190 Deficiency of the Human Cysteine Protease Inhibitor Cystatin M/E Causes Hypotrichosis and Dry Skin

Cystatins comprise a large family of cysteine protease inhibitors that provide regulatory and protective functions against uncontrolled proteolysis by cysteine proteases. 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. We performed Exome and Sanger sequencing 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. We identified a homozygous mutation 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. Biophysical measurements of the skin and histological examination of patient skin biopsies revealed acanthosis and an abnormal collagen fibre density in the dermis. Immunostainings showed an increased number of proliferating cells and upregulated protein expression of several epidermal differentiation genes. The predicted truncated protein was found to be expressed in the stratum granulosum of patient skin. 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). 3D-protein structure prediction using published X-ray crystal structures confirmed the disturbance of the protease/inhibitor binding sites of legumain and cathepsins L and V in the p.Gln121* variant. The herein characterized autosomal recessive hypotrichosis syndrome indicates an important role of human cystatin M/E in epidermal homeostasis and hair follicle morphogenesis.