Molecular diagnosis of pediatric patients with citrin deficiency in China: SLC25A13 mutation spectrum and the geographic distribution

Abstract

Citrin deficiency (CD) is a Mendelian disease due to biallelic mutations of SLC25A13 gene. Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is the major pediatric CD phenotype, and its definite diagnosis relies on SLC25A13 genetic analysis. China is a vast country with a huge population, but the SLC25A13 genotypic features of CD patients in our country remains far from being well clarified. Via sophisticated molecular analysis, this study diagnosed 154 new CD patients in mainland China and identified 9 novel deleterious SLC25A13 mutations, i.e. c.103A > G, [c.329 - 154_c.468 + 2352del2646; c.468 + 2392_c.468 + 2393ins23], c.493C > T, c.755 - 1G > C, c.845_c.848 + 1delG, c.933_c.933 + 1insGCAG, c.1381G > T, c.1452 + 1G > A and c.1706_1707delTA. Among the 274 CD patients diagnosed by our group thus far, 41 SLC25A13 mutations/variations were detected. The 7 mutations c.775C > T, c.851_854del4, c.1078C > T, IVS11 + 1G > A, c.1364G > T, c.1399C > T and IVS16ins3kb demonstrated significantly different geographic distribution. Among the total 53 identified genotypes, only c.851_854del4/c.851_854del4 and c.851_854del4/c.1399C > T presented different geographic distribution. The northern population had a higher level of SLC25A13 allelic heterogeneity than those in the south. These findings enriched the SLC25A13 mutation spectrum and brought new insights into the geographic distribution of the variations and genotypes, providing reliable evidences for NICCD definite diagnosis and for the determination of relevant molecular targets in different Chinese areas.

Figure 1. Native places of the 274 Chinese patients with citrin deficiency.

By the end of February in 2016, 274 Chinese patients from 26 provinces, municipalities and autonomous regions in China were diagnosed. This figure was generated by means of the software WPS Office PowerPoint 2016, which was freely available at http://www.wps.cn/product/wps2016/. The base map was created by incrementally assembling the outlines of the Chinese administrative regions, which could be downloaded via the URL link http://www.pptstore.net/ppt_yuansu/12145.html, as a free network resource.

Figure 2. Novel mutations and ASVs identified by direct DNA sequencing analysis and cDNA analysis.

The figure (a) showed the segmental DNA sequencing results of the 8 novel micro mutations. The figure (b) illustrated the cDNA sequencing results of the aberrant transcripts r.845_848delG, r.755_756delAG and r.933_934insGCAG, respectively, which were transcribed from the mutated alleles harboring c.845_c.848 + 1delG (C0221), c.755 − 1G > C (C0257) and c.933_c.933 + 1insGCAG (C0364), respectively.

Figure 3. Identification of the mutation [c.329−154_468 + 2352del2646bp; c.468 + 2392_468 + 2393ins23bp] in patient C0360.

(a) High-frequency mutation screening revealed the maternally-inherited mutation IVS16ins3kb. (b) Electrophoresis of the LA-PCR products with the primers covering exon 5 revealed an unexpected band of 1162 bp in size, which was inherited from the father. (c) Sanger sequencing of the 1162 bp product uncovered a 2646 bp deletion (sequences in blue), which spanned the entire exon 5 (capitals) and partial sequences of the introns 4 and 5. Meanwhile, a 23 bp insertion (sequences in red) which was 40nt behind the breakpoint was also discovered. Underlined were the primers for LA-PCR screening.

Figure 4. Functional analysis of the novel missense mutation c.103A > G (p.M35V).

After growing for 96 hours, the growth ability of the yeast strain pYX212-mutant (p.M35V) was not significantly different (P = 0.341) from that transfected with the empty vector pYX212 (vector). However, both of them had significantly lower (P = 0.000) growth ability in comparison to pYX212-citrin (citrin), the yeast strain transfected with normal citrin recombinant. The results in each group were means ± SD of six repeated experiments.