VAMP8/endobrevin is overexpressed in hyperreactive human platelets: suggested role for platelet microRNA.

BACKGROUND: Variation in platelet reactivity contributes to disorders of hemostasis and thrombosis, but the molecular mechanisms are not well understood. OBJECTIVES: To discover associations between interindividual platelet variability and the responsible platelet genes, and to begin to define the molecular mechanisms altering platelet gene expression. SUBJECTS/METHODS: Two hundred and eighty-eight healthy subjects were phenotyped for platelet responsiveness. Platelet RNA from subjects demonstrating hyperreactivity (n=18) and hyporeactivity (n=11) was used to screen the human transcriptome. RESULTS: Distinctly different mRNA profiles were observed between subjects with differing platelet reactivity. Increased levels of mRNA for VAMP8/endobrevin, a critical v-SNARE involved in platelet granule secretion, were associated with platelet hyperreactivity (Q=0.0275). Validation studies of microarray results showed 4.8-fold higher mean VAMP8 mRNA levels in hyperreactive than hyporeactive platelets (P=0.0023). VAMP8 protein levels varied 13-fold among platelets from these normal subjects, and were 2.5-fold higher in hyperreactive platelets (P=0.05). Among our cohort of 288 subjects, a VAMP8 single-nucleotide polymorphism (rs1010) was associated with platelet reactivity in an age-dependent manner (P<0.003). MicroRNA-96 was predicted to bind to the 3'-untranslated regionof VAMP8 mRNA and was detected in platelets. Overexpression of microRNA-96 in VAMP8-expressing cell lines caused a dose-dependent decrease in VAMP8 protein and mRNA, suggesting a role in VAMP8 mRNA degradation. CONCLUSIONS: These findings support a role for VAMP8/endobrevin in the heterogeneity of platelet reactivity, and suggest a role for microRNA-96 in the regulation of VAMP8 expression.

Molecular diagnosis of cystic fibrosis in Indian patients--a preliminary report.

AIM: The aim of the study was to screen for the common deltaF508 mutation and the poly T polymorphism and to determine their frequency in the cystic fibrosis transmembrane conductance regulator (CFTR) gene among the suspected CF cases referred to our clinical care centre for sweat chloride tests. METHODOLOGY: Sweat and EDTA blood samples were obtained from 23 clinically suspected cystic fibrosis (CF) cases. Sweat was estimated by pilocarpine iontophoresis procedure. Poly T polymorphism was detected by the multiplex-PCR based on ARMSTM technique and deltaF508 mutation by PCR-mediated site-directed mutagenesis method. RESULTS: Five cases, mainly with respiratory abnormalities and followed by steatorrhea had elevated sweat chloride levels (> 60 mmol/l), three of them, each with nutritional, respiratory and pancreatic abnormalities were borderline (40-60 mmol/l) and the remaining 15 clinically suspected CF cases had normal sweat chloride levels (< 40 mmol/l). The 9T variant was frequently observed (75%) in cases with elevated sweat chloride, including those exhibiting borderline values; with no 5T variant. The 7T was the most common variant (77%) observed in the cases with normal sweat chloride, with only one 5T variant (33%). Of the five cases with high sweat chloride, four cases were homozygous for deltaF508, whereas one was heterozygous with borderline sweat chloride, thus showing an overall frequency of 56.25% in the CF chromosome. DeltaF508 was found to be present with the 9T variant in all the instances. CONCLUSION: The presence of the 9T variant along with elevated sweat chloride levels can be used to predict a high risk of the individual harboring the severe deltaF508 mutation. It would be advisable to test for to the deltaF508 mutation along with the sweat chloride estimation in all the critically suspected CF cases diagnose CF with a higher degree certainty.

Identification of two LDL receptor mutations causing familial hypercholesterolemia in Indian subjects.

Familial hypercholesterolemia (FH) is a genetic disorder caused by numerous mutations in the low-density lipoprotein receptor (LDLR) gene. Mutational analyses of Indians in South Africa suggest the possibility of a high frequency of FH in India. This study aimed at identifying mutations in exons 3, 4, 9, and 14 of the LDLR gene among Indians and at eventually developing population-directed molecular-based screening assays. DNA samples from 25 hypercholesterolemic patients with clinical features of FH and 25 normal controls were analyzed for four known point mutations: W66G (exon 3), E207K (exon 4), E387K (exon 9), and P664L (exon 14), which are those most reported among Indian immigrants in South Africa. Subsequently, samples were screened for other mutations by modified heteroduplex analysis in these exons. Point mutations predicted to be common among Indians were absent in the samples analyzed. Heteroduplex analysis and sequencing revealed the presence of novel insertion mutations in two patients. Both mutations are single-nucleotide "G" insertions at position 242 in exon 3 and position 397 in exon 4. The observed mutations are predicted to cause a translational frameshift, encoding truncated LDLR proteins due to premature termination. The mutant proteins are likely to be degraded intracellularly and, therefore, are pathogenic.