Previous results from Harvey et al have shown that there are associations between the methylation of two specific CpG loci within the CDKN2A gene at birth (CpGs -845 and -907) and offspring childhood bone outcomes at 4 and 6 years. These results showed that there is a significant negative association with both CpG -845 and -907 and offspring total bone area, total BMC and areal BMD at both 4 and 6 years of age in the SWS cohort (unpublished data).
However, as this work by Harvey et al compared methylation at birth with childhood bone outcomes, the associations between CDKN2A methylation in SWS umbilical cord tissue and offspring bone outcomes at birth was not determined. This will allow for direct comparisons to be made between the SWS cohort and the MAVIDOS samples, the latter only having DXA bone outcome data at birth available at present. Linear regression analysis explored the associations between methylation of each of the individual CpG sites in CDKN2A and offspring bone outcomes at birth in the SWS cohort revealed significant associations between the two after adjusting for sex, age at time of DXA and gestational age (Table 7.1).
There were significant negative associations between the methylation of CpGs -796, -845 and -907 and offspring total BMC at birth after adjusting for gestational age, sex and age at time of DXA (p<0.041 for all). The methylation of CpGs -852 and -887 showed a weak trend towards significance with total BMC, which was also in a negative direction (p<0.089 for both).
Linear regression analysis also determined significant associations between the methylation of CpGs -796 and -907 and offspring total BA at birth (p<0.03 for both), after adjusting for gestational age, sex and age at time of DXA. These associations were also in a negative direction. Trends towards significance were also seen between total BA and the methylation of CpGs -854, -852 and -887 (p<0.091 for all), which were also in a negative direction.
The methylation of CpG -845 was also significantly associated with total BMD at birth after adjusting for gestational age, sex and age at time of DXA (p=0.035), whilst a trend towards significance was seen between total BMD and the methylation of CpG -887 (p=0.083). Both of these associations were in a negative direction.
There were no significant associations between the methylation of any of the CpGs in the CDKN2A region of interest and scBMC. Figure 7.1 visually demonstrates some of the significant associations between specific CDKN2A CpG methylation in umbilical cord and offspring total BMC, total BA and total BMD at birth using scatter plots.
176 Baby DXA: Total BMC (g), adjusted for gestational age, sex
and age
Baby DXA: Total BA (cm sq), adjusted for gestational age, sex
and age n b p-value n b p-value CDKN2A CpG -769 292 -0.0001022 0.225 292 -0.1653897 0.232 CDKN2A CpG -793 290 -0.0001142 0.174 290 -0.1769149 0.198 CDKN2A CpG -796 246 -0.000229 0.035 246 -0.3848732 0.029 CDKN2A CpG -836 334 -0.0001204 0.114 334 -0.1980114 0.113 CDKN2A CpG -845 330 -0.0001644 0.040 330 -0.250933 0.055 CDKN2A CpG -852 330 -0.0001242 0.088 330 -0.2020511 0.090 CDKN2A CpG -861 321 -0.0001167 0.162 321 -0.1653978 0.227 CDKN2A CpG -887 312 -0.0001556 0.052 312 -0.2472543 0.059 CDKN2A CpG -907 266 -0.0002086 0.030 266 -0.3470719 0.028
Baby DXA: Total BMD (g/cm sq), adjusted for gestational age, sex
and age
Baby DXA: scBMC (g), adjusted for gestational age, sex and age
n b p-value n b p-value CDKN2A CpG -769 292 -0.0001437 0.315 291 -0.0000066 0.676 CDKN2A CpG -793 290 -0.0001968 0.169 289 -0.0000167 0.289 CDKN2A CpG -796 246 -0.0001885 0.309 245 -0.0000036 0.861 CDKN2A CpG -836 334 -0.0001583 0.223 333 -0.0000091 0.529 CDKN2A CpG -845 330 -0.0002875 0.035 329 -0.0000149 0.322 CDKN2A CpG -852 330 -0.000149 0.232 329 -0.0000066 0.634 CDKN2A CpG -861 321 -0.000209 0.151 320 -0.0000224 0.167 CDKN2A CpG -887 312 -0.0002383 0.083 311 -0.000008 0.599 CDKN2A CpG -907 266 -0.0002502 0.122 265 -0.0000045 0.787
Table 7.1: Associations between CDKN2A CpG methylation in umbilical cord tissue and offspring bone outcomes at birth in SWS cohort. There are significant negative associations between methylation of CpGs
within CDKN2A and total BMC, total BA and total BMD (p<0.05; highlighted in orange), determined by linear regression and adjusting for gestational age, sex and age at time of DXA. There are also some negative trends towards significance (p<0.1) between CpGs within the CDKN2A region of interest and bone outcomes at birth (p<0.1; highlighted in blue). ‘n’ is number of subjects and ‘b’ is beta value. Linear regression analysis carried out by Phil Titcombe.
177
Figure 7.1: The methylation of CpGs -796, -845 and -907 in CDKN2A is shown plotted against total bone mineral content (BMC), total bone area (BA) and total bone mineral density (BMD)at birth. As the
percentage methylation of each of these CpGs increases, total BMC, total BMD and total BA decreases at birth (adj. p<0.04). Linear regression analyses of methylation versus bone outcome produce a negative beta value, demonstrating a negative association between methylation of each of the CpGs and the bone outcome represented on the above graphs. p-values across all six graphs are adjusted for gestational age, sex and age at time of DXA in the linear regression model.
178 7.3.1.2 MAVIDOS umbilical cord
To see if the associations seen in the SWS cohort can be replicated, associations between methylation of CpGs within the CDKN2A locus and offspring bone outcomes at birth were investigated in the MAVIDOS cohort. In this cohort, the methylation of CpGs in CDKN2A in offspring umbilical cord was not significantly associated with total BMC, total BA, total BMD or size corrected BMC (scBMC) at birth in MAVIDOS samples when adjusted for sex, age at time of DXA and gestational age (Table 7.2). There were weak negative associations between the methylation of CpG -796, -887 and -907 and total BMC (p= 0.08, 0.083 and 0.099 respectively). CpG -796 also showed a weak negative association with total BMD (p<0.1) and CpG -887 a weak negative association with total BA (p=0.093). There were no associations between any of the CpGs and scBMC.
179
Baby DXA: Total BMC (g), adjusted for gestational age,
sex and age
Baby DXA: Total BA (cm sq), adjusted for gestational age,
sex and age
n b p-value n b p-value CDKN2A CpG -769 136 -0.074 0.376 136 -0.008 0.975 CDKN2A CpG -793 119 -0.127 0.142 119 -0.364 0.195 CDKN2A CpG -796 106 -0.304 0.08 106 -0.589 0.289 CDKN2A CpG -836 147 -0.081 0.325 147 -0.116 0.669 CDKN2A CpG -845 144 -0.046 0.633 144 0.000248 0.999 CDKN2A CpG -852 142 -0.04 0.657 142 -0.024 0.935 CDKN2A CpG -861 137 -0.017 0.87 137 -0.056 0.867 CDKN2A CpG -887 146 -0.159 0.083 146 -0.505 0.093 CDKN2A CpG -907 144 -0.155 0.099 144 -0.414 0.181
Baby DXA: Total BMD (g/ cm sq), adjusted for gestational
age, sex and age
Baby DXA: scBMC (g), adjusted for gestational age,
sex and age
n b p-value n b p-value CDKN2A CpG -769 136 -0.000235 0.13 136 -0.025 0.57 CDKN2A CpG -793 119 -0.000175 0.272 119 0.008 0.857 CDKN2A CpG -796 106 -0.001 0.099 106 -0.095 0.294 CDKN2A CpG -836 147 -0.000195 0.204 147 -0.002 0.963 CDKN2A CpG -845 144 -1.38E-04 0.436 144 0.017 0.728 CDKN2A CpG -852 142 -1.12E-04 0.504 142 0.029 0.53 CDKN2A CpG -861 137 -4.09E-06 0.983 137 0.036 0.479 CDKN2A CpG -887 146 -0.000188 0.27 146 0.041 0.368 CDKN2A CpG -907 144 -0.000229 0.189 144 0.027 0.574
Table 7.2: The association between methylation of CDKN2A in umbilical cord DNA and bone outcomes at birth in MAVIDOS cohort. There are no significant associations between methylation of any of the CpGs
within the CDKN2A region of interest and bone outcomes at birth after correcting for gestational age, sex and age at time of DXA. There are some trends towards significance (p=0.051-0.1) in a negative direction between CpGs -796, -887 and -907 and total BMC, CpG -887 and total BA and CpG -796 and total BMD. ‘n’ denotes number of subjects and ‘b’ denotes beta value. Associations determined by linear regression model.
180
7.3.2 Is the methylation of RXRA associated with offspring bone outcomes at birth?