Serine Protease Activity and Residual LEKTI Expression Determine Phenotype in Netherton Syndrome

Abstract

Mutations in the SPINK5 gene encoding the serine protease (SP) inhibitor, lymphoepithelial-Kazal-type 5 inhibitor (LEKTI), cause Netherton syndrome (NS), a life-threatening disease, owing to proteolysis of the stratum corneum (SC). We assessed here the basis for phenotypic variations in nine patients with "mild", "moderate", and "severe" NS. The magnitude of SP activation correlated with both the barrier defect and clinical severity, and inversely with residual LEKTI expression. LEKTI co-localizes within the SC with kallikreins 5 and 7 and inhibits both SP. The permeability barrier abnormality in NS was further linked to SC thinning and proteolysis of two lipid hydrolases (beta-glucocerebrosidase and acidic sphingomyelinase), with resultant disorganization of extracellular lamellar membranes. SC attenuation correlated with phenotype-dependent, SP activation, and loss of corneodesmosomes, owing to desmoglein (DSG)1 and desmocollin (DSC)1 degradation. Although excess SP activity extended into the nucleated layers in NS, degrading desmosomal mid-line structures with loss of DSG1/DSC1, the integrity of the nucleated epidermis appears to be maintained by compensatory upregulation of DSG3/DSC3. Maintenance of sufficient permeability barrier function for survival correlated with a compensatory acceleration of lamellar body secretion, providing a partial permeability barrier in NS. These studies provide a mechanistic basis for phenotypic variations in NS, and describe compensatory mechanisms that permit survival of NS patients in the face of unrelenting SP attack.

Filaggrin Genotype in Ichthyosis Vulgaris Predicts Abnormalities in Epidermal Structure and Function

“…Specifically, the increase in pH should decrease activities of ceramidegenerating hydrolases, important for extracellular lipid organization. 55,75 Two of these hydrolytic enzymes exhibit acidic pH optima [␤-glucocerebrosidase (␤-GlcCer'ase) and acid sphingomyelinase (aSMase)], whereas two others [group 1 secretory phospholipase A 2 (sPLA 2 ) and steroid sulfatase (SSase)], display neutral-to-alkaline pH optima, suggesting that the altered pH gradient in IV could account for the observed delay in lipid processing in IV. In addition, decreased generation of acidic FLG products could account for the observed decrease in corneodesmosome density, if serine proteases are inhomogenously activated.…”

“…74 Furthermore, we observed an inverse relationship between FLG gene dose and skin surface pH in all FLGdeficient patients, with double-allele subjects displaying the greatest elevations in skin surface pH ( Figure 2E). This increase in skin surface pH could reduce the activity of ceramide-generating hydrolases, 55,75 accounting for the observed delay in maturation of extracellular lamellar bilayers in IV ( Figure 5, D and E). The increased pH in FLGdeficient SC could also impact SC function by altering TJ function and by favoring corneodesmosome proteolysis, which could further compromise barrier function.…”

“…We used an algorithmic sequence similar to that previously applied to Harlequin ichthyosis, 54 epidermolytic ichthyosis, 36 autosomal recessive (lamellar) ichthyosis, 34 loricrin keratoderma, 35 Netherton syndrome, 55 and neutral lipid storage disease. 56 Skin samples, derived from punch biopsies, were minced to Ͻ0.5 mm 3 fragments, rinsed three times in 0.1 mol/L cacodylate buffer, and pre-fixed in half-strength Karnovsky's fixative, followed by postfixation in reduced 1% osmium tetroxide (OsO 4 ) containing 1.5% ferrocyanide or in 0.2% ruthenium tetroxide (RuO 4 ).…”

A synonymous mutation in SPINK5 exon 11 causes Netherton syndrome by altering exonic splicing regulatory elements

“…5 Genotype-phenotype correlation in NS is largely incomplete; however, a correlation between the SPINK5 mutation position, LEKTI domain deficiency and disease severity has been suggested. 8,11,12 In our patient, translation of the aberrant transcript deleted of exon 11 is predicted to result in a truncated protein lacking inhibitory domains 5-15. This, combined with the effect of the c.153delT mutation, would be expected to result in a more severe NS phenotype than the one we actually observed in our patient.…”

“…5,6 The SPINK5 mutation database in NS comprises 62 different mutations, [7][8][9][10] and the genotype-phenotype correlation suggests that downstream mutations may partly allow for residual expression of functional LEKTI fragments, which results in less severe phenotypes. 11,12 Here we report on functional characterization of the c.891C4T variant identified in the SPINK5 gene in an Italian patient with NS. We use patient keratinocytes, minigene constructs and bioinformatic predictors to demonstrate that this synonymous change (p.Cys 297Cys), despite being distant from the acceptor splice site, induces an incomplete exon skipping by affecting a mechanism underlying splicing regulation.…”