Abstract
Citrin deficiency caused by SLC25A13 genetic mutations is an autosomal recessive disease, and four prevalent mutations including c.851_854del, c.1638_1660dup, IVS6+5G>A, and IVS16ins3kb make up >80% of total pathogenic mutations within the Chinese population. However, suitable assays for detection of these mutations have not yet been developed for use in routine clinical practice. In the current study, a real-time PCR-based multicolor melting curve analysis (MMCA) was developed to detect the four prevalent mutations in one closed-tube reaction. The analytical and clinical performances were evaluated using artificial templates and clinical samples. All four mutations in the test samples were accurately genotyped via their labeling fluorophores and Tm values, and the standard deviations of Tm values were indicated to be <0.2°C. The limit of detection was estimated to be 500 diploid human genomes per reaction. The MMCA assay of 5,332 healthy newborns from southern China identified a total of 107 SLC25A13-mutation carriers, indicating a carrier rate of 2%. The genotypes of 107 carriers and 112 random non-carriers were validated using direct sequencing and Long-range PCR with 100% concordance. In conclusion, the assay developed in this study may potentially serve as a rapid genetic diagnostic tool for citrin deficiency.
Highlights
The SLC25A13 gene is localized on chromosome 7q21.3 and encodes citrin, which is a calcium-stimulated mitochondrial aspartate/glutamate carrier [1]
The artificial templates of 15 SLC25A13 genotypes were detected using the multicolor melting curve analysis (MMCA) assay, and the mutations were individually identified by their labeling fluorophores and Tm values
The results indicated that 107 newborns were heterozygous for one of the four prevalent mutations, including c.851_854del that was identified in 77 newborns, c.1638_1660dup that was identified in 16 newborns, IVS6+5G>A that was identified in seven newborns and IVS16ins3kb that was identified in seven newborns (Table 3 and Supplementary Table 4)
Summary
The SLC25A13 gene is localized on chromosome 7q21.3 and encodes citrin, which is a calcium-stimulated mitochondrial aspartate/glutamate carrier [1]. Citrin deficiency that is caused by biallelic SLC25A13 mutations is an autosomal recessive disease and can present as neonatal intrahepatic cholestasis (NICCD; OMIM, 605814) in infants or as adult-onset citrullinemia type II (CTLN2; OMIM, 603471) in adolescents and adults [4, 5]. Infants with NICCD are characterized by intrahepatic cholestasis, hepatomegaly, diffuse fatty liver, variable liver dysfunction, and hyperammonemia [6, 7]. In early infancy, these symptoms overlap with those of other cholestatic liver diseases, such as neonatal hepatitis and biliary atresia, making it difficult for clinicians to obtain a prompt, and accurate diagnosis. Patients with CTLN2 are characterized by exhibiting adult-onset symptoms, hyperammonemia, and a number of
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