Circulating Variants of SARS-CoV-2 Among Macedonian COVID - 19 Patients in the First Year of Pandemic.

Genomic epidemiology has proven to be a useful tool for investigating pandemic outbreaks and tracking pathogen spread and evolution. This study describes the circulation of SARS-CoV-2 strains in N. Macedonia during a period of one year, encompassing three waves of the COVID-19 pandemic. A certain percentage (2-3%) of positive cases were continuously selected and analyzed by whole genome sequencing (WGS) technology. Using this approach, a total of 337 SARS-CoV-2 genomes were sequenced and 26 different lineages belonging to 7 clades were detected. During the first wave of the pandemic, the most dominant lineage was B.1.1, followed by B.1.1.70, which became the most dominant in the second wave. The B.1.1.7 lineage completely overpassed all other variants in the third wave. Our study strengthens the notion that the progression of COVID-19 pandemic is associated with emergence of new SARS-CoV-2 variants with increased virulence. The measure of the impact of this viral dynamic on the spread of the pandemic should be evaluated in association with other factors that might influence the transmission.

Alström Syndrome with Early Vision and Hearing Impairement.

Alström syndrome (ALMS) is an autosomal recessive disorder characterized by multiple organ involvement, including progressive cone-rod dystrophy, sensorineural hearing loss, childhood obesity, and type 2 diabetes mellitus. Pathogenic variants in the ALMS1 gene are the known cause for the occurrence of this devastating condition. Here we report on a 12 year old boy referred to the University Clinic with early signs of impaired hearing and vision, obesity, and scoliosis. Central vision was first affected, followed by peripheral vision. In addition, his weight began increasing after the age of two years, reaching 78 kg at a height of 157 cm (BMI 31.64). No polydactyly was present. His mental development was normal in spite of his hearing and vision impairments. There was acanthosis nigricans on the neck. ECG and the cardiac ultrasound were normal. At the age of 12 years, his testicles are 12 ml and his pubertal status is P2 A2. OGTT revealed impaired glucose tolerance with elevated insulin concentrations 121ulU/mL (reference range 2,00-29,1 ulU/mL). Renal function was unaffected, liver functions were normal. Uric acid and lipids were within normal plasma concentrations. A Whole Exome Sequencing was performed and a homozygous ALMS1 pathogenic, frameshift gene variant (LRG_741t1(ALMS1):c.4156dup; p.Thr1386AsnfsTer15) was determined as the cause of the disease. Both parents were carriers for the variant. The absence of mental retardation and polydactyly differentiates Alström and Bardet-Biedle syndrome.

Recent Common Origin, Reduced Population Size, and Marked Admixture Have Shaped European Roma Genomes.

The Roma Diaspora-traditionally known as Gypsies-remains among the least explored population migratory events in historical times. It involved the migration of Roma ancestors out-of-India through the plateaus of Western Asia ultimately reaching Europe. The demographic effects of the Diaspora-bottlenecks, endogamy, and gene flow-might have left marked molecular traces in the Roma genomes. Here, we analyze the whole-genome sequence of 46 Roma individuals pertaining to four migrant groups in six European countries. Our analyses revealed a strong, early founder effect followed by a drastic reduction of ∼44% in effective population size. The Roma common ancestors split from the Punjabi population, from Northwest India, some generations before the Diaspora started, <2,000 years ago. The initial bottleneck and subsequent endogamy are revealed by the occurrence of extensive runs of homozygosity and identity-by-descent segments in all Roma populations. Furthermore, we provide evidence of gene flow from Armenian and Anatolian groups in present-day Roma, although the primary contribution to Roma gene pool comes from non-Roma Europeans, which accounts for >50% of their genomes. The linguistic and historical differentiation of Roma in migrant groups is confirmed by the differential proportion, but not a differential source, of European admixture in the Roma groups, which shows a westward cline. In the present study, we found that despite the strong admixture Roma had in their diaspora, the signature of the initial bottleneck and the subsequent endogamy is still present in Roma genomes.

Orthodontic Treatment of a Periodontally - Affected Adult Patient (Case Report).

BACKGROUND: The advanced periodontal disease is characterised by a strongly pronounced loss of attachment and reduction of the alveolar bone support, which leads to luxation, migration of the teeth, functional discomfort and poor facial aesthetics. CASE PRESENTATION: The aim of this paper is to present the case of a 26-year-old female patient, registered at the Clinic of Periodontology with highly expressed gingivitis, unsatisfactory periodontal status, presence of diastemas between the frontal teeth and attachment loss of 5-6 millimetres in different areas. We conducted a thorough classic periodontal treatment, as well as training for proper maintenance of oral hygiene, with frequent professional oral-prophylactic sessions, complemented with orthodontic treatment. Fixed orthodontic appliances were installed, and mild forces were applied for gradual levelling of the teeth, with constant control of the periodontal status. After 20 months of treatment, the patient was in retention. CONCLUSION: Orthodontic therapy of periodontally-affected teeth can begin only after exhaustive administration of a periodontal treatment. Orthodontic treatment as an addition to the periodontal restoration must be gradual with mild forces for an optimal dental response, thus helping to improve function, facial aesthetics and psychological confidence of adult patients.

Multilevel regression modeling for aneuploidy classification and physical separation of maternal cell contamination facilitates the QF-PCR based analysis of common fetal aneuploidies.

BACKGROUND: The quantitative fluorescent polymerase chain reaction (QF-PCR) has proven to be a reliable method for detection of common fetal chromosomal aneuploidies. However, there are some technical shortcomings, such as uncertainty of aneuploidy determination when the short tandem repeats (STR) height ratio is unusual due to a large size difference between alleles or failure due to the presence of maternal cell contamination (MCC). The aim of our study is to facilitate the implementation of the QF-PCR as a rapid diagnostic test for common fetal aneuploidies. METHODS: Here, we describe an in-house one-tube multiplex QF-PCR method including 20 PCR markers (15 STR markers and 5 fixed size) for rapid prenatal diagnosis of chromosome 13, 18, 21, X and Y aneuploidies. In order to improve the aneuploidy classification of a given diallelic STR marker, we have employed a multilevel logistic regression analysis using "height-ratio" and "allele-size-difference" as fixed effects and "marker" as a random effect. We employed two regression models, one for the 2:1 height ratio (n = 48 genotypes) and another for the 1:2 height ratio (n = 41 genotypes) of the trisomic diallelic markers while using the same 9015 genotypes with normal 1:1 height ratio in both models. Furthermore, we have described a simple procedure for the treatment of the MCC, prior DNA isolation and QF-PCR analysis. RESULTS: For both models, we have achieved 100% specificity for the marker aneuploidy classification as compared to 98.60% (2:1 ratio) and 98.04% (1:2 ratio) specificity when using only the height ratio for classification. Treatment of the MCC enables a successful diagnosis rate of 76% among truly contaminated amniotic fluids. CONCLUSIONS: Adjustment for the allele size difference and marker type improves the STR aneuploidy classification, which, complemented with appropriate treatment of contaminated amniotic fluids, eliminates sample re-testing and reinforces the robustness of the QF-PCR method for prenatal testing.

Y-chromosome haplogroup architecture confers susceptibility to azoospermia factor c microrearrangements: a retrospective study.

AIM: To assess the association between azoospermia factor c microrearrangements and semen quality, and between Y-chromosome background with distinct azoospermia factor c microrearrangements and semen quality impairment. METHODS: This retrospective study, carried out in the Research Center for Genetic Engineering and Biotechnology "Georgi D. Efremov," involved 486 men from different ethnic backgrounds referred for couple infertility from 2002-2017: 338 were azoospermic/oligozoospermic and 148 were normozoospermic. The azoospermia factor c microrearrangements were analyzed with sequence tagged site and sequence family variant markers, quantitative fluorescent polymerase chain reaction, and multiplex ligation probe amplification analysis. The Y-haplogroups of all participants were determined with direct single nucleotide polymorphism typing and indirect prediction with short tandem repeat markers. RESULTS: Our participants had two types of microdeletions: gr/gr and b2/b3; three microduplications: b2/b4, gr/gr, and b2/b3; and one complex rearrangement gr/gr deletion + b2/b4 duplication. Impaired semen quality was not associated with microrearrangements, but b2/b4 and gr/gr duplications were significantly associated with haplogroup R1a (P<0.001 and P=0.003, respectively) and b2/b3 deletions with haplogroup E (P=0.005). There were significantly more b2/b4 duplication carriers in Albanians than in Macedonians with haplogroup R1a (P=0.031). CONCLUSION: Even though azoospermia factor c partial deletions/duplications and Y-haplogroups were not associated with impaired semen quality, specific deletions/duplications were significantly associated with distinct haplogroups, implying that the Y chromosome background may confer susceptibility to azoospermia factor c microrearrangements.

The Effects of NBF Gingival Gel Application in the Treatment of the Erosive Lichen Planus: Case Report.

The therapy of erosive lichen planus (ELP) has been particular problem in the treatment of oral lesions. This case of ELP in male patient 29 years old was treated with topic application of the NBF gingival gel, three times a day after meal, previously rinsed with Clorhexidine gluconate 0.12%. After 5 days of treatment, initial improvements were recorded, and after two weeks of application of the NBF gingival gel we observed significant improvement. Clinical monitoring after the fifth day showed mild epithelialization of the eroded mucosa, yet still present erythematous base of the lesion. After the second week the erythema area was significantly reduced and the eroded surfaces of the mucosa were minimal, measured less than 0.5 mm. After the third week there were no erosions to detect on the oral mucosa, yet still present vague redness, which completely pulled after the fourth week. Treatment ended after the fifth week when the topical application of the NBF gingival gel was terminated, and therapy was done, and clinically achieved effects remained stable even after the third month of the treatment. Topic application of the NBF gingival gel with ELP patients showed positive clinical effects in relatively short time period.

Loss of Y Chromosome in Peripheral Blood of Colorectal and Prostate Cancer Patients.

BACKGROUND: Although age-related loss of chromosome Y (LOY) in normal hematopoietic cells is a well-known phenomenon, the phenotypic consequences of LOY have been elusive. However, LOY has been found in association with smoking, shorter survival and higher risk of cancer. It was suggested that LOY in blood cells could become a predictive biomarker of male carcinogenesis. AIMS, METHODS & FINDINGS: To investigate the association of LOY in blood cells with the risk for development of colorectal (CC) and prostate cancers (PC), we have analyzed DNA samples from peripheral blood of 101 CC male patients (mean age 60.5±11.9 yrs), 70 PC patients (mean age 68.8±8.0 yrs) and 93 healthy control males (mean age 65.8±16.6 yrs). The methodology included co-amplification of homologous sequences on chromosome Y and other chromosomes using multiplex quantitative fluorescent (QF) PCR followed by automatic detection and analysis on ABI 3500 Genetic Analyzer. The mean Y/X ratio was significantly lower in the whole group of cancer patients (0.907±0.12; p = 1.17x10-9) in comparison to the controls (1.015±0.15), as well as in CC (0.884±0.15; p = 3.76x10-9) and PC patients (0.941±0.06; p = 0.00012), when analyzed separately. Multivariate logistic regression analysis adjusting for LOY and age showed that LOY is a more significant predictor of cancer presence than age, and that age probably does not contribute to the increased number of subjects with detectable LOY in cancer patients cohort. CONCLUSION: In conclusion, our results support the recent findings of association of LOY in blood cells with carcinogenesis in males.

White spot lesions: prevention and management during the orthodontic treatment.

The formation of white spot lesions, or enamel demineralization, around fixed orthodontic attachments is a common complication during and following fixed orthodontic treatment, which marks the result of a successfully completed case. This article is a contemporary review of the risk factors and preventive methods of these orthodontics scars. Preventive programmes must be emphasized to all orthodontic patients. The responsibility of an orthodontist is to minimize the risk of the patient having decalcification as a consequence of orthodontic treatment by educating and motivating the patients for excellent oral hygiene practice. Prophylaxis with topical fluoride application should be implemented: high-fluoride toothpastes, fluoride mouthwashes, gels and varnishes during and after the orthodontic treatment, especially for patients at high risk of caries.

Speech Perception Outcomes after Cochlear Implantation in Children with GJB2/DFNB1 associated Deafness.

BACKGROUND: Cochlear implants (CI) for the rehabilitation of patients with profound or total bilateral sensorineural hypoacusis represent the initial use of electrical fields to provide audibility in cases where the use of sound amplifiers does not provide satisfactory results. AIMS: To compare speech perception performance after cochlear implantation in children with connexin 26-associated deafness with that of a control group of children with deafness of unknown etiology. STUDY DESIGN: Retrospective comparative study. METHODS: During the period from 2006 to, cochlear implantation was performed on 26 children. Eighteen of these children had undergone genetic tests for mutation of the Gap Junction Protein Beta 2 (GJB2) gene. Bi-allelic GJB2 mutations were confirmed in 7 out of 18 examined children. In order to confirm whether genetic factors have influence on speech perception after cochlear implantation, we compared the post-implantation speech performance of seven children with mutations of the GBJ2 (connexin 26) gene with seven other children who had the wild type version of this particular gene. The latter were carefully matched according to the age at cochlear implantation. Speech perception performance was measured before cochlear implantation, and one and two years after implantation. All the patients were arranged in line with the appropriate speech perception category (SPC). Non-parametric tests, Friedman ANOVA and Mann-Whitney's U test were used for statistical analysis. RESULTS: Both groups showed similar improvements in speech perception scores after cochlear implantation. Statistical analysis did not confirm significant differences between the groups 12 and 24 months after cochlear implantation. CONCLUSION: The results obtained in this study showed an absence of apparent distinctions in the scores of speech perception between the two examined groups and therefore might have significant implications in selecting prognostic indicators of speech perception following cochlear implantation.

Therapeutic effect of proaftol in treatment of recurrent aphthous stomatitis.

UNLABELLED: Recurrent aphthous stomatitis (RAS) is a quite frequent, painful, ulcerative disease that affects the lining of the oral cavity and has an unknown etiology. The aim of this article is to examine the impact of the medication proaftol on epithelization speed and severity of pain in patients with RAS. In this randomized, double-blind, placebo-controlled study respondents were divided into two groups where one group was treated with proaftol spray and the other with a placebo. Aphthae considered for treatment had a diameter of 5-6 mm. The participants were given instruction on the use of the spray, two sprayings on the place of the aphtae 3-4 times a day. We examined two parameters in the symptomatology of RAS-lesion size (mm) and pain intensity (noted on four subjective levels: 0-no pain, 1-discomfort, 2-moderate pain, 3-severe pain). These parameters were noted on the baseline, the third, the fifth and the eighth days of examination. RESULTS: A significant faster reduction of the dimension of aphthous ulcers in patients treated with proaftol 3rd day p<0.001, 5th day p<0.0006, 8th day full epithelization in the control group. The magnitude of pain in the experimental compared with the control group on 3rd, 5th and 8th day was also significantly reduced: p<0.0001, p<0.007, p<0.007 respectively. CONCLUSION: The use of proaftol positively influences the rate of epithelization and reduction of subjective feeling of pain in patients with RAS. The action of propolis should be the goal of studies with a larger number of subjects.

"Balkan journal of medical genetics"--facts, editorial policies, practices and challenges.

The Balkan Journal of Medical Genetics (BJMG) is an international, open access journal that publishes scientific papers covering different aspects of medical genetics. It is published by the Macedonian Academy of Sciences and Arts twice a year in both printed and electronic versions. BJMG is covered by many abstracting and indexing databases, including PubMed Central and Thomson Reuters. Although there are many journals in the field of medical genetics, only a few come from regions outside Western Europe and North America. Being one of these few journals, BJMG aims to promote genetics and research on this topic in the Balkan countries and beyond. BJMG's ultimate goal is to raise the scientific quality and metrics of the journal and provide a better place for BJMG in the community of scientific journals.

Evaluation of buffer capacity of saliva in caries-free and caries-active children.

OBJECTIVES: The aim of this study was to evaluate the relationship between the physicochemical properties of saliva, such as buffering capacity, in caries-free and caries-active children. MATERIAL AND METHOD: Eighty children aged 12-14 years were included in this study. They were divided into two groups: a caries-free and a caries-active group, with 40 children in each group. Material for biochemical testing of saliva--a sample was taken from all subjects at different time intervals: 5, 30 and 60 minutes after the daily meal. The examined parameters were followed in the same examinees in a sample of saliva taken in the morning before consuming any food or implementation of oral hygiene: they represent basic information compared with the results of the examination. The concentration of salivary bicarbonates was determined by the enzyme method of continuous measurement. RESULTS: Salivary concentration of bicarbonates, measured fasting in the morning (basic values) in the caries-free group, ranging in limits from 6.60 to 9.70 mmol/l, and significantly lower values in the caries-active group (from 1.00 to 3.80 mmol/l). The same was the case with the concentration of salivary bicarbonates in different time intervals after taking the meal--5, 30 and 60 minutes in the examined groups. By increasing the time interval after taking a meal, the concentration of salivary bicarbonates continuously and significantly declined compared to its baseline concentration. The largest decrease of concentration of bicarbonate in terms of its basic value in both examined groups (caries-free and caries-active) took place during the 60 minutes after having the meal. CONCLUSION: Salivary bicarbonates to maintaining the acidobasic balance of saliva, and thus affects the incidence of caries. The positive effect of salivary bicarbonates was confirmed by the values found in this study: the caries-free group presented a higher concentration of bicarbonate than the basic values. The value of bicarbonate in saliva in adolescents can serve as a parameter for determining the risk of caries, and this, in turn, can be used in the planning and implementation of appropriate caries-preventive measures.

Prevalence of hepatitis C virus genotypes in risk groups in the Republic of Macedonia: a 5 years survey.

The prevalence of hepatitis C virus (HCV) genotypes depends on geographical location. HCV genotyping is important for epidemiological investigations and treatment management. The aim of this study was to determine the HCV genotype prevalence in the most prominent risk groups in the Republic of Macedonia in the last 5 years and to evaluate its association with patient's age, gender, and mode of transmission. A total of 1,167 HCV positive patients, divided into three risk groups (intravenous drug use, chronic hemodialysis, and other risk factor), were genotyped using an in-house ASO hybridization method with genotype-specific oligonucleotide probes. The genotypes 1, 2, and 3 were present with 52.2%, 0.6%, and 47.0%, respectively. Genotype 1 was most prevalent in hemodialysis (89.0%) and other risk factor group (53.8%). It was found associated independently with hemodialysis, age >40 and female gender. Genotype 3 predominated in intravenous drug users (64.0%) and was associated significantly also with age ≤40 and male gender. Multivariable logistic regression analysis pointed out hemodialysis (P < 0.0001, Exp (B) = 12.0) as a positive predictor factor for genotype 1 and age ≤40 (P = 0.021, Exp (B) = 1.8) and intravenous drug use (P < 0.0001, Exp (B) = 8.4) as a positive predictor factors for genotype 3. In conclusion, the main transmission route of HCV infection in the Republic of Macedonia is intravenous drug use, followed by hemodialysis. HCV genotypes 1 and 3 dominate in these two most prominent risk groups in the Republic of Macedonia.

Molecular characterization of cystinuria in south-eastern European countries.

Cystinuria is an autosomal recessive disorder caused by defective transport of cystine and dibasic amino acids in the proximal renal tubules and small intestine. So far, more than 128 mutations in SLC3A1 gene, and 93 in SLC7A9 gene have been described as a cause of cystinuria. We present a molecular characterization of the cystinuria in 47 unrelated south-east European families. The molecular methodology included direct sequencing, single strand conformational polymorphism, and restriction fragment length polymorphism. A total of 93 (94.9 %) out of 98 unrelated cystinuric chromosomes have been characterized. Mutations in SLC3A1 gene account for 64.3 % and in SLC7A9 gene for 30.6 % of the cystinuric chromosomes. Ten different mutations in SLC3A1 gene were found, and two of them were novel (C242R and L573X), while in SLC7A9 gene seven mutations were found, of which three were novel (G73R, V375I and c.1048_1051delACTC). The most common mutations in this study were T216M (24.5 %), M467T (16.3 %) and R365L (11.2 %) in SLC3A1 and G105R (21.4 %) in SLC7A9 gene. A population specificity of cystinuria mutations was observed; T216M mutation was the only mutation present among Gypsies, G105R was the most common mutation among Albanians and Macedonians, and R365L among Serbs. The results of this study allowed introduction of rapid, simple and cost-effective genetic diagnosis of cystinuria that enables an early preventive care of affected patients and a prenatal diagnosis in affected families.