Hypermethylation-associated downregulation of microRNA-4456 in hypersexual disorder with putative influence on oxytocin signalling: A DNA methylation analysis of miRNA genes

Figures & data

Table 1. Clinical characteristics of patients with hypersexual disorder and healthy volunteers.

	Patients	Healthy volunteers	Statistics (t-test, Kruskall-Wallis, Chisq.test), p value
N	60	33
Age (years)	39.4 (11.9)	37.4 (11.3)	ns
Men:Women, (n (%))	54 (90.0): 6 (10.0)	33 (100.0): 0 (0.0)	ns
Scandinavian descent (n(%))*	43 (71.7)	23 (69.7)	ns
Diagnosis depression (n(%))	9 (0.15)	0 (0.0)	4.83E-02
DST non-suppressors (n(%))	16 (26.7)	3 (9.1)	5.78E-02
CTQ Total	40.42 (11.89)	32.85 (9.39)	2.22E-04
TSH (mE/L)/T4 (nmol/L)	0.019 (0.0098)	0.027 (0.033)	ns
HBA1C (mml/mol)	32.97 (5.70)	32.82 (3.96)	ns
Cortisol (nmol/L)	467.85 (132.33)	474.49 (148.32)	ns
DST Cortisol (nmol/L)	100.4 (101.3)	62.54 (48.54)	ns
ACTH (pmol/L)	6.36 (3.05)	5.82 (2.99)	ns
DST ACTH (pmol/L)	2.11 (1.58)	1.26 (0.90)	8.10E-03
Testosteron (nmol/L)	14.02 (5.86)	14.24 (4.37)	ns
TNF-alpha (ng/L)	7.26 (1.83)	5.84 (2.41)	4.39E-06

Values are shown as mean (SD) unless otherwise specified. P-values were calculated by means of unpaired t-tests, Kruskall-Wallis’ test or chi-squared tests, contrasting values for patients with hypersexuality disorder and healthy volunteers. A one-tailed p-value <0.05 was considered significant.

*As a proxy for ethnicity, subjects born in a Scandinavian country (e.g., Denmark, Finland, Iceland, Norway or Sweden) and with biological parents born in the same country, were stratified as being of ‘Scandinavian descent’.

Abbreviations: CTQ, childhood trauma questionnaire; DST, dexamethasone suppression test; ACTH, ACTH levels after the dexamethasone suppression test; DST Cortisol, cortisol levels after the dexamethasone suppression test; DST non-suppressors, non-suppression status defined as DST cortisol levels >138 nmol/l

Table 2. Hypersexuality associated methylation changes in miRNA-associated CpG sites.

			% DNA Methylation (SD)
Gene	Transcript	Illumina ID	Patients	Healthy Volunteers	logFC	ES	SE	p	p (FDR)	pBC	pBC (FDR)
MIR708	NR_030598	cg18222192	50.1 (3.0)	51.5 (3.7)	−0.11	−0.11	2.35E-02	7.04E-06	4.14E-02	1.31E-05	5.81E-02
MIR4456	NR_039661	cg01299774	51.3 (3.4)	48.7 (4.2)	0.12	0.12	2.64E-02	9.36E-06	4.14E-02	8.97E-06	5.81E-02
MIR3141	NR_036094	cg05945665	65.3 (1.6)	66.1 (2.3)	−0.09	0.08	2.01E-02	3.75E-05	8.13E-02	6.72E-05	1.17E-01
Mir_584	.	cg27571590	73.3 (1.2)	74.0 (1.8)	−0.06	0.06	1.53E-02	4.78E-05	8.13E-02	3.66E-04	2.70E-01
MIR4710	NR_039860	cg16761754	38.5 (9.1)	34.0 (12.7)	0.35	−0.09	1.97E-02	4.88E-05	8.13E-02	7.73E-05	1.17E-01
MIR4278	NR_036242	cg08313382	58.5 (1.8)	57.6 (2.7)	0.08	0.09	2.27E-02	5.99E-05	8.13E-02	1.19E-04	1.17E-01
MIR345	NR_029906	cg02540736	53.2 (1.4)	54.1 (2.5)	−0.06	−0.07	2.08E-02	6.43E-05	8.13E-02	1.42E-03	4.69E-01
MIR4281	NR_036239	cg20960405	23.6 (1.9)	23.0 (2.8)	0.09	0.09	2.30E-02	9.29E-05	8.28E-02	1.07E-04	1.17E-01
Mir_1302	.	cg08241360	59.0 (4.1)	57.3 (6.5)	0.12	−0.03	1.11E-02	9.92E-05	8.28E-02	1.06E-02	6.71E-01
MIR4441	NR_039643	cg20871441	73.5 (1.0)	73.8 (1.0)	−0.03	0.04	1.19E-02	1.01E-04	8.28E-02	1.85E-03	4.69E-01

Analysis by multiple linear regression models of methylation M-values to a binary outcome variable of Hypersexuality (Y/N), adjusting for Depression (Y/N), dexamethasone suppresser (Y/N), CTQ total score and TNF-alpha (ng/L). 8,852 miRNA associated CpG-sites were analysed. Standard errors were calculated manually by multiplying the square root of the posterior values for sigma^2 with the standard deviation. P-values were corrected for multiple testing using the false discovery rate (FDR)-method. Correction for inflation and estimated bias was made using the package ‘Bacon’ for R statistics.

Abbreviations: logFC, log fold change of M-values; % DNA Methylation (SD), mean beta value percent DNA methylation (standard deviation); ES, effect size; SE, standard error; p, p-value; p (FDR), FDR-adjusted p-value; p_BC, p-values after bacon adjustment for inflation and bias; p_BC (FDR), FDR-adjusted p-values after bacon adjustment for inflation and bias.

Table 3. Methylation/transcription correlations in HD patients and healthy volunteers of candidate CpG sites and MIR708 and MIR4456.

	MIR708				MIR4456
	Coef.	Std. Error	t value	p.val	Coef.	Std. Error	t value	p.val
Intercept	0.64	0.08	7.62	1.82E-10	0.79	0.06	13.5	< 2e-16
cg18222192^a/cg01299774^b	0.63	0.40	1.56	0.13	−0.72	0.25	−2.84	5.76E-03
Disease state^c	−0.13	0.10	−1.40	0.17	−0.29	0.07	−3.94	1.78E-04
Interaction term^d	−0.06	0.47	−0.12	0.90	0.90	0.33	2.75	7.37E-03

Multiple linear regressions whereby the expression of each miRNA was correlated to M values of the CpG site intraindividually, adjusting for a categorical variable of disease state and an interaction term between methylation and disease state. miRNA expression levels were treated as dependent variable and DNA methylation as independent variable.

^acg18222192 in the case of MIR708

^bcg01299774 in the case of MIR4456

^cA categorical variable of disease state (hypersexuality disorder or healthy volunteer)

^dInteraction term between CpG site methylation and a categorical variable of disease state

Abbreviations: coef., regression coefficient; p, p-value

Table 4. Gene set overrepresentation analysis of MIR4456 putative binding targets – KEGG defined pathways.

KEGG Pathway (Homo sapiens)	Set size	Count	%	p-value	q-value	source
Oxytocin signaling pathway	159	24	15.2%	7.29E-06	1.82E-04	KEGG
cAMP signaling pathway	200	26	13.0%	5.12E-05	4.14E-04	KEGG
Glutamatergic synapse	114	18	15.8%	5.92E-05	4.14E-04	KEGG
Circadian entrainment	96	16	16.7%	7.76E-05	4.14E-04	KEGG
Adrenergic signaling in cardiomyocytes	149	21	14.1%	8.28E-05	4.14E-04	KEGG

The online web tool ComIR’ was used to computationally predict putative gene targets of MIR4456. Using the online web tool ‘ConsensusPathDB-human’, 1142 identified genes were investigated to see if there was a statistically significant abundance of genes involved in specific KEGG-defined pathways. We specified a ‘minimum overlap with input list’ of 15 genes and a stringent p-value cut-off of 0.0001.

Abbreviations: Count, number of candidate genes in a particular pathway; %, percentage of candidate genes in a particular pathway

Figure 1. Boxplot diagram of MIR4456 normalized expression values in HD and healthy volunteers.