X连锁显性遗传Alport综合征女性患者临床表型与不同组织X染色体失活方式的关系

目的探讨X连锁显性遗传Alport综合征(XLAS)女性患者临床表型的差异与不同组织X染色体失活方式的关系。方法以确诊的36例XLAS女性患者为研究对象,以尿蛋白作为判断临床表型严重程度的指标,检测所有患者外周血和其中12例患者皮肤组织的X染色体失活方式。采用限制性内切酶HpaⅡ酶消化后PCR扩增雄性激素受体基因第1外显子CAG重复序列多态性的方法检测X染色体失活。结果随着尿蛋白水平的增加,XLAS女性患者外周血中COL4A5致病等位基因所在X染色体失活比例的平均值降低,二者呈负相关(r=-0.543,P=0.002);XLAS女性患者尿蛋白水平与皮肤成纤维细胞中COL4A5致病等位基因所在X染色体失活比例无相关性(r=-0.131,P=0.701)。结论X染色体的失活方式也许能解释XLAS女性患者的临床表型差异,通过分析外周血中X染色体失活方式也许能够早期预测XLAS女性患者的预后。     

X连锁显性遗传Alport综合征的产前基因诊断

目的Alport综合征（As）是最常见的遗传性肾脏疾病,预后较差,无特异有效的治疗。x连锁显性（XLAS）是其主要遗传方式,因COL4A5基因突变或COL4A5和COL4A6两个基因突变所致。通过对两个XLAS家系进行遗传咨询和基因诊断,并对其中一个家系实施产前基因诊断,探讨AS产前基因诊断的方法及临床应用价值。方法对两个XLAS家系进行详细的遗传咨询后,采用巢式PCR扩增外周血淋巴细胞COL4A5 mRNA的整个编码区序列筛查两个家系的基因突变,然后扩增COMA5相应的外显子从基因组DNA水平进一步证实突变。产前基因诊断通过羊水细胞cDNA和DNA两个水平来检测胎儿突变情况,通过PCR扩增Y染色体性别决定基因SRY联合核型分析检测胎儿性别,通过三个x染色体微卫星标记（AR、DXS6797和DXS6807）连锁分析除外羊水中母体细胞污染的可能。结果突变筛查显示家系一为新发突变,先证者COL4A5第2696～2705位缺失GTATGATGGG共10个碱基,但母亲外周血基因组DNA不携带该缺失突变,通过遗传咨询,该家系未进行羊水穿刺和产前基因诊断。家系二COL4A5基因第4271位G被A取代,1424位G〉E,家系其他患者包括孕母均携带该突变。对家系二孕母进行了羊水穿刺和产前基因诊断。羊水细胞cDNA和DNA水平检测均显示胎儿无突变,PCR扩增SRY基因和羊水细胞核型分析均显示胎儿为男性。连锁分析显示羊水细胞中无母体细胞污染,且间接提示胎儿从母体遗传了携带正常COL4A5等位基因的x染色体。结论基于外周血淋巴细胞cDNA水平的COL4A5基因突变检测技术可快速用于XLAS产前基因诊断时家系基因突变筛查,有利于客观进行遗传咨询;产前基因诊断时联合eDNA和DNA两个水平检测胎儿基因突变情况更准确可信,PCR扩增SRY基因可以快速鉴定胎儿性别,x染色体微卫星标记连锁分析不但可以排除羊水中母体细胞污染,而且可以间接证明胎儿是否携带致病等位基因。     

X连锁Alport综合征女性患者临床表型差异的可能机制

Alport综合征(Alport syndrome,AS)是以血尿、感音神经性耳聋和进行性肾功能减退为临床特点的遗传性肾脏疾病,X连锁显性遗传(X-linked Alport syndrome,XLAS)为其主要遗传方式,因COL4A5和(或)COL4A6基因突变所致。X连锁Alport综合征女性患者临床表型差异很大,轻者无症状或仅表现为镜下血尿,重者有慢性肾功能衰竭,尤其是来自同一家系的女性患者临床表型可以明显不同,这种现象不能完全用COL4A5基因突变类型来解释。近年来,研究显示XLAS女性患者临床表型的差异与COL4A5突变mRNA及基底膜a5(Ⅳ)链的表达量相关,而COL4A5突变mRNA及基底膜a5(Ⅳ)链的表达量不同的机制可能与X染色体失活有关,其他表观遗传学调控方式也可能参与其中。该文就X连锁Alport综合征女性患者临床表型差异的可能机制进行了文献综述。     

Rett综合征X染色体失活方式及失活X染色体起源研究

目的研究Rett综合征（RTT）患儿和其母亲X染色体失活（XCI）方式,患儿失活X染色体来源,探讨RTT XCI与基因型、表型和遗传方式之间的关系。方法对55例RTT患儿、53例RTT患儿的母亲、48例正常女性对照,提取其周围血白细胞DNA,经甲基化敏感性限制性核酸内切酶HpaⅡ消化,对消化前后雄激素受体（AR）基因片段行PCR扩增和基因扫描分析。结果RTT患儿、RTT患儿母亲、正常对照AR基因片段杂合率分别为82％、77％、83％。RTT患儿XCI分布方式与患儿母亲及对照组相比,差异有统计学意义（P〈0．05）,RTT患儿母亲与正常对照组相比,差异无统计学意义（P〉0．05）。与患儿母亲及对照组相比,RTT患儿在50：50～59：41区段内患儿人数显著减少,差异有统计学意义（P〈0．05）。RTT患儿非随机失活在XCI≥65：35和≥80：20范围与母亲及对照组相比人数增多,但差异无统计学意义（P〉0．05）。在21例非随机失活的患儿中,18例倾向于父源X染色体失活,占85．7％,3例倾向于母源。在同一突变位点可以观察到各种XCI方式,T158M高度非随机失活率略高,R133C无高度非随机失活。非典型RTT比典型RTT高度非随机失活率增高,其中保留语言型与先天型相比,高度非随机失活率略高。结论RTT患儿XCI分布方式与及母亲及对照组相比,完全随机失活人数显著减少,但在非随机失活人数上差异无统计学意义。母亲作为携带者传递致病基因不是RTT主要的遗传方式。RTT患儿非随机失活的X染色体主要起源于父亲。RTT患儿的XCI方式与临床表型有一定相关性,但XCI并不能完全解释表型。     

X-连锁迟发性脊椎骨骺发育不良基因逃避X染色体失活与临床表型的关系

目的探讨X-连锁迟发性脊椎骨骺发育不良(SEDL)基因逃避X染色体失活(XCI)及与临床表型的关系。方法从SEDL基因剪接受体突变(IVS2-2A→C)所致SEDL患者、女性致病基因携带者和健康对照者外周血中提取总RNA,进行RT-PCR.对扩增产物应用非变性聚丙烯酰胺凝胶电泳(PAGE)进行分析。结果SEDL患者和对照者分别存在2种RT-PCR产物,女性携带者扩增出上述4种转录物。家系凋查未发现该家族女性携带者有任何临床表现。结论首次从人体细胞证实SEDL基因逃避XCI。女性致病基因携带者不发病可能与该基因逃避XCI有关。     

分子遗传学技术诊断MECP2重复综合征四例分析北大核心CSCD

目的 用多重连接探针扩增技术（MLPA）和微阵列比较基因组杂交技术（array-CGH）研究4例以运动、智力发育落后为主要表现的患儿甲基化CpG结合蛋白2基因（MECP2）基因突变特点.方法 取北京大学第一医院2012年6月至2014年4月收治的4例患儿及其中例2、例4母亲的外周血,提取基因组DNA;先对患儿用MLPA方法进行微缺失和微重复检测,然后用array-CGH进行分析进一步确定重复片段的大小;同时对2例患儿的母亲进行array-CGH和X染色体失活分析（XCI）.结果 4例患儿均表现为严重肌张力低下,运动、智力发育落后和语言发育障碍,除例2之外,另3例患儿婴儿期均反复发生肺炎.MLPA显示4例患儿均存在染色体Xq28重复;array-CGH检测显示4例患儿Xq28区域存在重复,4例患儿重复片段大小分别为14.931 Mb、0.393 Mb、0.482 Mb、0.299 Mb,经与UCSC（http：//genome.ucsc.edu/）数据库比对,4例患儿的重复片段均包含MECP2和宿主细胞因子C1基因（HCFC1）.例2和例4患儿的母亲存在Xq28重复,其中例4患儿母亲的重复片段起止位点和大小与患儿完全相同,例2母亲重复片段为0.343 Mb,小于患儿,近着丝粒断点与患儿不同,远端断点与患儿相同.X染色体失活分析发现母亲二条X染色体活性比例为0∶100,存在重复的一条X染色体完全失活,并且将发生重复的这条X染色体遗传给了患儿.结论 对于运动智力发育落后、肌张力低下、语言发育障碍和反复发生感染的患儿进行MLPA和array-CGH联合检测是诊断MECP2重复综合征的有效且特异的方法.     

Alport综合征的产前基因诊断

Alport综合征（AS）是最常见的遗传性。肾脏疾病,临床主要表现为血尿和进行性肾功能减退,伴随感音神经性耳聋和眼部异常等。目前已证实AS存在三种遗传方式：X连锁显性遗传（XLAS）、常染色体隐性遗传（ARAS）及常染色体显性遗传（ADAS）。其中,XLAS最常见,约占80％～85％,因COL4A5基因突变或COL4A5和COL4A6两个基因突变所致。ARAS约占AS的15％,因COL4A3或COL4A4基因突变所致。[第一段]     

发根脆性X智力低下蛋白检测法诊断脆性X综合征

目的：至今已有多种筛查和诊断脆性X综合征(fragile X syndrome,FXS)的方法,以PCR法和Southern印迹方法应用最广,然而每种方法均存在各自的局限性。该研究探讨发根脆性X智力低下蛋白（fragile X mental retardation protein,FMRP）检测在诊断或筛查FXS中的可靠性,以建立一种快速、简便、价廉且可靠的诊断FXS的方法。方法：采用发根FMRP免疫组化的检测方法对80例健康儿童、40例不明原因智力低下儿童、已确诊FXS家系成员12例进行检查; 用7-deza-dGTP PCR 法进行对照,探讨其对诊断FXS的应用价值。结果：在80例健康儿童中,发根FMRP的表达率均在80%以上。40例不明原因智力低下患儿中,2例确诊为FXS患儿的发根FMRP表达率分别为10%和0,另38例非FXS患者发根FMRP的表达率均在80%以上。在FXS家系调查中,确诊的2例FXS患者的发根FMRP表达率均为0。结论：发根FMRP检测诊断FXS具有快速、简便、价廉、可靠等特点,值得进一步推广应用。［中国当代儿科杂志,2009,11（10）：817-820］     

良性家族性婴儿癫痫两高发家系临床特征及染色体研究

探讨两癫痫高发家系的遗传方式、临床特点、脑电图及染色体情况。方法采用系谱分析其遗传方式，分别分析发病年龄、发作方式、治疗及预后、身心发育情况、脑电图及实验室检查结果。检查其染色体核型及姊妹染色体交换情况。结果两家系遗传方式均为常染色体显性遗传。39例患者中的29例发病年龄在出生后3个月～1岁。家系Ⅰ的患者主要失神发作（17／23人），家系Ⅱ的患者以部分发作为主（11／16人）。两家系中33例患者（包括20例未接受抗癫痫治疗者）发作于3～5岁时消失。4例脑电图轻度异常．其他人均正常。9例采用美解眠诱发的患者．2例出现癫痫波。体检及神经系统检查、实验室检查、智能测验、染色体数目及形态均正常，家系Ⅰ姊妹染色体互换率（SCE），高于对照组（P＜0．05），家系ⅡSCE与对照组间无显著性差异（P＞0．05）。结论两家系患者的临床特征及辅助检查结果符合良性家族性婴儿癫痫的诊断标准，其特点为婴儿期首发的无热惊厥，身心发育正常．脑电图检查发作间期正常，有类似发作的家族史。     

1个X-连锁鱼鳞病家系的基因突变鉴定与产前诊断

X-连锁鱼鳞病 （XLI）是一种类固醇硫酸酯酶缺乏的代谢性疾病,常于出生时或生后不久发病,编码类固醇硫酸酯酶的基因 （STS）位于X染色体短臂上,STS基因发生缺失或突变时可导致此病的发生。本研究收集一个家系的临床表型资料,其中先证者,男,足月顺产,11岁,全身皮肤干燥、粗糙、呈黑褐色鳞片状,主要累及腹部和肢体伸侧。采集家系中各成员的外周血提取DNA,采用多重连接依赖式探针扩增 （MLPA）技术对家系各成员的X染色体上的STS基因拷贝数进行检测,用全基因组芯片进一步明确X染色体微缺失片段的大小,随后采用MLPA技术对先证者母亲再生育进行产前诊断。结果发现家系中先证者及2个患者均为STS缺失的男性半合子,基因芯片鉴定出Xp22.31存在缺失,缺失大小为1.6Mb （chrX：6,516,735-8,131,442）,另鉴定出2个女性家庭成员为携带者。先证者母亲再生育产前诊断结果证实胎儿为携带者。本研究表明该XLI家系存在STS基因缺失,该缺失引发出XLI特有的皮肤病变,MLPA是XLI分子诊断与产前诊断的便捷可靠技术。     

Alport综合征的诊治研究进展

Alport综合征(Alport syndrome,AS)是一种以血尿、进行性肾功能减退,常伴有神经性耳聋和眼部病变为临床特征的遗传性肾小球基底膜疾病,其发病机制为编码Ⅳ型胶原基因突变.AS的诊断需结合临床表现、肾脏病理改变和免疫荧光学检查及基因诊断等方面综合判断.目前AS尚无根治措施.随着AS发病机制的不断明确,基因...     

X-chromosome inactivation and human genetic disease

The inactivation of one X-chromosome in females in early development is the process by which the effective dosage of X-linked genes is equalized between XX females and XY males. The mechanism that brings this about is the subject of intense research. The X-linked gene Xist is a key player, which is necessary but not sufficient for the initiation of X-inactivation. It codes for an untranslated RNA that coats the inactive X-chromosome, which takes on properties characteristic of heterochromatin, but how this change in chromatin is brought about remains unknown. Because of X-inactivation, females heterozygous for X-linked genes are mixtures of two types of cells and show a variable phenotype. The proportion s of the two types of cells can depart from equality due to cell selection either at the tissue or whole organism level. In rare cases, changes in the Xist gene can cause skewing of X-inactivation. A few genes escape from X-inactivation either wholly or partially.
Conclusion : X-chromosome inactivation is a physiological mechanism that equalizes gene-dosage effects on the sex chromosomes. The occurrence of this normal process affects the phenotype seen in females carrying X-linked mutant genes or chromosome anomalies.     

X-chromosome inactivation and human genetic disease

The inactivation of one X-chromosome in females in early development is the process by which the effective dosage of X-linked genes is equalized between XX females and XY males. The mechanism that brings this about is the subject of intense research. The X-linked gene Xist is a key player, which is necessary but not sufficient for the initiation of X-inactivation. It codes for an untranslated RNA that coats the inactive X-chromosome, which takes on properties characteristic of heterochromatin, but how this change in chromatin is brought about remains unknown. Because of X-inactivation, females heterozygous for X-linked genes are mixtures of two types of cells and show a variable phenotype. The proportions of the two types of cells can depart from equality due to cell selection either at the tissue or whole organism level. In rare cases, changes in the Xist gene can cause skewing of X-inactivation. A few genes escape from X-inactivation either wholly or partially. CONCLUSION: X-chromosome inactivation is a physiological mechanism that equalizes gene-dosage effects on the sex chromosomes. The occurrence of this normal process affects the phenotype seen in females carrying X-linked mutant genes or chromosome anomalies.     

91例儿童Alport综合征临床、病理特点及误诊分析

目的探讨儿童Alport综合征(AS)临床、病理特点和诊治情况,以提高对AS的认识。方法收集确诊的91例AS患儿临床资料进行回顾性分析。结果 91例患儿均有血尿,86例伴有蛋白尿。61例X连锁显性遗传AS(XL-AS)患儿有阳性家族史。肾活检的82例患儿中74例有轻度或轻-中度系膜增生,48例系膜区少量免疫复合物,53例肾小球基底膜(GBM)有变薄、增厚和撕裂。63例进行了肾组织Ⅳ型胶原α3、α5链免疫荧光检测,确诊AS 58例,其中53例符合XL-AS,5例符合常染色体隐性遗传AS。91例AS患儿中,58例通过肾组织Ⅳ型胶原α3、α5链免疫荧光确诊,21例通过电镜确诊,1例通过皮肤活检确诊;12例基因诊断确诊。发现6个COL4A5基因新突变。45例曾被误诊其他疾病,其中41例接受过激素和/或免疫抑制剂治疗。结论儿童AS临床表现缺乏特异性,特征性GBM电镜改变仅见于部分患儿,本区域儿童AS误诊误治率仍较高。COL4A5基因新突变比例较高。     

Clinical and pathological characteristics of Alagille syndrome in Chinese children

Background  Alagille syndrome (AS) is regarded as the most common cause of chronic cholestasis in childhood associated with specific phenotypic features in western countries. This study was undertaken to investigate the significance of AS in Chinese children with chronic cholestasis and to describe its clinical and histological features. Methods  From October 2004 to January 2007, 157 children who presented with conjugated jaundice from less than 3 months of age were admitted to a tertiary hospital in Shanghai. Investigations of the heart, spine, eyes and kidneys were conducted in 13 children who experienced prolonged cholestasis beyond 1 year of age after exclusion of biliary atresia and familial progressive intrahepatic cholestasis type 1 or 2. In patients with interlobular bile duct paucity, AS was diagnosed if 3 or more of the following 5 major features were present: cardiac murmur, posterior embryotoxon, butterfly-like vertebrae, renal abnormalities and characteristic faces. In patients without interlobular bile duct paucity or who did not receive liver biopsy, 4 or more features were required for the diagnosis. Results  Of the 13 children, 6 were diagnosed with AS at ages ranging from 1 year and 7 months to 3 years and 11 months. Jaundice was noticed in early infancy and then pruritus developed in all the 6 patients, of whom 5 presented with acholic stool and 4 had been misdiagnosed as having presumed biliary atresia by hepatobiliary scintigraphy or laparoscopic cholangiography. Biochemical examinations demonstrated increased concentration of total bile acid and hyperlipidemia. Interlobular bile duct paucity was demonstrated histologically in 5 patients who received liver biopsy. Vertebral abnormalities, heart murmur, characteristic faces and failure to thrive were found in all the 6 patients. Two patients had evidence of renal involvement. Micropenis, empty scrotum, and gall stone were seen in 1 patient. Conclusion  AS is also an important cause of prolonged cholestasis in Chinese children. It is difficult to differentiate AS from biliary atresia. Liver biopsy and spine X-ray may be helpful in the early detection of AS.     

Evolution of renal pathology in Fabry disease

Fabry disease is a rare lysosomal storage disorder which results from deficient activity of the enzyme α-galactosidase A. The resultant deposition and progressive accumulation of glycosphingolipids in all types of body tissue leads to severe clinical manifestations involving the heart, CNS and kidney. Renal manifestations are observed relatively early in the course of the disease, and progression to end-stage renal failure is common in hemizygous males in the third to fifth decades of life.
Renal biopsy specimens reveal evidence of diffuse intracytoplasmic glycosphingolipid accumulation, mainly affecting podocytes and epithelial cells of distal tubules, which are strikingly enlarged and vacuolated. On electron microscopy the deposits appear as typical osmiophilic inclusion bodies in the cytoplasm of all kinds of renal cells, and show a characteristic 'onion skin' or 'zebra' appearance. These pathological features are also evident in heterozygous females. Deposits occur before the development of renal impairment. As patients age, the disease progresses in cells throughout the kidney, and is associated with increasing glycosphingolipid accumulation.
Conclusion : The age-related evolution of renal pathology in Fabry disease is closely correlated with progressive intracellular deposition of glycosphingolipid and ultimately leads to end-stage renal failure.     

Evolution of renal pathology in Fabry disease

Fabry disease is a rare lysosomal storage disorder which results from deficient activity of the enzyme alpha-galactosidase A. The resultant deposition and progressive accumulation of glycosphingolipids in all types of body tissue leads to severe clinical manifestations involving the heart, CNS and kidney. Renal manifestations are observed relatively early in the course of the disease, and progression to end-stage renal failure is common in hemizygous males in the third to fifth decades of life. Renal biopsy specimens reveal evidence of diffuse intracytoplasmic glycosphingolipid accumulation, mainly affecting podocytes and epithelial cells of distal tubules, which are strikingly enlarged and vacuolated. On electron microscopy the deposits appear as typical osmiophilic inclusion bodies in the cytoplasm of all kinds of renal cells, and show a characteristic 'onion skin' or 'zebra' appearance. These pathological features are also evident in heterozygous females. Deposits occur before the development of renal impairment. As patients age, the disease progresses in cells throughout the kidney, and is associated with increasing glycosphingolipid accumulation. CONCLUSION: The age-related evolution of renal pathology in Fabry disease is closely correlated with progressive intracellular deposition of glycosphingolipid and ultimately leads to end-stage renal failure.     

Screening System for Asymptomatic Renal Disease in Children in Japan

The introduction in Japan of routine urinalysis for pre-school and school age children has greatly facilitated the discovery of renal disease in asymptomatic children. Over the past 8 years at Kitasato University Hospital, we have studied 113 cases of chronic progressive renal disease based on renal biopsy.
Thirty-one of these 113 patients were found by routine urinalysis at area schools. It is noteworthy that many of the asymptomatic children were found to have renal diseases such as MPGN, FGS, and IgA nephropathy.Although it is difficult to decide whether or not medical management is required for asymptomatic children, the histological findings may give pertinent guidance for planning an appropriate therapeutic program in the early stages of various types of renal disease. This early detection may enable us to observe these potentially progressive renal diseases from the early phase and to provide clues for the investigation of the pathogenesis of these renal diseases.     

Effect of metformin and rosiglitazone in a prepubertal boy with Alström syndrome

Alstr?m syndrome (AS) is an autosomal recessive disorder characterized by progressive pigmentary retinopathy, sensorineural hearing loss, fatty liver infiltration, obesity, insulin resistance and early-onset type 2 diabetes mellitus (DM2). Early onset of insulin resistance and DM2 are key components of this syndrome. AIM: To study the effect of early initiation of the insulin sensitizer metformin combined with rosiglitazone in a patient with AS with impaired glucose tolerance. PATIENT: An 8 year-old boy with AS presented with acanthosis nigricans and insulin resistance at the age of 6 years. He had progressive excessive weight gain from 9 months of age. By the age of 1 year he developed photosensitivity, blindness and nystagmus. At the age of 5.5 years, his body mass index (BMI) was above the 95th percentile. He developed impaired glucose tolerance at 6 years of age and treatment with metformin was initiated. After 8 months of treatment with metformin he developed DM2. The dose of metformin was increased, and rosiglitazone added. METHODS: A 2-hour oral glucose tolerance test (OGTT) and a rapid intravenous glucose tolerance test (IVGTT) were performed before treatment was initiated, after treatment with metformin and at the end of 1 year of combination therapy with metformin and rosiglitazone to calculate quantitative insulin sensitivity check index (QUICKI) and acute insulin response (AIR). For mutation analysis, all exons and splice site sequences of the ALMS1 gene were amplified and sequenced. RESULTS: Metformin treatment alone at the stage of impaired glucose tolerance did not prevent progression to DM2. However, metformin at a higher dose and in combination with rosiglitazone resulted in improvement of pancreatic beta-cell function, shown by markedly improved first-phase insulin response to glucose measured by AIR. The patient was found to have two heterozygous nonsense mutations in ALMS1, 8008 C-->T Ter, R2670X, and 11449 C-->T Ter, Q3817X. These alterations cause premature stops and result in a truncated ALMS1 protein. CONCLUSION: We suggest that early initiation of combined therapy comprising a high dose of metformin plus rosiglitazone may be valuable in managing insulin resistance and DM2 in children with AS.