key: cord-0858844-5e1wafza
authors: Mukhi, Benudhar; Gupta, Himanshu; Wassmer, Samuel C.; Anvikar, Anupkumar R.; Ghosh, Susanta Kumar
title: Haplotype of RNASE 3 polymorphisms is associated with severe malaria in an Indian population
date: 2020-10-28
journal: Mol Biol Rep
DOI: 10.1007/s11033-020-05934-1
sha: 54d62876b035f6cf6a9b315b264c362d7addea57
doc_id: 858844
cord_uid: 5e1wafza

Severe malaria (SM) caused by Plasmodium falciparum (Pf) infection has been associated with life-threatening anemia, metabolic acidosis, cerebral malaria and multiorgan dysfunction. It may lead to death if not treated promptly. RNASE 3 has been linked to Pf growth inhibition and its polymorphisms found associated with SM and cerebral malaria in African populations. This study aimed to assess the association of RNASE 3 polymorphisms with SM in an Indian population. RNASE 3 gene and flanking regions were amplified followed by direct DNA sequencing in 151 Indian patients who visited Wenlock District Government Hospital, Mangalore, Karnataka, India. Allele, genotype and haplotype frequencies were compared between patients with SM (n = 47) and uncomplicated malaria (UM; n = 104). Homozygous mutant genotype was only found for rs2233860 (+ 499G > C) polymorphism (< 1% frequency). No significant genetic associations were found for RNASE 3 polymorphism genotypes and alleles in Indian SM patients using the Fisher's exact test. C-G-G haplotype of rs2233859 (− 38C > A), rs2073342 (+ 371C > G) and rs2233860 (+ 499G > C) polymorphisms was correlated significantly with SM patients (OR = 3.03; p = 0.008) after Bonferroni correction. A haplotype of RNASE 3 gene was found associated with an increased risk of SM and confirming that RNASE 3 gene plays a role in susceptibility to SM. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11033-020-05934-1) contains supplementary material, which is available to authorized users.

Malaria remains to be a major public health problem in low and middle-income countries, especially in the sub-Saharan region. The World Health Organization (WHO) estimated that 228 million cases of malaria and 405,000 related deaths occurred globally in 2018 [1] . 19 sub-Saharan African countries and India were responsible for carrying approximately 85% of the worldwide burden [1] . Plasmodium falciparum (Pf) malaria is a complex disease with a wide spectrum of clinical manifestations ranging from uncomplicated (UM) to severe malaria (SM). SM is defined by life-threatening anemia, metabolic acidosis, cerebral malaria (CM), and multiorgan system involvement [2] . Sequestration of Pf-parasitized erythrocytes within the microvasculature of vital organs in the human host is considered a key pathogenic event leading to SM [3, 4] . P. falciparum erythrocyte membrane protein 1 (PfEMP1) is encoded by the multicopy var gene family and mediates the sequestration of parasitized erythrocytes to host receptors [5, 6] .

Both parasite and host factors have been identified as significant contributors to SM [3] . This includes parasite genes, and var groups A, B, as well as domain cassette (DC) 8, DC11 and DC13 have been shown to be associated with SM [7, 8] . Similarly, among host genes, polymorphisms in intercellular adhesion molecule 1 (ICAM-1), cluster of differentiation 36 (CD36), tumor necrosis factoralpha (TNF-α), Interferon-gamma (IFN-γ), interleukin-1β, complement receptor-1 (CR-1), ATP binding cassette Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1103 3-020-05934 -1) contains supplementary material, which is available to authorized users. subfamily B member 1 (ABCB1) and adenosine A2a receptor (ADORA2A) have linked to the development of SM [9] [10] [11] [12] [13] [14] [15] [16] . Ribonuclease 3 (RNASE 3), which encodes eosinophil cationic protein (ECP), an important protein produced by eosinophils during inflammation and infection [17, 18] , was found to increase susceptibility to SM [19] [20] [21] [22] . Indeed, SM patients had higher ECP levels and hypereosinophilia compared to UM patients [21] . In addition, another study demonstrated that ECP can suppress the growth of Pf in in vitro [22] . Above findings led to several genetic studies of the RNASE 3 gene in African populations [19, 20] , which all showed an association between RNASE 3 polymorphisms (+ 371C > G and + 499G > C) and SM [19, 20] , further confirming its role in severity of the disease. The minor allele frequencies of + 371C > G and + 499G > C polymorphisms were more than 0.20 in African populations [19, 20] . These RNASE 3 polymorphisms have also been associated with other disease susceptibility such as allergic asthma [23] and parasitic-helminth infection [24] .

Here, we conducted a case-control study to assess the association of RNASE 3 polymorphisms with SM in India, as the same polymorphism can have heterogeneous effect in two populations due to genetic differences [25] . RNASE 3 polymorphism alleles, genotypes and haplotypes frequencies were compared between falciparum malaria patients with SM and UM.

All subjects were recruited from the Department of Medicine, Wenlock District Government Hospital, Mangalore, Karnataka, India. Prior written informed consent was obtained from each adult patient, or informed assent from a parent or legal guardian if the individual was ≤ 18 years. The research and ethics committee of the Kasturba Medical College (KMC) under Manipal Academy of Higher Education, Mangalore, Karnataka, India, approved the study (IEC KMC MLR 03-16/49). The Institutional review board of ICMR-National Institute of Malaria Research, New Delhi, India also reviewed and approved the study (ECR/NIMR/ EC/2012/39). Patient data obtained in this study was kept confidential and unique laboratory code was used for laboratory and dataset analyses.

For this case-control study, we used a convenience sampling method, whereby all available malaria subjects in a specific area are included. We enrolled patients with SM and UM caused by P. falciparum who were admitted or visited to the Department of Medicine, Wenlock District Government Hospital, the largest health facility in Mangalore and the main malaria referral hospital in the region, Karnataka, India, from July 2015 to December 2018. UM patients were used as controls to understand the role of RNASE 3 polymorphisms in severe malaria. SM was defined based on the modified WHO criteria [26] . A total of 151 patients were recruited (age range: 1-75 years), including 19 (12.6%) children (≤ 18 years, median: 12; interquartile range: 8) and 132 (87.4%) adults (> 18, median: 32; interquartile range: 22) participants; 19 patients were female (12.6%). All the participants were from the same Tuluva ethnic group, as determined on the basis of shared history, food habit, language and habitat region. Patients with axillary temperature > 37.5 °C and confirmed mono-infection of Pf by expert microscopy and rapid diagnostic tests (RDTs) were included in the study. P. falciparum positive patients with other Plasmodium species, HIV, HBsAg, HCV, pneumonia, bacterial meningitis, sepsis and tuberculosis co-infections were excluded from the study.

Giemsa-stained thick and thin blood smears were air-dried, and tested for the presence of Pf parasites under a light microscope fitted with a 100× oil immersion lens and a 10× eyepiece (Zeiss Primo Star, Germany), and parasitemia was quantified as previously described [27] . In addition, the National Vector Borne Disease Control Programme (NVBDCP) approved RDT kits were used as per the manufacturer's instructions to confirm Plasmodium infections. These kits were FalciVax™ Rapid Test for Malaria Pv/Pf No.: 05FK80) targeting both P. vivax-specific pLDH and P. falciparum-specific HRP-2 antigens. All the cases were successfully treated with the artemisinin-based combination therapy (ACT) as prescribed by the National Vector Borne Control Programme.

Patients positive for mono-Pf infections were subjected to venipuncture; 4 ml of whole blood was collected in EDTA Vacutainers (BD Vacutainer®) for hematological tests, and a further 4 ml blood was taken for biochemical liver and kidney function tests using Clot Activator Vacutainers (BD Vacutainer®). DxH 800 Hematology (Beckman Coulter) and Cobas® 6000 (Roche) analyzers were used for hematological and biochemical tests, respectively.

EDTA blood collected for each patient was sent to Chromous Biotech, Pvt. LTD, Bangalore, India (https ://www. chrom ous.com/index .php?q=chrom ous-biote ch/about -us), for DNA isolation, RNASE 3 gene amplification and DNA Sanger sequencing. In brief, 100-250 μl of blood was used for DNA isolation using the Chromous Biotech DNA extraction kit (Cat. No.: RKN25/26). Finally, DNA was eluted in 35 μl of elution buffer available in the kit. RNASE 3 gene was amplified using two primer sets (Set-1: Fw: 5′-TCC AGC AAG AGT GGT GGA TGA GAT -3′ and Rv: 5′-CTG TTG TCA CAT GCA ACT ACA TAG -3′; Set-2: Fw: 5′-TTG CCA TCC AGC ACA TCA GTC TGA and Rv: 5′-CTG GTT CCA CCT CTA TTA CGA TTG C-3′) covering a 2047 bp region (chr14: 20890932-20892978, including upstream and downstream sequences) of the RNASE 3 gene and targeting more than 150 reported polymorphisms (as per the Ensembl release 101; Human GRCh38.p13). In brief, RNASE 3 gene fragments were amplified separately in 50 μl reactions including 200 ng of each forward and reverse primers of set-1 and set-2, 5 μl of 10 × PCR buffer, 2 μl of dNTPs (10 mM), 2 μl of template DNA and 3 units of Taq® DNA polymerase, reaction volume was raised by PCR-grade water. The PCR reaction was performed with the initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 52 °C for 30 s, 72 °C for 2 min, followed by final extension at 72 °C for 7 min. DNA sequencing [28] was performed using an ABI Prism (Applied Biosystems, USA) 3500 genetic analyzer for automated DNA sequencer using ABI Prism Big-Dye Terminator v3.1 cycle sequencing kit. The direct DNA sequencing was performed for the two amplicons described above, of 1177 bp and 1180 bp long regions of RNASE 3 containing all the reported gene polymorphisms. The variations in the sequences were identified by sequence alignment using NCBI blast with reference sequence NC_000014.9.

To compare continuous data and categorical data between groups, respectively, we performed Mann Whitney U test and Fisher's exact test. Odds ratio (OR), 95% confidence interval (95% CI) and p value for the mutant allele of each RNASE 3 polymorphisms were calculated using an online version of MedCalc software (https ://www.medca lc.org/ calc/odds_ratio .php). The SHEsis (https ://analy sis.bio-x. cn/SHEsi sMain .htm), an online software tool, was used for haplotype and linkage disequilibrium (LD) analyses. The software uses a partition-ligation-combination-subdivision expectation maximization algorithm for haplotype inference with multiallelic markers for haplotype analysis. All haplotypes with a frequency below 0.03 were discarded [29, 30] .

We defined statistical significance as p < 0.05. Bonferroni correction was applied for multiple comparisons.

All study participants (n = 151) were positive for P. falciparum mono-infection, and no other species including P. vivax, P. malariae, P. ovale or P. knowlesi were identified. Among participants, median (± IQR) parasitemia was 39,446 ± 52,106 parasites/µL and median (± IQR) age was 27 ± 21 years. Thrombocytopenia (< 150,000 platelets/µL) was found in 60.9% of patients (92/151), and 10.6% (16/151) had severe thrombocytopenia (< 50,000 platelets/µL). 31.1% (47/151) and 68.9% (104/151) of all patients were diagnosed with SM and UM, respectively. Comparisons between the demographic, hematological and biochemical laboratory findings of the participants with SM and UM was performed and are shown in Table 1 . Statistically significant differences were only found for age, parasitemia, red blood cell counts, urea, bilirubin, AST, ALT, albumin levels and ratios of albumin and globulin (Table 1) . Among patients with SM, 10.6% (5/47) and 23.4% (11/47) of patients had multiple organ dysfunction and splenomegaly, respectively. Information on symptoms of severity of the patients as per the WHO criteria [26] is presented in Table 2 .

We successfully amplified 151 samples for the RNASE 3 gene followed by direct DNA sequencing, covering more than 150 polymorphisms. All the reported polymorphisms were present in the wild type form except for three polymorphisms [rs2233860 (+ 499G > C), rs2233859 (− 38C > A) and rs2073342 (+ 371C > G)], and no novel polymorphisms were identified in our samples. Among + 499G > C, − 38C > A and + 371C > G polymorphisms (Fig. S1) , a homozygous mutant genotype was only found for + 499G > C polymorphism, only 0.7% (1/151) of patients possessed a mutant genotype (CC). However, − 38C > A and + 371C > G polymorphisms were either present in the form of homozygous wild-type or heterozygous genotypes in the studied participants. Thus, − 38C > A, + 371C > G and + 499G > C were further considered for odds ratio, haplotype and linkage disequilibrium analyses. The minor allele and genotype frequencies of the entire studied population are shown in Table 3 .

Odds ratio was calculated for different genetic models, neither alleles nor genotypes of − 38C > A, + 371C > G and + 499G > C polymorphisms were found associated with SM and UM patients ( Table 4 ).

The haplotype analysis was performed for three (− 38C > A, + 371C > G and + 499G > C) polymorphisms of RNASE 3 gene by using SHEsis. Among the 8 possible haplotypes comprising the three polymorphic loci, C-G-G haplotype was associated with SM patients (OR = 3.03; p = 0.008) after Bonferroni correction. No, haplotypes were found associated with UM. The results of the haplotype analysis are shown in Table 5 .

LD was estimated for the three polymorphisms using the D′ values. The analysis showed strong linkage disequilibrium (D′ = 1 or > 0.75) between rs2233859 (− 38C > A), rs2073342 (+ 371C > G) and rs2233860 (+ 499G > C) polymorphisms in UM patients. However, no complete linkage between the three polymorphisms was observed in the SM group due to absence of LD between the polymorphisms rs2233859 and rs2073342 (D′ = 0.57). The results of the linkage analysis are shown in Fig. S2 .

In this first study aiming to decipher the effect of RNASE 3 polymorphisms on SM susceptibility in India, we used the direct DNA sequencing of clinical samples collected using a convenience sampling method from Pf-positive patients residing in Mangalore, Karnataka, on the Western coast of India. We show evidence for the absence of homozygous mutant genotypes for RNASE 3 reported polymorphisms, except for 3′ UTR + 499G > C polymorphism, which was only found in one patient. No significant genetic association was found for genotypes and alleles of RNASE 3 polymorphisms with SM in the studied Indian population. This contrasts markedly with two reports on African populations, which showed an association between RNASE 3 polymorphisms and SM susceptibility [19, 20] . In Ghana, + 371C > G polymorphism was found associated with CM, a neurological type of SM, and + 499G > C polymorphism was associated with SM in a Senegalese population [19, 20] . No such associations were found in the present study. These differing results may be due to several reasons. First, the same polymorphism can have heterogeneous effect in two geographically and genetically distinct populations, despite the endemicity of P. falciparum at both sites [25] . Second, the sample size used in the present study was small compared to previous studies [19, 20] . Third, the present study did not include participants with CM and no past history of malaria, which could have provided higher granularity in our association study. Indeed, the SM patients enrolled in the Senegalese study consisted primarily of CM and severe anemia cases [20] ; it is therefore possible that the reported association was driven by a high number of CM patients, which would be in line with the findings from Ghana [19] . It has to be noted that Mangalore city alone contributes about 72% of total malaria in Karnataka. In the last 5 years, the local district health department instead of the local municipal authority governs the malaria control operation. Following this, active and passive fever surveys through digital surveillance devices are in function throughout the city [31] . Thus, prompt diagnosis and treatment are provided. This may be one of the reasons for not finding CM cases in the present study. However, further studies including large groups of different SM subtypes are needed to test this hypothesis in Indian populations.

We show that in SM patients from India, there is a lack of homozygous mutant genotypes of + 371C > G and + 499G > C polymorphisms. This absence may indicate that mutant alleles may have a deleterious effect on the Indian population. This could be explained by the presence of heterozygous genotypes in our cohort, which may be to balance the deleterious effect of these mutant alleles, especially for + 371C > G missense-polymorphism that has been associated with SM in Ghanaian population [19] and with parasitic-helminth infections in Ugandan population [24] . The + 371C > G polymorphism, resulting in an Arg/ Thr substitution, drastically reduces the protein cytotoxicity. The change in cytotoxic activity could be due to the substitution, which creates a potentially new glycosylation site in ECP [24, [32] [33] [34] . According to the dbSNP database, a 0.73 minor allele (G) frequency of + 371C > G polymorphism has been found in Asia, suggesting that it may increase in Indian population in the future.

Haplotype analysis can provide pivotal evidence on human evolution, and the identification of genetic variants causing specific human traits through linkage disequilibrium [35] . C-G-G haplotype of − 38C > A, + 371C > G and + 499G > C polymorphisms were found associated with SM in this study. However, no complete linkage between the three polymorphisms was observed in the SM group. Therefore, in the absence of homozygous genotype for the mutant alleles of − 38C > A and + 371C > G polymorphisms, any conclusion on the association of C-G-G haplotype with SM would be speculative. Further genetic analyses involving a larger sample size in India are warranted to further explore the possible association we describe here.

SM predominantly affected adults in this study (median age 36 years, interquartile range 26 years), indicating a probable age shift in anti-malarial immunity [36] . This may have resulted from the recent decrease in transmission due to India's largest national malaria control program [37] . Higher bilirubin, AST and ALT levels were found in SM patients, confirming the association between hepatic injury and severe Pf infection described in previous studies [38] [39] [40] [41] . In addition, low albumin levels, albumin:globulin ratio, and high urea levels were noted in SM patients compared to UM, confirming the presence of liver and kidney injuries ( Table 2) . Parasitemia was also found significantly increased in SM patients. However, peripheral parasitemia may not be a true indicator of disease severity due to the sequestration of parasitized erythrocytes in the microvasculature of vital organs [42] .

This study has a few limitations. In the era of next-generation sequencing and genome wide association studies, we performed a case-control study to assess the role of RNASE 3 polymorphisms in SM susceptibility in an Indian population using Sanger DNA sequencing. This option was chosen as case-control studies using Sanger sequencing assays are less time consuming, cost-effective, reliable, and can be useful for recognizing associating factors of disease outbreaks, as well as current cases, and allow the assessment of multiple risk factors at once [43] . Another limitation was the lack of CM patients included in the study, which may explain the discrepancies between our findings and the ones reported in Ghana and Senegal. Lastly, ECP levels were not assessed in plasma samples collected from study participants due to limited funding.

In conclusion, we tested the hypothesis if RNASE 3 polymorphisms have a role in determining the SM susceptibility in an Indian population, and demonstrated that the homozygous mutant genotype of RNASE 3 polymorphisms (+ 371C > G and + 499G > C), which were shown to be associated with CM and SM in the population of Ghana and Senegal, respectively, were absent in our cohort of SM patients.

However, C-G-G haplotype (− 38C > A, + 371C > G and + 499G > C polymorphisms) frequency was significantly higher in SM patients compared to UM, suggesting its association with an increased risk of SM, and further confirming a role of RNASE 3 gene in SM.

Investigating the pathogenesis of severe malaria: a multidisciplinary and cross-geographical approach

The pathogenic basis of malaria

Clinical significance of sequestration in adults with severe malaria

Adhesion of Plasmodium falciparum-infected erythrocytes to human cells: molecular mechanisms and therapeutic implications

Severe malaria is associated with parasite binding to endothelial protein C receptor

Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children

Cytoadhesion to gC1qR through Plasmodium falciparum Erythrocyte Membrane Protein 1 in severe malaria

Variations in host genes encoding adhesion molecules and susceptibility to falciparum malaria in India

Tumour necrosis factor alpha promoter polymorphism, TNF-238 is associated with severe clinical outcome of falciparum malaria in Ibadan southwest Nigeria

IFN-gamma and TNF associated with severe falciparum malaria infection in Saudi pregnant women

Polymorphisms in interleukin-1beta and interleukin-1 receptor antagonist genes and malaria in Ghanaian children

Polymorphic variability in the interleukin (IL)-1beta promoter conditions susceptibility to severe malarial anemia and functional changes in IL-1beta production

Complement receptor 1 variants confer protection from severe malaria in Odisha, India

Genetic and epigenetic changes in host ABCB1 influences malaria susceptibility to Plasmodium falciparum

Categorical complexities of Plasmodium falciparum malaria in individuals is associated with genetic variations in ADORA2A and GRK5 genes

Eosinophil cationic protein (ECP): molecular and biological properties and the use of ECP as a marker of eosinophil activation in disease

Structural determinants of the eosinophil cationic protein antimicrobial activity

Polymorphisms in the RNASE3 gene are associated with susceptibility to cerebral malaria in Ghanaian children

Genetic variants of RNASE3 (ECP) and susceptibility to severe malaria in Senegalese population

Increased eosinophil activity in acute Plasmodium falciparum infection-association with cerebral malaria

Killing of Plasmodium falciparum by eosinophil secretory products

Associations of ECP (eosinophil cationic protein)-gene polymorphisms to allergy, asthma, smoke habits and lung function in two Estonian and Swedish sub cohorts of the ECRHS II study

The 434(G>C) polymorphism within the coding sequence of Eosinophil Cationic Protein (ECP) correlates with the natural course of Schistosoma mansoni infection

No gene is an island: the flip-flop phenomenon

Guidelines for the treatment of malaria

Association between inflammatory cytokine levels and anemia during Plasmodium falciparum and Plasmodium vivax infections in Mangaluru: a Southwestern Coastal Region of India

DNA sequencing with chain-terminating inhibitors

SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci

A partition-ligation-combination-subdivision EM algorithm for haplotype inference with multiallelic markers: update of the SHEsis

Indigenously developed digital handheld Android-based Geographic Information System (GIS)-tagged tablets (TABs) in malaria elimination programme in Mangaluru city

The functional heterogeneity of eosinophil cationic protein is determined by a gene polymorphism and post-translational modifications

A SELDI-TOF MS study of the genetic and post-translational molecular heterogeneity of eosinophil cationic protein

Protein post-translational modification in host defense: the antimicrobial mechanism of action of human eosinophil cationic protein native forms

Haplotype-association analysis

Immunity to malaria in an era of declining malaria transmission

Malaria elimination in India-The way forward

Malarial hepatitis: a heterogeneous syndrome

Jaundice in Plasmodium falciparum

Hepatocyte dysfunction and hepatic encephalopathy in Plasmodium falciparum malaria

Malarial hepatitis

Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2

Control Studies. StatPearls

We thank all study participants as well as physi-