Major depressive disorder is associated with mitochondrial ND6 T14502C mutation in two Han Chinese families

Abstract

BACKGROUND

Globally, the World Health Organization ranks major depressive disorder (MDD) as the leading cause of disability. However, MDD molecular etiology is still poorly understood.

AIM

To explore the possible association between mitochondrial ND6 T14502C mutation and MDD.

METHODS

Clinical data were collected from two pedigrees, and detailed mitochondrial genomes were obtained for the two proband members. The assessment of the resulting variants included an evaluation of their evolutionary conservation, allelic frequencies, as well as their structural and functional consequences. Detailed mitochondrial whole genome analysis, phylogenetic, and haplotype analysis were performed on the probands.

RESULTS

Herein, we reported the clinical, genetic, and molecular profiling of two Chinese families afflicted with MDD. These Chinese families exhibited not only a range of onset and severity ages in their depression but also extremely low penetrances to MDD. Sequence analyses of mitochondrial genomes from these pedigrees have resulted in the identification of a homoplasmic T14502C (I58V) mutation. The polymorphism is located at a highly conserved isoleucine at position 58 of ND6 and distinct mitochondrial DNA (mtDNA) polymorphisms originating from haplogroups M10 and H2.

CONCLUSION

Identifying the T14502C mutation in two individuals with no genetic relation who exhibit symptoms of depression provides compelling evidence that this mutation may be implicated in MDD development. Nonetheless, the two Chinese pedigrees that carried the T14502C mutation did not exhibit any functionally significant mutations in their mtDNA. Therefore, the phenotypic expression of the T14502C mutation related to MDD may be influenced by the nuclear modifier gene(s) or environmental factors.

Key Words: Major depressive disorder; Mitochondrial DNA; ND6 T14502C; Mutation; Haplogroup; Chinese

Core Tip: In this study, we report the clinical, genetic and molecular characterization of two Chinese families with major depressive disorder (MDD). Sequence analyses of mitochondrial genomes from these pedigrees revealed homoplasmic T14502C (I58V) mutation. This observation of the T14502C mutation in two genetically unrelated individuals who suffer from depression strongly suggests that this mutation might play a role in the development of MDD.

INTRODUCTION

Major depressive disorder (MDD) is a widespread mental health condition defined by a significant change in mood accompanied by a loss of interest, motivation, or pleasure. Moreover, individuals with MDD may show other symptoms, including changes in appetite, sleep patterns, pain perception, energy levels, and symptoms related to cognitive functions or content[1]. MDD is associated with a significant decrease in functioning, a disruption of normal work-related activities, and substantial public health expenditures. The prevalence of MDD is indeed on the rise globally, and it significantly impacts people’s lives[2-4]. However, MDD molecular etiology is still poorly understood[5]. Extensive studies have shown that the molecular mechanism of depression is highly complex, which is multi-mechanism and multi-pathway rather than a single pathway[6,7]. According to the available literature, various hypotheses and pilot studies have indicated the significant involvement of innate immunity, inflammation, oxidative stress, and neurotrophic factors for synapse function and mitochondrial dysfunction in MDD[8-12].

The mitochondria are essential organelles in eukaryotic cells and are primarily responsible for producing adenosine-triphosphate (ATP) through the oxidative phosphorylation pathway. In addition, they perform crucial functions in numerous metabolic, regulatory, and developmental mechanisms[13]. Several studies have investigated the association between MDD and mitochondria. The role of mitochondria is crucial in the proper functioning of neurons, and it has been observed that patients with MDD experience impaired mitochondrial function[14-16]. Due to the fact that mitochondrial DNA (mtDNA) inheritance is exclusively through the maternal lineage, several studies have demonstrated the presence of “matrilineal inheritance” in families affected by depression. For instance, a significantly high incidence of depression was reported in mothers with probable maternal inheritance[17]. Additionally, a slight maternal bias in susceptibility toward developing depression was discovered [18].

Patients with MDD have been observed to exhibit a higher incidence of mtDNA deletion mutations compared to control groups or to display mtDNA mutations similar to those observed in mitochondrial disorders[19]. Our previous familial study found that MDD may be associated with mitochondrial T3394C mutations[20]. In this study, we aimed to explore the possible association between mitochondrial ND6 T14502C mutation and MDD. This study was to conduct a characterization of the clinical, genetic, and molecular features of two Chinese families that may have inherited MDD maternally. The ND6 gene mutation T14502C was identified through molecular analysis in two Chinese families. Furthermore, the research employed polymerase chain reaction (PCR) amplification of fragments that covered the entire mitochondrial genome, followed by DNA sequence analysis, for a better understanding of the role of mitochondrial haplotype in the phenotypic expression of the T14502C mutation in the two Chinese families.

MATERIALS AND METHODS

Patients and subjects

Two Han Chinese families (Figure 1) were identified through the Psychiatric Clinic of Ningbo Kangning Hospital, Zhejiang Province. Structured clinical interviews were utilized to establish MDD diagnosis for the probands who met diagnostic and statistical manual of mental disorders diagnostic criteria[21]. Additionally, a board-certified psychiatrist conducted a separate diagnostic evaluation to verify the diagnosis. The exclusion criteria of this study included substance-induced psychotic disorder, eating disorder, anxiety disorder, post-traumatic stress disorder, or bipolar disorder. Upon elucidating the objective of the present investigation and obtaining written informed consent, the following information was gathered: Age, gender, family history of MDD, history of suicide attempt, psychotic symptoms, and the Hamilton depression rating scale (HDRS)[22]. The probands diagnosed with MDD scored a minimum of 17 on the 17-item HDRS. The degree of MDD was defined based on HDRS, with the following classification: Normal < 7; Mild = 7-17; Moderate = 18-24; Severe > 24.

Figure 1 Two Chinese pedigrees with major depressive disorder. Patients are indicated by filled symbols. Arrow denotes the probands.

The Ningbo Kangning Hospital Ethics Committee approved protocols requiring informed consent, blood samples, as well as clinical assessments from the family members who participated in this study. Members belonging to these pedigrees were extensively interviewed to ascertain their personal or familial MDD medical histories and other clinical abnormalities. The 99 control DNA samples were obtained from the health screening clinic at Ningbo Kangning Hospital with the aim of conducting mtDNA mutation screening. The participants were interviewed to determine the absence of any significant psychiatric disorder in their medical history and to confirm that they were not currently under the influence of psychotropic medication. Upon study entry, all participants were confirmed to be medically healthy except for MDD, with no history of chronic medical conditions, absence of acute infection, and non-pregnant status.

Mutational analysis of the mitochondrial genome

Pure-gene DNA isolation kits (Gentra Systems) were utilized for isolating the genomic DNA from the obtained whole blood samples. The PCR amplification of complete mitochondrial genomes of the two probands was carried out using 24 overlapping fragments and sets of light (L)- and heavy (H)-strand oligonucleotide primers, as described earlier[23]. The fragments were purified and sequenced directly through the Big Dye Terminator Cycle sequencing reaction kit in an applied biosystems 3700 automated DNA sequencer. Subsequently, these results were compared to the updated consensus ambridge sequence (GenBank accession number: NC012920)[24]. The Seqweb program GAP program was utilized for alignments of DNA and protein sequences. The same method was used to analyze blood samples from 99 normal controls.

Phylogenetic analysis and haplogroup analyses

Four vertebrate mtDNA sequences were used in the interspecific analysis, including Homo sapiens, mouse, bovine, and Xenopus laevis[25]. The evaluation of conservation was conducted through a comparative analysis of four different vertebrate species. All mtDNA sequences from the two Chinese probands with the T14502C mutation were assigned to Asian mitochondrial haplogroups through the nomenclature of mitochondrial haplogroups[26].

RESULTS

Clinical presentation

The proband (II-2) of the family NB053, aged 43 years, as indicated in Table 1, reported experiencing depression and sought treatment at the Psychiatric Clinic at the Ningbo Kangning Hospital. HDRS indicated a score of 26, and the participant did not report any previous suicide attempts, thus exhibiting a typical clinical feature of MDD. No further anomalies were detected during the psychiatric assessment. Additionally, there was no significant medical history present in her records. The family originates from Zhejiang Province in Eastern China. Nonetheless, MDD was not observed in this family’s other five matrilineal relatives.

Table 1 Summary of clinical molecular data for two probands carrying the ND6 T14502C mutation.

Subject	Gender	Age of test (years)	Age of onset (years)	First episode	History of suicide attempts	Psychotic symptoms	HDRS	Level of depression	MtDNA haplogroup
NB053-II-2	F	43	43	Yes	No	Yes	26	Severe	M10
2NB038-II-3	M	53	52	Yes	No	No	23	Moderate	H2

F: Female; M: Male; HDRS: Hamilton depression rating scale; MtDNA: Mitochondrial DNA.

The proband (III-3) in the 2NB038 pedigree, aged 53 years, came to the Psychiatric Clinic at Ningbo Kangning Hospital. He was diagnosed with MDD one year ago. The participant had a score of 23 on the HDRS with no history of suicide attempts, indicating typical clinical features of MDD. Additionally, there was no significant medical history present in the records. The family originates from Zhejiang Province in Eastern China. MDD was not observed in any of the other matrilineal relatives.

In addition, no substantiated evidence indicates that any individual belonging to these families exhibited any other identifiable factor that could be attributed to MDD. Comprehensive family medical histories of the participants revealed no additional clinical abnormalities (diabetes, hearing impairment, and vision problems).

MtDNA analysis

In order to clarify the molecular basis of MDD, an analysis of mutations in the mitochondrial genome was conducted on the probands. The results of the conducted PCR amplification of fragments revealed that the substitution of valine with isoleucine (I58V) at amino acid position 58, caused by the T-to-C transition at position 14502 (T14502C) in the ND6 gene, has been detected in the subjects. The results also revealed that isoleucine at position 58 in ND6 is highly conserved among 22 organisms (Figure 2). Examination of allele frequency related to the T14502C mutation demonstrated that only one individual (a 45-year-old female) out of a cohort of 99 non-associated Chinese control subjects exhibited the T14502C mutation.

Figure 2 Alignment of partial ND6 polypeptides from different species. The arrow indicates the isoleucine at position 58 (I58) at the N-terminus of ND6 polypeptide, corresponding to the T14502C mutation.

In addition to the T14502C mutation that was found to be identical among the subjects, as presented in Table 2, they showed different mtDNA polymorphisms. The mitochondrial genomes exhibit several nucleotide changes, including 17 variants in the D-loop, 4 variants in the 12S rRNA gene, 5 variants in the 16S rRNA gene, 2 variants in the tRNA gene, 28 silent variants, and 13 missense mutations (1 novel/12 known) in the polypeptide-encoding genes. These missense mutations are GA4569G (Glu34 Lys), TA4573T (Met35 Lys) and A5505G (Ile346Val) in the ND2 gene; T8618C (Ile31Thr), A8701G (Thr59Ala) and A8860G (Thr112Ala) in the ATP6 gene; A10398G (Thr114Ala) in the ND3 gene; C14766T (Thr7Ile), T15071C (Tyr109His), A15326G (Thr194Ala) and G15617A (Val291Ile) in the Cytb gene. Within these, 17 mutations were identified in both probands. Moreover, we conducted a phylogenetic analysis of the variants in RNAs and polypeptides, along with sequences from other organisms, including bovines, mice, and Xenopus laevis. All variants, except for T14502C, did not demonstrate evolutionary conservation. The haplogroup affiliation of each mtDNA was established by utilizing the mtDNA sequence variations among two Chinese probands, depending on the nomenclature of mitochondrial haplogroups. The mtDNA samples obtained from the pedigrees NB053 and 2NB038 have been identified as belonging to the Eastern Asian haplogroups M10 and H2, respectively.

Table 2 Mitochondrial DNA mutations in two Chinese pedigrees with major depressive disorder.

Gene	Position	Replacement	Conservation1 (H/B/M/X)	CRS2	NB053	2NB038	Previously reported3
D-loop	73	A to G		A	G	G	Yes
	195	T to C		T		C	Yes
	263	A to G		A	G	G	Yes
	309	C to CCT		C	CCT		Yes
	310	T to C		T	C	TC	Yes
	408	T to A		T	A		Yes
	459	C to T		C	T		Yes
	489	T to C		T	C		Yes
	561	A to C		A	C		Yes
	567	A to ACCCC		A	ACCCC		Yes
	16176	C to T		C		T	Yes
	16223	C to T		C	T	T	Yes
	16232	C to T		C		T	Yes
	16311	T to C		T	C		Yes
	16355	C to T		C		T	Yes
	16362	T to C		T		C	Yes
	16519	T to C		T	C	C	Yes
12S rRNA	750	A to G	A/A/A/-	A	G	G	Yes
	813	A to G	A/A/C/T	A		G	Yes
	884	T to C	T/C/T/C	T	C		Yes
	1438	A to G	A/A/A/G	A	G	G	Yes
16S rRNA	1673	T to C	T/T/T/T	T	C		Yes
	2706	A to G	A/G/A/A	A	G	G	Yes
	3106	CN to C	C/-/T/-	CN	C	C	Yes
	3167	T to TC	T/T/C/C	T	TC		Yes
	3173	G to A	G/A/A/A	G	A		Yes
ND1	4140	C to T		C	T		Yes
ND2	4569	GA to G (Glu34 Lys)	E/E/E/E	GA	G		No
	4573	TA to T (Met35 Lys)	M/M/F/I	TA	T		Yes
	4769	A to G		A	G	G	Yes
	5505	A to G (Ile346Val)	I/L/-/-	A	G		Yes
CO1	5824	G to A		G		A	Yes
	6674	T to C		T		C	Yes
	7028	C to T		C	T	T	Yes
CO2	7975	A to G		A		G	Yes
tRNALys	8296	A to G	A/A/A/A	A		G	Yes
ATP6	8618	T to C (Ile31Thr)	I/F/F/I	T		C	Yes
	8701	A to G (Thr59Ala)	T/S/L/Q	A	G		Yes
	8793	T to C		T	C		Yes
	8860	A to G (Thr112Ala)	T/A/A/T	A	G	G	Yes
	8928	T to C		T		C	Yes
	9027	C to T		C	T		Yes
	9180	A to G		A		G	Yes
CO3	9540	T to C		T	C		Yes
ND3	10289	A to G		A		G	Yes
	10398	A to G (Thr114Ala)	T/T/T/A	A	G		Yes
	10400	C to T		C	T		Yes
ND4	10873	T to C		T	C		Yes
	10908	T to C (Phe50Ser)	F/L/K/H	T		C	Yes
	11581	C to A		C		A	Yes
	11719	G to A		G	A	A	Yes
ND5	12549	C to T		C	T		Yes
	12705	C to T		C	T	T	Yes
	13152	A to G		A	G		Yes
	13422	A to G		A	G		Yes
ND6	14220	A to G		A		G	Yes
	14311	T to C		T		C	Yes
	14431	T to C		T		C	Yes
	14502	T to C (Ile58Val)	I/I/I/I	T	C	C	Yes
CYTB	14766	C to T (Thr7Ile)	T/S/T/S	C	T	T	Yes
	14783	T to C		T	C		Yes
	15040	C to T		C	T		Yes
	15043	G to A		G	A		Yes
	15071	T to C (Tyr109His)	Y/F/F/Y	T	C		Yes
	15301	G to A		G	A		Yes
	15326	A to G (Thr194Ala)	T/M/I/I	A	G	G	Yes
	15514	T to C		T		C	Yes
	15617	G to A (Val291Ile)	V/V/V/V	G	A		Yes
tRNAThr	15900	T to C	T/C/T/T	T		C	Yes

¹Conservation of amino acids for polypeptides or nucleotides for RNAs in human, bovine, mouse, and xenopus laevis.

²Cambridge reference sequence.

³See online mitochondrial genome databases http://www.mitomap.org and http://www.genpat.uu.se/mtDB/.

H: Human; B: Bovine; M: Mouse; X: Xenopus laevis; CRS: Cambridge reference sequence.

DISCUSSION

Herein, a clinical, genetic, and molecular analysis was conducted on two Chinese families with MDD. The two probands under study were middle-aged individuals who presented with MDD for the first time. Neither of them exhibited suicidal tendencies and both experienced episodes of moderate and severe depression. The pedigrees indicate that MDD is only present in the maternal lineage as a clinical phenotype, which implies that the molecular basis for this disorder is the mtDNA mutation. Distinct sets of mtDNA polymorphisms were identified through sequence analysis of the complete mitochondrial genomes in these pedigrees, along with the identical T14502C (I58V) mutation in the ND6 gene. This mutation has been associated with the occurrence of mitochondrial diseases[27-32]. The T14502C mutation resulted in the change of highly conserved isoleucine at position 58 with valine (I58V) in the ND6 gene, the core component of 58 subunits of complex I. Several studies have suggested that the dysfunction of complex I is involved in the impairment of cellular energy metabolism in the context of a depressive episode[33]. The conserved coefficient of this amino acid was 100% in 22 species. Among them, I58V is located in the third transmembrane region, which is a hydrophobic region. Changes in amino acids may affect the three-dimensional structure of proteins, thus affecting the function of ND6 protein, reducing the activity of nicotinamide adenine dinucleotide-coenzyme Q reductase, and affecting the production of mitochondrial ATP, the primary source of energy, leading to the occurrence of diseases.

Although both families exhibited typical clinical symptoms of MDD, there were variations in terms of penetrance, psychotic symptoms, and age of onset. The penetrance rates observed in the two families were 16.7% and 10.0%, respectively. The present study reveals a low penetrance of MDD among the two Chinese families with the T14502C mutation. Furthermore, the presence of this mutation in only one of 99 controls suggests that the T14502C mutation[34], analogous to other mutations, is inadequate to induce the clinical phenotype. Therefore, the phenotypic expression of the T14502C mutation requires the presence of modifying factors such as nuclear background, environmental factors, and mitochondrial haplotypes. In particular, research has demonstrated that mitochondrial haplotypes affect primary mtDNA mutation-related MDD penetrance and expressivity[35]. These mitochondrial genomes of the NB053 and 2NB038 pedigrees belong to the Eastern Asian haplogroups M10 and H2, respectively.

The homogeneous mutation T14502C has been identified in families with MDD despite having diverse genetic backgrounds. The T14502C mutation may serve as a molecular foundation for the pathogenesis of MDD, as indicated by the low penetrance of MDD in families with this mutation. However, the mutation appears inadequate to induce the phenotypic expression of MDD. This implies that additional mediators contributed to the pathogenesis of these families in a synergistic manner. Furthermore, the complete mitochondrial genome of both families was observed to not exhibit additional mutation sites that were highly conserved and functional. This observation implies that the polymorphic sites that are unique to mitochondrial haplomorphism may not have a significant impact on the pathogenesis of the two families affected by the T14502C mutation and suffering from MDD.

Furthermore, the potential implication of mtDNA epigenetics in the development of MDD remains inadequately explored, and further investigation is warranted to elucidate its role in disease etiology and therapeutic interventions. Therefore, nuclear modifier gene (s) or environmental factor (s) may be involved in these Chinese individuals’ phenotypic expression of MDD-associated T14502C mutation. The ND6 T14502C mutation may represent a mitochondrial gene mutation site correlated to MDD. The results of our study may offer novel perspectives on the pathophysiological mechanisms underlying MDD and provide essential data on therapeutic approaches and interventions for this condition.

CONCLUSION

In this study, we report the clinical, genetic and molecular characterization of two Chinese families with MDD. Identifying the T14502C mutation in two individuals with no genetic relation who exhibit symptoms of depression provides compelling evidence that this mutation may be implicated in MDD development. Nonetheless, the two Chinese pedigrees that carried the T14502C mutation did not exhibit any functionally significant mutations in their mtDNA. Therefore, the phenotypic expression of the T14502C mutation related to MDD may be influenced by the nuclear modifier gene (s) or environmental factors.

ACKNOWLEDGEMENTS

We would like to thank all patients who participated in the study. We are grateful to all the psychiatrists and nurses who participated in our current study and those research staff that contributed to the subjects’ diagnosis and clinical assessments.