Effect of the introgression of Atlantic brown trout, Salmo trutta, into Adriatic trout, Salmo farioides in a stream at the drainage area of the Adriatic Sea basin of Montenegro

Background. The diversity of wild trout stocks in Montenegro is compromised by insufficiently controlled stocking. Adriatic and Mediterranean areas show a high degree of endemism of the salmonid species, with numerous native Salmo taxa described. The invasive effect of brown trout, Salmo trutta Linnaeus, 1758, of the Atlantic haplogroup on the endemic Adriatic trout, Salmo farioides Karaman, 1938, in the drainage area of the Adriatic Sea basin is lower compared to the effect inflicted by S. trutta has on Salmo labrax Pallas, 1814 of the Danubian haplogroup. The presently reported study was intended to describe the genetic structure of the population of Adriatic trout, Salmo farioides, from the Mrtvica River, a right tributary of the Morača River, Montenegro, in order to get an insight into the spread of non-native strains and their introgression into the native Adriatic trout gene pool. Materials and methods. Trout specimens sampled from the lower section of the Mrtvica River from 2004 to 2007, and from its upper section in the spring of 2014, were analysed for their mtDNA haplotypes. Nuclear DNA markers (microsatellites, LDH-C1* gene) were also included in genetic analyses since mtDNA is inherited only maternally. After statistical analyses, relations between individuals from the upper and lower Mrtvica River were reconstructed. Results. Using both LDH-C1* and seven microsatellite loci with several alleles exclusive to S. trutta, an introgression was detected only in the upper reaches of the Mrtvica River, where only stream-dwelling trout form occurs. In the lower reaches no introgression was detected at all, as revealed by the absence of both the Atlantic mitochondrial haplotype and microsatellite alleles exclusive to S. trutta. Conclusion. The allelic frequency at seven microsatellite loci of Salmo spp. from the two river sections revealed very different population structure, as a consequence of a low rate of gene flow between them. In addition to the physical barrier that prevents Salmo sp. from the lower Mrtvica River to migrate to the upper river section, it seems that the opportunity to mate with the large pool of conspecifics, including lake-dwelling S. farioides (i.e., Salmo cf. dentex), prevents Salmo trutta from surviving at the lower stretch and spread to the rest of the Morača River and Lake Skadar systems

One year of newborn screening for spinal muscular atrophy – results of a Serbian pilot project

Spinal muscular atrophy (SMA) is the most common genetic cause of death in childhood. Innovative therapies show the greatest benefit only when administered in the presymptomatic period, making newborn screening an ethical and medical priority in many countries. In 2022 Centre for Human Molecular Genetics initiated a feasibility study of the newborn screening for SMA in close collaboration with the University Children's Hospital Tirsova, Association SMA Serbia and with financial support from Novartis Gene Therapies, Roche and Biogen/Medis Pharma aiming to screen up to 8000 babies from the Obstetrics and Gynaecology Clinic Narodni Front during one year. A total of 6950 newborns were tested and SMA was confirmed in two unrelated newborns from families with no history of SMA. A 16-month old sibling of the first baby was also tested, even though he was completely asymptomatic, and SMA was also confirmed. Average time between birth and the first screen-positive result was 5 days, and 8 days between birth and final confirmation of diagnosis. All three children received modifying therapies in less than 10 days from final diagnosis. So far, no false-negatives have been reported among the newborns who tested negative in the screening. As pioneers and leaders in this field, we created synchronised work at different levels of healthcare system, established screening and diagnostic algorithms and follow-up protocols. We are currently involved in scaling up screening to include an additional maternity hospital and preparing the ground for the implementation of the newborn screening for SMA as the official national screening program.BOOK OF ABSTRACTS: 8th CONGRESS OF SERBIAN NEUROSCIENCE SOCIETY with international participation 31 May – 2 June 2023. Belgrade, Serbi

Repeat Interruptions Modify Age at Onset in Myotonic Dystrophy Type 1 by Stabilizing DMPK Expansions in Somatic Cells

CTG expansions in DMPK gene, causing myotonic dystrophy type 1 (DM1), are characterized by pronounced somatic instability. A large proportion of variability of somatic instability is explained by expansion size and patient’s age at sampling, while individual-specific differences are attributed to additional factors. The age at onset is extremely variable in DM1, and inversely correlates with the expansion size and individual-specific differences in somatic instability. Three to five percent of DM1 patients carry repeat interruptions and some appear with later age at onset than expected for corresponding expansion size. Herein, we characterized somatic instability of interrupted DMPK expansions and the effect on age at onset in our previously described patients. Repeat-primed PCR showed stable structures of different types and patterns of repeat interruptions in blood cells over time and buccal cells. Single-molecule small-pool PCR quantification of somatic instability and mathematical modeling showed that interrupted expansions were characterized by lower level of somatic instability accompanied by slower progression over time. Mathematical modeling demonstrated that individual-specific differences in somatic instability had greater influence on age at onset in patients with interrupted expansions. Therefore, repeat interruptions have clinical importance for disease course in DM1 patients due to stabilizing effect on DMPK expansions in somatic cells

Long read sequencing – the next level in genomic research

Sekvenciranje dugih fragmenata ili treća generacija sekvenciranja u realnom vremenu produkuje očitavanja pojedinačnih molekula DNK dužine od 1 kb do nekoliko Mb sa očuvanim epigenetičkim oznakama. Dostupne tehnologije su sekvenciranje pojedinačnih molekula u realnom vremenu (eng. single-molecule real-time sequencing, PacBio) i sekvenciranje kroz proteinske nanopore (Oxford Nanopore Technologies). PacBio tehnologija zasnovana je na detekciji ugradnje nukelotida od strane pojedinačnog molekula DNK polimeraze u realnom vremenu, korišćenjem fluoresecencije kao surogat markera. PacBio HiFi očitavanja su dužine ~15 kb sa tačnošću >99,9%. Oxford Nanopore tehnologija izvodi sekvencu nukleotida iz promena u intenzitetu jonske struje dok DNK prolazi kroz stohastički senzor – proteinsku nanoporu.Može sekvencirati fragmente DNK u rasponu od pet redova veličina (20 bp do nekoliko Mb) sa tačnošću dupleks očitavanja >99,9% kada se koriste R10.4.1 nanopore. Sa elektronskim „čitanjem” nukleinskih kiselina, inovacije kao što su minijaturni uređaj veličine dlana sa cenom <1000 dolara, sekvenciranje na terenu, digitalno obogaćivanje ciljnih sekvenci (adaptivno uzorkovanje) i direktno sekvenciranje RNK, postali su stvarnost. Sekvenciranje dugih fragmenata omogućilo je kompletiranje sekvence genoma čoveka, objavljivanje drafta ljudskog pangenoma i ubrzalo je sekvenciranje genoma eukariota. Od uvođenja metode 2011. godine sekvencirano je ~1000 od 1065 genoma deponovanih u NCBI bazi. Puni potencijal metode u izučavanju transkriptoma i epigenoma biće vidljiv u godinama koje slede. Sekvenciranje dugih fragmenata postaje osnova precizne medicine efikasne za sve ljudske populacije i očuvanja biodiverziteta, i zavredelo je da bude metoda 2022. godine prema časopisu Nature Methods.Long read or third-generation sequencing produces reads from 1 kb to several Mb in length with preserved epigenetic marks, at the single-molecule level and in real-time. Single-molecule real-time sequencing (PacBio) and protein nanopore sequencing (Oxford Nanopore Technologies) are available technologies. PacBio technology is based on monitoring the nucleotide incorporation by a single DNA polymerase molecule in real time using fluorescence as a surrogate marker. PacBio HiFi reads are ~15 kb in length with >99.9% accuracy. Oxford Nanopore sequencing infers nucleotide sequence from the changes in ion current intensity while DNA passes through a stochastic sensor – a protein nanopore. It can sequence DNA fragments ranging in five orders of magnitude (20 bp to several Mb), with duplex read accuracy >99.9% when using R10.4.1 nanopores. Innovations such as a miniature device of the palm-size with a price <1000 dollars, sequencing in the field, digital enrichment of target sequences (adaptive sampling) and direct RNA sequencing have become a reality with the electronic „reading“ of nucleic acids. Long read sequencing enabled completing the human genome sequence and releasing a draft of the human pangenome reference. It has also accelerated genome sequencing of eukaryotic species. Out of 1065 genomes deposited in the NCBI database, ~1000 were sequenced since its development. The full potential of the method in studying transcriptome and epigenome will be visible in the years to come. Long read sequencing is becoming the basis of precision medicine effective for all human populations and biodiversity conservation and was announced as the method of the year 2022 according to Nature Methods

Genetic risk factors in patients with Myasthenia gravis

Myasthenia gravis (MG) is a rare autoimmune disease mediated by antibodies against components of the neuromuscular junction, particularly the acetylcholine receptor (AChR). The prevalence of MG in Belgrade has been estimated at 189 cases per 1,000,000 inhabitants, which is among the highest prevalence reported to date. Genetic studies have mainly pointed to specific HLA alleles associated with MG. However, CTLA-4 and TNFRSF11A, playing a role in the immune response, have recently been associated with MG in genome-wide association studies. Since CTLA-4 and TNFRSF11A promote other autoimmune diseases, the main objective of this casecontrol study was to determine the association between these candidate genes and the risk for developing MG in Serbian population. Genotyping of rs231735 and rs231770 within the CTLA-4 gene and rs4263037 within TNFRSF11A in 447 AChR-MG patients and 447 individually sex- and age-matched controls revealed no association with MG (p=0.344, p=0.923 and p=0.557, respectively). However, when stratifying patients into those with early-onset (n=183) and late-onset MG (n=264), we found an association of minor rs231735 allele T with early-onset MG under the recessive genetic model (OR=0.548, 95% CI=0.339-0.888, p=0.014, p10e6 permutation=0.014). Haplotype analysis revealed that individuals with the GC haplotype rs231735-rs231770 had a higher risk for developing earlyonset MG (OR =1.360, p=0.027, p10e6 permutation =0.027). Considering the sufficient statistical power of the study (>90%) and the selection criteria for controls, our results suggest that the CTLA-4 may be associated with early-onset MG in Serbian population. Analysis of additional variants is needed to understand the association of CTLA-4 with MGBOOK OF ABSTRACTS: 8th CONGRESS OF SERBIAN NEUROSCIENCE SOCIETY with international participation 31 May – 2 June 2023. Belgrade, Serbi

Revolutionizing Spinal Muscular Atrophy Prevention in Serbia: Implementing a Mandatory Statewide Newborn Screening

Spinal muscular atrophy (SMA) is the prevalent genetic cause of childhood mortality. Pioneering treatments yield utmost advantages only within the presymptomatic phase, underlining the medical and ethical significance of newborn screening. In 2022, the Centre for Human Molecular Genetics initiated a pilot study of the newborn screening for SMA, working closely alongside the University Children’s Hospital Tirsova and Association SMA Serbia. The aim was to lay the foundation for the implementation of statewide newborn screening for SMA in Serbia by conducting screening for ~8000 infants from the Obstetrics and Gynaecology Clinic Narodni Front over the course of a year. In the subsequent year, we expanded the initiative to include another maternity hospital located outside Belgrade, introducing sample shipment via postal services and extending screening accessibility to a greater number of infants. In the initial year, 6 950 newborns underwent testing, revealing SMA in two unrelated infants. Subsequently, an older sibling of the first newborn, although asymptomatic at the time, was also tested at the age of 16 months, and SMA diagnosis was confirmed in this child as well. All three children received therapeutic interventions in <1 month from birth. To date, they have exhibited no signs of SMA, and there have been no false negative outcomes among the newborns who tested negative during the screening. In the second year, an additional 5 000 newborns underwent testing. As frontrunners in this field in Serbia, we orchestrated harmonized efforts across various tiers of healthcare, established screening and diagnostic algorithms and follow-up protocols. Our extensive efforts were primarily aimed at elevating awareness among all stakeholders about the critical importance of early disease detection. In this transformative journey, we transitioned from being isolated individuals and visionaries who championed a singular idea to an entire community and nation that now acknowledges the paramount significance of newborn screening. As a result, a total of 11 950 infants underwent testing during the 17-month pilot project, culminating in the rapid incorporation of newborn screening for SMA into the national screening program, effective as of September 14th 2023. Timely detection and treatment can transform SMA into a manageable condition, and there is substantial evidence supporting its inclusion in state-wide screening programs.4th International Congress on Spinal Muscular Atrophy, from 14th to 16th March 2024 in Ghent, Belgiu

Neurofilament as a biomarker of response to genetically designed therapies for spinal muscular atrophy

Considering the substantial impact of genetic therapies for spinal muscular atrophy (SMA), longitudinal follow-up of patients undergoing treatment is crucial to effectively monitor treatment response. While functional rating scales are commonly used as primary outcome measures, they may not fully capture all the therapeutic benefits. To address this limitation, the phosphorylated neurofilament heavy chain (pNFH) protein has emerged as a promising biomarker for evaluating treatment response. pNF-H is a neuron- specific filament that exhibits increased levels in the cerebrospinal fluid (CSF) and plasma in the presence of neuronal degeneration. Our study includes individuals treated with Nusinersen (CSF and plasma samples) and Risdiplam (plasma), as well as age- and sex-matched control subjects (CSF and plasma). By examining the dynamics of pNF-H levels in these groups, we sought to identify significant differences indicative of treatment response. Before treatment, SMA individuals typically exhibit higher levels of pNF-H compared to non-SMA individuals. Elevated levels of pNF-H are associated with more severe clinical manifestations of the disease. During Nusinersen treatment, a notable decline in pNF-H levels during the first 2 months can be observed. Current findings suggest that genetic therapies have a notable impact on reducing pNF-H levels over time. By examining the changes in pNF-H levels, our study offers valuable insights into the underlying biochemical alterations associated with these therapies. Furthermore, it supports the use of pNF-H as a complementary measure to functional rating scales and as a potential biomarker for evaluating treatment effectiveness and monitoring disease progression in SMA

Mutation rates of 22 autosomal STR LOCI in a European population from central Balkan, Republic of Serbia

Analysis of short tandem repeats (STRs) is a tool for human DNA identification, including paternity and criminal investigations. Locus-specific STR mutation rates are critical for interpretation of DNA identification in practice. Accumulating evidence suggests that STR mutation rates are populationspecific. However, STR mutation rates based on trios have not been analyzed in European population yet. In this study, mutation rates of 20 CODIS and two additional autosomal STR loci (Penta E and Penta D) were determined from 1279 cases of paternity testing in the Serbian population, and the relationships between STR mutation rates and sex, allele length, and heterozygosity were investigated. A total of 63 mutations were observed at 18 loci in a total of 28278 allele transmissions, including 62 (98.4%) single-step and 1 (1.6%) two-step mutations. The average mutation rate was 1.7×10-3 (95% CI: 1.3-2.3×10-3), whereas locus-specific mutation rates ranged from 5.6×10-3 (D12S391) to 0.6×10-3 (D2S1338, D5S818, and D21S11). No mutation was observed at the TPOX, TH01, D16S539, and D22S1045 loci. The mutation rate of paternal origin (2.4×10-3) was eight times higher than that of maternal origin (0.3×10-3), and long alleles mutated more frequently than short and medium alleles (χ2=4.436 and χ2=4.646, respectively, p<0.05). No differences were found when analyzing overall expansion versus contraction (χ2=0.999, p=0.317). Among short alleles two expansions and no contraction were detected, while among long ones three expansions and nine contractions were observed. Furthermore, we found no correlation between locus-specific mutation rates and corresponding heterozygosity (rs=-0.188, p=0.559). Comparisons between populations revealed statistically significant differences in locus-specific mutation rates of 13 CODIS STRs between Serbian population and Chinese or Brazilian. Our results show that STR mutation rates depend on sex, allele size and population, and provide useful data on STR mutation rate based on trios for human DNA identification in European populations.Book of abstracts: International Conference of Biochemists and Molecular Biologists in Bosnia and Herzegovina - ABMBBIH May, 202

Phosphorylated neurofilament heavy chain in cerebrospinal fluid and plasma as a Nusinersen treatment response marker in childhood-onset SMA individuals from Serbia

IntroductionBiomarkers capable of reflecting disease onset and short- and long-term therapeutic effects in individuals with spinal muscular atrophy (SMA) are still an unmet need and phosphorylated neurofilament heavy chain (pNF-H) holds significant promise.MethodsWe conducted a longitudinal prospective study to evaluate pNF-H levels in the cerebrospinal fluid (CSF) and plasma of 29 individuals with childhood-onset SMA treated with Nuinersen (SMA type 1: n = 6, 2: n = 17, 3: n = 6). pNF-H levels before and during treatment were compared with the levels of controls (n = 22), patients with Duchenne muscular dystrophy (n = 17), myotonic dystrophy type 1 (n = 11), untreated SMA individuals with chronic type 3 disease (n = 8), and children with presymptomatic SMA (n = 3).ResultsSMA type 1 showed the highest mean CSF pNF-H levels before treatment initiation. All Nusinersen-treated individuals (types 1, 2, and 3) showed significantly elevated mean baseline CSF pNF-H compared to controls, which inversely correlated with age at disease onset, age at first dose, disease duration and the initial CHOP INTEND result (SMA type 1 and 2). During 22 months of treatment, CSF pNF-H levels declined during loading doses, stabilizing at reduced levels from the initial maintenance dose in all individuals. Baseline plasma pNF-H levels in type 1 and 2 SMA were significantly increased compared to other cohorts and decreased notably in type 1 after 2 months of treatment and type 2 after 14 months. Conversely, SMA type 3, characterized by lower baseline pNF-H levels, did not show significant fluctuations in plasma pNF-H levels after 14 months of treatment.ConclusionOur findings suggest that CSF pNF-H levels in untreated SMA individuals are significantly higher than in controls and that monitoring of CSF pNF-H levels may serve as an indicator of rapid short-term treatment response in childhood-onset SMA individuals, irrespective of the subtype of the disease, while also suggesting its potential for assessing long-term suppression of neurodegeneration. Plasma pNF-H may serve as an appropriate outcome measure for disease progression and/or response to treatment in types 1 and 2 but not in type 3. Presymptomatic infants with SMA may show elevated pNF-H levels, confirming early neuronal degeneration

Phosphorylated neurofilament heavy chain in cerebrospinal fluid and plasma as a Nusinersen treatment response marker in childhood-onset SMA individuals from Serbia

IntroductionBiomarkers capable of reflecting disease onset and short- and long-term therapeutic effects in individuals with spinal muscular atrophy (SMA) are still an unmet need and phosphorylated neurofilament heavy chain (pNF-H) holds significant promise.MethodsWe conducted a longitudinal prospective study to evaluate pNF-H levels in the cerebrospinal fluid (CSF) and plasma of 29 individuals with childhood-onset SMA treated with Nuinersen (SMA type 1: n = 6, 2: n = 17, 3: n = 6). pNF-H levels before and during treatment were compared with the levels of controls (n = 22), patients with Duchenne muscular dystrophy (n = 17), myotonic dystrophy type 1 (n = 11), untreated SMA individuals with chronic type 3 disease (n = 8), and children with presymptomatic SMA (n = 3).ResultsSMA type 1 showed the highest mean CSF pNF-H levels before treatment initiation. All Nusinersen-treated individuals (types 1, 2, and 3) showed significantly elevated mean baseline CSF pNF-H compared to controls, which inversely correlated with age at disease onset, age at first dose, disease duration and the initial CHOP INTEND result (SMA type 1 and 2). During 22 months of treatment, CSF pNF-H levels declined during loading doses, stabilizing at reduced levels from the initial maintenance dose in all individuals. Baseline plasma pNF-H levels in type 1 and 2 SMA were significantly increased compared to other cohorts and decreased notably in type 1 after 2 months of treatment and type 2 after 14 months. Conversely, SMA type 3, characterized by lower baseline pNF-H levels, did not show significant fluctuations in plasma pNF-H levels after 14 months of treatment.ConclusionOur findings suggest that CSF pNF-H levels in untreated SMA individuals are significantly higher than in controls and that monitoring of CSF pNF-H levels may serve as an indicator of rapid short-term treatment response in childhood-onset SMA individuals, irrespective of the subtype of the disease, while also suggesting its potential for assessing long-term suppression of neurodegeneration. Plasma pNF-H may serve as an appropriate outcome measure for disease progression and/or response to treatment in types 1 and 2 but not in type 3. Presymptomatic infants with SMA may show elevated pNF-H levels, confirming early neuronal degeneration