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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 [
      • Fischer T.
      • Gedde-Dahl Jr., T.
      Epidermolysis bullosa simplex and mottled pigmentation: a new dominant syndrome. I: clinical and histological features.
      ]. 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 [
      • 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.
      ]. 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) [
      • 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.
      ]. 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 [
      • Bchetnia M.
      • Tremblay M.L.
      • Leclerc G.
      • Dupérée A.
      • Powell J.
      • McCuaig C.
      • et al.
      Expression signature of epidermolysis bullosa simplex.
      ]. 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 nameCytoband
      Gene location obtained from National Center for Biotechnology Information public database (http://www.ncbi.nlm.nih.gov).
      pFlc
      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.
      Cluster 1: Lipid biosynthetic process
      8144917NM_000237LPLLipoprotein lipase8p220.0024.639
      7936322NM_020918GPAMGlycerol-3-phosphate acyltransferase, mitochondrial10q25.23E−043.665
      7948344NM_201648GLYATGlycine-N-acyltransferase11q12.10.0113.02
      8063590NM_002591PCK1Phosphoenolpyruvate carboxykinase 1 (soluble)20q13.310.0022.953
      7929816NM_005063SCDStearoyl-CoA desaturase (delta-9-desaturase)10q24.310.0252.586
      8003635NM_172367TUSC5Tumor suppressor candidate 517p13.30.0032.496
      7938629NM_000922PDE3BPhosphodiesterase 3B, cGMP-inhibited11p15.10.0022.473
      8037186NM_005357LIPELipase, hormone-sensitive19q13.20.0282.296
      7990391NM_000499CYP1A1Cytochrome P450, family 1, subfamily A, polypeptide 115q24.16E−052.188
      7940762NM_001142535LGALS12Lectin, galactoside-binding, soluble, 1211q130.0042.178
      7962312NM_005164ABCD2ATP-binding cassette, sub-family D (ALD), member 212q11-q120.0312.015
      8106448NM_003719PDE8BPhosphodiesterase 8B5q13.30.0062.004
      Cluster 2: Immune response
      8037283NM_002780PSG4Pregnancy specific beta-1-glycoprotein 419q13.20.0372.453
      7996022NM_002990CCL22Chemokine (C C motif) ligand 2216q130.0312.296
      7951297NM_002426MMP12Matrix metallopeptidase 12 (macrophage elastase)11q22.30.0132.242
      8014316NM_002985CCL5Chemokine (C C motif) ligand 517q11.2-q125E−042.205
      8135909NM_000230LEPLeptin7q31.30.0082.061
      Cluster 3: Keratinization and skin pigmentation
      7920201NM_001017418SPRR2BSmall proline-rich protein 2B1q21-q220.0272.573
      7942991NM_000372TYRTyrosinase (oculocutaneous albinism IA)11q14-q210.0362.108
      Cluster 4: Cell growth and proliferation
      8106025NM_018429BDP1B double prime 1, subunit of RNA polymerase III transcription initiation factor IIIB5q132E−064.314
      8085244NM_022094CIDECCell death-inducing DFFA-like effector c3p25.30.0063.471
      8176532NM_003308TSPY1Testis specific protein, Y-linked 1Yp11.20.028−3.348
      8055992NM_145259ACVR1CActivin A receptor, type IC2q24.10.0023.029
      7915468NM_199342CCDC23Coiled-coil domain containing 231p34.20.0022.728
      8112961NM_001025RPS23Ribosomal protein S235q14.20.002−2.652
      8119595NM_198486RPL7L1Ribosomal protein L7-like 16p21.10.0162.56
      8180270NM_031859PCDHA10Protocadherin alpha 105q315E−042.42
      7909441NM_015714G0S2G0/G1switch 21q32.20.0192.355
      8141791NM_001097615POLR2J3Polymerase (RNA) II (DNA directed) polypeptide J37q22.10.0322.319
      8027323NM_033468ZNF257Zinc finger protein 25719q130.018−2.319
      8085360NM_003256TIMP4TIMP metallopeptidase inhibitor 43p250.0022.241
      7907271NM_001460FMO2Flavin containing monooxygenase 2 (non-functional)1q24.30.0032.178
      8173120NM_002625PFKFB16-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 1Xp11.210.0022.111
      8031398NM_017852NLRP2NLR family, pyrin domain containing 219q13.420.0442.092
      7963826NM_006741PPP1R1AProtein phosphatase 1, regulatory (inhibitor) subunit 1A12q13.20.0242.084
      8135378NM_002736PRKAR2BProtein kinase, cAMP-dependent, regulatory, type II, beta7q220.0052.052
      7907572NM_020318PAPPA2Pappalysin 21q23-q250.0022.041
      7941444NM_001335CTSWCathepsin W11q13.15E−042.016
      Cluster 5: Other functions
      7946033NM_000518HBBHemoglobin, beta11p15.50.014−6.749
      8074980NM_000853GSTT1Glutathione S-transferase theta 122q11.230.029−4.419
      8005231NR_026809FAM106AFamily with sequence similarity 106, member A17p11.20.0233.489
      8110417NM_001079527FAM153CFamily with sequence similarity 153, member C5q35.30.0053.279
      7902400NR_002748SNORD45BSmall nucleolar RNA, C/D box 45B1p31.10.007−3.208
      8139100M30894TARPTCR gamma alternate reading frame protein7p15-p147E−043.039
      7991766NM_000558HBA1Hemoglobin, alpha 116p13.30.021−2.879
      8005204NM_014695CCDC144ACoiled-coil domain containing 144A17p11.23E−042.659
      8044351CR590757RPL22P15Ribosomal protein L22 pseudogene 152q130.008−2.445
      7986598NM_001001413GOLGA6L1Golgin A6 family-like 115q11.20.028−2.364
      7919146NR_003366ANKRD20BAnkyrin repeat domain 20B2q11.10.015−2.197
      8083240NM_031850AGTR1Angiotensin II receptor, type 13q240.0242.084
      7925749NM_001001821OR2T34Olfactory receptor, family 2, subfamily T, member 341q440.028−2.067
      8156253NM_015667FAM75A7Family with sequence similarity 75, member A79q120.01−2.025
      a 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.
      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 [
      • Bchetnia M.
      • Tremblay M.L.
      • Leclerc G.
      • Dupérée A.
      • Powell J.
      • McCuaig C.
      • et al.
      Expression signature of epidermolysis bullosa simplex.
      ]. 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. [
      • 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.
      ] 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. [
      • 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.
      ] 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. [
      • 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.
      ] and Wagner et al. [
      • 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.
      ] 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) [
      • 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.
      ] 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 [
      • Kwon 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.
      ,
      • Tomita Y.
      The molecular genetics of albinism and piebaldism.
      ].
      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 [
      • 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.
      ]. 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.

      Acknowledgements

      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.

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        Expression signature of epidermolysis bullosa simplex.
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