Effect of an intronic variant within Zinc finger protein 384 gene on pre-mRNA splicing in a Maltese family with osteoporosis

Abstract

INTRODUCTION: Osteoporosis is a skeletal disease with a strong genetic basis. A study on an extended Maltese family with a highly penetrant form of osteoporosis, revealed the presence of the rs146089604 variant (c.686+32G>A) in intron 7 of the Zinc finger protein 384 (ZNF384) gene, predicted to affect pre-messenger RNA (mRNA) splicing. The aim of this study was to assess the functional effect of the variant using an exon-trapping vector transfected in three human cell types.

METHODS: The target DNA region harbouring G or A allele was inserted in the p.SPL3 vector, creating mini-gene constructs that were transfected in human kidney-derived cells (HEK-293) and two human osteoblasts-derived cells (SaOS-2 and h-FOB). Extracted mRNA was converted into complementary DNA (cDNA), amplified by PCR and sequenced to determine the transcript size and identify any splicing variants.

RESULTS: Mini-gene construct with the alternative A allele lead to exon 8 and part of intron 8 to be retained, both of which were spliced off in the presence of the G allele. These results were observed for constructs transfected in the osteoblasts-derived cell lines. In HEK-293 cells, no difference in transcript size was seen for the G or A allele, suggesting different splicing mechanisms.

CONCLUSION: Observations may indicate that the ZNF384 rs146089604 could be a causal variant contributing to osteoporosis. ZNF384 transactivates type I collagen and matrix metalloproteinases, and suppresses bone morphogenic protein (BMP) and Wnt signalling resulting in reduced bone volume and strength. Thus, impaired ZNF384 splicing could alter the protein’s function affecting bone homeostasis.