Alopecia areata susceptibility variant in MHC region impacts expressions of genes contributing to hair keratinization and is involved in hair loss

Background: Alopecia areata (AA) is considered a highly heritable, T-cell-mediated autoimmune disease of the hair follicle. However, no convincing susceptibility gene has yet been pinpointed in the major histocompatibility complex (MHC), a genome region known to be associated with AA as compared to other regions. Methods: We engineered mice carrying AA risk allele identified by haplotype sequencing for the MHC region using allele-specific genome editing with the CRISPR/Cas9 system. Finally, we performed functional evaluations in the mice and AA patients with and without the risk allele. Findings: We identified a variant (rs142986308, p.Arg587Trp) in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene as the only non-synonymous variant in the AA risk haplotype. Furthermore, mice engineered to carry the risk allele displayed a hair loss phenotype. Transcriptomics further identified CCHCR1 as a novel component interacting with hair cortex keratin in hair shafts. Both, these alopecic mice and AA patients with the risk allele displayed morphologically impaired hair and comparable differential expression of hair-related genes, including hair keratin and keratin-associated proteins (KRTAPs). Interpretation: Our results implicate CCHCR1 with the risk allele in a previously unidentified subtype of AA based on aberrant keratinization in addition to autoimmune events. 10 ng of total RNA from human hair follicle was processed for use on the microarray using the GeneChip WT pico Reagent Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The resultant single-strand cDNA was fragmented and labeled with biotin, then hybridized to the GeneChip Human Gene 2.0 ST Array. The arrays were washed, stained and scanned using the Affymetrix 450 Fluidics Station and GeneChip Scanner 3000 7G (Thermo Fisher Scientific) according to the manufacturer’s recommendations.

背景：斑秃（Alopecia areata, AA）是一种遗传度极高、由T细胞介导的毛囊自身免疫性疾病。然而，相较于其他基因组区域，目前尚未在主要组织相容性复合体（major histocompatibility complex, MHC）这一已知与AA相关的基因组区域中精准定位到确凿的易感基因。
方法：本研究通过CRISPR/Cas9系统开展等位基因特异性基因组编辑，构建了携带经单倍型测序鉴定得到的MHC区域AA风险等位基因的工程化小鼠。随后，对携带与未携带该风险等位基因的小鼠及AA患者开展功能学评估。
结果：本研究于卷曲螺旋α螺旋杆状蛋白1（coiled-coil alpha-helical rod protein 1, CCHCR1）基因中鉴定出一处变异位点（rs142986308，p.Arg587Trp），该位点为AA风险单倍型中唯一的非同义变异。进一步实验显示，携带该风险等位基因的工程化小鼠呈现脱发表型。转录组学分析进一步证实，CCHCR1是毛干中与毛发皮质角蛋白相互作用的新型组分。上述脱发模型小鼠及携带风险等位基因的AA患者均表现出形态学受损的毛发，且在毛发相关基因（包括毛发角蛋白及角蛋白关联蛋白，keratin-associated proteins, KRTAPs）的差异表达模式上具有一致性。
解读：本研究结果表明，携带风险等位基因的CCHCR1通过异常角化过程，结合自身免疫事件，参与了此前未被明确分型的AA亚型的发病机制。
实验流程：按照制造商说明书，使用GeneChip WT pico Reagent Kit（赛默飞世尔科技，Thermo Fisher Scientific）处理10 ng人毛囊总RNA，以用于基因芯片实验。将所得单链cDNA进行片段化并以生物素标记，随后与GeneChip Human Gene 2.0 ST Array进行杂交。根据制造商推荐流程，使用Affymetrix 450 Fluidics Station及GeneChip Scanner 3000 7G（赛默飞世尔科技，Thermo Fisher Scientific）对芯片进行洗涤、染色及扫描。