Comprehensive analysis of mitochondrial DNA variants, mitochondrial DNA copy number and oxidative damage in psoriatic arthritis.

Growing evidence suggests that abnormalities in mitochondrial DNA (mtDNA) are involved in the pathogenesis of various inflammatory and immuno-mediated diseases. The present study analysed the entire mitochondrial genome by next-generation sequencing (NGS) in 23 patients with psoriatic arthritis (PsA) and 20 healthy controls to identify PsA-related variants. Changes in mtDNA copy number (mtDNAcn) were also evaluated by quantitative polymerase chain reaction (qPCR) and mtDNA oxidative damage was measured using an 8-hydroxy-2'-deoxyguanosine assay. NGS analysis revealed a total of 435 variants including 187 in patients with PsA only and 122 in controls only. Additionally, 126 common variants were found, of which 2 variants differed significantly in their frequencies among patients and controls (P<0.05), and may be associated with susceptibility to PsA. A total of 33 missense variants in mtDNA-encoded genes for complexes I, III, IV and V were identified only in patients with PsA. Of them, 25 variants were predicted to be deleterious by affecting the functions and structures of encoded proteins, and 13 variants were predicted to affect protein's stability. mtDNAcn analysis revealed decreased mtDNA content in patients with PsA compared with controls (P=0.0001) but the decrease in mtDNAcn was not correlated with patients' age or inflammatory biomarkers (P>0.05). Moreover, a higher level of oxidative damage was observed in patients with PsA compared with controls (P=0.03). The results of the present comprehensive analysis of mtDNA in PsA revealed that certain mtDNA variants may be implicated in the predisposition/pathogenesis of PsA, highlighting the importance of NGS in the identification of mtDNA variants in PsA. The current results also demonstrated that decreased mtDNAcn in PsA may be a consequence of increased oxidative stress. These data provide valuable insights into the contribution of mtDNA defects to the pathogenesis of PsA. Additional studies in larger cohorts are needed to elucidate the role of mtDNA defects in PsA.

Investigation of germline variants in Bahraini women with breast cancer using next-generation sequencing based-multigene panel.

Germline variants in BRCA1 and BRCA2 (BRCA1/2) genes are the most common cause of hereditary breast cancer. However, a significant number of cases are not linked to these two genes and additional high-, moderate- and low-penetrance genes have been identified in breast cancer. The advent of next-generation sequencing (NGS) allowed simultaneous sequencing of multiple cancer-susceptibility genes and prompted research in this field. So far, cancer-predisposition genes other than BRCA1/2 have not been studied in the population of Bahrain. We performed a targeted NGS using a multi-panel covering 180 genes associated with cancer predisposition to investigate the spectrum and frequency of germline variants in 54 women with a positive personal and/or family history of breast cancer. Sequencing analysis revealed germline variants in 29 (53.7%) patients. Five pathogenic/likely pathogenic variants in four DNA repair pathway-related genes were identified in five unrelated patients (9.3%). Two BRCA1 variants, namely the missense variant c.287A>G (p.Asp96Gly) and the truncating variant c.1066C>T (p.Gln356Ter), were detected in two patients (3.7%). Three variants in non-BRCA1/2 genes were detected in three patients (1.85% each) with a strong family history of breast cancer. These included a monoallelic missense variant c.1187G>A (p.Gly396Asp) in MUTYH gene, and two truncating variants namely c.3343C>T (p.Arg1115Ter) in MLH3 gene and c.1826G>A (p.Trp609Ter) in PMS1 gene. Other variants of uncertain significance (VUS) were also detected, and some of them were found together with the deleterious variants. In this first application of NGS-based multigene testing in Bahraini women with breast cancer, we show that multigene testing can yield additional genomic information on low-penetrance genes, although the clinical significance of these genes has not been fully appreciated yet. Our findings also provide valuable epidemiological information for future studies and highlight the importance of genetic testing, and an NGS-based multigene analysis may be applied supplementary to traditional genetic counseling.

Mitochondrial DNA copy number in autism spectrum disorder and attention deficit hyperactivity disorder: a systematic review and meta-analysis.

Background: Several reports suggest that altered mitochondrial DNA copy number (mtDNA-cn), a common biomarker for aberrant mitochondrial function, is implicated in autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD), but the results are still elusive. Methods: A meta-analysis was performed to summarize the current indication and to provide a more precise assessment of the mtDNA-cn in ASD and ADHD. A search in the MEDLINE-PubMed, Scopus, and EMBASE databases was done to identify related studies up to the end of February 2023. The meta-analysis was conducted according to recommendations of the Cochrane Handbook of Systematic Reviews. Results: Fourteen studies involving 666 cases with ASD and ADHD and 585 controls were collected and judged relevant for the systematic review and meta-analysis. The pooled results by a random effects meta-analysis was reported as a geometric mean of the estimated average response ratio and 95% confidence interval. Overall analysis of studies reported differences in mtDNA-cn in blood samples (k = 10) and non-blood samples (brain tissues and oral samples; k = 4) suggested significantly higher mtDNA-cn in patients compared to controls (p = 0.0275). Sub-analysis by stratifying studies based on tissue type, showed no significant increase in mtDNA-cn in blood samples among patients and controls (p = 0.284). Conversely, higher mtDNA-cn was observed in non-blood samples in patients than in controls (p = 0.0122). Further stratified analysis based on blood-cell compositions as potential confounds showed no significant difference in mtDNA-cn in peripheral blood samples of patients comparted to controls (p = 0.074). In addition, stratified analysis of aged-matched ASD and ADHD patients and controls revealed no significant difference in mtDNA-cn in blood samples between patients and controls (p = 0.214), whereas a significant increase in mtDNA-cn was observed in non-blood samples between patients and controls (p < 0.001). Finally, when the mtDNA-cn was analyzed in blood samples of aged-matched patients with ASD (peripheral blood, leukocytes, and PBMCs) or ADHD (peripheral blood), no significant difference in mtDNA-cn was observed between ASD patients and controls (p = 0.385), while a significant increase in mtDNA-cn was found between ADHD patients and controls (p = 0.033). Conclusion: In this first meta-analysis of the evaluation of mtDNA-cn in ASD/ADHD, our results show elevated mtDNA-cn in ASD and ADHD, further emphasizing the implication of mitochondrial dysfunction in neurodevelopmental disorders. However, our results indicate that the mtDNA-cn in blood is not reflected in other tissues in ASD/ADHD, and the true relationship between blood-derived mtDNA-cn and ASD/ADHD remains to be defined in future studies. The importance of blood-cell compositions as confounders of blood-based mtDNA-cn measurement and the advantages of salivary mtDNA-cn should be considered in future studies. Moreover, the potential of mtDNA-cn as a biomarker for mitochondrial malfunction in neurodevelopmental disorders deserves further investigations.

Mitochondrial DNA haplogroup analysis in Saudi Arab patients with multiple sclerosis.

Previous studies have suggested that mitochondrial DNA (mtDNA) variants are associated with multiple sclerosis (MS), a complex neurodegenerative immune-mediated disease of the central nervous system. Since mtDNA is maternally inherited without recombination, specific mtDNA variants defining genetic background are associated with the susceptibility to human diseases. To assess the contribution of mtDNA haplogroups to the predisposition of MS in an Arab population, we analysed sequencing data of mitochondrial genomes from 47 native Saudi Arab individuals including 23 patients with relapsing-remitting MS (RRMS) and 24 healthy controls. All patients and controls could be classified into ten haplogroups. The European-specific haplogroup U was more prevalent in patients than in the controls (26.1% vs. 4.2%), whereas haplogroup T was only present in patients and haplogroups HV and N were only found in controls. Haplogroup U was significantly association with increased risk of MS (odds ratio = 6.26, p<0.05), although the association did not maintain significance after adjustment for multiple comparisons. Haplotype U was more prevalent in patients with younger age of onset (p = 0.006), but there was no relationship between haplotype U and disease severity, disease duration or EDSS and age-matched carriers and non-carriers of haplogroup U (p>0.05). Definition site of haplogroup U include the variant m.12308A>G in MT-TL2 gene which was found to affect highly conserved position within the variable arm of tRNALeu(CUN) and thus may impact mitochondrial protein synthesis, and two other variants namely m.11467A>G in MT-ND4 gene and m.12372G>A in MT-ND5 gene which were previously linked with mitochondrial function. Despite the small number of subjects, which may limit the statistical power of the study, our results showed for the first time a possible contribution of haplogroup U to the predisposition to MS in an Arab population. These findings warrant further validation in a large cohort to distinguish a genuine effect specific to MS from a chance finding due to small sampling.

Analysis of the entire mitochondrial genome reveals Leber's hereditary optic neuropathy mitochondrial DNA mutations in an Arab cohort with multiple sclerosis.

Several mitochondrial DNA (mtDNA) mutations of Leber's hereditary optic neuropathy (LHON) have been reported in patients with multiple sclerosis (MS) from different ethnicities. To further study the involvement of LHON mtDNA mutations in MS in the Arab population, we analyzed sequencing data of the entire mitochondrial genome from 47 unrelated Saudi individuals, 23 patients with relapse-remitting MS (RRMS) and 24 healthy controls. Ten LHON mutations/variants were detected in the patients but were absent in the controls. Of them, the common primary pathogenic mutation m.14484T>C and the rare mutation m.10237T>C were found in one patient, whereas the rare mutation m.9101T>C was found in another patient. The remaining were secondary single nucleotide variants (SNVs) found either in synergy with the primary/rare mutations or individually in other patients. Patients carrying LHON variants also exhibited distinct mtDNA variants throughout the mitochondrial genome, eight were previously reported in patients with LHON. Moreover, five other LHON-related SNVs differed significantly in their prevalence among patients and controls (P < 0.05). This study, the first to investigate LHON mtDNA mutations/variants in a Saudi cohort may suggest a role of these mutations/variants in the pathogenesis or genetic predisposition to MS, a possibility which needs to be explored further in a large-scale.

Leukocyte mitochondrial DNA copy number is a potential non-invasive biomarker for psoriasis.

Abnormalities in the mitochondria have been linked to psoriasis, a chronic immune-mediated inflammatory skin disease. The mitochondrial DNA (mtDNA) is present in thousands of copies per cell and altered mtDNA copy number (mtDNA-CN), a common indicator of mitochondrial function, has been proposed as a biomarker for several diseases including autoimmune diseases. In this case-control study, we investigated whether the mtDNA-CN is related to psoriasis, correlates with the disease duration and severity, and can serve as a disease biomarker. Relative mtDNA-CN as compared with nuclear DNA was measured by a quantitative real-time polymerase chain reaction in peripheral blood buffy coat samples from 56 patients with psoriasis and 44 healthy controls. The receiver operating characteristic (ROC) curve analysis was performed to evaluate the value of mtDNA-CN as a biomarker. We found that the mtDNA-CN was significantly decreased in patients with psoriasis compared to healthy controls (93.6±5.3 vs. 205±71; P = 0.04). Sub-group analyses with stratification of patients based on disease duration under or over 10 years and disease severity indicated that the mtDNA-CN was significantly lower in patients with longer disease duration (74±4.3 in disease duration >10 years vs. 79±8.3 in disease duration <10 years, P = 0.009), and higher disease severity (72±4.3 in moderate-to-severe index vs. 88.3 ± 6 in mild index, P = 0.017). Moreover, the mtDNA-CN was negatively correlated with the disease duration and disease severity (r = -0.36, P = 0.006; r = -0.41, P = 0.003 respectively). The ROC analysis of mtDNA-CN showed an area under the curve (AUC) of 0.84 (95% confidence interval: 0.69-0.98; P = 0.002) for differentiating patients from healthy controls. Our study suggests that low mtDNA-CN may be an early abnormality in psoriasis and associates with the disease progression. Our study also suggests that mtDNA-CN may be a novel blood-based biomarker for the early detection of psoriasis.

Next-generation sequencing of the whole mitochondrial genome identifies functionally deleterious mutations in patients with multiple sclerosis.

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system with genetics and environmental determinants. Studies focused on the neurogenetics of MS showed that mitochondrial DNA (mtDNA) mutations that can ultimately lead to mitochondrial dysfunction, alter brain energy metabolism and cause neurodegeneration. We analyzed the whole mitochondrial genome using next-generation sequencing (NGS) from 47 Saudi individuals, 23 patients with relapsing-remitting MS and 24 healthy controls to identify mtDNA disease-related mutations/variants. A large number of variants were detected in the D-loop and coding genes of mtDNA. While distinct unique variants were only present in patients or only occur in controls, a number of common variants were shared among the two groups. The prevalence of some common variants differed significantly between patients and controls, thus could be implicated in susceptibility to MS. Of the unique variants only present in the patients, 34 were missense mutations, located in different mtDNA-encoded genes. Seven of these mutations were not previously reported in MS, and predicted to be deleterious with considerable impacts on the functions and structures of encoded-proteins and may play a role in the pathogenesis of MS. These include two heteroplasmic mutations namely 10237T>C in MT-ND3 gene and 15884G>C in MT-CYB gene; and three homoplasmic mutations namely 9288A>G in MT-CO3 gene, 14484T>C in MT-ND6 gene, 15431G>A in MT-CYB gene, 8490T>C in MT-ATP8 gene and 5437C>T in MT-ND2 gene. Notably some patients harboured multiple mutations while other patients carried the same mutations. This study is the first to sequence the entire mitochondrial genome in MS patients in an Arab population. Our results expanded the mutational spectrum of mtDNA variants in MS and highlighted the efficiency of NGS in population-specific mtDNA variant discovery. Further investigations in a larger cohort are warranted to confirm the role of mtDNA MS.

Whole blood transcriptomic analysis reveals PLSCR4 as a potential marker for vaso-occlusive crises in sickle cell disease.

Sickle cell disease, a common genetic blood disorder, results from a point mutation in the ß-globin gene affecting the configuration of hemoglobin, predisposing to painful vaso-occlusive crisis (VOC) and multi-organ dysfunctions. There is a huge variation in the phenotypic expressions of SCD and VOC owing to genetic and environmental factors. This study aimed to characterize the whole blood gene expression profile using Microarray technology in Bahraini patients with SCD determining the differentially expressed genes in steady-state (n = 10) and during VOC (n = 10) in comparison to healthy controls (n = 8). Additionally, the study intended to identify potential genetic marker associated with hemolysis. The analysis identified 2073 and 3363 genes that were dysregulated during steady-state and VOC, respectively, compared to healthy controls. Moreover, 1078 genes were differentially expressed during VOC compared to steady state. The PLSCR4 gene was almost 6-fold up-regulated in microarray, 4-fold in polymerase chain reaction, and a mean protein concentration of 0.856 ng/ml was observed in enzyme-linked immunosorbent assay during VOC compared to steady-state (0.238 ng/ml) (p < 0.01). Amongst these genes, PLSCR4 is involved in erythrocyte membrane deformity thus, predisposing to hemolysis, adhesion, and thrombosis. In conclusion, PLSCR4 may serve as a potential biomarker for VOC and future large-scale validation are recommended.

Dysregulation of human beta-defensin-3 expression in the peripheral blood of patients with sepsis.

OBJECTIVES: Sepsis is a serious medical condition caused by the body's systemic inflammatory response to infections. The antimicrobial peptides, human beta-defensins, play a key role in modulating host immune responses, and aberrant expression of human beta-defensins has been implicated in many infections and inflammatory diseases. However, little is known about the expression of human beta-defensin-3 in systemic infectious diseases. METHODS: We investigated the gene expression and protein level of human beta-defensin-3 in peripheral whole blood from 107 participants-67 patients with sepsis and 40 healthy controls-and evaluated the feasibility of human beta-defensin-3 as an indicator for sepsis. Total RNA was extracted from peripheral blood samples, and relative mRNA expression of human beta-defensin-3 was determined by reverse transcription-quantitative polymerase chain reaction. Plasma concentration of human beta-defensin-3 was measured by enzyme-linked immunosorbent assay. Pearson's correlation analysis was performed to assess the relationship between human beta-defensin-3 mRNA and protein levels. Receiver operating characteristic analysis was performed to evaluate the value of human beta-defensin-3 as a biomarker for sepsis. RESULTS: Human beta-defensin-3 mRNA expression was significantly downregulated in sepsis patients compared to controls (p = 0.001). The mean fold change of mRNA expression (±standard error) was 0.82 ± 0.63 in sepsis patients and 1.39 ± 1.09 in controls. Plasma concentration of human beta-defensin-3 (pg/mL) was significantly lower in sepsis patients compared to healthy controls (p = 0.039). The mean protein concentration (±standard error) was 539.6 ± 39.4 in sepsis patients and 715.5 ± 53 in controls. There was a significant correlation between human beta-defensin-3 mRNA expression and the corresponding protein level in sepsis patients (r = 0.358, p = 0.04), but not in healthy controls (r = 0.124, p = 0.51). For discriminating sepsis patients from healthy controls, the area under the receiver operating characteristic curve was 0.722 (95% confidence interval: 0.597-0.847, p = 0.002) for human beta-defensin-3 mRNA and 0.689 (95% confidence interval: 0.557-0.827, p = 0.009) for human beta-defensin-3 protein. CONCLUSION: This is the first study to show the downregulation of human beta-defensin-3 gene expression and protein level in sepsis, which may contribute to the complex immunological imbalance in sepsis. The significant correlation between human beta-defensin-3 mRNA expression and protein concentration suggests that mRNA expression could be used to predict protein level. Our study also showed a potential role of human beta-defensin-3 as a blood-based biomarker for sepsis. More studies on the clinical significance of human beta-defensin-3 in sepsis could further support a biomarker development.

Next-generation sequencing of the whole mitochondrial genome identifies novel and common variants in patients with psoriasis, type 2 diabetes mellitus and psoriasis with comorbid type 2 diabetes mellitus.

Recent studies have shown the role of mitochondrial DNA (mtDNA) variants in the pathogenesis of both psoriasis (Ps) and type 2 diabetes (T2D) amongst different ethnicities. However, no studies have investigated the mtDNA variants present in patients with Ps, T2D, and both Ps and T2D (Ps-T2D) in the Arab population. The entire mitochondrial genomes of Kuwaiti subjects with Ps, T2D, Ps-T2D and healthy controls were sequenced using Ion Torrent next-generation sequencing. A total of 36 novel mutations and 51 previously reported mutations were identified in the patient groups that were absent in the controls. Amongst the novel mutations, eight were non-synonymous and exhibited amino acid changes. Of these, two missense mutations (G5262A and A12397G) in the ND genes were detected in the Ps group and a C15735T missense mutation in the CYB gene was detected in Ps-T2D. Other known sequence variations were seen more frequently in all or certain patient groups compared with the controls (P<0.05). Additionally, the A8701G missense mutation in the ATPase 6 gene missense mutation was also observed in a higher frequency in the Ps group compared with the control. The present study is the first to perform a complete mitochondrial genome sequence analysis of Kuwaiti subjects with Ps, T2D and Ps-T2D, and both novel and known mtDNA variants were discovered. The patient-specific novel non-synonymous mutations may be co-responsible in the determination of these diseases. The higher frequency of certain mtDNA variants in the patients compared with the controls may suggest a role in predisposing patients to these diseases. Further functional analyses are required to reveal the role of the identified mutations in these disease conditions.

Expression and clinical significance of miR-1 and miR-133 in pre-diabetes.

Pre-diabetes represents an intermediate state of altered glucose metabolism between normal glucose levels and type 2 diabetes mellitus (T2D), and is considered a significant risk factor for the development of T2D and related complications. Early detection of pre-diabetes may allow for the use of timely and effective treatment strategies to prevent its progression. Circulating microRNAs (miRNAs/miRs) that reflect changes in diabetes-related tissues, including the pancreas, liver, skeletal and heart muscle, and adipose tissue are promising biomarkers of disease progression. In our previous study, it was demonstrated that the cardiac and skeletal muscle specific miR-1 and miR-133 are upregulated in the blood of patients with T2D and cardiovascular disease. Since both miRNAs have been shown to be implicated in insulin resistance, an important feature of pre-diabetes, we hypothesised that their expression may be increased prior to clinical diagnosis of T2D, and may thus serve as biomarkers for pre-diabetes. The expression levels of circulating miRNAs were evaluated by reverse transcription-quantitative PCR in whole blood samples from 55 subjects, including 28 pre-diabetes individuals with impaired fasting glucose (FG) and impaired glucose tolerance, and 27 healthy controls. The individuals with pre-diabetes exhibited significantly higher expression levels of miR-1 and miR-133 compared with the controls (P<0.05). Target prediction search revealed that both miR-1 and miR-133 target several pathways involved in the pathophysiology of diabetes. Pearson's correlation analysis revealed that the two miRNAs were positively correlated with blood glucose parameters, including FG, 2-h oral glucose tolerance test and glycated haemoglobin A1c levels, as well as with insulin resistance (P<0.05). Multivariate logistic regression analysis revealed that the two miRNAs were significantly and directly associated with pre-diabetes, and this association remained significant after adjustment for several confounding variables (P<0.05). Moreover, linear regression analysis showed that the Homeostatic Model Assessment-Insulin Resistance was the only significant predictor to be significantly associated with both miRNAs (P<0.05). In discriminating pre-diabetes individuals from healthy controls, the area under the curves (AUCs) of the receiver operating characteristic curves (ROCs) were 0.813 and 0.810 for miR-1 and miR-133 respectively (P<0.05). Despite the relatively small number of participants, the present study showed for the first time that circulating levels of miR-1 and miR-133 were increased in individuals with pre-diabetes, and they were associated with important features of pre-diabetes. Thus, they may serve as clinical biomarkers for the early-stages of T2D.

Mitochondrial DNA Copy Number in Peripheral Blood as a Potential Non-invasive Biomarker for Multiple Sclerosis.

The impaired mitochondrial function has been implicated in the pathogenicity of multiple sclerosis (MS), a chronic inflammatory, demyelinating, and neurodegenerative disease of the CNS. Circulating mtDNA copy number in body fluids has been proposed as an indicator for several neurodegenerative diseases, and the altered cerebrospinal fluid mtDNA has been shown as a promising marker for MS. The aim of this study was to determine changes and biomarker potential of circulating mtDNA in peripheral blood in MS. The mtDNA copy number was quantified by real-time PCR in blood samples from 60 patients with relapsing-remitting MS (RRMS) and 64 healthy controls. The RRMS patients had significantly lower circulating mtDNA copy number compared to controls. Subgroup analysis with stratification of RRMS patients based on disease duration under or over 10 years revealed that the mtDNA copy number was significantly lower in the group with longer disease duration. A negative correlation was observed between mtDNA copy number and disease duration. The ROC curve analysis indicated a significant ability of mtDNA copy number to separate RRMS patients from controls with an AUC of 0.859. This is the first study to measure peripheral blood mtDNA copy number in MS patients. Current data suggest that the reduction in peripheral blood mtDNA copy number may be an early event in MS and correlate with the disease progression. The findings of this study indicate that circulating blood-based mtDNA copy number may be a potential non-invasive candidate biomarker for mitochondria-mediated neurodegeneration and MS. This can put forward the clinical applicability over other invasive markers.

Four novel mutations in the mitochondrial ND4 gene of complex I in patients with multiple sclerosis.

Multiple sclerosis (MS) is an immune-mediated neurological, inflammatory disease of the central nervous system. Recent studies have suggested that genetic variants in mitochondrial DNA (mtDNA)-encoded complexes of respiratory chain, particularly, complex I (NADH dehydrogenase), contribute to the pathogenicity of MS among different ethnicities, and targeting mitochondrial function may represent a novel approach for MS therapy. In this study, we sequenced ND genes (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6) encoding subunits of complex I in 124 subjects, 60 patients with relapsing-remitting MS and 64 healthy individuals, in order to identify potential novel mutations in these patients. We found several variants in ND genes in both the patients and controls, and specific variants only in patients with MS. While the majority of these variants were synonymous, 4 variants in the ND4 gene were identified as missense mutations in patients with MS. Of these, m.11150G>A was observed in one patient, whereas m.11519A>C, m.11523A>C and m.11527C>T were observed in another patient. Functional analysis predicted the mutations, m.11519A>C, m.11523A>C and m.11150G>A, as deleterious with a direct impact on ND4 protein stability and complex I function, whereas m.11527C>T mutation had no effect on ND4 protein stability. However, the 3 mutations, m.11519A>C, m.11523A>C and m.11527C>T, which were observed in the same patient, were predicted to cause a cumulative destabilizing effect on ND4 protein, and could thus disrupt complex I function. On the whole, this study identified 4 novel mutations in the mtDNA-encoded ND4 gene in patients with MS, which could lead to complex I dysfunction, and further confirmed the implication of mtDNA mutations in the pathogenicity of MS. The identified novel mutations in patients with MS may be ethnic-related and may prove to be significant in personalized treatment.

Effects of enhanced environment and induced depression on cuprizone mouse model of demyelination.

Impairment in cognition and motor activity are commonly encountered in patients affected by multiple sclerosis (MS), and depression is believed to be a contributing factor. The aim of the present study was to investigate the impact of induced depression on a cuprizone mouse model of demyelination and the effectiveness of enhanced environment (EE) as a method of intervention. C57BL/6 male mice were divided into five groups: Cuprizone only (Cup-O), cuprizone undergoing depression (Cup-Dep), cuprizone housed in EE (Cup-EE), cuprizone housed in EE and undergoing depression (Cup-ED) and the control (n=9-10 per group). Depression was induced by repeated open-space forced swim. Neurobehavioral tests were conducted following a 6-week period of 0.2% cuprizone-enriched diet. The Cup-EE group performed significantly better in terms of cognition and motor functions, when compared with the Cup-O group, as evidenced by the Morris water maze (MWM; P<0.001) and rotarod performance test (P<0.05) results. Conversely, the Cup-Dep group exhibited a significant decline in performance in the MWM (P<0.001) and rotarod performance test (P<0.05), when compared with the Cup-O group. The Cup-ED group had comparable results to those of the Cup-O group, indicating a reversal of the induced depression effects. Open field test results failed to show an anxiety-like behavior in the cuprizone mouse model. It was therefore concluded that EE can improve MS-associated cognitive and motor deficits. Insights gained from these results facilitate the exploration of non-medical modes of intervention as an emerging adjuvant therapy in MS.

Upregulation of Circulating Cardiomyocyte-Enriched miR-1 and miR-133 Associate with the Risk of Coronary Artery Disease in Type 2 Diabetes Patients and Serve as Potential Biomarkers.

Circulating miRNAs are increasingly suggested as clinical biomarker for diseases. We evaluated the expression of circulating cardiomyocyte-enriched miR-1 and miR-133 by real-time PCR in blood from patients with type 2 diabetes (T2D) without and with coronary artery disease (CAD) and healthy controls, investigated their association with the risk of CAD risk and their potential as biomarkers. The two miRNAs were upregulated in patients with T2D and CAD compared with controls, associated with CAD risk and remained significant after adjustment for multiple confounders. LDL-C was a positive predictor for miR-1 and miR-133, and mean blood pressure was also a positive predictor for miR-133. Both miRNAs strongly distinguished CAD from controls. miR-1 significantly distinguished CAD from T2D with higher diagnostic ability than miR-133, whereas the miR-1/miR-133 combination improved the diagnostic value. Upregulation of circulating miR-1 and miR-133 associate with the risk of CAD in T2D patients and may serve as diagnostic biomarkers.

Circulating matrix metalloproteinases and their tissue inhibitors as markers for ethnic variation in pelvic floor tissue integrity.

The purpose of the present study was to measure the plasma levels of matrix metalloproteinases (MMP)-2 and -9 and their tissue inhibitors (TIMP)-1 and -2, as surrogate biomarkers for pelvic floor tissue integrity in young, healthy multi-ethnic women. This was hoped to elucidate ethnic vulnerability to support-related pelvic floor dysfunctions. The plasma levels of MMP-2 and -9 and TIMP-1 and -2 were measured by sandwich ELISA in nulliparous, young (18-29 years) women volunteers (n=85) from five ethnic groups [n=17/group; Bahrainis, other Arabs, Filipinos, Indians/Pakistanis and Caucasians (Italians)] and compared with levels in Italians as the reference group. It was identified that the levels of plasma MMP-2 were significantly higher in Italians than in Bahrainis (P<0.001) and Filipinos (P<0.001), but significantly lower than in Indians/Pakistanis (P=0.013); whereas, the levels of plasma MMP-9 were significantly higher in Italians than in Bahrainis (P=0.009) and Indians/Pakistanis (P<0.015). The levels of plasma TIMP-2 were significantly lower in Italians than in Indians/Pakistanis (P=0.003), but the levels of plasma TIMP-1 were significantly higher in Italians than in all other groups (P<0.05) excluding Bahrainis. Although MMP-2 correlated negatively with TIMP-2 and MMP-9 correlated positively with TIMP-1, both correlations were not significant (r=0.071, P=0.533 and r=0.197, P=0.8, respectively). In all ethnic groups, MMP-9 level correlated positively with BMI (r=0.26, P=0.02), and TIMP-2 level with age (r=0.23, P=0.045). Overall, the trends for higher levels of MMP-2 and -9 and lower levels of TIMP-2 in the plasma of Caucasian women may indicate a greater tendency for collagenolysis and weaker connective tissue with increased risk of developing pelvic floor dysfunctions, and thus may potentially serve as biomarkers for pelvic floor tissue integrity.

Peripheral blood mitochondrial DNA copy number as a novel potential biomarker for diabetic nephropathy in type 2 diabetes patients.

The mitochondrial DNA copy number (mtDNA-CN) is a surrogate measure of mitochondrial function and altered mtDNA-CN reflects the oxidant-induced cell damage. A previous study by our group demonstrated that a reduction in the renal mtDNA-CN is implicated in the pathogenesis of diabetic nephropathy (DN), a leading cause of end-stage renal disease in diabetic patients. In the present study, it was investigated whether the mtDNA-CN in the peripheral blood may be utilized as a biomarker for DN in type 2 diabetes (T2D) patients. The study included 50 non-diabetic and 100 diabetic subjects. The diabetic subjects were sub-divided based on their albumin-to-creatinine ratio (ACR) into T2D patients with normoalbuminuria (n=50), DN patients with microalbuminuria (n=29) and DN patients with macroalbuminuria (n=21). The mtDNA-CN was measured in the peripheral blood by real-time polymerase chain reaction analysis. Patients with DN had a lower mtDNA-CN than patients with T2D and healthy controls (P<0.05). A sub-group analysis with stratification by the ACR indicated that a decreased mtDNA-CN was associated with the severity and the presence of DN, as it was lower in DN patients with macroalbuminuria than in DN patients with microalbuminuria and T2D patients with normoalbuminuria (P<0.01). The area under the receiver operating characteristic curve (AUC) for mtDNA-CN was 0.916 (sensitivity, 86% and specificity, 74%) and 0.961 (sensitivity, 96% and specificity, 88%) for differentiating DN patients from T2D patients without DN and from healthy controls, respectively. Furthermore, the AUC of mtDNA-CN for differentiating DN patients with microalbuminuria from those with macroalbuminuria was 0.895 (sensitivity, 83% and specificity, 85%). Multivariate analysis revealed that the mtDNA-CN was significantly associated with the occurrence and progression of DN, even after adjustment for age, mean blood pressure, glycated haemoglobin A1c and total cholesterol (P<0.05). In patients with DN, a decreased mtDNA-CN was negatively correlated with albuminuria and conventional risk factors for DN, and was positively correlated with the estimated glomerular filtration rate. The present results therefore suggest the utilization of circulating mtDNA-CN as a novel biomarker for the early diagnosis of DN and indicate the significance of decreased mtDNA-CN as another independent risk factor for DN.

Blood-based microRNAs as diagnostic biomarkers to discriminate localized prostate cancer from benign prostatic hyperplasia and allow cancer-risk stratification.

Prostate cancer (PCa) is the second most diagnosed malignancy, and the leading cause of cancer-associated mortality among males. Prostate-specific antigen (PSA) has long been used for the detection of PCa. However, PSA levels increase in PCa and benign prostatic hyperplasia (BPH), and are associated with a poor disease outcome. Circulating microRNAs (miRNAs) have been determined to be highly stable in the circulation, and could be utilized as biomarkers to improve disease diagnosis and management. In the present study, the effectiveness of four PCa-associated miRNAs in the discrimination of PCa from BPH and the risk-stratification of PCa was assessed. The study included 100 participants: 35 patients with localized PCa, 35 patients with BPH and 30 healthy subjects. Patients with PCa were categorized based on their tumor stage (T), PSA level and Gleason score (GS) into low-(T 1/2, PSA <10 ng/ml or GS ≤7) and high-risk groups (T 3/4, PSA >20 ng/ml or GS ≥8). Reverse transcription-quantitative polymerase chain reaction was employed to assess the miRNA expression in peripheral blood samples. Significantly reduced expression of miR-15a, miR-126, miR-192 and miR-377 was observed in patients with PCa compared with patients with BPH and healthy subjects. In addition, the expression of the four miRNAs was lower in high-risk PCa patients than in low-risk PCa patients, with miR-126 being the most downregulated. The expression of the four miRNAs was also significantly and independently associated with PCa. Receiver operating characteristic curve analysis revealed a significant ability of the miRNAs to distinguish patients with PCa from those with BPH, patients with PCa from controls and low-risk PCa from high-risk PCa. These data suggested that expression of these miRNAs in the blood circulation may be promising, non-invasive biomarkers for the early detection of localized PCa, and for PCa risk stratification. Further validations of the clinical implementation of these results are warranted in a larger cohort.

Expression of microRNA­377 and microRNA­192 and their potential as blood­based biomarkers for early detection of type 2 diabetic nephropathy.

The increased incidence of diabetic nephropathy (DN) in type 2 diabetes (T2D) requires novel markers for the early detection of DN. Previously, microRNAs (miRs) have been demonstrated to be promising disease biomarkers. The present study evaluated the biomarker potential of DN­associated miR­377 and miR­192 in the early stages of DN. The study included 85 participants: 55 patients with T2D (30 without DN and 25 with DN) and 30 healthy controls. The patients with T2D were classified according to albumin­to­creatinine ratio and were split into three groups: Normoalbuminuric group (n=30), microalbuminuric group (n=15) and macroalbuminuric group (n=10). Reverse transcription­quantitative polymerase chain reaction analysis was used to evaluate blood miR expression. It was observed that there was higher miR­377 expression and lower miR­192 expression in T2D patients with and without DN compared with healthy controls (P<0.05). miR­377 was higher in the normoalbuminuric group and gradually increased in the microalbuminuric and macroalbuminuric groups (P<0.05), whereas miR­192 was lower in the macroalbuminuric group compared with the normoalbuminuric group (P<0.05). Regression analysis revealed direct associations between the two miRs and albuminuria (P<0.05). miR­377 was independently associated with DN risk, even following multivariable adjustment, and albuminuria was the only predictor of miR­377 (P<0.001). In discriminating overall patients from healthy subjects, ROC analysis revealed areas under the curve (AUCs) of 0.851 for miR377 and 0.774 for miR­192 (P<0.001). In discriminating the normoalbuminuric group from the microalbuminuric/macroalbuminuric groups, the AUCs were 0.711 (P=0.008) and 0.70 (P=0.049) for miR­377 and miR­192, respectively. In patients with microalbuminuria and macroalbuminuria, miR­377 correlated positively with albuminuria and negatively with renal function, whereas miR­192 correlated negatively with albuminuria and positively with renal function (P=0.001), and the two miRs were correlated with known risk factors of DN (P<0.05). The results suggested that blood­based miR­377 and miR­192 may serve as potential biomarkers for early detection of DN. Further validation studies are required with larger sample sizes.

Evaluation of Two-Diabetes Related microRNAs Suitability as Earlier Blood Biomarkers for Detecting Prediabetes and type 2 Diabetes Mellitus.

Increased the incidence of prediabetes and type 2 diabetes (T2D) worldwide raises an urgent need to develop effective tools for early disease detection to facilitate future preventive interventions and improve patient's care. We evaluated the suitability of diabetes-related miR-375 and miR-9 as earlier biomarkers for detecting prediabetes and T2D.TaqMan-based RT-qPCR was used to quantify the expression of miRNAs in peripheral blood of 30 prediabetes patients, 30 T2D patients and 30 non-diabetic healthy controls. Compared to controls, miR-375 and miR-9 were expressed at higher levels in prediabetes patients and progressively more enriched in T2D patients. Both miRNAs were directly associated with the presence of prediabetes and T2D independently of known risk factors to T2D and miR-375 was independently associated with the development of T2D. Both miRNAs were positively correlated with the glycemic status and other T2D risk factors. The ROC analysis indicated good diagnostic abilities for miR-375 to distinguish overall patients from control and prediabetes from T2D patients. Whereas, miR-9 showed lower values and borderline significance in discriminating the subject groups. The combination of miRNAs enhanced the predictability to discriminate patients from control. These results suggest that miR-375 and miR-9 are associated with the susceptibility to developing T2D and miR-375 alone or in combination with miR-9 could serve as biomarkers for early detection of prediabetes and T2D.