地成人型多囊肾病家系成员进行症前基因诊断

目的 探讨对中国成人型囊肾病（ＡＰＫＤ）家系成员进行症前基因诊断的方法。方法 应用ＰＣＲ技术扩增与ＰＫＤＩ位点紧密连锁的微小卫星体ＤＮＡ遗传传标记，基因连锁的家系分析。结果 对一个ＡＰＫＤ家系中的无临床症，且Ｂ超检查呈阴性结果的６个孩子做了症前基因诊断；５个孩子（５－１５岁）携带ＰＫＤＩ基因；仅１个１１岁女孩是正常的个体。结论 能够应用ＰＣ译中国的成人型多囊肾病家系成员快速，准确地做出症前基因诊断     

成人型多囊肾病的基因诊断

应用人１６号染色体上α－珠蛋白基因３’ＨＶＲ（Ｄ１６ｓ８５）为探针，对三个成人型多囊肾病（ＡＰＫＤ）家系进行限制性片段长度多态性（ＲＦＬＰ）连锁分析，结果三个家系均对α－珠蛋白基因３’ＨＶＲ提供信息，并对家系３中一成员成功地进行了症状前诊断，确定该成员（１０岁）携带致病基因的风险为５％。说明α－珠蛋白基因３’ＨＶＲ探针可用于中国人群中ＡＰＫＤ的症状前诊断和产前诊断，同时证明此法是目前成人型多囊肾病症状前诊断和产前诊断最有效的手段。     

基因连锁分析评估成人型多囊肾的B超早期改变

成人型多囊肾 ,即常染色体显性多囊肾 (ADPKD) ,发病率约为 1/ 10 0 0 ,是最常见的遗传性疾病之一 ,多于 4 0岁以后出现临床症状。早期诊断 ,防止并发症的发生与发展 ,可减缓疾病进展。由于基因诊断难以普遍开展 ,我们试图在基因诊断基础上 ,总结早期患者的B超改变。现报告如下。材料与方法　 10个成人型多囊肾家系 ,共 95例 ,其中 <30岁者 4 6例 ,要求每个家系至少有两代人及两名以上先证者。家系成员抽静脉血 0 .5ml,提取基因组DNA ,TE溶解 ,- 2 0℃保存备用。合成三对引物 :SM6、CW 2、CW 4。PCR扩增 ,产物行聚丙烯酰胺凝胶电泳 …     

成人多囊肾病的临床特征及预后

成人多囊肾病(APKD)是常染色体显性遗传性疾病,其表现各样,可从出生时即出现囊性肾直至>80岁大多数患者常于50-60岁发现,约有25%发展至终未期肾病(ESRD)。作者对136例APKD患者作回顾性研究。APKD的诊断,为尿路造影或超声检查证实双侧有多囊改变。并有常染色体显性遗传相关疾病的家族史。在无症状患者,每个肾至少有3个囊,方可诊断APKD。平均随访6年3个月(范围2～10年)。37例患者在失去随访前观察2～9年(平均4年9个月)。慢性肾衰(CRF)为血清肌酐(Scr)>1.5mg/dl。所有患者从出生起都有发展至ESRD的危险,2例死于CRF。高血压为收缩压或舒张压分别>150mmHg或100mmHg。结果:107例患者(58例男性,49例女性)发病率     

64个常染色体显性多囊肾病家系的基因检测及产前诊断

目的检测和分析64个无亲缘关系的常染色体显性多囊肾病(autosomal dominant polycystic kidney disease, ADPKD)家系的基因变异类型, 探讨多种基因分析技术的检测效能和基因变异特点。方法该研究为横断面研究, 回顾性分析2017年12月至2020年8月就诊于郑州大学第一附属医院肾脏内科或遗传与产前诊断中心的64个ADPKD家系的临床资料, 采集先证者和家系成员血样, 应用二代测序对先证者进行基因检测, 对筛选出的可疑变异通过多重连接依赖式探针扩增或长片段PCR结合Sanger测序进一步验证。抽取高风险家系胎儿绒毛或羊水样本, 排除母源污染后进行产前基因诊断。结果 64个ADPKD家系中有57个家系(89.06%)检测到PKD1/PKD2基因变异, 其中51个家系(79.69%)共检出49种PKD1/PKD2基因致病性/可能致病性变异, 包含14种(28.57%)无义变异、14种(28.57%)移码变异、11种(22.45%)错义变异、5种(10.20%)剪接变异和5种(10.20%)缺失变异, PKD1和PKD2基因变异分别占87.76%(43...     

SM7微卫星标记快速多态分析行Ｉ型常染色体显性遗传多囊肾？…

应用聚合酶链反应（ＰＣＲ）技术对４个Ｉ型常染色体显性遗传多囊肾病（ＡＤＰＫＤ Ｉ）家系２４名个体的ＳＭ７（一种与ＡＤＰＫＤ Ｉ基因紧密连锁的微卫星标记）多态性进行连锁分析，其中３个家系得到了明确结果，ＰＣＲ扩增条带清晰，致病基因连锁关系明确。认为本法是一种快速、廉价、有效的ＡＤＰＫＤ Ｉ基因诊断方法。     

Apolipoprotein E Guangzhou(arginine 150 proline):一种新的脂蛋白肾病相关的基因突变

目的通过分析1个包含4个脂蛋白肾病患者的家系所有成员的载脂蛋白E (apoE)基因,寻找脂蛋白肾病相关的apoE基因突变。方法收集该家系全部成员的病史,所有成员进行尿常规、血肌酐、血脂、血清脂蛋白检查。抽提基因组DNA,PCR法扩增所有成员的apoE第4外显子。割胶纯化PCR产物后直接测序,并将纯化的PCR产物亚克隆至pMD 18-T载体,挑菌落测序。结果4例脂蛋白肾病患者和1例无症状直系亲属均携带一种新的apoE点突变的杂合子,150号密码子发生点突变:CGC→CCC(由精氨酸变成脯氨酸),伴有血浆apoE的升高。结论apoE点突变apoE Guangzhou(arginine 150 proline)可能是该家系脂蛋白肾病发病的原因。     

四例脂蛋白肾病患者载脂蛋白E基因突变筛查

目的 通过对4例脂蛋白肾病患者及家系成员载脂蛋白E(apoE)基因的分析， 研究脂蛋白肾病的发病机制。 方法 调查患者家系情况，对其中2例患者的家系成员进行尿常规筛查及血肌酐、血脂和血清脂蛋白的检测。PCR法扩增4例患者apoE基因的外显子， DNA测序， 发现突变后，寻找限制性内切酶酶切位点。使用聚合酶链反应-限制性酶切片段长度多态性(PCR-RFLP)的方法筛查正常对照及其家系成员。 结果 4例脂蛋白肾病患者中有2例携带杂合的apoE基因缺失突变 (142-144-0)，2例患者携带杂合的apoE点突变 (Arg25Cys)，2种突变均可见于尿液检查正常的亲属，并均表现为apoE升高。 结论 4例脂蛋白肾病的患者中发现两种apoE基因的突变，apoE Arg25Cys和apoE(142-144-0)。结合既往的研究结果，apoE(142-144-0)同时见于5例(本研究2例，前期研究3例)患者，为中国脂蛋白肾病患者常见的致病性基因突变。患者家系成员中的携带者可以不表现出肾脏病。     

成人多囊肝病

1856年Bristowe首先记述了并发多囊肾的成人多囊肝病(APLD),但直到最近,APLD和成人多囊肾病(APKD)才被认为是一种单基因显性遗传病。有关APKD的基因位于人第16对染色体,然而有些作者指出APLD可以单独发病,这样其基因传递的确切模式还有待于阐明。93%以上的APLD患者合并有APKD,但APKD仅34～78%伴发肝囊肿。本组患者肝内的囊肿大小不一,大者可含数升液体,肝脏增大有时可达盆腔。尽管肝内有大量囊肿形成,CT显示肝实质的体积仍可维持。在疾病早期,有时较难与单纯性肝囊肿(亦可多发)鉴别,因二者     

常染色体显性遗传型多囊肾病的诊断及治疗现况

常染色体显性遗传型多囊肾病 (ａｕｔｏｓｏｍａｌｄｏｍｉｎａｎｔｐｏｌｙｃｙｓｔｉｃｋｉｄｎｅｙｄｉｓｅａｓｅ ,ＡＤＰＫＤ)是一种最常见的单基因遗传性肾病 ,发病率约为 1 / 40 0～ 1 / 1 0 0 0 〔1〕。ＡＤＰＫＤ多在 30岁～ 5 0岁之间发病 ,因此过去常称为“成人型多     

改良聚合酶链反应法快速诊断常染色体显性遗传型多囊肾病

目的：检测微小卫星体ＳＭ７快速诊断常染色体显性遗传型多囊肾病（ＡＤＰＫＤ）。方法应用改良二温式聚合酶链反应（ＰＣＲ）法扩增与ＰＫＤ１位点紧密连锁的微小卫星体ＳＭ７,并对１０个家系进行基因连锁分析。结果在１０个家系１０３个家庭成员中,２９例先证者,９例无症状者通过基因连锁分析得到确诊,结论改良二温式ＰＣＲ法检测微小卫星体ＳＭ７可早期快速诊断ＡＤＰＫＤ。     

A novel frameshift mutation (2436insT) produces an immediate stop codon in the autosomal dominant polycystic kidney disease 2 (PKD2) gene.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder that can be caused by mutations in at least three different genes. Several mutations have been identified in PKD1 and PKD2 genes. Most of the mutations found in PKD2 gene are predicted to cause premature termination of the protein. METHODS: We analysed an Argentinian family characterized previously as PKD2. The PKD2 gene was amplified from genomic DNA using 17 primer pairs and the products were analysed by heteroduplex analysis. PCR products that showed a variation by heteroduplex analysis were sequenced directly. The mutation was confirmed by sequencing relatives. The segregation of the mutation in this family was verified by restriction endonuclease digestion of PCR products obtained from genomic DNA of all family members. Results and conclusions. Here, we report a novel mutation present in an Argentinian family characterized as PKD2 by linkage analysis. The mutation, shared by all affected members of the family, is a thymidine insertion at position 2436 of the gene, which results in a translation frameshift and creates an immediate stop codon. This mutation is expected to lead to a truncated protein that lacks the interacting domain with the PKD1 gene product. The thymidine insertion abolished a Ddel restriction site, allowing a rapid test for detection of PKD2 carriers in the family.     

Mutations of the PKD2 gene in Taiwanese patients with autosomal dominant polycystic kidney disease

BACKGROUND: Mutation analysis in the context of clinical phenotypes helps clarify the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). Over 78 PKD2 gene mutations have been reported in the literature, but few have been described from an Asian population. This study attempted to characterize PKD2 mutations and their clinical implications among Taiwanese. METHODS: Twenty unrelated ADPKD patients with uncharacterized genotypes were screened for mutations in the PKD2 gene via single-strand conformation polymorphism (SSCP) of PCR products from genomic DNA, using previously reported PCR conditions and primers. RESULTS: This study identified two novel mutations (C681A and 2136-2137delG) and one mutation (C2407T) previously reported in a Cypriot family. Overall, we found PKD2 mutations in 15% (three out of 20) of the ADPKD patients screened. The mutations included two nonsense mutations (Y227X and R803X) and one frameshift mutation (712-715X) that could all lead to premature termination of translation. The locations of mutations in this study spanned the entire PKD2 gene on exons 2, 11, and 13 without clustering and did not influence the renal disease severity. CONCLUSIONS: The study identified two novel mutations and one recurrent mutation of the PKD2 gene in 20 Taiwanese patients. The characteristics of the mutations in this study resemble those reported among Western populations.     

Thirteen novel mutations of the replicated region of PKD1 in an Asian population

BACKGROUND: Mutations of PKD1 are thought to account for approximately 85% of all mutations in autosomal dominant polycystic kidney disease (ADPKD). The search for PKD1 mutations has been hindered by both its large size and complicated genomic structure. To date, few mutations that affect the replicated segment of PKD1 have been described, and virtually all have been reported in Caucasian patients. METHODS: In the present study, we have used a long-range polymerase chain reaction (PCR)-based strategy previously developed by our laboratory to analyze exons in the replicated region of PKD1 in a population of 41 unrelated Thai and 6 unrelated Korean families with ADPKD. We have amplified approximately 3.5 and approximately 5 kb PKD1 gene-specific fragments (5'MR and 5'LR) containing exons 13 to 15 and 15 to 21 and performed single-stand conformation analysis (SSCA) on nested PCR products. RESULTS: Nine novel pathogenic mutations were detected, including six nonsense and three frameshift mutations. One of the deletions was shown to be a de novo mutation. Four potentially pathogenic variants, including one 3 bp insertion and three missense mutations, were also discovered. Two of the nonconservative amino acid substitutions were predicted to disrupt the three-dimensional structure of the PKD repeats. In addition, six polymorphisms, including two missense and four silent nucleotide substitutions, were identified. Approximately 25% of both the pathogenic and normal variants were found to be present in at least one of the homologous loci. CONCLUSION: To our knowledge, this is the first report of mutation analysis of the replicated region of PKD1 in a non-Caucasian population. The methods used in this study are widely applicable and can be used to characterize PKD1 in a number of ethnic groups using DNA samples prepared using standard techniques. Our data suggest that gene conversion may play a significant role in producing variability of the PKD1 sequence in this population. The identification of additional mutations will help guide the study of polycystin-1 and better help us to understand the pathophysiology of this common disease.     

Facilitated diagnosis of the contiguous gene syndrome: tuberous sclerosis and polycystic kidneys by means of haplotype studies

Tuberous sclerosis (TSC) and autosomal dominant polycystic kidney disease (ADPKD) are genetically heterogeneous diseases. The major gene for ADPKD (PKD1) lies adjacent to the TSC2 gene on chromosome 16p13. Some reports in the literature referred to an unusual presentation of TSC with enlarged cystic kidneys at birth, but it was not until the localization of the TSC2 and PKD1 genes that it was possible to analyze the interaction between both genes. We describe a case of a child with TSC and enlarged cystic kidneys. The study of genetic marker segregation in the family pointed to the presence of a deletion involving the 3' region of PKD1. A further study of the region showed a deletion of 40 kb involving both PKD1 and TSC2. We suggest that an additive or synergistic effect between PKD1 and TSC2 may cause this renal phenotype. A contiguous gene syndrome involving PKD1 and TSC2 should be suspected in children with TSC and enlarged polycystic kidneys at birth. The first approach to identify a deletion of both genes could be the analysis of the segregation of PKD1 and TSC2 markers in the family.     

Identification of gene mutations in autosomal dominant polycystic kidney disease through targeted resequencing

Mutations in two large multi-exon genes, PKD1 and PKD2, cause autosomal dominant polycystic kidney disease (ADPKD). The duplication of PKD1 exons 1-32 as six pseudogenes on chromosome 16, the high level of allelic heterogeneity, and the cost of Sanger sequencing complicate mutation analysis, which can aid diagnostics of ADPKD. We developed and validated a strategy to analyze both the PKD1 and PKD2 genes using next-generation sequencing by pooling long-range PCR amplicons and multiplexing bar-coded libraries. We used this approach to characterize a cohort of 230 patients with ADPKD. This process detected definitely and likely pathogenic variants in 115 (63%) of 183 patients with typical ADPKD. In addition, we identified atypical mutations, a gene conversion, and one missed mutation resulting from allele dropout, and we characterized the pattern of deep intronic variation for both genes. In summary, this strategy involving next-generation sequencing is a model for future genetic characterization of large ADPKD populations.     

A complete mutation screen of the ADPKD genes by DHPLC

BACKGROUND: Genetic analysis is a useful diagnostic tool in autosomal dominant polycystic kidney disease (ADPKD), especially when imaging results are equivocal. However, molecular diagnostics by direct mutation screening has proved difficult in this disorder due to genetic and allelic heterogeneity and complexity of the major locus, PKD1. METHODS: A protocol was developed to specifically amplify the exons of PKD1 and PKD2 from genomic DNA as 150 to 450 bp amplicons. These fragments were analyzed by the technique of denaturing high-performance liquid chromatography (DHPLC) using a Wave Fragment Analysis System (Transgenomics) to detect base-pair changes throughout both genes. DHPLC-detected changes were characterized by sequencing. RESULTS: Cost effective and sensitive mutation screening of the entire coding regions of PKD1 and PKD2 by DHPLC was optimized. All base-pair mutations to these genes that we previously characterized were detected as an altered DHPLC profile. To assess this method for routine diagnostic use, samples from a cohort of 45 genetically uncharacterized ADPKD patients were analyzed. Twenty-nine definite mutations were detected, 26 PKD1, 3 PKD2 and a further five possible missense mutations were characterized leading to a maximal detection rate of 76%. A high level of polymorphism of PKD1 also was detected, with 71 different changes defined. The reproducibility of the DHPLC profile enabled the recognition of many common polymorphisms without the necessity for re-sequencing. CONCLUSIONS: DHPLC has been demonstrated to be an efficient and effective means for gene-based molecular diagnosis of ADPKD. Differentiating missense mutations and polymorphisms remains a challenge, but family-based segregation analysis is helpful.     

Characterization of large rearrangements in autosomal dominant polycystic kidney disease and the PKD1/TSC2 contiguous gene syndrome

Large DNA rearrangements account for about 8% of disease mutations and are more common in duplicated genomic regions, where they are difficult to detect. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2. PKD1 is located in an intrachromosomally duplicated region. A tuberous sclerosis gene, TSC2, lies immediately adjacent to PKD1 and large deletions can result in the PKD1/TSC2 contiguous gene deletion syndrome. To rapidly identify large rearrangements, a multiplex ligation-dependent probe amplification assay was developed employing base-pair differences between PKD1 and the six pseudogenes to generate PKD1-specific probes. All changes in a set of 25 previously defined deletions in PKD1, PKD2 and PKD1/TSC2 were detected by this assay and we also found 14 new mutations at these loci. About 4% of the ADPKD patients in the CRISP study were found to have gross rearrangements, and these accounted for about a third of base-pair mutation negative families. Sensitivity of the assay showed that about 40% of PKD1/TSC contiguous gene deletion syndrome families contained mosaic cases. Characterization of a family found to be mosaic for a PKD1 deletion is discussed here to illustrate family risk and donor selection considerations. Our assay improves detection levels and the reliability of molecular testing of patients with ADPKD.