Genome-wide association studies reveal susceptibility loci for noninfectious claw lesions in Holstein dairy cattle. Genome-wide association studies reveal susceptibility loci for noninfectious claw lesions in Holstein dairy cattle

Sole ulcers (SU) and white line disease (WLD) are noninfectious claw lesions that arise due to compromised horn production and are common causes of lameness in dairy cattle, imposing welfare and profitability concerns. The low to moderate heritability estimates of SU and WLD susceptibility indicate that genetic selection could reduce their prevalence. To identify loci associated with SU and WLD susceptibility, generalized linear mixed model (GLMM) regression and random forest (RF) genome-wide association studies (GWAS) were performed. Cows from five commercial dairies in California were classified as sound controls having no instances of lameness and above six years of age (n = 102) or cases having SU (n = 152), WLD (n = 117), SU and/or WLD (SU+WLD, n = 198), or any type of noninfectious claw lesion (n = 217). Top SNPs were defined as those passing Bonferroni-corrected suggestive and significance thresholds in the GLMM analysis or those that a validated RF model considered important. Effects of top SNPs were quantified using Bayesian estimation. Linkage disequilibrium (LD) blocks defined by top SNPs were explored for candidate genes and previously identified, functionally relevant quantitative trait loci. The GLMM GWAS revealed regions of association on BTA8 for SU and BTA13 common to WLD, SU+WLD, and noninfectious claw lesions. Bayesian estimation of effect sizes indicated that these SNPs had effects significantly different from zero, indicating their small but notable contribution to susceptibility. Promising candidate genes identified in these regions were involved in wound healing, skin lesions, bone growth and mineralization, adipose tissue, and keratinization. The LD block defined by the most significant SNP on BTA8 for SU included a SNP previously reported to be associated with SU. The RF models were overfitted, indicating that SNP effects were very small and thereby preventing meaningful interpretation of SNPs with the highest importance values and downstream analyses. These findings suggested that variants associated with a variety of physiological systems may contribute to susceptibility for noninfectious claw lesions, demonstrating the complexity of genetic predisposition. Overall design: Whole blood was collected from 326 cows (cases with any noninfectious claw lesion n = 222, controls n = 104) from the coccygeal vein in the tail. Blood samples were centrifuged, and genomic DNA was extracted from the resulting buffy coat layer using the QIAGEN QIAamp DNA Blood Mini Kit . DNA samples were genotyped on the Illumina BovineHD BeadChip, and Illumina’s GenCall algorithm was used to call genotypes. Of the 326 samples, seven were removed during quality filtering (cases n = 5, controls n = 2), leaving 319 cows for analysis (cases n = 217, controls n = 102).

蹄底溃疡（sole ulcers, SU）与白线病（white line disease, WLD）均为因角质生成受损引发的非感染性蹄部病变，是奶牛跛行的常见诱因，会对动物福利与养殖经济效益造成负面影响。蹄底溃疡与白线病易感性的遗传力估计值处于低至中等水平，提示通过遗传选育可降低二者的发病率。为筛选与蹄底溃疡、白线病易感性相关的遗传位点，本研究开展了基于广义线性混合模型（generalized linear mixed model, GLMM）回归与随机森林（random forest, RF）的全基因组关联分析（genome-wide association studies, GWAS）。
研究对象为来自美国加州5家商业化奶牛场的奶牛，按表型分为两类：一是无跛行记录且年龄超过6岁的健康对照（n=102）；二是患病个体，包括仅患蹄底溃疡（n=152）、仅患白线病（n=117）、同时或分别罹患两种病变（SU+WLD，n=198），以及罹患任意类型非感染性蹄部病变的个体（n=217）。本研究将两类单核苷酸多态性（single nucleotide polymorphism, SNP）定义为显著SNP：一是在GLMM分析中通过邦弗罗尼校正的提示性阈值与显著性阈值的位点，二是经验证的RF模型判定为具有重要效应的位点。采用贝叶斯估计法对显著SNP的效应值进行量化分析。针对由显著SNP定义的连锁不平衡（linkage disequilibrium, LD）区块，本研究进一步筛选候选基因以及此前已被报道的功能相关数量性状位点（quantitative trait loci, QTL）。
基于GLMM的全基因组关联分析结果显示，蹄底溃疡相关的关联区域位于牛8号染色体（BTA8），而白线病、SU+WLD以及所有非感染性蹄部病变共享的关联区域则位于牛13号染色体（BTA13）。效应值的贝叶斯估计结果显示，这些SNP的效应值显著非零，表明其对疾病易感性虽影响较小，但仍具有不可忽视的贡献。在上述关联区域中筛选到的潜在候选基因，功能涉及伤口愈合、皮肤病变、骨骼生长与矿化、脂肪组织代谢以及角质化过程。针对蹄底溃疡最显著关联SNP所定义的BTA8连锁不平衡区块中，包含一个此前已被报道与蹄底溃疡相关的SNP。随机森林模型存在过拟合现象，这表明SNP的效应值极小，因此无法对重要性得分最高的SNP进行有效解读，也无法开展后续分析。本研究结果表明，与多种生理系统相关的遗传变异可能参与了非感染性蹄部病变的易感性调控，印证了该类疾病遗传易感性的复杂性。
整体实验设计：从326头奶牛（罹患任意类型非感染性蹄部病变的病例组n=222，对照组n=104）的尾静脉采集全血样本。将血液样本离心后，使用QIAGEN QIAamp DNA Blood Mini试剂盒从离心得到的棕黄层（白细胞层）中提取基因组DNA。采用Illumina BovineHD高通量基因芯片对DNA样本进行基因分型，并使用Illumina GenCall算法进行基因型判读。在质量过滤阶段剔除了7份样本（病例组n=5，对照组n=2），最终共319头奶牛纳入后续分析（病例组n=217，对照组n=102）。