Potential Roles of mtDNA Mutations in PCOS-IR: A Review.

Polycystic ovary syndrome (PCOS) is the most common heterogeneous endocrine disease that affecting females in reproductive age. Insulin resistance (IR), an important molecular basis for PCOS, accounts for at least 75% of women carrying this syndrome. Although there have been many studies on PCOS-IR, the detailed mechanisms are not fully understood. As essential hub for energy generation, mitochondria are critical to insulin secretion and normal function, whereas mutations in mitochondrial DNA (mtDNA) result in mitochondrial dysfunctions contributing to the pathophysiology of PCOS-IR via the regulation of balance of oxidative stress (OS), energy deficiency, or hormone metabolism. In the current review, we summarize the clinical and molecular features of PCOS-IR and discuss molecular mechanisms related to mtDNA mutations.

Leber's Hereditary Optic Neuropathy: the roles of mitochondrial transfer RNA variants.

Leber's Hereditary Optic Neuropathy (LHON) was a common maternally inherited disease causing severe and permanent visual loss which mostly affects males. Three primary mitochondrial DNA (mtDNA) mutations, ND1 3460G>A, ND4 11778G>A and ND6 14484T>C, which affect genes encoding respiratory chain complex I subunit, are responsible for >90% of LHON cases worldwide. Families with maternally transmitted LHON show incomplete penetrance with a male preponderance for visual loss, suggesting the involvement of secondary mtDNA variants and other modifying factors. In particular, variants in mitochondrial tRNA (mt-tRNA) are important risk factors for LHON. These variants decreased the tRNA stability, prevent tRNA aminoacylation, influence the post-transcriptionalmodification and affect tRNA maturation. Failure of mt-tRNA metabolism subsequently impairs protein synthesis and expression, folding, and function of oxidative phosphorylation (OXPHOS) enzymes, which aggravates mitochondrial dysfunction that is involved in the progression and pathogenesis of LHON. This review summarizes the recent advances in our understanding of mt-tRNA biology and function, as well as the reported LHON-related mt-tRNA second variants; it also discusses the molecular mechanism behind the involvement of these variants in LHON.

Elevated serum levels of galectin-3 binding protein are associated with insulin resistance in non-diabetic women after menopause.

OBJECTIVE: Galectin-3 binding protein (Gal-3BP) is one of the major fucosylated glycoprotein family members and has recently been implicated in non-alcoholic fatty liver disease, hyperlipaemia and coronary artery disease. Here, we analysed the serum concentrations of Gal-3BP in menopausal women to evaluate the association of circulating Gal-3BP and insulin resistance in females after menopause. MATERIALS AND METHODS: We evaluated serum levels of Gal-3BP in sixty-two non-diabetic women with menopausal status for at least one year. The clinical features, biochemical profiles and homeostasis model assessment of insulin resistance (HOMA-IR) indices were obtained routinely. RESULTS: Gal-3BP levels increased in women with higher HOMA-IR indices and were positively correlated with HOMA-IR indices. The Gal-3BP level was also an independent risk factor for a high HOMA-IR index and showed the most influence on the HOMA-IR index compared to fasting plasma glucose, triglyceride, age and body mass index. The cut-off value of the serum Gal-3BP level was 2234.32 ng/ml, with areas under the ROC curve (AUCs) of 0.68 (HOMA-IR index 1.5), 0.81 (HOMA-IR index 2.0) and 0.93 (HOMA-IR index 2.5). CONCLUSION: Serum levels of Gal-3BP are associated with impaired insulin sensitivity in non-diabetic menopausal women.

The Mitochondrial tRNAHis G12192A Mutation May Modulate the Clinical Expression of Deafness-Associated tRNAThr G15927A Mutation in a Chinese Pedigree.

BACKGROUND: Mutations in mitochondrial tRNA (mt-tRNA) genes have been found to be associated with both syndromic and non-syndromic hearing impairment. However, the pathophysiology underlying mt-tRNA mutations in clinical expression of hearing loss remains poorly understood. OBJECTIVE: The aim of this study was to explore the potential association between mttRNA mutations and hearing loss. METHODS AND RESULTS: We reported here the molecular features of a pedigree with maternally transmitted non-syndromic hearing loss. Among 12 matrilineal relatives, five of them suffered variable degree of hearing impairment, but none of them had any medical history of using aminoglycosides antibiotics (AmAn). Genetic screening of the complete mitochondrial genomes from the matrilineal relatives identified the coexistence of mt-tRNAHis G12192A and mt-tRNAThr G15927A mutations, together with a set of polymorphisms belonging to human mitochondrial haplogroup B5b1b. Interestingly, the G12192A mutation occurred 2-bp from the 3' end of the TψC loop of mt-tRNAHis, which was evolutionarily conserved from various species. In addition, the well-known G15927A mutation, which disrupted the highly conserved C-G base-pairing at the anticodon stem of mt-tRNAThr, may lead to the failure in mt-tRNA metabolism. Furthermore, a significant decreased in ATP production and an increased ROS generation were observed in polymononuclear leukocytes (PMNs) which were isolated from the deaf patients carrying these mt-tRNA mutations, suggested that the G12192A and G15927A mutations may cause mitochondrial dysfunction that was responsible for deafness. However, the absence of any functional mutations/variants in GJB2, GJB3, GJB6 and TRMU genes suggested that the nuclear genes may not play important roles in the clinical expression of non-syndromic hearing loss in this family. CONCLUSION: Our data indicated that mt-tRNAHis G12192A mutation may increase the penetrance and expressivity of deafness-associated m-tRNAThr G15927A mutation in this family.

Impacts of macrophage colony-stimulating factor (M-CSF) on the expression of natriuretic peptide precursor type C (NPPC) and regulation of meiotic resumption.

In mammalian follicles, oocytes are arrested at the diplotene stage of prophase I until meiotic resumption following the LH surge. C-type natriuretic peptide (CNP), encoded by natriuretic peptide precursor type C (NPPC), was found to be reduced by the LH surge in the follicle, and then lead to meiotic resumption by decreasing the level of cAMP in the oocyte. As a wide-spread cytokine, macrophage colony-stimulating factor (M-CSF) takes part in the oocyte development to maturation and ovulation. Our study describes the expression curve of M-CSF and its receptor and investigates the impact on the levels of CNP/NPPC to explore the possible mechanism for meiotic resumption in both vivo and vitro. The result shows after the LH/HCG surge, the expressions of M-CSF and its receptors decline significantly inside ovarian follicles, thus leading to transduction of a range of signals. Consequently, the expression of CNP reaches the peak at 2 h and immediately declines to a relatively low level.

Premature ovarian insufficiency may be associated with the mutations in mitochondrial tRNA genes.

Premature ovarian insufficiency (POI) is a common endocrine disorder featured by the triad constituting of amenorrhea for at least four months, to date, the molecular pathogenesis of POI is largely undetermined. Despite several investigations have reported an increase in reactive oxygen species (ROS) content in idiopathic POI, the role of mitochondrial DNA (mtDNA) mutations/variants in the progression of POI has not been widely investigated. The current study aimed to explore the association between mt-tRNA mutations/variants and POI; we first used the PCR-Sanger sequencing to detect the mutations/variants in mt-tRNA genes from 50 POI patients and 30 healthy subjects. In addition, we evaluated the mitochondrial functions by using trans-mitochondrial cybrid cells containing these potential pathogenic mt-tRNA mutations. Consequently, five mutations: tRNALeu(UUR) C3303T, tRNAMet A4435G, tRNAGln T4363C, tRNACys G5821A and tRNAThr A15951G were identified. Notably, these mutations occurred at the extremely conserved nucleotides of the corresponding mt-tRNAs and may result the failure in mt-tRNA metabolism and subsequently lead to the impairment in mitochondrial protein synthesis. Furthermore, biochemical and molecular analyses of the cybrid cells containing these mutations showed a significantly lower level of ATP production when compared with the controls, whereas the ROS levels were much higher in POI patients carrying these mt-tRNA mutations, strongly indicated that these mt-tRNA mutations may cause the mitochondrial dysfunction, and play active roles in the progression and pathogensis of POI. Together, this study shaded additional light on the molecular mechanism of POI that was manifestated by mt-tRNA mutations.

Serum retinol binding protein 4 and galectin-3 binding protein as novel markers for postmenopausal nonalcoholic fatty liver disease.

OBJECTIVE: To investigate the differential protein expression before and after menopause in women with nonalcoholic fatty liver disease (NAFLD) and to explore novel markers for menopausal NAFLD. METHODS: Eight serum samples collected from pre- or post-menopausal women with NAFLD were analysed by iTRAQ 2D-LC-MS/MS. Two protein candidates were selected and verified by enzyme-linked immunosorbent assay (ELISA) in serum samples collected from a one hundred and fifty-three female population subsequently, including 51 in post-menopausal status with NAFLD, 41 in pre-menopausal with NAFLD, 19 healthy individuals in post-menopausal status and 42 healthy pre-menopausal women. RESULTS: A total of one hundred and sixty-seven proteins exhibiting significant changes were characterized, among which sixty-five were up-regulated and one hundred and two were down-regulated. Of those altered proteins, the expression of serum retinol binding protein 4 (RBP4) and galectin-3 binding protein (LGALS3BP) was obviously increased in the post-menopausal patient group compared to the other. ELISA validations for the two proteins were consistent with the proteomic profiling. CONCLUSIONS: Serum RBP4 and LGAL3BP were up-regulated after menopause under NAFLD conditions, which suggested the two proteins may be potential markers as NAFLD in postmenopausal population.

Mitochondrial tRNALeu(UUR) C3275T, tRNAGln T4363C and tRNALys A8343G mutations may be associated with PCOS and metabolic syndrome.

Polycystic ovary syndrome (PCOS) is a very prevalent endocrine disease affecting reproductive women. Clinically, patients with this disorder are more vulnerable to develop type 2 diabetes mellitus (T2DM), cardiovascular events, as well as metabolic syndrome (MetS). To date, the molecular mechanism underlying PCOS remains largely unknown. Previously, we showed that mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) mutation was an important cause for PCOS. In the current study, we described the clinical and biochemical features of a three-generation pedigree with maternally transmitted MetS, combined with PCOS. A total of three matrilineal relatives exhibited MetS including obesity, high triglyceride (TG) and Hemoglobin A1c (HbA1c) levels, and hypertension. Whereas one patient from the third generation manifestated PCOS. Mutational analysis of the whole mitochondrial genes from the affected individuals identified a set of genetic variations belonging to East Asia haplogroup B4b1c. Among these variants, the homoplasmic C3275T mutation disrupted a highly evolutionary conserved base-pairing (28A-46C) on the variable region of tRNALeu(UUR), whereas the T4363C mutation created a new base-pairing (31T-37A) in the anticodon stem of tRNAGln, furthermore, the A8343G mutation occurred at the very conserved position of tRNALys and may result the failure in mitochondrial tRNAs (mt-tRNAs) metabolism. Biochemical analysis revealed the deficiency in mitochondrial functions including lower levels of mitochondrial membrane potential (MMP), ATP production and mtDNA copy number, while a significantly increased reactive oxygen species (ROS) generation was observed in polymononuclear leukocytes (PMNs) from the individuals carrying these mt-tRNA mutations, suggesting that these mutations may cause mitochondrial dysfunction that was responsible for the clinical phenotypes. Taken together, our data indicated that mt-tRNA mutations were associated with MetS and PCOS in this family, which shaded additional light into the pathophysiology of PCOS that were manifestated by mitochondrial dysfunction.

Macrophage colony-stimulating factor (M-CSF) is an intermediate in the process of luteinizing hormone-induced decrease in natriuretic peptide receptor 2 (NPR2) and resumption of oocyte meiosis.

BACKGROUND: Luteinizing hormone (LH) regulation of the ligand, natriuretic peptide precursor type C, and its receptor, natriuretic peptide receptor 2 (NPR2), is critical for oocyte maturation; however, the mechanism is not fully understood. Macrophage colony-stimulating factor (M-CSF) has recently been shown to be involved in oocyte maturation and ovulation. In the present study we determined whether or not M-CSF plays a role in the intermediate signal that mediates LH regulation of NPR2 in resumption of oocyte meiosis. METHODS: Immature female C57BL/6 mice were injected i.p. with 5 IU of equine chorionic gonadotropin (eCG) to stimulate follicle development. After 44-48 h, the eCG-stimulated mice were injected i.p. with an ovulatory dose of 5 IU of human chorionic gonadotropin (hCG). The ovaries were excised at selected times. Pre-ovulatory follicles (POFs) and cumulus-oocyte complexes were cultured in different media. Immunohistochemical and quantitative real-time PCR analyses were used to assess the expression of M-CSF, M-CSF receptor (M-CSF-R), and NPR2. The presence of germinal vesicle breakdown (GVBD) was examined under a stereomicroscope to morphologically evaluate resumption of oocyte meiosis. RESULTS: NPR2 was mainly expressed in cumulus cells of pre-ovulatory follicles, while M-CSF and M-CSF-R were expressed in both mural granulosa and cumulus cells. The levels of M-CSF/M-CSF-R and NPR2 decreased within 4 h after treatment of hCG. M-CSF not only reduced the expression of NPR2 mRNA via its receptor (M-CSF-R), but also increased the proportion of GVBD in oocytes. CONCLUSION: M-CSF serves as an intermediate signal, thus inducing a vital decrease in the NPR2 levels in cumulus cells, and regulates the process of LH-induced resumption of meiosis.

Mutations in mitochondrial tRNA genes may be related to insulin resistance in women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a very common endocrine disorder affecting women of reproductive age. Insulin resistance (IR), a central component of this disease, occurs in 30%-40% of women with PCOS. To date, the molecular mechanism underlying PCOS-IR remains largely unknown. Most recently, increasing evidence has shown that mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) mutations plays important roles in the pathogenesis of PCOS-IR. To identify the contribution of mitochondrial tRNA (mt-tRNA) mutations in this disease, we screened 80 women with PCOS-IR and 50 healthy control participants for mt-tRNA mutations. After genetic amplification and direct sequencing, we identified nine mt-tRNA mutations that were potentially associated with PCOS-IR: mt-tRNALeu(UUR) A3302G and C3275A mutations, mt-tRNAGln T4363C and T4395C mutations, mt-tRNASer(UCN) C7492T mutation, mt-tRNAAsp A7543G mutation, mt-tRNALys A8343G mutation, mt-tRNAArg T10454C mutation and mt-tRNAGlu A14693G mutation. These mutations were localized at evolutionarily conserved nucleotides and altered the secondary structure of mt-tRNAs, thus resulting in failure of mt-tRNA metabolism. Moreover, molecular and biochemical analysis revealed that levels of 8-OHdG, malondialdehyde and reactive oxygen species were increased in patients with PCOS-IR carrying these mt-tRNA mutations compared with in healthy control participants, whereas superoxide dismutase levels, mitochondrial copy number, membrane potential and ATP levels were significantly reduced. Taken together, our data indicate that mt-tRNAs are key locations for pathogenic mutations associated with PCOS-IR. Mitochondrial dysfunction caused by mt-tRNA mutations may be involved in the pathogenesis of PCOS-IR. Thus, our findings provide novel insight into the pathophysiology of this disorder.

Mitochondrial COI/tRNASer(UCN) G7444A mutation may be associated with hearing impairment in a Han Chinese family.

Mutations in mitochondrial genome have been found to be associated with hearing loss. Of these, the mitochondrial 12S rRNA and tRNASer(UCN) are the hot spots for pathogenic mutations associated with deafness. To understand the putative role of mitochondrial DNA (mtDNA) mutations in hearing loss, we recently initiated a mutational screening for the mtDNA mutations in Hangzhou area from Zhejiang Province. In this study, we described a maternally inherited Han Chinese family with high penetrance of hearing loss, notably, the penetrances of hearing loss in this family were 80% and 40%, when the aminoglycoside was included or excluded. Three matrilineal relatives in this pedigree exhibited different levels of hearing loss with different age at onset. In addition, sequence analysis of the complete mitochondrial genome showed the presence of the well-known C1494T mutation in 12S rRNA gene and the G7444A mutation in the COI/tRNASer(UCN). The C1494T mutation had been reported to be a pathogenic mutation associated with aminoglycoside-induced and non-syndromic hearing loss. While the G7444A mutation was considered as a secondary mutation associated with deafness. However, the lack of functional variants in GJB2 and TRMU genes suggested that nuclear modified genes may not play important roles in deafness expression. Thus, the combination of G7444A and C1494T mutations in mitochondrial genome may account for the high penetrance of hearing loss in this Chinese family.

Allele-specific PCR for detecting the deafness-associated mitochondrial 12S rRNA mutations.

Mutations in mitochondrial 12S rRNA (MT-RNR1) are the important causes of sensorineural hearing loss. Of these mutations, the homoplasmic m.1555A>G or m.1494C>T mutation in the highly conserved A-site of MT-RNR1 gene has been found to be associated with both aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. Since the m.1555A>G and m.1494C>T mutations are sensitive to ototoxic drugs, therefore, screening for the presence of these mutations is important for early diagnosis and prevention of deafness. For this purpose, we recently developed a novel allele-specific PCR (AS-PCR) which is able to simultaneously detect these mutations. To assess its accuracy, in this study, we employed this method to screen the frequency of m.1555A>G and m.1494C>T mutations in 200 deafness patients and 120 healthy subjects. Consequently, four m.1555A>G and four m.1494C>T mutations were identified; among these, only one patient with the m.1494C>T mutation had an obvious family history of hearing loss. Strikingly, clinical evaluation showed that this family exhibited a high penetrance of hearing loss. In particular, the penetrances of hearing loss were 80% with the aminoglycoside included and 20% when excluded. PCR-Sanger sequencing of the mitochondrial genomes confirmed the presence of the m.1494C>T mutation and identified a set of polymorphisms belonging to mitochondrial haplogroup A. However, the lack of functional variants in mitochondrial and nuclear modified genes (GJB2 and TRMU) in this family indicated that mitochondrial haplogroup and nuclear genes may not play important roles in the phenotypic expression of the m.1494C>T mutation. Thus, other modification factors, such as environmental factor, aminoglycosides or epigenetic modification may have contributed to the high penetrance of hearing loss in this family. Taken together, our data showed that this assay is an effective approach that could be used for detection the deafness-associated MT-RNR1 mutations.

Point mutation in mitochondrial tRNA gene is associated with polycystic ovary syndrome and insulin resistance.

Polycystic ovarian syndrome (PCOS) is characterized by chronic anovulation, hyperandrogenism and polycystic ovaries. To date, the molecular mechanisms underlying PCOS have remained to be fully elucidated. As recent studies have revealed a positive association between mitochondrial dysfunction and PCOS, current investigations focus on mutations in the mitochondrial genome of patients with POCS. The present study reported a Chinese patient with PCOS. Sequence analysis of the mitochondrial genome showed the presence of homoplasmic ND5 T12338C and tRNASer (UCN) C7492T mutations as well as a set of polymorphisms belonging to the human mitochondrial haplogroup F2. The T12338C mutation is known to decrease the ND5 mRNA levels and to inhibit the processing of RNA precursors. The C7492T mutation, which occurred at the highly conserved nucleotide in the anticodon stem of the tRNASer (UCN) gene, is important for the tRNA steady­state level as well as the aminoacylation ability. Therefore, the combination of the ND5 T12338C and tRNASer (UCN) C7492T mutations may lead to mitochondrial dysfunction, and is likely to be involved in the pathogenesis of PCOS. The present study provided novel insight into the molecular mechanisms of PCOS.

The Mitochondrial tRNALeu(UUR) A3302G Mutation may be Associated With Insulin Resistance in Woman With Polycystic Ovary Syndrome.

The aim of this study was to investigate the role of mitochondrial DNA (mtDNA) mutations in polycystic ovary syndrome (PCOS) with insulin resistance (IR), and to explore the possible maternally effects on PCOS. We performed clinical, genetic, and molecular characterization of a Han Chinese family with maternally inherited IR, and we further investigated the possible relationship between mitochondrial genetic background, copy number, and IR. Most strikingly, members from the first and second generation of this family exhibited the type 2 diabetes mellitus (T2DM) with IR, while the member in the third generation of this family manifested the PCOS. Sequence analysis of the complete mitochondrial genome showed the presence of a homoplasmic A3302G in the acceptor arm of transfer RNA(Leu(UUR)) (tRNA(Leu(UUR))) gene. This mutation disrupted the highly conserved base pairing (2T-71A) and resulted a failure in mt-tRNA metabolism. Analysis of the mitochondrial copy number showed that the patients with PCOS and IR had lower copy number than the health controls, suggesting that mitochondrial dysfunction may be involved in the pathogenesis of IR. Taken together, the A3302G mutation was a pathogenic mutation associated with IR in this Chinese family.

Mitochondrial tRNAThr 15891C>G mutation was not associated with Leber's hereditary optic neuropathy in Han Chinese patients.

Mutations in mitochondrial DNA (mtDNA) were the most important causes of Leber's hereditary optic neuropathy (LHON). To date, approximately 25 LHON-associated mtDNA mutations have been identified in various ethnic populations. Three primary mutations, the 3460G > A, 11778G > A and 14484T > C, in genes encoding the subunits of respiratory chain complex I, were the most common LHON-associated mtDNA mutations. Moreover, secondary mutations in mt-tRNA genes have been reported increasingly to be associated with LHON, simply due to the high mutation rates of mt-tRNAs. There is a lack of functional analysis and a poor genetic evaluation of a certain mt-tRNA mutation, which failed to meet the classic pathogenicity scoring system. As a result, how to classify a pathogenic mutation in mt-tRNA gene became important for both geneticist and clinician to diagnosis the LHON or the suspicious of LHON. In this study, we reassessed the role of a point mutation in mt-tRNA(Thr) gene which had been reported to be a mutation associated with LHON, the pathogenicity of this mutation has been discussed in this context.

Polymorphisms of Genes Involved in the Folate Metabolic Pathway Impact the Occurrence of Unexplained Recurrent Pregnancy Loss.

Low levels of folate combined with high levels of homocysteine may cause unexplained recurrent pregnancy loss (URPL). However, the relationships between polymorphisms in genes of the folate metabolic pathway and URPL remain controversial. We conducted a case-control study to explore polymorphisms of the major folate pathway genes, including methylenetetrahydrofolate reductase (MTHFR) 677C>T, MTHFR 1298A>C, methionine synthase (MTR) 2756A>G, methionine synthase reductase (MTRR) 66A>G and reduced folate carrier 1 (RFC-1) 80A>G, and their associations with URPL. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the distributions of MTHFR, MTR and RFC-1 polymorphisms, and the results were validated using direct sequencing. The polymorphisms in MTRR were determined using direct sequencing. Haplotypes were analyzed using SHEsis, an online tool for biological analysis. We found that the MTHFR 677T allele and the 677T/1298A/2756A/66A/80G haplotype were risk factors for URPL, while the MTR 2756G allele and the 677C/1298A/2756A/66A/80A haplotype exhibited protective effects on susceptibility to URPL in a Chinese Han population from the Hangzhou area.

Development of a novel anti ESAT-6 monoclonal antibody for screening of Mycobacterium tuberculosis.

Tuberculosis (TB) is a major global public health problem with an estimated one-third of the world's population infected with Mycobacterium tuberculosis, thus necessitating fast and accurate diagnosis of TB. However, the diagnosis of latent infection remains difficult due to the lack of a simple, reliable test for M. tuberculosis infection. The objective of the current study was to generate and characterize a novel monoclonal antibody (mAb) that specifically target the early secretory antigenic target 6 (ESAT-6) protein for tuberculosis (TB) immunological diagnosis. The BALB/c mice were immunized by a peptide with 13 amino acids (amino acid residues 24 to 36) of ESAT-6 protein. Stable hybridomas cell lines were established and mAb was identified by Western Blot, immunoprecipitation (IP) and Enzyme linked immunosorbent assay (ELISA). In addition, this mouse mAb was used to coat plates, and biotin-labelled polyclonal antibodies were used to detect the antigens. Finally, the antibody was verified in 280 patient sputum culture supernatants and pleural effusion aspirates. The positive detection rate of the generated ESAT-6 mAb was 91% in the clinical diagnosis of tuberculosis pleural effusion, and this mAb had a sensitivity of 92.4% and a specificity of 100% in all M. tuberculosis cases. Our data indicated that the mAb generated in this study can potentially serve as a tool in the laboratory diagnosis of TB.

Analysis of mitochondrial DNA sequence variants in patients with polycystic ovary syndrome.

PURPOSE: To understand the role of mitochondrial DNA (mtDNA) mutations in patients with polycystic ovary syndrome (PCOS). METHODS: A total of 57 women with PCOS and 38 controls were recruited in this study, mutational analysis of mitochondrial genome was performed using polymerase chain reaction and under a direct sequence analysis. RESULTS: Sequence characterization of mitochondrial genome showed a distinct set of polymorphisms mainly focused on oxidative phosphorylation (OXPHOS) complex, in addition, six variants in mitochondrial tRNA genes, including tRNA(Gln), tRNA(Cys), tRNA(Asp), tRNA(Lys), tRNA(Arg) and tRNA(Glu) were also identified in PCOS patients. Interestingly, these variants occurred at highly conserved nucleotides of corresponding tRNAs, which are important for tRNA stability level and biochemical function. CONCLUSIONS: Mutations in mtDNA, especially the OXPHOS complex and tRNAs, may be associated with PCOS patients, thus, our results shed new insight into the pathogenesis of PCOS.

[Serum IL-18 levels in mice with collagen-induced arthritis treated by recombinant adenovirus containing mIL-18BP and mIL-4 fusion gene].

OBJECTIVE: To investigate serum IL-18 levels in mice with collagen-induced arthritis treated by recombinant adenoviral vector containing mIL-18BP and mIL-4 fusion gene (AdmIL-18BP/mIL-4). METHODS: Arthritis was induced by injection of collagen in male DBA-1/BOM mice. Mice with collagen-induced arthritis (CIA) were intra-articularly injected with 10(7)pfu/6µL of AdmIL-18BP/mIL-4; and in mice of control groups AdLacZ or PBS were used. The animals were sacrificed at week 1, 2 and 4 after treatment. Serum IL-18 levels were determined by ELISA at the different time points. RESULT: The mean serum levels of IL-18 at weeks 1, 2, and 4 after injection of AdmIL-18BP/mIL-4 were (36.5±5.4)ng/L, (32.5 ± 3.2) ng/L and (28.7 ±2.9)ng/L, respectively, which were significantly lower than those at the same time point of AdLacZ group [(66.2 ±5.1)ng/L, (69.2 ±4.2)ng/L and (77.7 ±3.9)ng/L] and PBS group [(67.3 ±7.1)ng/L, (71.9 ±1.8)ng/L and (78.7±4.1)ng/L] (P<0.01 at all time points). In the therapy group, there were no significant differences in the mean serum concentrations of IL-18 at all time points. CONCLUSION: The serum IL-18 levels in CIA mice are down-regulated by treatment of recombinant adenovirus containing mIL-18BP and mIL-4 fuse gene, which might be a promising therapeutic strategy for rheumatoid arthritis.

A 9-bp deletion homoplasmy in women with polycystic ovary syndrome revealed by mitochondrial genome-mutation screen.

Polycystic ovary syndrome (PCOS) is a complex and heterogeneous disorder presenting a challenge for clinical investigators. To investigate the association of a mitochondrial genetic basis with PCOS, we screened mutations of the whole mitochondrial genome in 57 women patients with PCOS and 38 healthy control individuals. Two-step PCR reactions were adopted to amplify and sequence the whole mitochondrial genome. A 9-bp deletion variant appeared in homoplasmy between PCOS patients and control individuals. In the 62 individuals with complete sequences, eight of 34 (23.5%) patients showed the 9-bp deletion, compared with only two of 28 (7.1%) in healthy controls. The 9-bp deletion variant in region V of mitochondrial DNA may be associated with the heterogeneous disorder PCOS.