Association study of MIF promoter polymorphisms with suicide completers in the Japanese population.

BACKGROUND: Numerous studies suggest that inflammation plays a key role in suicidal behavior. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, has received increasing attention in depression research. However, no study has investigated whether MIF has genetic involvement in completed suicide. In this study, we sought to explore the relationship between two functional polymorphisms on the MIF gene promoter (MIF-794CATT5-8 microsatellite and MIF-173G/C single-nucleotide polymorphism [SNP]) and completed suicide by using one of the largest samples of suicide completers ever reported.
METHODS: The subjects comprised 602 suicide completers and 728 healthy controls. We genotyped MIF-794CATT5-8 microsatellite by polymerase chain reaction-based size discrimination assay and MIF-173G/C SNP by TaqMan® SNP genotyping assay. The allele-, genotype-, or haplotype-based association analyses between the suicide completers and the controls were carried out with the χ2 test, the Cochran-Armitage trend test, or Fisher's exact test.
RESULTS: Analyses of allele or genotype frequency distributions of the polymorphisms studied here did not reveal any significant differences between the suicide completers and the controls. Haplotype analysis also revealed no association with completed suicide.
CONCLUSION: To our knowledge, this is the first study that has examined the genetic association between MIF and completed suicide. Our results suggest that the effects of MIF-794CATT5-8 microsatellite and MIF-173G/C SNP on the MIF gene promoter might not contribute to the genetic risk of completed suicide in the Japanese population.

Introduction

Suicide is a significant public health problem worldwide, with ~1 million suicides each year. A review of the literature showed that >2% of the current traffic accidents were suicide attempts, and this phenomenon may be underreported.1 The involvement of genetic factors in suicidal behavior is supported by family studies, twin and adoption studies, candidate gene analyses, and genome-wide association studies. Various studies have shown that numerous genes (eg, serotonin receptors and transporters, brain-derived neurotrophic factors [BDNF], or catechol-O-methyltransferase [COMT]) are linked to suicidal behavior, although conflicting reports exist.2,3 In addition, recent studies reported aberrant cytokine levels in blood, cerebrospinal fluid, and postmortem brain samples from suicidal completers.4 For instance, increased blood levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are associated with suicidal ideation and attempt.5,6 Alternatively, mRNA and protein levels of IL-1β, IL-6, and TNF-α were increased postmortem in the prefrontal cortexes of teenaged suicide completers.7

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine involved in the regulation of innate and adaptive immunity.8 MIF was originally identified as a product of T-lymphocytes on the basis of its ability to prevent random migration of macrophages.9,10 MIF is secreted in response to inflammatory stimuli, including microbial products and glucocorticoids.11,12 Upon release, MIF acts in an autocrine and paracrine manner to promote proinflammatory cytokine production, eg, IL-1β, IL-6, and TNF-α.13,14 MIF has also been shown to facilitate DNA damage response and cell cycle regulation.15 In addition, MIF production has been discovered in many other nonimmune cells ubiquitously, being highly expressed in the liver, kidneys, anterior pituitary, and brain.8,16 It is reported that MIF expression in the brain was seen in neurons of the cortex, hypothalamus, hippocampus, cerebellum, and pons,17 and in astrocytes and the subgranular zone of the hippocampus.18 Increasing evidence suggests a role for MIF in depression.19 Deletion of MIF resulted in increased depressive behaviors in rodent models.18 In addition, several studies have explored MIF as a biomarker in major depressive disorder (MDD) and other mood disorders. Some studies have identified increased serum MIF both in patients with MDD and in healthy subjects with depressive symptoms.20–22 Exhaustive serum proteomic profiling of MDD has identified MIF as one of the robust changed analytes in patients compared with controls.23,24 Furthermore, it has been established that MIF has an intricate relationship with the hypothalamic–pituitary–adrenal axis. For instance, Edwards et al25 found that elevated serum MIF was associated with depressive symptoms, reduced cortisol response to acute stress, and lower morning cortisol values. Alternatively, MIF was shown to specifically counteract the glucocorticoid-induced suppression of inflammatory cytokine secretion in activated macrophages (IL-1β, IL-6, and TNF-α).12 The hypothalamic–pituitary–adrenal axis has also gained interest as a neurobiological factor related to suicide, on the basis of the findings of studies investigating dexamethasone suppression test (DST) response, since nearly a 10-fold higher risk of completed suicide was found in DST nonsuppressors than in DST suppressors in a depressed cohort.2,26

From these findings, we hypothesized that MIF is involved in the genetic pathophysiology of suicide. The MIF gene promoter contains two functional variations: MIF-794CATT5–8 repeat (rs5844572) and MIF-173G/C single-nucleotide polymorphism (SNP) (rs755622), which have been well documented to affect the gene expression and protein levels of MIF.27,28 Indeed, many studies have examined the association between these two polymorphisms and various physical diseases.29–34 However, no previous study has investigated the role of both these polymorphisms in suicidal behavior. Hence, we investigated the association of the two functional polymorphisms on the MIF promoter in suicide using the large sample size of 602 suicide completers and 728 controls.

Materials and methods

Subjects

This study was approved by the Ethical Committee for Genetic Studies of Kobe University Graduate School of Medicine. The study population consisted of 602 suicide completers (408 males: mean age ± standard deviation (SD), 51.0±17.4 years; 194 females: 50.7±18.8 years) and 728 unrelated healthy volunteers (340 males: mean age ± SD, 53.3±18.8 years; 388 females: 54.5±18.3 years). All subjects were ethnically Japanese.

Autopsies on suicide victims were conducted at the Department of Legal Medicine, Kobe University Graduate School of Medicine. The definition of suicide was based on the results of medicolegal examination and the police investigation as required by Japanese law. The methods of suicide were neck hanging (399), jumping from heights (100), gas suffocation (29), drowning (16), jumping in front of a vehicle (9), drug overdose (8), self-inflicted penetrating wounds (8), self-burning (3), taking poison (2), other methods (10), and unknown (18). We subdivided suicide completers into violent suicide (neck hanging, jumping from heights, gas suffocation, drowning, jumping in front of a vehicle, self-inflicted penetrating wounds, and self-burning) and nonviolent suicide (taking poison and drug overdose), according to Dumais et al.35 The healthy controls were recruited with consent after the purpose and procedures of the study were explained. The psychiatric assessment of each healthy participant was conducted as previously described.36,37 Briefly, none of them manifested psychiatric problems in unstructured interviews using unnamed symptom checklists based on the Diagnostic and Statistical Manual of Mental Disorders 4th edition criteria by two psychiatrists. Demographics of suicide completers and healthy controls are shown in Table 1. Informed consent was obtained from all of the participants and from the families of the subjects used for postmortem blood experiments.

Table 1

Demographics of suicide completers and healthy controls

Subjects	Suicide(n=602)	Control(n=728)
Mean age ± SD	50.9±17.9	54.0±18.5
n (male:female)	408:194	340:388
Suicide methods
Neck hanging	399
Jumping from heights	100
Gas suffocation	29
Drowning	16
Jumping in front of vehicles	9
Drug overdosing	8
Self-inflicted penetrating wounds	8
Self-burning	3
Taking poison	2
Other	10
Unknown	18

Abbreviation: SD, standard deviation.

Blood and DNA sampling

Peripheral blood was obtained from all suicide completers and controls. Blood samples were stored at −80°C before use. DNA was extracted using the QIAamp DNA Blood Midi Kit (Qiagen Inc., Valencia, CA, USA). Each DNA sample was quantified and qualified using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

Genotyping for the MIF-794CATT5–8 repeat polymorphism (rs5844572)

Genomic DNA (100 ng) was subjected to polymerase chain reaction (PCR) for one cycle at 95°C for 10 minutes, followed by 40 cycles at 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds and final extension at 72°C for 7 minutes. The forward primer was 5′-TTG-CAC-CTA-TCA-GAG-ACC-3′ labeled with a carboxyfluorescein fluorescent dye, and the reverse primer was 5′-TCC-ACT-AAT-GGT-AAA-CTC-G-3′. The 25 μL PCR reaction volume contained 100 ng of genomic DNA, 12.5 μL of AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and 1.0 μL of forward and reverse primer. The amplification included an initial holding at 95°C for 10 minutes, followed by a three-step PCR program: 95°C for 30 seconds, 55°C for 30 seconds, and 40 cycles at 72°C for 30 seconds, and finally, 72°C for 7 minutes. The PCR product was appropriately diluted and supplemented with GeneScan 600 size standard (Applied Biosystems, Foster City, CA, USA). The mixture was then separated on a 50-cm polyacrylamide gel at 15,000 V for 45 minutes on an ABI 3130 capillary sequencer and analyzed with GeneMapper® 4.0 software (Applied Biosystems). The PCR product sizes were 207 bp, 211 bp, and 215 bp in length, and these corresponded to 5-, 6-, and 7-CATT repeats, respectively. 8-CATT repeats were not observed in this study.

Genotyping for the MIF-173G/C SNP (rs755622)

We selected the predesigned TaqMan SNP genotyping assays from the Applied Biosystems database (http://www.appliedbiosystems.com) for the MIF-173G/C SNP (rs755622). Genotyping was performed with a 7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer’s protocol.

Statistics

We used the Haploview version 4.2 software program (http://www.broad.mit.edu/mpg/haploview) to determine the Hardy–Weinberg equilibrium and allelic or haplotype frequencies for each of the MIF promoter polymorphisms.38 The allele-, genotype-, or haplotype-based association analyses between the suicide and control groups were carried out with the χ2 test, Cochran–Armitage trend test, or Fisher’s exact test, as appropriate, using Haploview software, R version 3.1.0 (The R Foundation for Statistical Computing, 2014, Vienna, Austria) and Excel 2010 (Microsoft, Redmond, WA, USA) with the Statcel3 add-on (OMS, Saitama, Japan). The power analysis was performed with the PS v2.1.3.1 program.39 Statistical significance for the two MIF polymorphisms study was defined as two-tailed P<0.05/2=0.025.

Results

MIF-794CATT5–8 repeat and MIF-173G/C SNP analyses

No deviation from Hardy–Weinberg equilibrium was seen in suicide completers and healthy controls for both polymorphisms. The MIF-794CATT5–8 repeat genotype and allele frequencies are shown in Table 2. Neither the genotype distribution nor the allelic frequency of the polymorphism was significantly associated with completed suicide. The MIF-173G/C SNP genotype and allele frequencies in suicide completers and healthy controls are shown in Table 3. Neither the genotype distribution nor the allelic frequency of the SNP was significantly associated with completed suicide. Subgroup analyses based on sex difference or methods of suicide (violent or nonviolent) also yielded no significant findings (Tables S1–S5).

Table 2

Association of MIF-794CATT5–8 repeat with suicide completers and controls

Polymorphism	Suicide(n=602)		Control(n=728)		P-valuea	Global P-valueb	OR (95% CI)	Power
	N	Frequency	N	Frequency
MIF-794CATT5–8
Genotype
55	86	0.143	105	0.145	0.943	0.409	0.99 (0.73–1.35)	0.050
56	231	0.384	251	0.345	0.141		1.18 (0.95–1.48)	0.313
57	77	0.128	95	0.130	0.889		0.98 (0.71–1.35)	0.051
66	111	0.184	158	0.217	0.140		0.82 (0.62–1.07)	0.319
67	83	0.138	93	0.128	0.587		1.09 (0.79–1.50)	0.084
77	14	0.023	26	0.036	0.185		0.64 (0.33–1.24)	0.277
Allele
CATT5	480	0.399	556	0.382	0.376	0.645	1.07 (0.92–1.25)	0.146
CATT6	536	0.445	660	0.453	0.675		0.97 (0.83–1.13)	0.070
CATT7	188	0.156	240	0.165	0.544		0.94 (0.76–1.15)	0.096

Notes:

Genotypic P-values and allelic P-values were tested with the χ2 test.

Global P-values were calculated by Fisher’s exact test.

Abbreviations: MIF, macrophage migration inhibitory factor; OR, odds ratio; CI, confidence interval.

Table 3

Association of MIF-173 SNP with suicide completers and controls

Polymorphism	Suicide(n=602)		Control(n= 728)		P-valuea	Global P-valueb	OR (95% CI)	Power
	N	Frequency	N	Frequency
MIF-173G/C
Genotype
GG	373	0.620	451	0.619	0.997	0.714	1.00 (0.83–1.21)	0.050
GC	203	0.337	237	0.326	0.653		1.05 (0.84–1.33)	0.071
CC	26	0.043	40	0.055	0.326		0.78 (0.47–1.29)	0.168
Allele
G	949	0.788	1,139	0.782	0.711		0.97 (0.80–1.16)	0.066
C	255	0.212	317	0.218

Notes:

Genotypic P-values and allelic P-values were tested with the χ2 test.

Global P-value was tested with the Cochran–Armitage test for trend.

Abbreviations: MIF, macrophage migration inhibitory factor; OR, odds ratio; CI, confidence interval.

MIF promoter haplotype analyses

Linkage disequilibrium between each CATT repeat and the −173C was measured as follows: CATT5 and −173C: D’ =0.495, r2=0.043; CATT6 and −173C: D’ =0.871, r2=0.170; CATT7 and −173C: D’ =0.984, r2=0.678. The CATT7 and −173C polymorphisms were observed in strong linkage disequilibrium in our samples. We then examined whether the different haplotypes of the MIF-794CATT5–8 repeat and the MIF-173G/C SNP would show different distribution patterns in suicide completers compared with controls. The reconstructed frequencies of each possible haplotype based on the observed genotype data are shown in Table 4. There were six haplotypes in this study, none of which showed different distributions between suicide completers and controls. Subgroup analyses based on sex difference or methods of suicide (violent or nonviolent) also showed no significance (Tables S1–S5).

Table 4

Association of haplotypes of MIF promoter polymorphisms with suicide completers and controls

Haplotype	Suicide(n=602)		Control(n=728)		P-valuea	Global P-valueb	OR (95% CI)	Power
	N	Frequency	N	Frequency
Haplotype
MIFCATT5-173G	427	0.355	496	0.341	0.442	0.631	1.06 (0.90–1.25)	0.118
MIFCATT6-173G	522	0.433	641	0.440	0.716		0.97 (0.83–1.13)	0.065
MIFCATT7-173G	4	0.003	1	0.001	0.129		4.14 (0.54–31.7)	0.229
MIFCATT5-173C	53	0.044	60	0.041	0.746		1.06 (0.73–1.55)	0.068
MIFCATT6-173C	14	0.012	19	0.013	0.802		0.92 (0.46–1.84)	0.055
MIFCATT7-173C	184	0.153	239	0.164	0.426		0.92 (0.75–1.13)	0.096

Notes:

P-values were tested with the χ2 test.

Global P-value was calculated by Fisher’s exact test or Cochran–Armitage test for trend, as appropriate.

Abbreviations: MIF, macrophage migration inhibitory factor; OR, odds ratio; CI, confidence interval.

Discussion

To our knowledge, this is the first study to investigate the association between suicide and two functional polymorphisms in the MIF gene promoter, which are known to affect the gene expression and protein levels of MIF. We also conducted subgroup analysis to investigate sex differences because males tend toward a higher suicide rate than females do among all age groups worldwide.40 Furthermore, we determined differences between violent and nonviolent suicides in this study because lethality associated with violent methods is considerably higher than that associated with nonviolent methods.41 Moreover, some previous studies showed differences between subjects with violent vs nonviolent methods of suicide in other polymorphisms (for the COMT and BDNF genes) related to completed suicide.42,43 As a result, no significant differences were found in any of the analyses, which indicated that MIF-794CATT5–8 repeat microsatellite and MIF-173G/C SNP are not associated with an increased risk of suicide in the Japanese population. Recent meta-analyses with MDD cases also failed to show the genetic involvement of MIF.44,45 Therefore, MIF might not be primarily involved in genetic susceptibility to suicide (and depression).

The limitations of the present study should be considered. First, the current sample provides low powers based on the observed frequencies of two polymorphisms. The number of subjects in the association study may not have been large enough to detect a significant difference, although our sample size of suicide completers was one of the largest ever reported. In fact, most previous candidate gene analyses and genome-wide association studies for completed suicide could not overcome the statistical limitation of small samples2,3,46 because it is extremely difficult to obtain tissue samples from suicide completers. Second, our study included only the Japanese population. The distribution of MIF polymorphisms determined by us was similar to that determined in the previous study for Japanese population by Llamas-Covarrubias et al,47 but not for Caucasian by Swanberg et al.48 Given that the MIF polymorphisms studied here differ among Caucasians, Africans, and Asians,49 our results might not be extrapolated to other populations. In addition, MIF protein levels in the brains of individuals displaying suicidal behavior should be further investigated to clarify the associations between MIF and suicide other than genetic susceptibility.

Conclusion

In conclusion, our results suggest that MIF-794CATT5–8 microsatellite and MIF-173G/C SNP microsatellite might not contribute to the genetic risk of completed suicide in the Japanese population.

Table S1

Association of MIF promoter polymorphisms with suicide completers and controls for males

Polymorphism	Male suicide(n=408)		Male control(n=340)		P-valuea	Global P-valueb	OR (95% CI)	Power
	N	Frequency	N	Frequency
MIF-794CATT5–8
Genotype
55	56	0.137	49	0.144	0.788	0.584	0.945 (0.625–1.429)	0.059
56	159	0.390	119	0.350	0.263		1.186 (0.880–1.599)	0.193
57	53	0.130	44	0.129	0.984		1.004 (0.654–1.542)	0.050
66	77	0.189	77	0.226	0.204		0.795 (0.557–1.133)	0.256
67	53	0.130	38	0.112	0.450		1.187 (0.761–1.850)	0.108
77	10	0.025	13	0.038	0.279		0.632 (0.274–1.460)	0.194
Allele
CATT5	324	0.397	261	0.384	0.601	0.875	1.057 (0.858–1.303)	0.080
CATT6	366	0.449	311	0.457	0.733		0.965 (0.787–1.184)	0.061
CATT7	126	0.154	108	0.159	0.815		0.967 (0.731–1.280)	0.058
MIF-173G/C
Genotype
GG	252	0.618	218	0.641	0.507	0.753	0.904 (0.671–1.218)	0.100
GC	136	0.333	102	0.300	0.330		1.167 (0.856–1.591)	0.154
CC	20	0.049	20	0.059	0.553		0.825 (0.436–1.560)	0.097
Allele
G	640	0.784	538	0.791	0.747		0.960 (0.748–1.231)	0.062
C	176	0.216	142	0.209
Haplotype
MIFCATT5-173G	283	0.347	234.1	0.344	0.920	0.769	1.011 (0.816–1.253)	0.052
MIFCATT6-173G	357.4	0.438	300.6	0.442	0.875		0.984 (0.801–1.207)	0.053
MIFCATT7-173G	2.1	0.003	0.1	0.000	0.208		17.54 (0.031–9.989)	0.000
MIFCATT5-173C	41	0.050	26.9	0.040	0.320		1.284 (0.781–2.112)	0.132
MIFCATT6-173C	8.6	0.011	10.4	0.015	0.413		0.686 (0.276–1.702)	0.111
MIFCATT7-173C	123.9	0.152	107.9	0.159	0.715		0.949 (0.717–1.257)	0.067

Notes:

P-values were tested with the χ2 test.

Global P-value was calculated by Fisher’s exact test or Cochran–Armitage test for trend, as appropriate.

Abbreviations: MIF, macrophage migration inhibitory factor; OR, odds ratio; CI, confidence interval.

Table S2

Association of MIF promoter polymorphisms with suicide completers and controls for females

Polymorphism	Female suicide(n=194)		Female control(n=388)		P-valuea	Global P-valueb	OR (95% CI)	Power
	N	Frequency	N	Frequency
MIF-794CATT5–8
Genotype
55	30	0.155	56	0.144	0.741	0.850	1.084 (0.670–1.755)	0.066
56	72	0.371	132	0.340	0.461		1.145 (0.799–1.639)	0.114
57	24	0.124	51	0.131	0.793		0.933 (0.555–1.568)	0.055
66	34	0.175	81	0.209	0.339		0.805 (0.517–1.255)	0.162
67	30	0.155	55	0.142	0.678		1.108 (0.684–1.794)	0.072
77	4	0.021	13	0.034	0.384		0.607 (0.195–1.888)	0.119
Allele
CATT5	156	0.402	295	0.380	0.470	0.762	1.096 (0.854–1.407)	0.112
CATT6	170	0.438	349	0.450	0.708		0.954 (0.746–1.219)	0.067
CATT7	62	0.160	132	0.170	0.656		0.928 (0.667–1.290)	0.070
MIF-173G/C
Genotype
GG	121	0.624	233	0.601	0.589	0.390	1.103 (0.774–1.572)	0.082
GC	67	0.345	135	0.348	0.951		0.989 (0.688–1.420)	0.050
CC	6	0.031	20	0.052	0.256		0.587 (0.232–1.487)	0.196
Allele
G	309	0.796	601	0.774	0.394		1.139 (0.845–1.536)	0.089
C	79	0.204	175	0.226
Haplotype
MIFCATT5-173G	143.7	0.370	262.6	0.338	0.280	0.519	1.150 (0.892–1.483)	0.187
MIFCATT6-173G	164.3	0.423	340.6	0.439	0.615		0.939 (0.734–1.201)	0.081
MIFCATT7-173G	2.1	0.005	1.1	0.001	0.233		3.834 (0.380–38.62)	0.071
MIFCATT5-173C	12.3	0.032	32.4	0.042	0.398		0.751 (0.385–1.465)	0.124
MIFCATT6-173C	5.7	0.015	8.4	0.011	0.564		1.362 (0.467–3.975)	0.092
MIFCATT7-173C	59.9	0.154	130.9	0.169	0.538		0.900 (0.645–1.256)	0.098

Notes:

P-values were tested with the χ2 test.

Global P-value was calculated by Fisher’s exact test or Cochran–Armitage test for trend, as appropriate.

Abbreviations: MIF, macrophage migration inhibitory factor; OR, odds ratio; CI, confidence interval.

Table S3

Association of MIF promoter polymorphisms with violent suicide completers and controls

Polymorphism	Violent suicide(n=568)		Control(n=728)		P-valuea	Global P-valueb	OR (95% CI)	Power
	N	Frequency	N	Frequency
MIF-794CATT5–8
Genotype
55	82	0.144	105	0.144	0.995	0.313	1.001 (0.733–1.368)	0.050
56	220	0.387	251	0.345	0.114		1.201 (0.957–1.509)	0.329
57	70	0.123	95	0.130	0.697		0.937 (0.673–1.303)	0.066
66	105	0.185	158	0.217	0.153		0.818 (0.621–1.078)	0.309
67	79	0.139	93	0.128	0.551		1.103 (0.799–1.522)	0.088
77	12	0.021	26	0.036	0.122		0.583 (0.291–1.165)	0.387
Allele
CATT5	454	0.400	556	0.382	0.357	0.554	1.078 (0.919–1.263)	0.152
CATT6	509	0.448	660	0.453	0.791		0.979 (0.838–1.144)	0.057
CATT7	173	0.152	240	0.165	0.387		0.910 (0.736–1.126)	0.148
MIF-173G/C
Genotype
GG	355	0.625	451	0.620	0.840	0.542	1.024 (0.816–1.284)	0.054
GC	190	0.335	237	0.326	0.734		1.041 (0.825–1.315)	0.063
CC	23	0.040	40	0.055	0.230		0.726 (0.429–1.227)	0.250
Allele
G	900	0.792	1,139	0.782	0.539		1.061 (0.878–1.283)	0.094
C	236	0.208	317	0.218
Haplotype
MIFCATT5-173G	406.8	0.358	496.7	0.341	0.369	0.547	1.077 (0.916–1.268)	0.145
MIFCATT6-173G	494.8	0.436	640.9	0.440	0.812		0.981 (0.839–1.148)	0.055
MIFCATT7-173G	4.1	0.004	1.2	0.001	0.113		4.391 (0.573–33.63)	0.057
MIFCATT5-173C	47.2	0.042	59.3	0.041	0.915		1.021 (0.691–1.509)	0.052
MIFCATT6-173C	14.2	0.013	19.1	0.013	0.900		0.952 (0.477–1.901)	0.050
MIFCATT7-173C	168.9	0.149	238.8	0.164	0.286		0.890 (0.718–1.103)	0.184

Notes:

P-values were tested with the χ2 test.

Global P-value was calculated by Fisher’s exact test or Cochran–Armitage test for trend, as appropriate.

Abbreviations: MIF, macrophage migration inhibitory factor; OR, odds ratio; CI, confidence interval.

Table S4

Association of MIF promoter polymorphisms with nonviolent suicide completers and controls

Polymorphism	Nonviolent suicide(n=10)		Control(n=728)		P-valuea	Global P-valueb	OR (95% CI)	Power
	N	Frequency	N	Frequency
MIF-794CATT5–8
Genotype
55	1	0.100	105	0.144	0.692	0.890	0.659 (0.083–5.258)	0.037
56	5	0.500	251	0.345	0.306		1.900 (0.545–6.626)	0.187
57	1	0.100	95	0.130	0.776		0.740 (0.093–5.910)	0.036
66	1	0.100	158	0.217	0.371		0.401 (0.050–3.188)	0.073
67	2	0.200	93	0.128	0.498		1.707 (0.357–8.161)	0.152
77	0	0.000	26	0.036	0.543		–	–
Allele
CATT5	8	0.400	556	0.382	0.868	1.000	1.079 (0.438–2.656)	0.055
CATT6	9	0.450	660	0.453	0.977		0.987 (0.406–2.396)	0.050
CATT7	3	0.150	240	0.165	0.859		0.894 (0.260–3.075)	0.045
MIF-173G/C
Genotype
GG	6	0.600	451	0.620	0.840	0.852	0.921 (0.258–3.294)	0.054
GC	4	0.400	237	0.326	0.874		1.381 (0.386–4.941)	0.089
CC	0	0.000	40	0.055	0.953		–	–
Allele
G	16	0.800	1,139	0.782	0.935		1.113 (0.370–3.353)	0.047
C	4	0.200	317	0.218
Haplotype
MIFCATT5-173G	6.8	0.340	496.6	0.341	0.980	0.971	0.995 (0.392–2.526)	0.050
MIFCATT6-173G	8.9	0.445	641.2	0.440	0.965		1.019 (0.419–2.476)	0.050
MIFCATT7-173G	0.0	0.000	1.0	0.001	0.907		–	–
MIFCATT5-173C	1.2	0.060	59.4	0.041	0.642		1.501 (0.233–9.675)	0.120
MIFCATT6-173C	0.1	0.005	18.8	0.013	0.744		0.384 (0.001–195.1)	0.004
MIFCATT7-173C	3.0	0.150	239.0	0.164	0.863		0.899 (0.261–3.090)	0.045

Notes:

P-values were tested with the χ2 test.

Global P-value was calculated by Fisher’s exact test or Cochran–Armitage test for trend, as appropriate.

Abbreviations: MIF, macrophage migration inhibitory factor; OR, odds ratio; CI, confidence interval.

Table S5

Association of MIF promoter polymorphisms with violent and nonviolent suicide completers

Polymorphism	Violent suicide(n=568)		Nonviolent suicide(n=10)		P-valuea	Global P-valueb	OR (95% CI)	Power
	N	Frequency	N	Frequency
MIF-794CATT5–8
Genotype
55	82	0.144	1	0.100	0.692	0.940	1.519 (0.190–12.15)	0.032
56	220	0.387	5	0.500	0.469		0.632 (0.181–2.209)	0.134
57	70	0.123	1	0.100	0.824		1.265 (0.158–10.13)	0.036
66	105	0.185	1	0.100	0.492		2.041 (0.256–16.29)	0.031
67	79	0.139	2	0.200	0.582		0.646 (0.135–3.099)	0.139
77	12	0.021	0	0.000	0.642		–
Allele
CATT5	454	0.400	8	0.400	0.998	1.000	0.999 (0.405–2.462)	0.050
CATT6	509	0.448	9	0.450	0.986		0.992 (0.408–2.413)	0.050
CATT7	173	0.152	3	0.150	0.978		1.018 (0.295–3.511)	0.049
MIF-173G/C
Genotype
GG	355	0.625	6	0.600	0.871	0.932	1.111 (0.310–3.982)	0.056
GC	190	0.335	4	0.400	0.664		0.754 (0.210–2.704)	0.085
CC	23	0.040	0	0.000	0.516		–	–
Allele
G	900	0.792	16	0.800	0.933		0.953 (0.316–2.878)	0.048
C	236	0.208	4	0.200
Haplotype
MIFCATT5-173G	406.0	0.357	6.8	0.340	0.858	0.973	1.080 (0.425–2.745)	0.050
MIFCATT6-173G	494.6	0.435	8.9	0.445	0.932		0.962 (0.395–2.341)	0.051
MIFCATT7-173G	4.3	0.004	0.0	0.000	0.800		–	–
MIFCATT5-173C	48.0	0.042	1.2	0.060	0.666		0.691 (0.107–4.475)	0.121
MIFCATT6-173C	14.4	0.013	0.1	0.005	0.760		2.555 (0.005–1.304)	0.003
MIFCATT7-173C	168.7	0.149	3.0	0.150	0.988		0.988 (0.287–3.409)	0.051

Notes:

P-values were tested with the χ2 test.

Global P-value was calculated by Fisher’s exact test or Cochran–Armitage test for trend, as appropriate.

Abbreviations: MIF, macrophage migration inhibitory factor; OR, odds ratio; CI, confidence interval.