Identification Of The Novel Mutation IVS4ins6kb And Its Application In The Molecular Diagnosis Of NICCD Patients: Based On CDNA Cloning Analysis Of SLC25A13Gene In Peripheral Blood Lymphocytes

Objective: Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency （NICCD） is anautosomal recessive disease resulting from mutations of SLC25A13gene, which encodescitrin, an aspartate/glutamate carrier. The identification of biallelic mutations in SLC25A13isconclusive. However, conventional SLC25A13genetic analysis, such as PCR/PCR-RFLP, andDNA direct sequencing, can not reveal all mutations. This study was aimed:1) to investigatethe sequence feature of the SLC25A13transcripts in human peripheral blood lymphocytes（PBLs）,2) to identify novel SLC25A13mutations in NICCD patients,3) to developcorresponding molecular approaches for the definite diagnosis of NICCD patients.Subjects and methods: The research subjects encompassed9suspected NICCD patientsalong with their parents, with only one SLC25A13mutation revealed by conventional DNAanalysis in every patient. Ten healthy volunteers were enrolled as normal control. To explorethe sequence feature of the SLC25A13transcripts, total RNA was extracted from the PBLs,cDNA synthesized via reverse transcription, the coding region of SLC25A13cDNA amplifiedby nest PCR, and then the PCR products cloned and sequenced. Based on the abnormalsplicing feature of SLC25A13cDNA in the patient C0054, the DNA span with unknownmutation was positioned and further narrowed down by means of single nucleotidepolymorphism （SNP） analyses. The novel mutation was finally identified by LA-PCRamplification and direct sequencing. An LA-PCR diagnostic approach was developed for thenovel mutation, and was used into the screening of the mutation in the remaining8familieswith suspected NICCD patients. The clinical presentations and biochemical alterations wereexplored in the NICCD patients with definite diagnosis.Results:①134cDNA clones were randomly selected from10healthy volunteers. A total of21alternative splice variants （ASVs） were identified. Among them, ASVs with exon4skipping took account for37.31%, followed by the normal transcript carrying entire ORF,27.61%, and the remaining19ASVs,35.08%.②Among the total27cDNA clones selectedrandomly from the patient C0054, only3clones carried the paternally-inherited1399C>Tmutation.11ASVs were identified from the remaining24maternally-inherited cDNA clones, and95.8%of the AVSs with maternal origin demonstrated exon5skipping （r.329₄68del）.③Seven SNPs within intron4and5, i.e. rs6465494（A/G）, rs4727337（A/G）, rs67843496（AA/--）, rs6943325（C/T）, rs13307036（C/T）, rs6465490（C/A） and rs4273779（G/A）, wereanalyzed by direct sequencing. The SNP rs67843496was heterozygous （AA/--） in the PCRproduct amplified with primers IVS4S3and IVS4A3, but homozygous （AA） with primersIVS4S3and5R.④The products of LA-PCR amplification separated by electrophoresis ofagarose gel demonstrated that the patient C0054and his mother both had a large insertion of6kb （IVS4ins6kb）. It was proved by direct sequencing to be a DNA transposal insertionderiving from16p11.2（chromosome16:33755032-33761088,6057bp） and locating beforethe dinucleotide AG at the3’ end of intron4.⑤The screening for the mutation IVS4ins6kb inthe9families with suspected NICCD patients revealed that there were5NICCD patientsconfirmed to have this novel mutation. Their clinical presentations and biochemicalalterations were similar to those reported in previous publications.Conclusions: The investigation of the sequence feature of the SLC25A13transcripts in PBLsof10healthy volunteers uncovered21novel ASVs, which enriched the scientific knowledgeon the transcriptional features of the SLC25A13gene in human PBLs. The identification ofthe novel mutation IVS4ins6kb expanded the mutation spectrum of SLC25A13gene, andsuggested that SLC25A13cDNA cloning and SNP analysis in human PBLs could beconsidered as feasible tools for the identification of large insertions. Moreover, the LA-PCRdiagnostic procedure established in this research provided valuable molecular tool not onlyfor the definite diagnosis of NICCD patients, but also for further molecular epidemiologicinvestigation on this mutation in Chinese population.