To explore characteristics of next-generation sequencing (NGS) data analysis of SLC25A13 gene IVS16ins3kb variation in children with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD).

On July 2017 and April 2020, two cases of clinically diagnosed NICCD (case 1 and case 2) who had only single locus heterozygous variation of SLC25A13 gene confirmed by single nucleotide variation (SNV), insertion and deletion variations, and copy number variation (CNV) analysis of NGS targeted exon capture sequencing data using genome analysis toolbox (GATK), XHMM, and CNVkit softwares were selected as research subjects. Clinical data of 2 children, including medical history, laboratory test results, genetic test results, treatment and prognosis were collected by retrospective analysis method. With use of Fujun Gene FLIMS system (Fujun Gene Dynamic Laboratory Information Management System 3.0), split read method was used to analyzed IVS16ins3kb variation in original data of NGS targeted exon capture sequencing (fastq format) in case 1 and case 2, and long and accurate PCR (LA-PCR) method was used to prove IVS16ins3kb variation. This study was approved by the Medical Ethics Committee of Affiliated Jinhua Hospital, Zhejiang University School of Medicine (Approval No. 2018-119).

①Medical history: case 1 (female) and case 2 (male) were treated in our hospital due to xanthochromia for more than 2 months and more than 4 months, respectively. Their ages at admission to hospital were 2-month-19-day and 4-month-16-day, respectively. Both two were full-term infants and had no family history of hereditary diseases. ②Laboratory test results, treatment and prognosis: citrulline, methionine and other amino acids of case 1 and case 2 were significantly increased by blood stasis tandem mass spectrometry genetic metabolic disease test. Serum ammonia and lactate concentrations were increased, and serum total bilirubin (STB), serum direct bilirubin (SDB), serum indirect bilirubin (SIB), aspartate aminotransferase (AST), γ-glutamyl transpeptidase, alkaline phosphatase were anomaly increased by ulnar vein blood biochemistry test. After 3 months of lactose-free formula feeding, blood biochemical indexes of case 1 and case 2 were significantly improved. ③NGS targeted exon capture sequencing and Sanger sequencing results: case 1 had a heterozygous frameshift, loss-of-function hot spot variation c. 852_855delTATG in exon 9 of SLC25A13 gene, and case 2 had a heterozygous frameshift, loss-of-function hot spot variation c. 1638_1660dup in exon 16 of SLC25A13 gene by SNV, insertion and deletion variations, and CNV analysis of NGS targeted exon capture sequencing data analyzed by GATK, XHMM, and CNVkit softwares. The above two variations both inherited from their fathers. ④Split read analysis results of original data of NGS targeted exon capture sequencing (fastq format) of two cases showed that one end of characteristic split read in intron 16 region of SLC25A13 gene was the DNA sequence of chromosome 6, and the other end was the DNA sequence of chromosome 7, structural variation occurred in characteristic split read in intron 16 region of SLC25A13 gene which might be IVS16ins3kb insertion variation of SLC25A13 gene in chromosome 7. ⑤Case 1 had c. 852_855delTATG + IVS16ins3kb compound heterozygous hot spot mutations of SLC25A13 gene, and case 2 had c. 1638_1660dup + IVS16ins3kb compound heterozygous hot spot mutations of SLC25A13 gene which were confirmed by LA-PCR method. Both of them were diagnosed as NICCD finally.

For children with clinically suspected NICCD who have a negative NGS result or detection of a single allelic variation of SLC25A13 gene, the original data of NGS (fastq format) can be further analyzed for IVS16ins3kb variation by split read method to reduce rate of missed diagnosis and misdiagnosis of NICCD.