Citrin deficiency is an autosomal recessive disorder caused by mutations in the SLC25A13 gene and has two disease outcomes: adult-onset type II citrullinemia and neonatal intrahepatic cholestasis caused by citrin deficiency. The clinical appearance of these diseases is variable, ranging from almost no symptoms to coma, brain edema, and severe liver failure. Genetic testing for SLC25A13 mutations is essential for the diagnosis of citrin deficiency because chemical diagnoses are prohibitively difficult. Eleven SLC25A13 mutations account for 95% of the mutant alleles in Japanese patients with citrin deficiency. Therefore, a simple test for these mutations is desirable. We established a 1-hour, closed-tube assay for the 11 SLC25A13 mutations using real-time PCR. Each mutation site was amplified by PCR followed by a melting-curve analysis with adjacent hybridization probes (HybProbe, Roche). The 11 prevalent mutations were detected in seven PCR reactions. Six reactions were used to detect a single mutation each, and one reaction was used to detect five mutations that are clustered in a 21-bp region in exon 17. To test the reliability, we used this method to genotype blind DNA samples from 50 patients with citrin deficiency. Our results were in complete agreement those obtained using previously established methods. Furthermore, the mutations could be detected without difficulty using dried blood samples collected on filter paper. Therefore, this assay could be used for newborn screening and for facilitating the genetic diagnosis of citrin deficiency, especially in East Asian populations.
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