INTRODUCTION

Autosomal dominant polycystic kidney disease (ADPKD)is a multisystem disorder characterized by the formation of cysts in the kidneys and other organs. ADPKD affects more than 12.5 million people worldwide[1].Approximately 50% of patients with ADPKD have endstage renal disease (ESRD) by 60 years of age. Dialysis and kidney transplantation are the only treatment options for patients with ESRD[2]. The reason for the absence of targeted treatment is insufficient understanding of the molecular mechanism of cystogenesis.

The molecular nature of the disease includes germline mutation of either the PKD1 (polycystin-1),PKD2 (polycystin-2) or GANAB gene[3,4]. Although the germline mutation is present in every cell of the body,formation of cysts is limited to a small number of nephrons. This means that the germline mutation is not in itself sufficient to produce cysts; a second somatic event is also required[5]. More evidence has accumulated in recent years showing that the primary cilium plays an important role in the development of ADPKD.

The primary cilium is a signaling organelle that extends from the surface of the plasma membrane of most mammalian cells. Assembly of cilia depends on cell cycle progression, because the centrioles regulating the cell cycle are essential components in the formation of the basal body of the cilia[6]. The maintenance and elongation of cilia is ensured by a process called intraflagellar transport (IFT). In this process, protein complexes known as IFT trains carry cargo along tracks that run along the length of the cilia. Different motor proteins power the IFT trains in different directions.Kinesin moves IFT trains from the base to the tip, while dynein moves them back in the opposite direction[7].

Many studies have shown that the loss of primary cilia promotes renal cyst formation in vivo[8,9]. However,the epithelial cells of renal cysts are characterized not only by an absence of cilia but also by excessively long cilia[10-12]. Recent studies have confirmed that kidney cysts occurred following inactivation of polycystins in otherwise intact cilia or following complete removal of cilia by inactivation of IFT proteins. In addition, a relationship was shown between cilia and polycystins that regulates the severity of ADPKD. According this model, the progression of cysts is regulated by the duration of the interval time between the initial loss of polycystins and the subsequent ablation of cilia. These studies identified a new mechanism of cystogenesis based on the evidence that the loss of renal cilia inhibits cyst growth if the cilia are disrupted at the same time as the polycystins[13,14]. In addition, it was shown that defective primary cilia or the inactivation of ciliary genes induces aberrant signaling pathways associated with proliferation, differentiation and development in various PKD mouse models[13-16]. The structural deformity or absence of primary cilia is thus a key driving force in the development of ADPKD.

The results of these studies were achieved in mouse models by targeted inactivation of various ciliary genes,especially genes encoding IFT proteins. However, the actual mutation profile of ciliary genes in human ADPKD tissues is unknown. The aim of our study was to identify the mutation state of genes encoding the structural ciliary components in tissues from patients with ADPKD obtained by nephrectomy through targeted nextgeneration sequencing analysis.

MATERIALS AND METHODS

Sample collection

In our study, we obtained archived FFPE samples of polycystic kidney tissues and matched normal controls from 7 patients with ADPKD (four women and three men) who had undergone radical nephrectomy.Four patients had nephrectomy for the occurrence of complications associated with the enlarged kidney involved the recurrent infection of the urinary tract,arterial hypertension, and chronic pain. Three patients required nephrectomy to provide the space for the kidney allograft. The FFPE samples were subjected to histopathological examination. The samples containing at least 50% epithelial cysts were considered suitable for genetic analysis. The genetic testing of the PKD1 and PKD2 genes had been carried out previously in our laboratory. The mutation in the PKD1 gene was confirmed for all of the patients. Our study was approved by an ethics committee and informed consent was provided by all patients at the inception of the study.

DNA extraction and sample quality control

DNA was extracted from the FFPE tissues using the commercially available blackPREP FFPE DNA kit(Analytik Jena, Germany) and from peripheral blood using the QIAamp DNA Mini kit (Qiagen, Valencia,California, United States) according to the manufacturer’s instructions. The extracted DNA specimens from FFPE were further quantified using the Qubit dsDNA HS assay kit (Life Technologies/Fisher Scientific, United States)and the Agilent NGS FFPE QC Kit according to the manufacturer’s instructions.

拜别雨鸾师父，三人来到王积薪的棋室，上官星雨觉得这一关应好过，没成想棋圣他老人家不玩长生谱，也不玩媪妇谱，偏要三人一道与他对弈四象数独棋，这是他当年过昆仑山与痴和尚赌钱时所创，三人由青龙、白虎、朱雀、玄武里迷迷瞪瞪地挣脱出来，又与王积薪考较了一番他由长生谱里领悟出来的长生真气，总算是被他老人家重拿轻放，放出了棋室。

Design of the gene panel

Candidate genes were selected using a gene list in the SysCilia Gold Standard (SCGC) Version 1 database[17]. A panel of 191 genes was designed using the web-based tool SureDesign (Agilent Technologies, United States).The regions of interest included coding regions with 10 base pair (bp) upstream and 10 bp downstream for capture of splicing donor and acceptor sites. Overall, we analyzed genes encoding the proteins of basal bodies (31 genes), centrioles (25 genes), centrosomes (22 genes),IFT (24 genes), transition zone (17 genes), axoneme(9 genes), ciliary membrane (8), cilioplasma (5 genes),ciliary proteins located in the nucleus (5 genes), plasma membrane proteins (5 genes), ciliary tip (4 genes),regulatory ciliary proteins in the Trans-Golgi network (3 genes), ciliary root (2 genes) and ciliary proteins with non-specific localization (31 genes).

Library preparation and variant calling

Sequencing libraries were prepared using the Agilent SureSelectXT Custom 0.5 Mb up to 2.9 Mb according to the manufacturer’s instructions. The sequencing analyses were performed on the HiSeq 2500 sequencing system (Illumina, United States). Data were analyzed using a software package that was commercially available - NextGENe™ (SoftGenetics, United States).Only variants with a minimum of 50-fold coverage for at least 80% of the targeted bases were included into analysis. Variants were marked as potential errors if they exhibited strong strand bias (＜ 0.10), low depth of coverage (＜ 10), low-quality score (＜ 30) or low average quality (＜ 2.0). The functional analysis of variants was performed by using Geneticist Assistant software (SoftGenetics, United States). This software used combined computational prediction methods(SIFT, Polyphen2, LRT, MutationTaster, ANNOVAR,FATHMM, CADD and Mutation Assessor) to calculate the mean pathogenicity score of identified variants. The pathogenic variants detected by NGS were verified by using the Sanger sequencing method. The germline origin of the pathogenic variants was excluded by analysis of DNA extracted from peripheral blood.

RESULTS

Analysis of sequencing data

In our study, we achieved a high quality of sequenced data. The mean total reads generated per sample was approximately 5000000, with more than 98% of reads aligned with the reference genome and more than 80%of reads mapping to targeted regions. In addition, 93%of the targeted regions were covered by more than 30 reads. Overall, 1440 variants were identified in 7 ADPKD samples. These variants represent disease-specific genetic changes without occurrence in matched normal kidney tissues. Based on the results of the functional analysis, 908 variants were classifying as benign. These variants were reported in known databases of somatic mutations and single nucleotide polymorphisms. Of these, 732 variants were classified as clearly benign. The remaining 176 variants were of unknown significance,but with high value of minor allele frequency, on the basis of which they were determined as polymorphisms.

在先生看来，翻译包含译材和译法，其中译法是关键，而译法又分为译笔和译名，其中译名格外重要。先生认为这里的名指一切词品，不限于名、静、动词，这实际上已将译名扩展为词语翻译。接下来，先生一一分析了五种译名的方法。音义分译始于佛经翻译，但佛经里这类译名很少，试而未效；音义兼译于译者两全其美，实则吃力不讨好；造译更加少见且极费力、不方便，此三者都不是译名的通行译法。剩下的音译与义译是主要译法,占译名的绝大多数,也是近年译名辩论的焦点。因此先生也重点考察了音译与义译及其关系。

In our study, we analyzed 191 structural and functional ciliary genes using next-generation sequencing analysis. The tissue samples used in this study were obtained from 7 patients with ADPKD who underwent nephrectomy. Each sample contained polycystic kidney tissue and matched normal kidney tissue.All analyzed samples were formalin-fixed and paraffin-embedded. The germline origin of the ident ifi ed variants was excluded by analysis of DNA extracted from peripheral blood.

Analysis of disease-related pathogenic variants

The pathogenic variants were further analyzed based on their occurrence in individual ADPKD samples. The mutation profile of each analyzed sample was unique.Each sample had identified pathogenic variants in 5 to 15 genes, which varied from each other. However,the pathogenic variants in 5 genes were present in the vast majority of the ADPKD samples. Of these, 4 included genes encoded the centrosomal protein HTT(Huntingtin), the subdistal appendage of centriole ODF2(outer densefiber protein 2), the distal appendage of centriole CEP89 (centrosomal protein of 89 kDa) and a component of centriolar satellite PCM1 (pericentriolar material 1). The most affected gene was PCM1, in which pathogenic variants were present in all samples of ADPKD. The pathogenic variants in the HTT, ODF2 and CEP89 genes were identified in 5 of the 7 ADPKD samples. Another gene whose pathogenic variants affected all of the ADPKD samples was the KIF19 (KinesinFamily Member 19) gene. This gene encodes a key regulator of ciliary length located on the very top of the primary cilia in the ciliary tip included in anterograde IFT.In addition, we identified only one type of the pathogenic variant in these 5 genes (Table 2). In most cases, it was a frameshift mutation resulting in a premature stop codon and truncation of protein. According to the results of the predictive software, all detected variants negatively affected the function of the encoded proteins.

REFERENCES

22 de Almeida RM, Clendenon SG, Richards WG, Boedigheimer M,Damore M, Rossetti S, Harris PC, Herbert BS, Xu WM, Wandinger-Ness A, Ward HH, Glazier JA, Bacallao RL. Transcriptome analysis reveals manifold mechanisms of cyst development in ADPKD. Hum Genomics 2016; 10: 37 [PMID: 27871310 DOI: 10.1186/s40246-016-0095-x]

DISCUSSION

In the present study, we identified genetic changes in the structural ciliary genes in human ADPKD tissues.The most frequently affected genes encoded the centriolar and centrosomal proteins PCM1, ODF2, HTT and CEP89, which are essential for ciliogenesis. Recent studies have confirmed that the loss of these proteins specifically blocks ciliogenesis at the step of centriole-tomembrane docking. Undocked centrioles lose the signs of cilia assembly, even when they were previously under the influence of signals that support ciliogenesis[18,19].Thus, disruption of the function of these genes may be the cause of cilia loss in the epithelial cells of renal cysts.The most commonly affected gene in this group was PCM1, which is also essential for the correct localization of several centrosomal proteins and for anchoring microtubules to the centrosome. It is generally known that centrosome dysfunction is linked to aneuploidy and chromosomal instability[20]. Many studies have shown increased incidence of chromosome imbalances and abnormal chromosome segregation in ADPKD tissues[21,22]. Loss of function of the PCM1 gene may therefore be a key factor in these processes.

Another pathogenic variant, which was present in all of the analyzed ADPKD samples, was identified in the KIF19 gene. This gene encodes a member of the kinesin superfamily protein included in anterograde IFT, which is localized to ciliary tips. The results of a recent study have provided evidence that KIF19 is a key determinant of the optimal length of cilia. The length of cilia was abnormally extended by knockdown Kif19 in a mouse model[23]. Many studies have revealed that the change in the length of the primary cilium is an important trigger of various pathological processes in ADPKD[24,25].The epithelial cells of renal cysts usually show an absence or shortening of the primary cilium. However,stages of interstitialfibrosis and end-stage renal disease are associated with the elongation of the primary cilia[26,27]. In our study, we analyzed the mutation profile in the nephrectomized ADPKD tissues withdrawn at the end stage of renal disease. Given that we identified the pathogenic variant of the KIF19 gene in all analyzed ADPKD tissues, it may represent a significant event in the progression of the disease. However, these claims will require further analysis.

An interesting result of our study was the low frequency of mutations in the genes encoding IFT proteins.Kidney cysts arise in most mouse models following the disruption of cilia by targeted inactivation of genes encoding IFT components such as the heterotrimeric kinesin components KIF3a and the IFT proteins IFT20 and IFT88. However, genetic changes in these genes were not present in any analyzed ADPKD sample. We confirmed the presence of the pathogenic variants in only two genes (IFT172 and IFT80) of the total number of analyzed IFT genes. In addition, the pathogenic variants in these genes were detected in only one ADPKD sample. The results of our study showed that the loss of the primary cilia in the human ADPKD tissues may be predominantly caused by defects of centrosomal proteins and KIF19 protein. However, this claim requires confirmation by functional analysis of the use of animal models. It is also necessary to verify the results by analyzing a larger number of samples.

In our study, we identified the simultaneous occurrence of genetic changes in various ciliary genes. Each ADPKD tissue had pathogenic variants in 5 to 15 genes.The occurrence of multiple structural defects of the primary cilium may be due to several factors. Firstly,we analyzed the tissues at an advanced stage of the disease, in which genetic changes could have been accumulated. However, disturbances of centrosomal proteins may also be the cause of the multiple defects in the ciliary structure. An interesting finding was the presence of only one pathogenic variant in each individual gene. To determine whether they are “hotspot”mutations representing secondary somatic events in the development of the disease will require further analysis.

The results of our study may be limited by the relatively small number of analyzed samples. The reason for this small number is the fact that the majority of ADPKD patients do not require native nephrectomy,and cystic kidneys are not generally biopsied for technical and ethical reasons. However, this is thefirst study examining the complex mutational status of the structural and functional ciliary genes in human ADPKD tissues.

In conclusion, our study revealed genetic defects of ciliary genes that can lead to loss of the primary cilium in human ADPKD tissues. In addition, we identified unique genetic findings associated with the disease,which may play a significant role in the pathogenesis of the disease. However, other functional analyses are necessary to confirm this hypothesis. The results of our work thus provided valuable indications for the direction of further research in the area of molecular pathogenesis of ADPKD.

ARTICLE HIGHLIGHTS

Research background

The primary cilia and polycystins plays an important role in the regulating the severity of autosomal dominant polycystic kidney disease (ADPKD). While the loss of cilia or polycystins alone results in the development and progression of renal cysts, renal cilia involution reduces the progression of cyst growth induced by the inactivation of polycystins. The epithelial cells of renal cysts usually show various structural deformities of the primary cilium involve an absence,shortening or elongation. These structural changes can be caused by genetic defects of ciliary proteins. Mutation profile of ciliary genes in human ADPKD tissues is unknown. Revealing a genetic basis for ciliogenesis defects may identify causative factors of disease progression and the potential molecular targets for the development of new therapies of ADPKD.

Research motivation

Genetic defects of various ciliary genes whose inactivation leads to the development and progression of ADPKD have been identified in mouse models.However, recent studies have confirmed that the animal models of ADPKD incompletely mimic the human disease. Therefore, it is important to detect genetic abnormalities that can affect ciliogenesis directly in ADPKD human tissues.

Research objectives

The main objectives of this study is to identify the genetic defects of ciliary genes causing the loss of primary cilium in ADPKD human tissues. The results of our study are important indicators for directing further analysis.

Research methods

The other 532 identified variants represented novel genetic changes without record in databases. The results of the functional prediction determined 52 variants as clearly pathogenic. For the other 480 variants, benign status was clearly confirmed in all predictive software.Identified pathogenic variants were present in 39 genes encoding the various structural components of the primary cilium. An overview of these genes is shown in Table 1. The most common pathogenic variants affected the proteins of basal bodies (10 genes), centrosomes(7 genes), centrioles (6 genes) and ciliary proteins with non-specific localization (5 genes). The proteins in other parts of the cilium were rarely mutated.

Research results

We ident ifi ed unique of mutation profile in each of analyzed ADPKD samples,which was characterized by the presence of pathogenic variants in 5 to 15 ciliary genes. The most frequently identified defects were found in genes encoding centrosomal proteins and kinesin family member 19, which are important for ciliogenesis. In addition, pathogenic variants in the PCM1 and KIF19 genes were found in all ADPKD samples.

由于污泥取自北京市高碑店污水处理厂二沉池，活性污泥刚开始需要对制浆造纸废水有一定的适应期，所以刚启动时，在培养好的污泥中加入混凝-加核絮凝组合工艺处理后废水，将其配成浓度为20%的水样。条件不变继续培养污泥，然后隔天加入废水，依次配制成40%、60%、80%、100%，使污泥逐渐适应该麦草浆废水，依GB/T 11914—1989中的方法测定废水CODCr，计算其去除率。

Research conclusions

Our study had found that the human ADPKD tissues are characterized by the presence of several genetically altered ciliary proteins that plays an important role in ciliogenesis. The structural and functional disturbance of the primary cilium can be induced by mutations of these proteins. An interestingfinding of our study was that the mutations in genes encoding the proteins of intraflagellar transport (IFT) were rarely mutated. These genes are considered candidate genes related to ADPKD in mouse models. Centrosomal proteins and kinesin family member 19 are the most commonly mutated ciliary proteins in renal epithelial cells derived from human ADPKD cysts. Consistent with finding of recent studies, we can confirm that animal models not completely mimic the human disease. Mouse models induce polycystic kidney disease by genetic inactivation of one ciliary gene especially genes encoding IFT proteins.However, the genes encoding the proteins of IFT are rarely mutated in the human renal cystic cells. This study offered new insight into comprehensive mutation profile of ciliary genes in human ADPKD tissues. The results of our study suggested that the loss of the primary cilia in the human ADPKD tissues may be predominantly caused by defects of centrosomal proteins and kinesin family member 19. The defects of centrosomal proteins may be also the cause of chromosome imbalances, which are often present in human ADPKD tissues.The genetic defects of the KIF19 gene may be cause of the primary cilium elongation, which is a characteristic feature of the end-stage renal disease. This is thefirst study used of archived formalin-fixed and paraffin embedded tissues(FFPE) of ADPKD in order to determine mutation profile of ciliary genes by nextgeneration sequencing methods. An interestingfinding was the presence of only one somatic pathogenic variant in each individual ciliary gene. To determine whether they are “hotspot” mutations representing secondary somatic events in the development of the disease will require further analysis. Somatic mutations in genes encoding centrosomal proteins and KIF19 were present in all analyzed ADPKD samples. These mutations were present exclusively in polycystic kidney tissues and did not occur in matched control so we can assume their effect on cystogenesis. If our hypotheses will be confirmed by further studies,the identified ciliary proteins may represent potential molecular targets for the development of new treatments.

Research perspectives

Mutation profile of ciliary genes can be analyzed directly from archived FFPE tissues of ADPKD by NGS. Thefirst step, it is necessary to verify the results on a larger number of samples and matched tissues controls. Further research should be focused on the functional analysis of identified genetic variants.Consequently, it will be necessary to determine whether the inactivation of these genes will lead to a change in the structure of renal cilia and affect the development or progression of ADPKD.

Interestingly, the pathogenic variants in genes encoding proteins of retrograde IFT and other structural ciliary genes were detected in only one of the ADPKD samples. The coverage of protein-coding regions and theflanking regions of all analyzed IFT genes showed high values in each of the ADPKD samples.

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肇庆中院经审理认为，被告人邓强为国家工作人员，利用职务便利，为他人谋取利益，非法收受他人财物，数额巨大，其行为已构成受贿罪。被告人邓强在接受调查期间除如实交代收受林中伟贿赂的事实外，还主动交代了办案机关尚未掌握的同类犯罪事实，且归案后已退清受贿的赃款，依法可对其从轻处罚。

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4 Porath B, Gainullin VG, Cornec-Le Gall E, Dillinger EK, Heyer CM, Hopp K, Edwards ME, Madsen CD, Mauritz SR, Banks CJ,Baheti S, Reddy B, Herrero JI, Bañales JM, Hogan MC, Tasic V,Watnick TJ, Chapman AB, Vigneau C, Lavainne F, Audrézet MP,Ferec C, Le Meur Y, Torres VE; Genkyst Study Group, HALT Progression of Polycystic Kidney Disease Group; Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease, Harris PC. Mutations in GANAB, Encoding the Glucosidase IIα Subunit,Cause Autosomal-Dominant Polycystic Kidney and Liver Disease.Am J Hum Genet 2016; 98: 1193-1207 [PMID: 27259053 DOI:10.1016/j.ajhg.2016.05.004]

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将异形刀片加工成形，安装到实际割草车上开展试验研究，验证其割草效果。虽刀片扭矩有一定下降，但刀口的切割速度依然很高，实际割草效果仍能达到要求。经试验测试结果表明，优化后刀片上的扭矩减小了18%左右，与仿真计算的误差小于5%，证明仿真结果有效。另外刀片功率减小了216 W，节能约8.5%，达到了满意的节能效果。图15(a)是试验用割草车,图15(b)是优化后的异形刀片。

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8 Pazour GJ, Dickert BL, Vucica Y, Seeley ES, Rosenbaum JL,Witman GB, Cole DG. Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J Cell Biol 2000; 151: 709-718[PMID: 11062270]

如何确保学校近6万名师生的正常伙食供应，确保食品卫生安全呢？武汉理工大学后勤集团总经理、武汉理工大学食品药品安全工作站站长赵高山总结的六大机制很是到位。

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汉语不同于印欧语系的语言，印欧语系的语言主要用形态变化来表示语法意义，而汉语有大量的虚词，我们可以通过对某些虚词的研究了解汉语中虚词的发展特点。语气词对句子的语音起调节作用，表达不同的语气和感情色彩，战国楚简的出现，为我们研究语气词的发展提供了重要线索。

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19 Wang L, Lee K, Malonis R, Sanchez I, Dynlacht BD. Tethering of an E3 ligase by PCM1 regulates the abundance of centrosomal KIAA0586/Talpid3 and promotes ciliogenesis. Elife 2016; 5: 5[PMID: 27146717 DOI: 10.7554/eLife.12950]

预锯缝是指与下层接缝对应，预先在加铺层锯缝，放置加铺层其他地方开裂的一种技术。从理论的角度来说，这种技术仅适用于预防半刚性基层的反射裂缝，并在其中能够获得较好的效果。这主要是由于这种裂缝具有间距大且数量少的特点。加厚面罩对于较小面罩底部拉应力以及延长反射裂缝的时间具有重要的作用。但这2种方法均存在一定的局限，因而在实际的应用过程中，应综合采用多种技术，以预防反射裂缝的发生。

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2010年七夕情人节那天，两人的聊天记录长达十八页。李辉嗟叹：“我改变不了她，怎么办？她完全不是我想象的样子……”吴霞也很失落地说：“每次情人节、女人节、圣诞节、结婚纪念日，我都好像和自己心爱的人一起过，可江帆大多数时候都抛下我，陪客户应酬。到底是钱重要，还是我重要？江帆也不禁汗颜：“我对妻子实在呵护不够，她出轨我有责任。”

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ARRIVE guidelines statement: In our study, ARRIVE Guidelines have been adopted.

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