Modeling Lysosomal Storage Disorders in an Innovative Way: Establishment and Characterization of Stem Cell Lines from Human Exfoliated Deciduous Teeth of Mucopolysaccharidosis Type II Patients.

Among the many lysosomal storage disorders (LSDs) that would benefit from the establishment of novel cell models, either patient-derived or genetically engineered, is mucopolysaccharidosis type II (MPS II). Here, we present our results on the establishment and characterization of two MPS II patient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population has been isolated from LSD patient samples obtained from the dental pulp. Taking into account our results on the molecular and biochemical characterization of those cells and the fact that they exhibit visible and measurable disease phenotypes, we consider these cells may qualify as a valuable disease model, which may be useful for both pathophysiological assessments and in vitro screenings. Ultimately, we believe that patient-derived dental pulp stem cells (DPSCs), particularly those isolated from human exfoliated deciduous teeth (SHEDs), may represent a feasible alternative to induced pluripotent stem cells (iPSCs) in many labs with standard cell culture conditions and limited (human and economic) resources.

Help Comes from Unexpected Places: How a Tiny Fairy and a Tropical Fish may help us Model Mucopolysaccharidoses.

INTRODUCTION: When it comes to disease modeling, countless models are available for Lysosomal Storage Diseases (LSD). Historically, two major approaches are well-established: in vitro assessments are performed in patient fibroblasts, while in vivo pre-clinical studies are performed in mouse models. Still, both platforms have a series of drawbacks. Thus, we implemented two alternative and innovative protocols to mimic a particular sub-group of LSDs, the Mucopolysaccharidoses both in vitro and in vivo. METHODS: The first one relies on a non-invasive approach using dental pulp stem cells from deciduous teeth (SHEDs). SHEDs are multipotent neuronal precursors that can easily be collected. The second uses a state-of-the-art gene editing technology (CRISPR/Cas9) to generate zebrafish disease models. RESULTS: Even though this is an ongoing project, we have already established and characterized two MPS II and one MPS VI SHED cell models. These cells self-maintain through several passages and can give rise to a variety of cells including neurons. Furthermore, all MPS-associated sub-cellular phenotypes we have assessed so far are easily observable in these cells. Regarding our zebrafish models, we have successfully knocked down both naglu and hgsnat and the first results we got from the behavioral analysis are promising ones, as we can observe altered activity and sleep patterns in the genetically modified fish. For this particular approach we chose MPS III forms as our target disorders, since their neurological features (hyperactivity, seizures and motor impairment) and lifespan decrease would be easily recognizable in zebrafish. CONCLUSION: Now that these methods are well-established in our lab, their potential is immense. On one hand, the newly developed models will be of ultimate value to understand the mechanisms underlying MPS sub-cellular pathology, which have to be further elucidated. On the other hand, they will constitute an optimal platform for drug testing in house. Also noteworthy, our models will be published as lab resources and made available for the whole LSD community.

Citrullinemia and What Else?

INTRODUCTION: Citrullinemia type I (CTLN1) is a rare autosomal recessive metabolic disorder. Symptoms typically include vomiting, lethargy, seizures and coma. In neonatal presentation, death occurs in days if untreated. Survivors may evolve with neurocognitive dysfunction. RESULTS/CASE REPORT: Two 10 years old, non-identical, twin sisters (S1; S2) with CTLN1 were born after a 36W gestation: S1 by eutocic delivery and S2 by cesarean section with nuchal cord (Apgar score 5/10). On day four, S2 presented hyperammonemia with coma. S1 had no complications. Diagnosis followed that of S2. Neurocognitive development was monitored at 3 months - 4 years of age with Griffiths Scales: global development quotient kept within the average, but S2 had a deficit in language and eye and hand coordination. At 5 years, the neurocognitive abilities were evaluated using Wechsler Preschool and Primary Scale of Intelligence - Revised (WPPSI-R). S2 revealed difficulties in verbal area (vocabulary, comprehension and memorizing sentences), with a lower average verbal intelligence quotient (IQ). S1 had high average IQ. Due to learning difficulties, S2 was reassessment at 8 years old with Wechsler Intelligence Scale for Children - Third edition (WISC-III): full-scale IQ -"extremely low". CONCLUSION: These non-identical twin sisters share the same citrullinemia type 1 causing variants in the ASS1 gene. Nevertheless, their clinical presentation and neurocognitive evolution are diverse. Other factors, like the different genetic background and perinatal issues such as the type of delivery and its circumstances and the neonatal coma episode of S2 may explain the dissimilar evolution. Maximum ammonium levels (and its duration) are critical for the patients' neurodevelopment: 131 in S1 and 546 umol/l in S2.

Combined Oxidative Phosphorylation Deficiency Type-13 with Perinatal Presentation: A Case Report.

INTRODUCTION: Polynucleotide phosphorylase is involved in RNA processing in mitochondria. Biallelic variants in PNPT1 cause mitochondrial RNA import protein deficiency and heterogeneous clinical manifestations. CASE REPORT: The patiest was the first child of remote consanguineous parents, born at 35 weeks by caesarean section due to fetal growth restriction. Apgar index was 9/10/10. Birth weight, length and head circumference were at 3rd, <3rd and 10th percentiles, respectively. In the first hours of life, respiratory distress, hypoglycaemia and seizures ensued. She started invasive mechanic ventilation, phenobarbital and was transferred to ICU. Physical examination showed minor facial dysmorphisms, brief eye-opening, hypotonia and hyporeflexia. Electroencephalogram showed immature pattern and multifocal paroxysmal activity. MRI at D8 of life showed severe reduced brain volume. Normal aminoacid screen was also observed. Expanded newborn screening was negative. Mitochondrial organic aciduria was seen. WES showed a homozygotic likely pathogenic variant in the PNPT1 gene. MRI at 6-months showed brain atrophy, thin corpus callosum, reduced brainstem volume. Bilateral and symmetrical lesions in globi pallidi, compatible with Leigh síndrome were observed. Currently, at 14 months, no neurodevelopment progress, dystonia, visual deficit, sensorineural deafness, hypertrophic cardiomyopathy and microcephaly are observed. CONCLUSION: The early and severe Leigh-like presentation of our patient expands the phenotype spectrum of this disease. As far as we know, this is the first reported case of PNPT1 mutation with onset in the perinatal period. Moreover, hypertrophic cardiomyopathy has not yet been described in association with mutation of the PNPT1 gene. WES was the key for early diagnosis in this patient. It should be done in all children with severe clinical presentation of unknown origin.

Neutrophil/lymphocyte and monocyte/lymphocyte indexes as potential predictors of relapse at 1 year after diagnosis of pediatric multiple sclerosis: a single-center, exploratory and proof-of-concept study.

Introduction: Early identification of patients with a more unfavorable outcome in Multiple Sclerosis (MS) is crucial to optimize individualized treatment. Neutrophil-lymphocyte index (NLI) and monocyte-lymphocyte index (MLI) have been considered as potential biomarkers for disease prognosis. Our study aims to investigate the usefulness of NLI and MLI as predictors of relapse, disability progression, and lesion accumulation on magnetic resonance imaging (MRI) 1 year after diagnosis and treatment initiation, in pediatric-onset MS. Methods: A retrospective single-center study was conducted, including patients with diagnosis of MS established in pediatric age (<18 years old), at least 1-year of follow-up, and a complete blood count (CBC) performed at diagnosis. We collected the nearest-to-diagnosis NLI and MLI, as well as clinical and imaging variables, at diagnosis and 12 months later. Our cohort was further dichotomized into two groups, based on the presence of relapses. Statistical significance was considered for p < 0.05. Results: Eighteen patients (n = 18) were included. The relapsing group had higher mean, minimum, and maximum values for both NLI (5.17 ± 5.85, range: 1.57-11.92) and MLI (0.35 ± 0.22, range: 0.19-0.59), compared to the non-relapsing group (2.19 ± 1.63, range: 1.12-7.32 for NLI, and 0.24 ± 0.09, range: 0.14-0.44 for MLI). A higher percentage of patients in the relapsing group had increased NLI (>1.89, 66.7%) and MLI (>0.21, 66.7%) values than those in the non-relapsing group (46.7%). Patients who presented new T2-hyperintense lesions on MRI after 1 year of follow-up also had higher mean, minimum, and maximum values of both biomarkers. Patients who did not achieve No Evidence of Disease Activity-3 (NEDA-3) state exhibited higher values for both ratios. However, in our sample, no statistically significant correlations were found between MLI and NLI values and the clinical and imaging variables considered. Conclusion: The ease of obtaining NLI and MLI from routine blood tests renders them useful biomarkers as a screening tool in longitudinal follow-up. Our study was based on a very small sample size, but it allowed us to verify the feasibility of the protocol used. It is intended to involve other centers in the next phase of this work, testing the possible usefulness of the indices under analysis on a larger sample.

Multiple sclerosis under the age of ten: the challenge of a rare diagnosis in a special population - a case series.

Introduction: Multiple sclerosis (MS) is a chronic inflammatory demyelinating and degenerative disease of the central nervous system which, when it begins before the age of 18, is defined as paediatric MS. Most common clinical presentations include long tract involvement, brainstem/cerebellum syndromes, optic neuritis and acute disseminated encephalomyelitis. Paediatric-onset MS typically has a more inflammatory-active course and a higher lesion burden in imaging studies, but an extensive post-relapse recovery, with a slower long-term disability progression. The first demyelinating clinical attack occurs before 10 years old in less than 1% of patients, and, in this special population, the condition has particularities in clinical presentation, differential diagnosis, diagnostic assessment, current treatment options and outcome. Clinical cases: We present the cases of four Caucasian children (2 girls) diagnosed with relapsing-remitting MS before the age of ten, with a mean age at the time of the first relapse of 7.4 ± 2.4 years. Clinical presentation included optic neuritis, myelitis, brainstem syndrome, and acute disseminated encephalomyelitis. Baseline MRI identified several lesions, frequently large and ill-defined. Two patients were included in clinical trials and two patients remain in clinical and imaging surveillance. Conclusion: Diagnosis of MS before the age of 10 years is rare, but it has significant long-term physical and cognitive consequences, as well as a substantial impact on the current and future quality of life of the child and family. Early and correct diagnosis is essential. Prospective, randomized, large cohort studies are needed to assess the efficacy and safety of disease-modifying treatments in children under the age of ten.

Premature closure of ductus arteriosus after a single dose of diclofenac during pregnancy.

A male term neonate was admitted to the neonatal intensive care unit in the first hours of life with central cyanosis. Echocardiogram showed severe biventricular hypertrophy, markedly right-sided, tricuspid regurgitation, a patent foramen ovale and a closed ductus arteriosus (CDA). The mother recalled being treated with a single dose of intravenous diclofenac for low back pain 2 weeks earlier. The newborn was started on propranolol with symptomatic improvement and was discharged on day 10. At 1-month follow-up, he showed complete resolution of ventricular hypertrophy and suspended propranolol. In the literature, of the 22 cases of CDA after intrauterine exposure to diclofenac, 11 resolved in utero, 3 required ventilatory and inotropic support and 1 evolved to persistent pulmonary hypertension. In this case, a thorough anamnesis was key to identify the probable cause of an otherwise unexplained transient ventricular hypertrophy. This case also alerts to the fetal risks of non-steroidal anti-inflammatory drugs during the third trimester, requiring close monitoring.

Heterogeneity and heterotypic continuity of emotional and behavioural profiles across development.

PURPOSE: To identify emotional and behavioural symptoms profiles from early childhood to adolescence, their stability across development and associated factors. METHODS: Our sample included 17,216 children assessed at ages 3, 5, 7, 11 and 14 years from the UK Millennium Cohort Study. We used latent profile and latent transition analysis to study their emotional and behavioural profiles from early childhood to adolescence. We included sociodemographic, family and parenting variables to study the effect on latent profile membership and transitions. RESULTS: The number and specific profiles of emotional and behavioural symptoms changed with the developmental stage. We found a higher number of profiles for ages 3, 5, and 14, suggesting greater heterogeneity in the presentation of emotional and behavioural symptoms in early childhood and adolescence compared to late childhood. There was greater heterotypic continuity between ages 3 and 5, particularly in transitions from higher to lower severity profiles. Children exposed to socioeconomic disadvantages were more likely to belong or transition to any moderate or high emotional and behavioural symptoms profiles. Maternal psychological distress and harsh parenting were associated with internalizing and externalizing profiles, respectively. Higher levels of internalizing and externalizing symptoms across development were associated with lower mental wellbeing and higher rates of self-harm and substance use in adolescence. CONCLUSION: Emotional and behavioural symptoms develop early in life, with levels of heterogeneity and heterotypic stability that change throughout development. These results call for interventions to prevent and treat paediatric mental illness that consider the heterogeneity and stability of symptoms across development.

Emotional and behavioural pathways to adolescent substance use and antisocial behaviour: results from the UK Millennium Cohort Study.

This study examines the emotional and behavioural pathways to adolescent substance use and antisocial behaviour. Using a sample of 17,223 participants from the UK Millennium Cohort Study, we applied parallel-process growth mixture modelling on emotional and behavioural symptoms in those aged 3-14 and employed latent class analysis to identify patterns of substance use and antisocial behaviours at age 14. We then performed a multinomial regression analysis to explore the association between emotional and behavioural trajectories and patterns of adolescent substance use and antisocial behaviours, including sociodemographic, family, and maternal factors. We found five trajectories of emotional and behavioural symptoms and four classes of adolescence substance use and antisocial behaviour. Children and adolescents in the 'high externalising and internalising' and 'moderate externalising' trajectories were more likely to belong to any problematic behaviour class, especially the 'poly-substance use and antisocial behaviours' class. Inclusion in the 'moderate externalising and internalising (childhood limited)' class was associated with higher odds of belonging to the 'alcohol and tobacco' class. These associations remained significant after adjusting for important sociodemographic and contextual factors, such as maternal substance use, poverty, and parental status. Interventions on adolescent health promotion and risk behaviour prevention need to address the clustering of substance use and antisocial behaviour as well as the significant influence of early and chronic internalising and externalising symptoms on the aetiology of these behaviours.

Adolescent internet addiction - role of parental control and adolescent behaviours.

INTRODUCTION: Excessive Internet use can negatively affect academic performance, family relationships and emotional development among the youth. Such issues have been identified as Internet addiction (IA). We aimed to determine the prevalence of IA among Portuguese adolescents and assess how parental control can relate to IA. METHODS: An observational cross-sectional study was performed at public schools within a Portuguese region, using Young's Internet Addiction Test survey. General sociodemographic and emotional well-being data were obtained. A descriptive and bivariate analysis was done among Internet-addicted and average users, followed by a logistic regression analysis. Adjusted odds ratios (aORs) were computed with two-sided P values < .05 for statistical significance. RESULTS: A total of 1916 eligible responses were obtained. Mean age was 15 ± 1.8 years, with a slight predominance of female (53.3%) participants. In our sample, 16.5% were deemed Internet addicted and less likely to have any parental control over Internet use (aOR 0.74, P <.05). Moreover, 28% of the Internet-addicted users were less likely to have control over time spent online (aOR 0.72, P < .05), and close to half were unlikely to have online content restrictions (aOR 0.56, P < .01). CONCLUSIONS: Our findings reported a significant rate of Internet-addicted youth. IA was negatively related to parental control. Whenever any kind of parental control over Internet use was reported, IA was less likely to occur. Healthcare professionals should be aware of the risks of IA in adolescents to improve its prevention and intervention.

Early Individual and Family Predictors of Weight Trajectories From Early Childhood to Adolescence: Results From the Millennium Cohort Study.

Background: Early infancy and childhood are critical periods in the establishment of lifelong weight trajectories. Parents and early family environment have a strong effect on children's health behaviors that track into adolescence, influencing lifelong risk of obesity. Objective: We aimed to identify developmental trajectories of body mass index (BMI) from early childhood to adolescence and to assess their early individual and family predictors. Methods: This was a secondary analysis of the Millennium Cohort Study and included 17,165 children. Weight trajectories were estimated using growth mixture modeling based on age- and gender-specific BMI Z-scores, followed by a bias-adjusted regression analysis. Results: We found four BMI trajectories: Weight Loss (69%), Early Weight Gain (24%), Early Obesity (3.7%), and Late Weight Gain (3.3%). Weight trajectories were mainly settled by early adolescence. Lack of sleep and eating routines, low emotional self-regulation, child-parent conflict, and low child-parent closeness in early childhood were significantly associated with unhealthy weight trajectories, alongside poverty, low maternal education, maternal obesity, and prematurity. Conclusions: Unhealthy BMI trajectories were defined in early and middle-childhood, and disproportionally affected children from disadvantaged families. This study further points out that household routines, self-regulation, and child-parent relationship are possible areas for family-based obesity prevention interventions.

Clustering of health-related behaviours and its relationship with individual and contextual factors in Portuguese adolescents: results from a cross-sectional study.

BACKGROUND: Health behaviours are shaped early in life and tend to occur in complex specific patterns. We aimed to characterise these patterns among Portuguese adolescents and their association with individual and contextual factors. METHODS: This study was based in the Portuguese 2009/10 survey of Health Behaviour in School-Aged Children Study, comprising 4036 adolescents. Individuals were grouped using two-step cluster analysis based on 12 behaviours regarding diet, physical activity, screen use and substance use. The association between clusters and individual and contextual factors was analysed using multinomial regression. RESULTS: The median age was 13,6, and 54% were female. Overweight and obesity were highly prevalent (25%). We identified four behavioural clusters: "Active screen users", "Substance users", "Healthy" and "Inactive low fruit and vegetable eaters". Sociodemographics varied across clusters. The "Substance users" and "Active screen users" clusters were associated with poor family communication, academic performance and school attachment and violent behaviours, and the "Inactive low fruit and vegetable eaters" were associated with lower socioeconomic status. CONCLUSION: The understanding of these health-compromising patterns and their social determinants is of use to Public Health, allowing tailored health-promoting interventions. Further research is needed to understand how cluster membership evolves and its influence on nutritional status.

Gender-specific substance use patterns and associations with individual, family, peer, and school factors in 15-year-old Portuguese adolescents: a latent class regression analysis.

BACKGROUND: Adolescence is a critical period of vulnerability to substance use. Recent research has shown that gender differences in adolescence substance use are complex and in constant flux. The present study aims to investigate gender differences in substance use and initiation patterns in male and female adolescents, and to assess individual, family, peer, and school associated factors of these patterns. METHODS: We applied latent class regression analysis to a Portuguese representative population sample of 1551 15-year-old adolescents, drawn from the 2010 'Health Behavior in School-Aged Children' survey, to characterise different profiles of substance use and initiation for boys and girls, and to identify factors associated with latent class membership, stratifying the associations analysis by gender. RESULTS: Three common classes were found for both genders, specifically, Non-Users (boys [B] 34.42%, girls [G] 26.79%), Alcohol Experimenters (B 38.79%, G 43.98%) and Alcohol and Tobacco Frequent Users (B 21.31%, G 10.36%), with two additional unique classes: Alcohol Experimenters and Tobacco Users in girls (18.87%), and Early Initiation and Poly-Substance Users in boys (5.48%). Poor school satisfaction, bullying, fighting and higher family affluence scale score formed a common core of associated factors of substance use, although we found gender differences in these associations. In girls, but not in boys, family factors were associated with more problematic substance use. Not living with both parents was associated with girl's Alcohol and Tobacco Frequent Users (gATFU) class (OR 3.78 CI 1.18-12.11) and Alcohol Experimenters and Tobacco Users (AETU) class (OR 3.22 CI 1.4-7.44). Poor communication with mother was also associated with gATFU class membership (OR 3.82 CI 1.26-11.53) and AETU class (OR 3.66 CI 1.99-6.75). Additionally, a higher psychological symptoms score was associated with gATFU class membership (OR 1.16 CI 1.02-1.31). CONCLUSION: Although we found common patterns and associated factors between boys and girls, we report two unique patterns of substance use in boys and girls and specific associations between family, school and peers, and individual factors with these patterns. These findings underscore the need for substance use prevention and health promotion programmes that address potential differences in substance use patterns and associated factors.