Dystonin modifiers of junctional epidermolysis bullosa and models of epidermolysis bullosa simplex without dystonia musculorum.

The Lamc2jeb junctional epidermolysis bullosa (EB) mouse model has been used to demonstrate that significant genetic modification of EB symptoms is possible, identifying as modifiers Col17a1 and six other quantitative trait loci, several with strong candidate genes including dystonin (Dst/Bpag1). Here, CRISPR/Cas9 was used to alter exon 23 in mouse skin specific isoform Dst-e (Ensembl GRCm38 transcript name Dst-213, transcript ID ENSMUST00000183302.5, protein size 2639AA) and validate a proposed arginine/glutamine difference at amino acid p1226 in B6 versus 129 mice as a modifier of EB. Frame shift deletions (FSD) in mouse Dst-e exon 23 (Dst-eFSD/FSD) were also identified that cause mice carrying wild-type Lamc2 to develop a phenotype similar to human EB simplex without dystonia musculorum. When combined, Dst-eFSD/FSD modifies Lamc2jeb/jeb (FSD+jeb) induced disease in unexpected ways implicating an altered balance between DST-e (BPAG1e) and a rarely reported rodless DST-eS (BPAG1eS) in epithelium as a possible mechanism. Further, FSD+jeb mice with pinnae removed are found to provide a test bed for studying internal epithelium EB disease and treatment without severe skin disease as a limiting factor while also revealing and accelerating significant nasopharynx symptoms present but not previously noted in Lamc2jeb/jeb mice

Application of 4-D ultrasound-derived regional strain and proteomics analysis in

The comprehensive characterization of cardiac structure and function is critical to better understanding various murine models of cardiac disease. We demonstrate here a multimodal analysis approach using high-frequency four-dimensional ultrasound (4DUS) imaging and proteomics to explore the relationship between regional function and tissue composition in a murine model of metabolic cardiomyopathy (Nkx2-5183P/ þ ). The presented 4DUS analysis outlines a novel approach to mapping both circumfer- ential and longitudinal strain profiles through a standardized framework. We then demonstrate how this approach allows for spa- tiotemporal comparisons of cardiac function and improved localization of regional left ventricular dysfunction. Guided by observed trends in regional dysfunction, our targeted Ingenuity Pathway Analysis (IPA) results highlight metabolic dysregulation in the Nkx2-5183P/ þ model, including altered mitochondrial function and energy metabolism (i.e., oxidative phosphorylation and fatty acid/lipid handling). Finally, we present a combined 4DUS-proteomics z-score-based analysis that highlights IPA canonical pathways showing strong linear relationships with 4DUS biomarkers of regional cardiac dysfunction. The presented multimodal analysis methods aim to help future studies more comprehensively assess regional structure-function relationships in other pre- clinical models of cardiomyopathy

Functional analysis of Collagen 17a1: A genetic modifier of junctional epidermolysis bullosa in mice.

Previous work strongly implicated Collagen 17a1 (Col17a1) as a potent genetic modifier of junctional epidermolysis bullosa (JEB) caused by a hypomorphic mutation (Lamc2jeb) in mice. The importance of the noncollagenous domain (NC4) of COLXVII was suggested by use of a congenic reduction approach that restricted the modifier effect to 2-3 neighboring amino acid changes in that domain. The current study utilizes TALEN and CRISPR/Cas9 induced amino acid replacements and in-frame indels nested to NC4 to further investigate the role of this and adjoining COLXVII domains both as modifiers and primary risk effectors. We confirm the importance of COLXVI AA 1275 S/G and 1277 N/S substitutions and utilize small nested indels to show that subtle changes in this microdomain attenuate JEB. We further show that large in-frame indels removing up to 1482 bp and 169 AA of NC6 through NC1 domains are surprisingly disease free on their own but can be very potent modifiers of Lamc2jeb/jeb JEB. Together these studies exploiting gene editing to functionally dissect the Col17a1 modifier demonstrate the importance of epistatic interactions between a primary disease-causing mutation in one gene and innocuous 'healthy' alleles in other genes

Large deletion histology (all <i>Lamc2</i>^<i>wt/wt</i>).

(A-H) Em2 (B6-Col17a1em2/em2) male 43 weeks of age. (A) Locally extensive area of epidermal hyperplasia covered by degenerating inflammatory cells (mostly neutrophils) and desiccated proteinaceous material (scab). This covers an area of very mild dermal-epidermal separation (B) adjacent to a small ulcer (C) with transition to normal epidermis (D). (E, magnified in F) Focal area of epidermal hyperplasia overlying follicular dystrophy (FD) adjacent to a small area of dermal-epidermal separation (*). (G, magnified in H) A small area of dermis with fine dense regular collagenous connective tissue devoid of hair follicles (old mature scar) adjacent to more normal dense irregular collagenous connective tissue. Note the pigmentary incontinence (arrow, remnant of a dystrophic hair follicle). (I magnified in J and K) Normal skin from B6 wild type male mouse 43 weeks of age. Not the thin epidermis, tight dermal-epidermal junction, normal hair follicle, and uniform dense irregular collagenous connective tissue making up the dermis. (L) Em16 52-week-old male. Em16 rarely had foci of fibrosis (brackets) with underlying granulomatous inflammation. Note the multinucleated giant cell (arrow). (M magnified in N and O) Em15 male 52 weeks of age. Cutaneous ulcer and scar. (N) Epidermal hyperplasia overlying an area of fibrosis (scar formation). (O) Ulcer (left) with overlying scab formation. Mild fibrosis under mild epidermal hyperplasia with a mixed dermal inflammatory cell infiltrate.</p

DNA and cDNA sequence of <i>em1</i> and <i>em2</i>.

Previous work strongly implicated Collagen 17a1 (Col17a1) as a potent genetic modifier of junctional epidermolysis bullosa (JEB) caused by a hypomorphic mutation (Lamc2jeb) in mice. The importance of the noncollagenous domain (NC4) of COLXVII was suggested by use of a congenic reduction approach that restricted the modifier effect to 2–3 neighboring amino acid changes in that domain. The current study utilizes TALEN and CRISPR/Cas9 induced amino acid replacements and in-frame indels nested to NC4 to further investigate the role of this and adjoining COLXVII domains both as modifiers and primary risk effectors. We confirm the importance of COLXVI AA 1275 S/G and 1277 N/S substitutions and utilize small nested indels to show that subtle changes in this microdomain attenuate JEB. We further show that large in-frame indels removing up to 1482 bp and 169 AA of NC6 through NC1 domains are surprisingly disease free on their own but can be very potent modifiers of Lamc2jeb/jeb JEB. Together these studies exploiting gene editing to functionally dissect the Col17a1 modifier demonstrate the importance of epistatic interactions between a primary disease-causing mutation in one gene and innocuous ‘healthy’ alleles in other genes.</div