汉族人多囊肾病1型致病基因突变检测体系的建立及初步应用

目的 建立适合筛查汉族人多囊肾病1型致病基因（PKD1）突变的检测体系。方法 利用设计的82对引物[8对针对PKD15′端多拷贝区的长链聚合酶链式反应（PCR）引物和57对巢式PCR引物，17对针对3′端单拷贝区PCR引物]分别对PKD1的46个外显子进行扩增，扩增产物通过单链构象多态性（SSCP）分析筛检出异常条带后，再经测序确定基因突变位点。利用建立的PCR－SSCP检测体系对汉族人2个常染色体显性遗传性多囊肾病患者家系进行PKDA1突变检测，健康献血员为对照。结果 用82对PCR引物，可成功扩增PKD1各个外显子区域，并经测序证实为PKD1目的片段。将建立的SSCP－PCR基因突变检测体系，分别从2个汉族人常染色体显性多囊肾病（ADPKD）家系检测出PKD1基因Del 3 bp(G49761-G49763)和C47629T2个突变，其可分别导致编码产物第3827位缺失谷氨酸（Glu3827)和第3555位丝氨酸，而产生由苯丙氨酸(S3555F)替代的改变。结论 本研究建立的PCR－SSCP检测体系，可完成PKD1各外显子区域特异性扩增，并成功检测出汉族人2个ADPKD家系基因突变位点，不仅为PKD1基因突变的致病机理研究提供宝贵资料，而且为下一步汉族人多囊肾病的大规模基因突变筛查和临床诊断试剂盒的研制奠定了基础。     

A common polymorphism in exon 46 of the human autosomal dominant polycystic kidney disease 1 gene (PKD1)

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common single gene diseases in humans. We have identified a synonymous T to C transition polymorphism in exon 46 of the PKD1 gene (12838T→C, Pro4209Pro). The polymorphism was present with similar frequencies in ADPKD patients and unaffected individuals. The heterozygosity, determined in 89 Italian individuals, was 0·347. The frequency of the rarer allele was 0·222. This polymorphism is easy to determine as it abolishes a naturally occurring DdeI restriction site. The availability of an additional intragenic marker in the PKD1 gene will improve the accuracy of linkage studies in ADPKD families.     

中国汉族人群PKD2基因多态性检测

目的：检测中国汉族人群PKD2基因多态性。方法：选取50名健康志愿者，提取外周血白细囊DNA，应用聚合酶链反应-单链构象多态性分析技术(PCR-SSCP)进行多态性检测，取异常条带标本进行核苷酸序列测定，判别PKD2外显子基因多态性位点及类型。结果：从50名健康人中成功检测出2种多态性。第1种为PKD2外显子7的1716位碱基由鸟嘌呤置换为腺嘌呤，编码氨基酸仍为赣氨酸。第2种为PKD2外显子1的第420位碱基由鸟嘌呤置换为腺嘌呤，编码氨基酸仍为甘氨酸。结论：建立了PCR-SSCP直接检测我国汉族人PKD2基因多态性的方法，并成功检测出2种PKD2基因多态性，为开展常染色体显性遗传性多囊肾病基因诊断奠定了基础。     

Mutation analysis of autosomal dominant polycystic kidney disease genes in Han Chinese

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in two genes, PKD1 and PKD2. The complexity of these genes, particularly PKD1, has complicated genetic screening, though recent advances have provided new opportunities for amplifying these genes. In the Han Chinese population, no complete mutational analysis has previously been conducted across the entire span of PKD1 and PKD2. Here, we used single-strand conformation polymorphism (SSCP) analysis to screen the entire coding sequence of PKD1 and PKD2 in 85 healthy controls and 72 Han Chinese from 24 ADPKD pedigrees. In addition to 11 normal variants, we identified 17 mutations (12 in PKD1 and 5 in PKD2), 15 of which were novel ones (11 for PKD1 and 4 for PKD2). We did not identify any seeming mutational hot spots in PKD1 and PKD2. Notably, we found several disease-associated C-T or G-A mutations that led to charge or hydrophobicity changes in the corresponding amino acids. This suggests that the mutations cause conformational alterations in the PKD1 and PKD2 protein products that may impact the normal protein functions. Our study is the first report of screenable mutations in the full-length PKD1 and PKD2 genes of the Han Chinese, and also offers a benchmark for comparisons between Caucasian and Han ADPKD pedigrees and patients.     

Functional polycystin-1 dosage governs autosomal dominant polycystic kidney disease severity

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations to PKD1 or PKD2, triggering progressive cystogenesis and typically leading to end-stage renal disease in midlife. The phenotypic spectrum, however, ranges from in utero onset to adequate renal function at old age. Recent patient data suggest that the disease is dosage dependent, where incompletely penetrant alleles influence disease severity. Here, we have developed a knockin mouse model matching a likely disease variant, PKD1 p.R3277C (RC), and have proved that its functionally hypomorphic nature modifies the ADPKD phenotype. While Pkd1^+/null mice are normal, Pkd1^RC/null mice have rapidly progressive disease, and Pkd1^RC/RC animals develop gradual cystogenesis. These models effectively mimic the pathophysiological features of in utero–onset and typical ADPKD, respectively, correlating the level of functional Pkd1 product with disease severity, highlighting the dosage dependence of cystogenesis. Additionally, molecular analyses identified p.R3277C as a temperature-sensitive folding/trafficking mutant, and length defects in collecting duct primary cilia, the organelle central to PKD pathogenesis, were clearly detected for the first time to our knowledge in PKD1. Altogether, this study highlights the role that in trans variants at the disease locus can play in phenotypic modification of dominant diseases and provides a truly orthologous PKD1 model, optimal for therapeutic testing.     

Characterization of microsatellite markers to diagnose ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) maps to chromosome 16p13.3 (PKD1) and to chromosome 4q21-23 (PKD2), with the likelihood of a third unmapped locus. The size and genomic complexity of the PKD1 gene make it impractical to detect mutations for prenatal diagnosis. Therefore, pedigree-based linkage analysis remains useful for diagnosis of ADPKD. Since, the complete genome sequences of chromosome 16p13.3 and 4q21-23 including PKD1 and PKD2, respectively, were reported very recently, in order to do more precise diagnosis of ADPKD, we tried to find microsatellite markers. We performed database searches of 2000 kb of genome sequence across the 16p13.3 and the 4q21-23. To determine the distribution of alleles and the degree of polymorphism of the microsatellites, genotyping experiments were performed on 48 Korean individuals. We found novel 14 microsatellite markers around ADPKD that are more polymorphic and closer to PKD1 or PKD2 than the known markers. The novel microsatellite markers were applied to diagnose ADPKD families. These novel microsatellite markers are not only useful for presymptomatic and prenatal diagnosis of ADPKD, but also applicable in the study of positional cloning, human evolution and tumor biology.     

Autosomal dominant polycystic kidney disease: recent advances in pathogenesis and treatment

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder affecting 1 in 1,000 people in the general population and accounts for up to 10% of all patients on renal replacement therapy. Numerous fluid-filled epithelial cysts arise from different nephron segments as spherical dilatations or small out-pouchings, enlarge progressively and eventually become disconnected from the rest of the renal tubule. The development of cysts is accompanied by destruction of the renal parenchyma, interstitial fibrosis, cellular infiltration and loss of functional nephrons. ADPKD is not only a kidney disease but also a systemic disorder associated with intracranial arterial aneurysms, cardiac valvular defects, colonic diverticulosis and cyst formation in other organs such as the liver, spleen and pancreas. The identification of PKD1 and PKD2 together with the drive to elucidate the functions of their encoded proteins, polycystin-1 (PC1) and polycystin-2 (PC2), has led to an explosion of clinical and scientific interest in this common disorder. The aim of this review is to highlight recent advances in our understanding of ADPKD pathogenesis which are leading to exciting new treatment strategies.     

Molecular genetics of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a common Mendelian disorder, occurring in approximately 1 in 1000 births and accounting for 8% to 10% of cases of end-stage renal disease (ESRD). Mutations of 2 genes, PKD1 and PKD2, account for the disease in approximately 80% to 85% and 10% to 15% of families respectively. The gene products (polycystin 1 and 2) of PKD1 and PKD2 are plasma membrane proteins and components of a novel signalling pathway that regulates epithelial cell growth and differentiation. Significant inter- and intrafamilial renal disease variability in ADPKD has been well documented and is influenced by both germline and somatic genetic events. Specifically, genetic locus heterogeneity and 2 rare Mendelian syndromes have been shown to strongly influence the variability of interfamilial renal disease, and as-yet-unknown genetic and environmental factors likely modify both inter- and intrafamilial renal disease severity. Furthermore, individual cyst formation in ADPKD represents an aberration of monoclonal growth triggered by somatic PKD1 or PKD2 mutations within individual epithelial cells. Current studies are in progress to identify major genetic and environmental modifiers of renal disease variability. A thorough knowledge of these determinants will allow better patient risk assessment and development of mechanism-based therapy in ADPKD.     

Polycystin-1, the PKD1 gene product, is in a complex containing E-cadherin and the catenins

Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease characterized by cyst formation in kidney tubules and other ductular epithelia. Cells lining the cysts have abnormalities in cell proliferation and cell polarity. The majority of ADPKD cases are caused by mutations in the PKD1 gene, which codes for polycystin-1, a large integral membrane protein of unknown function that is expressed on the plasma membrane of renal tubular epithelial cells in fetal kidneys. Because signaling from cell-cell and cell-matrix adhesion complexes regulates cell proliferation and polarity, we speculated that polycystin-1 might interact with these complexes. We show here that polycystin-1 colocalized with the cell adhesion molecules E-cadherin and alpha-, beta-, and gamma-catenin. Polycystin-1 coprecipitated with these proteins and comigrated with them on sucrose density gradients, but it did not colocalize, coprecipitate, or comigrate with focal adhesion kinase, a component of the focal adhesion. We conclude that polycystin-1 is in a complex containing E-cadherin and alpha-, beta-, and gamma-catenin. These observations raise the question of whether the defects in cell proliferation and cell polarity observed in ADPKD are mediated by E-cadherin or the catenins.     

Identification and localization of polycystin,the PKD1 gene product.

Polycystin, the product of autosomal dominant polycystic kidney disease (ADPKD) 1 gene (PKD1) is the cardinal member of a novel class of proteins. As a first step towards elucidating the function of polycystin and the pathogenesis of ADPKD, three types of information were collected in the current study: the subcellular localization of polycystin, the spatial and temporal distribution of the protein within normal tissues and the effects of ADPKD mutations on the pattern of expression in affected tissues. Antisera directed against a synthetic peptide and two recombinant proteins of different domains of polycystin revealed the presence of an approximately 400-kD protein (polycystin) in the membrane fractions of normal fetal, adult, and ADPKD kidneys. Immunohistological studies localized polycystin to renal tubular epithelia, hepatic bile ductules, and pancreatic ducts, all sites of cystic changes in ADPKD, as well as in tissues such as skin that are not known to be affected in ADPKD. By electron microscopy, polycystin was predominantly associated with plasma membranes. Polycystin was significantly less abundant in adult than in fetal epithelia. In contrast, polycystin was overexpressed in most, but not all, cysts in ADPKD kidneys.     

The molecular genetic analysis of polycystic kidney disease]

Autosomal dominant polycystic kidney disease (ADPKD) is one of common single gene disorders. The development of molecular genetic techniques has shown that mutant PKD1 gene assigned to ADPKD was closely linked to alpha-globin on the short arm of chromosome 16. This location was established when genetic linkage was found between ADPKD and a highly polymorphic region at the 3' end of the alpha-globin cluster (3' HVR). The discover of genetic linkage markers such as 3' HVR probe has provided a diagnostic test in presymptomatic stage. We performed this diagnostic test using DNA probes in 3 patients with ADPKD of one Japanese family. They also showed PKD1 gene linkage as previously described by Reeders et al. Linkage analysis of the PKD1 gene might be available to diagnostic test of ADPKD. DNA diagnosis of ADPKD however has to be performed carefully because of an ethical standpoint.     

Usefulness of combined genetic data in Hungarian families affected by autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common hereditary diseases. Mutations of two known genetic loci (PKD1: 16p13.3 and PKD2: 4q21.2) can lead to bilateral renal cysts. The PKD1 locus is the more common (～85%), with a more severe phenotype. Because of the genetic complexity of ADPKD and the size and complexity of the PKD1 gene, pedigree-based linkage analysis is a useful tool for the genetic diagnosis in families with more than one subject affected. We tested linkage or non-linkage to the closely linked DNA markers flanking the PKD1 (D16S663 and D16S291) and one intragenic D16S3252 and PKD2 (D4S1563 and D4S2462) in 30 ADPKD-affected families, to determine the distributions of alleles and the degree of microsatellite polymorphisms (in 91 patients and 125 healthy subjects). To characterize the markers, used heterozygosity levels, polymorphism information content and LOD scores were calculated. The D16S663 marker included 12 kinds of alleles, while D16S291 had 10 alleles and D16S3252 had 8. D4S1563 had 12 alleles and D4S2462 had 11. In a search for a common ancestral relationship, we considered the patients’ alleles with the same repeat number. Only one haplotype was detected in more than one (2) unrelated families. The calculated two-point LOD scores indicated a linkage to PKD1 in 22 families (74%). In four families (13%) with a linkage to PKD2, the patients reached the end-stage renal disease after the age of 65 years. One family was linked to neither gene (3%), and in three families (10%) a linkage to both genes was possible. In the latter three families, the numbers of analyzed subjects were small (4–5), and/or some markers were only partially or non-informative. However, the elderly affected family members exhibited the clinical signs of the PKD1 form in these cases. The new Hungarian population genetic information was compared with available data on other populations.     

一例PTEN新生杂合突变患儿临床表型与免疫特征分析

  目的  探讨一例PTEN杂合突变患儿的临床表型及免疫特征，丰富PTEN突变相关临床表型谱。  方法  收集患儿门诊及住院期间的病史资料、生化检查及影像学检查结果，抽取外周静脉血进行医学全外显子组综合检测，Sanger测序验证患儿及其父母PTEN基因突变位点，采用流式细胞术进行T细胞PI3K/Akt/mTOR通路磷酸化水平及T细胞亚群与其耗竭相关表面分子检测，采用蛋白质印迹法检测外周血单个核细胞PTEN蛋白表达水平，健康对照为患儿父亲。  结果  患儿以大头畸形(头围＞P99)、疣状表皮痣、精神运动发育迟缓、学语延迟为主要临床表现，PTEN基因(NM_000314.8) c.388C＞T(p.R130X)新生杂合突变，PTEN蛋白表达减少，血清IgA水平稍低(0.177 g/L)，精细免疫分型CD4⁺终末分化效应记忆T细胞、CD8⁺终末分化效应记忆T细胞、过渡性B细胞比例及绝对数均增加，但T细胞PI3K/Akt/mTOR通路磷酸化水平正常。患儿以PTEN错构瘤综合征相关表型为主要表现，无明显PI3Kδ过度活化综合征样表型。  结论  该患儿PTEN基因的突变位点为国内首例，有助于丰富临床医生对该疾病的认识，提高诊治水平。     

Failure to ubiquitinate c-Met leads to hyperactivation of mTOR signaling in a mouse model of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder that is caused by mutations at two loci, polycystin 1 (PKD1) and polycystin 2 (PKD2). It is characterized by the formation of multiple cysts in the kidneys that can lead to chronic renal failure. Previous studies have suggested a role for hyperactivation of mammalian target of rapamycin (mTOR) in cystogenesis, but the etiology of mTOR hyperactivation has not been fully elucidated. In this report we have shown that mTOR is hyperactivated in Pkd1-null mouse cells due to failure of the HGF receptor c-Met to be properly ubiquitinated and subsequently degraded after stimulation by HGF. In Pkd1-null cells, Casitas B-lineage lymphoma (c-Cbl), an E3-ubiquitin ligase for c-Met, was sequestered in the Golgi apparatus with α₃β₁ integrin, resulting in the inability to ubiquitinate c-Met. Treatment of mouse Pkd1-null cystic kidneys in organ culture with a c-Met pharmacological inhibitor resulted in inhibition of mTOR activity and blocked cystogenesis in this mouse model of ADPKD. We therefore suggest that blockade of c-Met is a potential novel therapeutic approach to the treatment of ADPKD.     

常染色体显性多囊肾病的预后评估及治疗

常染色体显性多囊肾病(autosomal dominant polycystic kidney disease, ADPKD)患病率为1‰~2‰, 属于罕见病, 临床主要表现为双侧肾囊肿且逐渐发展, 肾脏体积进行性增大, 肾功能逐步降低。PKD1基因突变约占81%, PKD2基因突变约占10.5%~22%。血管加压素(arginine vasopressin, AVP)和环磷酸腺苷(cyclic adenosine monophosphate, cAMP)信号通路在ADPKD囊肿发展过程中发挥重要作用。近年来发表的梅奥风险评估模型和PROPKD(predicting renal outcome in polycystic kidney disease)评分是ADPKD较好的预后评估模型, 已成为临床医生决策的重要依据。通过拮抗AVP受体, 抑制cAMP通路的托伐普坦已成为ADPKD首个特异治疗药物, 可有效抑制总肾脏体积的增长和保护肾功能。药物的长期安全性仍需进一步研究。     

Practical genetics for autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common mendelian disorder of the kidney and accounts for ~5% of end-stage renal disease in North America. It is characterized by focal development of renal cysts which increase in number and size with age. Mutations of PKD1 and PKD2 account for most cases. Although the clinical manifestations of both gene types overlap completely, PKD1 is associated with more severe disease than PKD2, with larger kidneys and earlier onset of end-stage renal disease. Furthermore, marked within-family renal disease variability is well documented in ADPKD and suggests a strong modifier effect from as yet unknown genetic and environmental factors. In turn, the significant inter- and intra-familial renal disease variability poses a challenge for diagnosis and genetic counseling. In general, renal ultrasonography is commonly used for the diagnosis, and age-dependent criteria have been defined for subjects at risk of PKD1. However, the utility of the PKD1 ultrasound criteria in the clinical setting is unclear since their performance characteristics have not been defined for the milder PKD2 and the gene type for most test subjects is unknown. Recently, highly predictive ultrasound diagnostic criteria have been derived for at-risk subjects of unknown gene type. Additionally, both DNA linkage and gene-based direct sequencing are available for the diagnosis of ADPKD, especially in subjects with equivocal imaging results, a negative or indeterminate family history, or in younger at-risk individuals being evaluated as potential living related kidney donor. This review will highlight the utility and limitations of clinical predictors of gene types, imaging- and molecular-based diagnostic tests, and present an integrated approach for evaluating individuals suspected to have ADPKD.     

Loss of the polycystic kidney disease (PKD1) region of chromosome 16p13 in renal cyst cells supports a loss-of-function model for cyst pathogenesis.

It is not known whether mutations in the PKD1 gene cause autosomal dominant polycystic kidney disease (PKD) by an activating (gain-of-function) or an inactivating (loss-of-function) model. We analyzed DNA from cyst epithelial cells for loss of heterozygosity (LOH) in the PKD1 region of chromosome 16p13 using microsatellite markers. 29 cysts from four patients were studied. Five cysts from three patients had chromosome 16p13 LOH. Four of the cysts had loss of two chromosome 16p13 markers that flank the PKD1 gene. In two patients, microsatellite analysis of family members was consistent with loss of the wild-type copy of PKD1 in the cysts. In the third patient, 16p13 LOH was detected in three separate cysts, all of which showed loss of the same alleles. Chromosome 3p21 LOH was detected in one cyst. No LOH was detected in four other genomic regions. These results demonstrate that some renal cyst epithelial cells exhibit clonal chromosomal abnormalities with loss of the wild-type copy of PKD1. This supports a loss-of-function model for autosomal dominant PKD, with a germline mutation inactivating one copy of PKD1 and somatic mutation or deletion inactivating the remaining wild-type copy.     

Autosomic dominant polycystic kidney disease and metformin: Old knowledge and new insights on retarding progression of chronic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common congenital kidney disorder, generally caused by mutations in the PKD1 and PKD2 genes, coding for polycystins 1 and 2. Its pathogenesis is accompanied by alterations of the cAMP, mTOR, MAPK/ERK, and JAK/STAT pathways. ADPKD is clinically characterized by the formation of many growing cysts with kidney enlargement and a progressive damage to the parenchyma, up to its complete loss of function, and the onset of end-stage renal disease (ESRD). The current aim of ADPKD therapy is the inhibition of cyst development and retardation of chronic kidney disease progression. Several drugs have been recently included as potential therapies for ADPKD including metformin, the drug of choice for the treatment of type 2 diabetes mellitus, according to its potential inhibitory effects on cystogenesis. In this review, we summarize preclinical and clinical evidence endorsing or rejecting metformin administration in ADPKD evolution and pathological mechanisms. We explored the biology of APDKD and the role of metformin in slowing down cystogenesis searching PubMed and Clinical Trials to identify relevant data from the database inception to December 2020. From our research analysis, evidence for metformin as emerging cure for ADPKD mainly arise from preclinical studies. In fact, clinical studies are still scanty and stronger evidence is awaited. Its effects are likely mediated by inhibition of the ERK pathway and increase of AMPK levels, which are both linked to ADPKD pathogenesis.