Genomic sequencing for newborn screening: current perspectives and challenges.

Traditional newborn screening (NBS) serves as a critical tool in identifying conditions that may impact a child's health from an early stage. Newborn sequencing (NBSeq), the comprehensive analysis of an infant's genome, holds immense promise for revolutionizing health care throughout the lifespan. NBSeq allows for early detection of genetic disease risk and precision personalized medicine. The rapid evolution of DNA sequencing technologies and increasing affordability have spurred numerous endeavors to explore the potential of whole-genome sequencing in newborn screening. However, this transformative potential cannot be realized without challenges. Ethical aspects must be carefully navigated to safeguard individual rights and maintain public trust. Moreover, genomic data interpretation poses complex challenges due to its amount, the presence of variants of uncertain significance, and the dynamic nature of our understanding of genetics. Implementation hurdles, including cost, infrastructure, and specialized expertise, also present barriers to the widespread adoption of NBSeq. Addressing these challenges requires collaboration among clinicians, researchers, policymakers, ethicists, and stakeholders across various sectors. Robust frameworks for informed consent, data protection, and governance are essential. Advances in bioinformatics, machine learning, and genomic interpretation are crucial for translation into actionable clinical insights. Scalability and improving downstream health care access are vital for equitability, particularly in underserved communities. By fostering interdisciplinary collaboration, advancing technology and infrastructure, and upholding ethical principles, we can unlock the full potential of NBSeq as a tool for precision medicine and pave the way toward a future where every child has the opportunity for a healthier, genomics-informed start to life.

Experience with comprehensive pharmacogenomic multi-gene panel in clinical practice: a retrospective single-center study.

AIM: To assess the prevalence of actionable pharmacogenetic interventions in patients who underwent pharmacogenetic testing with a multi-gene panel. METHODS: We retrospectively reviewed single-center electronic health records. A total of 319 patients were enrolled who underwent pharmacogenomic testing with the RightMed test panel using TaqMan quantitative real-time PCR method and copy number variation analysis to determine the SNPs in the 27 target genes. RESULTS: Actionable drug-gene pairs were found in 235 (73.7%) patients. Relevant guidelines on genotype-based prescribing were available for 133 (56.7%) patients at the time of testing. Based on the patients' genotype, 139 (43.6%) patients were using at least one drug with significant pharmacogenetic interactions. CONCLUSION: Two out of three patients had at least one drug-gene pair in their therapy. Further studies should assess the clinical effectiveness of integrating pharmacogenomic data into patients' electronic health records.

Composition of the immunoglobulin G glycome associates with the severity of COVID-19.

A large variation in the severity of disease symptoms is one of the key open questions in coronavirus disease 2019 (COVID-19) pandemics. The fact that only a small subset of people infected with severe acute respiratory syndrome coronavirus 2 develops severe disease suggests that there have to be some predisposing factors, but biomarkers that reliably predict disease severity have not been found so far. Since overactivation of the immune system is implicated in a severe form of COVID-19 and the immunoglobulin G (IgG) glycosylation is known to be involved in the regulation of different immune processes, we evaluated the association of interindividual variation in IgG N-glycome composition with the severity of COVID-19. The analysis of 166 severe and 167 mild cases from hospitals in Spain, Italy and Portugal revealed statistically significant differences in the composition of the IgG N-glycome. The most notable difference was the decrease in bisecting N-acetylglucosamine in severe patients from all three cohorts. IgG galactosylation was also lower in severe cases in all cohorts, but the difference in galactosylation was not statistically significant after correction for multiple testing.

Replication of 15 loci involved in human plasma protein N-glycosylation in 4802 samples from four cohorts.

Human protein glycosylation is a complex process, and its in vivo regulation is poorly understood. Changes in glycosylation patterns are associated with many human diseases and conditions. Understanding the biological determinants of protein glycome provides a basis for future diagnostic and therapeutic applications. Genome-wide association studies (GWAS) allow to study biology via a hypothesis-free search of loci and genetic variants associated with a trait of interest. Sixteen loci were identified by three previous GWAS of human plasma proteome N-glycosylation. However, the possibility that some of these loci are false positives needs to be eliminated by replication studies, which have been limited so far. Here, we use the largest set of samples so far (4802 individuals) to replicate the previously identified loci. For all but one locus, the expected replication power exceeded 95%. Of the 16 loci reported previously, 15 were replicated in our study. For the remaining locus (near the KREMEN1 gene), the replication power was low, and hence, replication results were inconclusive. The very high replication rate highlights the general robustness of the GWAS findings as well as the high standards adopted by the community that studies genetic regulation of protein glycosylation. The 15 replicated loci present a good target for further functional studies. Among these, eight loci contain genes encoding glycosyltransferases: MGAT5, B3GAT1, FUT8, FUT6, ST6GAL1, B4GALT1, ST3GAL4 and MGAT3. The remaining seven loci offer starting points for further functional follow-up investigation into molecules and mechanisms that regulate human protein N-glycosylation in vivo.

Early results of intra-articular micro-fragmented lipoaspirate treatment in patients with late stages knee osteoarthritis: a prospective study.

AIM: To analyze clinical and functional effects of intra-articular injection of autologous micro-fragmented lipoaspirate (MLA) in patients with late stage knee osteoarthritis (KOA). Secondary aims included classifying cell types contributing to the treatment effect, performing detailed MRI-based classification of KOA, and elucidating the predictors for functional outcomes. METHODS: This prospective, non-randomized study was conducted from June 2016 to February 2018 and enrolled 20 patients with late stage symptomatic KOA (Kellgren Lawrence grade III, n=4; and IV, n=16) who received an intra-articular injection of autologous MLA in the index knee joint. At baseline radiological KOA grade and MRI were assessed in order to classify the morphology of KOA changes. Stromal vascular fraction cells obtained from MLA samples were stained with antibodies specific for cell surface markers. Patients were evaluated at baseline and 12-months after treatment with visual analog scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and Knee Injury and Osteoarthritis Outcome Score (KOOS). RESULTS: Three patients (15%) received a total knee replacement and were not followed up completely. Seventeen patients (85%) showed a substantial pattern of KOOS and WOMAC improvement, significant in all accounts. KOOS score improved from 46 to 176% when compared with baseline, WOMAC decreased from 40 to 45%, while VAS rating decreased from 54% to 82% (all P values were <0.001). MLA contained endothelial progenitor cells, pericytes, and supra-adventitial adipose stromal cells as most abundant cell phenotypes. CONCLUSION: This study is among the first to show a positive effect of MLA on patients with late stages KOA.

Plasma N-glycome composition associates with chronic low back pain.

BACKGROUND: Low back pain (LBP) is the symptom of a group of syndromes with heterogeneous underlying mechanisms and molecular pathologies, making treatment selection and patient prognosis very challenging. Moreover, symptoms and prognosis of LBP are influenced by age, gender, occupation, habits, and psychological factors. LBP may be characterized by an underlying inflammatory process. Previous studies indicated a connection between inflammatory response and total plasma N-glycosylation. We wanted to identify potential changes in total plasma N-glycosylation pattern connected with chronic low back pain (CLBP), which could give an insight into the pathogenic mechanisms of the disease. METHODS: Plasma samples of 1128 CLBP patients and 760 healthy controls were collected in clinical centers in Italy, Belgium and Croatia and used for N-glycosylation profiling by hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) after N-glycans release, fluorescent labeling and clean-up. Observed N-glycosylation profiles have been compared with a cohort of 126 patients with acute inflammation that underwent abdominal surgery. RESULTS: We have found a statistically significant increase in the relative amount of high-branched (tri-antennary and tetra-antennary) N-glycan structures on CLBP patients' plasma glycoproteins compared to healthy controls. Furthermore, relative amounts of disialylated and trisialylated glycan structures were increased, while high-mannose and glycans containing bisecting N-acetylglucosamine decreased in CLBP. CONCLUSIONS: Observed changes in CLBP on the plasma N-glycome level are consistent with N-glycosylation changes usually seen in chronic inflammation. GENERAL SIGNIFICANCE: To our knowledge, this is a first large clinical study on CLBP patients and plasma N-glycome providing a new glycomics perspective on potential disease pathology.

Case report: State-of-the-art risk-modifying treatment of sudden cardiac death in an asymptomatic patient with a mutation in the SCN5A gene and a review of the literature.

Brugada syndrome is a rare hereditary disorder characterized by distinct ECG findings, complex genetics, and a high risk of sudden cardiac death. Recognition of the syndrome is crucial as it represents a paradigm of sudden death tragedy in individuals at the peak of their lives. Notably, Brugada syndrome accounts for more than 20% of sudden cardiac deaths in individuals with structurally normal hearts. Although this syndrome follows an autosomal dominant inheritance pattern, it is more prevalent and severe in males. Diagnosis is primarily based on the characteristic ECG pattern observed in the right precordial leads. Mutations in the SCN5A gene, resulting in loss of function, are the most common genetic cause. We presented a 36-year-old proband with a family history of sudden cardiac death. Although the patient was asymptomatic for Brugada syndrome, his father had experienced sudden death at the age of 36. The proband was admitted to St. Catherine's Specialty Hospital where blood was taken and subjected to next-generation sequencing (NGS) using a "Sudden cardiac death" panel. The analysis identified a pathogenic variant in the SCN5A gene [c.4222G > A(p.Gly1408Arg)], which is associated with autosomal dominant Brugada syndrome. Based on the positive genetic test result, the patient was referred for further examination. ECG with modified precordial lead positioning confirmed the presence of the Brugada phenotype, displaying the type-2 and type-1 ECG patterns. Therefore, we made the diagnosis and decided to implant an implantable cardioverter-defibrillator (ICD) based on the results of broad genetic NGS testing, diagnostic criteria (ECG), and considering the high burden of sudden cardiac death in the patient's family, as well as his concerns that limited his everyday activities. This case shows that genetics and personalized medicine hold immense potential in the primary prevention, diagnosis, and treatment of Brugada syndrome and sudden cardiac death.

Sudden Cardiac Death-A New Insight Into Potentially Fatal Genetic Markers.

Sudden cardiac death (SCD) is an unexpected and dramatic event. It draws special attention especially in young, seemingly healthy athletes. Our scientific paper is based on the death of a young, 23-year-old professional footballer, who died on the football field after a two-year history of cardiac symptoms. In this study we analyzed clinical, ECG and laboratory data, as well as results of genetic testing analysis in family members. To elucidate potential genetic etiology of SCD in this family, our analysis included 294 genes related to various cardiac conditions.

Pharmacogenomics at the center of precision medicine: challenges and perspective in an era of Big Data.

Pharmacogenomics (PGx) is one of the core elements of personalized medicine. PGx information reduces the likelihood of adverse drug reactions and optimizes therapeutic efficacy. St Catherine Specialty Hospital in Zagreb/Zabok, Croatia has implemented a personalized patient approach using the RightMed® Comprehensive PGx panel of 25 pharmacogenes plus Facor V Leiden, Factor II and MTHFR genes, which is interpreted by a special counseling team to offer the best quality of care. With the advent of significant technological advances comes another challenge: how can we harness the data to inform clinically actionable measures and how can we use it to develop better predictive risk models? We propose to apply the principles artificial intelligence to develop a medication optimization platform to prevent, manage and treat different diseases.

A 24-Month Follow-Up Study of the Effect of Intra-Articular Injection of Autologous Microfragmented Fat Tissue on Proteoglycan Synthesis in Patients with Knee Osteoarthritis.

Osteoarthritis (OA) is a widely prevalent disease worldwide, and with an increasingly ageing society, it has become a challenge for the field of regenerative medicine. OA is a disease process involving multiple joint tissues, including those not visible on radiography, and is a complex disease process with multiple phenotypes that require evaluation by a multimodality imaging assessment. The purpose of this study was to evaluate the effect of micro-fragmented fat tissue intra-articular injection 24 months after application in two ways: Indirectly using functional magnetic resonance imaging (MRI) assessment analyzing the glycosaminoglycans (GAG) content in cartilage by means of delayed gadolinium (Gd)-enhanced magnetic resonance imaging of cartilage (dGEMRIC), as well as clinical outcome on observed level of GAG using standard orthopedic physical examination including VAS assessment. In our previous study assessing comprehensive results after 12 months, the dGEMRIC results have drawn attention. The present study explores the long-term effect of intra-articular injection of autologous microfragmented adipose tissue to host chondrocytes and cartilage proteoglycans in patients with knee OA. A prospective, non-randomized, interventional, single-center, open-label clinical trial was conducted from January 2016 to April 2018. A total of 17 patients were enrolled in the study, and 32 knees were assessed in a 12-month follow-up, but only 10 patients of them with 18 knees are included in a 24-month follow-up. The rest of the seven patients dropped out of the study 12 months after follow-up: three patients underwent knee arthroplasty, and the remaining four did not fulfil the basic criteria of 24 months involvement in the study. Surgical intervention (lipoaspiration), followed by tissue processing and intra-articular injection of the final microfragmented adipose tissue product into the affected knee(s), was performed in all patients. Patients were assessed for a visual analog scale (VAS), dGEMRIC at the baseline, three, six, 12 and 24 months after the treatment. A magnetic resonance sequence in dGEMRIC due to infiltration of the anionic, negatively-charged contrast gadopentetate dimeglumine (Gd-DTPA2) into the cartilage indicated that the contents of cartilage glycosaminoglycans significantly increased in specific areas of the treated knee joint. Our results suggest that this method of single intra-articular injection of autologous microfragmented adipose tissue improves GAG content on a significant scale, with over half of the measurements suggesting relevant improvement 24 months after intra-articular injection opposed to the expected GAG decrease over the natural course of the disease.

Immunophenotyping of a Stromal Vascular Fraction from Microfragmented Lipoaspirate Used in Osteoarthritis Cartilage Treatment and Its Lipoaspirate Counterpart.

Osteoarthritis (OA) is a degenerative joint disease accompanied by pain and loss of function. Adipose tissue harbors mesenchymal stem/stromal cells (MSC), or medicinal signaling cells as suggested by Caplan (Caplan, 2017), used in autologous transplantation in many clinical settings. The aim of the study was to characterize a stromal vascular fraction from microfragmented lipoaspirate (SVF-MLA) applied for cartilage treatment in OA and compare it to that of autologous lipoaspirate (SVF-LA). Samples were first stained using a DuraClone SC prototype tube for the surface detection of CD31, CD34, CD45, CD73, CD90, CD105, CD146 and LIVE/DEAD Yellow Fixable Stain for dead cell detection, followed by DRAQ7 cell nuclear dye staining, and analyzed by flow cytometry. In SVF-LA and SVF-MLA samples, the following population phenotypes were identified within the CD45- fraction: CD31+CD34+CD73±CD90±CD105±CD146± endothelial progenitors (EP), CD31+CD34-CD73±CD90±CD105-CD146± mature endothelial cells, CD31-CD34-CD73±CD90+CD105-CD146+ pericytes, CD31-CD34+CD73±CD90+CD105-CD146+ transitional pericytes, and CD31-CD34+CD73highCD90+CD105-CD146- supra-adventitial-adipose stromal cells (SA-ASC). The immunophenotyping profile of SVF-MLA was dominated by a reduction of leukocytes and SA-ASC, and an increase in EP, evidencing a marked enrichment of this cell population in the course of adipose tissue microfragmentation. The role of EP in pericyte-primed MSC-mediated tissue healing, as well as the observed hormonal implication, is yet to be investigated.

The Effect of Intra-articular Injection of Autologous Microfragmented Fat Tissue on Proteoglycan Synthesis in Patients with Knee Osteoarthritis.

Osteoarthritis (OA) is one of the leading musculoskeletal disorders in the adult population. It is associated with cartilage damage triggered by the deterioration of the extracellular matrix tissue. The present study explores the effect of intra-articular injection of autologous microfragmented adipose tissue to host chondrocytes and cartilage proteoglycans in patients with knee OA. A prospective, non-randomized, interventional, single-center, open-label clinical trial was conducted from January 2016 to April 2017. A total of 17 patients were enrolled in the study, and 32 knees with osteoarthritis were assessed. Surgical intervention (lipoaspiration) followed by tissue processing and intra-articular injection of the final microfragmented adipose tissue product into the affected knee(s) was performed in all patients. Patients were assessed for visual analogue scale (VAS), delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) and immunoglobulin G (IgG) glycans at the baseline, three, six and 12 months after the treatment. Magnetic resonance sequence in dGEMRIC due to infiltration of the anionic, negatively charged contrast gadopentetate dimeglumine (Gd-DTPA2-) into the cartilage indicated that the contents of cartilage glycosaminoglycans significantly increased in specific areas of the treated knee joint. In addition, dGEMRIC consequently reflected subsequent changes in the mechanical axis of the lower extremities. The results of our study indicate that the use of autologous and microfragmented adipose tissue in patients with knee OA (measured by dGEMRIC MRI) increased glycosaminoglycan (GAG) content in hyaline cartilage, which is in line with observed VAS and clinical results.

Aptitude of Saccharomyces yeasts to ferment unripe grapes harvested during cluster thinning for reducing alcohol content of wine.

Among the viticultural techniques developed to obtain wine with reduced alcohol content, the use of unripe grapes with low sugar and high malic acid concentration, harvested at cluster thinning, was recently explored. So far, no studies have evaluated the fermentation performances of Saccharomyces in unripe grape musts, in terms of fermentation ability and reducing malic acid contents, to improve the quality of this low-alcohol beverage. In this work, we evaluated 24 S. cerevisiae strains isolated from Italian and Croatian vineyards with different fermentation aptitudes. Moreover, four S. paradoxus were considered, as previous works demonstrated that strains belonging to this species were able to degrade high malic acid amounts in standard musts. The industrial strain S. cerevisiae 71B was added as reference. Sugar and malic acid contents were modified in synthetic musts in order to understand the effect of their concentrations on alcoholic fermentation and malic acid degradation. S. cerevisiae fermentation performances improved when glucose concentration decreased and malic acid level increased. The conditions that simulate unripe grape must, i.e. low glucose and high malic acid content were found to enhance S. cerevisiae ability to degrade malic acid. On the contrary, S. paradoxus strains were able to degrade high amounts of malic acid only in conditions that resemble ripe grape must, i.e. high glucose and low malic acid concentration. In fermentation trials when low glucose concentrations were used, at high malic acid levels S. cerevisiae strains produced higher glycerol than at low malic acid condition. Malic acid degradation ability, tested on the best performing S. cerevisiae strains, was enhanced in fermentation trials when unripe grape must was used.

'Omics' biomarkers associated with chronic low back pain: protocol of a retrospective longitudinal study.

INTRODUCTION: Chronic low back pain (CLBP) produces considerable direct costs as well as indirect burdens for society, industry and health systems. CLBP is characterised by heterogeneity, inclusion of several pain syndromes, different underlying molecular pathologies and interaction with psychosocial factors that leads to a range of clinical manifestations. There is still much to understand in the underlying pathological processes and the non-psychosocial factors which account for differences in outcomes. Biomarkers that may be objectively used for diagnosis and personalised, targeted and cost-effective treatment are still lacking. Therefore, any data that may be obtained at the '-omics' level (glycomics, Activomics and genome-wide association studies-GWAS) may be helpful to use as dynamic biomarkers for elucidating CLBP pathogenesis and may ultimately provide prognostic information too. By means of a retrospective, observational, case-cohort, multicentre study, we aim to investigate new promising biomarkers potentially able to solve some of the issues related to CLBP. METHODS AND ANALYSIS: The study follows a two-phase, 1:2 case-control model. A total of 12 000 individuals (4000 cases and 8000 controls) will be enrolled; clinical data will be registered, with particular attention to pain characteristics and outcomes of pain treatments. Blood samples will be collected to perform -omics studies. The primary objective is to recognise genetic variants associated with CLBP; secondary objectives are to study glycomics and Activomics profiles associated with CLBP. ETHICS AND DISSEMINATION: The study is part of the PainOMICS project funded by European Community in the Seventh Framework Programme. The study has been approved from competent ethical bodies and copies of approvals were provided to the European Commission before starting the study. Results of the study will be reviewed by the Scientific Board and Ethical Committee of the PainOMICS Consortium. The scientific results will be disseminated through peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT02037789; Pre-results.

Bacterial communities associated with the production of artisanal Istrian cheese.

In this work we report on the main bacterial microflora typical for fermentation and ripening of traditional Istrian cheese. Samples from milk as well as Istrian cheese were analyzed during the ripening process by using culture independent molecular fingerprinting methods as well as culture based approaches. Our results indicate changes in bacterial diversity pattern during the ripening process. Differences in bacterial diversity at the same ripening stage among different farms investigated were comparably low. Sequence analysis of the most prominent bands of denaturing gradient gel electrophoresis fingerprints revealed dominance of Lactococcus lactis subs. lactis in all samples and a strong presence of Enterococcus spp. which was also confirmed by plate count analysis.