ØEpidermolysis bullosa ØInherited ichthyoses • Group of diseases sharing two characteristic features - fragility of the skin and blistering. • The clinical features have a broad range of severity - from isolated nail dystrophy through relatively mild, localised blistering of extremities to generalized blistering and mutilation; adding to cutaneous complexity is finding of extracutaneous manifestations. Epidermolysis bullosa, dystrophic localisation: legs diagnosis: Epidermolysis Bullosa Hereditaria localisation: arms diagnosis: Epidermolysis Bullosa Hereditaria •Epidermolysis bullosa (EB) ebs-1 4 RTEmagicC_epidermolysis_bullosa_feet 15f1 • EB simplex – vznik puchýřů na úrovni bazálních keratinocytů – KRT5 (keratin 5), KRT14 (keratin 14), DSP (desmoplakin), DST (dystonin), JUP (junction plakoglobin), PKP1 (plakophilin), EXPH5 (exophilin 5), PLEC (plectin), TGM5 (transglutaminase 5) • EB junkční – vznik puchýřů na úrovni bazální membrány – COL17A1 (collagen, type XVII, alpha 1), LAMA3 (laminin, alpha 3), LAMB3 (laminin, beta 3), LAMC2 (laminin, gamma 2), ITGA6 (integrin, alpha 6), ITGB4 (integrin, beta 4), ITGA3 (integrin, alpha 3) • EB dystrofická – vznik puchýřů pod bazální membránou, na úrovni kotvících fibril – COL7A1 (collagen, type VII, alpha 1) •Epidermolysis bullosa, klasifikace •In 1960s, transmission electron microscopy was first applied to subdivide EB into three main categories. •In 1980s, the discovery and development of antibodies to different skin proteins permitted further insight into the pathophysiology of EB by immunofluorescence mapping of protein. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author •In 1990s, molecular genetic analysis of genes associated with particular types of EB was introduced. •Epidermolysis bullosa, diagnostics epidermolysis%20bullosa%202 • Mapping of type IV collagen (the lamina densa) to the roof of a blister in an EB patient’s skin, provided a rapid diagnosis of dystrophic EB. • • Mapping of type IV collagen to the floor of blister in an EB patient´s skin, provided diagnosis of EB simplex or junctional EB. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com •Focal separation within the epidermal basement membrane delineated by type IV collagen (stained red) and bullous pemphigoid antigen 2 (stained green). Nuclei (counterstained blue) outline the overlying epidermis as well as selected cells in the dermis. •Epidermolysis bullosa, immunofluorescence mapping •EBS •EBJ •EBD •A •B •D •C •E •F •A) normal skin, antibody to the type IV collagen, staining at the dermal–epidermal junction. B) patient, naturally occurring blister and the dermal–epidermal junction, labelling maps to the roof of the split, indicating sublamina densa plane of cleavage → EBD. C) normal skin, antibodies to the laminin, linear immunoreactivity at the dermal–epidermal junction. D) patient, the laminin labelling is present but maps to the base of the split → EBJ. G) Diagnosis of Dowling–Meara EB simplex by transmission electron microscopy. •G •Histopathology 2010, 56. Recessively inherited dystrophic epidermolysis bu... Dominantly inherited dystrophic epidermolysis bul... •Epidermolysis bullosa, diagnostika v EB Centru FNB •1. Klinické příznaky •(FNB, Pediatrická klinika) •2. Transmisní elektronová mikroskopie •(FNuSA, PAU) •3. Imunofluorescenční antigenní mapování •(FNuSA, PAU) •4. DNA nalýza (PCR-sekvenční analýza) •(FNB, CMBGT) •Dystrophic epidermolysis bullosa (DEB) •The COL7A1 gene (3p21, 118 exons); procollagen VII alpha chain. •DEB is inherited in both autosomal dominant (DDEB) and autosomal recessive manner (RDEB). •Dystrophic epidermolysis bullosa (DEB) •COL7A1 missense and nonsense mutations in DEB patients. The red lettering signifies dominant and the black signifies recessive. •N. Dang 2008 • DDEB usually involves glycine substitutions within the triple helical domain of COL7A1. •The clinical features of DEB have a broad range of severity: • The severe RDEB, associated with premature termination codon (PTC) mutations on both COL7A1 alleles. • The milder RDEB, caused by compound heterozygous mutations: one PTC mutation and one missense mutation (frequently glycine substitutions). •COL7A1 missense and nonsense mutations in DEB patients. The red lettering signifies dominant and the blue signifies recessive. N. Dang 2008 •Dystrophic epidermolysis bullosa (DEB) • COL7A1 - procollagen VII alpha 1 chain. Each proa1(VII) chain contains a central triple helical collagenous domain flanked by amino-terminal (NC-1) and carboxy-terminal (NC-2) non-collagenous domains. • The triple helical domain consists of a repeating Gly-X-Y sequence. • Three proa1(VII) chains folded into monomer. Two monomers form an antiparallel dimer, from which the NC-2 propeptides are removed proteolytically. Finally, the mature dimers laterally aggregate into anchoring fibrils. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author •Dystrophic epidermolysis bullosa (DEB) http://jmg.bmj.com/content/44/3/181/F1.large.jpg •Anchoring fibril assembly. • I: proa1(VII) are synthesized. • II, III: three chains assemble into the type VII collagen molecule. • IV, V: two type VII collagen molecules form the antiparallel dimer, the NC-2 domains are removed, and association of the monomers is stabilized by intermolecular disulphide bonds. • VI: a large number of dimer molecules assemble into anchoring fibrils and the NC-1 domain keeps the adhesive property at both ends. • •PTC mutation are associated with nonsense mediated mRNA decay (NMD). Missense mutations alter homotrimer formation. •Glycine substitutions often happen in triple helix region of COL7A1 affecting correct folding and secretion of the type VII collagen. •Uitto J., 2011 •Dystrophic epidermolysis bullosa (DEB) An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of referred object is ch12f52a.jpg. •Structure of collagen • Three procollagen VII alpha 1 chains coil around one another in characteristic triple helix structure. • The AA sequence of collagen triple helix domain consists of Gly-X-Y repeats. • The strict preservation of Gly in every third position is required for close packing of three supercoiled helices that make up the collagen triple helix structure. Gly residues are buried at the center of the triple helix, in a position that accommodates no other residue. • In contrast, the X and Y positions are exposed on the surface. The X position is frequently occupied by proline and the Y position by hydroxyproline. •Dystrophic epidermolysis bullosa (DEB) •Epidermolysis bullosa simplex •Three main autosomal dominant subtypes of EBS are distinguished based on the severity of blistering: • EBS localized - blistering is usually limited to the hands and feet. • EBS another generalized - more widespread blistering is observed but it is usually milder than in the most severe variant. • EBS Dowling-Meara - the most severe variant, characterized by generalized herpetiform blistering especially in the neonatal and infant periods. • • These subtypes of EBS are caused by mutations in either the keratin 5 (KRT5) or the keratin 14 (KRT14) genes. Most keratin mutations are inherited in an autosomal dominant manner. Epidermolysis bullosa simplex, Weber-Cockayne sub... Epidermolysis bullosa simplex, Koebner subtype. P... Ruptured bulla and newly erupted bulla of the leg... •Epidermolysis bullosa simplex (EBS) • Keratins are a group of structural proteins that polymerize to form keratin intermediate filaments. • • A characteristic feature of all intermediate filament proteins is the central alpha-helical rod domain, which is divided into four helices (1A, 1B, 2A and 2B) by three short nonhelical linker domains and terminated by globular head domains. • Keratins are divided into type I (KRT9–KRT24) or type II (KRT1–KRT8) proteins according to their physical and chemical properties, and the basic structural unit of intermediate filaments is a heterodimer of one type I keratin and its corresponding type II partner. • • Keratins 5 and 14 are natural partners that dimerize by coiled-coil interactions. http://www.biology.iupui.edu/biocourses/biol540h/images/grim_fil_struct_lab.jpg •Epidermolysis bullosa simplex (EBS) •Kirfek J., Cell. Mol. Life Sci. 60 (2003) •1A, 1B, 2A, 2B: a-helical parts; L1, L12, L2: non-a-helical parts, also called ‘linkers’. The a-helical segments exhibit a heptad substructure (abcdefg), where the a and d positions are occupied by apolar AAs. These hydrophobic AAs generate a surface that is wound around the axis of a single right-handed a-helix in a left-handed manner, ultimately leading to superhelix, i. e. coiled-coil formation of two such molecules. The phasing of the heptads is broken in the middle of segment 2B giving rise to a ‘stutter’. The stutter represents a helical segment which is not engaged in coiled-coil formation. The formation of heterodimers in which type I and type II polypeptide chains align in parallel and in exact axial register is the first step of keratin filament assembly. Two heterodimers associate, forming tetramer units aligned in an antiparallel manner. •The image illustrates the cell in EBS patients. It displays what a normal cell cytoskeleton ought to look like and what happens in EB disease states. normal cell and mutated cell •Epidermolysis bullosa simplex (EBS) From 75 probands with EBS: •26 EBS (34.7%), a mutation in KRT5 or KRT14, AD - the rate of de novo KRT5 and KRT14 mutations is 32.0% •40 superficial EBS/acral peeling skin sy., mutations in TGM5 (53.3%), AR •1 EBS with muscular dystrophy, mutations in PLEC (1.3%), AR •8 without a detectable causal mutation (10.7%) •Results of DNA diagnostics of EBS in CMBGT, FNB •Similar clinical features in the acral peeling skin syndrome (APSS) and localized EBS in young children. (a) Blisters (black arrows) and erosions (white arrows) on the palms and soles of the 2-year-old patient with APSS, and (b) of a 2-year-old patient with localized EBS heterozygous for the keratin 14 mutation. •TGM5 – acral peeling skin syndrome •TGM5 – AR – APSS •KRT14 – AD – EBS •D. Kiritski 2010 From 61 probands with DEB: •21, dominant DEB (34.4%), a mutation in COL7A1 - the rate of de novo COL7A1 mutations is 21.1% •39, recessive DEB (63.9%), mutations in COL7A1 •1 with recessive DEB, only one mutation identified (1.6%). At present, we have 57 living DEB patients in the Czech Republic Þ the estimated prevalence of DEB 5.4 cases per million inhabitants in the overall Czech population. •Results of DNA diagnostics of DEB in CMBGT, FNB 2005: DNA diagnostics of epidermolysis bullosa (EB) ØDystrophic EB - PCR and direct sequencing of COL7A1 ØSimplex EB - PCR and direct sequencing of KRT5 and KRT14 •DNA diagnostics of genodermatoses (skin and connective tissue disorders) in CMBGT Genodermatoses are a large group of clinically and genetically heterogeneous disorders. •DNA diagnostics of genodermatoses in CMBGT 2012: DNA diagnostics of ichthyoses ØAutosomal recesive congenital ichthyoses – PCR and direct sequencing of TGM1, ALOX12B, ALOXE3, CYP4F22, NIPAL4 ØRecesive X-linked ichthyosis – MLPA and direct sequencing of STS •Obr. 3: Long-Range PCR NEMO genu. 1 - 3 probandi, 4 – pozitivní kontrola s delecí 2,6 kb, 5 - negativní kontrola PCR-fragment analysis of NEMO MLPA of STS 2012: DNA diagnostics of incontinentia pigmenti – PCR and fragment analysis, PCR and direct sequencing of NEMO/IKBKG •DNA diagnostics of genodermatoses in CMBGT 2014 Ø„Sequence Capture“ and targeted sequencing of 100 genes associated with genodermatoses •Epidermis is separated from dermis by the basement membrane. •Keratinocytes, which compose epidermis, proliferate within the basal cell layer. Basal keratinocytes express KRT5, KRT14 and KRT15. •As basal keratinocytes commit to terminal differentiation, they switch off the expression of KRT5, KRT14 and KRT15 and induce the expression of KRT1, KRT10 and KRT2. •As differentiation proceeds, keratinocytes progress upwards through the different epidermal layers becoming anucleated and increasingly compacted in size, before being lost from the skin surface by desquamation. •A. Sandilands, Journal of Cell Science 2009 •Epidermal differentiation •EB junctional (COL17A1, LAMA3, LAMB3, LAMC2, ITGA6, ITGB4, ITGA3) •EB simplex (KRT5, KRT14, DSP, DST, JUP, PKP1, EXPH5, PLEC) •EB superficialis (TGM5) •ARCI (TGM1, ABCA12, NIPAL4, CYP4F22, ALOX12B, ALOXE3, PNPLA1, LIPN, CERS3) •Keratinopatic ichthyoses (KRT1, KRT2, KRT10) •Ichthyosis vulgaris (FLG) •EB dystrophic (COL7A1) •RXLI (STS) ØEpidermolysis bullosa – clinically and genetically heterogeneous group of disorders characterised by fragility of the skin and blistering. ØIchthyoses – clinically and genetically heterogeneous group of disorders characterized by dry, thickened, scaly or flaky skin; in many types there is cracked skin, which is said to resemble the scales on a fish. ØCommon ichthyoses (ichthyosis vulgaris - FLG, autosomal semidominant; recessive X-linked ichthyosis - STS) ØKeratinopathic ichthyoses (KRT1, KRT10, KRT2) – autosomal dominant ØAutosomal recessive congenital ichthyoses (TGM1, ABCA12, NIPAL4, CYP4F22, ALOX12B, ALOXE3, PNPLA1, LIPN, CERS3) •Inherited ichthyoses Clinically and genetically heterogeneous group of disorders characterized by scaling of the skin. Øaffects 1 in 2000 - 6000 males Øcaused by deficiency of the steroid sulfatase enzyme (a mutation in the STS gene) •STS is secreted into intercellular space of stratum corneum. •STS degrades cholesterol sulphate, generating cholesterol. The progressive decline in cholesterol sulphate permits corneodesmosome degradation leading to normal desquamation. •Recessive X-linked ichtyosis (RXLI), STS •P.M. Elias, J Lipid Res. 2008 •Cholesterol sulfotransferase (SULT2B1b) generates cholesterol sulfate in s. granulosum. •Steroid sulphatase (Ssase, STS) desulfated cholesterol sulfate back to cholesterol in s. corneum. •Disruption of this cycle accounts for abnormal desquamation and disruption of the skin barrier function. Øthe most common form of ichthyosis, affecting 1 in 250 people Øassociated with null mutations (mutations creating premature termination codon) in the FLG gene Øautosomal semidominant inheritancce with incomplete penetrance and variable expressivity • •disease severity can vary considerably even within affected families •IV can be asymptomatic, although patients may complain of dry, rough skin and cosmetic embarrassment •individuals with two FLG null mutations have a more severe phenotype than those with one FLG null mutation. •Ichthyosis vulgaris (IV), FLG •Diagrammatic representation of the FLG gene structure •S. J. Brown, J Invest Dermatol. 2012 •The FLG gene structure •The profilaggrin N-terminal domain plays role during terminal epidermal differentiation - the N-terminal domain is cleaved and translocates to the nucleus where it plays role in enucleation of keratinocytes in stratum corneum. •The precise function of the profilaggrin C-terminal domain is unclear but it is known to be required for the processing of profilaggrin to filaggrin. •10, 11 or 12 nearly identical filaggrin repeats (324 AA) have keratin binding properties. The large (>400kDa) insoluble profilaggrin is degraded to produce monomeric filaggrin in stratum corneum and then further proteolyzed to release amino acids. •FLG •A. Sandilands, Journal of Cell Science 2009 •Diagram summarizing the known and possible functions of profilaggrin, filaggrin, and amino •acids released by filaggrin proteolysis. •S. J. Brown, J Invest Dermatol. 2012 •Keratin-filaggrin degradation products •Results of DNA diagnostics of ARCI in CMBGT From 42 probands with ARCI: •14 (33.3%), ALOX12B •9 (21.4%), ALOXE3 •7 (16.6%), NIPAL4 •4 (9.5%), CYP4F22 •3 (7.1%), TGM1 •2 (4.8%), ABCA12 •3 (7.1%) without causal mutations V. Oji, 2009 ARCI phenotypes: HI (harlequin ichthyosis), LI (lamellar ichthyosis), CIE (congenital ichthyosiform erythroderma) •Lipids are packaged into lamellar bodies in stratum granulosum and then delivered by exocytosis to intercellular space of stratum corneum. •After further conversion lipids are organized into layers, constituting barrier against water loss. •Intercellular lipid layers in the stratum corneum •Akiyama M. 2006 •ABCA12 - keratinocyte transmembrane lipid transporter - transport lipids in lamellar granules •ABCA12 is a member of a large superfamily of ATP-binding cassette transporters, which bind and hydrolyze ATP to transport various molecules across a membrane. •ABCA12 gene mutations underlie ARCI (HI, LI, CIE). A, Model of formation of normal intercellular lipid layers and cornified cell envelope in stratum corneum. B, Loss-of-function mutations in ABCA12 lead to defective lipid transport via lamellar granules and malformation of intercellular lipid layers, resulting in loss of epidermal barrier function. •Intercellular lipid layers in the stratum corneum, ABCA12 •Akiyama M. 2006 Intercellular lipid layers and cornified cell envelope formation: (a)Model of formation of the normal intercellular lipid layers and cornified cell envelope in the stratum corneum. (b)ABCA12 dysfunction leads to defective intercellular lipid layers. (c)Transglutaminase 1 (TGM1) deficiency results in a malformed cornified cell envelope and subsequent defective intercellular lipid layers. TGM1 function: cross-link of proteins •Akiyama M. 2006 Peter Krieg, BBA - Molecular and Cell Biology of Lipids •Lipoxygenase 12R (12R-LOX, ALOX12B), lipoxygenase-3 (eLOX-3, ALOXE3) - The oxygenation of ceramide EOS → ω-hydroxyceramide (ω-OH-OS) - ω-hydroxyceramide is further hydrolyzed to sphingosine and ω-hydroxy-very long chain fatty acid (ω-OH-VLC-FA) - The ω-OH derivatives are crosslinked by TGM1 to glutamines of proteins of the cornified cell envelope (CE). The corneocyte is surrounded by an inner protein (cornified cell envelope, CE) and an outer cornified lipid envelope (CLE), a lipid monolayer covalently bound to the CE. EOS, O-linoleoyl-ω-hydroxyacyl-sphingosine; ox-EOS, 9R,10R-epoxy-11E-13R-hydroxylinoleoyl-ω-hydroxyacyl-sphingosine; ω-OH-OS, ω-hydroxyacyl-sphingosine (ω-hydroxyceramide); ω-OH-VLC-FA, ω-hydroxy-very long chain fatty acid; CE, cornified cell envelope; CLE, cornified lipid •‘‘Self-healing collodion baby’’ or “self-improving collodion ichthyosis” – the patients born as collodion babies upon shedding of the membrane acquire a nearly normal skin showing no or only mild signs of ichthyosis. •The TGM1 mutation p.Asp490Gly – a chelation of water molecules locks the mutated enzyme in an inactive trans conformation under elevated hydrostatic pressure in utero; after birth, the water molecules are removed and the enzyme to isomerize back to an active cis form, explaining the dramatic improvement in the phenotype. •The ALOX12B mutation p.Tyr521Cys – protein misfolding negatively affects the activity of mutated enzyme only under in utero conditions, thus explaining a dramatic improvement after birth. •Self-healing collodion baby Fetuses affected with HI start developing ichthyotic phenotype in amniotic fluid where stratum corneum barrier function is not required. •Intercellular lipid layers in the stratum corneum, ABCA12 M. Akiyama, HUMAN MUTATION 2010 A: Lipid contents in LG are secreted to intercellular space forming intercellular lipid layers which are important for epidermal barrier function. B: ABCA12 mutations disrupt lipid transport in LG and cause intracellular lipid accumulation. C: Disruption of epidermal barrier function and impaired epidermal differentiation coordinately cause ichthyosis phenotype. •ABCA12 – Adenosine triphosphate-binding cassette A12 ØABCA12 is a keratinocyte lipid transporter. ØABCA12 mutations underlie 3 major clinical types - harlequin ichthyosis; congenital ichthyosiform erythroderma; lamellar ichthyosis (according to the type of mutations). A, Model of formation of normal intercellular lipid layers and cornified cell envelope in the stratum corneum. B, Loss-of-function mutations in ABCA12 lead to defective lipid transport via lamellar granules and malformation of intercellular lipid layers, resulting in loss of epidermal barrier function and abnormal hyperkeratosis. Akiyama M. 2006 •ABCA12 mutations ØCollodion baby → congenital ichthyosiform erythroderma ØSequence Capture and targeted resequencing •ABCA12: c.5641C>T, p.(Arg1881*) •ABCA12: c.69G>A, p.(Pro23Pro) ??? Nucleotide change Sequence MaxEnt Scan NNSplice NetGene2 HSF-MaxEnt HSF-Matrices WT CCGgtgagt 11.64 0.99 0.67 10.9 94.09 c.69G>A CCAgtgagt 8.33 0.58 0.58 8.28 83.51 % Change -28.56% -41.41% -13.43% -24.04% -11,24% •Bioinformatic predictions of splice site scores in the mutation c.69G>A localised in the last nucleotide of ABCA12 exon 1: GAAGAGTTGATTGAGAAGTGCCTCTTGGTTAAGGATTAACCACAGGGAAAAATCCAGCAGAAACAGAAGAACTGTGGGTTTCTTACCCCAGCCCTCAAG GAAGCTATGCCGTGAAAGGGGTACTGATACACTGACATACAGCAAGTTGGACGGGGCATCAGTTCTTCATTTGTGGAGTGGAGAAAAGAAGAGGAAATC TCTCATTTGGGGCATTTGAAGGATGGCTTCCCTGTTTCATCAGCTTCAGATCCTGGTCTGGAAAAATTGGCTAGGTGTAAAAAGGCAGCCGgtgagtta aaaaaaagtgtgggagtatgggtcatggggcaatacgccaggtaatcctaaaatgtgatcttaatgagaagtgaaaacagaagagtttaaaggcatttt ccaaagcaggaagtaagatatttagaattcatccccctatctgtcttatagctaatggagcgagtcttaacatgcccccaaaagcagcctttt •Ultrastructural and immunohistochemical analysis of the ABCA12-patient´s skin Patient skin tissue Control skin tissue ØImmunohistochemical analysis of ABCA12 protein (partial deficit of ABCA12) performed by K. Veselý and M. Hermanová (FNuSA) C:\Users\20376\AppData\Local\Microsoft\Windows\Temporary Internet Files\Content.IE5\UTKIND9G\DSC_0006.JPG ØUltrastructural analysis (numerous droplets of neutral lipids in massively thickened stratum corneum) •Functional analysis of the ABCA12 mutation performed by K. Hrnčířová, P. Souček, T. Freiberger (CEITEC) Results: c.69G>A changes in a part of mRNA the splicing pattern – as a result of weakening of the 5´wt donor splice site wt and mutant ABCA12 exon 1 together with a part of intron 1 cloned into the pET01 vector → transfection of HeLa cells → analysis of splicing patern by qPCR ØSplicing mini gene assay C:\Users\20376\AppData\Local\Microsoft\Windows\Temporary Internet Files\Content.IE5\UTKIND9G\frequency.jpg Úprava komerčního vektoru •vektor pET01 Exontrap (MoBiTec) •Functional analysis of the ABCA12 mutation Úprava komerčního vektoru •vektor pET01 Exontrap (MoBiTec) •odstraněno •Functional analysis of the ABCA12 mutation Úprava komerčního vektoru •vektor pET01 Exontrap (MoBiTec) •Functional analysis of the ABCA12 mutation •1. exon genu ABCA12 (napojen přímo na 1. exon vektoru pET) + část 1. intronu ABCA12 (o délce 200bp) •varianta pacient a wt exon •exon 1 ABCA12 •část intronu 1 ABCA12 Úprava komerčního vektoru •Functional analysis of the ABCA12 mutation GAAGAGTTGATTGAGAAGTGCCTCTTGGTTAAGGATTAACCACAGGGAAAAATCCAGCAGAAACAGAAGAACTGTGGGTTTCTTACCCCAGCCCTCAAG GAAGCTATGCCGTGAAAGGGGTACTGATACACTGACATACAGCAAGTTGGACGGGGCATCAGTTCTTCATTTGTGGAGTGGAGAAAAGAAGAGGAAATC TCTCATTTGGGGCATTTGAAGGATGGCTTCCCTGTTTCATCAGCTTCAGATCCTGGTCTGGAAAAATTGGCTAGGTGTAAAAAGGCAGCCGgtgagtta aaaaaaagtgtgggagtatgggtcatggggcaatacgccaggtaatcctaaaatgtgatcttaatgagaagtgaaaacagaagagtttaaaggcatttt ccaaagcaggaagtaagatatttagaattcatccccctatctgtcttatagctaatggagcgagtcttaacatgcccccaaaagcagcctttt •exon 1 ABCA12 •intron 1 ABCA12 •mutace G>A Úprava komerčního vektoru •Functional analysis of the ABCA12 mutation Aberantní sestřih mRNA GAAGAGTTGATTGAGAAGTGCCTCTTGGTTAAGGATTAACCACAGGGAAAAATCCAGCAGAAACAGAAGAACTGTGGGTTTCTTACCCCAGCCCTCAAG GAAGCTATGCCGTGAAAGGGGTACTGATACACTGACATACAGCAAGTTGGACGGGGCATCAGTTCTTCATTTGTGGAGTGGAGAAAAGAAGAGGAAATC TCTCATTTGGGGCATTTGAAGGATGGCTTCCCTGTTTCATCAGCTTCAGATCCTGGTCTGGAAAAATTGGCTAGGTGTAAAAAGGCAGCCAgtgagtta aaaaaaagtgtgggagtatgggtcatggggcaatacgccaggtaatcctaaaatgtgatcttaatgagaagtgaaaacagaagagtttaaaggcatttt ccaaagcaggaagtaagatatttagaattcatccccctatctgtcttatagctaatggagcgagtcttaacatgcccccaaaagcagcctttt •červeně místo mutace – u pacienta G na A – poslední nukleotid 1. exonu ABCA12 •oranžově nově využité místo (po sestřihu zůstává z prvního exonu 119bp) •modře – začátek translace proteinu ABCA12 •podtržené – vystřižená oblast exonu 1 při využití kryptického místa (nejkratší PCR produkt o velikosti 269bp) •Functional analysis of the ABCA12 mutation •Výsledný konstrukt: •primer pET-1A •primer pET-1B •Transfekce HeLa buněk, kultivace •Izolace RNA (24 hodin po transfekci) •Reverzní transkripce, semikvantitativní PCR, vyhodnocení •Nesetřižená mRNA •Správně setřižená mRNA •Aberantně setřižená mRNA •Functional analysis of the ABCA12 mutation