Genetics of Digitocutaneous Dysplasia

The development of the human skeleton is a precisely controlled process. The study of Mendelian diseases involving abnormalities in the skeleton enables the idenfication of factors critical to skeletal development. Digitocutaneous dysplasia (DCD) is a rare genetic syndrome exhibiting abnormalities in the skeleton that are most pronounced in the hands and feet. This condition is caused by a single mis-splicing event that results in the in-frame deletion of 16 amino acids from the encoded filamin A protein (FLNA; NP_001104026.1:p.Val1724_Thr1739del). Tellingly, this region of FLNA has been shown to interact with the extracellular calcium-sensing receptor (CaSR). Activation of CaSR initiates downstream mitogen-activated protein kinase (MAPK) signaling that can trigger osteoblast recruitment and differentiation. This project aimed to explore the pathomechanisms that lead to the skeletal abnormalities in DCD. This was achieved using two approaches, one examining the effects of this FLNA variant on osteoblast function, and the other investigating the possible involvement of CaSR. Two immortalised cell lines, MC3T3-E1 and U2OS, were chosen to model osteoblasts. Given the murine origin of MC3T3-E1, the nucleotide sequence of mouse Flna was compared with that of human FLNA. It was discovered that rodents had what was equivalent to the pathogenic variant responsible for the mis-splicing of FLNA in humans. However, no mis-splicing was found in the mouse following the investigation of a publicly available mouse development RNA-seq dataset. Disrupting the splice site of interest in MC3T3-E1 using antisense oligonucleotides resulted in a mis-splicing event different to that associated with DCD. Taken together, these findings demonstrate that the regulation of splicing at this site differ between mice and humans. Antisense oligonucleotides were also transfected into human U2OS cells but no mis-splicing was induced. Subsequently, a transient transfection approach to introduce the misspliced form of FLNA via a plasmid vector was initiated. A satisfactory transfection efficiency was unable to be achieved and consequently, no clear results were obtained. Meanwhile, the expression of CASR was investigated using reverse transcription PCR. CASR was detected in mouse bone but not in two osteoblastic cell lines, MC3T3-E1 and U2OS. The apparent absence of CASR in these cell lines was thought to be due to this method lacking the sensitivity to detect very the low expression of this gene common in in vitro cultures. The experiments utilising antisense oligonucleotides along with the exploration of interspecific differences in the nucleotide sequence of FLNA has led to the uncovering of putative factors that influence mis-splicing at this site. The finding of Casr expression in bone has called attention to the possible role that osteocytes may have in the pathogenesis of DCD. Overall, this project has laid a foundation for further work and opened up some new avenues that can be explored in the future to better understand the various aspects of this disease