RESUMO
[This corrects the article DOI: 10.1371/journal.pone.0238697.].
RESUMO
Niemann-Pick type C disease is a lysosomal storage disease affecting primarily the nervous system that results in premature death. Here we present the first report and investigation of Niemann-Pick type C disease in Australian Angus/Angus-cross calves. After a preliminary diagnosis of Niemann-Pick type C, samples from two affected calves and two obligate carriers were analysed using single nucleotide polymorphism genotyping and homozygosity mapping, and NPC1 was considered as a positional candidate gene. A likely causal missense variant on chromosome 24 in the NPC1 gene (NM_174758.2:c.2969C>G) was identified by Sanger sequencing of cDNA. SIFT analysis, protein alignment and protein modelling predicted the variant to be deleterious to protein function. Segregation of the variant with disease was confirmed in two additional affected calves and two obligate carrier dams. Genotyping of 403 animals from the original herd identified an estimated allele frequency of 3.5%. The Niemann-Pick type C phenotype was additionally confirmed via biochemical analysis of Lysotracker Green, cholesterol, sphingosine and glycosphingolipids in fibroblast cell cultures originating from two affected calves. The identification of a novel missense variant for Niemann-Pick type C disease in Angus/Angus-cross cattle will enable improved breeding and management of this disease in at-risk populations. The results from this study offer a unique opportunity to further the knowledge of human Niemann-Pick type C disease through the potential availability of a bovine model of disease.
Assuntos
Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Células Cultivadas , Toxina da Cólera/metabolismo , Colesterol/metabolismo , DNA Complementar/genética , Modelos Animais de Doenças , Fibroblastos/patologia , Gangliosídeo G(M1)/metabolismo , Homozigoto , Mutação/genética , Proteína C1 de Niemann-Pick/química , Proteína C1 de Niemann-Pick/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Polissacarídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Improvements to restriction fragment length polymorphism (RFLP)-based genotyping assays currently used for detection of mutations responsible for bovine ferrochelatase and myophosphorylase deficiencies, and equine hyperkalemic periodic paralysis (HYPP) are described. Reports of sporadic inhibition of restriction enzyme activity suggest a critical factor in RFLP-based genotyping assays should be assurance that restriction enzymes perform to specification with every sample. The RFLP genotyping assays that use either a mismatched recognition sequence in one or both of the oligonucleotides, or incorporate a second native site within the PCR amplicon, provide the mechanism by which efficiency of restriction enzymes can be assessed with every sample. The outcome is confirmation of the activity of the discriminating enzyme regardless of genotype.