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Genetic and Molecular Studies of Two Hereditary Skin Disorders
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Monogenic disorders, i.e., disorders caused by mutations in a single gene, are rare and clinically heterogeneous conditions. Identification of the genetic cause of monogenic traits can bring new insights into molecular pathways and disease mechanisms. The aims of the present study were to identify the mutant genes in two autosomal recessive skin disorders and to characterize the functions of the mutated genes.  In order to identify candidate genes for the two disorders whole-genome SNP analysis, homozygosity mapping and gene sequencing were used.

Autosomal recessive congenital ichthyosis (ARCI) is a group of disorders characterized by extensive scaling and redness of the skin.  A subgroup of ARCI patients (n=27) was selected based on specific ultrastructural aberrations in their skin, revealed by electron microscopy. Mutations were identified in the Ichthyin gene in 93% of the selected patients, indicating a strong association between mutant Ichthyin and the specific morphological abnormalities. Ichthyin mRNA levels were shown to increase during keratinocyte differentiation in cells from healthy and affected individuals. Electron microscopy revealed a localization of ichthyin protein to keratins and desmosomes in epidermis. Staining of epidermal lipids identified aberrant lipid aggregates in skin sections of patients with Ichthyin mutations, indicating a role for Ichthyin in epidermal lipid metabolism.

In twelve KLICK syndrome patients with ichthyosis, palmoplantar keratoderma and keratotic striae on joints, a single-nucleotide deletion was identified in the 5’ region of the proteasome maturation protein (POMP) gene.  The deletion caused an increase in the proportion of POMP transcripts with long 5’ UTR’s in patient keratinocytes.  Immunohistochemical analysis of differentiated skin cell layers revealed aberrant expression of POMP, proteasome subunits and the skin protein filaggrin in patients. CHOP expression, associated with endoplasmic reticulum stress, was increased in the same layers. siRNA silencing of POMP in cell cultures reduced proteasome subunit levels and induced expression of CHOP.  The results indicate that the mutation in KLICK patients causes POMP and proteasome insufficiency with subsequent cellular stress.

This study conclusively contributes to the understanding of epidermal physiology and the pathogenesis of two inherited skin diseases.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 57 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 657
Keyword [en]
Monogenic disorder, autosomal recessive congenital ichthyosis, KLICK syndrome, Ichthyin, POMP, proteasome, epidermal differentiation
National Category
Medical Genetics
Research subject
Clinical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-149185ISBN: 978-91-554-8038-7OAI: oai:DiVA.org:uu-149185DiVA: diva2:405933
Public defence
2011-05-06, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskiölds väg 20, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2011-04-14 Created: 2011-03-15 Last updated: 2011-05-05Bibliographically approved
List of papers
1. Congenital ichthyosis: mutations in ichthyin are associated with specific structural abnormalities in the granular layer of epidermis
Open this publication in new window or tab >>Congenital ichthyosis: mutations in ichthyin are associated with specific structural abnormalities in the granular layer of epidermis
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2007 (English)In: Journal of Medical Genetics, ISSN 0022-2593, E-ISSN 1468-6244, Vol. 44, no 10, 615-620 p.Article in journal (Refereed) Published
Abstract [en]

Background: Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of skin disorders. Several mutant genes have been identified in ARCI, but the association between genotype and phenotype is poorly understood.

Methods: To investigate genotype–phenotype correlations in ARCI, we selected 27 patients from 18 families with specific ultrastructural features of the epidermis. The characteristic findings using electron microscopy (EM) were abnormal lamellar bodies and elongated membranes in the stratum granulosum, classified as ARCI EM type III. DNA samples from a subset of affected individuals were screened for homozygous genomic regions, and a candidate gene region was identified on chromosome 5q33. The region coincides with the ichthyin gene, previously reported as mutated in ARCI.

Results: Mutation screening of ichthyin revealed missense or splice-site mutations in affected members from 16 of 18 (89%) families with characteristics of ARCI EM type III. In a control group of 18 patients with ARCI without EM findings consistent with type III, we identified one patient homozygous for a missense mutation in ichthyin.

Discussion: Our findings indicate a strong association between ultrastructural abnormalities in the granular layer of epidermis and ichthyin mutations. The results also suggest that EM provides a tool for specific diagnosis in a genetically homogenous subgroup of patients with ARCI.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-12690 (URN)10.1136/jmg.2007.050542 (DOI)000249889300002 ()
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2011-05-05Bibliographically approved
2. Ichthyin Localizes to Keratins and Desmosomes in Epidermis and is Involved in Lipid Metabolism
Open this publication in new window or tab >>Ichthyin Localizes to Keratins and Desmosomes in Epidermis and is Involved in Lipid Metabolism
(English)Manuscript (preprint) (Other academic)
Keyword
Autosomal recessive congenital ichthyosis, Ichthyin, epidermal lipid metabolism
National Category
Medical Genetics
Research subject
Clinical Genetics
Identifiers
urn:nbn:se:uu:diva-149899 (URN)
Available from: 2011-03-24 Created: 2011-03-24 Last updated: 2011-05-05
3. A single-nucleotide deletion in the POMP 5' UTR causes a transcriptional switch and altered epidermal proteasome distribution in KLICK genodermatosis
Open this publication in new window or tab >>A single-nucleotide deletion in the POMP 5' UTR causes a transcriptional switch and altered epidermal proteasome distribution in KLICK genodermatosis
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2010 (English)In: American Journal of Human Genetics, ISSN 0002-9297, E-ISSN 1537-6605, Vol. 86, no 4, 596-603 p.Article in journal (Refereed) Published
Abstract [en]

KLICK syndrome is a rare autosomal-recessive skin disorder characterized by palmoplantar keratoderma, linear hyperkeratotic papules, and ichthyosiform scaling. In order to establish the genetic cause of this disorder, we collected DNA samples from eight European probands. Using high-density genome-wide SNP analysis, we identified a 1.5 Mb homozygous candidate region on chromosome 13q. Sequence analysis of the ten annotated genes in the candidate region revealed homozygosity for a single-nucleotide deletion at position c.-95 in the proteasome maturation protein (POMP) gene, in all probands. The deletion is included in POMP transcript variants with long 5' untranslated regions (UTRs) and was associated with a marked increase of these transcript variants in keratinocytes from KLICK patients. POMP is a ubiquitously expressed protein and functions as a chaperone for proteasome maturation. Immunohistochemical analysis of skin biopsies from KLICK patients revealed an altered epidermal distribution of POMP, the proteasome subunit proteins alpha 7 and beta 5, and the ER stress marker CHOP. Our results suggest that KLICK syndrome is caused by a single-nucleotide deletion in the 5' UTR of POMP resulting in altered distribution of POMP in epidermis and a perturbed formation of the outermost layers of the skin. These findings imply that the proteasome has a prominent role in the terminal differentiation of human epidermis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-127209 (URN)10.1016/j.ajhg.2010.02.018 (DOI)000276716800010 ()20226437 (PubMedID)
Available from: 2010-07-08 Created: 2010-07-08 Last updated: 2016-09-09Bibliographically approved
4. siRNA silencing of proteasome maturation protein (POMP) activates the unfolded protein response and constitutes a model for KLICK genodermatosis
Open this publication in new window or tab >>siRNA silencing of proteasome maturation protein (POMP) activates the unfolded protein response and constitutes a model for KLICK genodermatosis
2012 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 7, no 1, e29471- p.Article in journal (Refereed) Published
Abstract [en]

Keratosis linearis with ichthyosis congenita and keratoderma (KLICK) is an autosomal recessive skin disorder associated with a single-nucleotide deletion in the 5'untranslated region of the proteasome maturation protein (POMP) gene. The deletion causes a relative switch in transcription start sites for POMP, predicted to decrease levels of POMP protein in terminally differentiated keratinocytes. To investigate the pathophysiology behind KLICK we created an in vitro model of the disease using siRNA silencing of POMP in epidermal air-liquid cultures. Immunohistochemical analysis of the tissue constructs revealed aberrant staining of POMP, proteasome subunits and the skin differentiation marker filaggrin when compared to control tissue constructs. The staining patterns of POMP siRNA tissue constructs showed strong resemblance to those observed in skin biopsies from KLICK patients. Western blot analysis of lysates from the organotypic tissue constructs revealed an aberrant processing of profilaggrin to filaggrin in samples transfected with siRNA against POMP. Knock-down of POMP expression in regular cell cultures resulted in decreased amounts of proteasome subunits. Prolonged silencing of POMP in cultured cells induced C/EBP homologous protein (CHOP) expression consistent with an activation of the unfolded protein response and increased endoplasmic reticulum (ER) stress. The combined results indicate that KLICK is caused by reduced levels of POMP, leading to proteasome insufficiency in differentiating keratinocytes. Proteasome insufficiency disturbs terminal epidermal differentiation, presumably by increased ER stress, and leads to perturbed processing of profilaggrin. Our findings underline a critical role for the proteasome in human epidermal differentiation.

National Category
Medical Genetics
Research subject
Clinical Genetics
Identifiers
urn:nbn:se:uu:diva-149416 (URN)10.1371/journal.pone.0029471 (DOI)000301123400059 ()
Available from: 2011-03-19 Created: 2011-03-19 Last updated: 2012-04-18Bibliographically approved

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