Development of a quantitative real-time PCR-based newborn screening system for citrin deficiency using dried blood spots

Citrin deficiency is an autosomal recessive metabolic disorder caused by pathogenic variants in SLC25A13 , which manifests as an age-dependent spectrum ranging from neonatal intrahepatic cholestasis (NICCD) to adolescent and adult citrin deficiency (AACD, formerly CTLN2). Early detection through newborn screening (NBS) would be a prerequisite for early treatment, but conventional NBS based on citrulline and other metabolite levels has only limited sensitivity. This study aimed to develop a simple and accurate quantitative real-time PCR (qPCR)–based newborn screening (NBS) method using dried blood spots (DBSs) as a first-tier screening test to detect the six most common SLC25A13 variants in Japan. DBS samples from 1055 healthy newborns and 19 patients with confirmed variants were analyzed. Four variants (c.674C > A, c.852_855delTATG, c.1177 + 1G > A, and c.1311 + 1G > A) were screened using the TaqMan™ SNP genotyping system, and two large insertion/deletion variants (c.1638_1660dup and c.1750_1751[insNM_138459.3:2672_24;1750 + 72_1751-4dup]) were screened using a SYBR™ Green–based qPCR system with variant-specific primers. Results of qPCR were confirmed by Sanger sequencing. In all 19 known patients with citrin deficiency, the assay correctly identified the pathogenic alleles. Among 1055 newborns, heterozygous variants were detected in 19 cases (five c.852_855delTATG, thirteen c.1177 + 1G > A, and one c.1311 + 1G > A). The overall allele frequency (0.0090) was consistent with that reported in the Japanese genome database (jMorp, 0.0102), corresponding to an estimated carrier frequency of approximately 1 in 56 individuals. The assay reliably distinguished wild-type, heterozygous, and homozygous genotypes using a single DBS punch. We established a cost-effective, rapid, and reliable qPCR-based NBS system that detects the six most prevalent SLC25A13 variants using DBSs. This method is compatible with current NBS workflows and could improve early detection and intervention for citrin deficiency in Japan and, with adaptation of the target variants, in any other (not only) East Asian population. • Developed a qPCR-based newborn screening method to detect the six most prevalent SLC25A13 variants in Japan. • Successfully analyzed 1055 newborn dried blood spots and 19 genetically confirmed patient samples. • Allele frequencies matched Japanese genomic data, confirming reliability of the assay. • Method is rapid, cost-effective, and compatible with existing newborn screening workflows for citrin deficiency.