Ocular Gene Therapy in a Patient with Dystrophic Epidermolysis Bullosa.

Dystrophic epidermolysis bullosa is a rare genetic disease caused by damaging variants in COL7A1, which encodes type VII collagen. Blistering and scarring of the ocular surface develop, potentially leading to blindness. Beremagene geperpavec (B-VEC) is a replication-deficient herpes simplex virus type 1-based gene therapy engineered to deliver functional human type VII collagen. Here, we report the case of a patient with cicatrizing conjunctivitis in both eyes caused by dystrophic epidermolysis bullosa who received ophthalmic administration of B-VEC, which was associated with improved visual acuity after surgery.

Genomics-based treatment in a patient with two overlapping heritable skin disorders: Epidermolysis bullosa and acrodermatitis enteropathica.

Next-generation sequencing (NGS) is helpful in diagnosing complex genetic disorders and phenotypes, particularly when more than one overlapping condition is present. From a large cohort of 362 families with clinical manifestations of skin and mucosal fragility, referred by several major medical centers, one patient was found by NGS to have two overlapping heritable skin diseases, recessive dystrophic epidermolysis bullosa (RDEB; COL7A1 mutations) and acrodermatitis enteropathica (AE; SLC39A4 mutations). The pathogenicity of the variants was studied at gene expression as well as ultrastructural and tissue levels. Although there is no specific treatment for RDEB except avoiding trauma, supplementation with oral zinc (3 mg·kg-1 ·day-1 ) for the AE resulted in rapid amelioration of the skin findings. This case demonstrates the power of NGS in identifying two genetically unlinked diseases that led to effective treatment with major clinical benefits as an example of genomics-guided treatment.

Molecular pathology of the basement membrane zone in heritable blistering diseases:: The paradigm of epidermolysis bullosa.

Epidermolysis bullosa (EB), a phenotypically heterogeneous group of skin fragility disorders, is characterized by blistering and erosions with considerable morbidity and mortality. Mutations in as many as 18 distinct genes expressed at the cutaneous basement membrane zone have been shown to be associated with the blistering phenotype, attesting to the role of the corresponding proteins in providing stable association of the epidermis to the dermis through adhesion at the dermo-epidermal basement membrane zone. Thus, different forms of EB have been highly instructive in providing information on the physiological functions of these proteins as integral components of the supramolecular adhesion complexes. In addition, precise information of the underlying genes and distinct mutations in families with EB has been helpful in subclassification of the disease with prognostic implications, as well as for prenatal testing and preimplantation genetic diagnosis. Furthermore, knowledge of the types of mutations is a prerequisite for application of allele-specific treatment approaches that have been recently developed, including read-through of premature termination codon mutations and chaperone-facilitated intracellular transport of conformationally altered proteins to proper physiologic subcellular location. Collectively, EB serves as a paradigm of heritable skin diseases in which significant progress has been made in identifying the underlying genetic bases and associated aberrant pathways leading from mutations to the phenotype, thus allowing application of precision medicine for this, currently intractable group of diseases.

Early onset collagen VI myopathies: Genetic and clinical correlations.

OBJECTIVE: Mutations in the genes encoding the extracellular matrix protein collagen VI (ColVI) cause a spectrum of disorders with variable inheritance including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate phenotypes. We extensively characterized, at the clinical, cellular, and molecular levels, 49 patients with onset in the first 2 years of life to investigate genotype-phenotype correlations. METHODS: Patients were classified into 3 groups: early-severe (18%), moderate-progressive (53%), and mild (29%). ColVI secretion was analyzed in patient-derived skin fibroblasts. Chain-specific transcript levels were quantified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and mutation identification was performed by sequencing of complementary DNA. RESULTS: ColVI secretion was altered in all fibroblast cultures studied. We identified 56 mutations, mostly novel and private. Dominant de novo mutations were detected in 61% of the cases. Importantly, mutations causing premature termination codons (PTCs) or in-frame insertions strikingly destabilized the corresponding transcripts. Homozygous PTC-causing mutations in the triple helix domains led to the most severe phenotypes (ambulation never achieved), whereas dominant de novo in-frame exon skipping and glycine missense mutations were identified in patients of the moderate-progressive group (loss of ambulation). INTERPRETATION: This work emphasizes that the diagnosis of early onset ColVI myopathies is arduous and time-consuming, and demonstrates that quantitative RT-PCR is a helpful tool for the identification of some mutation-bearing genes. Moreover, the clinical classification proposed allowed genotype-phenotype relationships to be explored, and may be useful in the design of future clinical trials.

Se-methylselenocysteine alters collagen gene and protein expression in human prostate cells.

The anti-cancer activity of selenium is dose-dependent and species-specific but the mechanism is unclear. Se-methylselenocysteine (MSC), found in selenium-enriched alliums, is one of the most potent forms. We exposed two human prostate cell lines (LNCaP clone FGC and PNT1A) to nutritionally relevant doses of MSC and selenite, ranging from deficient to the equivalent of selenium supplementation in humans. The cells were adapted for one month to attain steady-state selenium status. Two microarray platforms, an in-house printed microarray (14,000 genes) and the Affymetrix U133A array (22,000 genes) were used to probe the molecular effects of selenium dose and form and several selenium-responsive genes were identified, many of which have been ascribed to cancer cell growth and progression. In response to MSC supplementation, the expression of 23 genes changed significantly, including several collagen genes. Quantitative RT-PCR assays were designed and optimized for four of the collagen genes to validate array data. Significant decreases in expression of collagen type I alpha 1 (COL1A1), COL1A2 and COL7A1 genes were observed in cells adapted to MSC supplementation compared to the control and selenite exposed cells. There were significant increases in genes encoding other types of collagen, including significant increases in COL6A1 and COL4A5 in response to MSC dose. Functional changes in collagen type I protein expression in response to MSC were confirmed by ELISA. This study reveals for the first time that MSC can alter the expression of several types of collagen and thus potentially modulate the extracellular matrix and stroma, which may at least partially explain the anti-cancer activity of MSC.

The protease domain of procollagen C-proteinase (BMP1) lacks substrate selectivity, which is conferred by non-proteolytic domains.

Procollagen C-proteinase (PCP) removes the C-terminal pro-peptides of procollagens and also processes other matrix proteins. The major splice form of the PCP is termed BMP1 (bone morphogenetic protein 1). Active BMP1 is composed of an astacin-like protease domain, three CUB (complement, sea urchin Uegf, BMP1) domains and one EGF-like domain. Here we compare the recombinant human full-length BMP1 with its isolated proteolytic domain to further unravel the functional influence of the CUB and EGF domains. We show that the protease domain alone cleaves truncated procollagen VII within the short telopeptide region into fragments of similar size as the full-length enzyme does. However, unlike full-length BMP1, the protease domain does not stop at this point, but degrades its substrate completely. Moreover, the protease domain cleaves other matrix proteins such as fibronectin, collagen I and collagen IV, which are left intact by the full-length enzyme. In addition, we show for the first time that thrombospondin-1 is differently cleaved by both BMP1 and its catalytic domain. In summary, our data support the concept that the C-terminal domains of BMP1 are important for substrate recognition and for controlling and restricting its proteolytic activity via exosite binding.