Characterization of a Double Heterozygote HbE/ß(+) Thalassemia IVS 1-1 [G>T] in a Juvenile Diabetic.

The present case report describes the molecular and proteomic based study of Hb variant HbE associated with ß(+) thalassemia IVS 1-1 G>T, in a juvenile diabetic patient. Given the ethnic origin and mobility of the variant hemoglobin at alkaline pH, HbE would be suspected. But hematologically and clinically abnormality being detected, HPLC and Electrophoresis not being able to characterize due to retention time and band being in region of HbA2, respectively, further characterization of hemoglobinopathy was made using MALDI and IVS 1-1 G>T being validated by reverse dot blot hybridization. Capillary electrophoresis was also employed in order to separate HbE and HbA2 bands. This case report being first of its kind, wherein a HbE/ß(+) thalassemia has been characterized using multiple techniques.

Identification of a Rare Hemoglobin Variant HbJ-Rajappen [alpha90 (FG2) Lys → Thr] Using Mass Spectrometry.

Hemoglobin J-Rajappen (alpha)90 Lys â†’ Thr is an alpha chain variant found in heterozygous state and presents normal hematological blood picture. Due to the ambiguity in results obtained while analyzing by HPLC and alkaline gel electrophoresis, we report this rare case of HbJ-Rajappen using non denaturing gel electrophoresis and matrix assisted laser desorption ionization mass spectrometry. Though HbJ-Rajappen has earlier been reported using different techniques, this is the first report being validated using mass spectrometry technique.

Co-inheritance of HbD (Iran)/Beta Thalassemia IVS1-5 (G > C) Trait in a Punjabi Lady with Diabetes.

The present report describes the molecular study of HbD (Iran) (beta) 22 Glu â†’ Gln associated with ß-Thalassemia IVS1-5 (G > C) found in India, and the first case in which mutation has been identified using mass spectrometry. Given the apparent ethnic origin and the mobility of the variant hemoglobin at alkaline pH, hemoglobin D-Punjab would be suspected, but HPLC excluded this possibility. Further characterization of hemoglobinopathy was made by using nondenaturing gel electrophoresis and matrix assisted laser desorption ionization mass spectrometry and IVS1-5 being validated by reverse dot blot hybridization followed by sequencing of the ß-globin gene.

Diagnostic Dilemma of HbA1c Detection in Presence of a Hemoglobinopathy: A Case Report.

We report a case of a diabetic, heterozygote with near normal hematology, marginally low level of hemoglobin A(2)(HbA(2)) having an increased level of hemoglobin F(HbF) that was pancellularly distributed among the red cells. BioRad DiaSTAT measurements gave a high glycated hemoglobin A1c(HbA1c) of 31.5% and the BioRad Variant analyzer recorded an HbA1c value which was very low, in discordance with the detected blood glucose levels. Flow cytometry and polymerization chain reaction (PCR) based studies were carried out which revealed the case to be that of the common hereditary persistence of fetal hemoglobin (HPFH)-3, an Asian Indian mutation. Fructosamine estimation and HbA1c by Boronate affinity chromatography were able to resolve the discordant value detected and was able to confirm the diabetes status. The case would have been a diagnostic dilemma, if reported without correlation.

The Diagnosis of α-Thalassaemia: A Case of Hemoglobin H -α Deletion.

We report a case of hemolytic anemia that was subsequently identified to be a case of α-thalassaemia harboring the common rightward 3.7 kb deletion/HbH. The diagnosis was based on sequential analyses using BioRad D10 HPLC, Alkaline gel electrophoresis, GPO α THAL-IC strips and the identification of the specific genetic lesion using an α Globin reverse dot blot hybridization assay. Supravital stain of RBCs helped in identifying classical HbH inclusions. In a background of a variable clinical presentation, lack of definitive hematological markers, and general under-diagnosis of α-thalassaemias we have used this case to highlight the features and sequence of techniques involved in identifying and characterizing an α-globin chain mutation, starting from a diffuse clinical history and presentation up to the identification of a specific genetic lesion involved.

Rho kinase inhibitor y27632 alters the balance between pluripotency and early differentiation events in human embryonic stem cells.

Human embryonic stem cells (hESC) differentiate spontaneously in culture and develop a complex microenvironment comprising of autologously derived niche that in turn supports their pluripotency. The basic hypothesis that we deal with is that hESCs undergoing differentiation, sequentially generate trophectoderm and endoderm lineages and thereafter influence further events through the production of growth factors. These factors control the fate of hESCs either by promoting or retarding the recruitment of new cells in the differentiation program. This scenario therefore represents an analog of the in vivo situation in which extra-embryonic tissues influence the behavior of the inner cell mass (ICM). The premise of the paper is the Rho kinase inhibitor Y27632 that can spatiotemporally alter this balance between pluripotency and differentiation. To evaluate the composition and inclination of lineage specification during spontaneous differentiation, we have studied the hESC colonies and their surrounding niche as interdependent entities. We show that the population of fibroblastic niche that surrounds hESC colonies co-expresses trophectoderm and niche cell markers including SSEA1, hCG, progesterone, HAND1, pSmad1 and FGFR1 as early as day 4. A sudden increase in the expression of GATA4 and AFP secretion indicated putative endoderm formation on day 6 in both control and Y27632 treated cultures. On day 6, 20 microM of Y27632 supplementation significantly reduced the trophectoderm-like niche population without affecting endoderm formation, enhanced the average size and number of hESC colonies, decreased IGF1 secretion thereby improving the pluripotency. Overall our findings support the afore mentioned hypothesis and demonstrate that closely packed epithelial trophectoderm-like cells bordering the hESC colonies present an initial and imminent localized niche which is spatiotemporally regulated. Such advances in understanding the behavior and modulation of hESC and its surrounding niche would facilitate better differentiation protocols for applications in regenerative medicine and drug screening.

Characterization of a hemoglobin variant: HbQ-India / IVS 1-1 [G>T]-ß-thalassemia.

Hemoglobin Q- India (alpha) 64 Asp → His is an alpha chain variant which is generally found in heterozygous state and presents normal hematological blood picture. Here we report a rare case of HbQ-India with a thalassemic phenotype that has been analyzed using a combination of mass spectrometry, gene sequencing and PCR analysis. This combined analyses revealed the HbQ variant to be associated with a beta chain mutation, IVS 1-1 [G>T]. Though HbQ has earlier been reported with thalassemic trait using different techniques, this is the first report of a compound α and ß chain Hb heterozygous mutant involving HbQ and IVS1-1 being validated using Mass Spectrometry and Reverse dot blot hybridization.

Propensity of human embryonic stem cell lines during early stage of lineage specification controls their terminal differentiation into mature cell types.

Human embryonic stem cells (hESCs) are able to stably maintain their characteristics for an unlimited period; nevertheless, substantial differences among cell lines in gene and protein expression not manifested during the undifferentiated state may appear when cells differentiate. It is widely accepted that developing an efficient protocol to control the differentiation of hESCs will enable us to produce adequate numbers of desired cell types with relative ease for diverse applications ranging from basic research to cell therapy and drug screening. Hence of late, there has been considerable interest in understanding whether and how hESC lines are equivalent or different to each other in their in vitro developmental tendencies. In this study, we compared the developmental competences of two hESC lines (HUES-9 and HUES-7) at molecular, cellular and functional levels, upon spontaneous differentiation without any added inducing agents. Both cell lines generated the three embryonic germ layers, extra-embryonic tissues and primordial germ cells during embryoid body (EB) formation. However HUES-9 showed a stronger propensity towards formation of neuroectodermal lineages, whereas HUES-7 differentiated preferentially into mesoderm and endoderm. Upon further differentiation, HUES-9 generated largely neural cells (neurons, oligodendrocytes, astrocytes and gangliosides) whereas HUES-7 formed mesendodermal derivatives, including cardiomyocytes, skeletal myocytes, endothelial cells, hepatocytes and pancreatic cells. Overall, our findings endorse the hypothesis that independently-derived hESCs biologically differ among themselves, thereby displaying varying differentiation propensity. These subtle differences not only highlight the importance of screening and deriving lines for lineage-specific differentiation but also indicate that individual lines may possess a repertoire of capabilities that is unique.

Electrospray mass spectrometric characterization of hemoglobin Q (Hb Q-India) and a double mutant hemoglobin S/D in clinical samples.

OBJECTIVES: The clinical analysis of hemoglobin by ion exchange chromatography can result in ambiguities in identification of the nature of the globin chain present in patient samples. LC/ESI-MS provides rapid and precise determination of globin chain masses. DESIGN AND METHODS: Hemolysate of hemoglobin Q-India and hemoglobin S/D/F have been analyzed using ESI-MS. Tandem-MS has been used to establish mutation in alpha chain of hemoglobin Q. RESULTS: The identification of hemoglobin Q-India is readily achieved by LC/ESI-MS, which establishes the presence of a mutant alpha chain differing in mass from normal alpha chain by 22 Da. The site of mutation has been identified by tandem-MS analysis of a tryptic fragment encompassing residues alphaV62-K90. LC/ESI-MS screening has also provide an example of simultaneous occurrence of mutant globin chains containing beta6E-->V (Hb S, sickle) and beta121E-->Q (Hb D) variant. Expression of gamma(G) globin chain is also demonstrated in this sample. CONCLUSIONS: The site of mutation in hemoglobin Q-India is identified as alpha64D-->H which differs from mutations alpha74D-->H in Hb Q-Thailand and alpha75D-->H in Hb Q-Iran. Mass spectrometric analysis of hemoglobins from a patient and her parents suggests inheritance of mutant beta globin genes from both parents.