Post-concussion symptoms and chronic pain after mild traumatic brain injury are modulated by multiple locus effect in the BDNF gene through the expression of antisense: A pilot prospective control study

Figures & data

Table 1. Details of candidate SNPs selected from the literature.

Genes	SNP	Chromosome	Location	MAF (%)	Phenotype	Reference
ApoE	rs429358	19	45411941	11.4	Neuropsychological assessment	46
	rs7412	19	45412079	8.6
ApoE promoter	rs405509	19	45408836	47.2	Concussion in athletes	47
	rs449647	19	45408564	18.1
PARP-1	rs1109032	1	226561403	17	Glasgow Coma Scale outcome	48
	rs3219090	1	226564691	31.4
	rs3219119	1	226556443	31.5
	rs2271347	1	226549498	23.6
IL-1a	rs1800587	2	113542960	27.8	Glasgow Coma Scale outcome	49
IL-1b	rs1143634	2	113590390	22.4	Glasgow Coma Scale outcome	50
	rs16944	2	113594867	31.6
IL-6	rs1800795	7	22766645	41.6	Brain injury severity	22
COMT	rs4680	22	19951271	49.9	Executive functions	23
p53	rs1042522	17	7579472	22.9	Glasgow Coma Scale outcome	51
DRD2/ANKK1	rs1800497	11	113270828	18.2	Cognitive outcome	25
BDNF	rs1519480	11	27675712	31.1	Memory and processing speed	52
	rs7124442	11	27677041	30
	rs6265	11	27679916	20.9
	rs11030101	11	27680744	45.9
	rs11030102	11	27681596	23.1
	rs11030104	11	27684517	21.7
	rs11030107	11	27694835	23.1
	rs7103411	11	27700125	21.3
	rs7127507	11	27714884	30
	rs7934165	11	27731983	47.4
	rs11030121	11	27736207	31.7
	rs12273363	11	27744859	20.3
	rs908867	11	27745764	8.4
NEFH	rs165602	22	29886043	13.7	Concussion in athletes	53
	rs3815335	22	29881468	30
NGB	rs3783988	14	77734580	22.5	Animal model of severe pathology	54

SNP = Single nucleotide polymorphism; MAF = minor allelic frequency; ApoE = apolipoprotein E; PARP-1 = poly[ADP ribose] polymerase 1; IL = interleukin; COMT = catechol-O-methyltransferase; DRD2 = dopamine receptor D2; ANKK1 = BDNF = brain-derived neurotrophic factor; NEFH = neurofilament heavy polypeptide; NGB = neuroglobin.

Table 2. Description of post-concussion symptoms.

		mTBI with pain (n = 65)	mTBI without pain (n = 29)	Controls (n = 22)	P value^b
(a) Patients’ self-report questionnaires in the acute phase^a
Demography	Gender (F/M)	(22/43)	(5/24)	(13/9)
	Age^c	38.04 (12.41)	38.88 (12.43)	31.9 (11.91)	0.055
Pain-related questionnaires	Pain VAS (100 mm)	51.30 (24.87)	23.37 (31.63)	0.59 (1.40)	<0.0001
	Pain Catastrophizing Scale^d	15.21 (12.26)	9.88 (7.46)	7.76 (5.78)	0.01
	Bodily pain SF-36^d	34.41 (17.76)	77.04 (19.68)	79.44 (23.06)	<0.0001
Post-concussion symptoms, psychological questionnaires, quality of life	Rivermead	21.98 (12.74)	13.17 (9.88)	—	0.003
	SF-36	47.05 (16.42)	72.09 (16.96)	84.94 (12.15)	<0.0001
	BDI-II^d	12.91 (8.55)	5.48 (4.33)	2.0 (4.29)	<0.0001
	BAI^d	10.17 (9.28)	3.67 (3.14)	2.18 (5.27)	0.003
	IES-R	20.16 (16.58)	9.35 (7.71)	—	0.006
Sleep–wake disturbances	Fatigue scale^d	3.4(1.3)	2.4(1.2)	2.5(1.2)	0.001
	Sleepiness scale^d	3.6(1.6)	2.5(1.2)	2.1(1.2)	<0.0001
	Mean reaction time^d	282.2(65.7)	241.0(18.2)	244.6(36.4)	<0.0001
		mTBI with chronic pain (n = 18)	mTBI without chronic pain (n = 18)	P value
(b) Patients’ self-report questionnaires in the chronic phase (n = 36)^e
Pain-related questionnaires	Pain VAS (100 mm)	36.18 (19.49)	6.25 (15.44)	<0.0001
	Pain Catastrophizing Scale	9.86 (9.35)	4.88 (5.01)	ns
	Bodily pain SF-36	52.93 (25.37)	77.88 (20.42)	0.01
Post-concussion symptoms, psychological questionnaires, quality of life	Rivermead	17.86 (11.88)	7.13 (6.80)	0.007
	SF-36	67.00 (19.55)	88.18 (6.94)	0.005
	BDI-II	10.22 (10.46)	2.55 (3.36)	0.02
	BAI	8.67 (12.24)	2.18 (3.60)	0.04
	IES-R	7.81 (15.28)	5.92 (6.92)	ns
Sleep–wake disturbances	Fatigue scale	2.0(1.3)	2.65(1.4)	ns
	Sleepiness scale	1.85(1.0)	2.6(1.4)	ns
	Mean reaction time	318.7(137.6)	24.9(19)	ns

^aAnalysis of variance between mTBI patients with pain and mTBI patients without pain and healthy controls in the acute posttrauma phase (at 6 weeks). Results are shown as mean (SD). Lower scores on the SF-36 and bodily pain SF-36 indicate better quality of life. Rivermead and IES-R were not administered to healthy controls.

^bThe P value refers to the analysis of variance between groups.

^cNo statistically significant difference in age among mTBIs but only between controls and mTBI.

^dNo statistically significant difference between mTBI without pain and healthy controls.

^eA Student’s t test comparison between mTBI patients with persistent and newly developed pain vs. mTBI patients without pain at one year posttrauma. Lower scores on the SF-36 and bodily pain SF-36 indicate better quality of life.

mTBI = mild traumatic brain injury; VAS = Visual Analogue Scale; SF-36 = Short-Form 36; BDI = Beck Depression Inventory–II; BAI = Beck Anxiety Inventory; IES-R = Impact of Event Scale–Revised.

Figure 1. (a) Alleles of SNPs are represented in a short stretch of DNA. Combinations of SNPs used to construct haplotypes are highlighted. (b) BDNF gene structure from refSeq. Full boxes are exons, lines represent introns. Numbers (1–13) represent SNP locations in the gene. (c) LD plot of the SNPs spanning the BDNF gene. The numbers correspond to D′ values between SNPs. One block of 69 kbp was constructed with Haploview 4.2 using the solid spine method.

Table 3. Haplotypes frequency distribution and association with pain status and all phenotypes^a.

		H1		H2		H3
		TCTTC		CCTCT		TTCTC
		0.47		0.27		0.21
		OR/beta	P value^b	OR/beta	P value^b	OR/beta	P value^b
mTBI with pain vs. mTBI without pain		1.80	0.46	2.83	0.006	0.55	0.09
Pain-related questionnaires	Pain VAS	−2.05	0.69	6.854	0.25	−4.68	0.53
	Pain Catastrophizing Scale	−1.285	0.42	5.495	0.004	−3.497	0.08
	Body pain SF-36	0.453	0.90	−14.6	0.007	17.85	0.001
Post-concussion symptoms, psychological questionnaires, quality of life	Rivermead	−2.897	0.29	6.453	0.008	−4.924	0.056
	SF-36	2.005	0.56	−11.71	0.004	13.25	0.002
	BDI-II	−1.019	0.44	3.287	0.04	−3.708	0.03
	BAI	−2.653	0.05	4.255	0.006	−2.814	0.08
	IES-R	−4.296	0.10	7.121	0.02	−4.873	0.15
Sleep–wake disturbances	Fatigue scale	−0.1307	0.59	0.553	0.04	−0.683	0.01
	Sleepiness scale	0.2238	0.24	0.155	0.51	−0.675	0.004
	Mean reaction time	−15.95	0.15	28.46	0.03	−2.621	0.85
			No effect		Deleterious		Protective

^aStatistically significant results are shown in bold. Haplotypes are constructed using rs7124442|rs6265|rs11030104|rs7127507|rs11030121. Frequencies of haplotype are shown under each haplotype. Haplotype H2 is deleterious as carriers report more pain, post-concussion symptoms, poor quality of life, posttraumatic stress, anxiety, and pain catastrophizing. On the other hand, H3 is protective with less pain, better quality of life, and less fatigue and sleepiness.

Shown are P values of association between the haplotypes and each phenotype. Significant P value set at ≤0.02. OR = odds ratio; mTBI = mild traumatic brain injury; VAS = Visual Analogue Scale; SF-36 = Short-Form 36; BDI = Beck Depression Inventory–II; BAI = Beck Anxiety Inventory; IES-R = Impact of Event Scale–Revised.

Table 4. Expression data for top five SNPs in BDNF in public databases and mTBI patients^a.

(a) eQTL data retrieved from DRG and GTeX databases
			DRG		Cortex		Nerve
Markers	SNP	MA	Beta	P value	Beta	P value	Beta	P value	Gene
H2	rs7124442	C	−0.038	0.002	−0.05	0.54	0.066	0.14	BDNF-AS
	rs7127507	C	−0.038	0.003	0.027	0.74	−0.077	0.09	BDNF-AS
	rs11030121	T	−0.040	0.002	0.062	0.46	−0.068	0.13	BDNF-AS
	rs7124442	C	0.043	0.017	0.13	0.14	−0.043	0.55	BDNF
	rs7127507	C	0.045	0.013	−0.15	0.10	0.021	0.77	BDNF
	rs11030121	T	0.047	0.010	−0.098	0.27	0.034	0.65	BDNF
H3	rs6265	T	−0.010	0.53	−0.331	0.003	−0.179	0.001	BDNF-AS
	rs11030104	C	−0.004	0.77	−0.335	0.002	−0.189	3.6E-04	BDNF-AS
	rs6265	T	−0.008	0.71	0.026	0.83	−0.409	5.7E-06	BDNF
	rs11030104	C	0.0003	0.98	0.065	0.57	−0.415	1.3E-06	BDNF
(b) BDNF-AS and BDNF expression from immortalized lymphoblastoid cell lines of patients
	H1		H2		H3
	TCTTC		CCTCT		TTCTC
Frequency of haplotypes	0.47		0.27		0.21
	Beta	P value	Beta	P value	Beta	P value
Delta Ct BDNF-AS	−0.62	0.11	−0.19	0.68	1.107	0.02
Delta Ct BDNF	−0.12	0.58	0.234	0.35	0.06	0.83

^aStatistically significant eQTLs are shown in bold. Three SNPs, markers of H2, control the expression of BDNF and BDNF-AS in opposite directions in DRG. The two SNP markers of H3 control the expression of BDNF-AS in the brain and nerves and BDNF only in nerves. Haplotypes are constructed using rs7124442|rs6265|rs11030104|rs7127507|rs11030121. Higher delta Ct represents lower mRNA expression.

SNP = single nucleotide polymorphism; BDNF = brain-derived neurotrophic factor; mTBI = mild traumatic brain injury; eQTL = expression quantitative trait loci; DRG = dorsal root ganglia; Minor allele (MA) = BDNF-AS = anti-sense of BDNF.

Figure 2. (a) qPCR results of BDNF and BDNF-AS mRNA expression relative to GAPDH (delta Ct) comparison between mTBI patients with pain and without pain and healthy controls. (b) qPCR results of BDNF and BDNF-AS mRNA expression relative to GAPDH (delta Ct) comparison between the three haplotypes H1 (n = 44), H2 (n = 25), and H3 (n = 20). *Corresponds to the significant group difference between BDNF-AS level of H3 compared to BDNF-AS levels of H1 and H2. Haplotypes are composed from rs7124442|rs6265|rs11030104|rs7127507|rs11030121.

Figure 3. Comparison between acute and chronic questionnaire (Rivermead, SF-36, Pain Catastrophizing Scale, Beck Depression Inventory, Beck Anxiety Inventory, Impact of Event Scale) scores, pain VAS, and mean reaction time on the PVT. A repeated measures ANOVA was used. Dotted lines represent mTBI patients with pain (n = 18); full lines represent mTBI patients without pain (n = 18).