No abstract

This study shows that tTTN-associated DCM is less severe at presentation and more amenable to standard therapy than LMNA mutation-induced DCM or iDCM.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes implicated in several dominant and recessive disease phenotypes. The canonical function of ARSs is to couple an amino acid to a cognate transfer RNA (tRNA). We identified three novel disease-associated missense mutations in the alanyl-tRNA synthetase (AARS) gene in three families with dominant axonal Charcot-Marie-Tooth (CMT) disease. Two mutations (p.Arg326Trp and p.Glu337Lys) are located near a recurrent pathologic change in AARS, p.Arg329His. The third (p.Ser627Leu) is in the editing domain of the protein in which hitherto only mutations associated with recessive encephalopathies have been described. Yeast complementation assays demonstrated that two mutations (p.Ser627Leu and p.Arg326Trp) represent loss-of-function alleles, while the third (p.Glu337Lys) represents a hypermorphic allele. Further, aminoacylation assays confirmed that the third mutation (p.Glu337Lys) increases tRNA charging velocity. To test the effect of each mutation in the context of a vertebrate nervous system, we developed a zebrafish assay. Remarkably, all three mutations caused a pathological phenotype of neural abnormalities when expressed in zebrafish, while expression of the human wild-type messenger RNA (mRNA) did not. Our data indicate that not only functional null or hypomorphic alleles, but also hypermorphic AARS alleles can cause dominantly inherited axonal CMT disease.

Longitudinal bone growth results from endochondral ossification, a process that requires proliferation and differentiation of chondrocytes. It has been shown that proper endochondral bone formation is critically dependent on the retinoblastoma family members p107 and p130. However, the precise functional roles played by individual E2F proteins remain poorly understood. Using both constitutive and conditional E2F1 transgenic mice, we show that ubiquitous transgene-driven expression of E2F1 during embryonic development results in a dwarf phenotype and significantly reduced postnatal viability. Overexpression of E2F1 disturbs chondrocyte maturation, resulting in delayed endochondral ossification, which is characterized by reduced hypertrophic zones and disorganized growth plates. Employing the chondrogenic cell line ATDC5, we investigated the effects of enforced E2F expression on the different phases of chondrocyte maturation that are normally required for endochondral ossification. Ectopic E2F1 expression strongly inhibits early-and late-phase differentiation of ATDC5 cells, accompanied by diminished cartilage nodule formation as well as decreased type II collagen, type X collagen, and aggrecan gene expression. In contrast, overexpression of E2F2 or E2F3a results in only a marginal delay of chondrocyte maturation, and increased E2F4 levels have no effect. These data are consistent with the notion that E2F1 is a regulator of chondrocyte differentiation.The vertebrate skeleton is formed by two distinct mechanisms of bone formation, intramembranous and endochondral ossification (23, 52). In the former, bone is generated directly from mesenchymal progenitor cells, whereas in the latter, a cartilage matrix precedes the formation of bone structures. The axial and appendicular skeletons develop through endochondral bone formation, while intramembranous ossification is mostly restricted to the skull and part of the clavicle. Endochondral ossification is especially important for longitudinal growth of the limbs, which requires the establishment of growth plates at the extreme ends of the long bones, and spatial and temporal control of chondrocyte differentiation (80). Growth plate chondrocytes form regular columns that sequentially and synchronously progress through proliferative, prehypertrophic, and terminally differentiating hypertrophic stages (13). Hypertrophic chondrocytes play a pivotal role in coordinating endochondral ossification, since they deposit the cartilage matrix that provides a template for invading osteoblasts to form trabecular bone at the site of the bone collar.The first step in the process of endochondral bone formation involves the chondrogenic differentiation of mesenchymal cells, and this is mainly regulated by transcription factors of the Sox family (Sox5, Sox6, and Sox9) (11, 67), which control the expression of cartilage-specific marker genes, including type II collagen [␣1 (II) collagen] and aggrecan genes (10,44,61). Sox Sox6 double-null mice develop severe chondrodysplasia characterized by t...