Rehan M. Faridi

Vascular endothelial growth factor gene polymorphisms in North Indian patients with end stage renal disease

CONTEXT Vascular endothelial growth factor (VEGF) is involved in the development and differentiation of the vascular system. VEGF is expressed constitutively by epithelial cells from embryonic to adult kidneys and may play a key role in progression of kidney diseases. It is required for the growth and proliferation of glomerular and peritubular endothelial cells. In the kidney VEGF expression is prominently found in glomerular podocytes and in tubular epithelial cells, while VEGF receptors are mainly seen on preglomerular, glomerular, and peritubular endothelial cells. OBJECTIVES We have investigated the role of VEGF gene polymorphisms (-2578C/A,-2549 18 bp I/D, -1154 G/A and +936 C/T) as a susceptibility marker for end stage renal disease (ESRD). PARTICIPANTS AND METHODS We genotyped VEGF gene polymorphism in three hundred patients and three hundred and fifty ethnically matched unrelated healthy controls free from any renal disease. These markers were studied using ARMS-PCR and PCR-RFLP methods. Patients were categorized on the basis of the histo-pathological subtypes into chronic glomerulonephritis (CGN=109), hypertensive nephrosclerosis (HTN=106) and chronic interstitial nephritis (CIN=60). RESULTS VEGF -2578C and -2549D alleles were found to be ESRD causative alleles. It was observed that there was significant differences in the frequencies of the T allele of +936C/T polymorphism among CGN, HTN and CIN respectively. VEGF -1154AA genotype and A allele were associated significantly with CGN. T-G-A-D, T-A-C-I,C-G-A-D,C-A-C-D,C-G-C-I,C-A-A-D and T-G-C-D were seven haplotypes concurred in all the ESRD patients irrespective of underlying disease. While C-G-C-D & C-G-A-I haplotypes showed risk association in CGN & CIN, C-A-C-I was observed to play predisposing role in HTN. CONCLUSION The results highlight the role of studied VEGF polymorphisms in end stage renal disease at large and subsequently in the three primary kidney diseases among the North Indian population.

Assessment of fidelity and utility of the whole-genome amplification for the clinical tests offered in a histocompatibility and immunogenetics laboratory

Increasing emphasis on the use of molecular tests in a histocompatibility and immunogenetics laboratory (HIL) poses a potential problem of lack of sufficient DNA to perform multiple genetic analyses. In this study, we report the feasibility, fidelity and utility of multiple displacement amplification (MDA) method to perform whole-genome amplification (WGA) to generate DNA specimens that can be analyzed by multiple molecular techniques and can be used for different clinical tests offered by an HIL. The MDA-generated DNA when compared with the native DNA showed 100% congruency in genotyping of 37 genes/loci using multiple downstream molecular techniques: sequence-based typing and sequence-specific primer-based typing for 5 human leukocyte antigen (HLA) class I and II genes (HLA-A, B, C, DRB1 and DQB1), luminex-based sequence-specific oligonucleotide (SSO) genotyping for a panel of 16 killer immunoglobulin-like receptor (KIR) genes and automated fragment size analysis for a panel of 15 short tandem repeat (STR) loci and amelogenin gene. For post-allogeneic hematopoietic cell transplantation (HCT) chimerism analysis, MDA-generated DNA appeared useful for enriching pre-transplant DNA but not for enriching post-transplant chimeric DNA. Overall, our results show that MDA-based WGA could generate DNA of high yield and fidelity that can be used for various clinical tests and research purposes.

Donor-Recipient Matching for KIR Genotypes Reduces Chronic GVHD and Missing Inhibitory KIR Ligands Protect against Relapse after Myeloablative, HLA Matched Hematopoietic Cell Transplantation

Background Allogeneic hematopoietic cell transplantation (HCT) can be curative for many hematologic diseases. However, complications such as graft-versus-host disease (GVHD) and relapse of primary malignancy remain significant and are the leading causes of morbidity and mortality. Effects of killer Ig-like receptors (KIR)-influenced NK cells on HCT outcomes have been extensively pursued over the last decade. However, the relevance of the reported algorithms on HLA matched myeloablative HCT with rabbit antithymocyte globulin (ATG) is used for GVHD prophylaxis remains elusive. Here we examined the role of KIR and KIR-ligands of donor-recipient pairs in modifying the outcomes of ATG conditioned HLA matched sibling and unrelated donor HCT Methods and Findings The study cohort consisted of 281 HLA matched sibling and unrelated donor-recipient pairs of first allogeneic marrow or blood stem cell transplantation allocated into ‘discovery’ (135 pairs) and ‘validation’ (146 pairs) cohorts. High resolution HLA typing was obtained from the medical charts and KIR gene repertoires were obtained by a Luminex® based SSO method. All surviving patients were followed-up for a minimum of two years. KIR and HLA class I distributions of HCT pairs were stratified as per applicable definitions and were tested for their association with cause specific outcomes [acute GVHD grade II-IV (aGVHD), chronic GVHD needing systemic therapy (cGVHD) and relapse] using a multivariate competing risks regression model as well as with survival outcomes [relapse-free survival (RFS), cGVHD & relapse free survival (cGRFS) and overall survival (OS)] by multivariate Cox proportional hazards regression model. A significant association between KIR genotype mismatching (KIR-B/x donor into KIR-AA recipient or vice versa) and cGVHD was found in both discovery (p = 0.001; SHR = 2.78; 95%CI: 1.50–5.17) and validation cohorts (p = 0.005; SHR = 2.61; 95%CI: 1.33–5.11). High incidence of cGVHD associated with KIR genotype mismatching was applicable to both sibling and unrelated donors and was specific to recipients who had one or two C1 bearing HLA-C epitopes (HLA-C1/x, p = 0.001; SHR = 2.40; 95%CI: 1.42–4.06). When compared with KIR genotype mismatched transplants, HLA-C1/x patients receiving grafts from KIR genotype matched donors had a significantly improved cGRFS (p = 0.013; HR = 1.62; 95%CI: 1.11–2.39). Although there was no effect of KIR genotype matching on survival outcomes, a significantly reduced incidence of relapse (p = 0.001; SHR = 0.22; 95%CI: 0.10–0.54) and improved relapse-free survival (p = 0.038; HR = 0.40; 95%CI: 0.17–0.95) was observed with one or more missing ligands for donor inhibitory KIR among the recipients of unrelated donor transplants. Conclusions The present study for the first time presents the beneficial effects of KIR genotype matching in reducing cGVHD in myeloablative transplant setting using HLA matched (sibling and unrelated) donors. The findings offer a clinically applicable donor selection strategy that can help control cGVHD without affecting the risk of relapse and/or identify patients at a high risk of developing cGVHD as potential candidates for preemptive therapy. The findings also affirm the beneficial effect of one or more missing inhibitory KIR ligands in the recipient in reducing relapse and improving a relapse free survival in unrelated donor transplants.

Killer immunoglobulin like receptor gene content diversity among Northern Indian population

Abstract Background: Genes encoding KIR receptors are clustered in one of the most variable regions of the human genome. KIR gene frequencies vary in worldwide populations and reveal high probability of individuals differing in their gene content. Aim: This study aimed to investigate KIR diversity among the northern Indian population who share features with either Western Eurasian or East Asian populations. It sought to decipher how northern Indians are associated phylogenetically with global populations whilst also focusing on differentiation of populations. Subjects and methods: This paper studied 867 northern Indians using PCR-SSP. Gene and genotypic frequencies were calculated, using statistical analyses. Findings were compared against 76 global populations of differing ethnicities. Results: This northern Indian population shared characteristics with Western Eurasian or Asian Indian populations, as is evident from genetic distance, clustered heatmap, phylogenetic assessment and principal component analysis. The findings are consistent with the demographic history of northern India, including specific features, such as presence of comparatively high KIR B-haplotype as compared to A-haplotype. Conclusion: KIR frequencies and profiles of northern Indians were more similar to Western Eurasians, Africans and Asian Indians. This may suggest that KIR genes are under constant evolutionary pressures and selection, which may be linked to different invading pathogens.