Pathogenesis-based therapy reverses cutaneous abnormalities in an inherited disorder of distal cholesterol metabolism

Abstract

Identification of the underlying genetic, cellular, and biochemical basis of lipid metabolic disorders provides an opportunity to deploy corrective, mechanism-targeted, topical therapy. We assessed this therapeutic approach in two patients with Congenital Hemidysplasia with Ichthyosiform erythroderma and Limb Defects (CHILD) syndrome, an X-linked dominant disorder of distal cholesterol metabolism. On the basis of the putative pathogenic role of both pathway-product deficiency of cholesterol and accumulation of toxic metabolic intermediates, we assessed the efficacy of combined therapy with lovastatin and cholesterol. We also evaluated the basis for the poorly understood, unique lateralization of the cutaneous and bone malformations of CHILD syndrome by analyzing gene activation in abnormal and unaffected skin. Ultrastructural analysis of affected skin showed evidence of both cholesterol depletion and toxic metabolic accumulation. Topical treatment with lovastatin/cholesterol (but not cholesterol alone) virtually cleared skin lesions by 3 months, accompanied by histological and ultrastructural normalization of epidermal structure and lipid secretion. The unusual lateralization of abnormalities in CHILD syndrome reflects selective clearance of keratinocytes and fibroblasts that express the mutant allele from the unaffected side. These findings validate pathogenesis-based therapy that provides the deficient end product and prevents accumulation of toxic metabolites, an approach of potential utility for other syndromic lipid metabolic disorders.

Figure 1. The Cholesterol Biosynthetic Pathway

HMG-CoA reductase is a rate-limiting step early in the cholesterol biosynthetic pathway, which is inhibited by statins. CHILD syndrome results from mutations in NAD(P)H steroid dehydrogenase-like (NSDHL), which prevent the post-lanosterol generation of cholesterol, potentially leading to pathway-product deficiency in addition to metabolite accumulation. Conradi-Hünermann-Happle syndrome is due to mutations in a gene encoding emopamil binding protein (EBP), which acts distal to NSDHL in cholesterol biosynthesis.

Figure 2. Clinical and Histologic Improvement with Topical Applications of Lovastatin/Cholesterol

a: Treated skin of Patient #1. b: Treated skin of Patient #2. c. Histopathologic evaluation of lesional skin of Patient #2 at baseline (left) showed marked epidermal acanthosis with psoriasiform hyperplasia. The granular layer was absent, and the stratum corneum showed parakeratosis and retention hyperkeratosis. Bar = 25 μm. By approximately 3 months after treatment initiation (right), the epidermis was of normal thickness with regular orthokeratinization and a normal granular layer. Bar = 7 μm.

Figure 3. Ultrastructural Normalization from Pathogenesis-Based Therapy in CHILD Syndrome

a and b: Pre-treatment biopsies show prominent abnormalities in epidermal lamellar body morphology (asterisks) and secretion in the stratum granulosum (SG). c: Impaired secretion results in entombment of unsecreted organelle contents within corneocytes (SC, solid arrows), reduced lamellar material in the SC interstices (open arrows), with massive distended foci of non-lamellar/amorphous material between corneocytes (asterisks). Post-treatment biopsies (d and e) show substantial improvement in lamellar body contents (e) and secretion, but some foci of non-lamellar material persist (d, open arrows). a, b, and e: osmium tetroxide post-fixation; c and d: ruthenium tetroxide post-fixation. Bars: a = 0.2 μm; b = 0.5 μm; c = 0.2 μm; d and e = 0.1 μm.

Figure 4. Ultrastructural Cytochemical Evidence of Improved Lamellar Body Secretion in Treated Skin

a: Pre-treatment biopsy, assessed with an ultrastructural marker of lamellar body contents, shows much retained (unsecreted) lamellar body contents within cornified cells (SC, arrows). Open arrows indicate focal sites where organelle contents have been secreted. b: Post-treatment biopsy reveals that most enzyme activity has been secreted (exteriorized) (arrows). Osmium tetroxide post-fixation. Bars: a: 0.5 μm; b = 0.25 μm.

Figure 5. Clinically ‘Unaffected’ Epidermis of CHILD Syndrome Shows Abnormalities of the Lamellar Body Secretory System

Most of the lamellar bodies in the stratum granulosum (SG) display abnormal internal contents (c, asterisks). Lamellar body secretion is partially impaired, with entombment of some organelles (a, open arrows) and retained acidic lipase activity in some corneocytes, as shown by ultrastructural cytochemistry (b, open arrows). Note also the inhomogeneous distribution of secreted enzyme activity (b, arrows). Bars: a = 0.1 μm, b = 0.5 μm, c = 0.2 μm.

Figure 6. NSDHL Gene Expression

a. Sequences of cDNA obtained from cultured keratinocytes and fibroblasts in unaffected (upper panel) and affected (lower panel) areas of the skin. There is clear exclusive expression of the mutant c.248G>A allele in keratinocytes taken from affected skin, whereas fibroblasts from this area express both the mutant and the normal allele. In the unaffected areas only the wild type is found in keratinocytes as well as fibroblasts. b. Genomic DNA sequences obtained from peripheral blood leukocytes, unaffected and affected keratinocytes respectively. All traces show the presence of both wild type and mutant alleles. Arrows point to the mutant residue.