Development of Real-time PCR Kit for Detection and Quantitation of Six Common Mutations A3243G, G3380A, A8344G, T8993G, T8993C, G11778A of Human Mitochondrial Genome

A procedure for production of a real-time PCR kit for detection and quantitation of 6 common mitochondrial genome mutations including A3243G, G3380A, A8344G, T8993G, T8993C, G11778A using fluorescent locked nucleic acid (LNA) Taqman probes was reported. The procedure consists of designing of specific primers and LNA probes, selection of master mixture components and real-time PCR thermal conditions. The produced kit had specificity of 100% and sensitivity ≥ 1% and remained fully active after 7 days of storage at 25 oC or 20 days at 4 oC or 6 months at -20 oC. The kit was used to analyze A3243G, G3380A, A8344G, T8993G, T8993C, G11778A mutations from 69 patients tentatively diagnosed with mitochondrial diseases and 3 cases of A3243G carriers (4.34%) was found. In these cases, the A3243G mutation was heteroplasmic, maternally inherited, and the heteroplasmy level was shown to be related to the symptome expression.tome expression.

Clinical Characteristics of Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes(MELAS)syndrome, a maternally inherited mitochondrial disorder, is characterized by its genetic, biochemical and clinical complexity. The most common mutation associated with MELAS syndrome is the mtDNA A3243G mutation in the MT-TL1 gene encoding the mitochondrial tRNA-leu(UUR), which results in impaired mitochondrial translation and protein synthesis involving the mitochondrial electron transport chain complex subunits, leading to impaired mitochondrial energy production. Angiopathy, either alone or in combination with nitric oxide (NO) deficiency, further contributes to multi-organ involvement in MELAS syndrome. Management for MELAS syndrome is amostly symptomatic multidisciplinary approach. In this article, we review the clinical presentations, pathogenic mechanisms and options for management of MELAS syndrome.

High Prevalence of Mitochondrial tRNA A3243G Mutation in Invasive Breast Cancer

Background & Aim: Mitochondria play vital roles in various cellular activities, such as energy production, maintaining the redox balance of the cell, the regulation of cellular proliferation and differentiation, and programmed cell death. Mitochondrial tRNA mutations are associated with many pathological conditions and numerous diseases have been associated with them. Therefore, the objectives of this study were to evaluate the presence and the frequency of mutations in the selected tRNAs in breast cancer patients and to correlate these mutations with the clinicopathological characters. Materials & Methods: The is a cross sectional study, where seventy seven breast tumors and adjacent non-tumorous (normal) tissue samples were analyzed by PCR- RFLP, and direct DNA sequencing. In this study three mt-tRNAs wre selected, two of them are the most important disease associated mt-tRNAs i.e tRNALue (UUR), tRNALys and one of the least pathogenic associated tRNA which is tRNAarg. Statistical analysis was used to determine the relationship between the detected mutations and the clinicopathologhical characters. Results: The tRNAleu (UUR) A3243G mutation occurring at the highly conserved location was detected in 4 samples (5.2%) of both breast cancer and adjacent non-tumorous tissue except one detected only in tumor tissue. Another A8343G mutation was detected in tRNAlys. There was no significant association between these mutations and clinicopathological parameters (P. value > 0.05). Conclusion: The detection of high frequent A3243G mutations in patients with invasive breast cancer suggests role of this mutation in the carcinogenesis. This mutation affects complex I, reduces translation of its core subunits, which can contribute to the impairment of oxidative phosphorylation and increase ROS production, leading to cancer progression.

Mitochondrial Diabetes: The Clinical Spectrum of MIDD and MELAS in Association With the A3243G Mutation

Introduction: Maternally inherited diabetes and deafness (MIDD) is a multisystem disorder characterized by insulinopenia and sensorineural hearing loss. This rare form of monogenic diabetes is most commonly associated with the A3243G mutation of mitochondrial DNA (mtDNA). The same mutation is seen in 80 percent of patients with MELAS (Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). MIDD and MELAS have overlapping features suggesting a continuum of expression for the A3243G mutation. Clinical Case: A 41-year-old male was referred for genetic testing and counseling by his ophthalmologist following detection of bilateral punched-out retinal pigment epithelium (RPE) lesions on routine exam. His medical history was significant for type 1 diabetes mellitus (T1DM) diagnosed at the age of 21 and bilateral sensorineural hearing loss diagnosed at the age of 38. The referral was triggered by patient reported family history of MELAS in two of his brothers who were both diagnosed at the age of 10 and died at the ages of 20 and 27. Whether the patient was previously tested during early childhood remains unclear. His medical history was negative for stroke-like episodes or seizures. Neurologic evaluation revealed mild fluent aphasia with paraphasic errors and some comprehension difficulties. MRI of the brain without contrast showed no focus of restricted diffusion. Mitochondrial DNA sequence analysis was performed. A pathogenic variant (m.3243 A>G) was detected in the MT-TL1 gene at approximately 25% heteroplasmy. The patient was advised to avoid metformin given increased risk for lactic acidosis. He was also instructed against use of statins. At the age of 45, the patient presented to the emergency department (ED) with a complaint of headache, vertigo and incoordination. Physical examination revealed left homonymous hemianopsia, right horizontal nystagmus and bilateral upper extremity dysmetria. MRI of the brain without contrast showed a large area of predominantly cortical restricted diffusion involving the right temporal and occipital region with associated T2/FLAIR hyper-intensity. Cross over between the PCA and MCA territories was suggestive of a stroke-like episode related to MELAS. The patient received a bolus of IV arginine at 0.5 g/kg followed by an IV infusion of 0.5 g/kg/day for 3 days. He was later transitioned to oral arginine 5g three times daily and was discharged to a rehab facility. Clinical Lesson: This case demonstrates the evolution of MIDD to MELAS, supporting the concept that both syndromes represent a spectrum of the same disease. A few case reports describe the progression of MIDD to MELAS. Why some patients develop MELAS and others develop MIDD is unclear but may be related to heteroplasmy. Early identification of MIDD and MELAS is crucial given associated comorbidities and unique management issues.

Heteroplasmy Detection of Mitochondrial DNA A3243G Mutation Using Quantitative Real-Time PCR Assay Based on TaqMan-MGB Probes

A point mutation of mitochondrial DNA (mtDNA) at nucleotide position 3243 A to G (mt.3243A>G) is involved in many common diseases, including maternally inherited diabetes and deafness (MIDD) and mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS). However, the mutant level of mt.3243A>G varies both among individuals and in different organs, tissues, and even cells of single individuals. For detection of this mutation, current methods have limited universality and sensitivity and may be not adequate for a routine clinical test. Here, we develop and evaluate a rapid TaqMan-MGB quantitative real-time PCR (qPCR) method for detecting and quantifying the heteroplasmy level of mt.3243A>G in single-tube analysis. With our method, the sensitivity of detection was as low as 0.1%, but the accuracy of quantification was reliable, down to 4%. All positives could be correctly identified, and the heteroplasmy levels determined by qPCR correlated well with the results from restriction fragment length polymorphism (RFLP) and pyrosequencing assays (r = 0.921~0.973 and 0.972~0.984). In addition, we demonstrated that the urinary sediments, leukocytes, or hair follicles might be ideal templates to detect and quantify the heteroplasmy of mt.3243A>G mutation; however, they should be optimized or retreated for further accurate quantification. Our study should allow rapid and high throughput diagnostic testing and can potentially be used to clarify the association between clinical phenotype and pathogenic mitochondrial mutations derived from various tissues.

Lower Circulating miR-122 Level in Patients with HNF1A Variant-Induced Diabetes Compared with Type 2 Diabetes

miR-122, the expression of which is regulated by several transcription factors, such as HNF1A, was recently reported to be associated with type 2 diabetes (T2DM) and hepatocellular carcinoma. HNF1A variants can cause diabetes and might be involved in the development of primary liver neoplasm. Differences in miR-122 expression among different types of diabetes have not been studied. This study aimed to investigate differences in serum miR-122 levels in Chinese patients with different forms of diabetes, including T2DM, type 1 diabetes (T1DM), HNF1A variant-induced diabetes (HNF1A-DM), glucokinase variant-induced diabetes (GCK-DM), and mitochondrial A3243G mutation-induced diabetes (MDM). In total, 12 HNF1A-DM patients, 24 gender-, age-, and body mass index-matched (1 : 2) T2DM patients and 24 healthy subjects were included in this study. In addition, 30 monogenic diabetes (11 GCK-DM and 19 MDM) and 17 T1DM patients were included. Fasted blood biochemistry and miR-122 were measured. The results showed that the HNF1A-DM patients had lower miR-122 levels [0.046 (0.023, 0.121)] than T2DM patients [0.165 (0.036, 0.939), P=0.02] and healthy controls [0.249 (0.049, 1.234), P=0.019]. The area under the curve of the receiver operating characteristic curve for miR-122 to discriminate HNF1A-DM and T2DM was 0.687 (95% CI: 0.52–0.86, P=0.07). There was no difference in serum miR-122 among HNF1A-DM, GCK-DM, MDM, and T1DM patients. Lower serum miR-122 is a unique feature of HNF1A-DM patients and might partially explain the increased risk for liver neoplasm and abnormal lipid metabolism in HNF1A-DM patients.