Cutaneous skeletal hypophosphatemia syndrome: clinical spectrum, natural history, and treatment.

Cutaneous skeletal hypophosphatemia syndrome (CSHS), caused by somatic RAS mutations, features excess fibroblast growth factor-23 (FGF23) and skeletal dysplasia. Records from 56 individuals were reviewed and demonstrated fractures, scoliosis, and non-congenital hypophosphatemia that in some cases were resolved. Phosphate and calcitriol, but not skin lesion removal, were effective at controlling hypophosphatemia. No skeletal malignancies were found. PURPOSE: CSHS is a disorder defined by the association of epidermal and/or melanocytic nevi, a mosaic skeletal dysplasia, and an FGF23-mediated hypophosphatemia. To date, somatic RAS mutations have been identified in all patients whose affected tissue has undergone DNA sequencing. However, the clinical spectrum and treatment are poorly defined in CSHS. The purpose of this study is to determine the spectrum of the phenotype, natural history of the disease, and response to treatment of hypophosphatemia. METHODS: Five CSHS subjects underwent prospective data collection at clinical research centers. A review of the literature identified 45 reports that included a total of 51 additional patients, in whom the findings were compatible with CSHS. Data on nevi subtypes, bone histology, mineral and skeletal disorders, abnormalities in other tissues, and response to treatment of hypophosphatemia were analyzed. RESULTS: Fractures, limb deformities, and scoliosis affected most CSHS subjects. Hypophosphatemia was not present at birth. Histology revealed severe osteomalacia but no other abnormalities. Skeletal dysplasia was reported in all anatomical compartments, though less frequently in the spine; there was no clear correlation between the location of nevi and the skeletal lesions. Phosphate and calcitriol supplementation was the most effective therapy for rickets. Convincing data that nevi removal improved blood phosphate levels was lacking. An age-dependent improvement in mineral abnormalities was observed. A spectrum of extra-osseous/extra-cutaneous manifestations that included both benign and malignant neoplasms was present in many subjects, though osteosarcoma remains unreported. CONCLUSION: An understanding of the spectrum, natural history, and efficacy of treatment of hypophosphatemia in CSHS may improve the care of these patients.

Human hypertension caused by mutations in WNK kinases.

Hypertension is a major public health problem of largely unknown cause. Here, we identify two genes causing pseudohypoaldosteronism type II, a Mendelian trait featuring hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Both genes encode members of the WNK family of serine-threonine kinases. Disease-causing mutations in WNK1 are large intronic deletions that increase WNK1 expression. The mutations in WNK4 are missense, which cluster in a short, highly conserved segment of the encoded protein. Both proteins localize to the distal nephron, a kidney segment involved in salt, K+, and pH homeostasis. WNK1 is cytoplasmic, whereas WNK4 localizes to tight junctions. The WNK kinases and their associated signaling pathway(s) may offer new targets for the development of antihypertensive drugs.

Mutations in ATP6N1B, encoding a new kidney vacuolar proton pump 116-kD subunit, cause recessive distal renal tubular acidosis with preserved hearing.

The multi-subunit H+-ATPase pump is present at particularly high density on the apical (luminal) surface of -intercalated cells of the cortical collecting duct of the distal nephron, where vectorial proton transport is required for urinary acidification. The complete subunit composition of the apical ATPase, however, has not been fully agreed upon. Functional failure of -intercalated cells results in a group of disorders, the distal renal tubular acidoses (dRTA), whose features include metabolic acidosis accompanied by disturbances of potassium balance, urinary calcium solubility, bone physiology and growth. Mutations in the gene encoding the B-subunit of the apical pump (ATP6B1) cause dRTA accompanied by deafness. We previously localized a gene for dRTA with preserved hearing to 7q33-34 (ref. 4). We report here the identification of this gene, ATP6N1B, which encodes an 840 amino acid novel kidney-specific isoform of ATP6N1A, the 116-kD non-catalytic accessory subunit of the proton pump. Northern-blot analysis demonstrated ATP6N1B expression in kidney but not other main organs. Immunofluorescence studies in human kidney cortex revealed that ATP6N1B localizes almost exclusively to the apical surface of -intercalated cells. We screened nine dRTA kindreds with normal audiometry that linked to the ATP6N1B locus, and identified different homozygous mutations in ATP6N1B in eight. These include nonsense, deletion and splice-site changes, all of which will truncate the protein. Our findings identify a new kidney-specific proton pump 116-kD accessory subunit that is highly expressed in proton-secreting cells in the distal nephron, and illustrate its essential role in normal vectorial acid transport into the urine by the kidney.

Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption.

Epithelia permit selective and regulated flux from apical to basolateral surfaces by transcellular passage through cells or paracellular flux between cells. Tight junctions constitute the barrier to paracellular conductance; however, little is known about the specific molecules that mediate paracellular permeabilities. Renal magnesium ion (Mg2+) resorption occurs predominantly through a paracellular conductance in the thick ascending limb of Henle (TAL). Here, positional cloning has identified a human gene, paracellin-1 (PCLN-1), mutations in which cause renal Mg2+ wasting. PCLN-1 is located in tight junctions of the TAL and is related to the claudin family of tight junction proteins. These findings provide insight into Mg2+ homeostasis, demonstrate the role of a tight junction protein in human disease, and identify an essential component of a selective paracellular conductance.

High-efficiency gene transfer and pharmacologic selection of genetically engineered human keratinocytes.

Low efficiencies of gene transfer to somatic cells have frustrated therapeutic gene delivery efforts in a wide array of tissues including the skin. Production of populations of keratinocytes in which all cells contain the desired therapeutic gene may be important in future genetic therapies. This may be the case in disorders such as epidermolysis bullosa and ichthyosis, where a failure to correct the vast majority of cells within tissue could perpetuate central disease features such as skin fragility and defective barrier function. We have refined retroviral gene transfer parameters to achieve significant improvements in gene delivery efficiencies to human keratinocytes compared to those previously reported. We have also generated retroviral vectors that allow rapid pharmacologic selection of human keratinocytes without interfering with the potential of these cells to regenerate epidermis in vivo--we determined that blasticidin is superior to the commonly used neomycin. The combined capabilities for efficient retroviral gene transfer and effective pharmacologic selection allow production of entirely engineered populations of human keratinocytes for use in future efforts to achieve effective cutaneous gene delivery.

Transglutaminase 1 expression in a patient with features of harlequin ichthyosis: case report.

Harlequin ichthyosis (HI) is a life-threatening disorder characterized clinically by massive generalized hyperkeratosis and ultrastructurally by an absence of lamellar bodies. However, infants who survive the perinatal period develop a phenotype resembling the nonbullous ichthyosiform erythrodermic (CIE) form of autosomal recessive ichthyosis. We studied a child with a severe hyperkeratotic skin disorder present at birth that developed into a CIE-like phenotype. Electron microscopy demonstrated an absence of lamellar bodies consistent with HI. Abnormalities of filaggrin and involucrin expression by immunostaining were evident. However, transglutaminase 1 (TGase1) was expressed in the epidermis in a pattern consistent with other diseases that involve epidermal acanthosis. Analysis of patient keratinocytes grown in vitro demonstrated expression of normal amounts of TGase1 mRNA and full length TGase1 protein, as well as normal levels of transglutaminase enzymatic activity.

Abnormal transglutaminase 1 expression pattern in a subset of patients with erythrodermic autosomal recessive ichthyosis.

An autosomal recessive ichthyosis characterized by collodian membrane at birth followed by generalized skin redness and fine, light-colored scales has been termed nonbullous congenital ichthyosiform erythroderma (CIE). CIE has often been classified together with the other major form of recessive ichthyosis without internal organ involvement, lamellar ichthyosis, which is characterized by minimal erythema and a coarser, darker scale pattern. Recently, autosomal recessive ichthyosis has been associated with keratinocyte transglutaminase (TGase1) defects in some patients. This group of diseases, however, is genetically heterogeneous and TGase1 abnormalities in CIE have not been clearly described. Therefore we examined TGase1 expression in five patients with CIE and three with classic lamellar ichthyosis. Although lamellar ichthyosis patients displayed no TGase1 expression, an abnormal intracellular accumulation of TGase1 was observed in four of five CIE patients. This finding was specific and was not observed in other skin disorders characterized by erythema and abnormal cornification, including erythrodermic psoriasis, atopic dermatitis, epidermolytic hyperkeratosis, and Netherton's syndrome. CIE keratinocytes with abnormal TGase1 localization expressed full-length TGase1 mRNA and protein but demonstrated transglutaminase activity intermediate between normal and the minimal activity seen in lamellar ichthyosis patient cells. The abnormal TGase1 expression pattern and CIE clinical features were recapitulated in epidermis regenerated in vivo on immune deficient mice from CIE patient keratinocytes. These studies describe a specific abnormality in TGase1 intrinsic to keratinocytes in a subset of CIE patients and suggest that this abnormality may be involved in the disordered epidermal differentiation seen in this disorder.

Direct cutaneous gene delivery in a human genetic skin disease.

The skin is an accessible somatic tissue for therapeutic gene transfer and, depending on therapeutic goals, a variety of cutaneous gene delivery approaches are currently available. Recent advances in direct injection of naked DNA into intact skin have shown promise and are less labor-intensive than approaches involving grafting of genetically modified cells. We have regenerated skin from transglutaminase 1 (TGase1)-deficient patients with the genetic skin disease lamellar ichthyosis (LI) on nude mice to examine the corrective impact of direct naked plasmid injection. Regenerated LI patient skin receiving repeated in vivo injections with a TGase1 expression plasmid displayed restoration of TGase1 expression in the correct tissue location in the suprabasal epidermis. Unlike LI skin regenerated from keratinocytes, first transduced in vitro with a retroviral expression vector for TGase1 prior to grafting, however, directly injected LI skin displayed a nonuniform TGase1 gene expression pattern. In further contrast, direct injection failed to correct the central histologic and functional abnormalities of the disease. These data demonstrate that partial restoration of gene expression can be achieved via direct injection of naked DNA in human genetic skin disease tissue but underscore the need for new advances to achieve efficient and sustained plasmid-based gene delivery to the skin.

A model of corrective gene transfer in X-linked ichthyosis.

Single gene recessive genetic skin disorders offer attractive prototypes for the development of therapeutic cutaneous gene delivery. We have utilized X-linked ichthyosis (XLI), characterized by loss of function of the steroid sulfatase arylsulfatase C (STS), to develop a model of corrective gene delivery to human skin in vivo. A new retroviral expression vector was produced and utilized to effect STS gene transfer to primary keratinocytes from XLI patients. Transduction was associated with restoration of full-length STS protein expression as well as steroid sulfatase enzymatic activity in proportion to the number of proviral integrations in XLI cells. Transduced and uncorrected XLI keratinocytes, along with normal controls, were then grafted onto immunodeficient mice to regenerate full thickness human epidermis. Unmodified XLI keratinocytes regenerated a hyperkeratotic epidermis lacking STS expression with defective skin barrier function, effectively recapitulating the human disease in vivo. Transduced XLI keratinocytes from the same patients, however, regenerated epidermis histologically indistinguishable from that formed by keratinocytes from patients with normal skin. Transduced XLI epidermis demonstrated STS expression in vivo by immunostaining as well as a normalization of histologic appearance at 5 weeks post-grafting. In addition, transduced XLI epidermis demonstrated a return of barrier function parameters to normal. These findings demonstrate corrective gene delivery in human XLI patient skin tissue at both molecular and functional levels and provide a model of human cutaneous gene therapy.

Sustainability of keratinocyte gene transfer and cell survival in vivo.

The epidermis is an attractive site for therapeutic gene delivery because it is accessible and capable of delivering polypeptides to the systemic circulation. A number of difficulties, however, have emerged in attempts at cutaneous gene delivery, and central among these is an inability to sustain therapeutic gene production. We have examined two major potential contributing factors, viral vector stamina and involvement of long-lived epidermal progenitor cells. Human keratinocytes were either untreated or transduced with a retroviral vector for beta-galactosidase (beta-Gal) at > 99% efficiency and then grafted onto immunodeficient mice to regenerate human epidermis. Human epidermis was monitored in vivo after grafting for clinical and histologic appearance as well as for gene expression. Although integrated vector sequences persisted unchanged in engineered epidermis at 10 weeks post-grafting, retroviral long terminal repeat (LTR)-driven beta-Gal expression ceased in vivo after approximately 4 weeks. Endogenous cellular promoters, however, maintained consistently normal gene expression levels without evidence of time-dependent decline, as determined by immunostaining with species-specific antibodies for human involucrin, filaggrin, keratinocyte transglutaminase, keratin 10, type VII collagen, and Laminin 5 proteins out to week 14 post-grafting. Transduced human keratinocytes generated multilayer epidermis sustained through multiple epidermal turnover cycles; this epidermis demonstrated retention of a spatially appropriate pattern of basal and suprabasal epidermal marker gene expression. These results confirm previous findings suggesting that viral promoter-driven gene expression is not durable and demonstrate that keratinocytes passaged in vitro can regenerate and sustain normal epidermis for prolonged periods.

Fas signal transduction triggers either proliferation or apoptosis in human fibroblasts.

Although shown to be highly expressed by the epidermis in inflammatory skin disease, the ability of the Fas protein to trigger apoptosis in the distinct cell subpopulations of cutaneous tissue, particularly with regard to receptor density and the degree of crosslinking, has not been fully characterized. We therefore determined the effect of Fas cross-linking in primary human dermal fibroblasts at both high and low levels of Fas receptor expression. First, we examined the effects of the anti-Fas monoclonal antibody, CH-11, on fibroblasts expressing low basal levels of Fas. In these cells Fas aggregation stimulated proliferation by 160 +/- 10% over untreated controls. In contrast, the same concentration of CH-11 had an inhibitory effect on epidermal keratinocyte growth. Because Fas is upregulated in inflamed skin, we next examined the effects of Fas cross-linking on fibroblasts expressing augmented levels ofFas. Fibroblasts were either transfected with plasmids for overexpression of full length or bioengineered Fas receptors or were transduced with a retroviral Fas expression vector. In these cells Fas oligomerization triggered the morphologic changes indicative of apoptosis regardless of whether or not the Fas-signaling domain was tethered to the plasma membrane. These studies indicate that Fas oligomerization in dermal fibroblasts may initiate dual signaling programs, either proliferation or apoptosis, and that the chosen outcome may depend upon the magnitude of Fas aggregation.

Transglutaminase 1 delivery to lamellar ichthyosis keratinocytes.

Therapeutic gene delivery in severe genetic skin disease may require production of a uniformly corrected population of cells capable of regeneration of normal skin elements when returned to the host. To achieve this, we have used lamellar ichthyosis (LI), a disorder of epidermal differentiation recently associated with defects in keratinocyte transglutaminase (TGase1), as a prototype. We have used a high-efficiency retroviral delivery approach to uniformly restore normal levels of TGase1 expression to primary keratinocytes from severely affected LI patients previously lacking TGase1. Delivered TGase1 was correctly targeted to membrane association and restored patient cell transglutaminase activity levels to normal. Corrected primary LI patient keratinocytes also demonstrated restoration of previously defective involucrin cross-linking and in vitro measures of cornification to levels found in normal cells. These results indicate that efficient TGase1 delivery to early passage keratinocytes can produce a population of corrected LI patient cells. The capability to produce such cells may provide a basis for future efforts at gene therapy for genetic skin disease.

Specific triggering of the Fas signal transduction pathway in normal human keratinocytes.

The epidermis is continually exposed to genotoxic injury and requires an efficient mechanism to eliminate genetically altered cells. The membrane receptor, Fas, initiates apoptosis in many cell types, including keratinocytes. Receptor cross-linking is the vital post-ligand binding step in Fas signal transduction, and we have utilized FK1012, capable of oligomerizing proteins engineered to contain the FK506 binding protein (FKBP), to trigger Fas via FKBP-linked receptor cytoplasmic domains in human keratinocytes. An FKBP chimera containing the Fas cytoplasmic domain targeted to the plasma membrane induced an up to 89% decrease in viability of keratinocytes, as reflected by the activity of constitutive promoters, in response to FK1012. Oligomerization of Fas, either with engineered Fas.FKBP by FK1012 or via antibody cross-linking of full-length Fas-induced cellular changes consistent with apoptosis. The lpr Fas point mutation abolished this effect. A Fas.FKBP construct unlinked to the membrane was fully active in this assay. Early developmental age or pre-treatment of cells with GM-CSF, TGF-beta, EGF, KGF, IFN-gamma, or phorbol ester failed to protect against Fas effects. These findings reveal that the Fas signal transduction pathway is active in keratinocytes, requires no induction, and dominantly overrides growth stimuli.

Corrective gene transfer in the human skin disorder lamellar ichthyosis.

Lamellar ichthyosis (LI) is a disfiguring skin disease characterized by abnormal epidermal differentiation and defective cutaneous barrier function. LI has been associated with loss of keratinocyte transglutaminase 1 (TGase1), an enzyme believed necessary for normal formation of the cornified epidermal barrier. Using LI as a prototype for therapeutic cutaneous gene delivery, we have used the human skin/immunodeficient mouse xenograft model to correct the molecular, histologic and functional abnormalities of LI patient skin in vivo. We have used TGase1-deficient primary keratinocytes from LI patients combined with high-efficiency transfer of functional TGase1 to regenerate engineered human LI epidermis on immunodeficient mice. Engineered LI epidermis displayed normal TGase1 expression in vivo, unlike unengineered LI epidermis where TGase1 was absent. Epidermal architecture was also normalized by TGase1 restoration, as was expression of the epidermal differentiation marker filaggrin. Engineered LI skin demonstrated restoration of cutaneous barrier function measures to levels seen in epidermis regenerated by keratinocytes from patients with normal skin, indicating functional correction in vivo of the proposed primary pathophysiologic defect in LI. These results confirm a major role for TGase1 in epidermal differentiation and demonstrate a potential future approach to therapeutic gene delivery in human skin.