Long noncoding RNAs: New evidence for overlapped pathogenesis between major depressive disorder and generalized anxiety disorder.

BACKGROUND: About half of patients with major depressive disorder (MDD) have clinically meaningful levels of anxiety. Greater severity of depressive illness and functional impairment has been reported in patients with high levels of anxiety accompanying depression. The pathogenesis for the comorbidity was still unsure. AIM: This study aimed to determine whether there would be molecular link for overlapped pathogenesis between MDD and anxiety disorder.
MATERIALS AND METHODS: Using long noncoding RNA (lncRNA) microarray profiling and reverse transcription polymerase chain reaction, six downregulated lncRNAs and three upregulated lncRNAs had been identified to be the potential biomarkers for MDD and generalized anxiety disorder (GAD), respectively. Then, the lncRNAs were cross-checked in forty MDD patients, forty GAD patients, and forty normal controls.
RESULTS: Compared with normal controls, six downregulated MDD lncRNAs also had a significantly lower expression in GAD (P < 0.01), and there was no significant difference between GAD and MDD (P > 0.05). In addition, three upregulated GAD lncRNAs had no different expression in MDD (P > 0.05), but there was remarkable difference between MDD and GAD (P < 0.01).
CONCLUSIONS: These results indicated that lncRNAs in peripheral blood mononuclear cells could be potential molecular link between MDD and GAD, which added new evidence to the overlapped pathogenesis and suggested that anxious depression could be a valid diagnostic subtype of MDD.

INTRODUCTION

Psychiatric comorbidity is defined as the presence, either simultaneously or in succession, of two or more specific disorders in an individual within a specified period. The Epidemiologic Catchment Area study and the National Comorbidity Study, both in the United States, found that 54% and 56%, respectively, of respondents with a lifetime history of at least one Diagnostic and Statistical Manual of Mental Disorders, 3rd Edition (DSM-III)/DSM-III-Revision disorder also met criteria for some other mental disorder.[1] A large number of studies conclude that generalized anxiety disorders (GADs) occur in patients with major depressive disorder (MDD) far more frequently than expected by chance.[2] Studies conducted in Europe and the USA have shown that comorbidity between MDD and anxiety disorders is the most prevalent comorbid condition among both genders (especially in women) and associated with younger age, lower educational attainment, and unemployment.[3] A community-based epidemiological survey in mainland China also reported that 63% of individuals with mood disorder had at least one type of anxiety disorder.[4] Comorbidity induces harmful consequences, such as more severe symptoms,[56] greater functional disability,[78] longer illness course,[1] higher rates of neuroticism,[9] earlier onset age, greater suicidal risk and poorer treatment response,[10] lower social competence,[11] and worse prognosis.[12]

In view of the prevalence and consequences of psychiatric comorbidity, identifying individuals at the greatest risk for comorbidity early becomes a public health priority. There was a key unanswered question related to the cause of comorbidity: the direction and mechanisms underlying causal links, as well as the potential spurious nature of such links.[11] Common genetic predisposition to MDD and anxiety[13141516] is well known, but the genetic factors explain only part of the co-occurrence of illness, and nonshared environment explains 40% covariance between MDD and GAD for female twins.[11] The relative contribution of genetic and nongenetic/environmental factors to this comorbidity has been examined in a series of population-based twin studies,[2] indicating that the interaction between genetic and environmental determinants plays an important role in the onset of comorbidity MDD and GAD, but they did not know the reason. With the development of RNA deep sequencing technology and bioinformatics, epigenetics can help to explain the interaction between genetic and environment. Epigenetics is defined as inheritable and reversible phenomena that affect gene expression without altering the underlying base-pair sequence[17] that environmental events and behavioral experience induce epigenetic changes at particular gene loci, and these changes help shape neuronal plasticity, function, and behavior, hence contributed to the pathogenesis of MDD.[18]

Long noncoding RNA (lncRNA), one major mechanism of epigenetics, is more than 200nt in length, not encoding proteins itself, but regulating gene expression in multilevel form of RNA, such as epigenetic regulation, transcriptional regulation, and posttranscriptional regulation. LncRNAs have been implicated in neurodevelopmental disorders such as Rett syndrome,[19] autism,[2021] schizophrenia (SZ),[22] and fragile X syndrome.[23] Using LncRNA microarray profiling and reverse transcription polymerase chain reaction (RT-PCR), the differentially expressed lncRNAs in MDD (downregulated TCONS_00019174, ENST00000566208, NONHSAG045500, ENST00000517573, NONHSAT034045, and NONHSAT142707)[24] and GAD (upregulated ENST00000505825, NONHSAG017299, and NONHSAT078768) compared with the normal control. In this study, all of the lncRNAs in MDD and in GAD were cross-checked again in another disease and controls, respectively, to ascertain whether there is the same lncRNAs expression both in MDD and GAD.

MATERIALS AND METHODS

Subjects

Forty MDD patients and forty GAD patients who met the criteria of the DSM-IV were enrolled from Changzhou Maternity and Child Health Care Hospital and No. 102 Hospital of the Chinese People's Liberation Army from May 2014 to February 2015. Diagnoses were independently made by two psychiatry attending doctors using the Chinese version of the modified Structured Clinical Interview for DSM-IV, patient version (SCID-I/P).[25] Both MDD patients and GAD patients were assessed using the 24-item Hamilton Depression Scale (HAMD-24)[26] and 14-item Hamilton Anxiety Scale (HAMA).[27] All the patients were first visitors or before any clinical treatment, drug naïve from any antidepressant for at least 3 months before study enrollment, no previous history of organic disease (such as heart disease, diabetes, and Parkinson's disease), and no other psychiatric disorders.

The forty healthy controls were recruited from the community nearby, having no family history of major psychiatric disorders (SZ, bipolar disorder, mania, anxiety, and substance abuse), without any history of severe traumatic events within 6 months. Controls were also assessed through the modified SCID-I/P, HAMD, and HAMA to rule out prior incidence of mental disease. MDD patients, GAD patients, and healthy controls were matched in gender, age, and ethnicity at a ratio of 1:1. The study was approved by the Ethical Committee for Medicine of Changzhou Maternity and Child Health Care Hospital and No. 102 Hospital of Chinese People's Liberation Army. All patients or their legal guardians provided written informed consent.

Reverse transcription polymerase chain reaction

Whole blood (5 ml) was collected in ethylenediaminetetraacetic acid tube, and peripheral blood mononuclear cells (PBMCs) were isolated from forty MDD patients, forty GAD patients, and forty controls. Total RNAs were extracted from the PBMCs using TRIzol (Invitrogen, Carlsbad, CA, USA) and the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, quantified by NanoDrop ND-2000 (Thermo Scientific, Delaware, ME, USA), DNase treated (TURBO DNase, Life Technologies), and reverse transcribed (Superscript III; Invitrogen). Then, six downregulated lncRNAs in GAD, three upregulated lncRNAs in MDD, and both of them in controls were performed through RT-PCR using Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems, Inc., USA). The method and process can be referred to our previous article.[24] Data were collected using the SDS 2.3 software (Applied Biosystems, Foster City, CA, USA) and DataAssist version 3.0 Software (Thermo Fisher Scientific, MA, USA). After normalized to β-actin, the expression levels of lncRNAs were calculated using the 2-ΔΔCt method.

Statistical analysis

All statistical analyses were carried out using DataAssist version 3.0 Software (Thermo Fisher Scientific, MA, USA), SPSS version 20.0 Software (Chicago, IL, USA), and GraphPad Prism 5 (Graphad Software Inc., San Diego, CA, USA). Demographic variables were compared between patients and matched controls with Chi-square test for qualitative variables and ANOVA for quantitative variables. The difference of six lncRNAs in GAD and three lncRNAs in MDD with healthy controls was analyzed by Wilcoxon rank sum test. P < 0.05 (two-tailed) was considered with statistical significance.

RESULTS

Demographic data of the major depressive disorder patients, generalized anxiety disorder patients, and normal controls

Using Chi-square test and ANOVA, there were no significant differences between patients and control group in age and sex distribution, but HAMD and HAMA scores were significantly different, as shown in Table 1.

Table 1

Clinical characteristics of MDD patients, GAD patients and normal controls

Differentially expressed long noncoding RNAs in major depressive disorder and generalized anxiety disorder

Using lncRNA microarray profiling and quantitative RT-PCR, six downregulated lncRNAs in MDD (TCONS_00019174, ENST00000566208, NONHSAG045500, ENST00000517573, NONHSAT034045, and NONHSAT142707) and three upregulated lncRNAs in GAD (ENST00000505825, NONHSAG017299, and NONHSAT078768) were found to be differentially expressed, as shown in Table 2.

Table 2

Six down-regulated lncRNAs in MDD and three up-regulated lncRNAs in GAD

Comparison of six downregulated major depressive disorder long noncoding RNAs in generalized anxiety disorder and controls

As shown in Figure 1, using Wilcoxon rank sum test, six downregulated lncRNAs in MDD were also significantly lower expressed comparing with normal controls (P < 0.01), and there was no significant difference between GAD and MDD (P > 0.05).

Figure 1

Comparison of the six downregulated long noncoding RNAs expression in generalized anxiety disorder and normal controls. NC = Normal control, GAD = Generalized anxiety disorder, MDD = Major depressive disorder

Comparison of the three upregulated generalized anxiety disorder long noncoding RNAs in major depressive disorder and controls

Using Wilcoxon rank sum test, there was no significant difference between three upregulated lncRNAs in MDD and in controls (P > 0.05). On the contrary, there was a significant difference between MDD and GAD (P < 0.01) [Figure 2].

Figure 2

Comparison of the three downregulated long noncoding RNAs expression in major depressive disorder and normal control. NC = Normal control, GAD = Generalized anxiety disorder, MDD = Major depressive disorder

DISCUSSION

Previous studies indicate that high levels of comorbidity existed between major depression and GAD, and anxiety symptoms can fluctuate over time. A follow-up study of individuals with uncomplicated GAD at baseline found that at the end of 3 years, the diagnosis of 24% of the participants had changed to depressive disorders and that of a further 16% had changed to depressive disorders comorbid with GAD.[28] Scholars had tried to find whether there were some unique genetic effects on anxiety or depressive disorders. Maron et al.[29] screened 90 SNPS in genes associated with the neurobiology of anxiety and found that polymorphisms in cholecystokinin-related genes and the serotonin 1A receptor (5-hydroxytryptamine receptor 1A) were associated with affective disorders. In a sample of Caucasian Vietnam veterans in Australia, the TaqI A allele of the dopamine receptor gene was associated with comorbid anxiety and depression.[30] PLXNA2 gene, which encodes for plexin 2A, had been implicated in comorbidity between depression and anxiety.[31] These studies also demonstrate that the genetic factors explain only part of the co-occurrence of illnesses. The paths from DNA to psychopathology were long and tortuous, involved the action, interaction, and correlation of many genes and environmental factors, so-called genotype × environment interaction and genotype-environment correlation,[16] whose effects change and/or accumulate through development as a result of endogenous mechanisms and the interplay between the person and the environment.[32] Early studies about the interaction between genes and environment are based on multivariate twin modeling,[1533] no further molecular biology research. With the development of RNA deep sequencing technology and bioinformatics, epigenetics became to fill the gaps in this area. Its molecular mechanism includes methylation of DNA, RNA interference, noncoding RNA (ncRNA), protein modification, and histone acetylation.

A significant number of brain-enriched or brain-specific lncRNAs are found adjacent to genes encoding transcriptional regulators and key drivers of neural development,[34] including those involved in the regulation of stem cell pluripotency, neuronal differentiation, and synaptogenesis.[19353637] In the present study, we found that the six downregulated lncRNAs expressed the same both in MDD and GAD, which illustrated that there could be overlapped pathogenesis between depression and anxiety, also directly providing evidence why part of anxiety patients would eventually transform into depression. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, our previous paper showed that the lncRNAs in this study were all involved in the following pathways: (1) Translational elongation, (2) protein transport, (3) ribosome activities, (4) RNA degradation, (5) DNA replication.[24] Besides, the ncRNAs expression in psychiatric disorders had a significant change after standard treatment, even returned to normal levels when symptoms remised,[243839] indicating lncRNAs could be objective molecular index to evaluate the therapeutic effect. In clinical treatment, antidepressants (paroxetine, sertraline, fluoxetine, venlafaxine hydrochloride, etc.,) can have therapeutic effect both in anxiety and depressive symptoms, but single use of anxiolytics or sedative/hypnotics (alprazolam, buspirone, etc.) has nothing to do with the core symptoms of depression. Thus, we speculated that when doctors are confused about the diagnosis and therapy, testing the lncRNAs expression in PBMCs can provide advice for them.

Comorbid mental disorders are so common that the rigid application of a diagnostic hierarchy will not adequately identify clinically important differences between patients. To improve the clinical characterization of depression and the effectiveness of treatments for anxiety disorders, clinicians must simultaneously assess the severity of both depressive and anxiety symptoms. If both two types of symptoms are prominent, revising their treatment regimens accordingly is needed. Based on this concept, although DSM-IV[40] did not recognize anxious depression as a diagnostic subtype, but in DSM-V, in the chapter on bipolar and related disorders and the chapter on depressive disorders, a specifier for anxious distress is delineated, the specifier is intended to identify patients with anxiety symptoms that are not part of the bipolar diagnostic criteria.[41] This study provided a theoretical support for the alteration.

CONCLUSIONS

In conclusion, this study found that lncRNAs in PBMCs might be molecular link between MDD and GAD, and the results were supportive of the view that anxious depression could be a valid diagnostic subtype of MDD and suggested the need for additional emphasis on the depression measurement of patients with anxiety disorder.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.