Rapid Genetic Diagnosis of Citrin Deficiency by Multicolor Melting Curve Analysis

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.

One-step genotyping of α-thalassaemia by multiplex symmetric PCR melting curve

“…There were three technical keys to the success of this multiplex symmetric PCR melting curve strategy, including enough templates amplification, efficient probe hybridisation and typical melting curves 19–21. For templates amplification, asymmetric PCR was always used to prepare enough single-strand templates.…”

Rare liver diseases are not rare in China

“…So far, over 80 pathogenic variants have been identified in China, most of which are coding sequence mutations. C.851_854del (p.R284fs286X), c.1638_1660dup (p.A554fs570X), IVS6+5G>A (p.A206fs212X) and IVS16ins3kb (p.A584fs585X) are widely reported in mainland China, whereas c.852_855delTATG from 18 patients is more prevalent in Hong Kong, indicating a regional difference between SLC25A13 mutation spectra 23 …”

“…22 The overall mutation carrier rates among newborns could be as high as 2% (107/5332) in southern China. 23 In addition, the mutation carrier rate is 1/51 in Guangdong…”

“…patients is more prevalent in Hong Kong, indicating a regional difference between SLC25A13 mutation spectra. 23 In China, most reported cases manifest as NICCD with age varying from 0.3 to 7.5 years, but few cases of CTLN2 are reported.…”

“…The gene frequency of homozygotes or compound heterozygotes for SLC25A13 in Japan is 1:17 000 based on the carrier (heterozygote) rate of 1:65 22 . The overall mutation carrier rates among newborns could be as high as 2% (107/5332) in southern China 23 . In addition, the mutation carrier rate is 1/51 in Guangdong Province with an estimated CD prevalence of 1/10 053, whereas a lower carrier frequency (1/95) is observed in Shaanxi Province with a theoretical incidence of 1/35 865, 24 showing a latitudinal gradient with a higher prevalence reported in lower latitudes.…”