Excessive glycine loop variations in the keratin 10 tail domain and implications for skin fragility.

Abstract

Keratins are vital structural proteins in the formation of intermediate filaments (IFs). Dysfunctional keratins lead to various inherited skin fragilities. The keratin tail domain is essential for the proper functioning of IFs, with glycine loop variability prominent in certain keratin tails. However, the effects of glycine loop variations on the functioning of IFs, and the pathogenicity of excessive variations, remain elusive. To clarify the pathogenic effects of excessive glycine loop variations. We performed whole-exome sequencing on a patient with epidermolysis bullosa (EB) lacking mutations in known EB-causing genes. Sanger sequencing was used to verify the identified variations in the patient, the patient's family members and a cohort of 319 healthy control individuals. The pathogenicity of the identified variations was evaluated using in vitro and in vivo IF assembly experiments, mouse models, cellular experiments and human skin three-dimensional models. We identified a compound heterozygous mutation in KRT10, comprising insertions of two and four tandem c.1654_1683 repeats at the c.1683_1684 position, which increased the number of glycine loops by two and four in the keratin 10 tail domain. Although insertions of one and two c.1654_1683 repeats were prevalent in healthy individuals (16.5% and 14.3%, respectively), four insertions were not detected. In vivo and in vitro experiments revealed that excessive glycine loops prevented the proper assembly of IFs. Experiments in mouse and three-dimensional human skin models confirmed that an overabundance of glycine loops led to an EB-like phenotype. In KRT10, the insertions of limited c.1654_1683 repeats were prevalent and harmless in the general population. However, excessive insertions, exemplified by compound heterozygous mutations with insertions of two and four c.1654_1683 repeats, could be pathogenic. This study was novel in highlighting the pathogenicity of excessive glycine loop extension in the keratin tail domain, establishing a paradigm for other human keratins with glycine-rich tail domains.