Priston M, Kozlowski K, Gill D, Letwin K, Buys Y, Levin A V, Walter M A, Héon E
Department of Ophthalmology and The Vision Science Research Program, University Health Network, The Toronto Western Hospital, Toronto, Ontario M5T 2S8, Canada.
Hum Mol Genet. 2001 Aug 1;10(16):1631-8. doi: 10.1093/hmg/10.16.1631.
The specific role of PITX2 in the pathogenesis of anterior segment dysgenesis has yet to be clearly defined. We provide here new insight into PITX2 pathogenesis through mutational and functional analyses. Three PITX2 mutations were found in a screen of 38 unrelated individuals affected with anterior segment anomalies (8%). All three mutations were found among the 21 individuals affected with Axenfeld-Rieger syndrome (ARS). We have identified two novel mutations, a valine-->leucine (V45L) missense mutation at position 45 within the PITX2 homeodomain, and a seven amino acid duplication (7aaDup) of residues 6-12 of the homeodomain. DNA-binding studies of the two mutant PITX2 proteins demonstrated a <10-fold reduction in the DNA-binding activity of the V45L mutant, and a >100-fold reduction in activity of the 7aaDup mutant. Luciferase reporter assays showed a >200% increase in PITX2 transactivation activity of the V45L mutant, while the 7aaDup mutant was unable to transactivate at detectable levels. Our analyses of the V45L PITX2 mutant reveal that the DNA-binding domain of PITX2 can influence transactivation activity independently of DNA binding. Furthermore, our findings expand the hypothesis that the amount of residual PITX2 activity underlies the variable severity of ocular phenotypes that result from PITX2 mutation. For the first time, we present evidence that increased PITX2 activity may underlie the severe ARS ocular phenotype. We conclude that increased activity of one PITX2 allele may be as physiologically disruptive as a mutation that nullifies a PITX2 allele, with either condition resulting in ARS.
PITX2在前段发育异常发病机制中的具体作用尚未明确界定。我们通过突变和功能分析，在此提供了有关PITX2发病机制的新见解。在对38名患有前段异常的无关个体（8%）进行的筛查中发现了三个PITX2突变。在21名患有Axenfeld-Rieger综合征（ARS）的个体中均发现了这三个突变。我们鉴定出两个新突变，一个是PITX2同源结构域内第45位的缬氨酸→亮氨酸（V45L）错义突变，另一个是同源结构域第6 - 12位残基的七个氨基酸重复（7aaDup）。对两种突变型PITX2蛋白的DNA结合研究表明，V45L突变体的DNA结合活性降低了不到10倍，而7aaDup突变体的活性降低了100倍以上。荧光素酶报告基因检测显示，V45L突变体的PITX2反式激活活性增加了200%以上，而7aaDup突变体在可检测水平下无法进行反式激活。我们对V45L PITX2突变体的分析表明，PITX2的DNA结合结构域可独立于DNA结合影响反式激活活性。此外，我们的研究结果扩展了这样一种假说，即残余PITX2活性的量是PITX2突变导致的眼部表型严重程度不同的基础。我们首次提供证据表明，PITX2活性增加可能是严重ARS眼部表型的基础。我们得出结论，一个PITX2等位基因活性增加在生理上可能与使一个PITX2等位基因无效的突变一样具有破坏性，这两种情况均可导致ARS。
