PURPOSE We aimed to assess the biological and clinical significance of the human cysteine protease inhibitor cystatin M/E, encoded by the CTS6 gene, in diseases of human hair and skin. METHODS Exome and Sanger sequencing was performed to reveal the genetic cause in two related patients with hypotrichosis. Immunohistochemical, biophysical, and biochemical measurements were performed on patient skin and 3D-reconstructed skin from patient-derived keratinocytes. RESULTS We identified a homozygous variant c.361C>T (p.Gln121*), resulting in a premature stop codon in exon 2 of CST6 associated with hypotrichosis, eczema, blepharitis, photophobia and impaired sweating. Enzyme assays using recombinant mutant cystatin M/E protein, generated by site-directed mutagenesis, revealed that this p.Gln121* variant was unable to inhibit any of its three target proteases (legumain and cathepsins L and V). Three-dimensional protein structure prediction confirmed the disturbance of the protease/inhibitor binding sites of legumain and cathepsins L and V in the p.Gln121* variant. CONCLUSION The herein characterized autosomal recessive hypotrichosis syndrome indicates an important role of human cystatin M/E in epidermal homeostasis and hair follicle morphogenesis.

中文翻译：

---

人半胱氨酸蛋白酶抑制剂胱抑素 M/E 的缺乏会导致少毛症和皮肤干燥。

目的 我们旨在评估由 CTS6 基因编码的人类半胱氨酸蛋白酶抑制剂 cystatin M/E 在人类头发和皮肤疾病中的生物学和临床意义。方法 进行外显子组和 Sanger 测序以揭示两名相关的少毛症患者的遗传原因。对患者皮肤和来自患者衍生角质形成细胞的 3D 重建皮肤进行免疫组织化学、生物物理和生化测量。结果 我们鉴定了一个纯合变体 c.361C>T (p.Gln121*)，导致 CST6 外显子 2 中的过早终止密码子与少毛症、湿疹、睑缘炎、畏光和出汗障碍相关。使用通过定点诱变产生的重组突变半胱氨酸蛋白酶抑制剂 M/E 蛋白的酶测定表明，这种 p。Gln121* 变体无法抑制其三种目标蛋白酶（legumain 和组织蛋白酶 L 和 V）中的任何一种。三维蛋白质结构预测证实了 p.Gln121* 变体中legumain 和组织蛋白酶 L 和 V 的蛋白酶/抑制剂结合位点的干扰。结论本文表征的常染色体隐性遗传性少毛综合征表明人胱抑素M/E在表皮稳态和毛囊形态发生中的重要作用。