An anti-oxidant, α-lipoic acid conjugated oleoyl-sn-phosphatidylcholineas a helper lipid in cationic liposomal formulations.

Development of safe non-viral carrier systems for efficient intra-cellular delivery of drugs and genes hold promise in the area of translational research. Liposome based delivery systems have emerged as one of the attractive strategies for efficient delivery of drugs and nucleic acids. To this end, number of investigations was carried on liposomal formulations using lipids for achieving higher efficiency in transfection with lower cytotoxicities. In our efforts to develop safer and efficient liposomal delivery systems, we synthesized a novel anti-oxidant lipid, α-lipoyl, oleyl-sn-phosphatidylcholine (LOPC) and used as a helper lipid in combination with a cationic amphiphile, Di-Stearyl Dihydroxy Ethyl Ammonium Chloride (DSDEAC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at varying concentrations of LOPC. DNA binding properties of the liposomal formulations (DS, DS LA1, DS LA2 and DS LA3) revealed that increasing the percentage of single aliphatic chain lipid LOPC, did not affect the DNA binding properties. But, transfection profiles of these liposomal formulations in 3 different cell lines (HeLa, HEK 293 and MCF7) showed difference in their efficacies. Results showed that optimal percentage of LOPC i.e. 25% in DSDEAC and DOPC at 1:1 molar ratio (DS LA1) enhanced transfection as compared to DSDEAC:DOPC alone. The endosomal escape studies with NBD labelled lysotracker and Rhodamine labelled liposomal formulations revealed that DS LA1 and DS LA2 facilitated the release of genetic cargo with a better efficiency than their counter parts. Reactive Oxygen Species (ROS), a key modulator of necroptosis were lowered with the treatment of DS LA1 than other liposomal formulations. Here in, we present a novel liposomal formulation using DSDEAC and DOPC at 1:1 molar ratio doped with 25-50% (mole ratio) LOPC as an efficient delivery system for enhanced transfection with quenching of ROS levels compared to formulations without LOPC.

Leukocyte Adhesion Deficiency-I: Clinical and Molecular Characterization in an Indian Population.

OBJECTIVE: To describe clinical and flow cytometric immunophenotyping details of 26 patients of Leukocyte adhesion deficiency-I (LAD-I) along with molecular characterization of 7 patients. METHODS: Diagnosis of LAD-I was suspected on the basis of clinical features, white blood cell count and absolute neutrophil counts and flow cytometric assessment of expression of CD18 and CD11(a, b, c) on leukocytes. Mutation analysis was performed using DNA PCR and conformation sensitive gel electrophoresis (CSGE) technique followed by sequencing. RESULTS: All the patients were symptomatic by the age of 6 mo, with history of recurrent bacterial infections involving skin, mucosa or umbilical cord (omphalitis) being the most frequent presenting symptoms. White blood cells (WBC) and absolute neutrophil counts (ANC) were markedly elevated, without any specific morphological findings. On flow cytometry, CD11a and CD11c showed moderate correlation with CD18 expression. Mutation analysis was performed in 7 patients and six different mutations (4 missense, 2 nonsense and 1 splice site) were identified, all of which were homozygous in nature. CONCLUSIONS: A presentation of repeated bacterial infections during infancy, especially omphalitis, with markedly elevated absolute neutrophil counts should trigger investigations for LAD-I including flow cytometric analysis of CD11/CD18 expression.

Carbonyl reductase 1 expression influences daunorubicin metabolism in acute myeloid leukemia.

PURPOSE: The present study aimed to investigate the role of expression of daunorubicin-metabolizing enzymes carbonyl reductase 1 and 3 (CBR1 and CBR3) on the in vitro cytotoxicity of daunorubicin in primary acute myeloid leukemia (AML) cells and the effect of genetic variants in CBR1 and CBR3 on the plasma pharmacokinetics of daunorubicin and daunorubicinol (DOL) in AML patients. METHODS: RNA expression of CBR1 and CBR3, intracellular daunorubicin and DOL levels, and in vitro cytotoxicity of daunorubicin were measured in bone marrow mononuclear cells of 104 adult AML patients. Plasma pharmacokinetics of daunorubicin and DOL was measured in 24 patients receiving daunorubicin-based induction chemotherapy for AML. RESULTS: Increased expression of CBR1 significantly reduced the in vitro cytotoxicity of daunorubicin and also positively correlated with intracellular DOL levels. Polymorphisms in CBR1 and CBR3 did not show any association with intracellular daunorubicin or DOL levels, but there was a trend towards significant increase in plasma daunorubicin systemic exposure in patients with a variant genotype for CBR1 polymorphism rs25678. CONCLUSIONS: This pilot study suggests that CBR1 RNA expression may be helpful in identifying AML patients at risk of developing resistance or toxicity to daunorubicin due to increased formation of DOL. Further confirmation of these findings in a larger sample pool would be required to determine the applicability of these results. Inhibition of CBR1 can be an option to improve the efficacy and prevent toxicity related to the treatment. Influence of daunorubicin and DOL plasma levels on clinical outcome, if any, remains to be evaluated.

A novel ß-globin gene mutation HBB.c.22 G>C produces a hemoglobin variant (Hb Vellore) mimicking HbS in HPLC.

Hemoglobinopathies are highly prevalent in Indian population. DNA analysis to detect causative mutations is required for identifying rare hemoglobin variants or when hematological results are discordant with the clinical phenotype. In this report, we describe a novel hemoglobin variant caused by a mutation in beta-globin gene, Codon 7 GAG→CAG (Glu→Gln) that elutes in the position of sickle haemoglobin (HbS) in cation exchange high performance liquid chromatography. This report highlights possible diagnostic pitfalls in interpreting data solely based on haemoglobin analysis and usefulness of mutation screening in definitive diagnosis of hemoglobinopathies.

A novel δ-globin gene mutation (HBD: c.323G>A) masking the diagnosis of ß-thalassemia: a first report from India.

An elevated HbA(2) (α2δ2) level (>3.5%) is a well-established diagnostic test for heterozygous ß-thalassemia. Mutations in the δ-globin gene can cause decreased expression of HbA(2), resulting in heterozygous ß-thalassemia with normal levels of HbA(2). In this report, we describe a novel missense mutation in δ-globin (HBD: c.323G>A, Gly > Asp) in an Indian family with heterozygous ß-thalassemia with normal HbA(2) levels.

Polymorphism in factor VII gene modifies phenotype of severe haemophilia.

The basis for 10-15% of patients with severe haemophilia having clinically mild disease is not fully understood. We hypothesized that polymorphisms in various coagulant factors may affect frequency of bleeding while functionally significant polymorphisms in inflammatory and immunoregulatory genes may also contribute to variations in the extent of joint damage. These variables were studied in patients with severe haemophilia, who were categorized as 'mild' (<5 bleeds in the preceding year, <10 World Federation of Haemophilia clinical and <10 Pettersson scores, n = 14) or 'severe' (all others, n = 100). A total of 53 parameters were studied in each individual for their association with the clinical severity. Age, F8:c activity and the incidence of thrombotic markers were comparable between the groups while the median number of bleeds, number of affected joints, clinical, radiological and functional joint scores (P < or = 0.001) and life-time clotting factor use (P < or = 0.007) were different. Patients with severe molecular defects had a 4.1-fold increased risk for a severe phenotype (95% CI: 1.18-14.42, P = 0.026) compared with other mutations. Of the polymorphisms studied, the FVII353Q (RR = 3.5, 95% CI: 1.04-12.05, P = 0.044) allele was associated with a severe phenotype. This data shows that apart from the F8/F9 genotype, functional polymorphisms in FVII gene affect the phenotype of patients with severe haemophilia.

Genotype phenotype correlation in Wilson's disease within families--a report on four south Indian families.

AIM: To study the genotype phenotype correlation in Wilson's disease (WD) patients within families. METHODS: We report four unrelated families from South India with nine members affected with WD. Phenotype was classified as per international consensus phenotypic classification of WD. DNA was extracted from peripheral blood and 21 exons of ATP7B gene and flanking introns were amplified by polymerase chain reaction (PCR). The PCR products were screened for mutations and the aberrant products noted on screening were sequenced. RESULTS: Four separate ATP7B mutations were found in the four families. ATP7B mutations were identical amongst affected members within each family. Three families had homozygous mutations of ATP7B gene while one family had compound heterozygous mutation, of which only one mutation was identified. We noted concordance between ATP7B gene mutation and Wilson's disease phenotype amongst members within each family. The age of onset of symptoms or of detection of asymptomatic disease, baseline serum ceruloplasmin and baseline urinary copper levels were also similar in affected members of each family. Minor differences in phenotype and baseline serum ceruloplasmin level were noted in one family. CONCLUSION: We report concordance between ATP7B mutation and WD phenotype within each family with > 1 member affected with WD. Homozygous ATP7B mutation was present in 3 of the 4 families studied. Our report supports allelic dominance as a determinant of WD phenotype. However, in one family with compound heterozygous mutation, there was a similar WD phenotype which suggests that there may be other factors determining the phenotype.

Analysis of beta globin mutations in the Indian population: presence of rare and novel mutations and region-wise heterogeneity.

Beta thalassaemia is a major public health problem in India. A comprehensive database of the spectrum of mutations causing beta thalassaemia in the Indian population is necessary. This study in which a large number of patients with beta thalassaemia including those from certain regions that were not explored earlier shows a great heterogeneity of mutations. Several novel and rare alleles that have not been reported earlier in the Indian population have been identified, and mutations differ in frequency in different regions of the country. This information on the spectrum of mutations has implications for the control of beta thalassaemia in a population with complex ethnic background and also on the genotype-phenotype correlation of the disease.

Developing an algorithm of informative markers for evaluation of chimerism after allogeneic bone marrow transplantation.

Analysis of chimerism by polymerase chain reaction amplification of STR or VNTR has become a routine procedure for the evaluation of engraftment after allogeneic stem cell transplantation. Knowledge of the frequency of different STR or VNTR alleles in unrelated individuals in a population is useful for forensic work. In the context of HLA identical sibling bone marrow transplantation the informativeness of these markers needs to be evaluated. We evaluated five STRs (THO1, VWA, FES, ACTBP2, and F13A1) and 1 VNTR (APOB) for informativeness in stem cell transplants from HLA identical sibling donors. All four markers used individually allowed us to discriminate 20-56% of the patient donor pairs. Using a combination of all these markers along with a polymorphic marker in the beta-globin gene and the sex chromosome specific amelogenin marker, we were able to discriminate 99% of the patient donor pairs. We have established an algorithm for evaluating chimerism following HLA identical sibling donor transplants in the Indian population using molecular markers in 310 patients. Analysis of heterozygote frequencies in different populations is similar suggesting that this algorithm can be used universally for transplant centers to evaluate chimerism following allogeneic bone marrow transplantation.

ATP7B mutations in families in a predominantly Southern Indian cohort of Wilson's disease patients.

OBJECTIVE: To analyze ATP7B mutations in Wilson's disease (WD) patients from the Indian subcontinent and to correlate these with WD phenotype. METHODS: We studied 27 WD patients from 25 unrelated families. Twenty-two families were from three southern Indian states - Tamil Nadu andhra Pradesh and Kerala. We applied conformation- sensitive gel electrophoresis (CSGE) to screen for the mutations in patients and their families. PCR products exhibiting aberrant patterns in CSGE were subjected to direct DNA sequencing. As siblings affected by WD within a family share identical ATP7B genotype, we compared WD phenotype among affected siblings within families. RESULTS: ATP7B mutations were detected in 22 of the 25 probands -13 were homozygotes and 9 were compound heterozygotes. Eleven novel mutations were detected. Only two common mutations were found: G3182A in 4 (16%) and C813A in 3 (12%) probands. 'Hot spots' for ATP7B mutations were exons 18 and 13. Lack of common dominant mutations prevented correlation of individual ATP7B mutations with WD phenotype. Symptomatic WD in a live sibling was not found in any family. In 8 families, a sibling died of presumed WD - in 6 of these, WD phenotype was identical to that in the proband. CONCLUSIONS: We describe the spectrum of ATP7B mutations including 11 novel mutations in Indian WD patients and document lack of a single dominant mutation. Identical WD phenotype among siblings in only 6 of 8 families with >1 child affected by WD suggests that factors other than ATP7B mutations influence WD phenotype.

Investigation of persistent hypochromic microcytosis unmasks hemoglobin Evanston [alpha 14 (A12) Try--> Arg] in a patient of cyclic thrombocytopenia preceding Takayasu's disease.

We report an unusual north Indian patient with Hemoglobin Evanston [alpha 14 (A12) Try --> Arg] who had acquired cyclic thrombocytopenia (10-1230 x 10(9)/l periodic oscillation of four week duration) which recovered without any specific therapy. She later developed Takayasu's disease and underwent three corrective stents. She is presently in clinical remission and is on regular follow up. To the best of our knowledge our patient is the first report of Hb Evanston from the indigenous population of India and highlights the need to look for point mutations in the alpha globin gene, which may interact with thalassemia or other hemoglobinopathies, in atypical cases. The association of these three disorders in our patient is possibly unrelated though an immune basis for the cyclic thrombocytopenia and Takayasu's disease is likely as seen in this report.

Cytochrome P4501A1 and glutathione S transferase gene polymorphisms in patients with aplastic anemia in India.

The etiology of acquired aplastic anemia (AA) in most patients remains unclear. It is believed that patients with a reduced ability to detoxify environmental toxins are at increased risk of developing AA. Cytochrome P450 (CYP450) and glutathione S transferase (GST) are the major phase I and phase II xenobiotic-metabolizing enzymes. We analyzed the impact of the polymorphisms in CYP4501A1 and GSTM1 and GSTT1 genes on the susceptibility and disease severity in 200 patients with AA and compared the frequency with the normal population. There was a significantly increased frequency of the CYP1A1m4 allele in AA patients compared with normal controls (odds ratio = 3.01; 95% confidence interval 1.76-5.17; p = 0.00001). None of the other CYP1A1 genotypes or the GST genotypes were significantly different between AA patients and controls. Altered metabolism of benzo(a)pyrene due to the polymorphism in the CYP1A1 gene might be an etiologic factor in the increased incidence of AA in these patients. The CYP1A1m4 allele may play a role in determining the risk of AA in India.

Identification of factor VIII gene mutations in 101 patients with haemophilia A: mutation analysis by inversion screening and multiplex PCR and CSGE and molecular modelling of 10 novel missense substitutions.

Haemophilia A (HA) is an X-linked bleeding disorder caused by diverse mutations in the human coagulation factor VIII (FVIII) gene. We have analysed DNA from 109 unrelated Indian patients with HA for their FVIII gene defects. Among these patients 89 (82%) had severe (FVIII:C <1%) HA, 11 (10%) had moderate (FVIII:C 1-5%) HA and nine (8%) had mild (FVIII:C 5-30%) HA. These patients were first screened for the common intron 22 and intron 1 inversions. Inversion negative samples were screened for point mutations by a multiplex PCR and conformation sensitive gel electrophoresis strategy. Mutations were identified in 101 of the 109 patients. These included two (2%) intron 1 and 51 (51%) intron 22 inversions, four (4%) gross deletions and 44 (43%) point mutations. Twenty-nine novel causative mutations, including 11 missense, seven frameshift, five nonsense mutations, three splice site defects and three gross deletions were detected. Ten of the novel missense mutations were studied by molecular modelling. Two different (Thr2253Pro and Pro1392fs) mutations were seen in four unrelated families and FVIII gene haplotyping suggested a common founder effect. Seven of these 109 patients had inhibitors. Among them, four had intron 22 inversions, one had a novel gross deletion (delexon 2-9) and one a nonsense mutation (Trp1535Stop). In one of these patients, no mutation could be identified in the FVIII gene. A Thr2253Pro novel mutation and an intron 22 inversion were identified in two female haemophiliacs. The data from this study suggests that the spectrum of gene defects in Indian patients with HA is as heterogeneous as reported in other populations.

Molecular genetics of hereditary prothrombin deficiency in Indian patients: identification of a novel Ala362 --> Thr (Prothrombin Vellore 1) mutation.

Prothrombin deficiency is a rare (1:200 000) autosomal recessive disorder caused by diverse mutations in prothrombin gene. We have studied the molecular basis of this disorder in four unrelated Indian patients. The diagnosis was based on prolonged prothrombin (PT) and activated partial thromboplastin times and low factor II coagulant activity (FII: C) measured using a PT based assay. FII: C levels ranged between 4.7% and 17.5%. Mutations were identified in all the four patients. Five different causative mutations including four (80%) missense and an in-frame deletion (20%) were identified. One of them was a novel, Ala362 --> Thr amino acid change affecting 'B' chain of -thrombin. This mutation was present in a compound heterozygous state with a previously reported Arg-1 --> Gln missense change affecting pro-peptide cleavage site. Ala362 --> Thr occurred at a codon, evolutionarily conserved in all the 24 different prothrombins or its related serine proteases studied. Molecular modeling of this mutation was found to cause a conformational change around the region involving a catalytic triad residue His363 and a cysteine residue at codon 364. The FII: C level in this patient was 17.5%. Three other previously reported mutations were also detected in the homozygous state: Arg271 --> Cys in Kringle-2 region, a Glu309 --> Lys in "A" chain of -thrombin and an in-frame deletion of 3 bp (AAG) leading to Del Lys301/302 in "A" chain of -thrombin. This is the first report of the molecular basis of prothrombin deficiency in Indian patients and we suggest the eponym 'Prothrombin Vellore 1' for Ala362 --> Thr mutation.

Six novel mutations including triple heterozygosity for Phe31Ser, 514delT and 516T-->G factor X gene mutations are responsible for congenital factor X deficiency in patients of Nepali and Indian origin.

Factor X (FX) deficiency is a rare (1 : 100000) autosomal recessive disorder caused by heterogeneous mutations in FX gene. We have studied the molecular basis this disease in six Indian and one Nepali patients. Diagnosis was confirmed by measuring the FX coagulant activity (FX: C) using a PT based assay. Six of them had a FX: C of < 1% and one patient had 24% coagulant activity. Mutations were identified in all the seven patients. These included eight (88.8%) missense and one frame-shift (11.2%) mutations of which six were novel. Three of the novel mutations, a Phe31Ser affecting 'Gla' domain and 514delT and 516T-->G mutations affecting Cys132 in 'connecting region' were identified in a triple compound heterozygous state in a Nepali patient presenting with a severe phenotype. Two other novel mutations, Gly133Arg, may affect the disulphide bridge between Cys132-Cys302 in the connecting region while Gly223Arg may perturb the catalytic triad (His236, Asp282 and Ser379). The other novel mutation, Ser354Arg, involves the replacement of a small-buried residue by a large basic aminoacid and is likely to have steric or electrostatic effects in the pocket involving Lys351-Arg347-Lys414 that contributes to the core epitope of FXa for binding to FVa. Three previously reported mutations, Thr318Met; Gly323Ser; Gly366Ser were also identified. This is the first report of the molecular basis of FX deficiency in patients from the Indian subcontinent.