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THE FREQUENCY OF DYSTROPHIN GENE MUTATION IN THE FAMILIES OF PATIENTS WITH DUCHENNE\BECKER MUSCULAR DYSTROPHY IN UZBEKISTAN POPULATION
Omonova Umida Tulkinovna, PhD of the Tashkent Pediatric Medical Institute E-mail: mbshakur@mail.ru
THE FREQUENCY OF DYSTROPHIN GENE MUTATION IN THE FAMILIES OF PATIENTS WITH DUCHENNE\BECKER MUSCULAR DYSTROPHY IN UZBEKISTAN POPULATION
Abstract: The article represents the data of molecular genetic researches performed among 91 patients with Duch-enne/Becker muscular dystrophy from 81 families. Results of the researches showed that in Uzbekistan population family cases of progressing Duchenne/Becker muscular dystrophy prevail over de novo mutation, and prolonged deletions were in the 3' area of dystrophine gene. According to indirect DNA-diagnostics the most informative ones for the families with high risk in Uzbekistan population are intra gene high polymorphic markers STR45 and STR49.
Keywords: progressing muscular dystrophy, molecular genetic researches, dystrophine.
Duchenne muscular dystrophy (ICD G71.0 - muscular dystrophy; synonyms - Duchenne pseudohypertrophic myodystrophy,
Duchenne progressing muscular dystrophy, Duchenne disease, Duchenne palsy) is the most malignant and the most widely spread form of myodystrophy. Population frequency of this pathology is 1:3500 boys and 21.7 per 100000 alive born boys [3; 5]. The group of dystrophic pathologies includes Duchenne/Becker progressing muscular dystrophies (D/BMD), representing allele variants conditioned by various mutations in dystrophine protein gene [1; 3]. Approximately 55-65% of all the cases of D/BMD were conditioned by the deletions of dystrophine gene with various lengths, 5-10% cases by duplications of a part of the gene, and the rest had spotty mutations [2; 4].
In the modern time there is scope of data relevant to deletion spectra in dystrophine gene in various populations in the world, and certain population differences were determined [2; 5]. Up to now molecular genetic studies of D/BMD were not performed in Uzbekistan. That's why for the development of the approaches of prenatal, differential, presymptomatic diagnostics and definition of mutant gene carriers it is topical to study molecular genetic nature of D/BMD in Uzbekistan.
The objective: study of genetic polymorphism of dystrophine gene among the patients with Duchenne/Becker muscular dystrophy and definition of the prevalence of dystrophine gene mutation carriers among the relatives in the families of the patients with D/BMD in Uzbekistan population.
Materials and methods. Molecular genetic researches were performed in scientific Research Institute of Hematology and Blood transfusion of the Ministry of Health of the Republic of Uzbekistan in the department of molecular medicine and cellular technologies. Molecular genetic researches were performed in two fields: multiplex PCR diagnostics for the detection of major deletions in the patients with D/BMD and performance of indirect DNA-diagnostics for the definition of dystrophine mutant gene carriers among women relatives of the patients with D/BMD. Genome DNA taken from the samples ofperipheral blood (Vacutainer Becton Dickinson International with EDTA) was isolated with the help of the DIAtom™ DNA Prep100 set for DNA isolation produced by "Center of Molecular genetics" Ltd., Moscow in compliance with the manual. Amplifica-
tion was performed using thermocyclers GeneAmp PCR-system 2720 (Applied Biosystems, USA) and Corbett Palm Cycler (Corbett Research, CG1-96 model, Australia) with the help of DMD-del and DMD-CA sets ("Center of Molecular genetics" Ltd., Moscow).
Direct DNA-diagnostics was done in 91 patients with D/BMD from 81 families; 84 (92.3%) patients with Duchenne muscular dystrophy, and 7 (8.6%) patients with Becker muscular dystrophy. Analysis was performed according to 20 exons of dystrophine gene -promoter area, 3, 4, 6, 8, 13, 17, 19, 32, 42, 43, 44, 45, 47, 48, 50, 51, 52, 53, 60 exons.
Indirect diagnostics was done in 21 families with D/BMD cases, using intra gene high polymorphic markers located in the 45th (STR-45), 49th (STR-49), 50th (STR-50) intrones of the gene.
Results and discussion. The studied pathology has X-reces-sive type of transmission. The basic group of the families consisted of non-relative couples and we did not reveal a direct correlation between the number of relative couples and the prevalence of the disease. According to the results of genealogical history study we revealed 5 families with close relative couples, and that was equal to 6.2% of the total number of the examined families; deletions were revealed in 4 cases of close relative couples (80%). Correspondingly, the number of non-relative couples was equal to 93.8% (76 families), and deletions were revealed in 59.2% cases (45 families).
All mutations of dystrophine gene revealed in 4 families with relative couples in each case were characterized by deletions of one exon in distal area of 3'-end (deletions of 48, 50, and 53 exons), and in 3 families out of 4 Duchenne muscular dystrophy was diagnosed for the first time in that generation.
According to the results of molecular diagnostics 33 patients (36.3%) from 32 семей (39.5%) had no deletions, while 58 patients (63.7%) from 49 families (60.5%) had major deletions of dystrophine gene with various lengths: from one to nine exons; in 65.3% there were verified long deletions; deletions of one exon were observed in 34.7% of the families. The basic deletion spectrum was located in distal part of dystrophine gene - 3'-end (deletion of 40-60 exons), that was equal to 81.6% (40 families, 47 patients).
Analysis of deletions most often revealed deletions in the distal part of dystrophine gene; and that was equal to 81.6% from the total number of the revealed mutations: 50 exon in 24 cases, 51 exon in
Section 3. Medical science
24 cases, 52 exon in 21 cases, 48 exon in 16 cases, 53 exon in 14 cases; there were no deletions in promoter area, 32. 42. And 60 exons.
In cases of maximal mutations more often we observed deletions of 19 (8 cases) and 17 (6 cases) exons.
Indirect diagnostics was performed in 21 families with the cases of progressing Duchenne/Becker muscular dystrophies using intra gene high polymorphic markers located in the 45th (STR-45), 49th (STR-49), 50th (STR-50) intrones of the gene. In 33.3% (7 families) out of 21 families direct DNA-diagnostics major deletions in dystrophine gene did not reveal, in 14.3% (3 families) - deletions in 5'-end were observed; and in 52.4% (11 families) we revealed deletions in 3'-end.
We performed 81 indirect DNA-diagnostics in 21 families with high risk of D/BMD. Among these families there were 8 (38.1%) non-informative in STR 45, STR 49, STR 50; in 13 (61.9%) families women relatives ofproband with D/BMD had confirmed the status of dystrophine gene mutation carriers.
On the basis of the Republican Center "Mother and child screening" the method of prenatal invasive diagnostics of Duchenne/Becker MD was implemented. 6 families with high risk of the pathology were checked; in 5 cases cordocentesis and in one case amniocentesis was performed.
According to the results of DNA-diagnostics we revealed 3 male fetus with indentified deletions in dystrophine gene and recommended to stop the pregnancy due to medical genetic indications. In the rest families pregnancy was prolonged with further postnatal checking of the born children for the confirmation of the absence of the damage in dystrophine gene.
Conclusions:
1. Results of proper molecular genetic researches showed that in Uzbekistan population family cases of Duchenne/Becker MD prevail over de novo mutations, and prolonged deletions in the 3'area of dystrophine gene.
2. According to the data of multiplex PCR in patients with Duchenne muscular dystrophy mutations were revealed in 16 exons out of 20 researches, and that served the basis for the application of the set for direct PCR-diagnostics of major deletions in dystrophine gene in Uzbekistan population.
3. According to the result of indirect DNA-diagnostics in the families with high risk in Uzbekistan population the most informative ones are intra gene high polymorphic markers STR45 and STR49.
References:
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2. Dadaly Y. L., Malmberg S. A., Podagova Y. V., Polyakov A. V., Petrukhin A. S. Characteristic clinical manifestations of Duchenne progressing muscular dystrophy in heterozygote carriers of mutations in dystrophine gene // Russian medical journal. - 2007. - No. 3. - P. 18-21 (in Russian).
3. Yevtushenko S. K., Shaymurzin M. R., Yevtushenko L. F. Pharmaceutical and non-pharmaceutical therapy of cardiomyopathy and pneumopathy in the cases of progressing neural muscular pathologies in children // Taurida medical biological bulletin. - 2009. - Vol. 12, -No. 2. - P. 46 (in Russian).
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