Monoclonal Antibodies to Anchoring Fibrils for the Diagnosis of Epidermolysis Bullosa

Murine monoclonal antibodies to human anchoring fibrils reacted with human and monkey cervix, tongue, esophagus, and vagina. Rat, mouse, and guinea pig tissues were negative. In 11 patients with dystrophic recessive epidermolysis bullosa there was no reaction by immunofluorescence and immunoelectron microscopy. Other forms of epidermolysis bullosa had normal reactivity

High-Molecular-Weight Human Epidermal Transglutaminase

Human stratum corneum was extracted in Tris-HCl containing EDTA and phenylmethylsulfonyl fluoride, separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transblotted to nitrocellulose papers and reacted with rabbit antihuman epidermal transglutaminase (ETG) antibody. Protein-bound antibody was detected with a multistep peroxidase procedure. Proteins with a molecular weight of 50,000 (50kDa) and 72,000 daltons (72kDa) were stained when anti-ETG Was used and not when second antibody alone or sera from nonimmunized animals were used. When ETG was treated with trypsin or organic solvents, there was no alteration in the mobility of the 50kDa ETC band, but there was complete disappearance of the 72kDa band. Antibody that bound 72kDa protein, when eluted from the blot, reacted with both 50kDa and 72kDa proteins; similarly, antibody that bound to the 50kDa protein, when eluted from the blot, reacted with both the 50kDa and 72kDa proteins. Partially purified 72kDa ETG activity was increased (3 to 16 times control levels) after heating at 56°C in the presence of calcium and dithiothreitol or by treatment with trypsin. These studies, in conjunction with the previous studies of ETG activation, are consistent with there being two forms of ETG. The different forms may play a role in regulating enzyme activity

Monoclonal Antibody to a 35 kD Epidermal Protein Induces Cell Detachment

A murine monoclonal antibody (ECS-1) was prepared from BALB/c mice immunized with trypsinized cultured human foreskin keratinocytes. The antibody showed a pattern suggestive of intercellular staining on the nucleated layers of normal human epidermis, adult palm, mouse lip epidermis, and cultured human keratinocytes. ECS-1 stained human fetal skin by 9 weeks estimated gestational age. ECS-1 reacted with a 35 kD protein extracted from neonatal foreskin epidermis and cultured human keratinocytes. The protein required Nonidet P-40 or sodium dodecyl sulfate and mercaptoethanol for solubilization. ECS-1 induced epidermal cell detachment which was enhanced by complement. ECS- 1 shares characteristics with human pemphigus antibodies

Mutations in KDSR Cause Recessive Progressive Symmetric Erythrokeratoderma

Supplemental Data Supplemental Data include five figures and three tables and can be found with this article online at http://dx.doi.org/10.1016/j.ajhg.2017.05.003. Supplemental Data Document S1. Figures S1–S5 and Tables S1–S3 Download Document S2. Article plus Supplemental Data Download Web Resources 1000 Genomes, http://www.internationalgenome.org/ ANNOVAR, http://annovar.openbioinformatics.org/en/latest/ BWA-MEM, http://bio-bwa.sourceforge.net/index.shtml Database of Genomic Variants, http://dgv.tcag.ca/dgv/app/home dbSNP, https://www.ncbi.nlm.nih.gov/projects/SNP/ Exome Aggregation Consortium (ExAC) Browser, http://exac.broadinstitute.org/ ExonPrimer, https://ihg.helmholtz-muenchen.de/ihg/ExonPrimer.html GenBank, https://www.ncbi.nlm.nih.gov/genbank/ Genome Analysis Toolkit (GATK), https://software.broadinstitute.org/gatk/ Integrative Genomics Viewer (IGV), http://software.broadinstitute.org/software/igv/ OMIM, https://www.omim.org/ SNPmasker, http://bioinfo.ebc.ee/snpmasker/ UCSC Genome Browser, https://genome.ucsc.edu/index.html Variant Effect Predictor, http://useast.ensembl.org/info/docs/tools/vep/index.html The discovery of new genetic determinants of inherited skin disorders has been instrumental to the understanding of epidermal function, differentiation, and renewal. Here, we show that mutations in KDSR (3-ketodihydrosphingosine reductase), encoding an enzyme in the ceramide synthesis pathway, lead to a previously undescribed recessive Mendelian disorder in the progressive symmetric erythrokeratoderma spectrum. This disorder is characterized by severe lesions of thick scaly skin on the face and genitals and thickened, red, and scaly skin on the hands and feet. Although exome sequencing revealed several of the KDSR mutations, we employed genome sequencing to discover a pathogenic 346 kb inversion in multiple probands, and cDNA sequencing and a splicing assay established that two mutations, including a recurrent silent third base change, cause exon skipping. Immunohistochemistry and yeast complementation studies demonstrated that the mutations cause defects in KDSR function. Systemic isotretinoin therapy has achieved nearly complete resolution in the two probands in whom it has been applied, consistent with the effects of retinoic acid on alternative pathways for ceramide generation