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      Modification of PCR Conditions and Design of Exon-Specific Primers for the Efficient Molecular Diagnosis of PKD1 Mutations

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          Abstract

          Background/Aims : Autosomal-dominant polycystic kidney disease (ADPKD) is a heterogeneous genetic disorder caused by mutations in the PKD1 and PKD2 genes. Currently, long-range PCR followed by nested PCR and sequencing (LRNS) is the gold standard approach for PKD1 testing. However, LRNS is complicated by the high structural and sequence complexity of PKD1, which makes the procedure for amplification and analysis of PKD1 difficult. Methods: Here in, we modified the PCR conditions and designed primers for efficient and specific amplification of both the long-range and individual exons of PKD1. Results: Using the modified system, seven long-range fragments were specifically amplified using two distinct sets of conditions, and all individual exon PCR assays were easily performed using a touch-down PCR method. Seven pathogenic or likely pathogenic variants, including two novel truncated frameshift indels and two novel likely pathogenic missense mutations, were identified in eight unrelated patients with or without histories of ADPKD disease (one variant was observed in two unrelated patients). Using combined bioinformatics tools, two indeterminate missense variants were identified in two sporadic patients. Conclusion: Four novel PKD1 variants were identified in this study. We demonstrated that the modified LRNS method achieves high sensitivity and specificity for detecting pathogenic variants of ADPKD.

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          Most cited references 29

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          Autosomal dominant polycystic kidney disease.

          Autosomal dominant polycystic kidney disease is the most prevalent, potentially lethal, monogenic disorder. It is associated with large interfamilial and intrafamilial variability, which can be explained to a large extent by its genetic heterogeneity and modifier genes. An increased understanding of the disorder's underlying genetic, molecular, and cellular mechanisms and a better appreciation of its progression and systemic manifestations have laid out the foundation for the development of clinical trials and potentially effective treatments.
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            PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.

            A second gene for autosomal dominant polycystic kidney disease was identified by positional cloning. Nonsense mutations in this gene (PKD2) segregated with the disease in three PKD2 families. The predicted 968-amino acid sequence of the PKD2 gene product has six transmembrane spans with intracellular amino- and carboxyl-termini. The PKD2 protein has amino acid similarity with PKD1, the Caenorhabditis elegans homolog of PKD1, and the family of voltage-activated calcium (and sodium) channels, and it contains a potential calcium-binding domain.
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              Unified criteria for ultrasonographic diagnosis of ADPKD.

              Individuals who are at risk for autosomal dominant polycystic kidney disease are often screened by ultrasound using diagnostic criteria derived from individuals with mutations in PKD1. Families with mutations in PKD2 typically have less severe disease, suggesting a potential need for different diagnostic criteria. In this study, 577 and 371 at-risk individuals from 58 PKD1 and 39 PKD2 families, respectively, were assessed by renal ultrasound and molecular genotyping. Using sensitivity data derived from genetically affected individuals and specificity data derived from genetically unaffected individuals, various diagnostic criteria were compared. In addition, data sets were created to simulate the PKD1 and PKD2 case mix expected in practice to evaluate the performance of diagnostic criteria for families of unknown genotype. The diagnostic criteria currently in use performed suboptimally for individuals with mutations in PKD2 as a result of reduced test sensitivity. In families of unknown genotype, the presence of three or more (unilateral or bilateral) renal cysts is sufficient for establishing the diagnosis in individuals aged 15 to 39 y, two or more cysts in each kidney is sufficient for individuals aged 40 to 59 y, and four or more cysts in each kidney is required for individuals > or = 60 yr. Conversely, fewer than two renal cysts in at-risk individuals aged > or = 40 yr is sufficient to exclude the disease. These unified diagnostic criteria will be useful for testing individuals who are at risk for autosomal dominant polycystic kidney disease in the usual clinical setting in which molecular genotyping is seldom performed.
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                Author and article information

                Journal
                KBR
                Kidney Blood Press Res
                10.1159/issn.1420-4096
                Kidney and Blood Pressure Research
                S. Karger AG
                1420-4096
                1423-0143
                2014
                December 2014
                30 November 2014
                : 39
                : 6
                : 536-545
                Affiliations
                aGraduate school, Southern Medical University; bKey Laboratory for Reproduction and Genetics of Guangdong Higher Education Institutes, Key Laboratory for Major Obstetric Diseases of Guangdong Province, Third Affiliated Hospital of Guangzhou Medical University; cDepartment of Clinical Laboratory, General Hospital of Guangzhou Military Command of People's Liberation Army, Guangzhou 510515, China
                Author notes
                *Xiaofang Sun and YuLing Shi, Third Affiliated Hospital of Guangzhou Medical University, Tel. +86-20-81292013;, Graduate school, Southern Medical University; General Hospital of Guangzhou Military, Command of People‘s Liberation Army, Tel. +86-20-88653462, Guangzhou 510515,, (China), E-Mail xiaofangsun@hotmail.com and yulingshi@163.com
                Article
                368464 Kidney Blood Press Res 2014;39:536-545
                10.1159/000368464
                25531466
                © 2014 S. Karger AG, Basel

                Open Access License: This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) ( http://www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Pages: 10
                Categories
                Original Paper

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