DNA methylation analysis of Homeobox genes implicates HOXB7 hypomethylation as risk factor for neural tube defects

Figures & data

Table 1 Background information of MMC patients included in the HumanMethylation450 BeadChip and Sequenom EpiTYPER

									MMC patient		Sibling
MMC patient	Type MMC	Hy/VP	ACM	Scoliosis	ADL	UI	Ethnicity	Maternal age (years) at birth of MMC patient	MTHFR 677C>T	Gender	MTHFR 677C>T	Gender	MMC patient versus sibling age (months)
1*^+	S	yes	1	yes	2	yes	Belgian	36	CC	F
2*^+	LS	yes	2	yes	3	yes	Belgian	29	CT	F
3*^+	LS	yes	2	yes	3	yes	Moroccan	32	CT	M
4*^+	LS	yes	1	yes	3	yes	Indonesian	26	CC	F
5*^+	LS	yes	2	no	2	yes	Belgian		CC	M	CC	F	−18
6*^+	LS	yes	2	yes	3	yes	Belgian	27	CC	F
7*^+	LS	yes	1	yes	3	yes	Belgian		CC	M
8*^+	S	yes	2	no	1	yes	Belgian	27	CC	F
9*	S	no	0	no	1	yes	Belgian		TT	M
10*	LS	yes	2	no	2	yes	Belgian	25	CC	M
11^+	LS	yes	2	yes	3	yes	Belgian	27	CC	M
12^+	LS	yes	2	yes	3	yes	Turkish	28	CC	F
13^+	S	yes	0	no	1	yes	Belgian	33	CT	M
14^+	LS	yes	2	yes	3	yes	Belgian		CC	F
15^+	LS	yes	2	yes	3	yes	Turkish	20	CT	F
16^+	S	yes	2	yes	3	yes	Belgian		TT	M
17^+	LS	yes	2	yes	3	yes	Belgian	25	CT	F
18^+	S	yes	2	yes	2	yes	Belgian	28	CC	M
19^+	LS	yes	2	no	1	yes	Belgian	36	CT	M
20^+	LS	yes	2	yes	1	yes	Belgian		CT	M
21^+	LS	yes	2	yes	3	yes	Belgian	36	CT	F
22^+	TL & LS	yes	2	yes	3	yes	Belgian	23	CC	M
23^+	S	no	0	yes	1	yes	Belgian	30	CT	M
24^+	LS	yes	2	yes	2	yes	Belgian	25	CT	F
25^+	LS	yes	2	no	2	yes	Belgian	34	CC	F
26^+	LS	no	0	no	1	yes	Belgian		CC	F
27^+	LS	yes	2	yes	3	yes	Belgian		CT	M	CC	F	−20
28^+	LS	yes	2	yes	3	yes	Moroccan		CT	F
29^+	LS	yes	2	yes	3	yes	Belgian	31	CC	M	CC	M	−19
30^+	LS	yes	0	no	1	yes	Mongolian		CC	F
31^+	LS	no	0	no	1	no	Belgian	20	TT	M	CC	M	−20
32^+	LS	yes	2	no	1	yes	Belgian	22	CC	M
33^+	LS	yes	2	yes	2	yes	Belgian	28	CT	M
34^+	LS	yes	2	yes	3	yes	Belgian		CT	F
35^+	S	yes	2	no	1	yes	Belgian	33	CT	M
36^+	LS	yes	2	no	3	yes	Belgian	27	CC	M
37^+	LS	yes	NA	yes	2	yes	Belgian	26	CT	F
38^+	LS	yes	2	yes	2	yes	Belgian		CT	F
39^+	LS	yes	2	yes	3	yes	Belgian	28	CT	F
40^+	LS	yes	2	no	1	yes	Belgian	32	CC	M
41^+	LS	no	NA	no	3	yes	Belgian	36	CC	M	CT	M	−26
42^+	L	yes	2	yes	3	yes	Ukrainian	20	CC	F
43^+	LS	yes	2	no	1	yes	Belgian	25	CC	F	TT	M	20
44^+	LS	yes	2	yes	3	yes	Belgian		CC	M
45^+	LS	yes	2	yes	3	yes	Belgian	29	CT	F
46^+	LS	yes	2	yes	3	yes	Belgian		CC	F
47^+	S	yes	2	no	1	yes	Moroccan	31	CT	F
48^+	LS	yes	2	yes	2	yes	Belgian	27	CT	F
49^+	S	yes	2	yes	3	yes	Belgian	34	CC	F
50^+	S	no	NA	no	1	yes	Belgian	36	CT	F	CC	F	17
51^+	LS	yes	2	no	3	yes	Belgian	30	CC	F
52^+	LS	yes	2	no	1	yes	Moroccan	24	CC	F
53^+	S	yes	2	no	1	yes	Belgian	28	CC	F
54^+	S	no	NA	no	1	yes	Belgian		CT	F
55^+	LS	yes	2	no	2	yes	Bosnian	31	CT	F	CC	M	53
56^+	S	no	0	no	1	yes	Belgian	30	CC	F
57^+	LS	yes	2	no	1	yes	Belgian	24	CC	F
58^+	S	no	2	no		yes	Moroccan	26	CC	F
59^+	LS	yes	2	yes	3	yes	Belgian	27	CC	F
60^+	LS	yes	2	no	1	yes	Turkish	35	CT	F	CC	F	−101
61^+	LS	yes	2	yes	3	yes	Belgian		CT	F
62^+	LS	yes	2	no	1	yes	Turkish	29	CT	F
63^+	LS	yes	2	yes	3	yes	Belgian	35	CT	F
64^+	L & CP	yes	2	no	2	yes	Belgian	40	CT	F	CT	F	−27
65^+	LS	yes	2	yes	2	yes	Belgian		CT	F
66^+	LS	yes	2	yes	2	yes	Belgian	24	CT	F
67^+	LS	yes	2	yes	2	yes	Belgian		CT	F
68^+	S	yes	2	no	1	no	Belgian		CT	M
69^+	LS	yes	2	yes	3	yes	Belgian	24	CT	M
70^+	LS	yes	2	yes	3	yes	Belgian	29	CC	M
71^+	LS	yes	2	yes	3	yes	Belgian	33	CC	F
72^+	LS	yes	2	yes	2	yes	Belgian	29	CC	M
73^+	LS	yes	2	yes	3	yes	Belgian	29	CT	F	CT	F	−24
74^+	LS	yes	2	yes	2	yes	Belgian		CT	M
75^+	LS	yes	2	no	1	yes	Belgian		CC	F
76^+	LS	yes	2	yes	2	yes	Belgian	31	CT	F
77^+	LS	yes	2	yes	3	yes	Belgian	26	CC	M
78^+	LS	yes	2	yes	3	yes	Belgian	30	CT	M
79^+	S	no	0	no	1	yes	Belgian		CT	M
80^+	LS	yes	2	no	1	yes	Belgian	30	CC	F
81^+	LS	yes	NA	no	2	yes	Turkish		CT	M
82^+	TL	yes	2	yes	3	yes	Belgian		TT	F
83^+	TL	yes	2	yes	1	yes	Belgian	24	TT	F
84^+	TL	yes	1	yes	3	yes	Belgian	37	CC	F
85^+	TL	yes	2	no	1	no	Belgian		TT	F

* Inclusion in HumanMethylation 450K BeadChip (10 MMC patients) and ⁺Sequenom EpiTYPER (83 MMC patients); MMC: myelomeningocele; M: male; F: female; S: Sacral; LS: Lumbosacral; TL: Thoracolumbal; CP: Cheilopalatoschisis; Hy/VP: presence of hydrocephaly and ventriculoperitoneal drain; ACM: Arnold Chiari Malformation: 1 = type 1, 2 = type 2; NA: Not Available; ADL: Activities of daily life: 1 = ambulatory, 2 = household ambulatory with wheelchair for longer distances, 3 = wheelchair dependent; UI: urinary incontinence; MTHFR 677C>T genotype (CC/CT/TT) in 85 MMC patients: 47% CC - 53% CT/TT vs. 40% CC - 60% CT/TT in 30 healthy unrelated controls (P-value = ns).

Table 2 Methylation of the HOX genes using the HumanMethylation450 BeadChip and analysis

						Ctrls (n = 6)		MMC (n = 10)
Cluster	Gene	Chr	Illumina ID	P-value <0.05	β-diff>0.05	Mean	SD	Mean	SD
A: Chr.7p14	HOXA2	7	cg06055873	0.016	−0.06	0.32	0.03	0.26	0.04
	HOXA2	7	cg19432993	0.031	−0.12	0.69	0.05	0.57	0.12
	HOXA2	7	cg06166490	0.016	−0.12	0.70	0.06	0.58	0.11
	HOXA2	7	cg04027736	0.022	−0.11	0.61	0.06	0.50	0.11
	HOXA2	7	cg00445443	0.042	−0.10	0.41	0.08	0.31	0.11
		7	cg15037137	0.007	−0.05	0.85	0.02	0.80	0.07
	HOXA4	7	cg25952581	0.042	−0.09	0.45	0.06	0.37	0.11
	HOXA4	7	cg17591595	0.042	−0.08	0.73	0.05	0.65	0.09
	HOXA11	7	cg24709033	0.011	−0.06	0.27	0.03	0.21	0.04
B: Chr.17q21	HOXB5	17	cg01405107	0.042	0.09	0.49	0.05	0.58	0.08
	HOXB6	17	cg09983216	0.016	−0.13	0.55	0.08	0.42	0.10
	HOXB7	17	cg11041817	0.007	−0.29	0.70	0.08	0.41	0.20
	HOXB7	17	cg22622477	0.007	−0.16	0.36	0.06	0.20	0.09
	HOXB7	17	cg07547765	0.007	−0.26	0.71	0.10	0.44	0.18
		17	cg19051015	0.022	−0.09	0.72	0.05	0.63	0.07
	HOXB9	17	cg15117739	0.007	−0.10	0.68	0.04	0.57	0.07
	HOXB9	17	cg12057127	0.042	−0.06	0.72	0.01	0.67	0.07
		17	cg20454400	0.031	−0.06	0.37	0.04	0.31	0.05
		17	cg16654603	0.011	−0.09	0.67	0.02	0.58	0.08
		17	cg02052915	0.016	−0.05	0.36	0.03	0.31	0.04
C: Chr.12q13		12	cg08299265	0.016	−0.06	0.39	0.04	0.33	0.03
		12	cg26643142	0.031	−0.06	0.35	0.03	0.30	0.05
	HOXC4	12	cg18473521	0.011	−0.12	0.43	0.08	0.31	0.07
D: Chr.2q31	HOXD9	2	cg04730882	0.005	−0.07	0.35	0.03	0.28	0.05
		2	cg07783843	0.011	−0.06	0.24	0.02	0.18	0.04
		2	cg05525812	0.007	−0.07	0.25	0.02	0.18	0.05

Nucleotide positions in accord to NCBI build 37/hg19. Selection is performed along both β-value > 0.05 difference and P-value < 0.05; calculated with Wilcoxon Rank-Sum test. The 3 probes in bold are located within the same CpG island at Chr17:46,685,244–46,685,449 within the HOXB7 gene body. This region was selected for the validation study using Sequenom EpiTYPER. β-diff: β-difference; Chr: chromosome; Ctrls: controls; MMC: myelomeningocele.

Figure 1. HOXB7 methylation studies by Sequenom EpiTYPER in MMC patients. (A) Localization of the studied amplicon (Chr17:46,685,144–46,685,550) within HOXB7 Exon 2. The amplicon covers 26 single CpGs and our assay provides data on 10 analytical CpG units. Nucleotide positions accord to the NCBI build 37/hg19. The CpG units studied by 450K Array (cg11041817, cg22622477 and cg07547765) and the in silico analysis (cg06493080, cg09357097) are also indicated. (B): Boxplot representing the methylation pattern of MMC patients and controls with box = 25th and 75th percentiles; bars = min and max values. The mean methylation level of each group is shown below the plot. (C): Methylation pattern for each CpG unit within the amplicon. Wilcoxon Rank-Sum test was performed. (D): Boxplot representing the methylation pattern of MMC patients and controls divided according to MTHFR 677C>T genotype with box = 25th and 75th percentiles; bars = min and max values. The mean methylation level of each group is shown below the plot.

Figure 2. HOXB7 methylation studies by Sequenom EpiTYPER in pairs of unaffected siblings vs. MMC patients. (A) Boxplot representing the methylation pattern of affected siblings and unaffected siblings with box = 25th and 75th percentiles; bars = min and max values. The mean methylation level of each group is shown below the plot. (B) Methylation pattern for each CpG unit within the amplicon. Wilcoxon Rank-Sum test was performed.

Figure 3. Phenotype analysis of Hoxb7a-overexpression in zebrafish embryos. (A) Phenotype analysis at 72 hpf of Hoxb7a mRNA injected zebrafish resulted in significant hypopigmentation and malformation in 66% of the injected zebrafish. These embryos had shorter anterior/posterior axes as well as crooked or bent tails. (B) Phenotype analysis after pax2a staining at 24 hpf resulted in about 63% embryos with a mild or severe affected phenotype after Hoxb7a overexpression compared to 13% in injected controls. (C) Pax2a staining after microinjection of different concentrations of mRNA. From left to right severe, mild affected and wild type (WT) embryos at 24 hpf. WT zebrafish show expression in the hindbrain, hindbrain-midbrain boundary, neural tube, mesoderm, optic stalk, otic vesicle, and pronephric duct. Microinjection 62.5 μM mRNA, 125 μM mRNA and 250 μM mRNA resulted in respectively 48%, 71% and 61% malformed zebrafish. There was no correlation between mRNA dosage and severity of malformation.