Identification of novel alleles and structural haplotypes of major histocompatibility complex class I and DRB genes in domestic cat (Felis catus) by a newly developed NGS-based genotyping method

Peripheral white blood cells (PWBCs) were obtained from 20 related domestic cats from two families and five unrelated cats (A6, A116, A165, A176 and A214). Each family was composed of ten cats representing three generations that were considered suitable for investigating the FLA allele and haplotype segregation. We first used the five unrelated cats to elucidate the alleles and specificity of expressed FLA-I loci in PWBCs and then used the 20 related cats for the NGS-based genotyping of expressible FLA-I and FLA-DRB loci. We have identified and presented novel alleles for the FLA-I and FLA-DRB genes and provided some further insights into the development of the NGS-based genotyping method at the RNA expression level, phylogenetic relationships and haplotype structures of FLA-I and FLA-DRB alleles and haplotypes in 20 cats from two families. This FLA polymorphism information could be useful as a standard internal control to establish the feline MHC genetic background for the benefit of biomedical research into disease associations such as infectious disease and autoimmune diseases in veterinary medical field.

本研究从两个家系的20只近缘家猫，以及5只无关家猫（编号A6、A116、A165、A176及A214）中采集了外周血白细胞（Peripheral white blood cells, PWBCs）样本。每个家系由覆盖三代的10只家猫组成，该家系结构适用于研究猫白细胞抗原（FLA）等位基因与单倍型的分离传递规律。本研究首先利用5只无关家猫阐明了外周血白细胞中表达型FLA-I基因座的等位基因及表达特异性，随后利用20只近缘家猫开展了可表达FLA-I及FLA-DRB基因座的基于下一代测序（Next Generation Sequencing, NGS）的基因分型分析。本研究已鉴定并报道了FLA-I及FLA-DRB基因的新型等位基因，并针对两个家系20只家猫的FLA-I、FLA-DRB等位基因及单倍型，从RNA表达层面的NGS基因分型方法开发、系统发育关系及单倍型结构等方面提供了新的研究视角。本研究获得的FLA多态性信息可作为标准内参，用于建立猫科动物主要组织相容性复合体（Major Histocompatibility Complex, MHC）的遗传背景，从而助力兽医学领域中感染性疾病、自身免疫性疾病等疾病关联相关的生物医学研究。