The study was conducted in the district of Muzaffarpur in Bihar State, India, where VL is highly endemic. VL cases and family members or controls were from villages located within a radius of ~100 km from the city of Muzaffarpur covering the districts of Muzaffarpur, Vaishali, Samastipur, Saran, Sheohar, East Champaran and Sitamarhi. Initially, families with at least two siblings affected with clinical VL were ascertained from medical records held in the Kala-Azar Medical Research Centre (KAMRC) in Muzaffarpur, India [18
]. This was later extended to collection of singleton cases plus parent (trios) (see Table ). The replication study comprised 958 unrelated cases and 1015 unrelated controls. The controls had no history of VL, or a family history of VL among first-, second- or third-degree relatives. Patients and controls were matched for self-reported age, sex, religion, caste and geographic district of recruitment (see Table ). Diagnosis of VL was based on presence of typical clinical features of VL i.e. fever with rigors and chills, splenomegaly, weight loss and pancytopenia followed by demonstration of parasites by parasitological methods (light microscopy, in vitro
culture) using splenic aspirates [19
]. Additional VL cases identified in the field were confirmed on the basis of proof of medical records of diagnosis and treatment issued from one of the local health clinics or private practice, and accompanied by demonstration of clinical response to anti-leishmanial treatment (typically with amphotericin B). An annual incidence rate of 2.49 clinical VL cases/1,000 persons has been reported in the region [20
]. L. donovani sensu strictu
(zymodeme MON-2) was confirmed as the causative agent of VL in the study region, in accordance with other reports on clinical isolates from kala-azar patients in the state of Bihar [21
]. Additional epidemiological and demographic details relating to the study site are described elsewhere [25
]. Informed written consent in Hindi was obtained from all participating individuals and from parents of children under 18 years old. Approval for the study was provided by the Ethical Committee of the Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. Collection of families for the primary study was undertaken between 2004 and 2006. The replication study collection was undertaken during 2009-2010. For the family-based primary study DNA was prepared from buccal swabs by whole genome amplification as described [18
], and SNPs genotyped using ABI predesigned Taqman assays (ABI, Mulgrave, Victoria, Australia). For the replication case-control study, genomic DNA was extracted from saliva using the Oragene technology (DNA Genotek, Ontario, Canada), and SNPs genotyped using Sequenom iPLEX platform (Sequenom, San Diego, CA). The GTn
repeat and IN/DELS were genotyped for all samples using ABI fragment analysis processed on an ABI3130 (Australia) or ABI3730 (India) Genetic Analyser.
Baseline characteristics of (A) families for the primary sample of Indian multicase VL families, and (B) the Indian case-control cohorts.
Family-based allelic association tests based on the TDT but generalized to allow analysis under additive and genotype-wise models of inheritance were performed within FBAT under the null hypothesis of "no linkage and no association" [26
]. TDT power approximations [28
] show that the 313 primary VL trios had ≥95% power to detect an odds ratio ≥2 at P
= 0.01 for markers with MAF ≥ 0.1, but only 49% power for an odds ratio of 1.5. Nevertheless, our primary sample was well-powered to detect effect sizes (odds ratios ≥ 2) equivalent to those observed in the earlier study of SLC11A1
and VL in Sudan [12
]. Robust association tests were performed to take account of multiple trios within a pedigree. Association tests for the replication case-control sample were undertaken using logistic regression analysis performed in PLINK [29
] or LOGIT (Stata) using an additive model and a genotypic test. The 941 cases and 992 controls which passed quality control (Hardy-Weinberg Equilibrium) had 100% power to detect associations with an odds ratio of 2 for markers with MAF ≥ 0.1 at P
= 0.001, and 93.5% power for odds ratio 1.5; MAF ≥ 0.1, P
Splenic biopsies were taken as part of routine diagnostic procedure at the Kala Azar Medical Research Centre, Muzaffarpur, Bihar State, India. Since the spleen is a major focus for parasite growth inside macrophages, this afforded an important opportunity to analyse gene expression in a primary site of infection. Pre- and post- treated patient's splenic samples were collected in 5 × RNA Later (Ambion) during 2009-2010, transported to Varanasi at 4°C and stored at -80°C until RNA was isolated. Details regarding age and sex (15 males, median age 16, range 7 to 45 years; 9 females, median age 10, range 8 to 30 years) splenic parasites (21 confirmed positive; 3 not done) and drug administered (19 Miltefosine; 1 Miltefosine + Paramomycin; 1Ambisome + Paramomycin; 3Amphomul) were recorded for each patient. Total RNA was isolated using RNeasy tissue kit (Qiagen) according to the manufacturer's instructions and eluted in 30 ul of RNase free water. Sample quality and integrity was assessed by ND-2000 spectrophotometer (Thermo Fischer Scientific) and agarose (Sigma Aldrich) gel electrophoresis. 500 ng of RNA was reverse transcribed using the High Capacity cDNA synthesis kit (Applied Biosystems). Taqman predesigned gene expression assay (Hs00184453_m1) was used to perform expression studies (7500 HT Real Time PCR system, ABI, Foster City CA, USA) with 18S rRNA (P/N 4319413E) used as an endogenous control to normalize the expression data. Experiments were performed on 24 paired pre- and post-treatment splenic aspirates from VL patients with appropriate no RT and no template controls included in each plate. All samples were run in duplicate. Results were analysed by 7500 software v.2.0.1 and Graph pad prism 5. Paired Student's T tests was used to test for significant differences between pre (Day-0) and post (D-30) expression levels for each genotype, i.e. 3/3, 3/2 and 2/2. One way ANOVA was used to test for differences between 3/3 vs 3/2 vs 2/2 groups at either Day-0 or Day-30.