探讨citrin缺陷所致新生儿肝内胆汁淤积症(NICCD)患儿SLC25A13基因IVS16ins3kb突变类型的第二代测序技术(NGS)数据分析特点。

选择2017年7月与2020年4月，浙江大学医学院附属金华医院儿科收治的符合NICCD临床诊断标准，采用染色体组分析工具箱(GATK)、XHMM、CNVkit软件，对患儿NGS靶向外显子捕获测序数据进行单核苷酸变异(SNV)、插入与缺失突变、拷贝数变异(CNV)分析，仅提示SLC25A13基因单个位点杂合突变的2例患儿(患儿1、2)为研究对象。采用回顾性分析方法，收集患儿1、2的临床病例资料，包括病史、实验室检查结果、基因检测结果、治疗与预后等。利用福君基因动态实验室信息管理系统3.0(FLIMS系统)，采用split read方法，对患儿1、2的NGS靶向外显子捕获测序原始数据(fastq格式)进行IVS16ins3kb突变类型分析，并采用长链、高保真PCR(LA-PCR)方法进行验证。本研究遵循的程序符合浙江大学医学院附属金华医院医学伦理委员会规定，并获得该委员会批准(审批文号：2018-119)。

①病史采集：患儿1(女性)与患儿2(男性)，分别因皮肤黄染2个月余与4个月余，于本院就诊，就诊时年龄分别为2个月+19 d、4个月+16 d，均为足月儿、均无家族遗传性疾病史。②实验室检查、治疗：入院时，对患儿1、2干血片采取串联质谱仪进行遗传代谢病检测结果显示，血液中瓜氨酸、甲硫氨酸等多种氨基酸含量显著增高；肘静脉血生化检查结果显示，血清氨、乳酸浓度及血清总胆红素(STB)、血清直接胆红素(SDB)、血清间接胆红素(SIB)、天冬氨酸氨基转移酶(AST)、γ-谷氨酰转移酶、碱性磷酸酶均异常增高。对患儿1、2均采取无乳糖配方奶喂养3个月后，血液生化指标均明显好转。③NGS靶向外显子捕获及Sanger测序法验证结果：采用GATK、XHMM、CNVki软件对患儿1、2的NGS靶向外显子捕获测序数据进行SNV、插入与缺失突变、CNV分析结果提示，分别存在SLC25A13基因9号外显子c.852_855delTATG、16号外显子c.1638_1660dup杂合移码、功能缺失性热点突变，经Sanger测序法验证，上述突变分别遗传自患儿1、2父亲。④使用split read方法，对患儿1、2的NGS靶向外显子捕获测序原始数据(fastq格式)进行分析发现，SLC25A13基因16号内含子区域特征性split read一端为6号染色体DNA序列，另一端为7号染色体DNA序列，SLC25A13基因16号内含子区域特征性split read发生结构变异，可能为7号染色体SLC25A13基因发生IVS16ins3kb插入突变。⑤采用LA-PCR方法验证结果提示，患儿1、2分别存在SLC25A13基因c.852_855delTATG+IVS16ins3kb及c.1638_1660dup+IVS16ins3kb复合杂合热点突变，均被确诊为NICCD患儿。

对于NGS结果呈阴性或检出SLC25A13基因单个位点杂合突变的临床疑似NICCD患儿，可采用split read方法，对其NGS原始数据(fastq格式)进一步进行IVS16ins3kb突变分析，降低对该病患儿漏诊及误诊率。

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.