Tabatha R. Simmons

Increasing Genotype-Phenotype Model Determinism: Application to Bivariate Reading/Language Traits and Epistatic Interactions in Language-Impaired Families

While advances in network and pathway analysis have flourished in the era of genome-wide association analysis, understanding the genetic mechanism of individual loci on phenotypes is still readily accomplished using genetic modeling approaches. Here, we demonstrate two novel genotype-phenotype models implemented in a flexible genetic modeling platform. The examples come from analysis of families with specific language impairment (SLI), a failure to develop normal language without explanatory factors such as low IQ or inadequate environment. In previous genome-wide studies, we observed strong evidence for linkage to 13q21 with a reading phenotype in language-impaired families. First, we elucidate the genetic architecture of reading impairment and quantitative language variation in our samples using a bivariate analysis of reading impairment in affected individuals jointly with language quantitative phenotypes in unaffected individuals. This analysis largely recapitulates the baseline analysis using the categorical trait data (posterior probability of linkage (PPL) = 80%), indicating that our reading impairment phenotype captured poor readers who also have low language ability. Second, we performed epistasis analysis using a functional coding variant in the brain-derived neurotrophic factor (BDNF) gene previously associated with reduced performance on working memory tasks. Modeling epistasis doubled the evidence on 13q21 and raised the PPL to 99.9%, indicating that BDNF and 13q21 susceptibility alleles are jointly part of the genetic architecture of SLI. These analyses provide possible mechanistic insights for further cognitive neuroscience studies based on the models developed herein.

The ZZ Domain of Dystrophin in DMD: Making Sense of Missense Mutations

Duchenne muscular dystrophy (DMD) is associated with the loss of dystrophin, which plays an important role in myofiber integrity via interactions with β‐dystroglycan and other members of the transmembrane dystrophin‐associated protein complex. The ZZ domain, a cysteine‐rich zinc‐finger domain near the dystrophin C‐terminus, is implicated in forming a stable interaction between dystrophin and β‐dystroglycan, but the mechanism of pathogenesis of ZZ missense mutations has remained unclear because not all such mutations have been shown to alter β‐dystroglycan binding in previous experimental systems. We engineered three ZZ mutations (p.Cys3313Phe, p.Asp3335His, and p.Cys3340Tyr) into a short construct similar to the Dp71 dystrophin isoform for in vitro and in vivo studies and delineated their effect on protein expression, folding properties, and binding partners. Our results demonstrate two distinct pathogenic mechanisms for ZZ missense mutations. The cysteine mutations result in diminished or absent subsarcolemmal expression because of protein instability, likely due to misfolding. In contrast, the aspartic acid mutation disrupts binding with β‐dystroglycan despite an almost normal expression at the membrane, confirming a role for the ZZ domain in β‐dystroglycan binding but surprisingly demonstrating that such binding is not required for subsarcolemmal localization of dystrophin, even in the absence of actin binding domains.

624. A Single Neonatal Delivery of an Exon 2 Directed AAV9.U7snRNA Vector Results in Long-Term Dystrophin Expression That Prevents Pathologic Features in the Dup2 Mouse

We recently identified an internal ribosome entry site (IRES) within exon 5 of the DMD gene. Mutations that truncate the reading frame 5’ of this exon can result in use of the IRES for alternate translational initiation beginning within exon 6 that results in expression of an N-truncated isoform. Despite lacking the calponin homology domain 1 (CH1) of the actin binding domain 1 (ABD1), this isoform is highly functional, as demonstrated by the minimal symptoms in patients who express it. Consistent with genotype-phenotype correlations in DMD patients, the IRES is not active in the presence of exon 2 duplication but is active when exon 2 is deleted. We developed an AAV9.U7snRNA vector to that truncates DMD reading frame by skipping of exon 2, and have shown that in a Duchenne muscular dystrophy (DMD) mouse model carrying a duplication of exon 2 (the Dup2 mouse), postnatal intramuscular (IM) or intravascular (IV) treatment results in functional and pathologic improvement in skeletal muscle. Relevant to efforts to identify and treat DMD patients at an earlier age, we sought here to determine whether earlier gene transfer might slow down or even prevent the development of pathology. Dup2 mice were injected via facial vein at postnatal day 1 (P1) with 1E12 total vector genomes of the AAV9.U7snRNA vector and sacrificed at either 1, 3, 6, or 12 months post-injection for evaluation of exon 2 skipping by RT-PCR, quantification of dystrophin expression, and characterization of histopathology. To model the applicability of this approach beyond exon 2 duplication patients, the same vector was used to treat 6 human patient fibroblast-derived transdifferentiated myoblasts (FibroMyoD cells) harboring various mutations within exons 1 to 4. In the Dup2 mouse, efficient skipping and abundant dystrophin expression were present up to one year following the single AAV injection. Dystrophic pathology was absent at all-time points; at one year, less than 1% of fibers showed central nucleation, in comparison to ~70% in untreated Dup2 mice. Two tests on the ex vivo diaphragm preparations: isometric force (providing assessment of strength), and eccentric contractions (evaluating sarcolemma stability) were performed at 3 and 6 months following P1 injection. Both tests demonstrated little to no difference between treated animals and wild type mice. In all FibroMyoD cultures, abundant exon 2 skipping and dystrophin expression were detected in myotubes at 14 days of culture after treatment. These results suggest that this exon-skipping vector offers a therapeutic approach not only to patients with exon 2 duplications but with all mutations within the first four DMD exons (~6% of patients), an area of the gene largely ignored by the current therapeutic approaches. This work strongly supports the idea that early treatment of these patients will have longstanding and significant benefit resulting in a better outcome.

60. Intramuscular and Systemic Induction of the N-Truncated Dystrophin By Out-Of-Frame Exon 2 Skipping Restores Muscle Function in the Dup2 Mouse, Providing Further Support for a Therapeutic Pathway for 5’ DMD Mutations

Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead the severe Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of DMD result in mild dystrophinopathy with expression of a N-truncated dystrophin. We have recently shown that this is due to activation of an internal ribosome entry site (IRES) within exon 5 resulting in translation from an exon 6 AUG codon.We demonstrated that this IRES is active in patients expressing the N-truncated dystrophin, raising the possibility of the therapeutic use of this isoform. To explore this we developed a novel out-of-frame exon-skipping approach that uses AAV-mediated U7snRNA to efficiently skip exon 2. By injecting this AAV vector into a DMD mouse model carrying a duplication of exon 2 (Dup2), this generates a truncated reading frame, leading to activation of the IRES and synthesis of the N-truncated isoform.We now demonstrate that despite lacking the first half of the canonical actin binding domain 1, this N-truncated protein is highly functional. Intramuscular injection of the AAV1.U7snRNA vector into Dup2 mice results in high levels of expression of the N-truncated isoform by 4 to 6 weeks post-injection, along with complete correction of the physiologic and pathologic features as measured by Evans blue dye uptake, hindlimb grip strength, tibialis anterior specific force, and force correction after eccentric contraction. Preliminary results supports that systemic delivery of AAV9.U7snRNA vector into Dup2 mice induce expression of this functional isoform into all muscle including heart and diaphragm, thereby improving muscle histopathology.Following treatment, a genome-wide normalized RPF-Seq data analysis (Ribosome Protected Fragment) was performed to check if the treatment restored the Haslett gene lists (gene altered in DMD) to a ‘non-dystrophic’ pattern. Our data clearly indicates that the treatment restored the global expression pattern to a more normal pattern. This level of correction to that of control mice supports the idea that this novel therapeutic approach should be beneficial for the 6% of patients with mutations within the first five exons of DMD.

G.P.94

Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead the severe Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of DMD result in mild dystrophinopathy with expression of a N-truncated dystrophin. We have recently shown that this is due to activation of an internal ribosome entry site (IRES) within exon 5 resulting in translation from an exon 6 AUG codon. We demonstrated that this IRES is active in patients expressing the N-truncated dystrophin, raising the possibility of the therapeutic use of this isoform. To explore this we developed a novel out-of-frame exon-skipping approach that uses AAV-mediated U7snRNA to efficiently skip exon 2. By injecting this AAV vector into a DMD mouse model carrying a duplication of exon 2 (Dup2), this generates a truncated reading frame, leading to activation of the IRES and synthesis of the N-truncated isoform. We now demonstrate that despite lacking the first half of the canonical actin binding domain 1, this N-truncated protein is highly functional. Intramuscular injection of the AAV1. U7snRNA vector into Dup2 mice results in high levels of expression of the N-truncated isoform by 4 to 6weeks post-injection, along with complete correction of the physiologic and pathologic features as measured by Evans blue dye uptake, hindlimb grip strength, tibialis anterior specific force, and force correction after eccentric contraction. Notably, utrophin levels remain unchanged. This level of correction to that of control mice supports the idea that this novel therapeutic approach should be beneficial for the 6% of patients with mutations within the first five exons of DMD. The efficiency of this treatment in inducing expression of the N-truncated dystrophin in 6 patient cell lines with different 5 ′ mutations is underway, and will be presented as well.