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As shown in the table in the main group SDR was 1.89 ± 0.06, PI — 0.98 ± 0.03, (p < 0.001), RI — 0.78 ± 0.01.
In the comparison group, the vascular resistance index in the MCA were within standard values and do not exceed 95 percentile:
SDR — 3.83 ± 0.10, PI — 1.61 ± 0.05, RI — 0.79 ± 0.01.
In the control group these figures were — 3.86 ± 0.10, 1.62 ± 0.03 and 0.81 ± 0.02 respectively.
Conclusion
As you can see from the above data, the restructuring of the fetal hemodynamics leads to increased cerebral blood flow (reduction of vascular resistance index in the middle cerebral artery). During comparative analysis of the vascular resistance in the UA there is increase up to 6 times in the main group relative to the comparison group. It should be emphasized that in this case combined disturbance in placental blood flow detected.
Thus, the Doppler study of blood flow parameters in the feta-placental link of the main group showed that the umbilical artery isolated increase in vascular resistance (SDR above 3.0) is occurs in 33.33 %, combined disturbance of blood flow in the umbilical artery and middle cerebral artery occurs in 26.67 % of observances. It should be noted that generally placental blood flow disturbances in the control group is detected overall in 10 %, and in the main group — 60 %, which is six times more often. Changes in feta-placental blood flow (increased rates of resistance index in umbilical artery and the decline in vascular resistance in the MCA), reflect the current fetal hypoxemia.
In general, as shown by our study, the frequency of adverse perinatal outcomes is increasing in the main group, which are exacerbating by the detection of changes in fetus hemodynamics during dopplerometry.
References:
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Raimova Malika Mukhamedjanovna, Tashkent State Dental Institute, Uzbekistan E-mail: malikamed-74@yandex.ru
Genetic associations of S282T polymorphism of the gene Nat2 with parkinsonism: clinical and molecular comparisons
Abstract: There have been carried out the molecular genetic studies of patients with Parkinson's disease (PD) and vascular parkinsonism (VP) among the people of Uzbek nationality. The association of different genotypes of S282T polymorphism of NAT2 gene with Parkinson's disease was studied. There were revealed significant differences in the frequency of the genotype m/m among the main and control groups (x2 = 4.9; p = 0.02). Homozygous for mutant alleles of the S282T polymorphism of NAT2 gene was reliably associated with increased risk of PD, the development of akinetic-rigid form and the rapid rate of progression.
Keywords: parkinsonism, Parkinson's disease, NAT2 gene.
Parkinson's disease (PD) is one of the four most common neurodegenerative disease of elders. In average, from 120 to 200 per 100 000 population worldwide is susceptible to this disease [1; 9; 10].
Over the past decade tremendous efforts have made to study the etiology and pathogenesis of Parkinson's disease. Numerous foreign and domestic researches have shown that the basis for the development of PD are complex interactions of genetic and environmental factors [2; 3; 4].
Alongside of the development of molecular genetic technologies, opportunities for the formalization of the genetic components of susceptibility to PD were opened, a significant number of data on the involvement of different polymorphic genes in the predisposition to the formation of PD were accumulated [4; 5; 6; 7; 8; 11].
However, despite the achievements of world scientific society in the human genome studies and the development ofhigh-resolution methods of DNA analysis, still we know relatively small number of genes, which in aggregate only partially explain some of the links in the pathogenesis of PD [6; 12; 13; 14].
Many works indicate the influence of NAT2 acetylation polymorphism on the development ofvarious diseases, including some of the neurological, in particular Parkinson's disease [3; 5; 6; 8]. One of the researches [3] informs about the increasing frequency of polymorphism in the "slow acetylators" in the PD group compared with the control group in the UK, but this relationship was not confirmed after the introduction of corrections for multiple comparisons. These results prompted us to further investigation of
Section 5. Medical science
association with PD Nat2 polymorphisms as a marker of increased risk of development of the disease.
We set a goal to search for associations of polymorphisms S282T of the gene Nat2 with parkinsonism (Parkinson's disease and vascular parkinsonism) in Uzbek people, as well as carrying out gene-phenotype associations in case of discovery of this connection.
Material and methods: We examined 200 patients with parkinsonism, out of which 140 patients with Parkinson's disease and 60 patients with vascular parkinsonism. As a control group of molecular genetic analysis was performed in 80 patients with chronic cerebral ischemia without signs of parkinsonism, comparable age and gender. Nationality all the patients were Uzbek, namely patient's relatives in three generations have been Uzbeks.
Material for DNA was 1 ml of blood from the cubital vein. For blood collection, storage and transportation, we used vacutainers or disposable plastic tubes with anticoagulant (conservator) with a volume 0.5 ml. The blood was stored at temperatures below -20 °C for further processing.
DNA isolation was carried out according to standard protocol for DNA isolation using a reagent kit Diatom™ DNA Prep 200 (production of "Izogen Laboratory" Ltd., Moscow, Russia).
Further, the supernatant with DNA was subjected to genotyp-ing by direct PCR amplification.
PCR analysis was performed using a set of reagents for PCR amplification of DNA GenePak™ PCR Core (production of "Izogen Laboratory" Ltd., Moscow, Russia). For these purposes, we used ready for the amplification tubes Master Mix containing Taq DNA polymerase inhibited for "hot start" in lyophilized dry condition, desoxynucleozodthreephosphates, and magnesium chloride with final concentrations 1 u, 200 mkM. and 2.5 mM, respectively, as well as optimized buffer system for standard PCR amplification. We added 5 ml. of a mixture of primers with final concentration of 0.5 mM, 10 ml. of PCR solvent, and 5 ml. of investigated DNA in test tubes Master Mix. For PCR amplification, we used GeneAmp® PCR System 9700 with a gold 96-cell block (Applied Biosystems).
Differentiation of DNA samples for the gene Nat2 was performed with primers 5 'gga-aca-aat-tgg-act-tgg 3' (F) and 5 'tct-agc-atg-aat-cac-tct-gc 3' (R).
Since analyzed mutation is a point nucleotide substitution, for its verification was used RFLP analysis — the analysis of restriction fragment length polymorphism (Restriction Fragment Length Polymorphism — RFLP analysis). For the analysis of the selected mutations we used Restrictase Fok1.
PCR amplification products were fractionated in 2-3 % agarose gel for 60-90 min. at a voltage of 100-120 V., colored with ethidium bromide, and visualized under UV light (Fig.1).
M — 1 234 56 78
766 bp 429 bp
337 bp 238 bp
Fig.1. The analysis of Nat2 gene in patients with PD in the DNA samples of 8 patients: M — molecular marker; — Control of the reaction; 1-8 — the samples of patients; 1, 2, 4-7 — genotype wt/m; 3 — genotype wt/wt; 8 — genotype m/m
Results of research and discussion
During carrying out of the molecular genetic studies to search for mutations in the gene NAT2 S282T homozygotes by mutant alleles were detected in patients with Parkinson's disease in 15 % (30 patients), which was reliably (p < 0.05) higher than in the control
group in which this mutation observed in 6.25 % of cases (5 patients). Study of the odds ratio (OR) showed that the OR > 1, i. e. S282T mutation in the gene NAT2 OR=2.8 (95 % CI 1.01 to 8.7) and that the chances of developing Parkinson's disease patients with mutations in the gene NAT2 S282T were increased in 2.8 times (Table 1).
Table 1. - The distribution of NAT2 genotypes in patients with parkinsonism and control sample
Genotype control (n = 80) patients (n = 200) x2 P OR
n % n %
wt/wt 45 56.25 78 39 6.2 0.01 0.49 (0.28-0.87)
wt/m 30 37.5 92 46 1.35 0.24 1.4 (0.8-2.5)
m/m 5 6.25 30 15 3.9 0.05 2.8 (1.01-8.7)
Note: wt — wild-type allele; m — mutant type allele; %2 — Pearson and high odds ratios marked in bold font.
Comparative analysis of gene NAT2 genotype frequencies between samples of patients with parkinsonism and the control group, we were able to obtain a statistically significant difference in genotype distribution in the case of wt/wt (p = 0.01, x2 = 6.2), with OR=0.49, which indicates that the wild-type allele is not associated with the development of parkinsonism. The table demonstrates that there is a quantitative predominance of genotype wt/m in the group with par-
distribution; p — significance level; OR — odds ratio, reliable parameters
kinsonism (46 %) compared with the control group (37.5 %), however, significant differences were not found between the groups (p=0.24).
Genotypes within which there is a wild-type allele, associated mainly with the phenotype of rapid acetylators, while homozygotes for mutant alleles — with a phenotype of slow acetylators.
We also investigated the frequency of occurrence of different genotypes of S282T polymorphism, depending on the clinical
form of parkinsonism. The analysis revealed significant differences in the incidence of genotype m/m in patients with PD (17.98 %) compared with the control group (6.25 %) (x2 = 4.9, p = 0.02). The value of OR = 3.26 indicates an association of genotype with the development of PD. In analyzing the distribution of the genotype wt/wt revealed that the "wild" genotype was significantly more common in the control group (56.25 %) compared with
PD (32.1 %) (x2 = 11.2, p = 0.001). The value of OR = 0.37 suggests an association of the genotype with resistance to the development of PD. In patients with VP frequency of occurrence of different genotypes of S282T polymorphism of NAT2 gene had no statistically reliable differences with the control group, but with the PD group had significant differences in genotype wt/wt, a value of OR = 0.39 (Table 2).
Table 2. - The distribution of NAT2 genotypes depending on the clinical forms of parkinsonism
Genotype Control (n = 80) Patients with PD (n = 140) Patients with VP (n = 60) P1 P2 P3
n % n % n %
wt/wt 45 56.25 45 32.1 33 55 0.001 0.1 0.005
wt/m 30 37.5 70 50 22 36.7 0.09 0.1 0.1
m/m 5 6.25 25 17.9 5 8.3 0.02 0.9 0.13
Note: wt — wild-type allele; m — mutant allele of type; P1 — reliability of differences between control subjects and patients with PD; P2 — reliability of differences between control subjects and patients with VP; P3 — reliability of differences between patients with PD and VP, reliable parameters marked in bold font.
We examined the association between genotype m/m with var- Conclusions: The study of S282T polymorphism of NAT2 gene
ious clinical features of Parkinson's disease. In this case two of them shows increase in frequency of genotype m/m in patients with PD were identified: association with the variant of the disease and the (x2 = 4.9; p = 0.02). Homozygous by mutant alleles of the S282T rate of progression: this genotype was associated with an akinetic- polymorphism of NAT2 gene is associated with increased risk of PD, rigid form of the disease (60 %) and rapid progression (48 %) development of its akinetic-rigid form and rapid progression.
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