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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

Genome-wide association studies (GWAS) have provided strong evidence that early- and late-onset MG have different genetic backgrounds. Recent in silico analysis based on GWAS results revealed rs231735 and rs231770 variants within CTLA-4 locus as possible MG causative genetic factors. We aimed to explore the association of rs231735 and rs231770 with MG in a representative cohort of Serbian patients. We conducted an age-, sex-, and ethnicity-matched case–control study. Using TaqMan allele discrimination assays, the frequency of rs231735 and rs231770 genetic variants was examined in 447 AChR-MG patients and 447 matched controls. There was no significant association of rs231735 and rs231770 with the entire MG cohort (P > 0.05). Nevertheless, when stratifying patients into early-onset (n = 183) and late-onset MG (n = 264), we found early-onset patients had a significantly lower frequency of the rs231735 allele T compared to controls (OR = 0.734, 95% CI = 0.575–0.938, p10e6 permutation < 0.05), and rs231735 genotype TT and rs231770 genotype TT had a protective effect on early-onset MG (OR = 0.548, 95% CI = 0.339–0.888, and OR = 0.563, 95% CI = 0.314–1.011, p10e6 permutation < 0.05). Consequently, we found that individuals with the rs231735-rs231770 haplotype GC had a higher risk for developing early-onset MG (OR = 1.360, P = 0.027, p10e6 permutation < 0.05). Our results suggest that CTLA-4 rs231735 and rs231770 may be risk factors only for patients with early-onset MG in Serbian population.

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 acknowledg

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 MG

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.

Introduction: Genome-wide association studies (GWAS) identify genomic loci that contain genetic determinants of complex diseases. Subsequent functional genomic approaches, such as bioinformatic finemapping and transcriptome-wide association studies (TWAS), can reveal potentially causal single nucleotide variants (SNVs) that can be tested on patient samples. We applied this approach to study causal SNVs for acetylcholine receptor (AChR) seropositive myasthenia gravis (MG). We focused on CHRNA1 and CHRNB1 loci, coding AChR subunits, and CTLA-4 locus, coding protein transmitting an inhibitory signal to T cells. Methods: CHRNA1 was fine-mapped by PAINTOR using data from GWAS summary statistics, 1000 genome and RegulomeDB. Alongside, rs4151121 identified by TWAS in CHRNB1, and rs231735 and rs231770 identified by fine-mapping in CTLA-4 were studied. SNVs were genotyped using allele discrimination assays in 447 Serbian AChR-MG patients (183 early-onset and 264 late-onset) and 447 sex- and age-matched controls. Results: CHRNA1 rs35274388 was fine-mapped as a potentially causal variant (PIP2=92%) exhibiting transcription factor binding and chromatin accessibility peaks. CHRNA1 rs35274388 minor allele A and CHRNAB1 rs4151121 minor allele G increased the risk for late-onset MG (OR=1.669, 95% CI=1.05-2.638, p=0.027, p10e6 permutation=0.031 and OR=1.322, 95% CI=1.063-1.644, p10e6 permutation=0.014, respectively). On the other hand, CTLA-4 rs231735 recessive genotype TT decreased, while rs231735- rs231770 haplotype GC increased the susceptibility to early-onset MG (OR=0.548, 95% CI=0.339-0.888, p=0.014, p10e6 permutation=0.014 and OR=1.360, p=0.027, p10e6 permutation=0.027, respectively). Conclusion: CHRNA1 rs35274388 and CHRNAB1 rs4151121 loci could be causal genetic factors for lateonset MG while CTLA-4 rs231735 and rs231770 could be causal genetic factors for early-onset MG in Serbian population.

Background: Spinal muscular atrophy (SMA) is the prevalent genetic cause of childhood mortality. Pioneering treatments yield utmost advantages only within the presymptomatic phase, underlining the significance of newborn screening. Materials and methods: 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. Results: In the initial year, 6950 newborns underwent testing, revealing SMA in two unrelated infants and in an asymptomatic 16-month old sibling of the first newborn. 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. 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. In the second year, we included a maternity hospital beyond Belgrade, introducing sample shipping via mail and extending screening accessibility to a greater number of infants. This resulted in 9800 infants undergoing testing within 16 months. Currently, we are actively preparing for the official incorporation of newborn screening for SMA into the national screening program. Conclusions: Timely detection and treatment can transform SMA into a manageable condition.

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.