Autosomal recessive congenital ichthyosis (ARCI), a phenotypically heterogeneous group of non‐syndromic Mendelian disorders of keratinization, is caused by mutations in as many as 13 distinct genes. We examined a cohort of 125 consanguineous families with ARCI for underlying genetic mutations. The patients’ DNA was analyzed with a gene‐targeted next generation sequencing panel comprising 38 ichthyosis associated genes. The interpretations of results of genomic data were assisted by genome‐wide homozygosity mapping and transcriptome sequencing. Sequence data analysis identified biallelic mutations in 106 families out of a total of 125 (85%), most of them (102, 96.2%) being homozygous, reflecting consanguinity in these families. Among the 85 distinct mutations in 10 different genes, 45 (53%) were previously unreported. Phenotype‐genotype correlations allowed assignment of specific genes in the majority of the families to a specific subtype of ARCI, lamellar ichthyosis (LI) versus congenital ichthyosiform erythroderma (CIE). Interestingly, mutations in several genes could give rise to an overlapping phenotype consistent with either LI or CIE. Also, this is the third report for SDR9C7 and SULT2B1, and fourth report for CERS3 mutations. Direct comparison of our results with previously published regional cohorts highlights the global mutation landscape of ARCI, however, population specific differences were noted.

Epidermolysis bullosa (EB) is caused by mutations in as many as 19 distinct genes. We have developed a next-generation sequencing (NGS) panel targeting genes known to be mutated in skin fragility disorders, including tetraspanin CD151 expressed in keratinocytes at the dermal-epidermal junction. The NGS panel was applied to a cohort of 92 consanguineous families of unknown subtype of EB. In one family, a homozygous donor splice site mutation in CD151 (NM_139029; c.351+2T>C) at the exon 5/intron 5 border was identified, and RT-PCR and whole transcriptome analysis by RNA-seq confirmed deletion of the entire exon 5 encoding 25 amino acids. Immunofluorescence of proband's skin and Western blot of skin proteins with a monoclonal antibody revealed complete absence of CD151. Transmission electron microscopy showed intracellular disruption and cell-cell dysadhesion of keratinocytes in the lower epidermis. Clinical examination of the 33-year old proband, initially diagnosed as Kindler syndrome, revealed widespread blistering, particularly on pretibial areas, poikiloderma, nail dystrophy, loss of teeth, early onset alopecia, and esophageal webbing and strictures. The patient also had history of nephropathy with proteinuria. Collectively, the results suggest that biallelic loss-of-function mutations in CD151 underlie an autosomal recessive mechano-bullous disease with systemic features. Thus, CD151 should be considered as the 20th causative, EB-associated gene.

Epidermolysis bullosa (EB), the paradigm of heritable skin fragility disorders, is associated with mutations in as many as 20 distinct genes. One of the clinical variants, recessive dystrophic EB (RDEB), demonstrates sub-lamina densa blistering accompanied by alterations in anchoring fibrils due to mutations in COL7A1. In this study, we characterized a patient with widespread connective tissue abnormalities, including skin blistering similar to that in RDEB. Whole exome sequencing, combined with genome-wide homozygosity mapping, identified a homozygous missense mutation in PLOD3 encoding lysyl hydroxylase 3 (LH3). No mutations in COL7A1, the gene previously associated with RDEB, were detected. The level of LH3 was dramatically reduced in the skin and fibroblast cultures from the patient. The blistering in the skin occurred below the lamina densa and was associated with variable density and morphology of anchoring fibrils. The level of type VII collagen expression in the skin was markedly reduced. Analysis of hydroxylysine and its glycosylated derivatives (galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine) revealed marked reduction in glycosylated hydroxylysine. Collectively, these findings indicate that PLOD3 mutations can result in a dystrophic EB-like phenotype in the spectrum of connective tissue disorders and add it to the list of candidate genes associated with skin fragility.

Epidermolysis bullosa (EB) is a heterogeneous group of heritable blistering diseases. We developed a next generation sequencing (NGS) panel covering 21 genes associated with skin fragility disorders, and it was applied to DNA from 91 probands with the diagnosis of EB. In one patient, novel homozygous mutations were disclosed in two different, unlinked EB-associated genes: EXPH5, chr11 g.108510085G > A; p.Arg1808Ter and COL17A1, chr10 g.104077423delT; p.Thr68LeufsTer106. Consequences of the COL17A1 mutation were examined by RNAseq which revealed a complex splicing pattern predicting synthesis of a truncated polypeptide (85%) or in-frame deletion of exon 4 (15% of transcripts). Transmission electron microscopy (TEM) and immunostaining revealed findings consistent with EB simplex (EBS) and junctional EB (JEB), and clinical examination revealed a complex phenotype with features of both subtypes. This case illustrates the power of next generation sequencing in identifying mutations in patients with complex EB phenotype, with implications for genotype-phenotype correlations, prenatal testing, and genetic counseling of families at risk for recurrence.

No abstract

Autozygosity mapping (AM) is a technique utilised for mapping homozygous autosomal recessive (AR) traits and facilitation of genetic diagnosis. We investigated the utility of AM for the molecular diagnosis of heterogeneous AR disorders, using epidermolysis bullosa (EB) as a paradigm. We applied this technique to a cohort of 46 distinct EB families using both short tandem repeat (STR) and genome-wide single nucleotide polymorphism (SNP) array-based AM to guide targeted Sanger sequencing of EB candidate genes. Initially, 39 of the 46 cases were diagnosed with homozygous mutations using this method. Independently, 26 cases, including the seven initially unresolved cases, were analysed with an EB-targeted next-generation sequencing (NGS) panel. NGS identified mutations in five additional cases, initially undiagnosed due to the presence of compound heterozygosity, deep intronic mutations or runs of homozygosity below the set threshold of 2 Mb, for a total yield of 44 of 46 cases (95.7%) diagnosed genetically.

Circulating microRNAs are beneficial biomarkers because of their stability and dysregulation in diseases. Here we sought to determine the role of miR-939, a miRNA downregulated in patients with complex regional pain syndrome (CRPS). Hsa-miR-939 is predicted to target several proinflammatory genes, including IL-6, VEGFA, TNFα, NFκB2, and nitric oxide synthase 2 (NOS2A). Binding of miR-939 to the 3′ untranslated region of these genes was confirmed by reporter assay. Overexpression of miR-939 in vitro resulted in reduction of IL-6, NOS2A and NFκB2 mRNAs, IL-6, VEGFA, and NOS2 proteins and NFκB activation. We observed a significant decrease in the NOS substrate l-arginine in plasma from CRPS patients, suggesting reduced miR-939 levels may contribute to an increase in endogenous NOS2A levels and NO, and thereby to pain and inflammation. Pathway analysis showed that miR-939 represents a critical regulatory node in a network of inflammatory mediators. Collectively, our data suggest that miR-939 may regulate multiple proinflammatory genes and that downregulation of miR-939 in CRPS patients may increase expression of these genes, resulting in amplification of the inflammatory pain signal transduction cascade. Circulating miRNAs may function as crucial signaling nodes, and small changes in miRNA levels may influence target gene expression and thus disease.

Biallelic mutations in the ITK gene cause a T-cell primary immunodeficiency with Epstein-Barr virus (EBV)–lymphoproliferative disorders. We describe a novel association of a homozygous ITK mutation with β–human papillomavirus (HPV)–positive epidermodysplasia verruciformis. Thus, loss of function in ITK can result in broad dysregulation of T-cell responses to oncogenic viruses, including β-HPV and EBV.