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 (WHO, 2019) . Nineteen sub-Saharan African countries and India were responsible for carrying approximately 85% of the worldwide burden (WHO, 2019) . 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 (Wassmer et al., 2015) . Sequestration of Pfparasitized erythrocytes within the microvasculature of vital organs in the human host is considered a key pathogenic event leading to SM (Miller et Waters et al., 1987) . Indeed, SM patients had higher ECP levels and hypereosinophilia compared to UM patients (Kurtzhals et al., 1998) . In addition, a parallel study demonstrated that ECP can suppress the growth of Pf in in vitro (Waters et al., 1987) . These findings led to several genetic studies of the RNASE 3 gene in African populations (Adu et al., 2011; Diop et al., 2018) , which all showed an association between RNASE 3 polymorphisms and SM (Adu et al., 2011; Diop et al., 2018) , further confirming its role in severity of the disease.
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 (Lin et al., 2007) . RNASE 3 polymorphism alleles, genotypes and haplotypes frequencies were compared between falciparum malaria patients with SM and UM. Finally, DNA was eluted in 35μl of elution buffer available in the kit. RNASE 3 gene was amplified using two primer sets (Set-1: Fw: 5'-TCCAGCAAGAGTGGTGGATGAGAT-3' and Rv: 5'-CTGTTGTCACATGCAACTACATAG-3'; Set-2: Fw: 5'-TTGCCATCCAGCACATCAGTCTGA and Rv: 5'-CTGGTTCCACCTCTATTACGATTGC-3') covering 2047bp region (chr14: 20890932 -20892978) of the RNASE 3 gene. In brief, RNASE 3 gene fragments were amplified separately in 50μl reactions including 200ng of each forward and reverse primers of set-1 and set-2, 5μl of 10x PCR buffer, 2μl of dNTPs (10mM), 2μl of template DNA and 3 units of Taq® DNA polymerase, reaction volume was raised by PCR-grade water. The reaction volume was prepared with PCR-grade water. The PCR reaction was performed with the initial denaturation at 94°C for 5min, followed by 35 cycles of 94°C for 30 sec, 52°C for 30 sec, 72°C for 2min, followed by final extension at 72°C for 7min. DNA sequencing (Sanger et al., 1977) was performed using ABI Prism (Applied Biosystems, USA) 3500 genetic analyzer automated DNA sequencer using ABI Prism BigDye Terminator v3.1 cycle sequencing kit. The direct DNA sequencing was performed for the two amplicons described above, of 1177bp and 1180bp 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 online version of MedCalc software (https://www.medcalc.org/calc/odds_ratio.php). The SHEsis (http://analysis.bio-x.cn/SHEsisMain.htm), an online software tool, was used for haplotype and . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint this version posted June 18, 2020. Table 2 .
We successfully amplified 151 samples for RNASE 3 gene followed by direct DNA sequencing. Among all the reported gene polymorphisms, homozygous mutant genotype was only found for rs2233860 polymorphism, only 0.7% (1/151) patients possessed a mutant genotype (CC). However, rs2233859 and rs2073342 polymorphisms were either present in the form of homozygous wild-type or heterozygous genotype in the studied participants (Table 3) . Thus, rs2233859, rs2073342 and rs2233860 were further considered for odds ratio, haplotype and linkage disequilibrium analyses.
Odds ratio was calculated for different genetic models, neither genotypes nor alleles of rs2233859, rs2073342 and rs2233860 polymorphisms were found associated with SM and UM patients ( Table 4 ).
The haplotype analysis was performed for three (rs2233859, rs2073342 and rs2233860) 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<0.001). 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 calculated D′ values. The analysis showed strong linkage disequilibrium (D′ = 1 or > 0.75) between rs2233859, rs2073342 and rs2233860 polymorphisms in UM patients. However, no complete linkage between the three . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 18, 2020. 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. 1 .
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 18, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 18, 2020. Haplotype analyses can provide pivotal evidence on human evolution, and the identification of genetic variants causing specific human traits through linkage disequilibrium (Liu et al., 2008) . C-G-G haplotype of rs2233859, rs2073342 and rs2233860 polymorphisms were found associated with SM (p<0.05) 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 rs2233859 and rs2073342 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 (Fowkes et al., 2016) . This may have resulted from the recent decrease in transmission due to India's largest national malaria control program (Ghosh and Rahi, 2019) . 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 (Anand et al., 1992; Chawla et al., 1989; Kochar et al., 2003a; 2003b ). 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 (Dondorp et al., 2005) .
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 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 (Tenny and Hoffman, 2020) . 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 have made an attempt to decipher the genetic association between RNASE 3 polymorphisms and SM in an India population, and demonstrated that the homozygous mutant genotype of RNASE 3 polymorphisms (rs2073342 and rs2233860), 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 of rs2233859, rs2073342 and rs2233860 polymorphisms were associated with a higher susceptibility to SM, further confirming a role of RNASE 3 gene in malaria severity.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 18, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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