Human Molecular Genetics, 2001, Vol. 10, No. 21 2453-2461
© 2001 Oxford University Press
Genetic bases of severe junctional epidermolysis bullosa presenting spontaneous amelioration with aging
INSERM U385, Faculté de Médecine, 06107 Nice Cedex 2 and 1Service de Dermatologie, Hôpital Necker, Enfants Malades, 75730 Paris 15, France
Received July 2, 2001; Revised and Accepted August 8, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Change of the clinical picture with aging is noted in some patients suffering from junctional epidermolysis bullosa (JEB), an inherited blistering disorder caused by extensive disadhesion of the epithelia. We have studied a patient born with severe JEB associated with absent expression of laminin 5. A remarkable reduction of the blistering tendency was observed with aging that correlated with a restored expression of immunoreactive laminin 5 molecules. Genetic analysis of the gene LAMB3 detected compound heterozygosity for the nonsense mutation R635X and a novel 2 bp deletion (1587delAG) resulting in a downstream premature termination codon. RT–PCR amplification of total RNA purified from skin biopsies demonstrated that the mutated ß3 mRNAs underwent rapid decay shortly after birth, and that illegitimate splicing of the mRNA carrying mutation 1587delAG generated a new internally shortened ß3 transcript with advancing age. Our genetic and biochemical data show that (i) the illegitimate splicing of the ß3 pre-mRNA results in synthesis and secretion of a laminin 5 heterotrimer with an internally deleted ß3 polypeptide, (ii) expression of the mutated ß3 polypeptide is up-regulated in the basal keratinocytes with high proliferative potential, (iii) absence of the N-terminal region of the ß3 rod domain II thought to stabilize the tertiary structure of the laminin 5 is not required for the assembly of the protein and (iv) the mutant laminin 5 retains its adhesive potential. Our results demonstrate that mRNA rescue may underlie the evolution of the clinical phenotype in inherited skin conditions.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The term epidermolysis bullosa (EB) refers to a group of inherited blistering diseases of the skin characterized by epithelial fragility to mechanical friction (1). EB is associated with molecular defects of the dermal–epidermal junction and has been divided into three major clinical subtypes according to the level of the blister plane as determined by ultrastructural and immunohistochemical analysis of the involved skin (2). In EB simplex, tissue separation occurs within the basal keratinocytes and is caused by genetic mutations in keratins 5 and 14 or in the hemidesmosomal component plectin. In dystrophic EB, the split localizes within the papillary dermis below the lamina densa of the basement membrane zone (BMZ), and results from mutations in collagen type VII. In junctional EB (JEB), blisters occur within the lamina lucida of the BMZ and are caused by mutations in the extracellular adhesion ligand laminin 5 and in integrin
6ß4 and collagen type XVII, the two transmembrane components of hemidesmosomes (HD).
Laminin 5 constitutes the major adhesion ligand of the basal cells in the stratified and transitional epithelia exposed to the external environment (3). In the epidermis, laminin 5 co-localizes with the anchoring filaments, the threadlike adhesion structures that span the lamina lucida and link the HD of the basal keratinocytes to the lamina densa of the BMZ (4). By bridging the cellular receptor integrin 6ß4 and collagen type VII, the major component of the anchoring fibrils of the papillary dermis, laminin 5 mediates the stable adhesion of the basal keratinocytes to the underlying chorion (5). Furthermore, interaction of the C-terminal G domain of laminin 5 with integrin 6ß4 triggers formation of HD (6).
Laminin 5 is synthesized by the basal keratinocytes as a heterotrimeric molecule composed of 3 (200 kDa), ß3 (140 kDa) and 
2 (155 kDa) chains encoded by the genes LAMA3, LAMB3 and LAMC2, respectively (7). The 3ß32 heterotrimer assembles by formation of a stable ß32 heterodimer that integrates the 3 chain via labile interactions (8). Upon secretion in the extracellular matrix, the 2 chain undergoes proteolytic processing that reduces the size of the polypeptide from 155 to 105 kDa (9,10). A distinct proteolytic processing also reduces the size of the 3 chain from 200 to 165 kDa (10). The assembled molecule displays the cross-shaped structure characteristic of laminins, with a globular domain formed by the C-terminal region of the 3 chain, a long rod-like domains formed by a coiled-coil helix resulting from interaction of domains I and II of each chain, and three N-terminal domains that in laminin 5 are truncated (11). Interchain ionic interactions and distal disulfide bonds stabilize the rod-like domain, while the N-terminal domains of the ß3 and 2 chains form two short arms visible by rotary shadowing analysis of purified laminin 5. The short arm of the ß3 chain consists of an intermediate domain III/V and an N-terminal globular domain VI. Domain III/V contains multiple cysteine-rich epidermal growth factor (EGF) modules the functions of which remain unknown. Domain VI is believed to mediate the interaction of laminin 5 with laminin 6 and/or collagen type VII (5,12,13).
In Caucasian patients with JEB, most of the genetic mutations so far identified in laminin 5 affect the LAMB3 gene. Mutation database analysis indicates that nonsense or frameshift mutations on both LAMB3 alleles that abolish laminin ß3 expression result in the severe and often lethal form of JEB [Herlitz JEB (H-JEB)]. Conversely, the combination of nonsense mutations with missense mutations or mutations in the splice site consensus sequences of LAMB3, which reduce the expression levels of the ß3 polypeptide, result in the mild manifestations of the condition [non-Herlitz JEB (n-HJEB)] (14). At the protein level, the clinical severity of JEB correlates well with the extent of laminin 5 assembly and deposition in the BMZ (15,16). It also correlates with the structural shape of the HD that, in H-JEB, appear either absent or reduced in number and structurally disorganized, while in n-HJEB they appear normally shaped (17).
In this study, we report the genetic basis of an uncommon form of JEB characterized by a marked improvement of skin adhesion with advancing age. Our results show that the general amelioration of the clinical picture is linked to activation of an illegitimate splicing event leading to expression of an aberrant laminin ß3 pre-mRNA that results in the synthesis of a partially functional laminin 5.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Clinical features
The proband, a 7-year-old Caucasian female, is the product of the union of unrelated and clinically unaffected parents that have an older healthy daughter. Shortly after birth, the patient developed extensive cutaneous blisters and erosions at sites exposed to friction (Fig. 1A). Electron microscopy of the skin revealed paucity of dysplastic HD in non-involved areas, and tissue separation at the basal cell/lamina lucida interface in the blisters (not shown). Immunostaining of non-blistered skin revealed an absence of reactivity to monoclonal antibody (mAb) GB3, specific to native laminin 5 (Fig. 1Cb) and mAb K140, specific to laminin ß3 chain (Fig. 1Ce). Staining of the laminin
3 and 2 chains and that of the major components of the HD were attenuated (not shown). Based on these findings the diagnosis of JEB was made.
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During the first months of life, the patient developed severe erosions of the skin and oral mucosa. Ulceration of the cornea and nail dystrophy was also observed. Nails were definitely lost at 9 months. The involvement of the patient’s skin then decreased progressively. At the age of 4, the proband presented an excellent general state and a normal growth rate (Fig. 1B). Sporadic blistering affecting the groin and the acral areas submitted to friction, corneal erosions and teeth with pitted enamel were observed. Ultrastructural examination of the proband’s skin showed well structured HD (not shown), and immunofluorescence staining of skin specimens revealed a substantial increase of immunoreactivity to a panel of anti-laminin 5 antibodies (Fig. 1Cc and f). The staining pattern of the dermal–epidermal junction was however discontinuous, with bright fluorescence patches at the tips of the dermal papillae alternating with zones of attenuated labeling at the deep rete ridges. Double immunostaining using monoclonal antibodies directed against the laminin
2 chain and the integrin ß1 subunit showed co-localization of the two polypeptides at the BMZ (Fig. 1D). In addition, staining with antibodies recognizing integrin 6ß4, plectin, type XVII collagen and the 230 kDa bullous antigen appeared enhanced with a patchy pattern (not shown).
Search for mutations in LAMB3
Because immunostaining of the laminin ß3 chain was undetectable in the patient’s skin at birth, we searched for genomic mutations in LAMB3. A search for sequence variations was initiated by PCR amplification of the exonic sequences of the gene (18). Direct sequencing of the PCR products covering all exons revealed a heterozygous AG deletion at position 1587 of exon 13 (GenBank accession no. L25541) (Fig. 2A). This 1587delAG deletion leads to a shift of the reading frame and results in a downstream premature termination codon (PTC) in exon 14. Presence of the PTC predicts a truncated ß3 polypeptide terminating at residue 524. The mendelian segregation of mutation 1587delAG in the proband’s family was assessed by allele-specific oligonucleotide (ASO) analysis of the genomic DNA obtained from the members of the proband’s family and unrelated controls. The proband, the father and the sister were heterozygous carriers for the mutation (Fig. 2B).
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Direct sequencing of the PCR-amplified fragment of DNA corresponding to exon 14 detected a heterozygous C
T transition (Fig. 2C). This base substitution corresponds to nucleotide 1903 of the ß3 cDNA sequence and leads to the nonsense mutation R635X in which the termination codon TGA replaces an arginine codon (CGA). The presence of transition 1903CT in the members of the proband family was verified by ASO analysis of the genomic DNA (Fig. 2D). The proband, the proband’s mother and the maternal grandfather were heterozygous for this mutation.
Expression of ß3 transcripts in the proband’s cultured keratinocytes
To examine the consequences of the mutations at the mRNA level, total RNA was purified from the proband’s cultured keratinocytes and used for RT–PCR amplification. The coding sequence of the ß3 mRNA was reverse transcribed and PCR-amplified using a pair of primers allowing production of a 738 bp cDNA fragment spanning the 3' end of exon 12 (120 bp), the entire exons 13 (112 bp) and 14 (379 bp) and the 5' region of exon 15 (127 bp). The agarose gel electrophoresis of the PCR products detected a slow migration band of the expected size and an additional fast migrating band of 
350 bp (Fig. 3A). Subcloning and amplification of these cDNAs into a bacterial vector, and direct nucleotide sequencing, identified four distinct species of laminin ß3 transcripts expressed in patient’s keratinocytes. Two cDNAs (736 and 738 bp, respectively) are produced by legitimate splicing of the aberrant ß3 pre-mRNAs transcribed from the two LAMB3 alleles. Both transcripts retain exon 14 (data not shown). The 738 bp cDNA identifies the maternal transcript carrying mutation 1903CT that leads to PTC, while the 736 bp cDNA identifies the paternal transcript that carries deletion 1587delAG leading to a downstream PTC at nucleotide 1602 (Fig. 3C). The other two ß3 cDNAs (359 and 357 bp, respectively) correspond to mRNA species internally deleted of exon 14, and generated by illegitimate splicing involving the acceptor splice site of exon 13 and the donor splice site of exon 15 of the LAMB3 alleles (Fig. 3B). Amplification of the 359 bp cDNA reveals that the maternal transcript carries an out-of-frame deletion of exon 14 which creates a downstream PTC within exon 15 (nucleotide 2117) (Fig. 3C), while amplification of the 357 bp cDNA shows that in the paternal transcript carrying deletion 1587delAG, the internal deletion of exon 14 restores the open reading frame (ORF) and allows expression of a shortened ß3 polypeptide (Fig. 3C). Further analysis by RT–PCR amplification of the ß3 RNA transcripts using a series of primer pairs sited between exons 11 and 16 excluded skipping of other exonic sequences (data not shown).
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Expression of an internally deleted laminin ß3 transcript in the proband’s skin
The expression products of the LAMB3 gene were then assessed using skin biopsy specimens obtained from the proband at birth and at the age of 4 (Fig. 4). Total RNA was extracted from skin sections and used for RT–PCR amplifications of the 738 bp ß3 cDNA fragment spanning from exons 12 to 15 that identifies the species of laminin ß3 transcripts synthesized by the proband’s keratinocytes ex vivo. Agarose gel electrophoresis of the PCR amplification products detected the 738 bp cDNA fragment in the skin of an unrelated healthy control, while no band was detectable in the proband’s skin at birth (Fig. 4). A unique fast-migrating cDNA fragment with an apparent mobility of 350 bp was amplified from the proband’s skin samples obtained at the age of 4. This fast-migrating cDNA fragment was cloned and amplified in bacteria. Direct DNA nucleotide sequencing of plasmid cDNAs isolated from 30 independent colonies revealed that all the bacteria had amplified the 357 bp ß3 cDNA that identifies the paternal ß3 transcript carrying mutation 1587delAG and the downstream in-frame skipping of exon 14 (Fig. 3B). These results indicate that no laminin ß3 transcript is readily detected in the proband’s skin at birth, while an mRNA encoding an aberrant laminin ß3 polypeptide is found at the age of 4. The abnormal polypeptide is internally shortened by 127 amino acids within domains II and III, and harbors a methionine and a proline residue that substitute the glycine and cysteine at positions 530 and 531, respectively.
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The proband’s keratinocytes express a mutant laminin 5 comprising an internally shortened laminin ß3 chain
To establish a possible correlation between the restored expression of the aberrant laminin ß3 chain and the favorable evolution of the condition, we assessed whether the shortened ß3 transcript encoded by the paternal LAMB3 allele incorporates into laminin 5 heterotrimers. Cell lysates and spent medium from cultures of the proband’s keratinocytes were immunoprecipitated using polyclonal antibodies (pAb) specific to each single chain of laminin 5 and mAb GB3. All the antibodies co-precipitated a polypeptide with the expected size (130 kDa) of the mutant ß3 chain internally deleted of 127 amino acids, and two polypeptides with the apparent molecular mass of 190 and 155 kDa characteristic of the unprocessed
3 and 2 chain, respectively (Fig. 5A). No band corresponding to the wild-type ß3 chain (145 kDa) was detected in the patient’s cells. In the spent medium, the ß3 mutant of 130 kDa co-precipitated with the processed 3 chain (165 kDa) and the unprocessed and processed forms of the 2 chain (155 and 105 kDa, respectively). By quantification of the autoradiograms, the mutant ß3 polypeptide was estimated to represent 15% of the amount of the wild-type counterpart secreted by the healthy control keratinocytes. These results demonstrate that the proband’s keratinocytes synthesize and secrete reduced amounts of a mutant laminin 5 comprising the aberrant 130 kDa ß3 chain.
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To determine whether the mutated laminin 5 exerts a functional effect on cell attachment to the culture substrate, we used a cell-detachment kinetic assay to evaluate the adhesive capacity of the patient’s keratinocytes compared to wild-type and laminin ß3-null keratinocytes (19). Exponentially growing cell cultures were treated with a trypsin/EDTA solution and the number of cells dislodged at increasing intervals of time was evaluated by direct counting (Fig. 5B). The results showed that attachment of the proband’s keratinocytes is sensibly enhanced compared with laminin 5-deficient counterparts. Indeed, 12% of the proband’s keratinocytes was dislodged after 4 min, compared with 55% of H-JEB cells. At 8 min, however, 90% of the proband’s keratinocytes were detached, compared with 40% of the wild-type cells. Taken together, these observations show that expression of the mutant laminin ß3 results in the secretion of partially functional laminin 5 molecules that enhance cell adhesion.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have investigated the genetic background of a rare form of JEB characterized by evolution from extreme skin fragility at birth to a blistering tendency that sensibly reduced with aging. The favorable course of the blistering condition, initially diagnosed as a lethal form of JEB, correlated with enhanced reactivity of the skin to antibodies directed against laminin 5.
Genetic analysis based on nucleotide sequencing of the cDNA encoding the laminin ß3 polypeptide revealed that the proband is a compound heterozygote for mutations R635X and 1587delAG in the LAMB3 gene. Both mutations hamper expression of the laminin ß3 polypeptide. R635X is a recurrent mutation detected in 45% of the LAMB3 alleles in Caucasian patients affected by the lethal form of JEB (20). In our proband, this mutation is generated by the base substitution 1903 CT in exon 14 of the maternal LAMB3 allele. Conversely, the 2 bp deletion 1587delAG disclosed in exon 13 of the paternal LAMB3 allele is a novel genetic mutation. This mutation induces a shift of the reading frame that results in a downstream PTC within the mRNA sequence encoded by exon 14. Consistent with the notion that the presence of a PTC causes the decay of aberrant RNA transcripts (21), the steady state level of the laminin ß3 messenger RNAs was extremely low in skin biopsies obtained shortly after the birth of the patient. The drastic reduction in the expression of LAMB3 explains the lack of immunoreactivity of the skin to the antibodies specific to the laminin ß3 chain and the native laminin 5. Absence of laminin 5 accounts for the severity of the condition.
By analyzing skin biopsy specimens obtained from the patient at the age of 4, we found that the paternal Lamb3 allele contributes to the favorable course of the disease. In fact, with advancing age, mutation 1587delAG in exon 13 becomes leaky, and illegitimate skipping of exon 14 restores the ORF of the mutant ß3 pre-mRNA and generates an internally shortened messenger RNA. Skipping of exons containing nonsense mutations has been observed in several conditions (22–24). Such a mechanism, which is thought to involve ribosome-mediate scanning of the pre-mRNAs transcripts for PTCs, allows translation of a functional polypeptide by rescuing the reading frame of mutant mRNA molecules (25). Indeed, in-frame skipping of exons containing PTCs in the LAMB3 gene has been currently observed in patients with mild JEB (26). In our proband, however, absence of mutant ß3 transcripts at birth suggests that activation of the cryptic splice site allowing skipping of exon 14 from the paternal ß3 pre-mRNA takes place a relatively long time after birth. A similar phenomenon of age-dependent activation of illegitimate mRNA splicing has been observed in analbuminemic rats carrying a mutation at a 5' splice site of the albumin gene (27). Age-dependent splicing modulation has also been found in a case of transient EB associated with pyloric atresia where the functional restoration of a mutated splice site of integrin ß4 pre-mRNA leads to expression of a wild-type ß4 polypeptide (28).
External signal modulating gene expression may act on the mRNA splicing machinery. We could not determine the nature of the factors that with age activate the illegitimate splicing of the paternal ß3 transcripts in our patient. We observed, however, that the deregulation of the splicing process occurs when the patient’s keratinocytes are expanded in culture. Intriguingly, immunostaining of the laminin ß3 polypeptide in the dermal–epidermal junction is discontinuous and corresponds to the staining pattern of integrin ß1. High expression of integrin ß1 is required for maintenance of epidermal stem cells, while progressive loss of its expression characterizes the transit-amplifying keratinocytes endowed with limited proliferative capacity and committed to differentiation (29,30). In light of these observations, expression of the mutated laminin 5 in our patient appears to correlate with the proliferative potential of the basal keratinocytes. The fact that corneal erosions do not take a favorable course with aging could be consistent with this idea. The persistent susceptibility to ocular lesions may in fact reflect a defective expression of laminin 5 in the transit amplifying basal cells with a central and paracentral localization in the cornea, i.e. the cells that have left the stem cell compartment (the limbus) and have lost their proliferative capacity (31).
It cannot be formally excluded that the irregular distribution of laminin 5 in the patient skin results from an enhanced local degradation of the protein in the extracellular matrix underlying the transit amplifying cells. The laminin 5 synthesized by the patient harbors a mutant 126 kDa ß3 polypeptide carrying a substitution of two amino acids at position 530–531 and an internal in-frame deletion of 127 amino acids spanning the N-terminus of the long arm and the proximal EGF-like repeat of the polypeptide (Fig. 6). We have shown that the proband’s keratinocytes synthesize the 126 kDa ß3 chain in vitro, and that the mutant ß3 chain associates with the 3 and 2 chains to form functional laminin 5 heterotrimers which are secreted in the spent medium. The altered structure of this mutant laminin 5 may modify the turnover of the protein in vivo, and/or affect its stability. Laminin 5 heterotrimers assembly proceeds intracellularly by formation of stable ß32 intermediates that associate with the 3 chain via labile interactions (8). The 3ß32 heterotrimer associates by specific interactions between the domains I and II of each chain to form the triple-helix coiled-coil long arm of laminin 5 (32). Disulfide bonds at each end of the long arm are thought to stabilize the heterotrimer. The mutant ß3 chain expressed by our patient lacks the two N-terminal cysteines of domain II that are required for binding to the 3 and 2 chains. Our results demonstrate that these cysteine residues are not required for the assembly of functional laminin 5 molecules, but do not provide information on the stability of the mutant laminin 5. The functional role of the EGF-like repeat lost by the mutant ß3 chain also remains unclear. These domains are thought to mediate specific interactions with the components of the extracellular matrix (32). Therefore, the absence of the sixth EGF-like domain of the ß3 short arm correlates well with both the reduced adhesion capacity of the patient’s keratinocytes observed in vitro and the occurrence of sporadic and localized blisters.
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In this patient, de novo synthesis of the laminin ß3 chain raised no detectable immune reaction against either the ß3 polypeptide or laminin 5. This observation implies that transfer of a curative ß3 transgene may present a limited risk of immune rejection in patients with mild JEB associated with expression of a defective laminin ß3 chain, which therefore opens interesting perspectives for a therapeutic approach of JEB based on gene transfers (19,33,34). However, this issue deserves further investigation, because transient synthesis of the laminin ß3 chain during the fetal life of our patient cannot be excluded, which could account for the tolerance to this protein expressed a relatively long time after birth.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Samples
The keratinocytes obtained from newborn skin biopsies were cultured on a feeder layer of irradiated mouse J2-3T3 fibroblasts (35). Total RNA was purified from frozen skin biopsies and from cultured keratinocytes using the RNable extraction kit (Eurobio, Les Ullis, France). Genomic DNA was extracted from the peripheral blood following standard techniques (36).
Immunohistochemistry
Indirect immunofluorescence analysis of frozen skin samples was performed as previously described (37). Expression of laminin 5 was investigated using the mAb GB3 raised against the native laminin 5 (38), mAb K140 specific to the laminin ß3 chain (9), and pAb SE85 and SE144, specific to the laminin 3 and 2 chains, respectively (15). Reactivity to integrin ß1 was assessed using mAb IOT29 (Immunotech, Marseille, France). Immunomapping of the HD components was performed using mAb HD121 directed against plectin (39), mAb GoH3 to integrin 6 (40), mAb 3E1 to integrin ß4 (Gibco BRL - Life Technologies, Cergy Pontoise, France), pAb FP1 and mAb 1A8C, specific to bullous pemphigoid antigen of 230 kDa (41), and mAb collagen XVII (42), respectively. Secondary antibodies were fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse Ig (Dako S.A., Trappes, France), goat anti-rat IgG (Cappel, ICN Biomedicals, Orsay, France) or swine anti-rabbit Ig (Dako S.A.). Double labeling was carried out using Texas red-conjugated goat anti-rabbit IgG (Cappel) and FITC-conjugated goat anti-mouse Ig (Dako S.A). Tissue sections and cell cultures were analyzed using a Zeiss Axiophot microscope.
Detection of genetic mutations
PCR reactions using 100 ng of genomic DNA as a template were performed following standard conditions (43). The pairs of primers were synthesized on the basis of the DNA sequences corresponding to the intron–exon boundaries of the gene LAMB3 (18) (GenBank accession nos U17744–U17759). Direct DNA sequence analysis of the amplification products was performed using the ABI Prism 310 automated sequencing system (Applied Biosystems, Foster City, CA). To detect mutation 1587delAG, the primers were (L) 5'-CCTATGCTGGGCCTTGACTT-3' and (R) 5'-ACTGACTCCGTGGGAAACGA-3', which amplify exon 13 (GenBank accession no. U17755). The primers used to identify mutation 1903CT were (L) 5'-GCTGCGACTTCTGTTATTCT-3' and (R) 5'-AAATGTAAGGAAGGACCAGC-3', which amplify exon 14 (GenBank accession no. U17756). The segregation of the genetic mutations in the kindred was determined using a standard ASO hybridization protocol (44). For mutation 1587delAG, the ASO were 5'-ACGTGGCCACAGGATGCC-3' (wild-type) and 5'-ACGTGGCCACGATGCCGA-3' (mutant). For mutation 1903CT, the ASO were 5'-GAGCAGATCCGAGCAGTTC-3' (wild-type) and 5'-GAGCAGATCTGAGCAGTTCT-3' (mutant).
RT–PCR analysis of laminin ß3 transcripts
Total RNA was purified from proband and control cultured keratinocytes and from frozen skin biopsies obtained from the proband at birth and at the age of 4 years. Total RNA was reverse-transcribed in a volume of 20 µl as recommended by the manufacturer (Promega, Madison, WI). The reaction products were used for PCR amplification of the 737 bp fragment of laminin ß3 cDNA (nucleotides 1366–2103; Genbank accession no. L25541) (12) comprising exons 13 and 14 of LAMB3. The primers were (L) 5'-AACGTGGTGGGTCCCAAATG-3' and (R) 5'-GTCCCTCTTCCTCTGATAC-3', and the PCR cycling conditions were: 94°C for 5 min, followed by 94°C for 45 s, 56°C for 45 s, 72°C for 50 s (35 cycles) and 72°C for 10 min. An actin cDNA fragment (nucleotides 363–1100) (GenBank accession no. AB004047), used as an internal control, was amplified using primers (L) 5'-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3' and (R) 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3', and PCR cycling conditions of 94°C for 5 min, 94°C for 45 s, 60°C for 45 s, 72°C for 50 s (35 cycles) and 72°C for 10 min. Five micrograms of the reaction products was run on 1% agarose gels. The amplified fragments of the ß3 cDNA were subcloned into the Topo cloning vector according to the recommendations of the manufacturer (Stratagene, La Jolla, CA) and submitted to direct DNA sequence analysis.
Immunoprecipitation
Radioimmunoprecipitation of cell extracts and spent culture medium has been described elsewhere (45). SDS–PAGE of the immunoprecipitates was performed using a 6.5% polyacrylamide gel under reducing conditions (46). The dried gels were exposed to X-ray films, using an intensifying screen. Quantitation of the autoradiograms was performed by densitometric scanning using the Bioprofil software program (Vilbert Lourmat, France).
Cell detachment assay
Keratinocytes (8 x 105) were seeded on plastic dishes and grown in a 3:1 mixture of Dulbecco modified Eagle’s medium and Ham’s F12 medium (Life Technologies, Cergy Pontoise, France) supplemented as described by Rheinwald and Green (35). Forty-eight hours after seeding, cells were rinsed twice, and treated with 0.05% trypsin, 2 mM EDTA at 37°C during 2, 4, 6, 8 and 10 min. The percentage of dissociation at each time was determined in triplicate.
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ACKNOWLEDGEMENTS |
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We acknowledge C.Prost for electron microscopic observations and A.Charlesworth for critical reading of the manuscript. This work was supported by grants from the Programme Hôspitalier de Recherche Clinique (France), the DEBRA Foundation (UK), the Association Française contre les Myopathies (France) and the Epidermolyse bulleuse Association d’Entraide (France).
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FOOTNOTES |
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+ To whom correspondence should be addressed at: INSERM U385, U.F.R de Médecine, Avenue de Valombrose, 06107 Nice cedex 2, France. Tel: +33 493 37 77 79; Fax: +33 493 81 14 04; Email: meneguzz@unice.fr
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REFERENCES |
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