Luis Castaño

Background/Objectives: Standard lipid profiling only partially captures the atherogenic risk conferred by lipoprotein heterogeneity. We investigated the association between olive oil consumption and both the standard lipid profile and the advanced lipoprotein subclass profile in a Spanish population-based cohort. Methods: In this cross-sectional analysis of the Di@bet.es study (n = 3123 after exclusions), 98 participants who reported never using olive oil were matched 1:1 by propensity score (nearest neighbor, caliper 0.2 SD; age, sex, BMI, sweets and carbohydrate intake, waist and hip circumference, physical activity, alcohol, and smoking) to 98 participants who always or usually used olive oil. Lipoprotein subclasses were quantified by ¹H-NMR spectroscopy (Liposcale^®). Groups were compared using Student’s t-test or the Mann–Whitney U test; p-values were adjusted with the Benjamini–Hochberg procedure. Results: Olive oil users had a lower concentration of medium VLDL particles (−28%; p = 0.002; adjusted p = 0.024). Total VLDL-P, VLDL-TG, total triglycerides, and HDL TG/C were nominally lower and HDL-c higher (p = 0.035–0.049), but none remained significant after adjustment (adjusted p ≥ 0.112). The derived TG/HDL-c ratio, a single unadjusted comparison, was also lower (p = 0.022). No differences in total cholesterol, LDL-c, or LDL and HDL particle number were found. Conclusions: Olive oil use was associated with a lower concentration of medium VLDL particles, without differences in LDL-c. Given the cross-sectional design and small matched sample, these exploratory findings do not establish causality or support clinical use of NMR profiling.

Context: Accurate type 1 diabetes (T1D) risk assessment can improve diagnosis and preventive care, reducing misdiagnosis and enabling preclinical interventions. Genetic risk scores (GRS) offer a promising tool, but population-specific validation is required. Objective: This study aims to assess the performance of established scores vs a novel Spanish-specific GRS for T1D differential diagnosis and risk stratification. Methods: From 2338 genotyped individuals, 2215 Spanish participants of European ancestry passed quality control: 816 T1D, 469 type 2 diabetes (T2D), 457 monogenic diabetes (MD), and 473 controls. A novel Spanish-specific GRS (T1D-SP-GRS) was developed and compared with HLA-DRB1 and 2 European-derived models (T1D-GRS and T1D-GRS2). The best-performing score was evaluated for T1D discrimination vs T2D and MD and for association with T1D-onset age. Results: T1D-GRS2 outperformed T1D-SP-GRS for T1D vs controls (AUC-ROC: 0.888 [95% CI: 0.867-0.908] vs 0.837 [0.797-0.877], P = .001), retaining high discrimination vs T2D and MD (0.886 [0.865-0.907]). In the combined pediatric-onset T1D and control cohort, the P50 cutoff yielded a sensitivity of 0.80 (95% CI: 0.76-0.84) and specificity of 0.81 (0.77-0.84); P25 prioritized sensitivity (0.97 [0.95-0.99]) while P75 specificity (0.96 [0.93-0.97]). A P90 threshold conferred 26-fold higher T1D odds vs controls and 18- and 17-fold vs T2D and MD (P < .001). Higher T1D-GRS2 in childhood- vs adulthood-onset T1D revealed an age-dependent risk profile primarily driven by HLA. Conclusions: T1D-GRS2 discriminates T1D from other diabetes etiologies, providing a complementary tool to support diagnostic accuracy. The higher genetic risk associated with a younger age at onset provides key insights for early-stage T1D screening and intervention strategies.

Aims: To investigate the association of post‐oral glucose tolerance test (OGTT) hypoglycaemia with clinical features, incident diabetes and mortality. Materials and Methods: This population‐based cohort study included 2924 adults (median age 49 years, 1680 women) without known diabetes who underwent a 75‐g OGTT as part of the Di@bet.es Study. Metabolic, demographic and lifestyle variables were collected. Serum glucose and insulin concentrations at 0, 30 and 120 min during the OGTT were obtained, allowing insulin resistance estimation using the HOMA‐IR and the Matsuda Index. Participants were grouped according to serum glucose at 120 min: < 70 mg/dL (post‐OGTT hypoglycaemia), 70 to < 140 mg/dL (normal result), 140 to < 200 mg/dL, and ≥ 200 mg/dL. The incidence of diabetes (median follow‐up, 7.5 years) and mortality (median follow‐up, 14.5 years) were assessed. Results: Participants with post‐OGTT hypoglycaemia ( n = 327, 11.1%) were younger, leaner, less insulin resistant and more frequently active smokers. Despite having the lowest serum glucose at 0, 30 and 120 min, and the lowest serum insulin at 0 and 120 min, these participants tended to have the highest serum insulin concentrations at 30 min. Participants with post‐OGTT hypoglycaemia had the lowest incidence of diabetes and mortality, although these associations were attenuated after adjusting for confounders (such as age, sex, body mass index and physical activity) and when compared specifically with the normal result group. Conclusions: Post‐OGTT hypoglycaemia is more frequent in smokers, younger individuals, and those with a favourable metabolic profile. Post‐OGTT hypoglycaemia acts as a marker of protection for incident diabetes and mortality. Preserved early insulin secretion together with good insulin sensitivity could explain post‐OGTT hypoglycaemia.

Imprinting disorders are caused by epigenetic alterations leading to abnormal gene expression from imprinted loci. Pseudohypoparathyroidism type 1B is characterized by methylation defects at the GNAS locus, affecting hormone responsiveness. Monozygotic twins discordant for imprinting disorders provide a unique opportunity to investigate the timing, maintenance, and tissue-specific distribution of epimutations and their relation to clinical expression. Most previous studies focus on infants, where fetal blood chimerism may influence epigenetic profiles, but data in adolescents remain scarce. We studied adolescent monozygotic twin sisters presenting clinical and biochemical discordance for pseudohypoparathyroidism type 1B. Both twins exhibited an identical, partial methylation defect at the GNAS locus in peripheral blood, consistent with a postzygotic epigenetic alteration occurring before embryonic splitting. Exclusion of known genetic causes, including uniparental disomy and structural variants, and absence of multilocus imprinting disturbances or maternal-effect gene mutations, were confirmed through comprehensive genomic and methylation analyses. The affected twin showed involvement of methylation abnormalities across all three germ layers derived tissues, whereas the unaffected twin presented very mild or absent defects in some tissues, possibly explaining the phenotypic divergence. The persistent concordance of partial methylation defects in blood despite phenotypic discordance suggests stable early epimutations maintained in hematopoietic progenitors, while tissue-specific mosaicism and differential methylation maintenance across organs critical to disease expression contribute to clinical differences. Advanced long-read sequencing demonstrated sensitivity for detecting partial and mosaic methylation patterns, rather than just complete alterations. This study underscores the complexity of epigenetic mosaicism in imprinting disorders, highlighting that early postzygotic partial epimutations may yield identical blood methylation profiles but divergent tissue distributions, leading to discordant clinical phenotypes in monozygotic twins. The adolescent age of our subjects challenges the fetal blood chimerism explanation for methylation concordance and emphasizes the importance of multi-tissue evaluation and sensitive molecular assays. These findings have important implications for diagnosis, monitoring, and genetic counseling in imprinting disorders, advocating for integrated approaches to assess epigenetic heterogeneity and phenotypic variability.

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It is well known that environmental factors influence the risk of type 2 diabetes mellitus (T2DM). Several studies have linked the xenobiotics present in tobacco or air pollutants to T2DM development, although the underlying mechanisms remain unclear. Surfactant protein D (SP-D), an immune component released into the bloodstream after lung injury, has been associated with metabolic diseases. The aim of this study was to investigate whether SP-D mediates the effects of smoking or air pollution exposure on T2DM risk in the Spanish adult population. Socio-demographic, lifestyle (including smoking status) and clinical data from 2155 participants from the Di@bet.es cohort were analyzed. Annual concentrations of PM₁₀, PM_2.5, SO₂, CO and NO₂ according to participants’ residential address codes were used to study air pollution exposure. T2DM was diagnosed at baseline and after 7.5 years of follow-up. SP-D serum levels were measured by ELISA and categorized as above or below the 25th percentile. Our results revealed a higher percentage of smokers in the high SP-D category; however, no associations were observed between air pollutants (PM₁₀, PM_2.5, SO₂, CO) and SP-D categories. Both smoking and elevated SP-D levels were found to increase the risk of T2DM independently. Mediation analysis indicated that SP-D mediates 14% of the effect of smoking on T2DM incidence in the Spanish adult population.

Programs for Undiagnosed Rare Diseases (URD) with anonymized data sharing are contributing to the earlier genetic diagnosis of patients and to the identification and characterization of novel genetic disorders. Recently, de novo pathogenic variants in two non-coding spliceosomal small nuclear RNAs (snRNAs), RNU4-2 and RNU2-2 , key regulators of gene expression during neurodevelopment, have been linked to neurodevelopmental disorders (NDD). With the aim to identify individuals with causal genetic variants in known and novel snRNA genes, we analyzed WGS and clinical data of 1,708 probands with undiagnosed NDD enrolled in the Spanish URD Programs. Selected individuals underwent in-depth re-phenotyping including facial mask analyses (Face2Gene), and blood-DNA methylation profiling was obtained (Infinium methylation Epicv2 microarrays). We identified 38 patients (2.22%) with de novo pathogenic variants in snRNA genes, 28 in RNU4-2 (1.64%), 6 in RNU2-2 (0.35%), and 4 in RNU5B-1 (0,23%). A clinically recognizable NDD (ReNU syndrome) is caused by RNU4-2 variants, with dysmorphic features making a disease-associated facial mask, RNU2-2 associates an early onset epileptic encephalopathy, and RNU5-B1 a less defined NDD with overlapping features. A DNA methylomic episignature was also identified for RNU4-2 , which does not overlap with the other conditions. We have also defined autosomal recessive inheritance in 8 additional patients (0.47%) with NDD and biallelic variants in three snRNA genes (3 in RNU4-2 , 3 in RNU2-2 , and 2 in RNU4-1 ). Our study has facilitated the quick diagnosis, management, and genetic counselling of 2.69% previously undiagnosed patients with NDD, further reinforcing the value and utility of the URD Programs. GRAPHICAL

Background and Aims: Metabolic dysfunction–associated steatotic liver disease (MASLD) affects a heterogeneous group of patients. Among them, those with a cholestatic profile show worse outcomes. Here, we investigated whether E2F2 is involved in MASLD-associated cholestasis and, if so, the role of miRNAs. Approach and Results: E2f2 -knockout ( E2f2 −/− ) and wild-type (WT) mice were fed a choline-deficient high-fat diet (ChD-HFD) or an HFD after injection of diethylnitrosamine (DEN-HFD) to induce metabolic dysfunction–associated steatohepatitis (MASH). E2F2 was overexpressed in the liver by AAV8. Cholestasis was induced by bile duct ligation or by a 3,5-diethoxycarbonyl-1,4-dihydrocollidine-enriched diet. microRNA sequencing was performed. Two biopsy-proven MASLD patient cohorts were used. E2F2 deficiency resulted in increased synthesis and excretion of cholesterol, phosphatidylcholine, and bile acids, reducing their storage in the liver while increasing their presence in feces. This was consistent with increased expression of genes involved in biliary lipid metabolism, reduced inflammation and fibrosis, and the generation of a distinct miRNA profile, thereby preventing MASH. Liver-specific induction of E2F2 in vivo hampered the transcriptional program involved in biliary lipid metabolism and upregulated miR-34a-5p, which was downregulated in E2f2 −/− mice. The protective effects observed in E2f2 −/− mice were lost when a miR-34a-5p mimic was used. Hepatic miR-34a-5p levels were elevated in patients with advanced fibrosis, inflammation, steatosis score, cholelithiasis, and increased serum bile acids and biliary lipids. E2f2 deficiency conferred protection against cholestatic liver injury. Conclusions: E2F2 deficiency protects against MASH and cholestasis, preventing cholesterol accumulation, fibrosis, and inflammation through modulation of miR-34a-5p. This could provide therapeutic benefits for patients with cholestatic MASH.

Introduction. This study examined the possible association between numeracy skills and glycemic outcomes in children with type 1 diabetes. Methods. The study used a cross-sectional design and collected data from 7 centres of the Hvidoere Study Group. HbA1c was measured centrally. Numeracy was assessed using the specific 5-item Diabetes Numeracy Test (DNT-5) and the international, general Wordless Mathematical Test (WMT). The HbA1c predictive multivariate generalised linear model was constructed using the adjusted R-squared index for model selection. Pearson's correlation coefficient was calculated between observed and predicted HbA1c levels in the training and testing datasets. Results. 306 adolescents aged 12-18 (mean age 14.96 ± 1.68) years and diabetes duration of 6.57 (± 3.75) participated in this study. Numeracy skills, as assessed by the WMT but not DNT 5, predicted the HbA1c levels after adjustment for sociodemographic and clinical factors. The correlation between observed and predicted HbA1c levels was consistent in both datasets and was 0.34 (N=155) and 0.37 (N=61) for the training and test datasets, respectively (p=0.412). The effect size for the WMT-based predictive model of HbA1c adjusted for clinical and socioeconomic factors was significantly higher (p<0.05) than the single-parameter-based model. Conclusion. Numeracy, as assessed by an international general math test, is a good predictor of HbA1c in children and adolescents with type 1 diabetes. The basic and short WMT is a potentially effective tool in personalised clinical pediatric diabetes practice. Therapy planning should consider adjusting therapy to compensate for lower numeracy skills and/or training to improve the patient’s numerical proficiency.