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.peer-reviewe
