Abstract
restriction, maxillary hypoplasia, prominent heels and a variable incidence of skeletal abnormalities such as slender long bones and relatively tall vertebrae. Affected patients have a relatively poor growth response to growth hormone therapy. Nonsense and missense mutations in the genes encoding Obscurin-like 1 (OBSL1) and Cullin7 (CUL7) are known to cause 3M syndrome. There are, however some patients with well-defined 3-M syndrome but no mutation identified in either OBSL1 or CUL7. Aim: To assess cell proliferation, apoptosis and intracellular signal transduction responses to growth hormone (GH) and insulin-like growth factor-1 (IGF-I) in three 3-M patient cell lines: OBSL1 and CUL7 mutants and a cell line with no mutations in either gene. Methods: Dermal fibroblast cell lines were established from 2 unaffected children as controls and 3 patients, one with a nonsense mutation in CUL7 (c.4191delC p.H1379HfsX11), one with a nonsense mutation in OBSL1 (c.1273insAp.T425NfsX40) and one with 3-M syndrome but no identified mutation. Cell proliferation was assessed using a WST8 assay over 3 days and by measuring EdU incorporation at 24 hours. Apoptosis was measured using an ELISA for cleaved caspase 3 (CC-3), an essential enzyme for cell death. To assess GH and IGF-I signal transduction, control and patient fibroblasts were stimulated with GH (200ng/ml) and IGF-I (100ng/ml) with lysates taken at 0, 5, 15 and 60 minutes and immunoblotted for activation of ERK1/2, STAT5b, IRS-1 and Akt. Results: Both the WST8 assay and EdU incorporation showed basal cell proliferation to be decreased across all 3-M cell lines in comparison to controls (p < 0.01 for WST8 assay (OBSL1 mutant 65%, CUL7 mutant 64% and unknown mutant 78% compared to controls on day 3) and p < 0.01 for EdU incorporation (OBSL1mutant 43%, CUL7 mutant 24% and unknown mutant 25%)). There was no significant difference in the levels of CC-3 between patients and controls (p = 0.425, OBSL1 mutant 123%, CUL7 mutant 106% and unknown mutant 107% compared to controls). Following IGF-I stimulation, all three 3-M cell lines displayed normal IRS1 activation, but two showed reduced Akt activation when compared to controls (CUL7 mutant p < 0.01, unknownmutant p =0.024). Following GH stimulation, Stat5b and ERK1/2 activation was normal in OBSL1 and CUL7 mutant cell lines. Conclusions: 3-M syndrome is associated with reduced cell proliferation across all three cell lines but normal rates of apoptosis, even though each cell line carries a mutation in a different gene. However, only two of the three cell lines had reduced Akt activation in response to IGF-I stimulation. This suggests that disruption to different ‘upstream’ molecular pathways may lead into a common ‘downstream’ pathway that generates a consistent 3-M phenotype.
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