Splicing modulation of integrin beta4 pre-mRNA carrying a branch point mutation underlies epidermolysis bullosa with pyloric atresia undergoing spontaneous amelioration with ageing.

A general improvement with ageing has been reported in a few cases of epidermolysis bullosa with pyloric atresia (PA-JEB), an autosomal recessive skin disease characterized by extensive disadhesion of epithelia. In a patient who improved from severe to mild PA-JEB, a search for mutations in the integrin beta4 gene (IGTB4) detected heterozygosity for a novel base substitution 3986-19T-->A in the putative branchpoint sequence of intron 31, and a point mutation 3802+1G-->A in the donor splice site of intron 30 previously associated with severe PA-JEB. Analysis of mRNA showed that the intronic mutation prevents legitimate splicing of the beta4 pre-mRNA. Functional splicing can be restored in vitro by seeding the proband's keratinocytes on feeders of irradiated fibroblasts. Study of mRNA in wild-type keratinocytes transfected with IGTB4 minigenes containing intron 31 with or without mutation 3986-19T-->A, confirmed the causative role of the intronic mutation in PA-JEB, and highlighted the influence of feeders on the maturation process of the mutated beta4 pre-mRNA. Our results show that in a context of overall reduction of the beta4 mRNA levels, activation of the legitimate splice site in the aberrant beta4 pre-mRNA underlies the transient severity of the condition. The results also point to the relevance which the interaction between epithelial and stromal cells may have in modulating expression of integrin receptors.

Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa.

Laminin-5 is composed of three distinct polypeptides, alpha3, beta3, and gamma2, which are encoded by three different genes, LAMA3, LAMB3, and LAMC2, respectively. We have isolated epidermal keratinocytes from a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, which led to complete absence of the beta3 polypeptide. In vitro, beta3-null keratinocytes were unable to synthesize laminin-5 and to assemble hemidesmosomes, maintained the impairment of their adhesive properties, and displayed a decrease of their colony-forming ability. A retroviral construct expressing a human beta3 cDNA was used to transduce primary beta3-null keratinocytes. Clonogenic beta3-null keratinocytes were transduced with an efficiency of 100%. Beta3-transduced keratinocytes were able to synthesize and secrete mature heterotrimeric laminin-5. Gene correction fully restored the keratinocyte adhesion machinery, including the capacity of proper hemidesmosomal assembly, and prevented the loss of the colony-forming ability, suggesting a direct link between adhesion to laminin-5 and keratinocyte proliferative capacity. Clonal analysis demonstrated that holoclones expressed the transgene permanently, suggesting stable correction of epidermal stem cells. Because cultured keratinocytes are used routinely to make autologous grafts for patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary human epidermal stem cells defective for a structural protein opens new perspectives in the long-term treatment of genodermatoses.

Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia.

Herlitz junctional epidermolysis bullosa (H-JEB) provides a promising model for somatic gene therapy of heritable mechano-bullous disorders. This genodermatosis is caused by the lack of laminin-5 that results in absence of hemidesmosomes (HD) and defective adhesion of squamous epithelia. To establish whether re-expression of laminin-5 can restore assembly of the dermal-epidermal attachment structures lacking in the H-JEB skin, we corrected the genetic mutation hindering expression of the beta 3 chain of laminin-5 in human H-JEB keratinocytes by transfer of a laminin beta 3 transgene. The transduced keratinocytes synthesized a recombinant beta 3 polypeptide that assembled with the endogenous laminin alpha 3 and gamma 2 chains into a biologically active laminin-5 that was secreted, processed and deposited into the extracellular matrix. Re-expression of laminin-5 induced cell spreading, nucleation of hemidesmosomal-like structures and enhanced adhesion to the culture substrate. Organotypic cultures performed with the transduced keratinocytes, reconstituted epidermis closely adhering to the mesenchyme and presenting mature hemidesmosomes, bridging the cytoplasmic intermediate filaments of the basal cells to the anchoring filaments of the basement membrane. Our results provide the first evidence of phenotypic reversion of JEB keratinocytes by somatic gene therapy and demonstrate that genetic treatment of the mild forms of skin blistering diseases and other inherited extracellular matrix pathologies is a realistic goal.

Functional Re-expression of laminin-5 in laminin-gamma2-deficient human keratinocytes modifies cell morphology, motility, and adhesion.

Herlitz junctional epidermolysis bullosa (H-JEB) is characterized by a reduced adherence of keratinocytes consequent to deficient expression of the extracellular adhesive ligand laminin-5. To complement the genetic defect causing H-JEB, we transferred an eukaryotic cassette expressing the cDNA for the gamma2 chain of laminin-5 into H-JEB keratinocytes in which the expression of the polypeptide is hampered by a homozygous mutation generating a premature termination codon. Transfection using adenovirus-polylysin-transferrin-DNA complexes resulted in a transient synthesis of the recombinant laminin gamma2 chain that associated with the endogenous alpha3 and beta3 chains to form laminin-5 molecules readily deposited on the tissue culture substrate. Furthermore, retroviral-mediated transduction of the gamma2 cDNA yielded persistent expression and polarized secretion of laminin-5. The protein incorporated into the basement membrane produced by the revertant cells inoculated subcutaneously in nude mice. In these transfectants, re-expression of laminin-5 induced changes in cell morphology and reorganization of focal adhesions that assumed the shape and distribution of the counterparts detected in normal keratinocytes. These observations correlated with an enhanced cell-substrate adhesion and a reduced motility of the transfected cells. Our results demonstrate that a restored expression of laminin-5 induces a phenotypic reversion of genetically altered H-JEB keratinocytes and open new perspectives to the analysis of the mechanisms regulating adhesion of epithelial cells.

Identification of a homozygous one-basepair deletion in exon 14 of the LAMB3 gene in a patient with Herlitz junctional epidermolysis bullosa and prenatal diagnosis in a family at risk for recurrence.

Herlitz junctional epidermolysis bullosa, a severe epidermal blistering disorder, is inherited in an autosomal recessive manner. It has recently been shown that, in kindreds with junctional epidermolysis bullosa, the disorder results from mutations in the gamma 2 chain of laminin-5, a basement membrane protein synthesized by the basal cells of stratifying squamous epithelia. In this report we describe a mutation identified in the beta 3 chain gene of laminin-5 in a family with Herlitz junctional epidermolysis bullosa. The disease is caused by a homozygous deletion of 1 bp that leads to a frameshift and premature termination codon. The segregation of the mutated allele in the family is consistent with the pathogenic role of the mutation. We also report a direct DNA-based prenatal exclusion of Herlitz junctional epidermolysis bullosa in a pregnancy at risk using a chorionic villus biopsy and allele-specific oligomer hybridization from polymerase chain reaction-amplified genomic DNA.

Identification of a homozygous exon-skipping mutation in the LAMC2 gene in a patient with Herlitz's junctional epidermolysis bullosa.

We describe a family with the Herlitz type of junctional epidermolysis bullosa, in which the disease is associated with a homozygous splice-site mutation in the gamma 2-chain gene (LAMC2) of laminin-5. The mutation consists of a G-to-T substitution resulting in the out-of-frame skipping of exon 7, a frame shift, and premature stop codon accompanied by a severe reduction in the level of mRNA from the mutant allele. The distribution of the wild-type and mutated gamma 2-chain alleles in family members implicates the mutation in the pathology and confirms the haplotypes of the healthy carriers previously determined by genetic linkage analysis. Our results confirm that the lethal Herlitz junctional epidermolysis bullosa phenotype is caused by mutations resulting in an altered synthesis of laminin-5.

Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the gamma 2 subunit of nicein/kalinin (LAMININ-5).

We have linked Herlitz's junctional epidermolysis bullosa (H-JEB) to the gene (LAMC2) encoding the gamma 2 subunit of nicein/kalinin, an isolaminin (laminin-5) expressed by basal keratinocytes. In four H-JEB kindreds, a maximum two-point lod score of 5.33 at theta = 0 was observed between a microsatellite near LAMC2 at 1q25-31 and the disease. In one family, a homozygous point mutation leading to a premature stop codon (CGA to TGA) was identified in exon 3 of the gene. The segregation of the mutated allele implicates the mutation in the pathology of the disorder and corroborates the linkage results.

The 100-kDa chain of nicein/kalinin is a laminin B2 chain variant.

We have isolated the basement membrane component nicein and performed rotary-shadow analyses using electron microscopy that showed the presence of two forms (I and II) of the protein. Molecular cloning of the cDNA that codes for the 100-kDa chain of the protein revealed that the sequence matches those independently identified for the 105-155-kDa subunit of kalinin, a recently identified basement membrane component. These data demonstrate that nicein and kalinin contain an identical chain. The length of the open reading frame in the cDNA (approximately 5200 nucleotides) and amino acid sequence obtained from the N-terminus of the 105-kDa kalinin chain showed the occurrence of a precursor polypeptide. This immature polypeptide is probably related to form I, observed by rotary shadowing, while the mature form is related to form II. It is noteworthy that nicein/kalinin subunits share discrete sequence similarities with the B2 chain of human laminin, but with a cleavage occurring within domain III that eliminates domains IV and V from the final product. The sequence of this subunit is nearly identical to that of B2t, a recently described polypeptide supposed to be related to a new laminin variant. Since nicein/kalinin expression is specifically impaired in the severe genodermatosis Herlitz junctional epidermolysis bullosa, the role and structure of this tissue-restricted laminin variant is crucial for the understanding of epidermal-dermal adhesion.

Maintenance of autonomous genetic elements in twelve transgenic mouse strains established after transfer of pPyLT1 DNA.

Maintenance and efficient meiotic segregation of autonomous genetic elements in four transgenic mouse strains established after micro-injection of plasmid pPyLT1 were previously reported. These findings are now extended to a total of twelve independent transgenic families. In spite of extensive rearrangements, half of these plasmids maintained the region of pBR322 necessary for shuttle transfer in E. coli. They all included mouse DNA sequences and the same, or closely related sequences were present in distinct mouse strains.

Germ line transmission of autonomous genetic elements in transgenic mouse strains.

Upon microinjection into fertilized mouse eggs of circular molecules of plasmid pPyLT1 carrying the gene encoding the large T protein of polyoma virus within bacterial vector sequences, autonomous circular plasmids were stably maintained in low copy numbers in transgenic strains. These plasmids could be rescued in E. coli by transfection. Integrated forms could be detected neither in somatic tissues, nor in spermatozoa. Efficiency of paternal or maternal transmission was close to 100%. The plasmids had lost or had extensively rearranged the polyoma sequences. In addition, they had acquired defined segments of genomic mouse DNA, which might be responsible for correct segregation of daughter copies at both mitosis and meiosis (centromeric function).