A Dominant-Negative Mutation of Mouse <i>Lmx1b</i> Causes Glaucoma and Is Semi-lethal via LBD1-Mediated Dimerisation

Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice.LIM同源域转录因子LMX1B的突变可引发指甲-髌骨综合征（nail-patella syndrome），这是一种常染色体显性多效性人类遗传病，患者表现为指甲、髌骨及肘关节发育不良，同时伴随其他骨骼异常，还可可变地并发肾病与青光眼。该疾病被认为属于单倍剂量不足型（haploinsufficient）疾病。针对小鼠的研究显示，在发育进程中，Lmx1b可调控肢体背腹轴模式形成，同时对肾脏与眼部发育、中脑-后脑边界建立以及特定神经元亚型的特化具有必需作用。完全缺失Lmx1b的小鼠会在出生时死亡。与人类的情况不同，携带Lmx1b无效等位基因的杂合小鼠并不会表现出突变表型。本研究报道了一种新型小鼠突变体Icst，其为N-乙基-N-亚硝基脲（N-ethyl-N-nitrosourea，ENU）诱导产生的错义突变：LMX1B同源域内发生V265D氨基酸替换，该突变会消除LMX1B的DNA结合能力，进而使其丧失激活其他基因转录的功能。尽管Icst纯合突变与Lmx1b无效等位基因纯合突变的表型完全一致，但杂合Icst突变可引发表型，而无效等位基因杂合则不会产生异常表型。杂合Icst突变会导致青光眼性眼部缺陷，并伴随半致死性，其诱因可能为肾功能衰竭。研究表明，相较于Icst突变，Lmx1b转基因能够更有效地挽救无效等位基因的表型缺陷。免疫共沉淀实验显示，野生型与Icst突变型LMX1B均能与LIM结构域结合蛋白1（LIM domain binding protein 1，LDB1）形成复合物，这使得Icst杂合子中具有功能活性的LMX1B水平低于无效等位基因杂合子。我们据此得出结论，Icst是Lmx1b的显性负效等位基因（dominant-negative allele）。该研究结果提示需重新评估指甲-髌骨综合征是否始终为单倍剂量不足型疾病。此外，Icst是一种罕见的人类青光眼模型：其致病基因在人类与小鼠中发生相同突变时，均可引发青光眼表型。