Characterisation of the human GFRa-3 locus and investigation of the gene in Hirschsprung disease
Characterisation of the human GFRa-3 locus and investigation of the gene in Hirschsprung disease
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DOI：
10.1136/jmg.37.9.674
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发表时间：
2000-09-01
影响因子：
4
通讯作者：
Mulligan, LM
中科院分区：
文献类型：
--
作者：
Onochie, CI;Korngut, LM;Mulligan, LM
背景-GDNF家族受体α（GFR α）蛋白是细胞外表面结合的分子，其在可溶性神经营养因子的GDNF家族与RET受体的结合中充当接头。这些分子对于许多神经嵴衍生细胞类型和肾脏的发育是必需的。RET和GDNF配体家族的两个成员的突变与先天性肠神经节缺失的先天性巨结肠病（HSCR）相关。GFR α家族的成员也是HSCR突变的候选者。一个这样的基因是GFR α-3，这是在周围神经系统和发育中的neuros. ESTA-我们已经表征了人类GFR α-3位点的结构，并调查了基因的序列变异在一个面板的HSCR patients. Methods-长距离PCR或PAC克隆的亚克隆被用来调查GFR α-3内含子-外显子边界。单链构象多态性（SSCP）分析和直接测序的组合被用来调查GFR α-3序列variants.Results-GFR α-3跨越8个编码外显子，并具有基因结构和组织类似的GFR α-1。我们在正常对照人群中发现了GFR α-3的三种多态性变异，其中一个亚组也发生在HSCR患者中。我们在GFR α-3的编码序列中没有检测到任何序列变异。我们在GFR α-3的5' UTR中发现了一个碱基替换，翻译起始位点上游15个碱基对。在内含子4（IVS 4 - 30 G>A）中在剪接分支位点和剪接受体位点之间鉴定出第二个取代。最后一个变异是在外显子7的剪接供体共有序列（IVS 7 + 4 ins GG）内插入2个碱基对。结论-我们没有检测到GFR α-3和HSCR表型变异之间的任何相关性。我们的数据表明，该基因的突变不是HSCR的原因。
Background-The GDNF family receptor alpha (GFR alpha) proteins are extracellular cell surface bound molecules that act as adapters in binding of the GDNF family of soluble neurotrophic factors to the RET receptor. These molecules are essential for development of many neural crest derived cell types and the kidney. Mutations in RET and in two members of the GDNF ligand family are associated with Hirschsprung disease (HSCR), a congenital absence of the enteric ganglia. Members of the GFR alpha family are also candidates for HSCR mutations. One such gene is GFR alpha-3, which is expressed in the peripheral nervous system and developing nerves.Objective-We have characterised the structure of the human GFR alpha-3 locus and investigated the gene for sequence variants in a panel of HSCR patients.Methods-Long range PCR or subcloning of PAC clones was used to investigate GFR alpha-3 intron-exon boundaries. A combination of single strand conformation polymorphism (SSCP) analysis and direct sequencing was used to investigate GFR alpha-3 sequence variants.Results-GFR alpha-3 spans eight coding exons and has a gene structure and organisation similar to that of GFR alpha-1. We identified three polymorphic variants in GFR alpha-3 in a normal control population, a subset of which also occurred in HSCR patients. We did not detect any sequence variants within the coding sequence of GFR alpha-3. We found a base substitution in the 5' UTR of GFR alpha-3, 15 base pairs upstream of the translation start site. A second substitution was identified in intron 4 (IVS4-30G>A) between the splice branch site and the splice acceptor site. The final variant was a 2 base pair insertion within the splice donor consensus sequence of exon 7 (IVS7+4ins GG).Conclusions-We did not detect any correlation between variants of GFR alpha-3 and the HSCR phenotype. Our data suggest that mutations of this gene are not a cause of HSCR.