Genomic profiling identifies novel mutations and SNPs in ABCD1 gene: a molecular, biochemical and clinical analysis of X-ALD cases in India

X-linked adrenoleukodystrophy (X-ALD) affects the nervous system white matter and adrenal cortex secondary to mutations in the ABCD1 gene that encode the peroxisomal membrane protein. We conducted a genomic and protein expression study of susceptibility gene with its clinical and biochemical analysis. To the best of our knowledge this is the first preliminary comprehensive study in Indian population that identified novel mutations and SNPs in a relatively large group. We screened 17 Indian indigenous X-linked adrenoleukodystrophy cases and 70 controls for mutations and SNPs in the exonic regions (including flanking regions) of ABCD1 gene by direct sequencing with ABI automated sequencer along with Western blot analysis of its endogenous protein, ALDP, levels in peripheral blood mononuclear cells. Single germ line mutation was identified in each index case in ABCD1 gene. We detected 4 novel mutations (2 missense and 2 deletion/insertion) and 3 novel single nucleotide polymorphisms. We observed a variable protein expression in different patients. These findings were further extended to biochemical and clinical observations as it occurs with great clinical expression variability. This is the first major study in this population that presents a different molecular genetic spectrum as compared to Caucasian population due to geographical distributions of ethnicity of patients. It enhances our knowledge of the causative mutations of X-ALD that grants holistic base to develop effective medicine against X-ALD.     

Trauma hemorrhagic shock-induced lung injury involves a gut-lymph-induced TLR4 pathway in mice

Background

Injurious non-microbial factors released from the stressed gut during shocked states contribute to the development of acute lung injury (ALI) and multiple organ dysfunction syndrome (MODS). Since Toll-like receptors (TLR) act as sensors of tissue injury as well as microbial invasion and TLR4 signaling occurs in both sepsis and noninfectious models of ischemia/reperfusion (I/R) injury, we hypothesized that factors in the intestinal mesenteric lymph after trauma hemorrhagic shock (T/HS) mediate gut-induced lung injury via TLR4 activation.

Methods/Principal Findings

The concept that factors in T/HS lymph exiting the gut recreates ALI is evidenced by our findings that the infusion of porcine lymph, collected from animals subjected to global T/HS injury, into naïve wildtype (WT) mice induced lung injury. Using C3H/HeJ mice that harbor a TLR4 mutation, we found that TLR4 activation was necessary for the development of T/HS porcine lymph-induced lung injury as determined by Evan''s blue dye (EBD) lung permeability and myeloperoxidase (MPO) levels as well as the induction of the injurious pulmonary iNOS response. TRIF and Myd88 deficiency fully and partially attenuated T/HS lymph-induced increases in lung permeability respectively. Additional studies in TLR2 deficient mice showed that TLR2 activation was not involved in the pathology of T/HS lymph-induced lung injury. Lastly, the lymph samples were devoid of bacteria, endotoxin and bacterial DNA and passage of lymph through an endotoxin removal column did not abrogate the ability of T/HS lymph to cause lung injury in naïve mice.

Conclusions/Significance

Our findings suggest that non-microbial factors in the intestinal mesenteric lymph after T/HS are capable of recreating T/HS-induced lung injury via TLR4 activation.     

Overexpression of microRNAs from the miR-17-92 paralog clusters in AIDS-related non-Hodgkin's lymphomas

Background

Individuals infected by HIV are at an increased risk for developing non-Hodgkin''s lymphomas (AIDS-NHL). In the highly active antiretroviral therapy (HAART) era, there has been a significant decline in the incidence of AIDS-associated primary central nervous system lymphoma (PCNSL). However, only a modest decrease in incidence has been reported for other AIDS-NHL subtypes. Thus, AIDS-NHLs remain a significant cause of morbidity and mortality in HIV infected individuals. Recently, much attention has been directed toward the role of miRNAs in cancer, including NHL. Several miRNAs, including those encoded by the miR-17-92 polycistron, have been shown to play significant roles in B cell tumorigenesis. However, the role of miRNAs in NHL in the setting of HIV infection has not been defined.

Methodology/Principal Findings

We used quantitative realtime PCR to assess the expression of miRNAs from three different paralog clusters, miR-17-92, miR-106a-363, and miR-106b-25 in 24 cases of AIDS-NHLs representing four tumor types, Burkitt''s lymphoma (BL, n = 6), diffuse large B-cell lymphoma (DLBCL, n = 8), primary central nervous system lymphoma (PCNSL, n = 5), and primary effusion lymphoma (PEL, n = 5). We also used microarray analysis to identify a differentiation specific miRNA signature of naïve, germinal center, and memory B cell subsets from tonsils (n = 4). miRNAs from the miR-17-92 paralog clusters were upregulated by B cells, specifically during the GC differentiation stage. We also found overexpression of these miRNA clusters in all four AIDS-NHL subtypes. Finally, we also show that select miRNAs from these clusters (miR-17, miR-106a, and miR-106b) inhibited p21 in AIDS-BL and DLBCL cases, thus providing a mechanistic role for these miRNAs in AIDS-NHL pathogenesis.

Conclusion

Dysregulation of miR-17-92 paralog clusters is a common feature of AIDS-associated NHLs.     

Safety and Immunogenicity of DNA and MVA HIV-1 Subtype C Vaccine Prime-Boost Regimens: A Phase I Randomised Trial in HIV-Uninfected Indian Volunteers

Study Design

A randomized, double-blind, placebo controlled phase I trial.

Methods

The trial was conducted in 32 HIV-uninfected healthy volunteers to assess the safety and immunogenicity of prime-boost vaccination regimens with either 2 doses of ADVAX, a DNA vaccine containing Chinese HIV-1 subtype C env gp160, gag, pol and nef/tat genes, as a prime and 2 doses of TBC-M4, a recombinant MVA encoding Indian HIV-1 subtype C env gp160, gag, RT, rev, tat, and nef genes, as a boost in Group A or 3 doses of TBC-M4 alone in Group B participants. Out of 16 participants in each group, 12 received vaccine candidates and 4 received placebos.

Results

Both vaccine regimens were found to be generally safe and well tolerated. The breadth of anti-HIV binding antibodies and the titres of anti-HIV neutralizing antibodies were significantly higher (p<0.05) in Group B volunteers at 14 days post last vaccination. Neutralizing antibodies were detected mainly against Tier-1 subtype B and C viruses. HIV-specific IFN-γ ELISPOT responses were directed mostly to Env and Gag proteins. Although the IFN-γ ELISPOT responses were infrequent after ADVAX vaccinations, the response rate was significantly higher in group A after 1^st and 2^nd MVA doses as compared to the responses in group B volunteers. However, the priming effect was short lasting leading to no difference in the frequency, breadth and magnitude of IFN-γELISPOT responses between the groups at 3, 6 and 9 months post-last vaccination.

Conclusions

Although DNA priming resulted in enhancement of immune responses after 1^st MVA boosting, the overall DNA prime MVA boost was not found to be immunologically superior to homologous MVA boosting.

Trial Registration

Clinical Trial Registry CTRI/2009/091/000051     

Correlation of G/A -22018 single-nucleotide polymorphism with lactase activity and its usefulness in improving the diagnosis of adult-type hypolactasia among North Indian children

Adult-type hypolactasia (AtH or lactase non-persistence) is the physiological decline in lactase activity that manifests in majority of the world’s population after weaning. Recently, various single-nucleotide polymorphisms (SNPs) upstream of lactase gene (LCT) have been suggested to be associated with AtH or the lactase persistent trait in different human populations. C/T -13910 SNP was found be completely associated with AtH in Finnish population, and G/A -22018 SNP was found to be strongly, but not completely, associated with AtH. The aim of this study was to correlate G/A -22018 SNP with intestinal lactase activity in North Indian children. These children were also genotyped for C/T -13910 SNP. We also examined the differences in milk consumption and milk-related clinical symptoms in children with different genotypes of G/A -22018 and C/T -13910 SNPs. Intestinal biopsies were obtained from 231 children aged 2–16 years undergoing routine endoscopy for various abdominal complaints. The biopsies were assayed for lactase, sucrase, and maltase activities and genotyped for G/A -22018 and C/T -13910 SNPs using restriction fragment length polymorphism and DNA sequencing analysis. There was a significant correlation between lactase activity and different genotypes of G/A -22018 SNP. Children with G/G -22018 genotype had low lactase activity. With a reference value of <10 U/g protein (lactase activity) to be indicative of AtH, the sensitivity and specificity of genetic test based on G/A -22018 SNP was 94.4 and 94.1 %, respectively. Furthermore, the consumption of milk was lower in children with G/G -22018 genotype. Flatulence was the only symptom significantly more frequent among the children with G/G -22018 genotype compared to those with G/A and A/A -22018 genotypes. However, most of the children with G/G -22018 genotype seem to tolerate small amounts of milk without any significant difference in gastrointestinal symptoms from those with G/A and A/A -22018 genotypes.     

Proteomic alterations of distinct mitochondrial subpopulations in the type 1 diabetic heart: contribution of protein import dysfunction

Diabetic cardiomyopathy is associated with increased risk of heart failure in type 1 diabetic patients. Mitochondrial dysfunction is suggested as an underlying contributor to diabetic cardiomyopathy. Cardiac mitochondria are characterized by subcellular spatial locale, including mitochondria located beneath the sarcolemma, subsarcolemmal mitochondria (SSM), and mitochondria situated between the myofibrils, interfibrillar mitochondria (IFM). The goal of this study was to determine whether type 1 diabetic insult in the heart influences proteomic make-up of spatially distinct mitochondrial subpopulations and to evaluate the role of nuclear encoded mitochondrial protein import. Utilizing multiple proteomic approaches (iTRAQ and two-dimensional-differential in-gel electrophoresis), IFM proteomic make-up was impacted by type 1 diabetes mellitus to a greater extent than SSM, as evidenced by decreased abundance of fatty acid oxidation and electron transport chain proteins. Mitochondrial phosphate carrier and adenine nucleotide translocator, as well as inner membrane translocases, were decreased in the diabetic IFM (P < 0.05 for both). Mitofilin, a protein involved in cristae morphology, was diminished in the diabetic IFM (P < 0.05). Posttranslational modifications, including oxidations and deamidations, were most prevalent in the diabetic IFM. Mitochondrial heat shock protein 70 (mtHsp70) was significantly decreased in diabetic IFM (P < 0.05). Mitochondrial protein import was decreased in the diabetic IFM with no change in the diabetic SSM (P < 0.05). Taken together, these results indicate that mitochondrial proteomic alterations in the type 1 diabetic heart are more pronounced in the IFM. Further, proteomic alterations are associated with nuclear encoded mitochondrial protein import dysfunction and loss of an essential mitochondrial protein import constituent, mtHsp70, implicating this process in the pathogenesis of the diabetic heart.     

MCT oil-based diet reverses hypertrophic cardiomyopathy in a patient with very long chain acyl-coA dehydrogenase deficiency

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is one of the genetic defects of mitochondrial fatty acid beta-oxidation presenting in early infancy or childhood. If undiagnosed and untreated, VLCAD deficiency may be fatal, secondary to cardiac involvement. We assessed the effect of replacing part of the fat in the diet of a 2 ½-month-old male infant, who was diagnosed with VLCAD deficiency,with medium-chain triglyceride (MCT) oil and essential fats. The patient presented with vomiting, dehydration, and was found to have persistent elevation of liver function tests, hepatomegaly, pericardial and pleural effusion, right bundle branch block, and biventricular hypertrophy. Because of the cardiomyopathy, hepatomegaly, and an abnormal acylcarnitine profile and urine organic acids, he was suspected of having VLCAD deficiency. This was confirmed on acyl-coA dehydrogenase, very long chain (ACADVL) gene analysis. He was begun on an MCT oil-based formula with added essential fatty acids, uncooked cornstarch (around 1 year of age), and frequent feeds. By 7 months of age, cardiomyopathy had reversed and by 18 months of age, all cardiac medications were discontinued and hypotonia had improved such that physical therapy was no longer required. At 5 years of age, he is at the 50^th percentile for height and weight along with normal development. Pediatricians need to be aware about the basic pathophysiology of the disease and the rationale behind its treatment as more patients are being diagnosed because of expansion of newborn screen. The use of MCT oil as a medical intervention for treatment of VLCAD deficiency remains controversial mostly because of lack of clear phenotype-genotype correlations, secondary to the genetic heterogeneity of the mutations. Our case demonstrated the medical necessity of MCT oil-based nutritional intervention and the need for the further research for the development of specific guidelines to improve the care of these patients.     

Atorvastatin inhibited Rho-associated kinase 1 (ROCK1) and focal adhesion kinase (FAK) mediated adhesion and differentiation of CD133CD44 prostate cancer stem cells

Prostate cancer has become a global health concern and is one of the leading causes of cancer death of men after lung and gastric cancers. It has been suggested that the 3-hydroxy-3-methyl-glutarylcoenzyme-CoA (HMG-CoA) reductase inhibitor atorvastatin shows anticancer activity in prostate cancer cell lines. To this end, we analyzed the influence of atorvastatin on the cell adhesion and differentiation of CD133⁺CD44⁺ cells derived from prostate cancer biopsies and peripheral blood. CD133⁺CD44⁺ cells were treated with atorvastatin (16–64 μM) for different time periods. Cell adhesion to endothelial cell monolayers and differentiation into prostate cancer cells were evaluated. α1, β1 and α2β1 integrins adhesion receptors and the downstream target of atorvastatin Rho-dependent kinase (ROCK) and focal adhesion kinase (FAK) were analyzed by Western blot. Further blocking studies with the ROCK inhibitor H1152, anti-FAK antibody and anti-integrin α1 and β1 antibodies were carried out. Atorvastatin treatment inhibited dose-dependently cell attachment to endothelium and differentiation. The inhibitory effect of atorvastatin on cell adhesion was associated with decreased expression of integrins α1 and β1 and phosphorylated MYPT1 and FAK. Furthermore, atorvastatin strongly reduced ROCK1 and FAK mediated differentiation of CD133⁺CD44⁺ cells, which was confirmed by antibody treatment. Atorvastatin modified the expression of cell adhesion molecules and differentiation markers. These beneficial effects of atorvastatin may be mediated by ROCK and FAK signaling pathway. The data presented may point to novel treatment options for prostate cancer.     

Identification and characterization of a mycobacterial NAD+-dependent alcohol dehydrogenase with superior reduction of diacetyl to (S)-acetoin

An enzyme capable of reducing acetoin in the presence of NADH was purified from Mycobacterium sp. B-009, a non-clinical bacterial strain of soil origin. The enzyme is a homotetramer and can be classified as a medium-chain alcohol dehydrogenase/reductase based on the molecular weight of the monomer. Identification of the structural gene revealed a limited distribution of homologous genes only among actinomycetes. In addition to its activity as a reductase specific for (S)-acetoin (EC 1.1.1.76), the enzyme showed both diacetyl reductase (EC 1.1.1.304) and NAD⁺-dependent alcohol dehydrogenase (EC 1.1.1.1) activities. (S)-Acetoin and diacetyl reductases belong to a group of short-chain alcohol dehydrogenase/reductases but do not have superior abilities to dehydrogenate monoalcohols. Thus, the purified enzyme can be readily distinguished from other enzymes. We used the dual functionality of the enzyme to effectively reduce diacetyl to (S)-acetoin, coupled with the oxidation of 1-butanol.     

Determination of common genetic variants in cytidine deaminase (CDA) gene in Indian ethnic population

Cytidine deaminase (CDA) is the major enzyme involved in metabolism of gemcitabine, a pyrimidine analog widely used for chemotherapy of solid tumors. While only low amounts of administered gemcitabine undergo intracellular phosphorylation into active forms and involve in antineoplastic activities, majority of it is rapidly inactivated by CDA and excreted to avoid drug toxicity. Knowledge of the genetic polymorphisms mildly effecting cellular activity of the enzyme CDA is therefore crucial to understanding drug-induced toxicities associated with gemcitabine. Functional significance and allele frequencies for common SNPs including 79A>C (*2) and 208G>A (*3) have been reported in various ethnic populations including Caucasian, African, Korean and Japanese. However, such studies have not been reported in any Indian sub-population. In the present study, conventional polymerase chain reaction (PCR) based amplification using gene specific primers and Sanger sequencing were performed to identify CDA variants in 50 healthy individuals from Indian sub-population. Established common variant 79A>C known to reduce CDA activity was observed at a frequency of 0.14 in the study cohort. In addition to other known variants, one novel variant, c.325−209T>C was detected at a frequency of 0.06. Genetic variants in CDA gene and their frequencies established in our study hold value in pharmacogenetics.