Computational protocol: Identification of novel mutations in Chinese Hans with autosomal dominant polycystic kidney disease

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Protocol publication

[…] Frame-shifting deletions or insertions, nonsense, typical splicing and in-frame changes of five or more amino acids were defined as pathogenic mutations in this study []. Pathogenic potential of missense, intronic changes and synonymous were evaluated using a method recommended by Tan et al.Firstly, gene variations were classified by analyzing recurrence as reported in the literature, ADPKD mutation database (PKDB) [] and Single Nucleotide Polymorphism database (dbSNP). Secondly, novel variations and previous unclassified variations were checked in family members available and 100 unrelated normal controls. Variations found in unaffected family members or unrelated normal controls were classified as polymorphisms. Then, the functional significance of the remaining unclassified variations was evaluated computationally using web-based software programs. SIFT and PolyPhen-2 were used to predict possible impact of substitutions on protein function and/or structure [-]. The Align-GVGD program was used to determine the Grantham Matrix Score (GMS) for evaluating evolutionary conservation (Grantham Variation[GV]) and chemical differences of resulting amino acid substitutions (the Grantham Distance[GD]) [-]. Potential splice-site effects were predicted using NNSplice and NetGene2 with default settings for missense, synonymous, and intronic changes [-].All variations analyzed by these web-based software programs were finally sorted into four categories: 1) probable pathogenic; 2) indeterminate; 3) probable polymorphism; and 4) polymorphism. Only gene variations that were unanimously predicted to be deleterious by SIFT, PolyPhen-2 and Align-GVGD or to affect splicing by NNSplice and NetGene2 were considered to be "probably pathogenic", if no other definite mutation was found in the same patient. If a definite mutation coexisted with a deleterious missense change or a likely atypical splicing variation in the same patient, the missense change and the atypical splicing variation were considered to be "indeterminate". Similarly, only variations that were scored as begin or predicted to have no effect on splicing by all corresponding applications were considered to be "polymorphisms". Otherwise, they were classified as "probable polymorphisms". […]

Pipeline specifications

Software tools SIFT, PolyPhen, Align-GVGD, NNSplice, NetGene2
Databases dbSNP PKDB
Application WGS analysis
Organisms Homo sapiens
Diseases Kidney Diseases