Characterization of point mutations in patients with X-linked ichthyosis. Effects on the structure and function of the steroid sulfatase protein.

X-linked ichthyosis is the result of steroid sulfatase (STS) deficiency. While most affected individuals have extensive deletions of the STS gene, point mutations have been reported in three patients (1). In this study, we identify an additional three point mutations and characterize the effects of all six mutations on STS activity and expression. All six are unique single base pair substitutions. The mutations are located in a 105-amino acid region of the C-terminal half of the polypeptide. Five of the six mutations involve the substitutions of Pro or Arg for Trp372, Arg for His444, Tyr for Cys446, or Leu for Cys341. The other mutation is in a splice junction and results in a frameshift causing premature termination of the polypeptide at residue 427. All the affected residues are conserved to some degree within the sulfatase family. The six mutations were reproduced in normal STS cDNA and transiently expressed in STS-deficient cells. All six mutant vectors direct the expression of STS protein that lacks enzymatic activity. The mutant polypeptides show a shift in mobility on SDS-PAGE and resistance to proteinase K digestion when translated in the presence of dog pancreas microsomes, indicating glycosylation and normal translocation.

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.

Characterization of the promoter region of human steroid sulfatase: a gene which escapes X inactivation.

The human X-linked steroid sulfatase gene (STS) was among the first genes shown to escape X inactivation. At least fourteen genes regulated in this fashion have now been recognized. They are dispersed into several regions of the X chromosome and may be controlled in a locus specific manner. Studies of the promoters of these genes could provide insights into the mechanism of X inactivation, however little information of this nature is currently available. For this reason we examined 5' flanking sequences of the human STS gene for promoter function. Four transcription start sites scattered over a 50bp region were identified. Functional domains of this TATA-less and GC poor promoter were identified by study of a series of terminal and internal deletions. A putative promoter sequence was identified which by itself exhibits little or no basal activity. However when combined with upstream regulatory elements, this segment showed weak but reproducible activity in a CAT (chloramphenicol acetyltransferase) reporter assay. Several regulatory domains acting as enhancers and repressors were subsequently identified. The relationship of this 5' sequence to the ability of the STS gene to escape X-inactivation is discussed.