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CLINICAL AND MOLECULAR-GENETIC FEATURES OF THE NEUROMUSCULAR PATHOLOGIES IN REPUBLIC OF MOLDOVA
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Introduction: Despite the fact that most hereditary diseases of the nervous system are quite rare, in overall, the subgroup of neurogenetic diseases is large, includes more than 2.400 diff erent diseases affl icting the less than one of the 2.000 peoples throughout the world. Problems arising in the study of hereditary diseases of the nervous system (HDNS), are very complex because of the diffi culty of diagnosis, due to the fact that clinical polymorphism (in the same as well as in diff erent families) and genetic heterogeneity is characteristic for these conditions.
Scope. To evaluate some epidemiological and clinical aspects of hereditary neuromuscular diseases (HNMD) in Moldova (1991-2016). To study molecular- genetic features of “core” neuromuscular diseases (DMD/B, SMA and hereditary motor sensory neuropathies IA type (HMSN, 1A)).
Material and methods. Th is work have been done on the based of the scientifi c department of National Centre of Reproductive Health and Medical Genetics and Institute of Mother and Child according to the referral. We used information about 1.556 aff ected and 20 kinds of hereditary diseases. Data collection began since 1991 and was conducted on a continual basis. Have been used the following methods: clinical pedigree analysis, biochemical, electrophysiological, and molecular-genetic (basic methods of identifying of mutations: PCR, multiplex PCR and restriction fragment length polymorphism (RFLP)).
Results: The prevalence of HNMD in RM was 23,5: 100.000 population. For the fi rst time defi ned proportion (58%) of HNMD in the structure of hereditary diseases of the nervous system in children. Th ree of the fi ve common groups of neurological pathology related to neuromuscular pathology and traditionally fall into the «core» of hereditary diseases of the nervous system in the Moldova. Th e incidence rate of DMD/B – 6.11 per 100.000, SMA – 3.8 per 100.000 and HMSN – 7.2 per 100.000 population of the RM. Th e association between the number of cases and the number of population in the districts was medium – Spearman ρ coeffi cient was from 0,46 to 0,5. Th e molecular-genetic analysis was carried out in 213 patients with DMD/BMD for direct search of extended deletions in the DMD gene using 3 sets of primers for MPCR. In our study, the percentage of deletions detection in patients with DMD/BMD is 85% (181 from 213) that is consistent with studies of the European population, such as Italian surveys – 78%, French surveys – 68%, Hungarian surveys – 73%, Belgium (Leuven) surveys – 81,8%. Th us, thanks to the introduction and modifi cation of existing methods of defi nition of deletions, we raised the percentage of detectable deletions in exons to 85%, and were able to wide the range of detectable deletions and to identify 6 rare variant of deletions and 16 double deletions, which are not described in the DMD mutations databases. In patients with SMA from Moldova may be observed the extensive deletions which are usually concentrated in hotspots of exons 7 and 8. Investigation of extended deletions by the PCR-RFLP was performed in 113 patients, and deletions of exon 7 have been found in 65,42% patients, the deletions of the exon 8 in 51.4 % patients. Deletions of exons 7 and 8 were revealed in 62 patients (35,5%). Th e possibility of using direct diagnosis, i. e. search of deletions in exons 7 and 8 in patients with SMA from RM is 85.0%. Th e prevalence of HMSN 1A duplications in patients from Moldova is 69%, according to various authors, from 34.3% (Spain, Barcelona) up to 85% (Israel). Analysis of markers D17S921, D17S122 and D17S834 in patients showed the following frequences: 23.88% duplications of D17S921 locus, 32.83% for D17S122 locus and 24.62% for D17S834 locus. It was found also that the same individual may have mutations in two loci.
Conclusions: Created Register HDNS where recorded data on all diagnosed cases and families burdened HDNS allows the use of information for the practical needs of medical and genetic counseling.. Based on the results of determining the spectrum is formed list of diseases for the introduction of new methods for molecular diagnostics.
Due to clinical polimorphysns and genetic heterogeneity, hereditary diseases of the nervous system (HDNS) are difficult for diagnosis and classification. Hereditary neuromuscular disease subgroup includes more than 2400 different diseases with incidence of 1:2000. The problems arises with HDNS are very complex due to difficult diagnosis and clinical polymorphism (i. e., in the same family as well as in different families) and genetic heterogeneity.
AIM OF THE STUDY
Monitoring the epidemiological situation with neuro-muscular disorders in the population of the Republic of Moldova for the past 25 years (1991- 2016) and assessment of genetic features of “core” neuro-muscular pathologies (Duchenne/Becker Muscular Dystrophy, Slinal Muscular Atrophy, Motor Sensory Hereditary Neuropathy Type IA);
MATERIALS AND METHODS
The study was carried out on the basis of the Institute of Mother and Child, Center of Reproductive Health and Medical Genetics, Laboratory of Medical Genetics, the Republic of Moldova. Conform to the aims proposed were used data of 1556 patients or individuals suspicion of hereditary pathologies of the nervous system. Data collection since 1991 have been made continuously to the present. To address the proposed objectives have been used the following methods: clinical-biochemical, electrophysiological investigations, family history and methods of molecular genetics to determine identification of mutations, i. e., polymerase chain reaction (PCR), multiplex PCR and restriction fragment length polymorphism analysis (RLFP). Statistical analysis of the data and build the graphs was carried out by applying the language and software R (www.r-project.org). Data on the population of each region represents the statistical data from the National Bureau of Statistics (www.statistica.md). The population of regions, with the exception of Transnistria, presented as for 2011. Names of each regions for the graphs were abbreviated as follows: AN – Anenii Noi BL - Balti BR - Briceni BS - Basarabeasca CA - Causeni CH - Cahul CL - Calarasi CM - Cimislia CN- Cantemir CR- Criuleni CS - Chisinau DB – Dubasari DD – Donduseni DR - Drochia ED - Edinet FL-Faleşti FR- Floresti GL - Glodeni HC - Hancesti IL- Ialoveni LE - Leova NS - Nisporeni OC - Ocniţa OR - Orhei RS - Rascani RZ - Rezina SD- Soldanesti SO- Soroca SR - Sangerei ST- Straseni SV- Stefan-Voda TL - Telenesti TR- Taraclia UG – Gagauz Yeri UN - Ungheni TN – Transnistria Due to the lack of data on the population of the regions in Transnistria they were grouped in a single region – Transnistria (TN): BD - Bender TH - Tiraspol TN1- Crigoriopol TN2 - Slobozia TN3 - Camenca RB - Rabnita Regions of Administrative Territorial Unit Gagauz Yeri include the following regions: CT – Comrat, CG - Ceadâr-Lunga, VL – Vulcăneşti. The prevalence of diseases has been reported as the number of patients per 100000.
For the first time was carried out continuous monitoring of nervous system disorders. Using clinical and electromyographic mathods of investigations of 645 patients from the Republic of Moldova with hereditary neuromuscular diseases, which were included in genetic registry we are revealed 58% of cases of neuromuscular hereditary diseases. The prevalence of these pathologies has been estimated as 23,5:100000 of population. The age of patients ranged from 3 months to 65 years, more than half were under the age of 25 years. According to the level of degeneration of the neuromuscular system, cases were divided into progressive muscular dystrophy, neurogenic muscular atrophy (amyotrophy) and hereditary neuropathies . Duchenne muscular dystrophy was revealed in 435 patients, with the prevalene of 11.7 per 100000 population. Hereditary neuromuscular disorders in this group represents 46% and includes X-linked pseudohypertrophy, affecting the muscles of the limbs and shoulder and facial area. In the subgroup of X-linked muscular dystrophy prevailed in children more often revealed pseudohypertrophy which progressing to Duchenne/Becker type of disorder (214 famililes, 238 patients) with the prevalence 6.11 to 100000 of population. The disease is characterized by continuos worsening with severe decreasing of motor activity. In all patients was detected an increasing the level of serum creatine kinase and lactate dehydrogenase 6-20 fold higher than normal vaslue. Electromyographic study (EMG) revealing the primary character of muscular pathology. Autosomal forms of Limb-girdle Muscular Atrophy are most prevalent in adolescents with autosomal recessive inheritance of Erb muscular dystrophy. The distribution of prevalences in the Republic of Moldova is 4.4 of 100000 population, were revealed 157 patients, aged between 6 and 35 years. In most cases, the disease progressed slowly, 5 women have given birth to healthy babies. The autosomal recessive form of hip-shoulder Leiden-Mobius dystrophy was established in 3 patients. The disease progressed rapidly, and at the age of 7-10 years has lead to immobility and appearances of deformations. Clinical manifestations of the disease is similar to the Duchenne Muscular Dystrophy. Limb-girdle Muscular Atrophy was esteblished in 21 pacienţi. The prevalence of this form was 0,58 of 100000 population. Spinal muscular atrophy was registered in 137 patients from the 106 families. The prevalence in the population of Moldova was 3.8 cases per 100000 of population. The prevalence of hereditary neuromuscular disease was 15%. In 97.1% of the observations, we can assume the autosomal recessive mode of inheritance, and in 2.9% the autosomal dominant mode (it was confirmed at the molecular level). Hereditary sensory-motor neuropathies were observed in 260 patients from the 140 families, distribution in Moldova being 7.2 to 100000 of population. Among the hereditary neuromuscular diseases, these pathologies consists 30%. Conform clinical, electrophysiological and genetic characteristics were identified 5 clinical forms of hereditary neuropathies: autosomal dominant CMT type I, autosomal dominant and autosomal recessive CMT type II, type III (Dejerine-Sottas) and X-linked type. Characterization of identified HDNS types and forms allow to make the analysis of distribution of prevalent neuromuscular pathologies in different regions of the Republic of Moldova over the past 20 years. Evaluation of the number of cases of CMT, SMA and DMD demonstrates the highest incidence for Chisinau municipality – 28, 28 and 51, respectively. For the period evaluated (lack of data in the register) were not detected cases of DMD, SMA or CMT in regions Basarabeasca, Vulcanesti, and Drochia (Fig. 1). Analysis of cumulative prevalence distribution in population show that in about 23% of the regions prevail DMD. SMA has been recorded in more than 4.5% of the regions. For 6.8% of regions have not been obtained data. Other districts are characterized by the high frequency of CMT . For the first time has been demonstrated the distribution of neuromuscular pathologies in Republic of Moldova. With average significance, it was shown the association between the number the cases of pathologies and the population of districts (ρ = 0.48). In this regard, epidemiological studies of HDNS in different regions and the creation of the HDNS registry in country are especially relevant for neurological and genetic health services. Register of hereditary diseases of the nervous system, created in 1991, where they were recorded all the data about the cases diagnosed and about families with hereditary pathology enables the use of the information for the purposes of practical medical genetic counseling.
Another objective of the study consisted in studying the incidence of deleţiilor in the dystrophin gene in patients with DMD from the Republic of Moldova and analysis of distributions of deletions in the DMD gene. Duplications and deletions occurs everywhere in the dystrophin gene. However, the deletions are tend to concentrate in two gene regions: in the “hot” region of 5 ‘ end (exons 2-20) and in central part of the gene (exons 44-55), i. e., in regions that contains from 1 up to a few deleted exons. According to data from world literature, 30% of deletions are located in the proximal part of the gene, and 70% in the distal part of the gene . Conform to our data, the percentage of determined deletions in patients with DMD/BMD in Moldova is rather high and is 85% (181 from 213), which corresponds to the data obtained in Europe population, especially those of Italy – 78% (Politano L., 1998), Hungary – 73% (Herczegfalvi A., 1999), Belgium (Leuven) – 81.8% (Dr. Gert Matthijs and Annemie Meertens, 2004), Canada – 73% (Stockley et al., 2006). However, in some populations the frequency of deletions’ detection in patients with DMD/BMD in patients from Moldova, e. g., by the data from Argentina there detected only 84 (48%) patients from 174 non-related individuals (Giliberto et al., 2004); in Chile – in 47% of patients with DMD/BMD have been identified extended deletions (P. Rocco et al, 2001); in Korea deletions have been detected in 58.6% cases ; in China in 65% of the patients had deletions (Lo et al., 2006); in Australia 58% patients presented deletions (Taylor et al. 2007) [http://www.dmd.nl/]. This is a good indicator in Moldova and there are obvious criteria for the clinical diagnosis and in introduction of advanced search engine of deletions in 32 exons of DMD gene. By mena of cluster analysis of all deleted exons deletaţi in patients were determined the contribution of each exon in the pathological process. The analysis showed what proximal part of the gene is implicated in minority of patients – 2,7%, 2,3%, and 2,6% for exons 3, 6, and 8 of DMD gene. The analysis showed the involvement of three central regions of the gene in pathologial process, namely, exons 45, 47, and 48 – 6,11%, 8,55%, and 10,08%, respectively, and other region with exons 50, 51, 52, and 53, with share of 7,02%, 4,27%, 3,97%, and 2,29%, respectively. Thus, in the group of patients with DMD in Moldova, as in previous research, was noted the prevalence of involvement of distal and central parts of DMD gene in the deletional process. The results of the analysis of deletions in patients from Moldova have been identified the deletions comprises single exon in 81 patients (44.75%), other patients had from two to 45 exons. Patients with deletion of single exon, were divided according to clinical forms as follows: 12 (14.8%) patients with MD Becker and 69 (85.2%) patients with MD Duchenne. First place by prevalence occupying 48 exon (10%), followed by 47 exon (8.5%) and 50 and 45 exons (7.02% and 6.11%, respectively). Analyzing the data concerning deletions depending on the presence or absence of frameshift have been identified in most cases in-frame deletions in 30 (37.0%) of investigated patients, and in 50 patients (61.7%) were revealed out-of frame deletions, and in single case was detected deletion of first exon, in Leiden database, this type of DMD deletion can have effects at the level of transcription (RNA) and translation (protein). Also it is need to note the presence of rare mutaitions in Moldavian sample: • Rare extended deletion involvong exon 19 ex01ex19del -> c.-244-?_2380+?del in a 6 years old boy. Analysis of this mutation showed difficulties in prognosis, as the process involves initial part of transcriptional region. Deletion comprising 45 exons (ex01ex45del -> c.-244-?_6614+?del) was diagnosed in 2 individuals, and present difficulties as inviolved the start of the gene (first exon). • Extended deletion which involved central and distal parts of Dystrophin gene, comprises thirty eight exons (ex03del -> c.94-?_186+?del & ex08ex44del -> c.650-?_6438+?del), and software identified the deletion as not producing frameshift, and determined in 4 years old children with DMD. In such a way, due to the implementation and modification of existing methods of determination of deletions, widening the range of exons from 13 (Beggs A.) and 17 (Abbs S.) until 23, then 36, we are increasing the rate of detection of deletions up to 78.4%, and having the possibility to determine 8 rare mutations and 10 double deletions.
An important direction initiated by our team in the following years (1995-1996) was studying the SMN1 gene whose mutations lead to one of the most common neuromuscular diseases – spinal muscular atrophy (Verding-Hoffman disease). The first diagnosis of the disease has been realized with the support of the Laboratory of Molecular Genetics, led by A. Poleacov (now Center for molecular genetics, RAMS), Moscow, Russian Federation. In line with scheme of the determination of the genetical nature of disease in a specific familes and keep in mind of the high frequency of deletions of exon 7 and/or exon 8 of SMN gene in patients with SMA I-III types, was carried out analysis of mutatios in patients with typical and atypical forms of SMA. It was used a variant of the method for detecting of mutations, developed in the diagnostic laboratory of Center of Molecular Genetics, Moscow, Russia. Identification of extended deletions using the PCR with RFLP analysis was used in 113 patients, and were determined deletions of exon 7 in 73 of patients (65,42%), deletion of exon 8 in 89 patients (51,4%). Deletion of exon 7 is more prevalent than deletion of exon 8. Deletions of exons 7 and 8 were determined in 62 patients (35,5%). The possibility to use the direct diagnosis, i. e., searching the deletions in exons 7 and 8 in patients with SMA in Moldova is 85,0%. Molecular-genetic study was carried out in 106 DNA samples obtained from the patients with HMSN 1A (Charcot-Marie disease) and in its relatives. Were investigated 106 patients from 79 families from Republic of Moldova. Family history in selected data showed in 56% of cases the presence of autosomal dominant pattern of inheritance. For linkage analysis with autosomal dominant loci kniwn as HMSN were used high polymorphic micro satellite markers linked to locus HMSN 1A 17p11.2-p12 (РМР22). The incidence of dupliocations in locus 17p11.2-p12 (РМР22) in patients from Moldova constitued 69%, conform different authors, from 34,3% (Spain, Barcelona) up to 85% (Israel). Analysis of markers D17S921, D17S122 and D17S834 in analysed patients shoiwed the following frequencies: 23,88% of duplication for locus D17S921, 32,83% – for locus D17S122 and 24,62% – for locus D17S834. Therefore, in was shown what in the same patient can be determined two mutations in two l oci. Conform to the data from European Consortium of Investigation of Charcot-Marie-Tooth disease, de novo duplication was noted in 76,5% of patients with HMSN I patients, in which parents have not clinical pr EMG signs of neuropathy. However, under the current conditions within the Republic of Moldova, some analysis cannot be done and that is why we have organized partnerships with various laboratories abroad, as well as Department of Molecular Neuroscience, UCL Institute of Neurology, UK; „Genomed”, Russia„ Genotek”, Russia, and Center of Molecular Genetics in Moscow, Russia etc. Thus, as an example of such cooperation we can present the case of a patient with ERB’s muscular dystrophy, which established a new type of mutation in the gene CAPN3 (c.550delA (4 exon) and c.1302C>T (10 exon) (Figure 2) which was not known at that time . In another case, two mutations have been detected in heterozygous status in exon 10 (c.1331delCinsTCGTACCCAG) which causes the deletion of amino-acid from 444 position of the protein and in exon 13 (c.1696G>A) within CAPN3 gene, where were not known at that time. Another patient with signs of motor sensory neuropathy clinic-sensorială, where we are not be able to determine any mutation from among those most commonly found in the population of the Republic of Moldova, that we analyze in cooperation with „Genomed” laboratory. Using the panel of „Neuro Muscular Diseases” it was determined the exon 11 mutation in the heterozygous state, described earlier in the gene SH3TC2 (chr5:148406435G>A, rs 80338933), which leads to the appearance of the site of premature termination of translation in codon 954 (p. Arg954Ter, NM_024577.3). This mutation in homozygous or compound heterozygous state along with other mutations has been described in patients with the Charcot-Marie Tooth disease type 4c OMIM:608206.0005). Has been identified mutation c.679C>T in heterozygous state, not described previously in exon 6 of that same gene SH3TC2 (chr5:148421031G>A, rs532463685), which causes the amino acid substitution at position 227 (pArg227Trp, NM_024577.3). In this regard, in accordance with the examples described, we note that sequencing method allows for an accurate diagnosis. It should be noted, that the present diagnostic panel for neuro-muscular diseases, which include genes responsible for 341 various hereditary diseases, makes it easier to establish a correct diagnosis, due to the fact that the clinical manifestations of various neuromuscular pathologies can be remarkably similar.
Created registry of HDNS contains the data about all the diagnosed cases and families with HDNS, and can later be used in the practice of medical genetics and counseling. Based on the results of the determination the range of diseases it allow to adopt new methods of molecular diagnostics. In this sense, the development in the Republic of Moldova the centralized system of medical assistance and genetics contribute to the improvement of quality of prenatal diagnosis and improving diagnostic service in the country. New modified set of primers (series 6, with 36 of exons) allowed the broadening the range of DMD gene deletions detected up to 85% and the discovery of 6 variants of the rare mutation, 16 double deletions and 3 new deletions that were missing in the database MDD-LOVD version v2.0 dated of August 5, 2016. The share of detection of deletions of exon 7 and 8 in SMN gene in patients from Moldova with spinal muscular atrophy is 85%. Family analysis in data selection showed what in 56% of cases the pattern of inheritance in CMT disease is autosomal dominant. The incidence of duplications NSME 1A in patients from the Republic of Moldova is 69%. Prenatal diagnosis is an importanr strategy and effective measure for prevention of the birth of children with hereditary single-gene diseases in families at risk. Development in the Republic of Moldova the centralized system for medical assistance and genetics contribute to the improvement of the quality of prenatal diagnosis and improve diagnostic service in the country