Background

Citrullinemia type I (CTLN1, MIM #215700) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate synthase (ASS). CTLN1 is characterized by life-threatening hyperammonemia and risk for resulting neurocognitive impairments. The diagnosis of CTLN1 is confirmed by the identification of biallelic pathogenic variants in the ASS1 gene. However, there are a small percentage of CTLN1 patients with a characteristic biochemical phenotype without identifiable variants in ASS1. We describe the molecular characterization of two related Romani children with biochemically diagnosed CTLN1, whose clinical genetic testing failed to detect any pathogenic variant in ASS1.

Methods

Genomic DNA was extracted from peripheral blood lymphocytes collected from both patients. Sanger sequencing was performed after PCR amplifications of 5′- and 3′-untranslated regions of the ASS1 gene. A luciferase reporter assay was performed using the human malignant melanoma A2058 cell line and the human liver cancer cell line HepG2.

Results

We interrogated the non-coding regions of ASS1 by targeted PCR amplification and identified a homozygous 477-bp microdeletion in the promoter region of the ASS1 gene in both patients. Heterozygosity of the variant was confirmed in their parents. Sanger sequencing confirmed the microdeletion contained the entire sequence of the non-coding exon 1 of ASS1 that includes promoter elements of GC-box, E-box, AP2-binding site, and TATA-box. Luciferase reporter assay using an expression plasmid containing the wild-type or mutant ASS1 sequences showed robust reporter expression from the wild-type sequence and significantly reduced expression driven by the mutant insert (3.6% in A2058 cells and 3.3% in HepG2 cells). These findings were consistent with the hypothesis that the microdeletion identified in the patients disrupted an essential promoter element and resulted in deficiency of ASS1 mRNA expression.

Conclusions

This is the first report of CTLN1 patients caused by a Romani microdeletion variant affecting the non-coding upstream sequence of ASS1. Ablation of the promoter sequence can cause CTLN1 by the reduction of ASS1 expression. Currently available clinical sequencing methods usually do not cover the promoter sequence including the non-coding exon of ASS1, highlighting the importance of evaluating this region in genetic testing for CTLN1.

中文翻译：

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ASS1 启动子序列中的一种新型罗马微缺失变异会导致 I 型瓜氨酸血症。

 背景

I 型瓜氨酸血症（CTLN1，MIM #215700）是一种常染色体隐性遗传尿素循环障碍，由精氨基琥珀酸合酶 (ASS) 缺乏引起。 CTLN1 的特点是危及生命的高氨血症和由此导致的神经认知障碍的风险。 CTLN1 的诊断通过ASS1基因中双等位基因致病变异的鉴定得到证实。然而，有一小部分 CTLN1 患者具有特征性生化表型，但ASS1中没有可识别的变异。我们描述了两名经生化诊断为 CTLN1 的相关罗姆儿童的分子特征，他们的临床基因测试未能检测到ASS1的任何致病性变异。

 方法

从两名患者的外周血淋巴细胞中提取基因组 DNA。对ASS1基因的 5'-和 3'-非翻译区进行 PCR 扩增后进行 Sanger 测序。使用人恶性黑色素瘤 A2058 细胞系和人肝癌细胞系 HepG2 进行荧光素酶报告基因测定。

 结果

我们通过靶向 PCR 扩增检测了ASS1的非编码区，并在两名患者的ASS1基因启动子区发现了纯合的 477 bp 微缺失。该变异的杂合性在其父母中得到证实。桑格测序证实，微缺失包含ASS1非编码外显子 1 的完整序列，其中包括 GC-box、E-box、AP2 结合位点和 TATA-box 启动子元件。使用含有野生型或突变型ASS1序列的表达质粒进行荧光素酶报告基因检测，显示野生型序列的报告基因表达强劲，并且突变插入片段驱动的表达显着降低（A2058 细胞中为 3.6%，HepG2 细胞中为 3.3%）。这些发现与患者体内发现的微缺失破坏了必需的启动子元件并导致ASS1 mRNA 表达缺陷的假设相一致。

 结论

这是首例由影响ASS1非编码上游序列的 Roman 微缺失变异引起的 CTLN1 患者的报告。启动子序列的消融可通过减少ASS1表达而引起 CTLN1。目前可用的临床测序方法通常不覆盖包括ASS1非编码外显子在内的启动子序列，凸显了评估该区域在CTLN1基因检测中的重要性。