Summary Defects in various erythrocyte membrane proteins genes (ankyrin, band‐3, β‐ and α‐spectrin and protein 4·2) can cause hereditary spherocytosis (HS). This molecular heterogeneity of HS, together with co‐inherited genetic modifiers, results in marked phenotypic variability among patients. We studied the molecular spectrum and genotype‐phenotype correlations in 73 families (with 113 patients) with HS. Deleterious variants including nonsense (42%), deletions (18%), splice site (20%), missense (10%) and duplication/insertion (10%) were found in 47 patients. The variants detected included sporadic and dominantly‐inherited defects in ANK1 (53·2%), SPTB (36·2%) and SLC4A1 (4·2%). Compound heterozygous variants in SPTA1 (6·4%) showed autosomal recessive inheritance. Alpha‐spectrin variants were associated with severe anaemia and splenectomy alleviated symptoms. Co‐inherited glucose‐6‐phosphate dehydrogenase (G6PD) deficiency was found in 15%. G6PD variants (n = 5) led to greater transfusion requirements (1‐8 times) in males with HS. Homozygosity (41%) for the promoter variant of UGT1A1 (Gilbert syndrome) led to a significantly higher mean bilirubin level (126·54 µmol/l) with a higher frequency of cholelithiasis (30%) (P < 0·001). This first‐ever south Asian study on the molecular spectrum of HS found ANK1 and SPTB genes variants to be the commonest with inheritance being sporadic/dominant. Next‐generation sequencing provided a relatively sensitive and rapid tool for molecular diagnosis with a diagnostic yield of 64·4%.

To study the effect of vitamin E (VE), copper (Cu) and zinc (Zn) supplementation on the in vitro phagocytic activity (PA) and lymphocyte proliferation response (LPR) of blood neutrophils and lymphocytes, thirty Sahiwal pregnant cows (six in each group) in their late gestation at 30 days before the expected date of calving were selected from the NDRI experimental herd and supplemented with various micronutrients from 30 days before calving to 45 days after calving. Cows were supplemented individually with VE (1000 IU/cow/day), Cu (20 ppm/cow/day) and Zn (80 ppm/cow/day) and also with a combination of VE, Cu and Zn to study cumulative effect of all micronutrients. One group without any supplementation acted as a control. Blood neutrophils and lymphocytes were isolated and studied for their PA and LPR. Supplementation of micronutrients like VE, Cu, Zn and a combination of all these nutrients significantly (p < 0.01) increased the PA of experimental cows as compared to control (unsupplemented) cows during the pre-partum period. During post-partum, all the micronutrients (VE, Cu, Zn and their combination) showed a significant (p < 0.01) increase in the PA of experimental cows as compared to control cows. Of all the groups, significant (p < 0.01) and maximum PA was observed in the combination group followed by Zn-supplemented group during both the pre- and post-partum period. A significant (p < 0.01) increase in LPR of B lymphocytes was observed in combination-supplemented group during the pre-partum period and during both the pre- and post-partum period in the Cu-supplemented group.

Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk-secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI-TOF/TOF MS and 1D-Gel-LC-MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT-PCR and Western blotting. The 1D-Gel-LC-MS/MS and 2DE-MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC-specific proteins that will help the researchers to understand the molecular events taking place during lactation.

EMA assay was efficient to diagnose cases of HS including postsplenectomy cases and shows no overlap with IDA, βTT, and AIHA.

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Reference genes are indispensable for normalizing mRNA levels across samples in real-time quantitative PCR. Their expression levels vary under different experimental conditions and because of several inherent characteristics. Appropriate reference gene selection is thus critical for gene-expression studies. This study aimed at selecting optimal reference genes for gene-expression analysis of reticulocytes and at validating them in hereditary spherocytosis (HS) and β-thalassemia intermedia (βTI) patients. Seven reference genes (PGK1, MPP1, HPRT1, ACTB, GAPDH, RN18S1, and SDHA) were selected because of published reports. Real-time quantitative PCR was performed on reticulocytes in 20 healthy volunteers, 15 HS patients, and 10 βTI patients. Threshold cycle values were compared with fold-change method and RefFinder software. The stable reference genes recommended by RefFinder were validated with SLC4A1 and flow cytometric eosin-5'-maleimide binding assay values in HS patients and HBG2 and high performance liquid chromatography-derived percentage of hemoglobin F in βTI. Comprehensive ranking predicted MPP1 and GAPDH as optimal reference genes for reticulocytes that were not affected in HS and βTI. This was further confirmed on validation with eosin-5'-maleimide results and percentage of hemoglobin F in HS and βTI patients, respectively. Hence, MPP1 and GAPDH are good reference genes for reticulocyte expression studies compared with ACTB and RN18S1, the two most commonly used reference genes.