Search

In this study, we analysed the mutation spectrum in subjects with suspected lipodystrophy using a targeted Next-generation sequencing (NGS) approach. Subjects with suspected lipodystrophy were for screened six genes (AGPAT2, BSCL2, LMNA, PPARG, ZMPSTE24, INSR) and the variants identified were confirmed through Sanger sequencing. The clinical and biochemical parameters were compared among the mutation positive and negative subjects. We identified eight individuals with pathogenic or likely pathogenic mutations, including both homozygous and heterozygous variants. Homozygous variants included  AGPAT2(NM_006412.4):c.493-2A>G, AGPAT2(NM_006412.4):c.254_258dup, and BSCL2(NM_001122955.4):c.570del, while heterozygous variants encompassed LMNA(NM_170707.4):c.1444C>T, LMNA(NM_170707.4):c.1456A>G, LMNA(NM_170707.4):c.1445G>A, and PPARG(NM_015869.5):c.949T>C mutations. In this cohort, three subjects were diagnosed with congenital generalized lipodystrophy, while the remaining five had familial partial lipodystrophy. Majority (7/8) of the patients with lipodystrophy had hepatic involvement. Notably, more than half of the subjects (5/8) achieved optimal glycemic control through insulin sensitizers (PPARγ agonist and Metformin). Interestingly, even with a limited gene panel test, mutation-positive individuals exhibited a higher prevalence of typical clinical features and biochemical characteristics associated with lipodystrophy compared to their mutation-negative counterparts. In subjects with lipodystrophy, targeted NGS based screening may establish a genetic diagnosis and aid in family screening and genetic counselling. Knowing the clinical and biochemical features typical to lipodystrophy may help in diagnosis especially in resource limited setting., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Bone marrow stromal cell antigen 2 (BST2) is an interferon induced host restriction factor for HIV-1 that blocks the release of nascent virions from infected T cells. We aimed to characterize BST2 gene variants in HIV-1 positive individuals in Indian cohort and study the association of these variants with disease progression in long term non progressors (LTNPs) and progressors. Archived samples of 32 LTNPs, 17 progressors, and 78 controls were screened for BST2 gene polymorphisms using Sanger's sequencing method. Frequency distribution, survival analysis and bioinformatics tools were used to study the association of BST2 variants with disease progression. Eighteen variants of BST2 gene were observed in Indian cohort. Intronic SNP rs919267T/C (OR = 4.489 [0.8494-27.03], p = .04157) and exonic SNP rs13485C/G (OR = 3.887 [0.8262-25.56], p = .0488) were found to be significantly associated with disease progression. Also, rs13485C/C genotype in combination with rs919267C/T (OR = 9.406 [1.384-111], p = .0085) and rs145303329 Δ19bp (OR = 3.887 [0.826-25.5], p = .048) were found to be significantly associated with disease progression. 19 bp indel rs145303329 and its allele c.1-443_1-442insCGCCCCCAGAC[C/T]CAGGCCC from BST2 promoter also showed association with disease progression (OR = 12.97 [0.9731-850.5], p = .026). Docking of AP2 repressor with above allele showed the total binding energy of LTNPs and progressors to be -2581.42 kcal/mol and -3563.27/-3562.84 kcal/mol respectively. We have identified the novel association of three BST2 gene SNPs; rs919267, rs13485 and indel rs145303329 from Indian cohort to be associated with the risk of HIV-1 disease progression for the first time., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: The presence of specific chemotherapeutic protocols for hairy cell leukemia (HCL) makes it essential to discriminate this entity from other lymphoproliferative disorders. Hence, awareness of the variations in clinical presentations and immunophenotypic aberrancies is requisite to ensure diagnostic accuracy., Materials and Methods: A retrospective study was carried out to analyze the clinical-pathological profile of patients with HCL diagnosed over a period of 81 months (2010-September 2017) in our institute. Flow cytometry was performed in all the patients, and further, BRAFV600E mutation analysis was performed by real-time polymerase chain reaction in a limited number of samples., Result: A total of 353 lymphoproliferative disorders were assessed during the period, of which 16 (4.5%) were diagnosed as HCL, which included 15 cases of classical HCL and single case of HCL-v. Striking male predominance was noted with a median age of 52 (range 22-90 years). 47% patients presented with pancytopenia, while 20% cases had leukocytosis. Three patients presented with bleeding diathesis in the form of melena and purpuric spots. The absence of splenomegaly was observed in 20% patients (4/15) while 2 (13.3%) cases had lymphadenopathy. Hypocellular marrow was observed in 13% cases. Bright expression of CD20/CD22 along with CD25/CD103/CD123/CD11c was noted in all the patients of classical HCL. Aberrant expression of CD23 and CD5 was seen in 33% ( n =5) and 6.7% ( n =1) cases respectively. CD200 was positive in all the 5/15 cases tested. The case of HCL-v presented with very high leukocyte count and exhibited a CD103/CD11c+ and CD123/CD25- profile. BRAFV600E, mutation was present in all the four patients tested who included patients with a hypocellular marrow and absent splenomegaly., Conclusion: HCL has characteristic profiles, yet it may exhibit unusual clinical and immunophenotypic presentations. Perspicacious use of flow cytometry and BRAFV600E mutation analysis will aid in the diagnosis in unprecedented cases., Competing Interests: None

Background: Acute leukemia is a heterogenous disease having diverse phenotypes. Immunophenotyping by flowcytometry is essential for diagnosis of myeloid and lymphoid subtypes. Aberrant phenotype incidence is controversial and dissimilar results have been reported by different groups., Objectives: Purpose of the study was to determine the incidence of aberrant phenotypes in North East Indian patients with acute leukemia., Methods: We analysed a total of 100 cases (AML = 36, ALL = 61, MPAL = 3) by multiparametric flow cytometry using an acute panel of monoclonal antibodies (MoAbs). The MoAbs were selected to identify differentiation-associated antigens of both myeloid and lymphoid lineages., Results: Aberrant phenotypes were found in 21 (58.3%) cases of AML, 36 (59.2%) cases of B-ALL and 6 (66.7%) cases of T-ALL. CD7 was the most frequent lymphoid associated antigen found in 33% of AML cases while CD117 was the myeloid antigen most frequently detected in ALL (54%) cases. Aberrant expression of CD 117 is highly significant by Fischer's exact test (P< 0.0001)., Conclusion: We conclude that aberrant phenotypes are present in a great majority of acute leukemia patients of North East India. Future studies will be directed to correlate of these markers with prognosis and therapeutic response.

Primary central nervous system-diffuse large B-cell lymphoma (PCNS-DLBCL) is a rare, extranodal malignant lymphoma carrying poor prognosis. The prognostic impact of tumor microenvironment (TME) composition and the PD-1/PD-L1 immune checkpoint pathway are still undetermined in PCNS-DLBCL. We aimed to quantify the tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs), and PD-L1 expression in the PCNSL and evaluated their prognostic significance. All patients with histopathologically diagnosed PCNS-DLBCL over a period of 7 years were recruited. Immunohistochemistry for CD3, CD4, CD8, FOXP3, CD68, CD163, PD-1, and PD-L1 was performed on the tissue microarray. Forty-four cases of PCNS-DLBCL, who satisfied the selection criteria, were included with mean age of 55 ± 12.3 years and male-to-female ratio of 0.91:1. The mean overall survival (OS) and disease-free survival (DFS) was 531.6 days and 409.8 days, respectively. Among TILs, an increased number of CD3+ T cells showed better OS and DFS, without achieving statistical significance. CD4 positive T-cells were significantly associated with the longer OS (p = 0.037) and DFS (p = 0.023). TAMs (68CD and CD163 positive) showed an inverse relationship with OS and DFS but did not reach statistical significance (p > 0.05). Increased PD-L1 expression in immune cells, but not in tumor cells, was associated with significantly better DFS (p = 0.037). The TME plays a significant role in the prognosis of PCNS-DLBCL. Increased number of CD4+ T cells and PD-L1-expressing immune cells is associated with better prognosis in PCNS-DLBCL. Further studies with larger sample size are required to evaluate the role of targeted therapy against the TME and immune check point inhibitors in this disease., (© 2021 Scandinavian Societies for Medical Microbiology and Pathology.)

Background: During infections involving intracellular pathogens, iron performs a double-edged function by providing the pathogen with nutrients, but also boosts the host's antimicrobial arsenal. Although the role of iron has been described in visceral leishmaniasis, information regarding its status in the dermal sequel, Post Kala-azar Dermal Leishmaniasis (PKDL) remains limited. Accordingly, this study aimed to establish the status of iron within monocytes/macrophages of PKDL cases., Methodology/principal Findings: The intramonocytic labile iron pool (LIP), status of CD163 (hemoglobin-haptoglobin scavenging receptor) and CD71 (transferrin receptor, Tfr) were evaluated within CD14+ monocytes by flow cytometry, and soluble CD163 by ELISA. At the lesional sites, Fe3+ status was evaluated by Prussian blue staining, parasite load by qPCR, while the mRNA expression of Tfr (TfR1/CD71), CD163, divalent metal transporter-1 (DMT-1), Lipocalin-2 (Lcn-2), Heme-oxygenase-1 (HO-1), Ferritin, Natural resistance-associated macrophage protein (NRAMP-1) and Ferroportin (Fpn-1) was evaluated by droplet digital PCR. Circulating monocytes demonstrated elevated levels of CD71, CD163 and soluble CD163, which corroborated with an enhanced lesional mRNA expression of TfR, CD163, DMT1 and Lcn-2. Additionally, the LIP was raised along with an elevated mRNA expression of ferritin and HO-1, as also iron exporters NRAMP-1 and Fpn-1., Conclusions/significance: In monocytes/macrophages of PKDL cases, enhancement of the iron influx gateways (TfR, CD163, DMT-1 and Lcn-2) possibly accounted for the enhanced LIP. However, enhancement of the iron exporters (NRAMP-1 and Fpn-1) defied the classical Ferritinlow/Ferroportinhigh phenotype of alternatively activated macrophages. The creation of such a pro-parasitic environment suggests incorporation of chemotherapeutic strategies wherein the availability of iron to the parasite can be restricted., Competing Interests: The authors have declared that no competing interests exist.

We have genotyped 53 individuals from three ethnic groups (Japanese, Chinese, Asian Indian) for an allotypic polymorphism of a widely expressed low affinity Fc receptor for IgG (Fc gamma RIIA). The method, requiring PCR amplification of genomic DNA and Southern analysis with allele specific oligonucleotide probes, detects a single nucleotide difference (G or A) at base 494 which results in an arginine (R) or histidine (H) at amino acid 131 of the Fc gamma RIIA protein. This polymorphism has been shown to determine the affinity of the receptor for hIgG2; Fc gamma RIIA-H131 has a high affinity for hIgG2, while Fc gamma RIIA-R131 binds hIgG2 weakly. We found that the Japanese and Chinese groups have an increased frequency of the H/H131 allotype (61 and 50% respectively) as compared to the Caucasian group (23%), in agreement with previously reported phenotype data. The genotype distribution of the Asian Indian group was not different from our Caucasian group. The shifts in frequency of the R131 and H131 alleles in different populations may have implications for disease susceptibility when the hIgG2 antibody isotype predominates.

In pregnant women, studies are lacking on the relationship of vegetable and animal flesh (poultry, red meat and seafood) intake with inflammation, especially in low- and middle-income countries. We conducted a cohort study of pregnant women receiving antenatal care at BJ Medical College in Pune, India. The dietary intake of pregnant women was queried in the third trimester using a validated food frequency questionnaire. Twelve inflammatory markers were measured in plasma samples using immunoassays. Only 12% of the study population were vegetarians, although animal flesh intake levels were lower compared to Western populations. In multivariable models, higher intakes of total vegetables were associated with lower levels of the T-helper (Th) 17 cytokine interleukin (IL)-17a ( p = 0.03) and the monocyte/macrophage activation marker soluble CD163 (sCD163) ( p = 0.02). Additionally, higher intakes of poultry were negatively associated with intestinal fatty-acid binding protein (I-FABP) levels ( p = 0.01), a marker of intestinal barrier dysfunction and Th2 cytokine IL-13 ( p = 0.03), and higher seafood was associated with lower IL-13 ( p = 0.005). Our data from pregnant women in India suggest that a higher quality diet emphasizing vegetables and with some animal flesh is associated with lower inflammation. Future studies should confirm these findings and test if modulating vegetables and animal flesh intake could impact specific aspects of immunity and perinatal health.

Polycystic ovary syndrome (PCOS), a gynaecological endocrine disorder affects 9% of Indian women and is linked to type II diabetes. The association of INSR (INSulin Receptor gene) variants (rs2059807 and rs1799817) with PCOS was established through genome-wide association studies, yet requires validation for the Indian population. This case-control study included 253 PCOS women and 308 age-matched control. The minor allele frequency of rs2059807 had an odds ratio of 13.5 and that of rs1799817 was 11.8. The cohort with rs2059807 MAF presented elevated levels of luteinising hormone [PCOS vs Control: 6.32 ± 2.26 mIU/mL vs 4.97 ± 3.27 mIU/mL], estradiol [116.01 ± 60.63 pg/mL vs 65.04 ± 44.98 pg/mL], and decreased HDL - C [50.4 ± 11.59 mg/dL vs 64 ± 15.49 mg/dL] showing disturbances in the hormonal patterns. The rs1799817 polymorphism cohort had elevated levels of serum insulin [17.99 ± 11.6 mIU/mL vs 11.67 ± 6.63 mIU/mL], blood glucose [199.15 ± 63.72 mg/dL vs 96.6 ± 24.3 mg/dL], and testosterone [0.91 ± 0.2 nmol/L vs 0.53 ± 0.16 nmol/L] thereby triggering metabolic dysfunction and predisposed to lifestyle disorder. Also, the SNPs were found to be in linkage equilibrium and contributed to the development of PCOS differentially., Competing Interests: Declaration of competing interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: The key to newer therapeutic and eradication approaches often lies in understanding slow disease progression in HIV infection. The paediatric population has been poorly studied in this regard. We aimed to describe a cohort of perinatally infected long-term nonprogressor (LTNP) children living with HIV in India and to evaluate the immune biomarkers of disease progression., Methods: LTNPs (ART-naïve, with a CD4 count ≥ 500 cells/μL at age ≥ 7 years) among the cohort of HIV-infected children were identified and monitored longitudinally, and their CD4 T-cell counts and plasma viral loads were measured every 6 months. The plasma monocyte/macrophage activation markers, namely soluble CD14 (sCD14), soluble CD163 (sCD163) and interferon-inducible protein-10 (IP-10) were measured by enzyme-linked immunosorbent assay (ELISA) in LTNPs and progressors. The Mann-Whitney U-test was used to compare the two groups and P values < 0.05 were considered statistically significant. Spearman's rank or Pearson's correlation coefficient (r) was calculated to determine the associations between variables., Results: Among 378 children living with HIV-1 surveyed in our cohort, 40 (10.6%) were LTNPs. Longitudinal analysis of the LTNP data showed that both CD4 count and viral load declined significantly with age (P < 0.0001 for both). Plasma sCD14 levels were significantly (P < 0.005) higher in progressors and sCD163 levels were significantly (P < 0.0001) higher in LTNPs., Conclusions: The prevalence of LTNPs in our cohort of perinatally infected children living with HIV was 10.6%. We observed a trend for associations between the increasing sCD163 monocyte/macrophage activation marker levels, declining CD4 counts and the gradual loss of nonprogressor status with age in the LTNPs. These findings underscore the need for early antiretroviral therapy in those children with proven slow disease progression., (© 2019 British HIV Association.)

Background: Manual thrombus aspiration during primary percutaneous intervention provides us with aspirated thrombus sample, that may contain material from the disrupted plaque. Immunohistopathological analysis of thrombus can yield valuable information about the clinical and cardiovascular outcomes and possible mechanisms of myocardial infarction., Material and Methods: We studied and analysed the immunohistopathological features of coronary thrombus aspirated from patients undergoing primary percutaneous coronary angioplasty. Immunohistological staining included markers namely CD68, SMA and CD34 for macrophages, smooth muscle actin and endothelium, respectively. Major adverse cardiac events, angiographic outcome and infarct size were also noted., Results: Fifty-three patients (Mean age - 51.3±13years; males-47) who underwent primary percutaneous coronary intervention with aspiration thrombectomy were enrolled. Thrombus was successfully aspirated in 40 of 53 patients (75.4%). Patients with successful thrombus aspiration had higher ST-segment resolution (≥50%) as compared to patients with failed thrombus aspiration. Presence of RBC-rich thrombus on microscopy was more commonly associated with post-procedure TIMI flow of <2 as compared to patients with fibrin-rich thrombus and a trend towards lower myocardial blush grade<2 (P=0.10), and a significantly higher final infarct size (37.5±5% vs 25±15%; P=0.04 of myocardium) on nuclear scan. Immunohistology revealed presence of plaque material in 72% (26/36) of the samples., Conclusions: Immunohistopathological evaluation of intracoronary thrombus may be of prognostic importance. High prevalence of plaque material in the aspirated intracoronary thrombus suggests plaque rupture as a possible etiology for vessel occlusion in these patients., Short Summary: Immunohistopathological evaluation of intracoronary thrombus reveals high prevalence of plaque material in the aspirated intracoronary thrombus suggesting plaque rupture as a possible etiology for vessel occlusion in Indian STEMI patients., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Genetic contributions towards Type 2 diabetes (T2D) have been assessed through association studies across different world populations with inconsistencies. The majority of the T2D susceptibility loci are common across different races or populations but show ethnicity-specific differences. The pathogenesis of T2D involves genetic variants in the candidate genes. The interactions between the genes involved in insulin signaling and secretory pathways are believed to play an important role in determining an individual's susceptibility towards T2D. Therefore, the present study was initiated to examine the differences, if any, in the contribution of polymorphisms towards T2D susceptibility in the background of different ethnic specifications. The present case-control study included a total of 1216 T2D cases and healthy controls from three ethnic groups (Jat Sikhs, Banias and Brahmins) of North-West India. Polymorphisms were selected on the basis of information available in the literature for INS (rs689), INSR (rs1799816) and PP1G.G (rs1799999) in context to T2D. The genotyping was done using PCR-RFLP method. Statistical analysis was done using SPSS 16.0. The analyses revealed that INS (rs689) polymorphism conferred risk towards T2D susceptibility in all the three ethnic groups whereas INSR (rs1799816) polymorphism conferred risk towards T2D in Brahmins only and PP1G.G (rs1799999) polymorphism indicated T2D risk in Jat Sikhs only. Furthermore, interaction analyses indicated the cumulative role of three genetic variants in modulating T2D susceptibility in the three ethnic groups. In conclusion, our results substantiated the evidences for the role of ethnicity in differential susceptibility to T2D in the background of same genetic variants.

Background Aims: A phase II clinical trial of an autologous dendritic cell (DC) formulation for the management of refractory solid malignant tumors was conducted across six sites in India with an objective to study safety and efficacy., Methods: A total of 51 patients with refractory cancer (either sex) with life expectancy ≥3 months, Eastern Cooperative Oncology Group score ≤2, available tumor tissue and adequate organ and bone marrow function were recruited. Monocytes obtained by leukapheresis, differentiated into DCs by cytokines and primed with autologous tumor lysate (fresh tissue biopsy or paraffin block). On the 8th day, mature DCs were analyzed for expression of CD40, CD80, CD83, CD86, DC205 and DC209. The treatment regime consisted of six doses (intravenous) over 14 weeks with 2 post-treatment follow-up visits, 6 weeks apart. Safety was assessed at all visits and responses were evaluated on days 58, 100 and 184 or at end of the study., Results: A total of 38 patients were evaluated for safety and efficacy. One adverse event classified as possibly related was an episode of rigors or chills with mild pyrexia during one infusion. Objective response rate by Response Evaluation Criteria In Solid Tumors was 28.9% (11/38) and immune-related response criteria was 42.1% (16/38); 90% confidence interval for objective response rate was (17.2, 43.3) and (28.5, 56.7) by Response Evaluation Criteria In Solid Tumors and immune-related response criteria, respectively. The median time to treatment progression was >9 weeks. Median overall survival was 397 days. An increase in the expression of interferon-γ was not significant., Conclusions: Therapy was safe. The responses, time to treatment progression and survival are encouraging for patients with aggressive refractory disease., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: E-cadherin (CDH1) plays an important role in intercellular adhesion, cell signaling, and cellular differentiation. Association of single-nucleotide polymorphisms (SNPs) of CDH1 has been identified in a number of epithelial malignancies; however, studies related to breast cancer are very few., Aim: To investigate the association between CDH1 SNPs and breast cancer risk in south Indian women., Methods: Genotyping of CDH1 functional SNPs (-347G/GA, -160C/A, and +54C/T) was carried out on genomic DNA of blood from breast cancer patients (n=202) and controls (n=250) of south Indian origin by PCR-sequencing and PCR-restriction fragment length polymorphism techniques. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were assessed by Haploview Software., Results: The frequencies of -160A/A genotypes (p=0.038) and -160A alleles (p=0.046) were significantly higher in patients compared to controls. In addition, the frequency of the -347GA/-160A/+54C haplotype was also significantly elevated in patients (p=0.0238). Strong LD was observed between -347G/GA and +54C/T loci (D'=0.44) in patients compared to controls., Conclusion: The CDH1 -160C/A polymorphism may constitute an inheritable risk factor for breast cancer in south Indian women.

Background and Objective: The INSR gene, which encodes the insulin receptor, is a candidate gene for type 2 diabetes (T2D). The objective of the present study was to sequence some of the crucial exons in the INSR gene such as exon 2, which encodes the insulin-binding domain of the INSR protein, and exons 17-21, which encode the protein tyrosine kinase domain for mutations/polymorphisms, and to study their association with T2D in the South Indian population., Subjects and Methods: The INSR gene was sequenced in 25 normal glucose-tolerant (NGT) and 25 T2D subjects, and the variant found was genotyped by polymerase chain reaction-restriction fragment length polymorphism in 1,016 NGT and 1,010 T2D subjects, randomly selected from the Chennai Urban Rural Epidemiology Study., Results: Only one previously reported polymorphism, His1085His [rs1799817, (C→T)], in exon 17 was detected by sequencing. The frequency of the "T" allele of the His1085His polymorphism was significantly lower in the T2D subjects (31%) compared with the NGT subjects (35%) and showed significant protection against diabetes (odds ratio 0.85, 95% confidence interval 0.75-0.97, P=0.019). Regression analysis according to a recessive model taking the CC+CT genotype as the reference showed that the TT genotype was protective against diabetes (odds ratio 0.71, 95% confidence interval 0.50-0.99, P=0.048). Adjusting this P value by the number of competing models (three) using Bonferroni's correction, we found that the association finding did not remain significant., Conclusions: The "T" allele of the His1085His polymorphism in the INSR gene shows significant protection against diabetes. This study gains importance because there are no data available to date on the role of INSR variants in T2D in the Indian population.

Background: In addition to standard risk criteria at diagnosis, minimal residual disease (MRD) following initiation of therapy is a well-recognized risk factor to predict relapse. Literature from developing countries addressing therapeutic or laboratory practices related to MRD, is largely lacking. In a first paper from India, we describe our experience in establishing a flow cytometry-based MRD assay for precursor B lineage ALL (BCP-ALL) with emphasis on the assay standardization and cost., Methods: Normal templates for B cell development were established in 10 control patients using CD45, CD11a, CD38, CD20, CD10, CD19, CD58, CD34, CD123, and CD22. BCP-ALL samples (n = 42) were characterized at diagnosis to identify a suitable marker for follow-up during mid (D+21) and end of induction (D+33). Both, multiparametric immunophenotyping and single marker detection of LAIP were used for data analysis., Results: In 95.2% of BCP-ALL at least two informative markers could be obtained when a minimum of four cocktail combinations were used. The combination CD20, CD10, CD45, and CD19 was the most useful (71.4%) followed by combinations containing CD38 (66.7%), CD22 (57.1%), CD11a (52.4%), and CD58 (33.3%). Using our approach, 60 and 47% of patients had detectable MRD at mid and end induction time points, respectively., Conclusion: We have described a relatively cost effective MRD panel which is applicable to over 90% of patients. We hope that this data would encourage more centers in India and other resource constrained health delivery systems to develop MRD assays., (Copyright © 2012 International Clinical Cytometry Society.)

In a first series from India, we report 9 cases of hepatosplenic T cell lymphoma (HSTCL) seen in 23 months accounting for 4.2% of all mature T-non-Hodgkin lymphomas (NHLs) in our institution. All patients presented with organomegaly, cytopenias and had evidence of bone marrow involvement. The tumor cells had a blastic (55%) morphology with predominantly intrasinusoidal (33.3%) or intrasinusoidal with an additional interstitial component (33.3%). On flow cytometry, the classical phenotype (CD3+, CD7+, CD4-, CD8-, CD5-, CD56+/-) was seen only in 4 patients. Unusual variations included CD45 (overexpression), CD7 (dim expression), CD3 (overexpression, heterogeneous and dim), CD2 (overexpression), CD5 (heterogeneous), CD8 (heterogeneous or dim or overexpression) and aberrant expression of CD19. Fluoresvent in situ hybridisation (FISH) and karyotyping was abnormal in 5 out of 7 patients evaluated. All of the 5 cases showed abnormalities in chromosome 7 (ring chromosome or isochromosome 7q). Five patients died of disease and related complications in a span of 1-3 months after diagnosis whereas 4 were alive at their last follow up out of which 2 had documented a relapse. In our series, HSTCL was characterized by typical clinical and variable immunophenotypic features and a dismal clinical outcome.

Though multiple studies link chromosomal regions 1q21-q23 and 20q13 with type 2 diabetes, fine mapping of these regions is yet to confirm gene(s) explaining the linkages. These candidate regions remain unexplored in Indians, which is a high-risk population for type 2 diabetes. Hypothesizing regulatory regions to have a more important role in complex disorders, we examined association of 207 common variants in proximal promoter and untranslated regions of genes on 1q21-23 and 20q13 with type 2 diabetes in 2115 North Indians. Further, top signals were replicated in an independent group of 2085 North Indians. Variants-rs11265455-SLAMF1 (odds ratios (OR)=1.32, P=1.1 × 10(-3)), rs1062827-F11R (OR=1.36, P=1.7 × 10(-3)) and rs12565932-F11R (OR=1.35, P=1.8 × 10(-3)) were top signals for association with type 2 diabetes whereas rs1333062-ITLN1 (OR=1.28, P=3.4 × 10(-3)) showed strongest association in body mass index-stratified analysis. Replication of these four variants confirmed associations of rs11265455-SLAMF1 (OR=1.27, P=9.1 × 10(-3)) and rs1333062-ITLN1 (OR=1.25, P=1.1 × 10(-3)) with type 2 diabetes. Meta-analysis further corroborated the association of rs11265455-SLAMF1 (OR random effect=1.29, P random effect=3.9 × 10(-5)) and rs1333062-ITLN1 (OR random effect=1.19, P random effect=1.8 × 10(-4)). In conclusion, the study demonstrates that variants of SLAMF1 and ITLN1, both implicated in inflammation, are associated with type 2 diabetes in Indians.

Background: Plasma cell leukemia (PCL) is a rare but aggressive subtype of plasma cell dyscrasia. It is known to present with highly variable morphological features and may mimic with other lymphoid neoplasms. Multicolor flow cytometry (MFC) with availability of newer markers is highly useful in the diagnosis of the plasma cell leukemia. We present an immunophenotypic profile in ten cases of PCL along with their clinical and laboratory findings., Materials and Methods: We retrospectively studied immunophenotypic profile of 10 cases of plasma cell leukemia (out of 4615 cases of hematolymphoid neoplasms) using five parameter, three color flow cytometric analysis. We also studied their clinical presentation and other laboratory findings., Results: Common clinical features at presentation were weakness, bone pain, anemia, thrombocytopenia and osteolytic lesions. Plasma cell population was identified on strong expression of CD38 and co-expression of CD38 and CD138. CD56 was expressed in 44% cases. CD19 and CD20 were negative in all cases. Surface light chain restriction was seen in 50% cases and in remaining 50% cases revealed cytoplasmic light chain restriction. CD117 was expressed in one out of two cases studied., Conclusions: MFC immunophenotyping is highly useful to differentiate Plasma cell leukemia from other chronic lymphoproliferative disorders with plasmacytoid morphology as well as from non-neoplastic reactive PC and co-expression of CD38 and CD138 is a best combination to identify the plasma cells by MFC.

Background: Primary central nervous system (CNS) lymphoma (PCNSL) is a rare malignant non-Hodgkin's lymphoma and it accounts for 1% of all intracranial tumors. Only a few PCNSL studies have been reported from India, and studies on prognostic factors determining outcome, or evaluation of the response to currently accepted treatment, are lacking., Aims: This study attempts to further delineate the clinical, radiological and pathological profile of PCNSL in India, to evaluate response to treatment and to assess usefulness of the International Extranodal Lymphoma Study Group (IELSG) score., Settings and Design: All patients with pathologically proven PCNSL admitted over three years at a large tertiary care institution were studied., Materials and Methods: Clinical features, IELSG prognostic score, imaging and pathological features, and response to treatment were evaluated. Results were analyzed using chi 2 test., Results: Of 26 patients found, all except two were immunocompetent. Median age at diagnosis was 59 years. Focal deficits (76.9%) and neuropsychiatric symptoms (57.6%) were the commonest presenting complaints. Except for one case, at least some contrast enhancement was seen in brain lesions of all patients. Pathological studies showed high grade diffuse large B-cell (DLBCL) histology in 96.2% of patients. Of 22 patients who received methotrexate (MTX) based chemotherapy with/without radiotherapy; six died, with a response rate of 72.7%. Median survival was 10 months. Median follow-up duration was 14.5 months. Four patients developed treatment-related cognitive decline. All six patients with IELSG score of 4/5 died, while all 16 patients with a score of 0-3 survived., Conclusions: PCNSL presents most commonly in the sixth decade with focal neurological deficit, behavioral symptoms and cognitive decline. High grade DLBCL is the commonest histological subtype. Steroids should ideally be withheld until biopsy as they may confound the diagnosis. Most immunocompetent patients respond well to high dose MTX-based chemotherapy with/without radiation. High IELSG scores correlate with worse prognosis in patients with PCNSL.

Objective: Polycystic ovary syndrome (PCOS) is a multigenic disorder, and insulin resistance is one of its hallmark features. Polymorphisms in exon 17 of insulin receptor (INSR) gene are reported to be associated with PCOS. We investigated this association in Indian women and its putative relationship with PCOS associated traits, which has not been explored so far., Methods: In this case control study, the polymorphisms were investigated by direct sequencing in 180 women with PCOS and 144 age matched controls. Clinical, anthropometric, biochemical, and hormonal parameters were also estimated., Results: The silent C/T polymorphism at His1058 in exon 17 of INSR was found to be present in our study population. The polymorphic genotype (CT+TT) was significantly associated with PCOS in lean women (chi(2)=8.493, df=1, P=0.004). It showed association with higher fasting insulin levels (P=0.02), homeostasis model assessment of insulin resistance (P=0.005), free androgen index (P=0.03), and lower quantitative insulin sensitivity check index (P=0.004) in lean PCOS women. No other novel or known polymorphism was identified in exon 17 in this cohort., Conclusions: The study shows significant association of C/T polymorphism at His1058 of INSR with PCOS in the lean rather than obese Indian women. Its association with indices of insulin resistance and hyperandrogenemia is also seen in the same group. The findings strengthen the concept that pathogenesis of PCOS is different in lean and obese women.

Wilson's disease (WD), a familial neurological disorder involving the brain and liver secondary to altered copper metabolism, is common in South India. In view of the paucity of studies on this condition, the pathomorphological features of eight cases of WD were studied in detail at autopsy (brain alone, 1; brain and liver biopsy, 1; brain and visceral organs, 6), and are described with a discussion of the differential features of the neurological and hepatic forms. Of the six patients presenting with neurological manifestations, five had central pontine myelinolysis, five had subcortical white matter cavitations, four had putaminal softening, and six had variable ventricular dilatation, unlike the hepatic form. The presence of Opalski cells and pontine myelinolysis appear to be specific to the neurological form of WD. Liver abnormalities were observed in all cases (cirrhosis, 6; steatosis, 4; chronic active hepatitis, 2). Contrary to the rubric 'hepatolenticular degeneration', involvement of the lenticular nucleus was not universal, and nor was the pathology restricted to these anatomical areas.

Background: Susceptibility/resistance to Plasmodium falciparum malaria has been correlated with polymorphisms in more than 30 human genes with most association analyses having been carried out on patients from Africa and south-east Asia. The aim of this study was to examine the possible contribution of genetic variants in the TNF and FCGR2A genes in determining severity/resistance to P. falciparum malaria in Indian subjects., Methods: Allelic frequency distribution in populations across India was first determined by typing genetic variants of the TNF enhancer and the FCGR2A G/A SNP in 1871 individuals from 55 populations. Genotyping was carried out by DNA sequencing, single base extension (SNaPshot), and DNA mass array (Sequenom). Plasma TNF was determined by ELISA. Comparison of datasets was carried out by Kruskal-Wallis and Mann-Whitney tests. Haplotypes and LD plots were generated by PHASE and Haploview, respectively. Odds ratio (OR) for risk assessment was calculated using EpiInfotrade mark version 3.4., Results: A novel single nucleotide polymorphism (SNP) at position -76 was identified in the TNF enhancer along with other reported variants. Five TNF enhancer SNPs and the FCGR2A R131H (G/A) SNP were analyzed for association with severity of P. falciparum malaria in a malaria-endemic and a non-endemic region of India in a case-control study with ethnically-matched controls enrolled from both regions. TNF -1031C and -863A alleles as well as homozygotes for the TNF enhancer haplotype CACGG (-1031T>C, -863C>A, -857C>T, -308G>A, -238G>A) correlated with enhanced plasma TNF levels in both patients and controls. Significantly higher TNF levels were observed in patients with severe malaria. Minor alleles of -1031 and -863 SNPs were associated with increased susceptibility to severe malaria. The high-affinity IgG2 binding FcgammaRIIa AA (131H) genotype was significantly associated with protection from disease manifestation, with stronger association observed in the malaria non-endemic region. These results represent the first genetic analysis of the two immune regulatory molecules in the context of P. falciparum severity/resistance in the Indian population., Conclusion: Association of specific TNF and FCGR2A SNPs with cytokine levels and disease severity/resistance was indicated in patients from areas with differential disease endemicity. The data emphasizes the need for addressing the contribution of human genetic factors in malaria in the context of disease epidemiology and population genetic substructure within India.

Type 1 diabetes (T1D) is a polygenic autoimmune disease. Susceptibility to T1D is strongly linked to a major genetic locus that is the major histocompatibility complex (MHC) and several other minor loci including insulin, CTLA4 that contribute to diabetes risk in an epistatic way. MHC harbours genes whose primary function is to govern immune responsiveness. Being the most polymorphic genomic region known in humans, MHC serves as a very exciting minigenome model for studying susceptibility to T1D. We have observed enormous diversity in HLA class I and class II genes in the north Indian population and identified several 'novel alleles' and 'unique haplotypes'. For example, multiple DR3+ve autoimmunity favouring haplotypes have been identified, some of which are unique to the Asian north Indian T1D patients. Our molecular studies have revealed that (i) the classical Caucasian autoimmunity favouring AH8.1 (HLA-A1 B8 DR3) is rare in the Indian population and has been replaced by a variant AH8.1v that differs from the Caucasian AH8.1 at several gene loci, (ii) AH8.2 (HLA-A26 B8 DR3) is the most common DR3 positive haplotype in this population and resembles the Indian AH8.1v rather than Caucasian AH8.1, and (iii) there are additional HLA-DR3 haplotypes HLA-A24 B8 DR3 (AH8.3), A3 B8 DR3 (AH8.4) and A31 B8 DR 3 (AH 8.5) that occur in the Indian population. The studies have led to a hypothesis that AH8.1 and AH8.1v might have co-evolved from a common ancestor but preferential divergence of AH8.2 over AH8.1 leading to survival advantage might have been driven by vigorous pathogenic challenges encountered by the Indian population. These studies have important implications in our understanding of disease pathogenesis, identification of high risk individuals, disease diagnosis, disease management and immunological therapeutic approaches.

Single nucleotide polymorphisms (SNPs) of the CTLA-4 gene have been associated with manifestation of type 1 diabetes in several populations. We assessed the association of five SNPs present in the CTLA-4 gene [-318C/T, -1661A/G and -1722C/T in the promoter region, +49A/G in exon 1 and CT60 in the 3' untranslated region (UTR) region] with type 1 diabetes in North Indian subjects. Genotyping was performed in the patients (n = 130) and the healthy control (n = 180) subjects by polymerase chain reaction-fragment length polymorphism analysis using MseI, BbvI, BstEII and NcoI restriction endonucleases for the -318, -1661, -1722, +49 and CT60 SNPs, respectively. The frequency of G alleles at -1661 locus was significantly higher in the patient group compared with the control subjects. Although the frequency of T alleles at -318 SNP was significantly higher in patients with type 1 diabetes compared with the controls, it did not remain significant after Bonferroni correction for the number of alleles tested. The frequencies of C/T alleles and genotypes at -1722C/T and G allele at +49A/G and CT60 SNPs were not significantly different between the patient and the control groups. Of the various possible haplotypes constructed using the five genetic loci tested (-318, -1661, -1722, +49, CT60), the frequency of 'TGTAG' haplotype was significantly higher in the patients when compared with the controls. The results of the present study indicate that the presence of G allele at -1661 locus at the CTLA-4 gene (IDDM12 locus) is associated with increased susceptibility to type 1 diabetes in North Indians, whereas A allele is protective.

Background: Enumeration of lymphocyte subsets has been widely used for the diagnosis and monitoring of several haematological and immunological disorders. Various studies have demonstrated age, sex and racial differences in lymphocyte subset expression. Reference values are not available for Indian children and there is a need for this information to replace commonly used, but inappropriate, adult lymphocyte subset ranges., Methods: One hundred thirty-eight healthy children between 3 and 15 years of age, attending a local government school in Chennai, South India were Included in the study. Haemoglobin levels, and total and differential cell counts were determined using an automated counter and lymphocyte subsets were analysed by flowcytometry., Results: The mean (SD) absolute lymphocyte count declined with age from 4338 (1031) at 3 years to reach a plateau of 3096 (914) at 11-13 years (p < 0.05). A significant decline was also observed in the absolute numbers of CD3+, CD4+, CD8+ and CD19+ cells. However, the percentage values of CD3+, CD4+, CD8+, CD16/56+ cells and the CD4/CD8 ratio remained fairly stable across the age range., Conclusion: Our data would prove useful in interpreting disease-related changes in lymphocyte subsets in Indian children of different age groups. Age-related decrease in the absolute lymphocyte count as well as numbers of CD4 and CD8 cells was found to occur between the ages of 3 and 11 years. A normogram relating age to CD4 count has been developed.

The HIV epidemic in the Asian subcontinent has a significant impact on India. Patients with AIDS have an increased risk of developing non-Hodgkin lymphoma (NHL). In this study, we have investigated the pattern of distribution of lymphoid neoplasms and also studied the Epstein-Barr virus (EBV)-association and p53 expression in 35 HIV-positive patients from India. The biopsy samples were studied for histology and for expression of CD20, CD3, CD15, CD30, light chains, CD138, bcl-6, epithelial membrane antigen, EBV-latent membrane protein-1, and p53 protein. In situ hybridization was performed with digoxigenin-labeled anti-sense EBV-encoded nuclear RNA-1 (EBER-1) probe. Polymerase chain reaction (PCR) was performed on DNA extracted from paraffin sections for EBV-subtype analysis. The 35 cases included 7 cases of Hodgkin disease (HD), 4 cases of plasmacytoma (PL), and 24 cases of NHL. Among the cases of NHL, 3 were Burkitt lymphoma (BL), 4 were diffuse large B-cell lymphoma (DLBL) of centroblastic type (CBL), 10 were DLBL of immunoblastic type (IBL), 4 were high-grade B-cell lymphoma (unspecified) and the rest were other subtypes. EBV-association was noted in all cases of HD, 2 of 3 BL, and 3 of 10 IBL. PCR analysis of the EBNA-3C gene revealed amplimers corresponding to type A. A p53 protein overexpression was noted in 6 of 10 IBLs, 1 of 3 BLs, 2 of 3 CBLs, and 5 of 7 cases of HD. This is the first reported study of lymphoid malignancies in HIV-positive individuals from India.

The beta-thalassaemias are a heterogeneous group of inherited disorders caused by mutations in and around the structural gene of the beta-chain of the adult haemoglobin (HbA). Studies at the gene level have identified a large number of beta-thalassaemia gene variations in different populations. These findings have implications for the use of molecular diagnosis for genetic counselling and prenatal detection of the beta-thalassaemias. In our unit, we initiated studies to investigate the molecular defects in beta-thalassaemias in Saudi Arabia using amplification-refractory mutation systems, dot blot analysis and restriction endonuclease analysis, and identified mutations producing beta(+)- and beta zero-thalassaemias. Twenty of the mutations encountered in the Asian, Mediterranean, Chinese and other Arab populations were investigated. The most commonly encountered mutations in Saudi beta-thalassaemia patients were IVS-I-110, IVS-II-1, CD 39, IVS-I-5 and IVS-I 3' end (-25), while frameshifts at CD 8/9, Cap+1 (A-->C) and CD 6 mutations were identified at a low frequency. These mutations account for 84.94% of the total beta-thalassaemia mutations. The remaining 15% remain unknown. This is the first report on the type and nature of mutations in Saudi beta-thalassaemia patients. It presents frequencies of twenty mutations and emphasises the need for further detailed investigations to clarify the whole spectrum of beta-thalassaemia mutations in the Saudi population.

Objective: To assess the prevalence of early T precursor‐acute lymphoblastic leukaemia (ETP‐ALL), study its clinicopathological features and devise a 'flow score' based on immunophenotypic profiles. Material Methods: This was a retrospective study where clinical and laboratory data of all consecutive T‐ALL cases were analysed to identify features differentiating ETP from non‐ETP‐ALL. The utility of a flow score based on the five commonly used markers in leukaemia panels for T‐ALL (CD34, CD8, CD5, CD13 and CD33) was evaluated to differentiate ETP from non‐ETP‐ALL. Results: Early T precursor‐acute lymphoblastic leukaemia constituted 24.2% (n = 29) of all T‐ALL cases. It was significantly more common in adults (30.2%) as compared to paediatric (17.5%) patients (P =.046). The median age of presentation was significantly higher than the non‐ETP group. (24 vs 19 years; P =.01). Patients with ETP‐ALL usually presented with organomegaly, lymphadenopathy, lower levels of haemoglobin, total leucocyte count, peripheral blood blast proportion and LDH levels as compared to non‐ETP‐ALL. The majority of ETP‐ALL cases had L2 morphology with a moderate amount of cytoplasm showing frequent blebbing. A flow score cut‐off value of ≥3 on ROC curve analysis had a sensitivity and specificity of 100% and 94.6% respectively. Conclusion: Early T precursor‐acute lymphoblastic leukaemia had unique clinical and laboratory features. The prevalence of this entity is more common in the adult population. A flow score based on a minimum of five widely used markers can confidently identify ETP‐ALL and should be included in the primary panel of markers used for flow cytometric analysis. [ABSTRACT FROM AUTHOR]