The proposed project is designed to provide me with the knowledge practical tools and experience in cellular biology to establish a strong, independent research program integrating dermatology, molecular genetics and cutaneous biology to study inherited skin disorders. The Department of Dermatology and Cutaneous Biology at Jefferson Medical College has an outstanding research program, and will provide laboratory facilities, equipment, resources and time to support this proposal.
The research aims to identify the spectrum of connexin defects involved in inherited skin disorders. The Department of Dermatology and Cutaneous Biology at Jefferson Medical College has an outstanding successful research program, and will provide laboratory facilities, equipment, resources and time to support this proposal.
The research aims to identify the spectrum of connexin defects involved in human inherited skin disorders, and to elucidate the role of connexins and gap junctional intercellular communication in normal and diseased skin. It is based on my extensive clinical linkage and molecular genetic studies of erythrokeratodermia variabilis (EKV) that recently resulted in the cloning of two novel human connexin genes, GJB3 and GJB5, and the disclosure of mutations in GJB3 as proximal cause of EKV. Moreover, I identified pathogenic mutations in GJB3 as proximal cause of EKV. Moreover, I identified pathogenic mutations in another connexin gene (GJB2) in a family with dominant palmoplantar keratoderma associated with deafness, and demonstrated in a collaborative study evidence for a dominant inhibitory effect of the mutant protein Cx26 on gap junction function. I now plan to clone the human GJB4 gene, and to screen the epidermally expressed connexin genes for mutations in EKV, other erythrokeratodermias and disorders of cornification, which will allow to define the spectrum of genodermatoses caused by connexin defects. In addition, the project focuses on the comprehensive characterization of the complex gap junction network of human skin, which might be formed by as many as 8 connexins, and its function in normal and disturbed epidermal differentiation. The development of antibodies against human Cx31 and rat Cx30.3 will facilitate immunohistochemical studies to evaluate the spatial and differential expression of all epidermal connexins. We will test the hypothesis that connexin mutations have a dominant- inhibitory effect on normal channel function, which is crucial for normal epidermal differentiation, with functional studies in different expression system. Constructs of normal and mutant connexin genes will e expressed in paired Xenopus oocytes, and their ability to form functional intercellular channel is measured by voltage clamp. Stable transfected HeLa cells will be used to study the intracellular sub-localization of different mutant Cx, while gap junctional intercellular communication is evaluated using the dye transfer technique. We expect that these approaches provide critical information on the patho-mechanism of connexin defects in skin.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08AR002141-05
Application #
6719674
Study Section
Arthritis and Musculoskeletal and Skin Diseases Special Grants Review Committee (AMS)
Program Officer
Moshell, Alan N
Project Start
2000-03-06
Project End
2005-02-28
Budget Start
2004-03-01
Budget End
2005-02-28
Support Year
5
Fiscal Year
2004
Total Cost
$121,631
Indirect Cost
Name
Thomas Jefferson University
Department
Dermatology
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
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Kellermayer, Richard; Keller, Matthew; Ratajczak, Paulina et al. (2005) Bigenic connexin mutations in a patient with hidrotic ectodermal dysplasia. Eur J Dermatol 15:75-9
Richard, Gabriele (2005) Connexin disorders of the skin. Clin Dermatol 23:23-32
Geyer, Adam S; Ratajczak, Paulina; Pol-Rodriguez, Marlyanne et al. (2005) Netherton syndrome with extensive skin peeling and failure to thrive due to a homozygous frameshift mutation in SPINK5. Dermatology 210:308-14
Shurman, Daniel L; Glazewski, Lisa; Gumpert, Anna et al. (2005) In vivo and in vitro expression of connexins in the human corneal epithelium. Invest Ophthalmol Vis Sci 46:1957-65

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