人羊水细胞中SLC25A13基因转录产物的克隆和序列分析

Cloning and sequence analysis of SLC25A13 transcripts in human amniocytes

摘要:

目的：克隆分析citrin蛋白编码基因SLC25A13在人羊水细胞中的mRNA编码区全长，并分析其转录子的序列特征，为从mRNA水平开展 Citrin 缺陷导致的新生儿肝内胆汁淤积症（NICCD）产前诊断提供实验依据。方法：选取1例接受 citrin 缺陷病产前诊断并已证实胎儿为851del4突变携带者的羊水标本；另1例取自无 citrin 缺陷病史患者的羊水细胞标本作为正常对照。抽提体外培养的羊水细胞总RNA，逆转录合成cDNA，通过巢式PCR扩增SLC25A13 cDNA编码区全长。PCR产物克隆后测序分析。结果：从2例羊水细胞标本中成功克隆到SLC25A13 cDNA编码区全长，并发现SLCA型转录子（正常型转录子mRNA）；在正常对照样本中发现 SLCB型转录子（外显子9和10之间CAG插入）；在851del4突变携带者标本中发现SLCC 型转录子（外显子5~11缺失），未发现含851del4突变等位基因转录产物。结论：SLC25A13 cDNA编码区全长可以从羊水细胞中扩增获得，而外显子5~11缺失型转录子是SLC25A13基因一种新的转录子。在正常对照和包含851del4突变的杂合子胎儿SLC25A13基因转录产物中正常mRNA占据优势，提示胎儿无罹患NICCD风险。这些转录特征可以为NICCD产前诊断提供实验依据。

Abstract:

OBJECTIVE: This research intends to amplify the entire coding region sequences of SLC25A13 mRNA which encodes citrin, and to investigate sequence features of the transcripts for this gene in cultured human amniocytes. This study will provide laboratory evidence for prenatal diagnosis of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) at mRNA level. METHODS: One amniocyte sample was collected from a pregnant woman who underwent prenatal diagnosis of citrin deficiency and whose fetus has proven a carrier of 851del4 mutation by genomic DNA analysis. Another amniocyte sample, as a control, was from a fetus without family history of citrin deficiency. Total RNA was extracted from cultured amniocytes, cDNA was synthesized, and then nested-PCR was performed to amplify the entire coding region sequences of SLC25A13. The PCR products were cloned and analyzed by sequencing. RESULTS: The entire coding region of SLC25A13 gene was successful amplified from two cultured human amniocytes. The splice variant of SLC25A13, SLCA (normal mRNA), was identified in the two samples. SLCB (CAG insertion between exon 9-10) was identified in the control. SLCC (exon 5-11 skipping), but not transcriptional product from the allele with 851del4 mutation, was identified in the 851del4 mutation carrier. CONCLUSIONS: This study demonstrated that the entire coding region of SLC25A13 cDNA can be successfully amplified from two cultured human amniocytes, and revealed exon 5-11 skipping as a novel SLC25A13 transcript. Normal mRNA predominated in the transcripts in normal control and 851del4 mutation carrier, suggesting that the two fetuses were not at risk for NICCD. These SLC25A13 transcription features provided laboratory evidence for prenatal diagnosis of NICCD.