Blanco-Sanchez, B. | Search bioRxiv

bioRxiv2020

COPB2 haploinsufficiency causes a coatopathy with osteoporosis and developmental delay

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures and developmental delay of variable severity. Because the role of COPB2 in bone has not been characterized, we studied the effect of COPB2 deficiency on skeletal development in mice and zebrafish. Copb2+/- mice showed low bone mass and decreased bone strength. In zebrafish, larvae carrying a copb2 heterozygous frameshift variant showed delayed mineralization. copb2-null embryos showed endoplasmic reticulum (ER) and Golgi disorganization, and embryonic lethality. COPB2 siRNA-treated fibroblasts showed delayed collagen trafficking with retention of type I collagen in the ER and Golgi, and altered distribution of Golgi markers. Our data suggest that COPB2 haploinsufficiency leads to disruption of intracellular collagen trafficking and osteoporosis, which may improve with ascorbic acid supplementation. This work highlights the role of COPI complex as a critical regulator of bone mass and identifies a new form of coatopathy due to COPB2 deficiency.

genetics↗

bioRxiv2019

Transgene-mediated skeletal phenotypic variation in zebrafish

When considering relationships between genotype and phenotype we frequently ignore the fact that the genome of a typical animal, notably including that of a fish and a human, harbors a huge amount of foreign DNA. Some of it, including the DNA of \"autonomous\" transposable elements, can spontaneously mobilize to occupy new chromosomal sites and take on new functions, presenting a challenge to the host organism and also possibly introducing new fuel for evolutionary change. Transposable elements are useful for introducing transgenes, integrating them into host genomes with high efficiency. Transgenesis has become very widespread in biological research, and in our society at large. This year the governments of both Canada and the United States have approved the first use of genetically engineered animals in food production, Atlantic salmon, Salmo salar. With the recent advent of amazing gene-editing technology, there is no doubt that the transgene industry will grow explosively in the coming years. The biology of transgenes needs to be included in our understanding of the genome. It is in this spirit that we have investigated an unexpected and novel phenotypic effect of the chromosomally integrated transgene fli1a-F-hsp70l:Gal4VP16. We examine larval fras1 mutant zebrafish (Danio rerio). Gal4VP16 is a potent transcriptional activator, and already well known for toxicity and mediating unusual transcriptional effects. In the presence of the transgene, phenotypes in the neural crest-derived craniofacial skeleton, notably fusions and shape changes associated with loss of function fras1 mutations, are made more severe, as we quantify by scoring phenotypic penetrance, the fraction of mutants expressing the trait. A very interesting feature is that the enhancements are highly specific for fras1 mutant phenotypes - occurring in the apparent absence of more wide-spread changes. Except for the features due to the fras1 mutation, the transgene-bearing larvae appear generally healthy and to be developing normally. The transgene behaves as a genetic partial dominant: A single copy is sufficient for the enhancements, yet, for some traits, two copies may exert a stronger effect. We made new strains bearing independent insertions of the fli1a-F-hsp70l:Gal4VP16 transgene in new locations in the genome, and observed increased severities of the same phenotypes as observed for the original insertion. This finding suggests that sequences within the transgene, e.g. Gal4VP16, are responsible for the enhancements, rather than effect on neighboring host sequences (such as an insertional mutation). The specificity, and biological action underlying the traits, are subjects of considerable interest for further investigation, as we discuss. Our findings show that work with transgenes needs to be undertaken with caution and attention to detail.

developmental biology↗