Identification of novel type VII collagen gene mutations resulting in severe recessive dystrophic epidermolysis bullosa.

In this work, we studied the proband in a small nuclear family of Chinese and Dutch/German descent and identified two novel mutations in the type VII collagen gene leading to recessive dystrophic epidermolysis bullosa, Hallopeau-Siemens variant (HS-RDEB). The maternal mutation is a single base pair deletion of a cytosine nucleotide in exon 26, designated 3472delC, resulting in a frameshift and a premature termination codon (PTC) within the same exon, 7 bp downstream of the site of the mutation. The paternal mutation is a G-->A transition located at the 5' donor splice site within intron 51, designated IVS51 + 1G-->A. This mutation leads to the activation of a cryptic splice site, 32 bp downstream of the mutation site and to subsequent aberrant out-of-frame splicing, resulting in two alternative mRNA transcripts and a downstream PTC. To our knowledge, these two mutations have not been previously reported. These findings extend the body of evidence for compound heterozygous mutations leading to HS-RDEB and provide the basis for prenatal diagnosis in this family.

A novel missense mutation in the COL7A1 gene underlies epidermolysis bullosa pruriginosa.

Epidermolysis bullosa (EB) pruriginosa is a subtype of dominant dystrophic EB (DDEB), characterized by severe pruritus and blistering localized to the extensor surface of the extremities. EB pruriginosa exhibits extensive clinical heterogeneity with variable expression and delayed age of onset. Mutations in the COL7A1 gene, especially in glycine residues within Gly-X-Y repeats, have been shown to cause this form of DDEB. Here, we report a novel COL7A1 mutation in a Taiwanese pedigree with EB pruriginosa. Using PCR and direct sequence analysis we have identified a G-->T transversion at nucleotide 7097 in exon 92 of COL7A1, converting a glycine residue to valine (G2366V). The mutation resides within a consecutive, uninterrupted stretch of 17 Gly-X-Y residues in the triple-helical domain of type VII collagen. Interestingly, an affected member of this family also displayed elevated IgE levels, previously reported in some patients with this disorder. Our finding further implicates COL7A1 mutation in the pathogenesis of EB pruriginosa and underscores the heterogeneous clinical symptoms of glycine mutations in DDEB.

Trans-species hair growth induction by human hair follicle dermal papillae.

A series of experimental bioassays has shown that the dermal papilla of the adult rodent vibrissa hair follicle retains unique inductive properties. In view of the many phenotypic and functional differences between specific hair follicle types, and the growing interest in hair follicle biology and disease, it remains important to establish that the human hair follicle dermal papilla has equivalent capabilities. In this study we tested the ability of human hair follicle papillae to induce hair growth when implanted into transected, athymic mouse vibrissa follicles. The implanted papillae that interacted with mouse follicle epithelium created new fibre-producing follicle end bulbs. The origin of the papillae in the recombinant structures was confirmed using laser capture microdissection and human specific gender determination by PCR. The demonstration that intact adult human dermal papillae can induce hair growth has implications for molecular analysis of basic hair growth mechanisms, particularly since the study involved common epithelial-mesenchymal signalling and recognition properties across species. It also improves the prospects for a cell-based clinical approach to hair follicle disorders.

Diagnosis of autosomal recessive lamellar ichthyosis with mutations in the TGM1 gene.

BACKGROUND: Autosomal recessive lamellar ichthyosis (ARLI) is a clinically and genetically heterogeneous disorder. In many cases, mutations in the transglutaminase 1 gene (TGM1) have been identified, however, other clinically indistinguishable cases have been linked to chromosomes 2, 3 and 19. Previous studies have failed to establish any correlation between clinical characteristics and genetic mutations. OBJECTIVES: To investigate the molecular basis of ARLI in 10 patients with the typical clinical presentation of the disorder. METHODS: We performed polymerase chain reaction and direct sequencing-based mutation screening in all of these patients, and TGM1 immunofluorescence microscopy and in vitro enzyme activity assays in selected patients. RESULTS: Mutation screening revealed 14 mutations, four of which have been previously described. While immunofluorescence microscopy was negative in patients with non-sense mutations or out-of-frame insertions or deletions, the results were variable in cases with mis-sense mutations and in cases with no mutations in the TGM1 gene. In vitro enzyme activity assays gave results consistent with the mutation data. CONCLUSIONS: Our findings support the importance of mutation screening in the evaluation of ARLI.

DNA based molecular analysis in the rapid diagnosis of Herlitz junctional epidermolysis bullosa.

The junctional form of epidermolysis bullosa (JEB) is an inherited blistering disease in which blisters occur at the level of the lamina lucida in the cutaneous basement membrane zone. Specific mutations have been detected in the genes encoding different components of the hemidesmosomal-anchoring filament complex. In the recessively inherited lethal (Herlitz) type of JEB (H-JEB), typically nonsense mutations or insertions or deletions are present on both alleles of any of the three genes encoding the polypeptide subunits of the anchoring filament protein, laminin 5. In this study, we searched for mutations in a proband who presented at birth with severe and extensive blistering. We detected a novel 1 bp deletion and a previously reported hotspot mutation (R635X) in the LAMB3 gene. This mutation combination established the diagnosis of H-JEB in this case, in which attempted diagnosis by skin biopsy had failed. The molecular analysis was performed shortly after birth while the patient was admitted to the intensive care unit, and the definitive molecular diagnosis allowed the parents and physicians to devise management plans.

Characterization of the desmosomal cadherin gene family: genomic organization of two desmoglein genes on human chromosome 18q12.

The human desmoglein genes, desmogleins 1--3, are members of the desmosomal cadherin superfamily, and encode critical components of the desmosome. These genes are tightly clustered within 150--200 kb of chromosome 18q12.1 and represent excellent candidate genes for genetic disorders of the epidermis linked to this region of the genome. Mutations in desmoglein 1 have already been implicated in the genetic disorder striate palmoplantar keratoderma. Similarly, a mutation in desmoglein 3 underlies the balding mouse phenotype, although no human mutations in desmoglein 3 have been identified to date. In this study, we have characterized the genomic organization of two of the three desmoglein genes mapped to chromosome 18q12. Comparison of their exon-intron structure reveals the high level of evolutionary conservation expected from these related genes. The identification of the genomic structure of the desmoglein genes will facilitate mutation detection in genodermatoses with desmosomal abnormalities resulting from underlying defects in these genes.

Structural analysis reflects the evolutionary relationship between the human desmocollin gene family members.

Desmocollins, members of the desmosomal cadherin family, are known to play an important role in desmosomal intercellular adhesion. The human desmosomal cadherin cluster is located on chromosome 18q12, and consists of three desmoglein and three desmocollin genes. The cDNAs of all six of these genes have been cloned and sequenced, however, the exon-intron organization was reported for only one human desmocollin gene, DSC2. We elucidated the exon-intron structures of the DSC1 and DSC3 genes using PCR amplification of genomic DNA and direct sequencing of BAC clones. The results suggest a strong evolutionary conservation between the genomic organization of the desmocollin genes.

Structural analysis and mutation detection strategy for the human LAMC3 gene.

Laminins are heterotrimeric extracellular matrix molecules, present in a wide range of basement membranes within human tissues. They consist of a combination of different alpha, beta, and gamma subunits. Three different gamma subunits have been described to date. Two of them, the gamma1 and gamma2 chains are constituents of basement membrane related laminins, while the gamma3 chain was detected in skin, heart, lung, reproductive tract, brain, and in the retina. Unlike other laminins, the expression of the gamma3 chain was localized to peripheral nerves and to the apical surface of ciliated epithelial cells and in the retina. To further investigate the function and the possible pathogenic role of laminin gamma3 in human disease, we elucidated the structure of the corresponding LAMC3 gene which encodes this polypeptide. Here we report the genomic organization of the LAMC3 gene and a mutation detection strategy for use in genetic studies.

Preimplantation genetic diagnosis in two families at risk for recurrence of Herlitz junctional epidermolysis bullosa.

The Herlitz type of junctional epidermolysis bullosa (H-JEB) is a severe inherited bullous disease which leads to the early demise of the affected newborn. Mutations in the genes encoding the 3 polypeptides of the anchoring filament protein laminin 5 underlie this condition. We studied 2 families with affected children who previously died from H-JEB. Mutation screening using heteroduplex analysis and direct sequencing of the PCR products revealed a previously described hotspot mutation in LAMB3 (R635X), and a novel delayed termination codon in LAMB3 in the first proband. In the second proband, we found a novel initiation codon mutation in LAMB3, and a novel 2 bp deletion in LAMB3. For preimplantation genetic diagnosis (PGD) in these families, we developed nested multiplex PCR assays, amplifying the mutations and informative intragenic polymorphisms in the probands. Single embryonic cells were biopsied from 8-cell embryos using standard techniques, and subjected to the multiplex PCR assay followed by restriction enzyme digestion. Embryos found not to carry either mutation were transferred to the mothers, and a pregnancy was established in the second family as evidenced by the elevated level of HCG, although the pregnancy did not persist. This study illustrates the feasibility of PGD for an inherited skin disorder for the first time.

Epidermolytic hyperkeratosis in a Hispanic family resulting from a mutation in the keratin 1 gene.

Epidermolytic hyperkeratosis (EHK; bullous congenital ichthyosiform erythroderma) is a genodermatosis resulting from mutations in either the keratin 1 (K1) or keratin 10 (K10) genes. It is characterized by erythroderma and blistering at birth, and the development of ichthyotic hyperkeratosis and palmoplantar keratoderma. A wide variety of mutations within the highly conserved helix initiation and termination motifs of the central rod domains of the K1 or K10 genes correlate with the highly variable phenotypic severity observed in EHK. We report a novel missense mutation designated L214P in a large Hispanic pedigree with EHK. The mutation is located in the highly conserved 1A segment of the alpha-helical rod domain. The presence of this mutation underscores the importance of sequence alterations located in the central rod domain in the pathogenesis of EHK.

Epidermolytic palmoplantar keratoderma in a Hispanic kindred resulting from a mutation in the keratin 9 gene.

Epidermolytic palmoplantar keratoderma (EPPK) is a localized keratinization disorder caused by mutations in the highly conserved coil 1A domain of the keratin 9 gene, KRT9. We present a Hispanic pedigree spanning three generations, with affected individuals in all generations. Using polymerase chain reaction amplification and direct sequencing we demonstrated a previously reported missense mutation in KRT9, which is expressed almost exclusively in the skin of palms and soles. The C-->T missense mutation R162W changes a basic amino acid (arginine) to a neutral amino acid (tryptophan). We describe this mutation in a Hispanic pedigree with EPPK for the first time, extending the finding of this mutation in other genetic backgrounds, and demonstrating the prevalence of this mutation in diverse populations.

Analysis of the desmoplakin gene reveals striking conservation with other members of the plakin family of cytolinkers.

Members of the plakin family of cytolinker proteins integrate filaments into cellular networks and anchor these networks to the plasma membrane. Their importance is supported by the existence of cell and tissue fragility disorders caused by mutations in certain family members. In this study, the human gene encoding desmoplakin (DSP) was characterized and its structure compared with the related family members: plectin, bullous pemphigoid antigen 1 (BPAG1), envoplakin (EVPL) and periplakin (PPL). Sequence analysis of genomic clones was carried out in combination with a PCR-based strategy to define intron-exon borders. DSP was mapped using the GB4 radiation hybrid mapping panel to the interval between markers D6S296 and AFM043 x f2, corresponding to cytogenetic band 6p24. In addition, the murine gene (Dsp) was mapped to mouse chromosome 13 by interspecific backcross mapping. DSP encompasses approximately 45 kb organized into 24 exons and 23 introns, and the pattern of intron-exon borders bears a striking resemblance to other members of the plakin family. Notable features include the fact that a single large exon encodes the entire C-terminus of each gene. In contrast, the N-termini comprise numerous smaller exons with conservation of many intron-exon borders. Detailed characterization and mapping of these genes will facilitate their further evaluation as targets of genetic disorders and provide insights into the evolutionary relationships among molecules in this emerging gene family.

Ichthyosis bullosa of Siemens resulting from a novel missense mutation near the helix termination motif of the keratin 2e gene.

Ichthyosis bullosa of Siemens (IBS) is an autosomal dominant disorder of keratinization. It is characterized by a mild epidermolytic ichthyosis which tends to localize to the flexures. Affected individuals are born with widespread blistering, which develops into large hyperkeratotic plaques over the extremities. Mutations in the K2e gene cause epidermolytic hyperkeratosis confined to the upper spinous and granular layers, as observed in IBS. In this report, we describe a novel mutation in the keratin 2e gene in a four-generation IBS kindred of German ancestry. The mutation resides within the 2B helix termination motif of the keratin 2e gene, and extends the body of evidence implicating keratin 2e gene mutations in IBS.

Identification of a de novo glycine substitution in the type VII collagen gene in a proband with mild dystrophic epidermolysis bullosa.

The dystrophic forms of epidermolysis bullosa result from different types and combinations of mutations in the type VII collagen gene (COL7A1). We describe a novel glycine substitution arising as a de novo mutation in a proband with a clinically mild form of dystrophic epidermolysis bullosa and no family history of any blistering disease. This report underscores the predominance of glycine substitutions in the dominantly inherited forms of dystrophic form epidermolysis bullosa, and heightens our awareness of unusual modes of inheritance. This information is critical for accurate genetic counseling and determination of recurrence risk in families with dystrophic EB.

Novel and de novo glycine substitution mutations in the type VII collagen gene (COL7A1) in dystrophic epidermolysis bullosa: implications for genetic counseling.

The dystrophic forms of epidermolysis bullosa (DEB) are due to mutations in the type VII collagen gene (COL7A1). In dominant DEB, a characteristic genetic lesion is a glycine substitution mutation within the collagenous domain of the protein. In this study, we have examined the molecular basis of six new families in which the proband has clinical features and/or ultrastructural findings consistent with DEB. The results revealed a glycine substitution mutation in all six families, four of which are novel and previously unpublished. In three families with clinically unaffected parents, de novo mutations G2043R and G2040V were found. These results emphasize the predominance of glycine substitution mutations in dominant DEB, and indicate that in some cases the phenotype is due to de novo dominant mutations.

Molecular analysis of the human laminin alpha3a chain gene (LAMA3a): a strategy for mutation identification and DNA-based prenatal diagnosis in Herlitz junctional epidermolysis bullosa.

Mutations in the genes (LAMA3, LAMB3, and LAMC2) encoding the subunit polypeptides of the cutaneous basement membrane zone protein laminin 5 have been reported in different forms of junctional epidermolysis bullosa (JEB), an inherited blistering skin disease. In this study, we present the complete exon-intron organization of the "a" transcript of the laminin alpha3 chain gene, LAMA3a, which is expressed primarily in the skin. We have performed fine-resolution mapping of this gene on chromosome 18q11.2 using a human-hamster radiation hybrid panel. We have also developed a mutation-detection strategy based on the exon-intron structure of LAMA3a. This strategy, based on PCR amplification of genomic sequences, followed by heteroduplex scanning and automated nucleotide sequencing, was used for successful mutation screening in a family with the lethal (Herlitz) type of JEB, and two novel LAMA3 mutations were identified in the proband. The mutations consisted of a single-base pair deletion in LAMA3a exon A11 on the paternal allele, designated 1239delC, and a two-base pair deletion in LAMA3a exon A23 on the maternal allele, designated 2959delGG. This information was also used for DNA-based prenatal testing in a subsequent pregnancy in this family. Collectively, these results attest to our expanding capability to elucidate the genetic basis of various forms of epidermolysis bullosa using molecular techniques.

Molecular basis of non-lethal junctional epidermolysis bullosa: identification of a 38 basepair insertion and a splice site mutation in exon 14 of the LAMB3 gene.

Epidermolysis bullosa (EB) is a group of genodermatoses characterized by fragility and easy blistering of the skin. In the junctional forms of EB (JEB), blisters occur at the level of the lamina lucida, and specific mutations have been detected in the genes encoding different components of the hemidesmosomal-anchoring filament complex. In the non-lethal form of JEB (NL-JEB), mutations in genes encoding two of the polypeptide chains of the anchoring filament protein laminin 5 have recently been described. In this study, we searched for mutations in a family using PCR amplification of exon 14 of LAMB3, the laminin 5 beta3 chain gene, followed by heteroduplex analysis and automated sequencing of the PCR products. We detected a novel combination of mutations in this family, consisting of an out-of frame insertion on one allele, and a splice site mutation on the other allele, representing the first report of a large insertion in LAMB3, together with a splice site mutation inherited in trans, which result in the NL-JEB phenotype.