Recurrent deletion in glycine decarboxylase gene and nonketotic hyperglycinemia Medical genetics, Research

甘氨酸脱羧酶基因(GLDC)突变导致非酮症高血糖(NKH)，这是一种以严重神经障碍为特征的先天性代谢错误。我们确定了GLDC及其假基因(PsiGLDC)的结构，并研究了它们在NKH分子分析中的表达。GLDC基因全长135 kb，由25个外显子组成。除内含子21的供体位点使用变异型GC而不是GT外，所有供体和受体位点都遵循典型的GT-AG规则。通过引物延伸分析，将转录起始点定位于翻译起始三联体上游163bp的残基。PsiGLDC基因没有内含子，与功能性GLDC编码区的同源性为97.5%，表明psiGLDC是一个加工过的假基因，起源于大约400~800万年前的GLDC转录本。RNA印迹分析表明，GLDC在人的肝、肾、脑和胎盘中均有表达。然后我们检查了一名NKH患者，他的淋巴母细胞中没有检测到GLDC mRNA。此例患者的GLDC功能基因外显子1~3未扩增，而对照组扩增。这些结果表明该患者存在较大的纯合缺失(至少30kb)。然后以psiGLDC为内对照，设计一种半定量聚合酶链式反应来估计GLDC等位基因的数量，并分别确认患者及其父母的缺失纯合性和杂合性。GLDC和psiGLDC的结构信息有助于NKH的分子分析。

Mutations in glycine decarboxylase gene (GLDC) cause nonketotic hyperglycinemia (NKH), an inborn error of metabolism characterized by severe neurological disturbance. We determined the structures of GLDC and its pseudogene (psiGLDC) and studied their expression in molecular analysis of NKH. The GLDC gene spanned a least 135 kb and consisted of 25 exons. All donor and acceptor sites adhered to the canonical GT-AG rule except for the donor site of intron 21, where a variant form GC was used instead of GT. The transcription initiation site was assigned to a residue 163 bp upstream from the translation initiation triplet by primer extension analysis. The psiGLDC gene had no intron and shared 97.5% homology with the coding region of functional GLDC, suggesting that psiGLDC ia a processed pseudogene that arose from the GLDC transcript about 4 to 8 million years ago. RNA blotting analysis revealed that GLDC is expressed in human liver, kidney, brain and placenta. We then examined a patient with NKH with no detectable GLDC mRNA in his lymphoblasts. Exons 1 to 3 of the functional GLDC gene from this patient were not amplified by PCR, whereas those from control subjects were amplified. These results suggest a large homozygous deletion (at least 30 kb) in the patient. We then devise a semi-quantitative PCR to estimate the number of GLDC alleles using psiGLDC as an internal control, and confirmed the homozygosity and heterozygosity of the deletion in the patient and his parents, respectively. Structural information of GLDC and psiGLDC should facilitate the molecular analysis of NKH.