Low vitamin D status is associated with inflammatory response in older patients with cerebral small vessel disease.

OBJECTIVES: This study aimed to determine the association of the NF-κB inflammatory signaling pathway with vitamin D status in older cerebral small vessel disease (SVD) patients. METHODS: We measured serum 25(OH)D, pro-and anti-inflammatory cytokines, and mRNA levels of the vitamin D-activating enzyme, CYP27B1, as well as NF-kB, COX-2, the chemokine-CCL2, IL-1ß, IL-6, TNF-α, TGF-ß, and IL-10, in cerebral SVD patients aged ≥60 years presenting with vascular dementia and age and gender-matched healthy controls. RESULTS: Low vitamin D status (insufficiency: serum 25(OH)D 12-20 ng/ml; deficiency: ≤12 ng/ml) was more prevalent among patients compared to controls. The mRNA levels of NF-kB, COX-2, CCL2, IL-1ß, and IL-6, and serum levels of pro-inflammatory cytokines (IL-1α, IL-1ß, IL-6, and TNF-α) were significantly higher in cases compared to controls. There was a significant correlation between CYP27B1 and NF-kB, COX-2, CCL2, and IL-1ß gene expression. Serum IL-1α, IL-1ß, and IL-6 concentrations and the expression of CCL-2, NF-kB2, and NF-kB3 genes were higher in vitamin D-deficient subjects compared to vitamin D-sufficient subjects. There was a significant negative correlation between serum 25(OH)D and IL-1α, IL-6, and TNF-α, and a positive correlation between 25(OH)D and IL-10. CONCLUSION: Low vitamin D is associated with an inflammatory response via NF-kB signaling, which could play a role in the etio-pathogenesis of SVD. Further large-scale studies are required to validate our findings.

Altered MicroRNA Expression in Intracranial Aneurysmal Tissues: Possible Role in TGF-ß Signaling Pathway.

The molecular mechanisms behind the rupture of intracranial aneurysms remain obscure. MiRNAs are key regulators of a wide array of biological processes altering protein synthesis by binding to target mRNAs. However, variations in miRNA levels in ruptured aneurysmal wall have not been completely examined. We hypothesized that altered miRNA signature in aneurysmal tissues could potentially provide insight into aneurysm pathophysiology. Using a high-throughput miRNA microarray screening approach, we compared the miRNA expression pattern in aneurysm tissues obtained during surgery from patients with aneurysmal subarachnoid hemorrhage (aSAH) with control tissues (GEO accession number GSE161870). We found that the expression of 70 miRNAs was altered. Expressions of the top 10 miRNA were validated, by qRT-PCR and results were correlated with clinical characteristics of aSAH patients. The level of 10 miRNAs (miR-24-3p, miR-26b-5p, miR-27b-3p, miR-125b-5p, miR-143-3p, miR-145-5p, miR-193a-3p, miR-199a-5p, miR-365a-3p/365b-3p, and miR-497-5p) was significantly decreased in patients compared to controls. Expression of miR-125b-5p, miR-143-3p and miR-199a-5p was significantly decreased in patients with poor prognosis and vasospasm. The target genes of few miRNAs were enriched in Transforming growth factor-beta (TGF-ß) and Mitogen-activated protein kinases (MAPK) pathways. We found significant negative correlation between the miRNA and mRNA expression (TGF-ß1, TGF-ß2, SMAD family member 2 (SMAD2), SMAD family member 4 (SMAD4), MAPK1 and MAPK3) in aneurysm tissues. We suggest that miR-26b, miR-199a, miR-497and miR-365, could target multiple genes in TGF-ß and MAPK signaling cascades to influence inflammatory processes, extracellular matrix and vascular smooth muscle cell degradation and apoptosis, and ultimately cause vessel wall degradation and rupture.

Late Onset Pompe Disease with Novel Mutations and Atypical Phenotypes.

BACKGROUND: Late onset Pompe disease (LOPD) is rare and generally manifests predominantly as progressive limb girdle muscle weakness. It is linked to the pathogenic mutations in GAA gene, which leads to glycogen accumulation in various tissues. MATERIALS AND METHODS: We describe the unusual clinical, biochemical, histopathological and genetic characteristics of 5 cases of LOPD. RESULTS: The first case had progressive anterior horn cell like disease (AHCD) that evolved later to classical limb girdle syndrome and respiratory failure, the second patient had rigid spine syndrome with gastrointestinal manifestations, the third had limb girdle weakness superimposed with episodic prolonged worsening and respiratory failure, the fourth had large fibre sensory neuropathy without primary muscle involvement and the fifth presented with classical limb girdle muscle weakness. Two homozygous missense mutations c.1461C > A (p.Phe487Leu) and c.1082C > T (p.Pro361Leu) in the GAA gene were identified in case 1 and 2 respectively. Case 3 was compound heterozygous with inframe c.1935_1940del (p.Val646_Cys647del) and an intronic splice effecting variant c.-32-13T > G. Compound heterozygous missense variants c.971C > T (p.Pro324Leu) and c.794G > A (p.Ser265Asn) were identified in case 4. Case 5 had a frameshift insertion c.1396dupG (p.Val466GlyfsTer40) and a synonymous splice affecting variant c.546G > T(p.Thr182=). CONCLUSION: We are describing for the first time from India on LOPD with unusual phenotypes identified. A high degree of clinical suspicion and diagnosing rare phenotypes of Pompe disease is imperative to consider early initiation of Enzyme Replacement Therapy (ERT).

Circulating MicroRNAs as Potential Molecular Biomarkers for Intracranial Aneurysmal Rupture.

INTRODUCTION: Diagnosis of the rupture of an intracranial aneurysm (IA) relies on sophisticated neuro-imaging studies, and molecular biomarkers to identify an IA or predict its rupture are still unavailable. OBJECTIVE: Our objective was to determine the plasma microRNA (miRNA) expression profile in patients with ruptured IA presenting as aneurysmal subarachnoid hemorrhage (aSAH) and identify potential biomarkers of aneurysmal rupture. METHODS: Plasma miRNA profiling was carried out using quantitative real-time polymerase chain reaction (qRT-PCR) in 20 patients with aSAH and 20 age- and sex-matched healthy controls. Eight differentially expressed miRNAs were validated by qPCR in a larger cohort of 88 patients with aSAH and 110 healthy controls. A receiver operating characteristic (ROC) curve was constructed to evaluate the overall performance of the miRNA-based assay. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to determine the potential pathway of miRNA-target genes. RESULTS: The miRNA profiles were clearly distinct in patients compared with controls. Validation studies showed that three upregulated miRNAs (miR-15a-5p, miR-34a-5p, miR-374a-5p) and five downregulated miRNAs (miR-146a-5p, miR-376c-3p, miR-18b-5p, miR-24-3p, miR-27b-3p) could distinguish patients with aSAH from healthy controls with high predicted probability (0.865 and 0.995, respectively). Further, the expression levels of the eight candidate miRNAs were significantly dysregulated only in aSAH cases and not in patients with SAH due to other causes. Plasma miR-146a-5p and miR-27b-3p were associated with clinical outcomes in patients with aSAH. Functional analysis of the eight differentially expressed miRNA showed that the target genes involved in signaling pathways were related to inflammation. CONCLUSIONS: Our study determined the plasma miRNA signature of ruptured IAs and identified eight candidate miRNAs that could be useful biomarkers for this condition. We hypothesize that these differentially expressed miRNAs may play pivotal roles in IA pathology.

Vitamin D receptor (VDR) gene polymorphism and vascular dementia due to cerebral small vessel disease in an Asian Indian cohort.

Vitamin D receptor (VDR) and its ligand Vitamin D, play a crucial role in regulating multiple pathways for maintaining vascular health. The present study aimed at evaluating whether single nucleotide polymorphisms in VDR gene were associated with susceptibility to vascular dementia (VaD) due to cerebral small vessel disease (SVD). A total of 644 subjects (302 patients diagnosed with cerebral SVD-associated VaD and 342, age- and gender-matched healthy controls) were genotyped for VDR gene variants, FokI, ApaI, TaqI and BsmI, by PCR-RFLP method. Among the 4 examined VDR variants, the presence of the minor allele (Ff+ff vs FF) of FokI variant increased the risk for cerebral SVD by 1.5-fold in men (p = 0.047). Serum 25-hydroxyvitamin D [25(OH)D] was lower in subjects having the FokI "ff" genotype compared to those with the "FF" genotype (p = 0.044). Moreover, in subjects with low serum 25(OH)D the presence of "ff" genotype increased the odds of SVD by 2.5 folds (p = 0.041). ApaI polymorphism decreased the risk of cerebral SVD in women. The distribution of TaqI and BsmI variants were not significantly different between patients and controls. Further studies in large cohorts are necessary to validate the role of FokI polymorphism in cerebral SVD and VaD etiopathogenesis.

Effect of temperature on lysosomal enzyme activity during preparation and storage of dried blood spots.

BACKGROUND: The use of dried blood spots (DBS) for the assay of lysosomal enzymes has facilitated the implementation of pilot studies for newborn screening for lysosomal storage disorders in various developed countries. The aim of the study was to determine the influence of ambient temperature during DBS preparation and storage on lysosomal enzyme activity in a developing, tropical country. METHODS: Blood samples from 12 healthy subjects collected on a S&S 903 filter paper were dried and stored at different temperatures for different periods of time. Activities of five lysosomal enzymes (acid α-glucosidase, acid α-galactosidase, acid ß-glucocerebrosidase, acid sphingomyelinase, and galactocerebrosidase) were determined by tandem mass spectrometric and fluorimetric (acid α-glucosidase and acid ß-glucocerebrosidase only) assays. RESULTS: The mean activities of all five enzymes decreased significantly when DBS was dried at temperatures above 24°C (P<.0001). DBS stored at 4°C, 24°C, 30°C, 37°C, and 45°C for 10 days and more, also showed significant reduction in activities of all five enzymes (P<.0001). CONCLUSION: The results highlight the importance of maintaining the correct ambient temperature during DBS preparation and storage to avoid false positive results when screening for lysosomal storage disorders.

Age and gender-specific reference intervals for lysosomal enzymes in dried blood spot samples: A study in Indian population.

OBJECTIVES: The study aimed to establish age and gender-specific reference values for the activities of lysosomal enzymes (acid α-galactosidase [GLA], acid ß-glucocerebrosidase [GBA], acid α-glucosidase [GAA], acid sphingomyelinase [ASM] and galactocerebrosidase [GALC]) in dried blood spots (DBS) of Indian population. DESIGN AND METHODS: A total of 3797 healthy Indian subjects (1456 females and 2341 males) aged from 2days to 60years were selected for the study. Activities of 5 lysosomal enzymes were determined by tandem mass spectrometry, for newborns (<30days), infants (>1month-1year), children (>1-5years) and (>5-18years) and adults (>18years).Variations in enzyme activities based on age and gender were studied. The reference interval was defined as the central 95% range, and was determined based on age and gender. RESULTS: Highly significant differences in activities were observed for GAA (p=0.001), GLA (p<0.0001), GBA (p<0.0001), ASM (p<0.0001) and GALC (p<0.0001), between different age groups. Comparison of activities between genders showed significant difference for ASM in children aged 1-5years (p=0.03) with higher activity in females, and for GLA in children aged 5-18years (p=0.004) where the activity was higher in males. Reference intervals decreased with age for all enzymes, except GAA. The ranges of GLA and GALC were higher in females, whereas GBA was higher in males. CONCLUSION: The study establishes age and gender-specific reference values for the screening and identification of lysosomal storage disorders in Indian population. Our data may facilitate establishment of mass screening programs for these disorders in India.

Vitamin D status and vascular dementia due to cerebral small vessel disease in the elderly Asian Indian population.

Vitamin D plays vital roles in human health and recent studies have shown its beneficial effect on brain functioning. The present study was designed to evaluate the association of vitamin D with vascular dementia (VaD) due to cerebral small vessel disease (SVD) in Asian Indian population. 140 VaD patients aged ≥ 60 years with neuroimaging evidence of SVD, and 132 age and gender-matched controls, were investigated. Vitamin D status was estimated by measuring serum 25-hydroxy vitamin D. Logistic regression model revealed that deficient levels of vitamin D (<12 ng/ml) were associated with 2.2-fold increase in odds of VaD after adjustment with covariates. Hypertension was independently associated with 11.3-fold increased odds of VaD. In hypertensives with vitamin D deficiency and insufficiency (12-20 ng/ml), the odds were increased to 31.6-fold and 14.4-fold, respectively. However, in hypertensives with vitamin D sufficiency (>20 ng/ml), the odds of VaD were increased by 3.8-fold only. Pearson correlation showed that serum vitamin D was inversely associated with systolic and diastolic blood pressure (r=-0.401 and -0.411, p<0.01, respectively) in vitamin D-deficient subjects. Since the combined presence of hypertension and vitamin D deficiency increases the probability of developing VaD, screening for vitamin D status in addition to regular monitoring of blood pressure, could reduce the risk of VaD associated with cerebral SVD in the elderly Asian Indian subjects.