GTPBP2 in-frame deletion in a canine model with progressive retinal degeneration

Progressive retinal atrophy (PRA), caused by aberrant functioning of rod/cone photoreceptors leads to blindness. Different types of photoreceptor abnormalities with different ages of onset and progression have been reported in dogs, even in the same breed, and new ones are constantly discovered in Labrador retriever. We collected samples from 3 affected dogs and 3 unaffected siblings, along with the unaffected parents of the same litter. Homozygosity mapping detected six candidate regions >1Mb. Whole-genome sequencing detected an homozygous 3-bp deletion in the coding region of GTPBP2, CFA12 - a GTP-binding protein (CFA12:12,246,348) c.1606_1608del, p.Ala536del).       
            The variant was absent from the online EVA database, the Dog Biomedical Variants Database, and the Dog10k variants database. Testing 75 healthy dogs from the same kennel detected wild type and carrier individuals. More than 600 Labradors from the general population (USA) were genotyped wild type for the variant. The variant was absent from the online EVA database, the Dog Biomedical Variants Database, and the Dog10k variants database. GTPBP2 is associated with Jaberi-Elahi syndrome in Homo sapiens, and splice variants in Mus musculus are associated with neurodegeneration; in both cases photoreceptor degeneration may be included in its manifestation.         
            Heterologous cellular systems were transfected with cDNA encoding WT or A536del mutant GTPBP2 protein and immunoblot analysis of total cell lysate with antibodies to GTPBP2 showed that the expression level of the GTPBP2 mutant protein A536del is slightly but not significantly reduced, compared to the WT form. Instead, the cellular distribution of GTPBP2 protein was investigated by using immunofluorescent methods and confocal analysis of cells transfected with WT or A536del GTPBP2 protein revealed that the WT form is diffuse throughout the cytoplasm of cells, while the mutant form resulted in the formation of cytosolic aggregates in almost 70-80% of cells. We therefore show a non-syndromic, retinal-specific phenotype occurring in a large animal model, associated with a deletion in a conserved Alanine, in a conserved interval outside the GTP domain of GTPBP2, suggesting a potentially novel role of the sequence on cellular localization of the protein.

Methods
Blood samples were gathered and DNA extracted as for standard protocol (BACC2 kit)

SNPdata:  genotype data Type from 220k CanineHD array (Illumina). 

Libraries of 300 bp insert size were prepared and illumina HiSeq2500 paired-end reads (2x100 bp) were collected (one lane per sample). Fastq files were generated using Casava 1.8. A total of 561.77 millions reads (100 bp paired-end reads) for the four libraries were collected for the sequenced dogs from a shotgun fragment library (corresponding to an average coverage of the genome of 28.2x and 27.6x for the cases, and 26.7x and 28.4x for the parents).

进行性视网膜萎缩（Progressive retinal atrophy, PRA）是由视杆/视锥光感受器功能异常引发的致盲性疾病。犬类中已报道存在多种不同发病年龄与进展进程的光感受器异常亚型，即便在同一犬种中亦是如此，而拉布拉多寻回犬中仍不断有新的亚型被发现。本研究收集了3只患病拉布拉多犬、3只未患病的同窝同胞，以及该窝未患病的双亲样本。纯合性定位分析筛选得到6个长度大于1Mb的候选区域。全基因组测序在犬12号染色体（CFA12）上的GTP结合蛋白GTPBP2（CFA12:12,246,348）的编码区中，检测到一处纯合的3碱基缺失变异c.1606_1608del，对应的蛋白变异为p.Ala536del。
该变异未见于在线EVA数据库、犬生物医学变异数据库以及Dog10k变异数据库。对同犬舍的75只健康犬进行基因分型检测，发现了野生型及携带者个体。对美国普通种群的600余只拉布拉多犬进行基因分型，结果均为该变异的野生型。该变异未见于在线EVA数据库、犬生物医学变异数据库以及Dog10k变异数据库。人类中，GTPBP2与Jaberi-Elahi综合征相关；而小家鼠（Mus musculus）中其剪接变异体与神经退行性疾病相关，两类病症的临床表现均可能包含光感受器退行性病变。
将编码野生型（Wild Type, WT）或A536del突变型GTPBP2蛋白的cDNA转染至异源细胞系统中，通过针对GTPBP2的抗体对全细胞裂解物进行免疫印迹分析，结果显示：与WT蛋白相比，A536del突变型GTPBP2蛋白的表达水平略有降低，但该差异无统计学显著性。随后通过免疫荧光法与共聚焦显微镜分析，对转染了WT或A536del GTPBP2蛋白的细胞中GTPBP2蛋白的细胞分布进行探究，结果发现：WT蛋白在细胞胞质内呈弥散分布，而突变型蛋白则在近70%-80%的细胞中形成胞质聚集物。综上，本研究在大型动物模型中发现了一种非综合征性、视网膜特异性的表型，该表型与GTPBP2的GTP结构域外保守区域内的保守丙氨酸缺失相关，提示该序列可能在蛋白的细胞定位过程中发挥此前未被报道的新功能。

Methods
血液样本采集与基因组DNA提取按照标准实验流程进行，使用BACC2试剂盒完成提取。
单核苷酸多态性（Single Nucleotide Polymorphism, SNP）数据：采用220K犬高密度基因分型芯片（CanineHD array，Illumina公司）获取基因型数据。
构建插入片段长度为300bp的测序文库，采用Illumina HiSeq2500平台进行双端测序（读长2×100bp），每个样本使用一个测序泳道。使用Casava 1.8软件生成FASTQ格式文件。本研究共为4个测序文库获取了5.6177亿条100bp双端测序读段，均来自鸟枪法片段文库；其中患病样本的基因组平均覆盖度分别为28.2×和27.6×，亲本样本的平均覆盖度分别为26.7×和28.4×。