Sex-Stratified Genome-Wide Association Study in the Spanish Population Identifies a Novel Locus for Lacunar Stroke.

BACKGROUND: Ischemic stroke (IS) represents a significant health burden globally, necessitating a better understanding of its genetic underpinnings to improve prevention and treatment strategies. Despite advances in IS genetics, studies focusing on the Spanish population and sex-stratified analyses are lacking. METHODS: A case-control genome-wide association study was conducted with 9081 individuals (3493 IS cases and 5588 healthy controls). IS subtypes using Trial of ORG 10172 in Acute Stroke Treatment criteria were explored in a sex-stratified approach. Replication efforts involved the MEGASTROKE, GIGASTROKE, and the UK Biobank international cohorts. Post-genome-wide association study analysis included: in silico proteomic analysis, gene-based analysis, quantitative trait loci annotation, transcriptome-wide association analysis, and bioinformatic analysis using chromatin accessibility data. RESULTS: Identified as associated with IS and its subtypes were 4 significant and independent loci. Replication confirmed 5p15.2 as a new locus associated with small-vessel occlusion stroke, with rs59970332-T as the lead variant (beta [SE], 0.13 [0.02]; P=4.34×10-8). Functional analyses revealed CTNND2 given proximity and its implication in pathways involved in vascular integrity and angiogenesis. Integration of Hi-C data identified additional potentially modulated genes, and in silico proteomic analysis suggested a distinctive blood proteome profile associated with the lead variant. Gene-set enrichment analyses highlighted pathways consistent with small-vessel disease pathogenesis. Gene-based associations with known stroke-related genes such as F2 and FGG were also observed, reinforcing the relevance of our findings. CONCLUSIONS: We found CTNND2 as a potential key molecule in small-vessel occlusion stroke risk, and predominantly in males. This study sheds light on the genetic architecture of IS in the Spanish population, providing novel insights into sex-specific associations and potential molecular mechanisms. Further research, including replication in larger cohorts, is essential for a comprehensive understanding of these findings and for their translation to clinical practice.

Ischaemic stroke patients present sex differences in gut microbiota.

BACKGROUND: Gut microbiota plays a role in the pathophysiology of ischaemic stroke (IS) through the bidirectional gut-brain axis. Nevertheless, little is known about sex-specific microbiota signatures in IS occurrence. METHODS: A total of 89 IS patients and 12 healthy controls were enrolled. We studied the taxonomic differences of the gut microbiota between men and women with IS by shotgun metagenomic sequencing. To evaluate the causal effect of several bacteria on IS risk, we performed a two-sample Mendelian randomisation (MR) with inverse-variance weighting (IVW) using genome-wide association analysis (GWAS) summary statistics from two cohorts of 5959 subjects with genetic and microbiota data and 1,296,908 subjects with genetic and IS data, respectively. RESULTS: α-Diversity analysis measured using Observed Species (p = 0.017), Chao1 (p = 0.009) and Abundance-based Coverage Estimator (p = 0.012) indexes revealed that IS men have a higher species richness compared with IS women. Moreover, we found sex-differences in IS patients in relation to the phylum Fusobacteria, class Fusobacteriia, order Fusobacteriales and family Fusobacteriaceae (all Bonferroni-corrected p < 0.001). MR confirmed that increased Fusobacteriaceae levels in the gut are causally associated with an increased risk of IS (IVW p = 0.02, ß = 0.32). CONCLUSIONS: Our study is the first to indicate that there are gut microbiome differences between men and women with IS, identifying high levels of Fusobacteriaceae in women as a specific risk factor for IS. Incorporating sex stratification analysis is important in the design, analysis and interpretation of studies on stroke and the gut microbiota.

Genetic Architecture of Ischaemic Strokes after COVID-19 Shows Similarities with Large Vessel Strokes.

We aimed to analyse whether patients with ischaemic stroke (IS) occurring within eight days after the onset of COVID-19 (IS-COV) are associated with a specific aetiology of IS. We used SUPERGNOVA to identify genome regions that correlate between the IS-COV cohort (73 IS-COV cases vs. 701 population controls) and different aetiological subtypes. Polygenic risk scores (PRSs) for each subtype were generated and tested in the IS-COV cohort using PRSice-2 and PLINK to find genetic associations. Both analyses used the IS-COV cohort and GWAS from MEGASTROKE (67,162 stroke patients vs. 454,450 population controls), GIGASTROKE (110,182 vs. 1,503,898), and the NINDS Stroke Genetics Network (16,851 vs. 32,473). Three genomic regions were associated (p-value < 0.05) with large artery atherosclerosis (LAA) and cardioembolic stroke (CES). We found four loci targeting the genes PITX2 (rs10033464, IS-COV beta = 0.04, p-value = 2.3 × 10-2, se = 0.02), previously associated with CES, HS6ST1 (rs4662630, IS-COV beta = -0.04, p-value = 1.3 × 10-3, se = 0.01), TMEM132E (rs12941838 IS-COV beta = 0.05, p-value = 3.6 × 10-4, se = 0.01), and RFFL (rs797989 IS-COV beta = 0.03, p-value = 1.0 × 10-2, se = 0.01). A statistically significant PRS was observed for LAA. Our results suggest that IS-COV cases are genetically similar to LAA and CES subtypes. Larger cohorts are needed to assess if the genetic factors in IS-COV cases are shared with the general population or specific to viral infection.

Biological Age Acceleration Is Lower in Women With Ischemic Stroke Compared to Men.

BACKGROUND: Stroke onset in women occurs later in life compared with men. The underlying mechanisms of these differences have not been established. Epigenetic clocks, based on DNA methylation (DNAm) profiles, are the most accurate biological age estimate. Epigenetic age acceleration (EAA) measures indicate whether an individual is biologically younger or older than expected. Our aim was to analyze whether sexual dichotomy at age of stroke onset is conditioned by EAA. METHODS: We used 2 DNAm datasets from whole blood samples of case-control genetic studies of ischemic stroke (IS), a discovery cohort of 374 IS patients (N women=163, N men=211), from GRECOS (Genotyping Recurrence Risk of Stroke) and SEDMAN (Dabigatran Study in the Early Phase of Stroke, New Neuroimaging Markers and Biomarkers) studies and a replication cohort of 981 IS patients (N women=411, N men=570) from BASICMAR register. We compared chronological age, 2 DNAm-based biomarkers of aging and intrinsic and extrinsic epigenetic age acceleration EAA (IEAA and extrinsic EAA, respectively), in IS as well as in individual IS etiologic subtypes. Horvath and Hannum epigenetic clocks were used to assess the aging rate. A proteomic study using the SOMAScan multiplex assay was performed on 26 samples analyzing 1305 proteins. RESULTS: Women present lower Hannum-extrinsic EAA values, whereas men have higher Hannum-extrinsic EAA values (women=-0.64, men=1.24, P=1.34×10-2); the same tendency was observed in the second cohort (women=-0.57, men=0.79, P=0.02). These differences seemed to be specific to cardioembolic and undetermined stroke subtypes. Additionally, 42 blood protein levels were associated with Hannum-extrinsic EAA (P<0.05), belonging to the immune effector process (P=1.54×10-6) and platelet degranulation (P<8.74×10-6) pathways. CONCLUSIONS: This study shows that sex-specific underlying biological mechanisms associated with stroke onset could be due to differences in biological age acceleration between men and women.

Genome-Wide Studies in Ischaemic Stroke: Are Genetics Only Useful for Finding Genes?

Ischaemic stroke is a complex disease with some degree of heritability. This means that heritability factors, such as genetics, could be risk factors for ischaemic stroke. The era of genome-wide studies has revealed some of these heritable risk factors, although the data generated by these studies may also be useful in other disciplines. Analysis of these data can be used to understand the biological mechanisms associated with stroke risk and stroke outcome, to determine the causality between stroke and other diseases without the need for expensive clinical trials, or to find potential drug targets with higher success rates than other strategies. In this review we will discuss several of the most relevant studies regarding the genetics of ischaemic stroke and the potential use of the data generated.

ICA1L Is Associated with Small Vessel Disease: A Proteome-Wide Association Study in Small Vessel Stroke and Intracerebral Haemorrhage.

Small vessel strokes (SVS) and intracerebral haemorrhages (ICH) are acute outcomes of cerebral small vessel disease (SVD). Genetic studies combining both phenotypes have identified three loci associated with both traits. However, the genetic cis-regulation at the protein level associated with SVD has not been studied before. We performed a proteome-wide association study (PWAS) using FUSION to integrate a genome-wide association study (GWAS) and brain proteomic data to discover the common mechanisms regulating both SVS and ICH. Dorsolateral prefrontal cortex (dPFC) brain proteomes from the ROS/MAP study (N = 376 subjects and 1443 proteins) and the summary statistics for the SVS GWAS from the MEGASTROKE study (N = 237,511) and multi-trait analysis of GWAS (MTAG)-ICH−SVS from Chung et al. (N = 240,269) were selected. We performed PWAS and then a co-localization analysis with COLOC. The significant and nominal results were validated using a replication dPFC proteome (N = 152). The replicated results (q-value < 0.05) were further investigated for the causality relationship using summary data-based Mendelian randomization (SMR). One protein (ICA1L) was significantly associated with SVS (z-score = −4.42 and p-value = 9.6 × 10−6) and non-lobar ICH (z-score = −4.8 and p-value = 1.58 × 10−6) in the discovery PWAS, with a high co-localization posterior probability of 4. In the validation PWAS, ICA1L remained significantly associated with both traits. The SMR results for ICA1L indicated a causal association of protein expression levels in the brain with SVS (p-value = 3.66 × 10−5) and non-lobar ICH (p-value = 1.81 × 10−5). Our results show that the association of ICA1L with SVS and non-lobar ICH is conditioned by the cis-regulation of its protein levels in the brain.

Human ApoA-I Overexpression Enhances Macrophage-Specific Reverse Cholesterol Transport but Fails to Prevent Inherited Diabesity in Mice.

Human apolipoprotein A-I (hApoA-I) overexpression improves high-density lipoprotein (HDL) function and the metabolic complications of obesity. We used a mouse model of diabesity, the db/db mouse, to examine the effects of hApoA-I on the two main functional properties of HDL, i.e., macrophage-specific reverse cholesterol transport (m-RCT) in vivo and the antioxidant potential, as well as the phenotypic features of obesity. HApoA-I transgenic (hA-I) mice were bred with nonobese control (db/+) mice to generate hApoA-I-overexpressing db/+ offspring, which were subsequently bred to obtain hA-I-db/db mice. Overexpression of hApoA-I significantly increased weight gain and the incidence of fatty liver in db/db mice. Weight gain was mainly explained by the increased caloric intake of hA-I-db/db mice (>1.2-fold). Overexpression of hApoA-I also produced a mixed type of dyslipidemia in db/db mice. Despite these deleterious effects, the overexpression of hApoA-I partially restored m-RCT in db/db mice to levels similar to nonobese control mice. Moreover, HDL from hA-I-db/db mice also enhanced the protection against low-density lipoprotein (LDL) oxidation compared with HDL from db/db mice. In conclusion, overexpression of hApoA-I in db/db mice enhanced two main anti-atherogenic HDL properties while exacerbating weight gain and the fatty liver phenotype. These adverse metabolic side-effects were also observed in obese mice subjected to long-term HDL-based therapies in independent studies and might raise concerns regarding the use of hApoA-I-mediated therapy in obese humans.

Sitosterolemia: diagnosis, investigation, and management.

Sitosterolemia is a rare autosomal recessively inherited disease caused by mutations affecting ABCG5 or ABCG8, which are located on human chromosome band 2p21. Around 100 cases have been reported in the literature. Sitosterolemic patients typically exhibit a 30-fold to 100-fold increase in plasma concentrations of plant sterols. The clinical manifestations include xanthomas, premature atherosclerosis, hemolytic anemia, and macrothrombocytopenia. It is noteworthy that abnormal hematological parameters may be the only clinical feature of sitosterolemic patients, suggesting that sitosterolemia may be more frequent than previously thought. Severe accumulation of plant sterols in mouse models of sitosterolemia induced complex cardiac lesions, anemia, and macrothrombocytopenia, disrupted adrenal and liver cholesterol homeostasis, and caused infertility and hypertriglyceridemia. It remains unclear whether all disease traits are present in sitosterolemic patients. The drug ezetimibe appears to be effective in reducing plasma plant sterol levels, promotes xanthoma regression, and improves the cardiovascular and hematological signs in sitosterolemic patients.

Identification of Genetic Loci Associated With Intracerebral Hemorrhage Using a Multitrait Analysis Approach.

BACKGROUND AND OBJECTIVES: Genome-wide association studies (GWASs) have only 2 loci associated with spontaneous intracerebral hemorrhage (ICH): APOE for lobar and 1q22 for nonlobar ICH. We aimed to discover new loci through an analysis that combines correlated traits (multi-trait analysis of GWAS [MTAG]) and explore a gene-based analysis, transcriptome-wide association study (TWAS), and proteome-wide association study (PWAS) to understand the biological mechanisms of spontaneous ICH providing potential therapeutic targets. METHODS: We use the published MTAG of ICH (patients with spontaneous intraparenchymal bleeding) and small-vessel ischemic stroke. For all ICH, lobar ICH, and nonlobar ICH, a pairwise MTAG combined ICH with traits related to cardiovascular risk factors, cerebrovascular diseases, or Alzheimer disease (AD). For the analysis, we assembled those traits with a genetic correlation ≥0.3. A new MTAG combining multiple traits was performed with those traits whose pairwise MTAG yielded new GWAS-significant single nucleotide polymorphisms (SNPs), with a posterior-probability of model 3 (GWAS-pairwise) ≥0.6. We perform TWAS and PWAS that correlate the genetic component of expression or protein levels with the genetic component of a trait. We use the ICH cohort from UK Biobank as replication. RESULTS: For all ICH (1,543 ICH, 1,711 controls), the mean age was 72 ± 2 in cases and 70 ± 2 in controls, and half of them were women. Replication cohort: 700 ICH and 399,717 controls. Novel loci were found only for all ICH (the trait containing lobar and nonlobar ICH), combining data of ICH and small vessel stroke, white matter hyperintensities volume, fractional anisotropy, mean diffusivity, and AD. We replicated 6 SNPs belonging to 2q33.2 (ICA1L, ß = 0.20, SE = 0.03, p value = 8.91 × 10-12), 10q24.33 (OBFC1, ß = -0.12, SE = 0.02, p value = 1.67 × 10-8), 13q34 (COL4A2, ß = 0.02, SE = 0.02, p value = 2.34 × 10-11), and 19q13.32 (APOC1, ß = -0.19, SE = 0.03, p value = 1.38 × 10-12; APOE, ß = 0.21, SE = 0.03, p value = 2.70 × 10-11; PVRL2:CTB-129P6.4, ß = 0.15, SE = 0.03, p value = 1.38 × 10-8); 2 genes (SH3PXD2A, Z-score = 4.83, p value = 6.67 × 10-7; and APOC1, Z-score: = 5.11, p value = 1.60 × 10-7); and ICA1L transcript (Z-score = 6.8, p value = 9.1 × 10-12) and protein levels (Z-score = -5.8, p value = 6.7 × 10-9). DISCUSSION: Our results reinforce the role of APOE in ICH risk, replicate previous ICH-associated loci (2q33 and 13q34), and point to new ICH associations with OBFC1, PVRL2:CTB-129P6.4, APOC1, and SH3PXD2A. Our study used data from European subjects, our main limitation. These molecules could be potential targets for future studies for modulating ICH risk.

A multitrait genetic study of hemostatic factors and hemorrhagic transformation after stroke treatment.

BACKGROUND: Thrombolytic recombinant tissue plasminogen activator (r-tPA) treatment is the only pharmacologic intervention available in the ischemic stroke acute phase. This treatment is associated with an increased risk of intracerebral hemorrhages, known as hemorrhagic transformations (HTs), which worsen the patient's prognosis. OBJECTIVES: To investigate the association between genetically determined natural hemostatic factors' levels and increased risk of HT after r-tPA treatment. METHODS: Using data from genome-wide association studies on the risk of HT after r-tPA treatment and data on 7 hemostatic factors (factor [F]VII, FVIII, von Willebrand factor [VWF], FXI, fibrinogen, plasminogen activator inhibitor-1, and tissue plasminogen activator), we performed local and global genetic correlation estimation multitrait analyses and colocalization and 2-sample Mendelian randomization analyses between hemostatic factors and HT. RESULTS: Local correlations identified a genomic region on chromosome 16 with shared covariance: fibrinogen-HT, P = 2.45 × 10-11. Multitrait analysis between fibrinogen-HT revealed 3 loci that simultaneously regulate circulating levels of fibrinogen and risk of HT: rs56026866 (PLXND1), P = 8.80 × 10-10; rs1421067 (CHD9), P = 1.81 × 10-14; and rs34780449, near ROBO1 gene, P = 1.64 × 10-8. Multitrait analysis between VWF-HT showed a novel common association regulating VWF and risk of HT after r-tPA at rs10942300 (ZNF366), P = 1.81 × 10-14. Mendelian randomization analysis did not find significant causal associations, although a nominal association was observed for FXI-HT (inverse-variance weighted estimate [SE], 0.07 [-0.29 to 0.00]; odds ratio, 0.87; 95% CI, 0.75-1.00; raw P = .05). CONCLUSION: We identified 4 shared loci between hemostatic factors and HT after r-tPA treatment, suggesting common regulatory mechanisms between fibrinogen and VWF levels and HT. Further research to determine a possible mediating effect of fibrinogen on HT risk is needed.

Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia.

During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia.

Causal role of lipid metabolome on the risk of ischemic stroke, its etiological subtypes, and long-term outcome: A Mendelian randomization study.

BACKGROUND AND AIMS: The lipid profile is consistently associated with coronary artery disease (CAD) and ischemic stroke (IS). However, the lipoprotein subfractions have not been deeply explored in stroke subtypes, especially in IS outcome. METHODS: We performed two-sample Mendelian randomization (MR) analysis using 92 lipid traits measured by nuclear magnetic resonance in 115,000 subjects from the UK Biobank. Data for genetic associations with IS, its subtypes, and long-term outcome (LTO) were obtained from three cohorts of European ancestry: GIGASTROKE (73,652 cases, 1,234,808 controls), GODS (n = 1791) and GISCOME (n = 6165). Results obtained using CARDIoGRAMPlusC4D were used to identify differences with CAD. RESULTS: Genetically determined low concentration of medium high-density lipoprotein (HDL) particles (odds ratio (OR) = 0.92, 95% CI 0.88-0.96; p = 3.6 × 10-4) and its cholesterol content (OR = 0.92, 95% CI 0.88-0.96; p = 1.9 × 10-4) showed causal associations with an increased risk of stroke. Genetic predisposition to high apolipoprotein (apo)B to apoA-I ratio was causally associated with an increased risk of IS (OR = 1.12, 95% CI 1.06-1.18, p = 1.1 × 10-4), and a highly suggestive association was found between non-esterified cholesterol in low-density lipoprotein (LDL) and increased risk of atherothrombotic stroke (LAS) (OR = 1.35, 95% CI 1.10-1.66; p = 4.0 × 10-3). Low cholesterol in small and medium LDL was suggestively associated with poor LTO. CONCLUSIONS: Our results support that low medium HDL concentration was causally associated with an increased stroke risk, while high levels of non-esterified cholesterol in LDL were suggestively associated with an increased risk of LAS and with a better LTO.

Peripheral effects of morphine and expression of µ-opioid receptors in the dorsal root ganglia during neuropathic pain: nitric oxide signaling.

BACKGROUND: The local administration of µ-opioid receptor (MOR) agonists attenuates neuropathic pain but the precise mechanism implicated in this effect is not completely elucidated. We investigated if nitric oxide synthesized by neuronal (NOS1) or inducible (NOS2) nitric oxide synthases could modulate the local antiallodynic effects of morphine through the peripheral nitric oxide-cGMP-protein kinase G (PKG)-ATP-sensitive K+ (KATP) channels signaling pathway activation and affect the dorsal root ganglia MOR expression during neuropathic pain. RESULTS: In wild type (WT) mice, the subplantar administration of morphine dose-dependently decreased the mechanical and thermal allodynia induced by the chronic constriction of the sciatic nerve (CCI), which effects were significantly diminished after their co-administration with different subanalgesic doses of a selective NOS1 (N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidine tris(trifluoroacetate) salt; NANT), NOS2 (L-N(6)-(1-iminoethyl)-lysine; L-NIL), L-guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ), PKG ((Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate; Rp-8-pCPT-cGMPs) inhibitor or a KATP channel blocker (glibenclamide). The evaluation of the expression of MOR in the dorsal root ganglia from sham-operated and sciatic nerve-injured WT, NOS1 knockout (KO) and NOS2-KO mice at 21 days after surgery demonstrated that, although the basal mRNA and protein levels of MOR were similar between WT and both NOS-KO animals, nerve injury only decreased their expression in WT mice. CONCLUSIONS: These results suggest that the peripheral nitric oxide-cGMP-PKG-KATP signaling pathway activation participates in the local antiallodynic effects of morphine after sciatic nerve injury and that nitric oxide, synthesized by NOS1 and NOS2, is implicated in the dorsal root ganglia down-regulation of MOR during neuropathic pain.

Genetic Determinants of Plasma Low-Density Lipoprotein Cholesterol Levels: Monogenicity, Polygenicity, and "Missing" Heritability.

Changes in plasma low-density lipoprotein cholesterol (LDL-c) levels relate to a high risk of developing some common and complex diseases. LDL-c, as a quantitative trait, is multifactorial and depends on both genetic and environmental factors. In the pregenomic age, targeted genes were used to detect genetic factors in both hyper- and hypolipidemias, but this approach only explained extreme cases in the population distribution. Subsequently, the genetic basis of the less severe and most common dyslipidemias remained unknown. In the genomic age, performing whole-exome sequencing in families with extreme plasma LDL-c values identified some new candidate genes, but it is unlikely that such genes can explain the majority of inexplicable cases. Genome-wide association studies (GWASs) have identified several single-nucleotide variants (SNVs) associated with plasma LDL-c, introducing the idea of a polygenic origin. Polygenic risk scores (PRSs), including LDL-c-raising alleles, were developed to measure the contribution of the accumulation of small-effect variants to plasma LDL-c. This paper discusses other possibilities for unexplained dyslipidemias associated with LDL-c, such as mosaicism, maternal effect, and induced epigenetic changes. Future studies should consider gene-gene and gene-environment interactions and the development of integrated information about disease-driving networks, including phenotypes, genotypes, transcription, proteins, metabolites, and epigenetics.

Pharmacogenetics studies in stroke patients treated with rtPA: a review of the most interesting findings.

Recombinant tissue-plasminogen activator (rtPA) is the only drug used during the acute phase of stroke. Despite its important benefits, a percentage of patients suffer symptomatic hemorrhagic transformations or a lack of early recanalization rates. These undesirable effects are associated with acute neurological and long-term functional deterioration. For the past 20 years, pharmacogenetic studies have tried to find the genetic risk factors associated with rtPA response. Most of these studies have used a gene-candidate strategy; however, recent genome-wide association studies have emerged indicating that genetic predisposition could modulate rtPA response. This review summarizes the most interesting findings in this field, including which genes and genetic variations are associated with hemorrhagic transformations and recanalization rates after thrombolytic therapy.

The role of nitric oxide in the local antiallodynic and antihyperalgesic effects and expression of delta-opioid and cannabinoid-2 receptors during neuropathic pain in mice.

Both delta-opioid receptor (DOPr) and cannabinoid-2 receptor (CB2R) agonists attenuate neuropathic pain, but the precise mechanism implicated in these effects is not completely elucidated. We investigated whether nitric oxide synthesized by neuronal (NOS1) or inducible (NOS2) nitric-oxide synthases could modulate DOPr and/or CB2R antiallodynic and antihyperalgesic effects through the peripheral nitric oxide-cGMP-protein kinase G (PKG) pathway activation and affect their expression during neuropathic pain. In wild-type (WT) mice at 21 days after chronic constriction of sciatic nerve, we evaluated the effects of [d-Pen(2),d-Pen(5)]-enkephalin (DPDPE); (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone (JWH-015); and a NOS1 [N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidine tris(trifluoroacetate) salt; NANT], NOS2 [l-N(6)-(1-iminoethyl)-lysine; l-NIL], l-guanylate cyclase [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ], or PKG [(Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate; Rp-8-pCPT-cGMPs] inhibitor administered alone or combined. Expression of DOPr and CB2R mRNA in the spinal cord and dorsal root ganglia of naive and nerve-injured WT, NOS1-knockout (KO), and NOS2-KO mice, also was assessed. The subplantar administration of NANT, l-NIL, ODQ, or Rp-8-pCPT-cGMPs dose-dependently inhibited neuropathic pain and enhanced the local effects of DPDPE or JWH-015. Moreover, although the basal levels of DOPr and CB2R mRNA were similar between WT and NOS-KO animals, nerve injury only decreased (DOPr) or increased (CB2R) their expression in the dorsal root ganglia of WT and NOS2-KO mice, and not in NOS1-KO mice. Results suggest that inactivation of the nitric oxide-cGMP-PKG peripheral pathway triggered by NOS1 and NOS2 enhanced the peripheral actions of DOPr and CB2R agonists and that nitric oxide synthesized by NOS1 is implicated in the peripheral regulation of DOPr and CB2R gene transcription during neuropathic pain.

Polygenic Markers in Patients Diagnosed of Autosomal Dominant Hypercholesterolemia in Catalonia: Distribution of Weighted LDL-c-Raising SNP Scores and Refinement of Variant Selection.

Familial hypercholesterolemia (FH) is associated with mutations in the low-density lipoprotein (LDL) receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin 9 (PCSK9) genes. A pathological variant has not been identified in 30-70% of clinically diagnosed FH patients, and a burden of LDL cholesterol (LDL-c)-raising alleles has been hypothesized as a potential cause of hypercholesterolemia in these patients. Our aim was to study the distribution of weighted LDL-c-raising single-nucleotide polymorphism (SNP) scores (weighted gene scores or wGS) in a population recruited in a clinical setting in Catalonia. The study included 670 consecutive patients with a clinical diagnosis of FH and a prior genetic study involving 250 mutation-positive (FH/M+) and 420 mutation-negative (FH/M-) patients. Three wGSs based on LDL-c-raising variants were calculated to evaluate their distribution among FH patients and compared with 503 European samples from the 1000 Genomes Project. The FH/M- patients had significantly higher wGSs than the FH/M+ and control populations, with sensitivities ranging from 42% to 47%. A wGS based only on the SNPs significantly associated with FH (wGS8) showed a higher area under the receiver operating characteristic curve, and higher diagnostic specificity and sensitivity, with 46.4% of the subjects in the top quartile. wGS8 would allow for the assignment of a genetic cause to 66.4% of the patients if those with polygenic FH are added to the 37.3% of patients with monogenic FH. Our data indicate that a score based on 8 SNPs and the75th percentile cutoff point may identify patients with polygenic FH in Catalonia, although with limited diagnostic sensitivity and specificity.

Molecular analysis of APOB, SAR1B, ANGPTL3, and MTTP in patients with primary hypocholesterolemia in a clinical laboratory setting: Evidence supporting polygenicity in mutation-negative patients.

BACKGROUND AND AIMS: Primary hypobetalipoproteinemia is generally considered a heterogenic group of monogenic, inherited lipoprotein disorders characterized by low concentrations of LDL cholesterol and apolipoprotein B in plasma. Lipoprotein disorders include abetalipoproteinemia, familial hypobetalipoproteinemia, chylomicron retention disease, and familial combined hypolipidemia. Our aim was to review and analyze the results of the molecular analysis of hypolipidemic patients studied in our laboratory over the last 15 years. METHODS: The study included 44 patients with clinical and biochemical data. Genomic studies were performed and genetic variants were characterized by bioinformatics analysis. A weighted LDL cholesterol gene score was calculated to evaluate common variants associated with impaired lipid concentrations and their distribution among patients. RESULTS: Twenty-three patients were genetically confirmed as affected by primary hypobetalipoproteinemia. In this group of patients, the most prevalent mutated genes were APOB (in 17 patients, with eight novel mutations identified), SAR1B (in 3 patients, with one novel mutation identified), ANGPTL3 (in 2 patients), and MTTP (in 1 patient). The other 21 patients could not be genetically diagnosed with hypobetalipoproteinemia despite presenting suggestive clinical and biochemical features. In these patients, two APOB genetic variants associated with lower LDL cholesterol were more frequent than in controls. Moreover, the LDL cholesterol gene score, calculated with 11 SNPs, was significantly lower in mutation-negative patients. CONCLUSIONS: Around half of the patients could be genetically diagnosed. The results suggest that, in at least some of the patients without an identified mutation, primary hypobetalipoproteinemia may have a polygenic origin.

The clinical laboratory and dyslipidemia.

The prevalence of dyslipidemia (alterations in lipoprotein metabolism) as a risk factor for cardiovascular disease continues to rise. Determination of circulating levels of lipids, lipoproteins and related proteins allows the diagnosis, treatment and followup of the various types of dyslipidemia. However, numerous factors influence the concentration of these constituents in the circulation. These factors depend both on the individual (habits, physical characteristics, presence of other risk factors) and on the sample and analytical methods employed and must be taken into account when assessing and interpreting laboratory results. Similarly, the cause of the different forms of dyslipidemia may lie not only in environmental but also in genetic factors. Consequently, the inclusion of genetic tests in the diagnosis of dyslipidemia is increasingly frequent.

A rare STAP1 mutation incompletely associated with familial hypercholesterolemia.

Autosomal dominant hypercholesterolemia, being referred to as familial hypercholesterolemia (FH), is mainly due to defective LDL receptor (LDLR) function, but is also associated with variants in genes encoding APOB (LDLR ligand) and PCSK9, the catabolic regulator of LDLR. The signal-transducing adaptor family member 1 (STAP1) gene has been recently linked to FH. We describe the case of a 56-year-old male patient found to have hypercholesterolemia at age 34, but who did not continue follow-up nor received treatment with lipid-lowering drugs. At age 55 he suffered a myocardial infarction. A systematic NGS analysis did not show point mutations in the LDLR, APOB, LDLRAP1, or PCSK9 genes, nor large rearrangements of the LDLR gene, but revealed the heterozygous missense variant rs199787258 of STAP1 (c.526C > T; p.Pro176Ser). This variant was also found in heterozygosis in the two siblings of the index case, who also had hypercholesterolemia, but did not cosegregate in his progeny. A bioinformatics analysis and available structural information predicts p.Pro176Ser as the most damaging of all STAP1 missense variants associated with familial hypercholesterolemia. Our findings confirm and extend the linkage between STAP1 variants and FH, and point to an important role of this adaptor protein within a signaling pathway that affects cholesterol homeostasis.