进行TRβ基因突变位点分析,研究基因突变情况。[方法] 三例临床确诊病人均来自我院住院病人,进行甲状腺功能、生化、垂体核磁共振及地塞米松抑制试验,明确甲状腺激素不敏感综合征诊断。获得知情同意书的患儿及其家系进行外周血DNA的TRβ基因突变位点分析。[结果] 三例患儿均存在甲状腺肿大、心悸、多汗高代谢症候群,一例存突眼体征。甲状腺功能共同特征显示变成了天冬氨酸（GAT）,但患儿母亲没有甲状腺疾病的临床表现。第一例患儿及父母未发现TRβ基因突变位点。三例患儿均给予溴隐亭治疗,心率及甲状腺肿大症状好血FT3、FT4升高明显,TSH正常或略升高。第一例患儿及家系成员进行TRβ基因突变位点分析,发现患儿及其母亲TRβ基因第9外显子突变第1235位碱基由胞嘧啶变成腺嘌呤,致第317位密码子从丙氨酸（GCT）转,未出现溴隐亭副作用发生。[结论] 发现一例目前未报道的中国人家系新的TRβ基因突变,提示甲状腺激素不敏感综合征可由TRβ基因突变所致。发现该例患儿及其母亲的TRβ基因第9外显子具相同突变位点（A317D）,但患儿母亲并没有甲状腺功能亢进症的临床表现,说明即使基因位点突变相同,所产生的临床表型也不尽相同,提示甲状腺激素不敏感综合征具多样的临床表现,临床工作中需仔细识别。国内首次尝试应用溴隐亭治疗甲状腺不敏感综合征可以获得满意临床效果,未出现副作用的发生,为临床治疗该病提供新的选择。     

1个肯尼迪病家系临床与分子遗传学特征

目的：探讨肯尼迪病的临床特点及分子遗传学特征。方法收集1个肯尼迪病家系的临床资料,分析患者的临床表现、血清雄激素、肌酶水平、神经电生理检查结果及雄激素受体基因第1外显子的分子生物学特点。结果该家系患者均青年起病,缓慢进展,表现为肢体无力、不自主抖动及肌肉萎缩等下运动神经元损害特点,其中除先证者未婚外家族中其他患者均正常生育,先证者的弟弟肌酸肌酶水平增高,先证者及其弟弟经肌电图检查均呈神经源性损害。经基因检测先证者及其弟弟的雄激素受体的CAG基因重复次数均为50次。结论肯尼迪病主要表现为下运动神经元损害并伴有感觉障碍,基因学检测是肯尼迪病的确诊依据。     

雄激素受体基因动态突变及线粒体基因分析诊断Kennedy病1例报告

目的通过对一例肌肉活检提示线粒体肌病的患者进行雄激素受体基因（androgen receptor gene，AR）进行动态突变分析和全线粒体（mtDNA）基因扫描明确诊断。方法使用PCR方法分析患者雄激素受体（androgen receptor gene，AR）基因的CAG重复序列数，使用Touch Down PCR方法分析患者全线粒体基因突变。结果该患者雄激素受体基因第一个外显子（Xqll-12）上存在46个三核普酸（CAG）重复，确诊Kennedy病。全线粒体（mtDNA）基因分析未显示线粒体疾病的相关致病突变。结论AR基因CAG动态突变分析和全mtDNA基因扫描是KD与线粒体脑肌病鉴别诊断或KD并发线粒体脑肌病诊断的重要依据。     

Leber病两家系分析

目的 探讨Leber病的临床特征和基因诊断方法.方法 采用聚合酶链反应,对两家系2例先证者目的DNA进行扩增,并基因测序,明确突变类型.结果 2例先证者均为Leber病患者,基因突变类型为G11778A.结论 基因检测明确两家系2例Leber病患者诊断.     

全基因组外显子测序技术在遗传性纤维蛋白原异常家系致病基因的克隆鉴定中的应用

目的研究采用全基因组外显子测序技术在遗传性纤维蛋白原异常家系致病基因鉴定中的应用价值。方法采集12例遗传性纤维蛋白原异常患者及其核心家庭成员外周血检测凝血指标,并提取其基因组DNA进行全基因组外显子测序,分析测序结果,探讨遗传性纤维蛋白原异常的分子机制。结果全基因组外显子测序结果显示:先证者A1、A4以及B2均为FGA基因g.1233GA突变,A1的妹妹、A4的父亲以及B2的母亲均携带有相同的突变;A2、A7、B4和B5均为FGG基因g.10819GA突变,家系成员中A2的母亲和外婆、A7的姐姐和女儿、B4的母亲和B5的母亲均携带有相同的突变;A3、A5、A6、B1和B3及其相关亲属共10例携带有FGB基因g.9692AG突变。先证者及家系主要成员中发生Fg基因突变的成员APTT、PT和TT均明显延长,但Fg活性显著降低。结论遗传性纤维蛋白原异常可由多种Fg基因外显子突变导致,FGB和FGG外显子突变较为常见。     

颅骨锁骨发育不全家系基因多态性与表型关系的研究

目的研究家族性颅锁骨发育不全家系基因型与临床表型的关系。方法提取临床收集的一个先天性颅锁骨发育不全家系中患者和健康成员外周血基因组DNA,PCR扩增RUNX2/CBFA1基因7个编码外显子及其侧翼内含子序列,分别进行正反向测序,对发现的突变位点行酶切分析验证。结果家系中两位患者（先证者及其父亲）显示cDNA 346T→A的杂合突变,使编码的色氨酸变成精氨酸（W 116R）,属错义突变。酶切结果进一步证实了该错义突变。测序还发现先证者父亲cDNA 198G→A的杂合突变,致第66位氨基酸的密码子GCG被GCA取代,但二者均编码丙氨酸,属同义突变。先证者及家系健康成员中未见此改变。结论 RUNX2/CBFA1基因346T→A杂合突变是该家系发病的分子基础。     

伴皮层下梗死和白质脑病的常染色体显性遗传性脑病的非典型临床表现

目的 分析1例伴皮层下梗死和白质脑病的常染色体显性遗传性脑病(CADASIL)家系的基因突变及临床表现特征.方法 记录该CADASIL家系先证者及其亲属的临床表现,并对NOTCH3基因突变区行基因检测.结果 先证者为老年男性患者,先后出现偏头痛、脑梗死发作,并伴有抑郁、焦虑等情感障碍,本例家系3代中共有10人有类似临床表现,但均无明显智能减退.头颅MRI显示双侧基底节区、皮质下及脑干多发缺血梗死灶.NOTCH3基因突变分析发现14号外显子c.2182 C＞T杂合突变,本例确诊为CADASIL.结论 本例CADASIL家系临床表现与经典的CADASIL有所不同,未能发现有明确的智能损害,推测与基因突变位点的变异有关.     

13例Wilson病患者基因8号外显子778密码子碱基突变的SSCP及酶切分析

目的 寻找山东籍Wilson病患者WND基因突变热点,以便易于用SSCP及酶切分析方法检出。使无先证者及独生子女家系易于作出基因诊断,同时可用于杂合子筛查。方法 PCR扩增WND基因8号外显子778密码子区域,SSCP检洲点突变,用MSPI内切酶进一步验证G-T突变的正确性。结果 13例Wilson患者,检出778密码子G-T纯合突变2例,杂舍突变7例,未突变4例,共计26条染色体,突变染色体11条,总染色体突变率为42.3％。结论 WND基因8号外显子,778密码子G-T突变是山东WD患者的一个突变热点。SSCP和酶切分析是诊断此位点突变的便捷方法,可用于WD患者及杂合子检出。     

遗传性高铁血红蛋白一家系的基因诊断

目的 检测Ⅰ型遗传性高铁血红蛋白血症(HM)先证者(L72P,长乐)家系成员的细胞色素b5还原酶(b5R)基因的突变。方法 PCR方法扩增正常人和HM先证者之兄、父、母的b5R基因组DNA片段(含第3和第4外显子);用限制性内切酶Apa Ⅰ分析扩增产物。结果PCR扩增产物用Apa Ⅰ酶切后,正常人为649和471 bp;先证者之兄为649、440和31bp,表明存在b5R基因第72密码子纯合子突变:其父、母均为649、471、440和31bp,表明存在b5R基因第72密码子杂合子突变。结论PCR结合Apa Ⅰ内切酶分析可用于b5R基因第72密码子突变的检测;PCR-RFLP方法是检测人群中b5R基因杂合子突变的简捷有效的方法。     

Androgen receptor: structure,role in prostate cancer and drug discovery

Androgens and androgen receptors (AR) play a pivotal role in expression of the male phenotype. Several diseases, such as androgen insensitivity syndrome (AIS) and prostate cancer, are associated with alterations in AR functions. Indeed, androgen blockade by drugs that prevent the production of androgens and/or block the action of the AR inhibits prostate cancer growth. However, resistance to these drugs often occurs after 2–3 years as the patients develop castration-resistant prostate cancer (CRPC). In CRPC, a functional AR remains a key regulator. Early studies focused on the functional domains of the AR and its crucial role in the pathology. The elucidation of the structures of the AR DNA binding domain (DBD) and ligand binding domain (LBD) provides a new framework for understanding the functions of this receptor and leads to the development of rational drug design for the treatment of prostate cancer. An overview of androgen receptor structure and activity, its actions in prostate cancer, and how structural information and high-throughput screening have been or can be used for drug discovery are provided herein.     

反复肺部感染的X-连锁无丙种球蛋白血症诊疗与基因报告分析

目的提高医务人员对X-连锁无丙种球蛋白血症的认识,防范此类病人可能并发的疾病,指导对该类病人的防治宣教。方法对2020年1月西南医科大学附属中医医院收治的1例长期反复呼吸道感染伴血清IgA、IgG、IgM明显降低的免疫缺陷青年病人采用芯片捕获高通量测序方法检测其全血,并对其检测结果进行分析解读。结果检测报告提示在受检者中检出Bruton酪氨酸激酶(BTK)基因的致病突变,从而导致无丙种球蛋白血症,为X染色体隐性遗传。结论该病人长期反复呼吸道感染和免疫球蛋白低下与BTK基因致病突变有密切关系,其相应核苷酸变化c.763C>T(p.Arg255Ter)为首次报道,对其父母和子女都应进一步开展免疫功能筛查和生活宣教。     

雄激素受体基因CAG重复序列多态性与男性急性心肌梗死的关系

目的:探讨男性雄激素受体(AR)基因CAG重复序列多态性与急性心肌梗死(AMI)的关系.方法:选择112例男性AMI患者(AMI组)和118例健康男性体检者(对照组),采用聚合酶链反应(PCR)和直接测序法测定其外周血AR基因CAG重复序列的长度.结果:AMI组和对照组CAG重复序列长度范围为12～33,平均值分别为23.09±3.52和20.33±3.65,2组间的差异有统计学意义(t=5.826,P＜0.01).与CAG重复序列长度＜23相比,≥23者发生急性心肌梗死的危险性增加了4.391倍,差异有统计学意义(P＜0.01).结论:男性AR基因CAG重复序列是急性心肌梗死的危险因素.     

DNA 聚合酶基因在范可泥贫血症家系突变状态中的影响研究

目的：通过对范可泥贫血症（ fanconi anemia, FA）家系DNA聚合酶基因突变分析,初步观察其稳定突变位点,为上述疾病易感基因风险突变位点的进一步确定奠定基础。方法 FA家系15例成员作为研究对象对家系先征者DNA聚合酶基因家族POLA、POLB、POLD1、POLD2、POLE、POLG测序,检测突变位点,并分析筛选有义突变位点;对家系成员在先征者有义突变区域直接测序。结果 FA患儿DNA聚合酶的外显子共有四个突变位点。其中, POLD119p13殮．3-13．4 EXON250403975 G＞A,密码子由CGU变为CAU,氨基酸由精氨酸变为组氨酸;POLE112p24．3 EXON43-44132688389 A＞G,密码子由GAG变为GGG,编码氨基酸由谷氨酸变为甘氨酸;家系中患儿的母亲、外祖父发生POLD1 EXON250403975 G＞A;家系中患儿的外祖母、外祖父、祖母、父亲、母亲、二伯、四伯、大姑、二姑、两个堂姐均发生了POLE1 EXON43-44132688389 A＞G。结论通过对FA家系成员在上述已经筛选出的两个突变位点进行测序,该家族成员广泛携带这两个突变位点。这两个基因的突变位点应得到足够的重视,其可能为FA疾病的稳定突变位点。     

Cellular and molecular mechanisms of action of linuron: an antiandrogenic herbicide that produces reproductive malformations in male rats.

Antiandrogenic chemicals alter sex differentiation by several different mechanisms. Some, like flutamide, procymidone, or vinclozolin compete with androgens for the androgen receptor (AR), inhibit AR-DNA binding, and alter androgen-dependent gene expression in vivo and in vitro. Finasteride and some phthalate esters demasculinize male rats by inhibiting fetal androgen synthesis. Linuron, which is a weak competitive inhibitor of AR binding (reported Ki of 100 microM), alters sexual differentiation in an antiandrogenic manner. However, the pattern of malformations more closely resembles that produced by the phthalate esters than by vinclozolin treatment. The present study was designed to determine if linuron acted as an AR antagonist in vitro and in vivo. In vitro, we (1) confirmed the affinity of linuron for the rat AR, and found (2) that linuron binds human AR (hAR), and (3) acts as an hAR antagonist. Linuron competed with an androgen for rat prostatic AR (EC(50) = 100-300 microM) and human AR (hAR) in a COS cell-binding assay (EC(50) = 20 microM). Linuron inhibited dihydrotestosterone (DHT)-hAR induced gene expression in CV-1 and MDA-MB-453-KB2 cells (EC(50) = 10 microM) at concentrations that were not cytotoxic. In short-term in vivo studies, linuron treatment reduced testosterone- and DHT-dependent tissue weights in the Hershberger assay (oral 100 mg/kg/d for 7 days, using castrate-immature-testosterone propionate-treated male rats; an assay used for decades to screen for AR agonists and antagonists) and altered the expression of androgen-regulated ventral prostate genes (oral 100 mg/kg/d for 4 days). Histological effects of in utero exposure to linuron (100 mg/kg/d, day 14-18) or DBP (500 mg/kg/d, day 14 to postnatal day 3) on the testes and epididymides also are shown here. Taken together, these results support the hypothesis that linuron is an AR antagonist both in vivo and in vitro, but it remains to be determined if linuron alters sexual differentiation by additional mechanisms of action.     

Immunisation with a Novel Hepatitis B Vaccine

The most critical problem in the treatment of prostate cancer is the emergence of hormone-refractory tumour during hormonal therapy. Although the vast majority of patients initially have an excellent response to androgen withdrawal, disease will often return later. There is no effective treatment for such hormone-refractory prostate cancer. Despite the fact that the mechanisms leading to transition from androgen-dependent to androgen-independent disease have been extensively studied, they have mostly remained unclear. It was earlier considered that the growth of hormone-refractory tumour is independent of androgens and androgen receptor (AR) signalling pathways. Recently, however, several studies have indicated that aberrations in the AR signalling pathway could actually be key mechanisms in androgen independence. For example, the revelations of AR gene amplification, mutations and cross-talk with the ERBB2 pathway suggest that AR is still operational in hormone-refractory tumours. Thus, AR is a putative new-old target for novel treatment modalities.     

Learning from estrogen receptor antagonism: structure-based identification of novel antiandrogens effective against multiple clinically relevant androgen receptor mutants

Current treatment strategy for advanced prostate cancer is to suppress androgen receptor (AR) by castration and antiandrogens. However, several clinically relevant AR mutations cause insensitivity to current antiandrogens and convert them into agonists. We aim to identify full AR antagonists even for AR mutants. As crystal structure of AR ligand-binding domain (LBD) at antagonistic form is not available, we decided to learn from estrogen receptor (ER) antagonism: (i) We built a structural model of wild-type AR-LBD complexed with antiandrogen bicalutamide (wild type/bicalutamide) using ERα-LBD/hydroxytamoxifen structure as the template for helix-12. (ii) By comparative structural analysis of 24 ERα-LBD complexes, we found residues D351 and L354 at helix-3 adopt unique conformations, and distance between them is a marker of ERα-LBD/antagonist complexes. The AR residues corresponding to D351 and L354 are E709 and L712, respectively. We found distance between E709 and L712 of the wild type/bicalutamide model is substantially different from that of AR-LBD/agonist complexes, suggesting this distance could be a marker of antagonistic AR-LBD, which was supported by molecular dynamics simulations. Based on the wild type/bicalutamide model, we discovered compound 3 is a novel antiandrogen effective against the wild type and T877A-, W741C-, and H874Y-mutated androgen receptors. We found compound 3 has dual functions, inhibiting androgen receptor and IKK(β) .