Infantile cortical hyperostosis (Caffey disease) is characterized by spontaneous episodes of subperiosteal new bone formation along 1 or more bones commencing within the first 5 months of life. A genome-wide screen for genetic linkage in a large family with an autosomal dominant form of Caffey disease (ADC) revealed a locus on chromosome 17q21 (LOD score, 6.78). Affected individuals and obligate carriers were heterozygous for a missense mutation (3040C→T) in exon 41 of the gene encoding the α1(I) chain of type I collagen (COL1A1), altering residue 836 (R836C) in the triple-helical domain of this chain. The same mutation was identified in affected members of 2 unrelated, smaller families with ADC, but not in 2 prenatal cases and not in more than 300 chromosomes from healthy individuals. Fibroblast cultures from an affected individual produced abnormal disulfide-bonded dimeric α1(I) chains. Dermal collagen fibrils of the same individual were larger, more variable in shape and size, and less densely packed than those in control samples. Individuals bearing the mutation, whether they had experienced an episode of cortical hyperostosis or not, had joint hyperlaxity, hyperextensible skin, and inguinal hernias resembling symptoms of a mild form of Ehlers-Danlos syndrome type III. These findings extend the spectrum of COL1A1-related diseases to include a hyperostotic disorder.
The bromodomain and extraterminal (BET) family of epigenetic reader proteins are key regulators of pathologic gene expression in the heart. Using mice carrying a human mutation in phospholamban (PLN R9C ) that develop progressive dilated cardiomyopathy (DCM), we previously identified the activation of inflammatory gene networks as a key early driver of DCM. We reasoned that BETs control this inflammatory process, representing a key node in the progression of genetic DCM. Using a chemical genetic strategy, PLN R9C or age-matched wild type mice were treated longitudinally with the BET inhibitor JQ1 or vehicle. JQ1 abrogated DCM, reduced cardiac fibrosis, and prolonged survival in PLN R9C mice by inhibiting inflammatory gene network expression at all disease stages. Cardiac fibroblast proliferation was also substantially reduced by JQ1. Interestingly, JQ1 had profound effects on pathologic gene network expression in cardiac fibroblasts, while having little effect on transcription in cardiomyocytes. Using co-immunoprecipitation, we identified BRD4 as a direct and essential regulator of NFB-mediated inflammatory gene transcription in cardiac fibroblasts. In this this model of chronic, heritable DCM, BETs activate inflammatory gene networks in cardiac fibroblasts via an NFB-dependent mechanism, marking them as critical effectors of pathologic gene expression.
Dilated cardiomyopathy (DCM) is a leading cause for heart failure and is associated with a rate of mortality of 20% within 5 years of diagnosis. The most common genetic causes for DCM are mutations of the sarcomere protein titin (encoded by
TTN
), which occurs in 10-20% of DCM cases. Dominant DCM mutations truncate titin (TTNtv) and result in haploinsufficiency. Thus, strategies to increase the expression of the wild type
TTN
allele could attenuate damaging effects of TTNtv. Utilizing bioinformatic tools, we identified a putative enhancer for
TTN
in its intron 1. We deleted a 658 bp region from intron 1 which encompasses the region of interest in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome editing to validate its function. Utilizing RNA sequencing and qPCR of RNA harvested from hiPSC-derived cardiomyocytes (hiPSC-CMs), we demonstrated that a homozygous deletion in this region leads to a drop in
TTN
expression compared to the wild type (WT) control (0.344-fold change, p < 0.001). To further characterize this region, we subdivided it into three parts which we called E1 (296 bp), E2 (206 bp), and E3 (139 bp). E1 includes a highly conserved region and a region of open chromatin as identified by the Assay for Transposase-Accessible Chromatin Sequencing (ATAC-Seq) performed on hiPSC-CMs. A homozygous E1 deletion resulted in a decreased
TTN
expression of 0.63-fold compared to the WT control (p < 0.001) when performing RNA sequencing on hiPSC-CMs. Both homozygous E2 and E3 deletions resulted in an increased
TTN
expression (1.56-fold change, p < 0.001; 1.19 fold change, p < 0.001). Utilizing a published sarcomere tracking platform, SarcTrack, to investigate hiPSC-CM physiology, we saw a decreased contractility of 6.6% in hiPSC-CMs carrying a homozygous E1 deletion compared to 10.1% in the WT control (p < 0.001). Cells carrying homozygous E2 or E3 deletions were hypercontractile (13.8%, p < 0.001; 13.7%, p < 0.001). Given our results, we hypothesize that
TTN
expression depends on the E1 region. If confirmed, we expect that increasing the activity of this enhancer using small molecules may provide a novel therapeutic target for DCM caused by TTNtv.
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