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1.
Arthritis Rheumatol ; 71(8): 1285-1296, 2019 08.
Article in English | MEDLINE | ID: mdl-30730609

ABSTRACT

OBJECTIVE: To identify methylation quantitative trait loci (mQTLs) correlating with osteoarthritis (OA) risk alleles and to undertake mechanistic characterization as a means of target gene prioritization. METHODS: We used genome-wide genotyping and cartilage DNA methylation array data in a discovery screen of novel OA risk loci. This was followed by methylation, gene expression analysis, and genotyping studies in additional cartilage samples, accompanied by in silico analyses. RESULTS: We identified 4 novel OA mQTLs. The most significant mQTL contained 9 CpG sites where methylation correlated with OA risk genotype, with 5 of the CpG sites having P values <1 × 10-10 . The 9 CpG sites reside in an interval of only 7.7 kb within the PLEC gene and form 2 distinct clusters. We were able to prioritize PLEC and the adjacent gene GRINA as independent targets of the OA risk. We identified PLEC and GRINA expression QTLs operating in cartilage, as well as methylation-expression QTLs operating on the 2 genes. GRINA and PLEC also demonstrated differential expression between OA hip and non-OA hip cartilage. CONCLUSION: PLEC encodes plectin, a cytoskeletal protein that maintains tissue integrity by regulating intracellular signaling in response to mechanical stimuli. GRINA encodes the ionotropic glutamate receptor TMBIM3 (transmembrane BAX inhibitor 1 motif-containing protein family member 3), which regulates cell survival. Based on our results, we hypothesize that in a joint predisposed to OA, expression of these genes alters in order to combat aberrant biomechanics, and that this is epigenetically regulated. However, carriage of the OA risk-conferring allele at this locus hinders this response and contributes to disease development.


Subject(s)
DNA Methylation/genetics , Osteoarthritis/genetics , Plectin/genetics , Quantitative Trait Loci/genetics , Receptors, N-Methyl-D-Aspartate/genetics , Adult , Aged , Alleles , Biomechanical Phenomena/genetics , Cartilage, Articular/metabolism , CpG Islands/genetics , Female , Genetic Predisposition to Disease/genetics , Genome-Wide Association Study , Genotype , Humans , Male , Membrane Proteins/genetics , Middle Aged , Polymorphism, Single Nucleotide , Risk Factors
2.
Hum Mol Genet ; 27(19): 3464-3474, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30010910

ABSTRACT

Osteoarthritis (OA) is a common, multifactorial and polygenic skeletal disease that, in its severest form, requires joint replacement surgery to restore mobility and to relieve chronic pain. Using tissues from the articulating joints of 260 patients with OA and a range of in vitro experiments, including CRISPR-Cas9, we have characterized an intergenic regulatory element. Here, genotype at an OA risk locus correlates with differential DNA methylation, with altered gene expression of both a transcriptional regulator (RUNX2), and a chromatin remodelling protein (SUPT3H). RUNX2 is a strong candidate for OA susceptibility, with its encoded protein being essential for skeletogenesis and healthy joint function. The OA risk locus includes single nucleotide polymorphisms (SNPs) located within and flanking the differentially methylated region (DMR). The OA association SNP, rs10948172, demonstrates particularly strong correlation with methylation, and two intergenic SNPs falling within the DMR (rs62435998 and rs62435999) demonstrate genetic and epigenetic effects on the regulatory activity of this region. We therefore posit that the OA signal mediates its effect by modulating the methylation of the regulatory element, which then impacts on gene expression, with RUNX2 being the principal target. Our study highlights the interplay between DNA methylation, OA genetic risk and the downstream regulation of genes critical to normal joint function.


Subject(s)
Core Binding Factor Alpha 1 Subunit/genetics , DNA Methylation/genetics , Osteoarthritis/genetics , Transcription Factors/genetics , Adult , Aged , Aged, 80 and over , CRISPR-Cas Systems , Female , Gene Expression Regulation/genetics , Genetic Predisposition to Disease , Genotype , Humans , Joints/physiopathology , Male , Middle Aged , Osteoarthritis/physiopathology , Polymorphism, Single Nucleotide , Regulatory Sequences, Nucleic Acid , Risk Factors
3.
Mol Ther Nucleic Acids ; 5: e307, 2016 Apr 05.
Article in English | MEDLINE | ID: mdl-27045209

ABSTRACT

Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction.

4.
Mol Ther ; 22(5): 929-39, 2014 May.
Article in English | MEDLINE | ID: mdl-24468915

ABSTRACT

Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvß8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy.


Subject(s)
Genetic Engineering , Genetic Therapy , Peptides/genetics , Skin Abnormalities/therapy , Capsid Proteins/genetics , Dependovirus/genetics , Genetic Vectors , Humans , Integrin alpha5/genetics , Keratinocytes/metabolism , Keratinocytes/pathology , Peptides/therapeutic use , Skin Abnormalities/genetics , Skin Abnormalities/pathology , Transduction, Genetic , Tropism
5.
Exp Dermatol ; 22(9): 601-3, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23947675

ABSTRACT

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by deficiency of type VII collagen due to COL7A1 mutations such as c.6527insC, recurrently found in the Spanish RDEB population. Assessment of clonal correction-based therapeutic approaches for RDEB requires large expansions of cells, exceeding the replication capacity of human primary keratinocytes. Thus, immortalized RDEB cells with enhanced proliferative abilities would be valuable. Using either the SV40 large T antigen or papillomavirus HPV16-derived E6-E7 proteins, we immortalized and cloned RDEB keratinocytes carrying the c.6527insC mutation. Clones exhibited high proliferative and colony-forming features. Cytogenetic analysis revealed important differences between T antigen-driven and E6-E7-driven immortalization. Immortalized cells responded to differentiation stimuli and were competent for epidermal regeneration and recapitulation of the blistering RDEB phenotype in vivo. These features make these cell lines useful to test novel therapeutic approaches including those aimed at editing mutant COL7A1.


Subject(s)
Collagen Type VII/genetics , Epidermolysis Bullosa Dystrophica/genetics , Epidermolysis Bullosa Dystrophica/therapy , Keratinocytes/metabolism , Mutation , Animals , Cell Line , Cell- and Tissue-Based Therapy , Epidermolysis Bullosa Dystrophica/pathology , Genetic Therapy , Heterografts , Homozygote , Humans , Keratinocytes/transplantation , Mice , Models, Genetic , Regeneration
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