Association between the single nucleotide variants of the mitochondrial cytochrome B gene (MT-CYB) and the male infertility.

BACKGROUND: Idiopathic male infertility can be attributed to genetic predispositions that affect sperm performance and function. Genetic alterations in the mitochondrial DNA (mtDNA) have been linked to certain types of male infertility and abnormal sperm function. Mutations in the mitochondrial cytochrome B (MT-CYB) gene might lead to some deficiencies in mitochondrial function. Thus, in the current study, we aimed to investigate the effect of mutations in the MT-CYB gene on sperm motility and male infertility. METHODS AND RESULTS: Semen specimens were collected from 111 men where 67 men were subfertile and 44 were fertile. QIAamp DNA Mini Kit and REPLI-g Mitochondrial DNA Kit from QIAGEN were used to isolate and amplify the mitochondrial DNA. Followed by PCR and Sanger sequencing for the target sequence in the MT-CYP gene. Sequencing of the MT-CYB gene revealed a total of thirteen single nucleotide polymorphisms (SNPs). Eight SNPs were non-synonymous variant (missense variant) including: rs2853508, rs28357685, rs41518645, rs2853507, rs28357376, rs35070048, rs2853506, and rs28660155. While five SNPs were Synonymous variant: rs527236194, rs28357373, rs28357369, rs41504845, and rs2854124. Among these SNPs, three variants showed a significant difference in the frequency of the genotypes between subfertile and fertile groups: rs527236194 (T15784C) (P = 0.0005), rs28357373 (T15629C) (P = 0.0439), and rs41504845 (C15833T) (P = 0.0038). Moreover, two SNPs showed a significant association between allelic frequencies of rs527236194 (T15784C) (P = 0.0014) and rs41504845 (C15833T) (P = 0.0147) and male subfertility. CONCLUSION: The current study showed a significant association between the MT-CYB gene polymorphisms and the development of male infertility. In particular, rs527236194, rs28357373 and rs41504845 variants were found to be the most related to the subfertility group. Further studies on larger and other populations are required to reveal the exact role of this gene in the development of male infertility. In addition, functional studies will be helpful to elucidate the molecular impact of the MT-CYP polymorphisms on mitochondrial function.

A Multi-copy Nucleic Acid-Based Diagnostic Test for Bovine Tropical Theileriosis.

BACKGROUND: Bovine tropical theileriosis (BTT) is a haemoprotozoan tick-borne disease that implicates huge losses to livestock in terms of considerable mortality and morbidity in tropical and subtropical regions of the globe. Currently available diagnostic methods have less specificity and sensitivity towards the detection of Theileria species. Therefore, an attempt was made to diagnose Theileria annulata by targeting a multi-copy gene, viz. mitochondrially encoded cytochrome b (MT-CYB) gene via polymerase chain reaction (PCR) in different agro-zones of India. METHODS AND RESULTS: 129 cattle blood samples were collected from major livestock rearing regions of India and processed for both molecular and microscopic techniques. Screening of Giemsa-stained thin blood smears was able to detect 14 samples (10.85%) as positive for T. annulata. However, the MT-CYB gene-based PCR assay detected 107 samples (82.94%) positive for T. annulata out of 129 samples. Furthermore, the MT-CYB gene-based PCR assay was standardized in terms of its sensitivity and specificity. Specificity of PCR assay was evaluated against other common haemoprotozoan parasites of tropical countries viz. Babesia bigemina, Anaplasma marginale and Trypanosoma evansi. The multi-copy MT-CYB gene-based PCR assay provided an optimum level of sensitivity (up to the level of 10 femtogram) and high specificity. Haematological examination (Hb, PCV and TLC) of 113 samples revealed significantly (p < 0.05) decreased Hb and PCV levels in positive animals in comparison with the control group of healthy animals. However, the control group had significantly higher (p < 0.001) TLC levels than the positive group. CONCLUSION: The MT-CYB gene-based PCR assay was found to be highly sensitive that can accurately detect the occurrence of T. annulata infection in carrier animals which are potential infection sources to healthier populations in naive demographic locations through infected ticks.

Mitochondrial Genome Study Identifies Association Between Primary Open-Angle Glaucoma and Variants in MT-CYB, MT-ND4 Genes and Haplogroups.

Background and purpose: Primary open-angle glaucoma (POAG) is an optic neuropathy characterized by death of retinal ganglion cells and atrophy of the optic nerve head. The susceptibility of the optic nerve to damage has been shown to be mediated by mitochondrial dysfunction. In this study, we aimed to determine a possible association between mitochondrial SNPs or haplogroups and POAG. Methods: Mitochondrial DNA single nucleotide polymorphisms (mtSNPs) were genotyped using the Illumina Infinium Global Screening Array-24 (GSA) 700K array set. Genetic analyses were performed in a POAG case-control study involving the cohorts, Groningen Longitudinal Glaucoma Study-Lifelines Cohort Study and Amsterdam Glaucoma Study, including 721 patients and 1951 controls in total. We excluded samples not passing quality control for nuclear genotypes and samples with low call rate for mitochondrial variation. The mitochondrial variants were analyzed both as SNPs and haplogroups. These were determined with the bioinformatics software HaploGrep, and logistic regression analysis was used for the association, as well as for SNPs. Results: Meta-analysis of the results from both cohorts revealed a significant association between POAG and the allele A of rs2853496 [odds ratio (OR) = 0.64; p = 0.006] within the MT-ND4 gene, and for the T allele of rs35788393 (OR = 0.75; p = 0.041) located in the MT-CYB gene. In the mitochondrial haplogroup analysis, the most significant p-value was reached by haplogroup K (p = 1.2 × 10-05), which increases the risk of POAG with an OR of 5.8 (95% CI 2.7-13.1). Conclusion: We identified an association between POAG and polymorphisms in the mitochondrial genes MT-ND4 (rs2853496) and MT-CYB (rs35788393), and with haplogroup K. The present study provides further evidence that mitochondrial genome variations are implicated in POAG. Further genetic and functional studies are required to substantiate the association between mitochondrial gene polymorphisms and POAG and to define the pathophysiological mechanisms of mitochondrial dysfunction in glaucoma.

Liver mitochondrial DNA damage and genetic variability of Cytochrome b - a key component of the respirasome - drive the severity of fatty liver disease.

BACKGROUND AND AIMS: The progression of nonalcoholic fatty liver disease (NAFLD) into severe histological forms (steatohepatitis - NASH) is paralleled by the occurrence of complex molecular processes. Mitochondrial dysfunction is a hallmark feature of advanced disease. Mitochondrially encoded cytochrome B (cytochrome b, MT-CYB), a member of the oxidative phosphorylation system, is a key component of the respirasome supercomplex. Here, we hypothesized that NAFLD severity is associated with liver tissue cytochrome b mutations and damaged mitochondrial DNA (mtDNA). METHODS: We included 252 liver specimens of NAFLD patients - in whom histological disease ranged from mild to severe - which were linked to clinical and biochemical information. Tissue molecular explorations included MT-CYB sequencing and analysis of differential mtDNA damage. Profiling of circulating Krebs cycle metabolites and global liver transcriptome was performed in a subsample of patients. Tissue levels of 4-hydroxynonenal - a product of lipid peroxidation and 8-hydroxy-2'-deoxyguanosine, a marker of oxidative damage - were measured. RESULTS: Compared to simple steatosis, NASH is associated with a higher level of MT-CYB variance, 12.1 vs. 15.6 substitutions per 103  bp (P = 5.5e-10). The burden of variants was associated with increased levels of 2-hydroxyglutarate, branched-chain amino acids, and glutamate, and changes in the global liver transcriptome. Liver mtDNA damage was associated with advanced disease and inflammation. NAFLD severity was associated with increased tissue levels of DNA oxidative adducts and lipid peroxyl radicals. CONCLUSION: NASH is associated with genetic alterations of the liver cellular respirasome, including high cytochrome b variation and mtDNA damage, which may result in broad cellular effects.

Phylogeography and Phylogenetic Evolution in Tibetan Sheep Based on MT-CYB Sequences.

To date, molecular genetics and population studies in Tibetan sheep (Ovis aries) have been limited, and little is known about the phylogenetic evolution and phylogeography of Tibetan sheep populations. The aim of the present research was to explore phylogeography and phylogenetic evolution of Tibetan sheep populations, on the basis of mitochondrial DNA (mtDNA) gene MT-CYB (1140 base pairs). Our dataset consisted of 641 MT-CYB sequences from the same amount of animals belonging to 15 populations of Tibetan sheep living in the Qinghai-Tibetan Plateau, China. Haplotype and nucleotide diversities were 0.748 ± 0.010 and 0.003 ± 0.001, respectively. The analysis of phylogeography revealed the presence of two formerly described haplogroups in 15 populations of Tibetan sheep, however only one haplogroup was present in Awang sheep. Moreover, 641 Tibetan sheep were distributed into a minimum of two clusters by clustering analysis. The 15 Tibetan sheep populations and 19 reference populations of 878 individuals were separated into six main groups based on their substitutions per site, from which we constructed a phylogenetic tree. Minor differences in branching order of various taxa between trees acquired from either gene were observed. This study provides insights on the origins and phylogenetic evolution of populations residing in the Qinghai-Tibetan Plateau, which will aid information of future conservation programs aimed at conserving this valuable genetic resource.

Evidence of diaphragmatic dysfunction with severe alveolar hypoventilation syndrome in mitochondrial respiratory chain deficiency.

Diaphragmatic dysfunction has been reported in congenital myopathies, muscular dystrophies, and occasionally, mitochondrial respiratory chain deficiency. Using a minimally invasive procedure in 3 young girls, 1 with a heteroplasmic MT-CYB mutation and 2 with biallelic pathogenic TK2 variants, we provided functional evidence of diaphragmatic dysfunction with global respiratory muscle weakness in mitochondrial respiratory chain deficiency. Analysis of respiratory muscle performance using esogastric pressures revealed paradoxical breathing and severe global inspiratory and expiratory muscle weakness with a sniff esophageal inspiratory pressure and a gastric pressure during cough averaging 50% and 40% of predicted values, respectively. This diaphragmatic dysfunction was responsible for severe undiagnosed nocturnal hypoventilation, requiring noninvasive ventilation. Our results underline the interest of this minimally invasive procedure for the evaluation of respiratory muscle performance and its potential value for the monitoring of future clinical trials in respiratory chain deficiency.

Fine-tuning of the respiratory complexes stability and supercomplexes assembly in cells defective of complex III.

The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649-15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII2, yielding a small amount of active CI+CIII2 supercomplex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII2 supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH2-linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII2, but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII2 and CIV2 and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion.

Mitochondrial DNA Mutations and Rheumatic Heart Diseases.

Acute rheumatic fever (ARF) is an autoimmune disease affecting the heart-valve endocardium in its final stage. Although rare in developing countries, ARF persists in third-world countries, particularly Senegal, where rheumatic heart diseases (RHDs) are the most common pediatric cardiovascular pathology. This study aimed to investigate mutations in MT-CYB in ARF and RHD in Senegalese patients. MT-CYB was amplified from blood samples from ARF patients at the Clinical of Thoracic and Cardiovascular Surgery of Fann National University Hospital Centre, Dakar, Senegal (control group, healthy individuals) and sequenced. More than half of the MT-CYB mutations (58.23%) were heteroplasmic. Transitions (61.67%) were more frequent than transversions (38.33%), and non-synonymous substitutions represented 38.33% of mutations. Unoperated RHD patients harbored frequent MT-CYB polymorphisms (7.14 ± 14.70 mutations per sample) and accounted for 72.73% of mutations. Paradoxically, subjects undergoing valvular replacement harbored infrequent polymorphisms (1.39 ± 2.97 mutations per patient) and lacked 36 mutations present in unoperated subjects. A genetic differentiation was observed between these two populations, and the mutations in operated subjects were neutral, while those in unoperated subjects were under positive selection. These results indicate a narrow link (perhaps even causal) between MT-CYB mutations and ARF and its complications (i.e., RHDs) and that these mutations are largely deleterious.

Somatic Mitochondrial Mutations in Oral Cavity Cancers among Senegalese Patients.

Background: Somatic mutations affecting the mitochondrial DNA (mtDNA) have been frequently observed in human cancers and proposed as important oncological biomarkers. However, the exact mtDNA mutations that is responsible for the pathogenesis of cancer remains unclear. The aim of this study was to investigate somatic mutations in the MT-CYB and D-Loop regions of mitochondrial DNA (mtDNA) in oral cavity cancers from Senegalese patients. Methods: MT-CYB and the D-Loop of mtDNA derived from 45 oral cavity cancer tissues and 21 control blood samples were assessed by PCR and sequencing. The sequences of MT-CYB and the D-Loop from cancerous tissues were compared with control sequences, and sequence differences were recognized as somatic mutations. Results: Overall, 389 somatic mtDNA mutations were identified, most of which (79.43%) were located in the D-Loop region. The majority of base substitution mutations were G-to-A (63.93%) and T-to-C (16.39%) transitions. In the protein-coding MT-CYB gene, 29 missense mutations were observed. The pathogenic mutation load of MT-CYB was 3.11%. Pathogenic mutations were carried by 25% of patients. pArg76Pro (pArg282Pro in rCRS) was novel and was the most common pathogenic mutation observed. Conclusion: These results strongly indicate that mtDNA mutations are a potential marker of oral cavity cancer.

Identification of a broad spectrum of mammalian and avian species using the short fragment of the mitochondrially encoded cytochrome b gene.

Mitochondrial DNA (mtDNA), especially the gene for cytochrome b (MT-CYB), has been found to be highly informative for species identification. In this study, we present the results of the analysis of a 127 bp long fragment of MT-CYB, amplified using universal primers, variable enough to be used for species identification and discrimination, even in highly degraded animal samples. The total number of analyzed species in this study was 30, including 17 mammalian and 13 bird species. Using a newly created primer pair, we successfully amplified and sequenced the target sequence in almost all tested species. The amplification was incomplete in just two species, and as a result, partial, but still variable sequences, were obtained. Using the target fragment we successfully identified all tested samples. Initial results suggested that the intraspecies genetic diversity of the target region, in all tested species, was low - from 0 to 4.72%. The interspecies genetic diversity of the target region, crucial for successful discrimination, showed relatively high diversity, ranging from 8.36% to 42.52%. Given its short length, the target region should be used for species determination, particularly in samples that are degraded or are low in DNA quantity.

Codon usage pattern of complex III gene of respiratory chain among platyhelminths.

Codon usage bias refers to the phenomenon where synonymous codons are used with unequal frequencies. To understand the patterns of codon usage in mitochondrial cytochrome B (MT-CYB) gene of phylum platyhelminthes we used bioinformatic approaches to analyze the protein coding sequences of five different classes - cestoda, monogenea, rabditophora, trematoda and turbellaria. It was found from nucleotide composition analysis that in all the classes, A/T-ended codons were preferred to G/C -ended codons. From box plot analysis GC1 was found to have highest response to codon usage bias. Correspondence analysis indicated that besides mutation other factors such as natural selection might also affect the codon usage pattern. Neutrality plot reveals that both mutation and natural selection played role in codon usage pattern in five classes of MT-CYB gene. Various factors namely nucleotide composition, natural selection and mutation pressure affected the codon usage pattern.

Human Mitochondrial Cytochrome b Variants Studied in Yeast: Not All Are Silent Polymorphisms.

Variations in mitochondrial DNA (mtDNA) cytochrome b (mt-cyb) are frequently found within the healthy population, but also occur within a spectrum of mitochondrial and common diseases. mt-cyb encodes the core subunit (MT-CYB) of complex III, a central component of the oxidative phosphorylation system that drives cellular energy production and homeostasis. Despite significant efforts, most mt-cyb variations identified are not matched with corresponding biochemical data, so their functional and pathogenic consequences in humans remain elusive. While human mtDNA is recalcitrant to genetic manipulation, it is possible to introduce human-associated point mutations into yeast mtDNA. Using this system, we reveal direct links between human mt-cyb variations in key catalytic domains of MT-CYB and significant changes to complex III activity or drug sensitivity. Strikingly, m.15257G>A (p.Asp171Asn) increased the sensitivity of yeast to the antimalarial drug atovaquone, and m.14798T>C (p.Phe18Leu) enhanced the sensitivity of yeast to the antidepressant drug clomipramine. We demonstrate that while a small number of mt-cyb variations had no functional effect, others have the capacity to alter complex III properties, suggesting they could play a wider role in human health and disease than previously thought. This compendium of new mt-cyb-biochemical relationships in yeast provides a resource for future investigations in humans.

Codon usage trend in mitochondrial CYB gene.

Here we reported the pattern of codon usage and the factors which influenced the codon usage pattern in mitochondrial cytochrome B (MT-CYB) gene among pisces, aves and mammals. The F1 axis of correspondence analysis showed highly significant positive correlation with nucleobases A3, C and C3 and significant negative correlation with T and T3 while F2 of correspondence analysis showed significant positive correlation with C and C3 and significant negative correlation with A and A3. From the neutrality plot, it was evident that the GC12 was influenced by mutation pressure and natural selection with a ratio of 0.10/0.90=0.11 in pisces, 0.024/0.976=0.0245 in aves and in mammals 0.215/0.785=0.273, which indicated that the role of natural selection was more than mutation pressure on structuring the bases at the first and second codon positions. Natural selection played the major role; but compositional constraint and mutation pressure also played a significant role in codon usage pattern. Analysis of codon usage pattern has contributed to the better understanding of the mechanism of distribution of codons and the evolution of MT-CYB gene.

Polymorphisms in the MT-ATP6 and MT-CYB genes in in vitro fertilization failure.

The purpose of this study was to investigate whether fertilization failure after in vitro fertilization could be explained by polymorphisms in MT-ATP6 and MT-CYB genes. We performed a prospective comparative study of 111 fresh IVF cycles in Han Chinese between July 2011 and February 2013. Human sperm mitochondrial DNA (mtDNA) variants in the MT-CYB and MT-ATP6 genes were screened by polymerase chain reaction (PCR) and direct sequencing. Forty-six couples had low fertilization rates (< or =30%) or total fertilization failure, and 65 controls with normal fertilization. One unreported point mutation (A15472G) was found in this study. There were 7 and 3 polymorphic sites in the MT-ATP6 and MT-CYB, respectively. Interestingly, the frequencies of points 8701 and 15301 homozygous variants in study group were significantly higher than those in control group. However, the frequencies of the points 8701, 9075 and 15,301 heterozygous variants in study group were significantly lower than those in control group (4.35% versus 16.92%, 15.22% versus 32.31% and 6.52% versus 33.84%, respectively, p < 0.05). In addition, the frequency in subjects harboring A8701G and G15301A variants in study group was significantly higher than that in control group (63.04% versus 33.85%, p < 0.05). This study suggests that, in part, polymorphisms in the MT-ATP6 and MT-CYB genes may contribute to the unexpected fertilization failure.

A novel in-frame 18-bp microdeletion in MT-CYB causes a multisystem disorder with prominent exercise intolerance.

A novel heteroplasmic mitochondrial DNA (mtDNA) microdeletion affecting the cytochrome b gene (MT-CYB) was identified in an Italian female patient with a multisystem disease characterized by sensorineural deafness, cataracts, retinal pigmentary dystrophy, dysphagia, postural and gait instability, and myopathy with prominent exercise intolerance. The deletion is 18-base pair long and encompasses nucleotide positions 15,649-15,666, causing the loss of six amino acids (Ile-Leu-Ala-Met-Ile-Pro) in the protein, but leaving the remaining of the MT-CYB sequence in frame. The defective complex III function was cotransferred with mutant mtDNA in cybrids, thus unequivocally establishing its pathogenic role. Maternal relatives failed to show detectable levels of the deletion in blood and urinary epithelium, suggesting a de novo mutational event. This is the second report of an in-frame intragenic deletion in MT-CYB, which most likely occurred in early stages of embryonic development, associated with a severe multisystem disorder with prominent exercise intolerance.

Association of mitochondrial letm1 with epileptic seizures.

Leucine zipper-EF-hand containing transmembrane protein 1 (Letm1) is a mitochondrial protein that is associated with seizure attacks in Wolf-Hirschhorn syndrome. This study aimed to investigate the expression pattern of Letm1 in patients with temporal lobe epilepsy (TLE) and pilocarpine-induced rat model of epilepsy, and to determine if altered Letm1 leads to mitochondrial dysfunction and increased susceptibility to seizures. Using immunohistochemical, immunofluorescent, western blotting, and transmission electron microscopic methods, we have found that Letm1 was significantly decreased in TLE patients, and gradually decreased in experimental rats from 1 to 7 days after onset of seizures. Letm1 knock-down by a lentivirus bearing LV-Letm1-sh resulted in mitochondrial swelling and decreased expression of Letm1 target protein mitochondrially encoded cytochrome B (MT-CYB). Behavioral study revealed that inhibition of Letm1 caused early onset of the first seizure, increased seizure frequency, and duration. However, administration of Letm1 homolog nigericin failed to prevent epilepsy. These results indicate that inhibition of Letm1 and mitochondrial dysfunctions contributes to the development of epileptic seizures. Appropriate Letm1 level may be critical for maintaining normal neuronal functions.

Molecular and bioenergetic differences between cells with African versus European inherited mitochondrial DNA haplogroups: implications for population susceptibility to diseases.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.