Despite unprecedented strategies to support its prevention as a public health imperative, suicide has, alarmingly, increased in recent years—now representing the second leading cause of death in teens and young adults in the U.S. as well as worldwide (68, 69). American Indians and Alaska Natives (AI/AN) demonstrate the highest burden of suicide compared to all other ethnic or racial groups, highlighting urgency to critically advance research and prevention (3–6). However, rates of suicide vary substantially between tribal groups and by distinct geographic region (2), underscoring the need to delineate risk and resilience factors in local communities to develop community-specific prevention and intervention efforts (10–12, 70).
The present study focused on an American Indian population living on eight contiguous reservations. This population demonstrates high rates of suicide as well as AUD (19, 43). In this study, we investigated potential genetic risk factors for suicidal behaviors by studying: (1) the possible genetic overlap of SB with AUD, and (2) the impacts of rare- and low-frequency genomic variants on SB. We identified five variants significantly associated with SB-AUD, two of which are intergenic and three are intronic. Two of the five variants, although not significantly associated with either SB or AUD severity on their own in this AI cohort, were significantly associated with the dual phenotype SB-AUD—suggesting the genetic correlations were induced by residual shared factors. Six genes, one lincRNA, and one canonical pathway, were found to be significantly associated with SB through rare and low-frequency variant analysis. Four additional genes, and two pathways, also were strongly associated with SB.
F-box gene FBXO11 significantly associated with SB-AUD in this AI population
The intronic variants significantly associated with SB-AUD are on genes AACSP1, ANK1, and FBXO11. AACSP1 is a pseudogene whose function is unclear, although it has been associated with an Alzheimer’s marker in an interaction analysis (71). ANK1 encodes ankyrin-1 protein active in red blood cells as well as brain and muscle. The gene has been linked to Alzheimer’s disease through epigenetic deregulation (72, 73) and appears to play a role in immunomodulation (74).
Rs184204326 on gene FBXO11 was the only common variant significantly associated with SB-AUD. FBXO11 encodes a member of the F-box protein family and is highly conserved in evolution. It is part of the SCF (SKP1-cullin-F-box) complex, which is responsible for ubiquitination and degradation of SCF substrates and plays an important role in the maintenance of genome stability (75). FBXO11 is also involved in regulating alternative splicing (76). While the gene is expressed in various tissues, it is particularly abundant in the brain. FBXO11 has not been previously associated with SB. However, variants on or near FBXO11 have been associated with a number or correlated behavior traits and disorders such as alcohol consumption (77), smoking initiation (77, 78), risk taking behaviors (79, 80), externalizing behaviors (81), educational attainment (82), insomnia (83), schizophrenia and depression (84, 85). De novo mutations on FBXO11 have also been found to cause intellectual disability with behavior problems as well as facial dysmorphisms (86) and other neurodevelopmental disorders (87). Variant rs77969729 on FBXO11 has been linked to Alzheimer’s disease in the UKBiobank (88). This variant is in LD with rs184204326 on FBXO11 in the AI cohort and associated with SB-AUD at p = 3.4E-7.
Rare mutations in PEDF are likely a risk factor for suicidal behaviors
Six genes and one lincRNA were found to be significantly associated with SB through rare variant analysis. The top gene SERPINF1 encodes serpin F1, also known as pigment epithelium-derived factor (PEDF). This gene has nine rare nonsynonymous or splicing site mutations, of which four are among the 0.1–0.5% most deleterious. Nearly 6% of individuals in the AI cohort carry some of these mutations. PEDF is a secreted glycoprotein and a potent inhibitor of angiogenesis (89). It also has neuroprotective effects and been implicated in depression (90, 91). Reduction in PEDF levels have been found in the plasmas of patients with major depressive disorder (MDD), as well as in the prefrontal cortex (PFC) of animal models exhibiting depressive-like behaviors (91). Conversely, overexpression of PEDF in the PFC has been shown to induce antidepressant “like” behaviors, by exerting effects on the tryptophan and glutamate in the PFC, with tryptophan being an essential amino acid precursor of serotonin, which is known to be associated with both depression and SB (92). Another study has shown that PEDF in the hippocampus has a similar effect on depressive phenotypes in animal models by contributing to the synaptic formation and Wnt signaling activation in that region (93). PEDF has thus been suggested as a biomarker and a novel therapeutic target for depression (90).
Additional genes significantly associated with SB in the AI cohort included ZNF30, CD34, SLC5A9, OPRD1, and HSD17B3. ZNF30 encodes a zinc finger protein. A microdeletion of five genes including ZNF30 results in chromosome 19q13.11 deletion syndrome that includes features such as: developmental delay, microcephaly and intellectual disabilities (94). The GWAS meta-analysis by the international suicide genetics consortium (ISGC) reported another zinc finger family gene (ZNF28) associated with SB and suicide death (31). CD34 is involved in the innate immune system. Its variants have been associated with Alzheimer’s disease (88) and risk-taking behavior (80). SCL5A9 is involved in sodium ion transport and is also known to be a sodium-dependent glucose transporter (95). This gene has been associated with amygdala volume (96) and metabolic measurements (97). Variants on or near HSD17B3 have been linked to smoking behavior (98), brain morphology measurements (99), memory performance (100) and Alzheimer's markers (71, 101).
Opioid receptor delta 1 (OPRD1) gene encodes a member of the opioid family of the G-protein coupled receptors (GPCR). Delta opioid receptors are involved in reward mediation and neuroprotection. OPRD1 is specifically involved in the opioid receptor signaling pathway and cellular response to hypoxia. Numerous candidate gene studies have implicated OPRD1 in addiction, including opioid, cocaine, and alcohol dependence (102, 103). Variants on OPRD1 have also been associated with schizophrenia (104) and educational attainment (82).
Hypoxia regulation is significantly associated with suicidal behaviors
Nonsyn rare variants in a pathway related to hypoxia inducible factor (HIF) regulation were significantly associated with SB (see Table 3). Nearly half of the individuals in the AI cohort have some of these rare mutations on their genes in this pathway. The network analysis of the top rare-variant genes associated with SB also found that those genes were enriched for response to decreased oxygen levels (see Fig. 3).
Chronic hypoxia is suggested to be a risk factor for suicide (64), and metabolic stress associated with hypoxia is a possible mechanism (105). Hypoxia is also hypothesized to increase the risk of suicide by reducing the synthesis of brain serotonin (66) or downregulating PEDF (106), which has a protective role in depression and is associated with SB in this AI cohort. HIFs are transcriptional factors that respond to reduced oxygen levels in cell and tissue. HIF-1 protects against hypoxia and reduces oxidative stress (107), while HIF-2α plays an important in the modulation of inflammatory responses (108). Recent studies have indicated that oxidative stress and abnormal energy metabolism in the brain play significant roles in the development of depression. Therefore, increasing HIF-1 activity has been suggested as a potential new therapeutic target for depression and suicide ideation (107). Gene expression analyses have demonstrated that patients with MDD exhibit increased expression of HIF-1 and its target genes, including VEGF and GLUT1 (SLC2A1) (65). Our network analysis of the top rare-variant genes associated with SB in the AI have also found enrichments in VEGF receptors signaling and angiogenesis.
Alcohol exposure also can alter expression of HIF. For instance, alcohol exposure can induce HIF-1α activation, however the dose and timing of alcohol exposure results in differential expression of HIF-1α in the brain and other organs (109). Both acute and chronic alcohol exposure have been found to increase HIF-1α expression in the brain cortex, whereas chronic binge alcohol exposure decreased HIF-1α expression (109). Another study found HIF3A can be epigenetically induced in the amygdala in animal models by acute alcohol exposure, and its epigenetic reprogramming was associated with the anti-anxiety effect of acute alcohol exposure (110). Nonsyn rare variants in HIF3A were strongly associated with SB (nominal p = 2.9E-05, FDR = 0.09) in the present study.
Vasopressin regulated water metabolism and hexose transport strongly associated with suicidal behaviors
Nonsyn rare variants in two additional pathways, vasopressin-regulated water reabsorption and cellular hexose transport, were found to be strongly associated with SB in the AI (FDR < 0.1). Vasopressin is an evolutionary ancient neuropeptide that is involved in regulating physiological processes such as renal water reabsorption and cardiovascular homeostasis. It also plays an important role in the modulation of emotional and social behaviors in the brain (111, 112), with vasopressin containing neurons most abundantly found in the hypothalamus. The vasopressin system is known to interact with the HPA axis (67). Indeed, cortisol response and stress reactivity within the HPA axis is well-established as an endophenotype for depression and SB, as increased cortisol level is associated with death by suicide (113, 114). HPA axis dysfunction has also been observed in those with a history of suicide attempts (114). In addition, changes in water and electrolyte metabolism have been reported in clinical studies of depressed patients (115, 116). Alcohol also uniquely interacts with the vasopressin system. Alcohol is a diuretic that promotes water loss by inhibiting the production of vasopressin. The HPA axis response to alcohol can be altered by manipulating the vasopressin system (117). The vasopressin system has thus become an emerging therapeutic target for stress and depression, as well as alcohol-related behaviors (67, 117). Gene families in the cellular hexose transport pathway mediate glucose absorption in the small intestine, glucose reabsorption in the kidney, glucose uptake by the brain across the blood-brain barrier, and glucose release by all cells in the body (118). Evidence suggests that disturbances in glucose metabolism may be associated with suicidal ideation and attempts (119). A recent study has found a significant association between blood glucose and suicide attempts in male patients with MDD (120). Taken together, our findings suggest that pathways underlying vasopressin-regulated water reabsorption and the cellular hexose transport system warrant additional investigation in association with SB.
Strengths and Limitations
The results of this study should be interpreted in light of several limitations. The analyses were not meant to generate a comprehensive model of suicide risk and AUD in this community group, but rather to determine whether specific genetic associations could be identified for suicidal thoughts and acts, and between SB and AUD phenotypes using genetic analyses. A larger sample, powered to access and assess additional variables associated with suicide risk, particularly according to the above systems, is recommended. Next, our findings may not generalize to other American Indians in the population from which the sample was drawn or be representative of all American Indians, as rates of AUD and suicide vary among tribes (17, 19, 121). In addition, we used retrospective data for lifetime measures of suicide risk, which are subject to recall bias and may include reporting bias for psychiatric symptoms. Lifetime suicidal ideation and behaviors were assessed using a subscale of the SSAGA as well as verified death records. Prospective investigations, using validated measures of suicidal symptom severity and intensity of suicidal ideation, and indexing the severity and lethality of SB, are recommended to further elucidate specific symptom relationships. Despite these limitations and in consideration of the current sample size, we were able to identify several genes and pathways implicated in SB and AUD severity. Our findings suggest that rare variant analysis in a high-risk population, enabled by whole-genome sequencing, has the potential to identify novel genetic factors influencing suicidal behaviors. Bivariate association analysis between comorbid disorders may further increase statistical power when the same loci potentially contribute to both disorders.
In conclusion, we conducted the first genome-wide bivariate association analysis for SB and AUD, and identified five genome-wide significant loci. We also conducted the first large-scale rare variant analysis and identified 11 novel genes and three pathways for SB. Of particular importance, this study represents the first investigation of genetic factors for SB in an American Indian population that has high risk for suicide. Although our findings may be population specific, the rare functional mutations relating to PEDF and HIF regulation may suggest an important mechanism underlying suicidal behaviors and a potential therapeutic target for treatment and intervention in the prevention of suicide.