Genomic locus modulating corneal thickness in the mouse identifies POU6F2 as a potential risk of developing glaucoma

Central corneal thickness (CCT) is one of the most heritable ocular traits and it is also a phenotypic risk factor for primary open angle glaucoma (POAG). The present study uses the BXD Recombinant Inbred (RI) strains to identify novel quantitative trait loci (QTLs) modulating CCT in the mouse with the potential of identifying a molecular link between CCT and risk of developing POAG. The BXD RI strain set was used to define mammalian genomic loci modulating CCT, with a total of 818 corneas measured from 61 BXD RI strains (between 60–100 days of age). The mice were anesthetized and the eyes were positioned in front of the lens of the Phoenix Micron IV Image-Guided OCT system or the Bioptigen OCT system. CCT data for each strain was averaged and used to QTLs modulating this phenotype using the bioinformatics tools on GeneNetwork (www.genenetwork.org). The candidate genes and genomic loci identified in the mouse were then directly compared with the summary data from a human POAG genome wide association study (NEIGHBORHOOD) to determine if any genomic elements modulating mouse CCT are also risk factors for POAG.This analysis revealed one significant QTL on Chr 13 and a suggestive QTL on Chr 7. The significant locus on Chr 13 (13 to 19 Mb) was examined further to define candidate genes modulating this eye phenotype. For the Chr 13 QTL in the mouse, only one gene in the region (Pou6f2) contained nonsynonymous SNPs. Of these five nonsynonymous SNPs in Pou6f2, two resulted in changes in the amino acid proline which could result in altered secondary structure affecting protein function. The 7 Mb region under the mouse Chr 13 peak distributes over 2 chromosomes in the human: Chr 1 and Chr 7. These genomic loci were examined in the NEIGHBORHOOD database to determine if they are potential risk factors for human glaucoma identified using meta-data from human GWAS. The top 50 hits all resided within one gene (POU6F2), with the highest significance level of p = 10−6 for SNP rs76319873. POU6F2 is found in retinal ganglion cells and in corneal limbal stem cells. To test the effect of POU6F2 on CCT we examined the corneas of a Pou6f2-null mice and the corneas were thinner than those of wild-type littermates. In addition, these POU6F2 RGCs die early in the DBA/2J model of glaucoma than most RGCs. Using a mouse genetic reference panel, we identified a transcription factor, Pou6f2, that modulates CCT in the mouse. POU6F2 is also found in a subset of retinal ganglion cells and these RGCs are sensitive to injury.

中央角膜厚度（Central corneal thickness, CCT）是遗传度最高的眼部性状之一，同时也是原发性开角型青光眼（Primary Open Angle Glaucoma, POAG）发病的表型风险因素。本研究采用BXD重组近交系（BXD Recombinant Inbred, RI），旨在鉴定调控小鼠中央角膜厚度的新型数量性状位点（Quantitative Trait Loci, QTLs），以期揭示CCT与POAG发病风险之间的分子关联。本研究使用BXD RI品系组来界定调控CCT的哺乳动物基因组位点，共对61个BXD RI品系（年龄介于60~100日龄）的818份角膜样本进行了检测。实验中对小鼠实施麻醉，将眼球置于Phoenix Micron IV图像引导光学相干断层扫描（Optical Coherence Tomography, OCT）系统或Bioptigen OCT系统的镜头前方。对每个品系的CCT数据取平均值，并借助基因网络数据库（GeneNetwork, www.genenetwork.org）的生物信息学工具，筛选调控该表型的QTLs。随后将小鼠中鉴定到的候选基因与基因组位点，与人类POAG全基因组关联研究（Genome Wide Association Study, GWAS）的汇总数据（NEIGHBORHOOD数据库）进行直接比对，以确定调控小鼠CCT的基因组元件是否同时为POAG的风险因子。本次分析共鉴定出1个位于13号染色体的显著QTL，以及1个位于7号染色体的提示性QTL。针对13号染色体（13~19 Mb区域）的显著位点，我们进一步开展了调控该眼部表型的候选基因鉴定。小鼠13号染色体该QTL区域内，仅Pou6f2基因携带非同义单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs）。该基因的5个非同义SNPs中，有2个会导致脯氨酸残基发生改变，这可能会引发蛋白质二级结构改变，进而影响蛋白功能。小鼠13号染色体峰值区域的7 Mb序列在人类基因组中分布于两条染色体：1号染色体与7号染色体。我们在NEIGHBORHOOD数据库中对这些基因组位点进行了分析，以确定其是否为基于人类GWAS元数据鉴定出的人类青光眼潜在风险因子。排名前50的关联位点均位于POU6F2基因内，其中SNP rs76319873的显著性最高，p值为10⁻⁶。POU6F2在视网膜神经节细胞（Retinal Ganglion Cells, RGCs）与角膜缘干细胞中均有表达。为验证POU6F2对CCT的调控作用，我们检测了Pou6f2基因敲除小鼠的角膜，发现其角膜厚度较野生型同窝小鼠更薄。此外，在DBA/2J青光眼小鼠模型中，Pou6f2相关的RGCs死亡时间早于多数其他RGCs。本研究借助小鼠遗传参考面板，鉴定出了调控小鼠CCT的转录因子Pou6f2。POU6F2同样在特定亚群的视网膜神经节细胞中表达，且这类RGCs对损伤敏感。