ALU-INSERTION Yb8NBC36 IN THE KCNJ6 GENE IS A RISK FACTOR FOR PARKINSON’S DISEASE
Gilyazova I1,*, Khidiyatova I1, Akhmetova V1, Baitimerov A2, Magzhanov R2, Khusnutdinova E1
*Corresponding Author: Dr. Irina Gilyazova, Department of Human Genomics, Institute of Biochemistry and Genetics, Ufa Science Center, Russian Academy of Sciences, 71 Prospekt Oktyabrya, Ufa 450054, Russia; Tel.: +7(3472)356088; Fax: +7(3472)356100; E-mail: gilyasova_irina@mail.ru
page: 43

MATERIALS AND METHODS

We examined 200 unrelated patients, aged 48-83 years (mean 62 years). Patients were examined clinically at the Department of Neurology, Bashkir State Medical Univer­sity, Ufa, Russia, with courses of neurosurgery and medi­cal genetics. All exhibited at least two of the four principal signs of PD: resting tremor, cogwheel rigidity, bradyki­nesia, or postural-reflex impairment. Exclusion criteria included prior history of multiple cerebrovascular events or other causes of parkinsonian symptoms (encephalitis, brain injury or tumor, etc.). The group of patients included 54 Russians, 100 Tatars, 18 Bashkirs and 28 patients who originated from mixed marriage families. The 259 controls (99 Russians, 89 Tatars, 71 Bashkirs), aged 46-82 years were matched for age, ethnic origin and area of residence. Neither control subjects nor their first-degree relatives had a lifetime history of any neurodegenerative disorder. All patients and healthy controls gave consent to being included in the study.

      Genotyping. DNA was extracted from peripheral blood leukocytes using a standard phenol-chloroform extraction [11]. The fragments of 742 bp [Alu-insertion (*I)] and 430 bp [Alu-deficient (*D)] were amplified using polymerase chain reaction (PCR) with the following prim­ers: 5’- CAA GTC GAG TCA GCT ATC CCT-3’ and 5’- ATA TAT TCT GGA TCC CAG TCC C-3’. Polymerase chain reaction was carried out in a total volume of 12.5 µ L containing 100 ng of genomic DNA, 0.25 µ M primers, 0.25 mM dNTPs, 0.5 units Taq DNA polymerase (Silex, Moscow, Russia) and 1.25 µ L 10X buffer (67 mM Tris-HCl, pH 8.8, 6.7 mM MgCl2, 16.6 mM (NH4)2SO4, 0.01% Tween-20). The conditions for genotyping consisted of an initial denaturation for 4 min. at 94°C, followed by 30 cycles of denaturation for 35 seconds at 94°C, annealing for 35 seconds at 59°C and extension for 50 seconds at 72°C. The PCR products were electrophoresed on a 2% agarose gel.

      Statistical Methods. The genotype and allele fre­quency distributions between PD patients and controls were compared using a chi-square test with Yates’s correc­tion [12,13]; p <0.05 was considered to be statistically significant. Odds ratios (OR) with 95% confidence inter­val (CI) were calculated [13,14].

 




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