Gene Expression Analysis of Epidermolysis Bullosa Simplex with Mottled Pigmentation

Epidermolysis bullosa simplex with mottled pigmentation (EBS-MP) is a subtype of epidermolysis bullosa simplex first reported in 1979 [[1]Fischer T. Gedde-Dahl Jr., T. Epidermolysis bullosa simplex and mottled pigmentation: a new dominant syndrome. I: clinical and histological features.Clin Genet. 1979; 15: 228-238Crossref PubMed Scopus (51) Google Scholar]. The disease has its onset in early childhood and manifests with either much localized skin blistering, resembling the Weber-Cockayne subtype of EBS, or with more extensive bulla formation as seen in the Koebner subtype of EBS. Associated features include palmoplantar keratoderma and reticular hyperpigmentation unrelated to the blistering. Histologically, intraepidermal cleavage and keratinocyte degeneration are constant features of EBS-MP and ultrastructural analysis of pigmented areas demonstrates abundant mature melanosomes within basal cells [[2]Uttam J. Hutton E. Coulombe P.A. Anton-Lamprecht I. Yu Q.C. Gedde-Dahl Jr., T. et al.The genetic basis of epidermolysis bullosa simplex with mottled pigmentation.Proc Natl Acad Sci USA. 1996; 93: 9079-9084Crossref PubMed Scopus (154) Google Scholar]. To date, EBS-MP disease was investigated at the genetic level in only 13 reports within the world including the current one. Most patients with EBS-MP have been shown to carry a dominant missense mutation (P25L) in the head domain of keratin 5 (K5) [[3]Glàsz-Bóna A. Medvecz M. Virágh Z. Hatvani Z. Blazsek A. Kárpáti S. Epidermolysis bullosa simplex with mottled pigmentation – mutation analysis proved the diagnosis in a four-generation pedigree.Eur J Dermatol. 2010; 20: 698-700PubMed Google Scholar]. Our present report used microarray analysis to compare the patterns of gene expression from skin biopsies (superficial 2-mm punch biopsies of buttocks normal appearing skin) of one EBS-MP patient versus seven normal subjects in an effort to better characterize new aspects of EBS-MP pathology. The studied EBS-MP patient was a 11-year-old male of French-Canadian Caucasian origin. He exhibited blistering of the skin since birth especially to the extremities, mottled pigmentation of the limbs and trunk (2–5 mm), mild nail dystrophy and punctuated hyperkeratosis especially on the soles. DNA sequencing of keratin 5 gene (KRT5) showed a C → T transition at nucleotide position 74 in the EBS-MP patient resulting in the substitution of leucine to proline residue at position 25 (P25L) in the head domain of K5. Since eleven seemingly unrelated EBS-MP families in the literature have exactly the same keratin mutation, this strongly suggests that the P25L mutation underlies both epidermolysis bullosa and the associated pigmentation. To screen for candidate genes that may contribute to the pathogenesis of EBS-MP, we compared the expression profile of skin tissue between the EBS-MP patient and seven volunteers (as EBS-MP is an orphan disease, we were unable to recruit more than one EBS-MP case from the same population). Gene expression was assessed on the Affymetrix Genechip Human Gene 1.0 ST microarrays as described previously [[4]Bchetnia M. Tremblay M.L. Leclerc G. Dupérée A. Powell J. McCuaig C. et al.Expression signature of epidermolysis bullosa simplex.Hum Genet. 2012; 131: 393-406Crossref PubMed Scopus (19) Google Scholar]. We found 52 differentially expressed genes (p value <0.05 and absolute fold change >2) including twelve genes involved in lipid biosynthesis (LPL, GPAM, GLYAT, PCK1, SCD, TUSC5, PDE3B, LIPE, CYP1A1, LGALS12, ABCD2, PDE8B), two genes in keratinization and skin pigmentation processes (SPRR2B, TYR), nineteen genes in cell growth and apoptosis (BDP1, CIDEC, TSPY1, ACVR1C, CCDC23, RPS23, RPL7L1, PCDHA10, G0S2, POLR2J3, ZNF257, TIMP4, FMO2, PFKFB1, NLRP2, PPP1R1A, PRKAR2B, PAPPA2, CTSW), five genes in immune response (PSG4, CCL22, MMP12, CCL5, LEP) and fourteen genes with predicted or unknown function. Summary of the data is presented in Table 1.Table 1List of genes differentially expressed in EBS-MP skin patient in comparison to healthy subjects.ClustersProbe setACCNUMGene symbolGene nameCytobandaGene location obtained from National Center for Biotechnology Information public database (http://www.ncbi.nlm.nih.gov).pFlcbFold-changes (Flc) are indicated for each probe set significantly under- or over-expressed (p<0.05; absolute Flc>2) by epidermal cells of the EBS-MP patient compared with control subjects. Positive data indicate genes that are overexpressed; negative data indicate genes that are underexpressed by epidermal cells of the patient.Cluster 1: Lipid biosynthetic process8144917NM_000237LPLLipoprotein lipase8p220.0024.6397936322NM_020918GPAMGlycerol-3-phosphate acyltransferase, mitochondrial10q25.23E−043.6657948344NM_201648GLYATGlycine-N-acyltransferase11q12.10.0113.028063590NM_002591PCK1Phosphoenolpyruvate carboxykinase 1 (soluble)20q13.310.0022.9537929816NM_005063SCDStearoyl-CoA desaturase (delta-9-desaturase)10q24.310.0252.5868003635NM_172367TUSC5Tumor suppressor candidate 517p13.30.0032.4967938629NM_000922PDE3BPhosphodiesterase 3B, cGMP-inhibited11p15.10.0022.4738037186NM_005357LIPELipase, hormone-sensitive19q13.20.0282.2967990391NM_000499CYP1A1Cytochrome P450, family 1, subfamily A, polypeptide 115q24.16E−052.1887940762NM_001142535LGALS12Lectin, galactoside-binding, soluble, 1211q130.0042.1787962312NM_005164ABCD2ATP-binding cassette, sub-family D (ALD), member 212q11-q120.0312.0158106448NM_003719PDE8BPhosphodiesterase 8B5q13.30.0062.004Cluster 2: Immune response8037283NM_002780PSG4Pregnancy specific beta-1-glycoprotein 419q13.20.0372.4537996022NM_002990CCL22Chemokine (C C motif) ligand 2216q130.0312.2967951297NM_002426MMP12Matrix metallopeptidase 12 (macrophage elastase)11q22.30.0132.2428014316NM_002985CCL5Chemokine (C C motif) ligand 517q11.2-q125E−042.2058135909NM_000230LEPLeptin7q31.30.0082.061Cluster 3: Keratinization and skin pigmentation7920201NM_001017418SPRR2BSmall proline-rich protein 2B1q21-q220.0272.5737942991NM_000372TYRTyrosinase (oculocutaneous albinism IA)11q14-q210.0362.108Cluster 4: Cell growth and proliferation8106025NM_018429BDP1B double prime 1, subunit of RNA polymerase III transcription initiation factor IIIB5q132E−064.3148085244NM_022094CIDECCell death-inducing DFFA-like effector c3p25.30.0063.4718176532NM_003308TSPY1Testis specific protein, Y-linked 1Yp11.20.028−3.3488055992NM_145259ACVR1CActivin A receptor, type IC2q24.10.0023.0297915468NM_199342CCDC23Coiled-coil domain containing 231p34.20.0022.7288112961NM_001025RPS23Ribosomal protein S235q14.20.002−2.6528119595NM_198486RPL7L1Ribosomal protein L7-like 16p21.10.0162.568180270NM_031859PCDHA10Protocadherin alpha 105q315E−042.427909441NM_015714G0S2G0/G1switch 21q32.20.0192.3558141791NM_001097615POLR2J3Polymerase (RNA) II (DNA directed) polypeptide J37q22.10.0322.3198027323NM_033468ZNF257Zinc finger protein 25719q130.018−2.3198085360NM_003256TIMP4TIMP metallopeptidase inhibitor 43p250.0022.2417907271NM_001460FMO2Flavin containing monooxygenase 2 (non-functional)1q24.30.0032.1788173120NM_002625PFKFB16-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 1Xp11.210.0022.1118031398NM_017852NLRP2NLR family, pyrin domain containing 219q13.420.0442.0927963826NM_006741PPP1R1AProtein phosphatase 1, regulatory (inhibitor) subunit 1A12q13.20.0242.0848135378NM_002736PRKAR2BProtein kinase, cAMP-dependent, regulatory, type II, beta7q220.0052.0527907572NM_020318PAPPA2Pappalysin 21q23-q250.0022.0417941444NM_001335CTSWCathepsin W11q13.15E−042.016Cluster 5: Other functions7946033NM_000518HBBHemoglobin, beta11p15.50.014−6.7498074980NM_000853GSTT1Glutathione S-transferase theta 122q11.230.029−4.4198005231NR_026809FAM106AFamily with sequence similarity 106, member A17p11.20.0233.4898110417NM_001079527FAM153CFamily with sequence similarity 153, member C5q35.30.0053.2797902400NR_002748SNORD45BSmall nucleolar RNA, C/D box 45B1p31.10.007−3.2088139100M30894TARPTCR gamma alternate reading frame protein7p15-p147E−043.0397991766NM_000558HBA1Hemoglobin, alpha 116p13.30.021−2.8798005204NM_014695CCDC144ACoiled-coil domain containing 144A17p11.23E−042.6598044351CR590757RPL22P15Ribosomal protein L22 pseudogene 152q130.008−2.4457986598NM_001001413GOLGA6L1Golgin A6 family-like 115q11.20.028−2.3647919146NR_003366ANKRD20BAnkyrin repeat domain 20B2q11.10.015−2.1978083240NM_031850AGTR1Angiotensin II receptor, type 13q240.0242.0847925749NM_001001821OR2T34Olfactory receptor, family 2, subfamily T, member 341q440.028−2.0678156253NM_015667FAM75A7Family with sequence similarity 75, member A79q120.01−2.025a Gene location obtained from National Center for Biotechnology Information public database (http://www.ncbi.nlm.nih.gov).b Fold-changes (Flc) are indicated for each probe set significantly under- or over-expressed (p < 0.05; absolute Flc > 2) by epidermal cells of the EBS-MP patient compared with control subjects. Positive data indicate genes that are overexpressed; negative data indicate genes that are underexpressed by epidermal cells of the patient. Open table in a new tab To confirm the validity of our GeneChip data, we performed real-time PCR (qRT-PCR) for the EBS-MP patient and one control paired according to age and gender. qRT-PCR was performed for three genes [Tyrosinase (oculocutaneous albinism IA) (TYR), chemokine (C C motif) ligand 22 (CCL22), and activin A receptor, type IC (ACVR1C)] as described previously [[4]Bchetnia M. Tremblay M.L. Leclerc G. Dupérée A. Powell J. McCuaig C. et al.Expression signature of epidermolysis bullosa simplex.Hum Genet. 2012; 131: 393-406Crossref PubMed Scopus (19) Google Scholar]. We found statistically significant increase in gene expression of these three genes in the EBS-MP patient as observed in our microarrays results. The twelve differentially expressed genes involved in lipid metabolism are all more expressed in the EBS-MP patient compared to the control subjects suggesting that they can be involved in maintaining cell and tissue integrity in this phenotype. The gene expression changes in the immune response cluster included chemokine (C C motif) ligand 22 (CCL22) and chemokine (C C motif) ligand 5 (CCL5) which might be the key factors in controlling chronic skin inflammation. This result is in accordance with the previous report of Roth et al. [[5]Roth W. Reuter U. Wohlenberg C. Bruckner-Tuderman L. Magin T.M. Cytokines as genetic modifiers in K5−/− mice and in human epidermolysis bullosa simplex.Hum Mutat. 2009; 30: 832-841Crossref PubMed Scopus (46) Google Scholar] showing a higher expression of certain inflammatory cytokines (MCP-1/CCL2, MIP-3a/CCL20, and MIP-3b/CCL19) in a mouse with KRT5 mutations and with Lu et al. [[6]Lu H. Chen J. Planko L. Zigrino P. Klein-Hitpass L. Magin T.M. Induction of inflammatory cytokines by a keratin mutation and their repression by a small molecule in a mouse model for EBS.J Invest Dermatol. 2007; 127: 2781-2789Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar] showing significant higher expression of proinflammatory cytokines IL-6 and IL-1b in the transcriptome of K5−/− mouse. Therefore, potential contribution of immunological processes to the pathogenesis of EBS-MP was suggested. Among the nineteen genes differentially expressed and involved in cell differentiation, proliferation and apoptosis, three show lower expression levels in EBS-MP in comparison to control subjects and all the other are higher expressed in the patient suggesting that keratinocyte differenciation is disturbed in EBS-MP phenotype. These altered clusters are probably not associated to the pigmentation abnormalities seen in the EBS-MP phenotype as they had been documented to be altered in the EBS phenotype. Liovic et al. [[7]Liovic M. D’Alessandro M. Tomic-Canic M. Bolshakov V.N. Coats S.E. Lane E.B. Severe keratin 5 and 14 mutations induce down-regulation of junction proteins in keratinocytes.Exp Cell Res. 2009; 315: 2995-3003Crossref PubMed Scopus (44) Google Scholar] and Wagner et al. [[8]Wagner M. Hintner H. Bauer J.W. Onder K. Gene expression analysis of an epidermolysis bullosa simplex Dowling-Meara cell line by subtractive hybridization: recapitulation of cellular differentiation, migration and wound healing.Exp Dermatol. 2012; 21: 111-117Crossref PubMed Scopus (23) Google Scholar] showed that essentially genes encoding proteins expressed in the keratin-interacting desmosomes and hemidesmosomes, tight junctions and gap junctions are modulated in cultured EBS-Dowling-Meara cells and that none of these genes was significantly changed in the mild form of EBS (EBS-loc) [[7]Liovic M. D’Alessandro M. Tomic-Canic M. Bolshakov V.N. Coats S.E. Lane E.B. Severe keratin 5 and 14 mutations induce down-regulation of junction proteins in keratinocytes.Exp Cell Res. 2009; 315: 2995-3003Crossref PubMed Scopus (44) Google Scholar] which is in accordance with our microarray results as our patient presents the EBS-loc subtype in association with the mottled pigmentation. The most interesting difference between EBS-MP patient versus the healthy controls skin microarrays profiles, with obvious links to the skin mottled pigmentation associated to the studied phenotype, is tyrosinase (oculocutaneous albinism IA, TYR) gene expression alteration. TYR showed expression level that is two-fold increased in skin tissue of our EBS-MP subject in comparison to normal subjects. TYR is the main enzyme of melanin biosynthesis and is responsible of various forms of albinism [9Kwon B.S. Haq A.K. Pomerantz S.H. Halaban R. Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus.Proc Natl Acad Sci USA. 1987; 84: 7473-7477Crossref PubMed Scopus (397) Google Scholar, 10Tomita Y. The molecular genetics of albinism and piebaldism.Arch Dermatol. 1994; 130: 355-358Crossref PubMed Scopus (25) Google Scholar]. The higher expression of TYR in the microarray data could explain the observed ultrastructural features of EBS-MP phenotype including accumulation of mature melanosomes in basal keratinocytes that demonstrate perinuclear vacuolization and cytolysis [[2]Uttam J. Hutton E. Coulombe P.A. Anton-Lamprecht I. Yu Q.C. Gedde-Dahl Jr., T. et al.The genetic basis of epidermolysis bullosa simplex with mottled pigmentation.Proc Natl Acad Sci USA. 1996; 93: 9079-9084Crossref PubMed Scopus (154) Google Scholar]. So, we think that skin pigmentation in the EBS-MP patient may be dependent on tyrosinase function and we generated a cellular model of the studied patient (immortalized keratinocytes) in order to validate this hypothesis and to be useful for further genetic investigations of this rare phenotype. In conclusion, these data, providing a global view of physiopathologic processes active in EBS-MP phenotype, reveal differences of gene expressions between EBS-MP and normal subjects and identify TYR as a relevant gene that may be involved in this disease. We thank the patients and their families for their enthusiastic collaboration. This work was supported by Le Groupe Riverin Inc and by the Campagne majeure de développement de l’Université du Québec à Chicoutimi. The authors thank Claude Belleville for her invaluable help in recruiting and evaluating the subjects for the study, and the technical staff at the McGill University and Génome Québec Innovation Centre for microarray hybridization as well as the technical staff at the Sequencing facility of the CHUL/CHUQ.