The major genetic risk factor for severe COVID-19 does not show any association among South Asian populations

With the growing evidence on the variable human susceptibility against COVID-19, it is evident that some genetic loci modulate the severity of the infection. Recent studies have identified several loci associated with greater severity. More recently, a study has identified a 50 kb genomic segment introgressed from Neanderthal adding a risk for COVID-19, and this genomic segment is present among 16% and 50% people of European and South Asian descent, respectively. Our studies on ACE2 identified a haplotype present among 20% and 60% of European and South Asian populations, respectively, which appears to be responsible for the low case fatality rate among South Asian populations. This result was also consistent with the real-time infection rate and case fatality rate among various states of India. We readdressed this issue using both of the contrasting datasets and compared them with the real-time infection rates and case fatality rate in India. We found that the polymorphism present in the 50 kb introgressed genomic segment (rs10490770) did not show any significant correlation with the infection and case fatality rate in India.

Since the beginning of COVID-19 pandemic, it has been observed that people with a different ethnic background and country or continent of origin have variable degrees of susceptibility 1,2 . Though there are a few well known factors for higher susceptibility, e.g. age and comorbidity 3,4 , the hospitalisation of younger healthy people has also been reported 5 . Recent genome wide association study has identified a gene cluster at chromosome 3 as well as the ABO gene at chromosome 9 associated with the severe risk factor for COVID-19 among Europeans 6 . Subsequently, the COVID-19 Host Genetics Initiative has corroborated this result 7 . The worldwide meta-analysis of the COVID-19 Host Genetics Initiative has identified 13 genetic loci associated with higher susceptibility or higher severity 8 .
Zeberg and Pääbo 9 have identified a risk haplotype of 50 kb size introgressed from Neanderthals, which they called the 'Neanderthal core haplotype' . This risk haplotype was found to be present with an allele frequency of 30% among South Asians, 8% in Europeans and 4% among admixed Americans. The peak carrier frequency was estimated among the Bangladeshi population, where 63% carried at least one copy of this haplotype. The study also cited twice the risk of mortality among people of Bangladeshi extraction living in the UK as opposed to the native population of Brittanic pedigree 10 .
Conversely, three of our studies on ACE2, the gateway of SARS-CoV-2, identified a haplotype, shared among South Asians and East Eurasians, likely protecting them from severe risk [11][12][13] . Additionally, the spatial distribution of this haplotype showed strong association with the low infection as well as low case fatality rate (CFR) 13  www.nature.com/scientificreports/ To resolve this discrepancy between the two sets of findings and the associated claims, we have extracted a SNP (rs10490770) reported to be associated with the high risk for COVID-19 9 , from our published and unpublished genome wide datasets (Supplementary Table S1), and looked for existing association with the state-wise COVID-19 data of India.

Materials and methods
Zeberg and Pääbo 9 have mainly discussed about the SNP rs35044562. However, they reported 12 other SNPs present in the 'Neanderthal core haplotype' that are in high linkage disequilibrium (r 2 > 0.98) (Supplementary Table S2). SNP rs10490770 showed high LD (r 2 = 0.99) with the SNP rs35044562. The genome-wide genotype data by Illumina tagged rs2285666 and rs10490770 SNPs in their panel. Therefore, we searched the genotype datasets generated by this platform. The frequency data for both of the SNPs from various Indian populations were extracted using Plink 1.9 14 , from 1000 genome project data phase 3 15 , data published by the Estonian Biocentre [16][17][18][19] and our newly genotyped samples for various Indian states and Bangladesh (Supplementary  Table S1). In addition to our previous study 13

Results and discussion
In contrast to the conclusions drawn by Zeberg and Pääbo 9 , our study on ACE2 identified a haplotype that is frequent among South Asians and East Eurasians [11][12][13] . This haplotype is derived by a polymorphism rs2285666 responsible for elevated expression of ACE2. We have found high inverse correlation of this haplotype with the state-wise cases as well as the case-fatality rate (CFR) among Indian populations 13 . This correlation was significant at various timelines of the pandemic in India (Table 1). We verified the statistical tests with the updated data up to December 2020 and found these data to be consistent with previous observations ( Fig. 1 and Supplementary  Fig. S1). Thus, it is likely that the ACE2 SNP rs2285666 has played a significant role in modulating the susceptibility to the disease among Indian populations. In our search of the SNPs reported to be associated with high risk by Zeberg and Pääbo 9 , we found rs10490770 from genome-wide datasets 17,18,[21][22][23] . We applied the same tests done for the ACE2 SNPs (Fig. 1). The state-wise frequency variation of this SNP did not show any association either with the number of cases or the CFR (Table 1 and Supplementary Fig. S1). We repeated these regression tests for the number of cases as well as the CFR data, obtained during all the three months. However, none of them showed any association with the rs10490770 (p > 0.3) ( Table 1). It is interesting to note that this SNP (rs10490770) has been found to be associated with disease severity in the global data 8 . However, the lack of association for rs10490770 with COVID-19 cases or CFR in India is striking and suggests instead a complex susceptibility response among Indian populations. Along with the complex genetic structure 11,24 , socio-economic status 25 and hygiene 26 may have contributed to such a complex scenario. Furthermore, a detailed clinical and genome-wide association study on Indian COVID-19 patients would be useful to resolve this complexity.
Zeberg and Pääbo 9 used the data of higher susceptibility to the disease among the Bangladeshi population living in UK 10 to support their findings. By considering the effect of sex, age, socio-economic deprivation and region, this report found that people of Bangladeshi origin had double the risk of mortality as compared to people of British origin. However, the higher mortality rate for Bangladeshi population in the UK needs more detailed investigation on comorbidity, genetic admixture as well as local environment and socio-economic circumstances in their particular British context. More importantly, a similar trend had also been observed among admixed Americans, where some of the same qualifications may apply mutatis mutandis [27][28][29] . Furthermore, it is notable that among the Bangladeshi samples analysed by us, the tribal populations of Bangladesh showed almost three times less frequency of rs10490770 (Supplementary Table S1). This is likely due to the different population histories of the caste and tribal populations of Bangladesh 30,31 . The Tibeto-Burman speakers of Bangladesh show a closer genetic affinity with the East and Southeast Asian populations, whereas the Indo-European speaking caste Table 1. Estimates of Pearson correlation coefficient for the rs2285666 and rs10490770 with the real-time COVID-19 cases as well as case fatality rate among Indian populations. The significant values are shown in bold letters. *Correlation is significant at the 0.05 level (2-tailed). **Correlation is significant at the 0.01 level (2-tailed). rs2285666 (T) rs10490770 (C) FreqMay2020 FreqAug2020 FreqDec2020 CFRAug2020 CFRAug2020 www.nature.com/scientificreports/ populations incline with the Indian populations. Therefore, it is advised explicitly to differentiate between the caste and tribal populations while making any statement about Bangladeshi populations. Significantly, our data also show that the incidence of the allele rs2285666 has been found to occur in the highest frequency of 100% in Indian populations such as the Nishi and Kokborok (Tripuri), who represent Trans-Himalayan language communities ( Supplementary Fig. S1 and Supplementary Table S1). As a linguistic phylum, the Trans-Himalayan language family is widespread in parts of eastern Eurasia and includes languages such as Tibetan, Burmese, Mandarin, Cantonese and Hokkien. Thus, our extensive analyses on real-time data did not show any association of rs10490770 with the state-wise infection rates as well as CFRs, suggesting that the risk allele for COVID-19 in Europe does not play a significant role in COVID-19 severity in South Asia.

Data availability
All datasets generated for this study are included in the article/Supplementary Material.  www.nature.com/scientificreports/