Employing real time PCR for the diagnosis of Huntington disease

Huntington disease [HD] is a dominantly inherited, neurodegenerative disease characterized by choreiform movement disturbances and dementia. The onset age of this disease is varied but usually is between the ages 40-50. Huntington's disease is caused by a triplet-repeat expansion in the IT15 gene [also known as huntingtin or HD] which is located on chromosome 4p3.1. Since many clinical picture of HD are indistinguishable from other distinct genetic disorders molecular test such as PCR is the only way to confirm the disease. The aim of this study was to introduce a new and fast technique for the diagnosis of Huntington disease. Blood specimens were collected from individuals suspected for Huntington disease and also people with no symptoms and family history of this disease. DNAs were extracted according to standard protocol. Using conventional PCR, patient positive for Huntington disease were diagnosed. Then employing real time PCR on the basis of difference between melting temperature [Tm] a new and fast diagnostic method was introduced. Among 29 patients suspected to be HD only 8 HD patients were confirmed using PCR and real time PCR. The numbers of CAG repeat were between 42-50 and melting temperatures were between 89-92. The concept of using melting temperature in real time PCR protocol presented in here could be employed for the rapid diagnosis of the diseases caused by the increased in triple repeat sequences. It is fast, robust and has the potential use for the prenatal diagnosis

Investigation of polymorphisms in non-coding region of human mitochondrial DNA in 31 Iranian hypertrophic cardiomyopathy [HCM] patients

The D-loop region is a hot spot for mitochondrial DNA [mtDNA] alterations, containing two hypervariable segments, HVS-I and HVS-II. In order to identify polymorphic sites and potential genetic background accounting for Hypertrophic CardioMyopathy [HCM] disease, the complete non-coding region of mtDNA from 31 unrelated HCM patients and 45 normal controls were sequenced. The sequences were aligned upon the revised Cambridge Reference Sequence [rCRS] and any incompatibilities were recorded as numerical changes in homoPolymeric C Tract [PCT], single base substitutions, insertions and deletions [Indels]. Nucleotide substitutions were found to make up the majority of the mutations, rather than indels. We drew significantly high transition rate [81.8%] versus lower frequency of transversions [18.2%]. 12 polymorphisms were identified in this study which had not been published in the MitoMap database. PCT changes at position 303-309 were detected in 83% of our samples. Our results suggest that an increased level of HVS-I and HVS-II substitutions may be an indicator of mitochondrial DNA instability. Furthermore, mtDNA mutations may play an important role in pathogenesis of cardiac arrest which has remained unexplained for long

Lack of association of mitochondria A3243G tRNA leu mutation in Iranian patients with type 2 diabetes

Many kinds of mutations in mitochondrial [mt] DNA have been reported to be related to the development of Diabetes Mellitus [DM], this type of diabetes has also been shown to be influenced by other genetic factors and/or environmental factors. Among them, tRNALeu[UUR] and its adjacent mtDNA NADH dehydrogenase subunit 1[ND1] region within the mt genome are linked to high susceptibility to DM. A point mutation at 3243 base pair [bp] in the mt tRNA Leu[UUR] is commonly referred to as a syndrome of mitochondrial myopathy, Encephalopathy, Lactic acidosis, and Stroke-like episodes [MELAS]. In the current study, we have assessed the frequency of the A3243G in Iranian diabetic type 2 patients. DNA was obtained from peripheral leukocytes of 154 patients with diabetes Mellitus type2 [l50 with type 2 and 4 with gestational diabetes] and 40 control subjects. Insulin concentration from patients' blood was measured using Radioimmunoassay procedure. Patients showed fasting blood sugar [FBS] between 150-230 mg/dl, body mass index [BMI] between 19-32 Kg/m2 and insulin concentration 0.9-2.35 mg/ml. PCR-RFLP, single strand conformation polymorphism [SSCP] and sequencing methods were used to detect the A3243G or other mutations in the mitochondrial tRNALeu [UUR] gene. A3243G mutation was not detected in patients. SSCP results showed a new pattern of PCR product in 6 patients. The C3316T transition mutation in the ND1 mitochondrial gene was confirmed in selected samples [n=6] by sequencing. No differences were observed between the two groups for C3316T and A3243G mutations [P=0.348]. The mt C3316T mutation did not have any effect on the clinical finding of type 2 diabetes carrying this mutation. These data together with clinical characteristics of the patients may suggest that the mt C3316T mutation might be a polymorphism in the Iranian population