Establishment And Application Of Diagnostic System For Gene Mutation Analysis Of Hereditary Polycystic Kidney Disease

Polycystic kidney disease (Polycystic Kidney Disease, PKD) is a common, monogenic disease with multi-systemic disorders. Depending on the different types of inheritance, PKD is inherited as an autosomal dominant polycystic kidney disease (Autosomal Dominant Polycystic Kidney Disease, ADPKD) or autosomal recessive polycystic kidney disease (Autosomal Recessive Polycystic Kidney Disease, ARPKD). The disease incidence in PKD is high. For example, ADPKD is the most common disease with an incidence of 1 in 400 to 1 in 1000 [1,2]. ARPKD has an incidence rate of 1 in 20000 live births. At present, there are about 12 million patients with polycystic kidney disease in the world. ADPKD is characterized by progressive development of renal cysts, which leads to about 50% of ADPKD patients eventually develop into end-stage renal disease (ESKD), leaving hemodialysis or kidney transplant as the only available treatment options at this stage [3]. In addition to renal cysts, ADPKD usually associated with a variety of additional cysts in the liver, seminal vesicles, pancreas and arachnoids membranes, and the intracranial aneurysm prevalence is about five times higher than the normal population. ARPKD is also known as infantile polycystic kidney disease. There is about 30-50% of the affected fetus dead because of the early onset of disease and oligoamnios. Thus PKD is a severe disease with significantly higher incidence and higher mortality rate than other disease [4]. Polycystic kidney disease, especially autosomal dominant polycystic kidney disease is often associated with a disease family history, and the affected individuals are burdened heavily both in mental and financial.To date, the mainly diagnosis methods of polycystic kidney disease are based on ultrasound, CT scan and other imaging tests. Although most of ADPKD patients are typically associated with disease family history and those patients are more easily to be diagnosed by imaging tests, genetic diagnosis of PKD is more important for a clear clarify the different type of PKD and plays a more critical role in prognosis and prenatal diagnosis of PKD. Previous studies have shown that the pathogenic mutations in ADPKD are usually caused by two genes, PKD1 and PKD2, which are located at 16p3.3 and 4q21 region, respectively. The protein produced by the two genes are polycystic protein 1 (Polycystin-1, PC1) and polycystin-2 (Polycystin- 2, PC2) [5,6], respectively. PC1 is a membrane protein with a function of regulating cell-cell interactions and PC2 is a nonspecific cation channel; ARPKD is caused by mutations in PKHD1 gene located at the 6p12 region. The gene product of PKHD1 is Fibrocystin/Polyductin (FPC).Point mutations are the major variations for ADPKD and ARPKD. Large deletion, duplication, or rearrangement events are very rare (3%-4%) reported in the PKD patients. Therefore, genetic diagnosis of polycystic kidney disease is mainly based on Sanger sequencing method. However, directly sequencing ADPKD mutations is more complicated than sequencing for mutations that cause other inherited diseases. The reasons for the difficult diagnosis of PKD, especially for ADPKD, are listed as followings:First, the disease is genetically heterogeneous with two causative genes, PKD1 and PKD2, mutations in the PKD1 gene account for 75% to 85% of ADPKD cases, whereas the remaining 15% of cases are due to mutations in PKD2. ADPKD cases caused by mutations in PKD1 appear to be more severe than those associated with PKD2 mutations. For example, an average time onset of renal failure occurs in patients with PKD1 mutations is 20 years earlier than the age of onset for those with PKD2 mutations [7,8]. Second, there is no hot spot mutations have been identified in the PKD1 and PKD2 genes, which means the entire protein-coding sequences of these two genes must be fully screened [9,10]. Third, the region between exon 1 and exon 33 of PKD1 has undergone intrachromosomal duplication throughout the human genome, which resulted in the generation of six homologous pseudogenes (PKD1P1-P6) that are proximally located between 13 and 16 Mb away from PKD1. These homologous pseudogenes share 98-99% sequence similarity with the PKD1 gene. In addition, the extreme enrichment of GC nucleotides (up to 70% or even 80%) was found in PKD1, which would further complicates the ability to perform efficient polymerase chain reaction (PCR) assays and sequencing reactions [11,12].Although there are variety of methods currently available for the gene mutation diagnosis of polycystic kidney disease, there are several technical flaws and shortcomings present in those methods, which hampered them for widely using. With the quickly development of next-generation sequencing technology (NGS), application of NGS in the diagnosis of genetic diseases is more and more frequently reported [13-15]. Since NGS technology can simultaneously processed massive DNA fragmentation, the efficiency of sequencing is much higher than the traditional Sanger sequencing method. In addition, low level mosaics mutations also can be identified by increasing the depth of the reads of sequencing. Therefore, the application of this new technology will be able to overcome the technical bottleneck of genetic diagnosis of polycystic kidney disease.ADPKD is a classical hereditary disease, and the onset age of ADPKD is always on 20-30 years old. Many patients been diagnosed after they got married. The risk ratio to inherited mutation to the next generation is 50%; therefore, those patients with ADPKD mutations have strongly willing to perform prenatal genetic diagnosis and hope to give a healthy birth. Therefore, prenatal diagnosis or preimplantation genetic diagnosis (PGD) for early diagnosis of ADPKD and other genetic diseases is a very important for early disease intervention. For proband mutation detection, prenatal diagnosis, or PGD for ADPKD patients, precise identify pathogenic allele is the most critical step, and entire exon and splicing site sequencing of PKD1, PKD2 and PKHD1 gene are required. In this study, we modified and established a new diagnostic system for quickly, efficiently and simplify genetic diagnosis of polycystic kidney disease. In addition, we also applied and assessed the next generation sequencing technology in the diagnostic of PKD mutations. In conclusion, we established an efficient, specifically and high-throughput diagnosis system for polycystic kidney disease and prenatal diagnosis application in this study.Chapter 1Establish the diagnosis system for polycystic kidney disease gene mutation detection and its application in prenatal diagnosisObjectivesThe purpose of this study is to establish a more convenient, efficient, specific and quickly method for gene mutation diagnosis of polycystic kidney disease. Using this system to identify genetic mutations of the probands and to apply this technique in prenatal diagnosis, which is useful for early prevention of polycystic kidney disease.By improving the traditional diagnostic methods, we want to simplify the reaction conditions as possible as we can. To achieve our aim, we designed specific primers for efficiently and specifically amplified whole PKD1 gene. A specific long-range PCR method was established, which is avoid contamination of pseudogene sequences, and the long-range PCR products can be used as templates for the following specific nested PCR; Different to the traditional method, we design individual exon-specific primers for the nested PCR step, which further reducing the non-specific products or whole genome contamination in the sequencing reaction. The established diagnosis system greatly improves the efficiency and accuracy for identification and prediction of PKD mutations.In this study we also established a unified touch-down amplification system for amplification all exon of PKD1, PKD2 and PKHD1 genes in a simple condition. Simple reaction condition for this reaction system will beneficial in the promotion and application of primary hospital. In addition, we also applied prenatal diagnosis for those PKD patients in this study. Whole genome chromosomal microarray analysis was also used for genetic prenatal diagnosis fetus with ultrasound abnormalities.Research MethodsCase Sources:1.32 cases of autosomal dominant polycystic kidney disease patients,4 cases of autosomal recessive polycystic kidney disease fetus and 42 fetus cases with ultrasound abnormalities in this study are come from the Third Affiliated Hospital of Guangzhou Medical University and General Hospital of Guangzhou Military Command of People’s Liberation Army. In addition,15 cases of ADPKD patients’ samples from Medical genetics Lab, Department of Health, HK were diagnosed using our system. All the probands were diagnosed as having PKD phenotype based upon renal ultrasound findings. All enrolled patients and their family members provided written informed consent;2. The mutation of probands was identified using our diagnosis system. Prenatal diagnosis was performed in pregnant women whom suffering from polycystic kidney disease or her husband is a polycystic kidney disease proband. Fetus who were suspected as ARPKD by ultrasound were also performed prenatal diagnosis, amniotic fluid or villi samples and their parents’ samples were collected for direct DNA detecting;3. Whole genome chromosomal microarray analysis was used for genetic prenatal diagnosis to the fetus with ultrasound abnormalities. Combined with polycystic kidney sequencing methods, we greatly improved prenatal genetic diagnosis system for detection fetus with PKD or other urinary abnormality.Experimental methods:1. Using online software PRIMER3, we directly designed all exons primers for PKD2 and PKHD1 genes. Via the free online website hosted by the University of California Santa Cruz (UCSC), we evaluated the targeted fragments of the PKD1 gene sequence using the BLAT program to find the minimal region that included nucleotide differences compared with the six pseudogenes, designed seven long-range PCR primers that specifically amplified the region between exon 1 and exon 34 of PKD1;2. In this study, the annealing temperature for all primers was designed near 60℃ to ensure that all fragment amplifications could be subjected to similar PCR conditions. To efficiently amplify the GC-rich long fragments, a long and accurate PCR kit was used in the present study; PCR products were verified by 0.5% agarose gel electrophoresis;3. PKD1 exon-specific PCR primers design:primer for individual exons amplification was also designed following the same principle applied during long-range primer design;4. For the individual exon amplifications, a selected touch-down PCR condition was the designed for quickly and specifically amplification of whole exons of PKD1, PKD2 and PKHD1 genes;5. Purification the amplified products and run sequencing PCR reactions using 3500XL Genetic Analyzer;6. Raw data were load into mutation Surveyor software for automatic analysis;7. Bioinformatics analysis:all sequencing results were evaluated via searching bioinformatics websites and databases, and interpretation of results was computationally evaluated using a combination of bioinformatics software;8. DNA-STR genotyping was performed for prenatal samples identification: During the chorionic villus sampling process, chorionic villi samples and maternal samples were identified by using the GoldeneyeTM DNA identification system. The mutation allele was directly sequenced.42 cases of fetus with urinary system abnormalities were analyzed by using whole genome chromosomal microarray.Results1. All exons primers of PKD2 and PKHD1 were specific designed, the annealing temperature of all primers are close to 60 ℃. In silico PCR predicted that all the primers are specific and unique;2. After inputting the targeted region sequence of PKD 1 into the BLAT program and identifying the mismatched sequence in the homologous pseudogenes, we designed seven long-range PCR primers (exon 1, exon 2-8, exon 9-12, exon 13-15, exon 15-21, exon 22-26, exon 27-34) that specifically amplified the region between exon 1 and exon 34 of PKD1. Individual exon primers were designed for the remaining exons (35-46 exons) because these exons have unique sequence;3. All products of long-range PCR were checked by electrophoresis. The size of each fragments consistent with the predicted size;4.56 primer pairs of primers that amplify individual PKD1 exons were designed following the same principle applied during long-range primer design; electrophoresis results shown that all these products are unique and specific;5. An optimized conditions were designed for amplification of individual exons, the touch-down PCR amplification system and conditions were established as a specific, efficient and easy method for all of the individual exons of all three genes amplification;6. To assess the effectiveness of the diagnosis system,10 ADPKD patients’ samples were first tested. According to the screening data generated from the 10 patients using our diagnosis system, all 10 patients presented disease-associated or likely disease-associated variants, demonstrating the high sensitivity and specificity of our system. Nine pathogenic or likely pathogenic variants, including two novel truncated frame shift indels and two novel likely pathogenic missense mutations, were identified in ten unrelated patients with ADPKD disease. Among those point mutations,7 were pathogenic mutations (one variant was observed in two unrelated patients),2 were predicted as likely pathogenic missense mutations. Additional 22 PKD patients’ samples were sequenced via our established system and 15 pathogenic mutations,7 likely pathogenic missense mutations were identified in this study;7. Genetic prenatal diagnosis was performed in 5 cases of ADPKD patients for directly detection of the mutated allele which had found in the proband. In addition, prenatal diagnosis of 4 cases with suspected ARPKD disease was also performed. STR results indicated that the fetus samples are not contaminated with their mother’s DNA.2 ADPKD cases shown abnormal results, which was coincident with the variants identified in their probands. Compound heterozygous mutations were identified in all ARPKD cases, with a 100% positive detection ratio. Whole chromosomal microarray results shown that 3 of 42 fetus cases have pathogenic copy number variations, who were shown urinary system abnormalities using ultrasound. The variants ratio is 7.14%.Conclusions1. In summary, we established a PKD diagnosis system for the efficient, sensitive and specific molecular diagnosis of PKD disease. Using exon-specific primers and modified PCR conditions, the pathogenic variant detection ratio exceeded 70%, and pathogenic and likely pathogenic variant detection ratio is about 90%;2. The established diagnosis system can efficiently, quickly and specifically detection all PKD1, PKD2 and PKHD1 gene mutations;3. Combined with information of ultrasound and bioinformatics tools, our system can provide accurate results for polycystic kidney disease genotype studying;4. Application of whole chromosomal microarray would improve the positive detection ratio in the genetic prenatal diagnosis procedure.Chapter 2Application of Next Generation sequencing method in genetic diagnosis of polycystic kidney diseaseObjectivesAlthough we have established a highly efficient and specific gene mutation detection system for polycystic kidney disease in the previous chapter, and this new system has greatly advantage when compared to the traditional detection methods, it still is a time-consuming, complex and hard work procedure. In addition, the traditional Sanger sequencing method cannot detect large deletion, duplication and complex rearrangement, and all these parts of variants only can be detected via Multiplex Ligation Dependent Probe Amplification (MLPA) method. Therefore, in this chapter, we apply high-throughput next generation sequencing technology (NGS) to identify PKD mutations. Whole exon sequencing and target gene NGS method were used to evaluation the genetic diagnosis results of PKD1, PKD2 and PKHD1 genes, including entire protein coding regions, splicing sites and other targeted gene sequence. The aim of this chapter is to establish a standard NGS procedure in clinical lab for quickly and high-throughput PKD gene mutation detecting, because this parallel sequencing method can performed with multiple samples and detected multiple genes, simultaneously. The application of NGS in PKD mutation diagnosis system significantly reduces the cost and save the time for PKD sequence analysis, which also greatly improve routine genetic diagnostics of PKD procedure. The results of NGS indicated that targeted NGS is an optima method for analyze gene variant profiles of monogenic diseases with high sensitivity and throughput.Research MethodsCase Sources5 cases of ADPKD and 2 cases of ARPKD DNA samples were collected in this chapter; all mutations of these samples have been identified via our diagnosis system. All samples are collected from the Third Affiliated Hospital of Guangzhou Medical University and General Hospital of Guangzhou Military Command of People’s Liberation Army. All enrolled patients and their family members provided written informed consent. Whole blood samples from those PKD probands were collected for NGS analysis. One library sample was captured by using both whole exome capture and targeted gene capture to compare the efficiency difference between these two methods for PKD mutation diagnosis. A case with low ratio mosaic variation was also detected by using NGS methodExperimental Methods1. Genomic DNA of whole blood samples were extracted;2. Using of the conventional diagnosis system which was established in the last chapter, all variants were double confirmed before NGS procedure;3. Fragment of genomic DNA:genomic DNA was sheared using Covaris S2 instrument, the genomic DNA was breaked into small fragments with a size of 200-300bp;4. Sample purification:Use Agencourt AMPure XP beads to purify all samples;5. Repair the ends, purification and add A-tailing on the 3 ’end of the DNA fragments;6. Ligate the indexing-specific paired-end adaptor;7. Amplify adaptor-ligated library;8. Assess library quantity via Qubit Fluorometer9. Pooling the samples and do whole exom capture for 72h;10. Targeted genes capture:pooled libraries were amplified by LM-PCR prior to the capture procedure and then those amplified libraries were captured by hybridized with custom capture array for 72h (NimbleGen, Roche);11. Purify and removing unbound sequence, the captured libraries were then eluted;12. Amplified captured libraries;13. Determine the quality and quantity of captured libraries prior to sequencing;14. Sequencing on Illumina HiSeq2000 platform for 90 cycles of sequencing per read to generate paired-end reads;15. Sequencing data analysis.Results1. All DNA samples were double checked the variants via our the previously established PKD diagnosis system;2. Quality assessment of libraries:quality of libraries was assessed by Tape Station 2200 instrument, the fragment size and molecular of libraries was all within the requirements;3. Pretreatment of sequencing data:the reads errors of low quality data were removed by application NGS software;4. NGS data analysis of target sequence capture method:there is a total of 173559 reads alignment to the targeted PKD1, PKD2 and PKHD1 genes, which was accounting for 0.229% of total alignment reads. The sequencing depth is from 0 (which means no valid sequence coverage) to the maximum depth of sequencing 2363. And the average depth is 190. Percent of region of interest with 5X coverage is about 85%;5. NGS data analysis of whole exon capture method:After removal of duplicate reads, there is only 14122 valid reads alignment to the targeted PKD1, PKD2 and PKHD1 genes, which was accounting for 0.027% of the total alignment reads. The sequencing depth is from 0 (which means no valid sequence coverage) to the maximum depth of sequencing 309. And the average depth is 28. Percent of region of interest with 5X coverage is about 55.4%;6. Coverage analysis:The average coverage of whole Exon sequencing in PKD genes regions is low, for example, the coverage of PKD1 exons is less than 40%; whereas the targeted capture method has a higher sequencing coverage, and the overall coverage of the three targeted genes is about 95%, the remaining 5% region of PKD1 exon can not cover entirely yet;7. The poor quality of sequencing data of whole exon capture method cannot effectively cover the whole targeted PKD genes region because most of reads matched to the repeated sequencing and those reads always been filtered out as sequencing errors or machine mistakes; whereas the quality of sequencing data of target sequence capture method is much better. It can detect all variations of the validation samples accurately;8. A low ratio mosaic variation was efficiently detected by using NGS method, and NGS method is more sensitive to quickly and accurately identify mosaics;9. Although it is not effective for PKD diagnosis when compared to targeted NGS method, whole exon sequencing is still sensitive to detect CNV and chromosomal aneuploidy.Conclusions1. In this study, we indicated that targeted NGS method is more suitable for PKD gene-based diagnostic NGS technology platform, while the whole exon capture method is has low sensitivity and poor data qualities;2. We have established an appropriate procedure for detection PKD gene mutation via using NGS method;3. For diagnostic of complex structural genes, or genes with high GC contents, NGS technology has its shortcomings. The uncovered region still needs using conventional Sanger sequencing for complement;4. Compared to the classical Sanger sequencing method, the advantages of NGS technology is it is a fast, low cost method with robust results. For large cohort study and analyze large genes, NGS technology is a preferred choice;5. Except for detecting point mutations and small insertions, deletions, NGS is also a good way to detect the gene copy number variation and chromosomal aneuploidy detection. It is sensitive to detect low ratio of mosaisiam; In conclusion, NGS is accuracy, sensitive and more suitable for hereditary disease detection.