Funding Information: This study was funded by the Ministry of Education and Science, Republic of Latvia, project ?Establishment of COVID-19 related biobank and integrated platform for research data in Latvia?, project No. VPP-COVID-2020/1-0016. We acknowledge The Boris and Inara Teterev Foundation for support to Riga Stradins University inpatient sample collection. The authors acknowledge the Latvian Biomedical Research and Study Centre and the Genome Database of the Latvian Population for providing the infrastructure, biological material, and data. Funding Information: This study was funded by the Ministry of Education and Science, Republic of Latvia, project “Establishment of COVID-19 related biobank and integrated platform for research data in Latvia”, project No. VPP-COVID-2020/1-0016. We acknowledge The Boris and Inara Teterev Foundation for support to Riga Stradins University inpatient sample collection. The authors acknowledge the Latvian Biomedical Research and Study Centre and the Genome Database of the Latvian Population for providing the infrastructure, biological material, and data.publishersversionPeer reviewe

Ansone, Laura

Birzniece, Liga

Briviba, Monta

Klovins, Janis

Kolesova, Oksana

Litvina, Helena Daiga

Megnis, Kaspars

Peculis, Raitis

Rescenko, Raimonds

Rovite, Vita

Rozentale, Baiba

Terentjeva, Anna

Viksna, Ludmila

Virologica Sinica

Funding Information: This study was funded by the Ministry of Education and Science, Republic of Latvia, project ?Establishment of COVID-19 related biobank and integrated platform for research data in Latvia?, project No. VPP-COVID-2020/1-0016. We acknowledge The Boris and Inara Teterev Foundation for support to Riga Stradins University inpatient sample collection. The authors acknowledge the Latvian Biomedical Research and Study Centre and the Genome Database of the Latvian Population for providing the infrastructure, biological material, and data. Funding Information: This study was funded by the Ministry of Education and Science, Republic of Latvia, project “Establishment of COVID-19 related biobank and integrated platform for research data in Latvia”, project No. VPP-COVID-2020/1-0016. We acknowledge The Boris and Inara Teterev Foundation for support to Riga Stradins University inpatient sample collection. The authors acknowledge the Latvian Biomedical Research and Study Centre and the Genome Database of the Latvian Population for providing the infrastructure, biological material, and data.Peer reviewe

Riga Stradins university

LETTERReplication of LZTFL1 Gene Region as a Susceptibility Locusfor COVID-19 in Latvian PopulationRaimonds Rescenko1 • Raitis Peculis1 • Monta Briviba1 • Laura Ansone1 • Anna Terentjeva2 • Helena Daiga Litvina1 •Liga Birzniece1 • Kaspars Megnis1 • Oksana Kolesova2 • Baiba Rozentale2 • Ludmila Viksna2 • Vita Rovite1 •Janis Klovins1Received: 30 March 2021 / Accepted: 24 August 2021 / Published online: 20 October 2021 Wuhan Institute of Virology, CAS 2021Dear Editor,Clinical outcomes of SARS-CoV-2 infection are highlyheterogeneous, ranging from symptoms-free infection todeath of the patient (Park et al. 2020). Although the out-come can be explained to some extent by age and comor-bidities, genetic factors have also been linked to theprognosis of SARS-CoV-2 infection (Hu et al. 2021; Liuet al. 2020; Maya et al. 2020; Pairo-Castineira et al. 2021;The Severe COVID-19 GWAS Group 2020; Zeberg andPääbo 2020). To this date, the association of nine singlenucleotide polymorphisms (SNPs) at genome-wide signif-icance level has been reported in the Genome WideAssociation Study (GWAS) catalog for severe COVID-19outcome phenotype (MONDO_0100096) (GWAS catalog2021). These SNPs were identified in two studies involving4378 COVID-19 patients of European, 176 admixedAmerican, and 62 of South Asian ancestry (Pairo-Casti-neira et al. 2021; The Severe COVID-19 GWAS Group2020). A leading association across these studies is foundin the 3p21.31 region, specifically near genes LZTFL1 andSLC6A20 (Pairo-Castineira et al. 2021; The SevereCOVID-19 GWAS Group 2020), and rs11385942 variantfrom this region also shows the lowest heterogenousP value in a worldwide cohort of 49 studies from 19countries (The COVID-19 Host Genetics Initiative andGanna 2021).While the primary attention in previous studies was toidentify genetic associations with the severity of theCOVID-19 disease, we decided to evaluate the alreadyreported genome-wide significant associations regardlessof the disease severity in a representative sample of 2692individuals from the population of Latvia. Additionally, weaimed to further examine the corresponding loci to identifynew associated polymorphisms in a region with positivesignals. The study included all 475 COVID-19 patientsrecruited to the Genome Database of Latvian Population(LGDB) (Rovite et al. 2018) from June 2020 to January2021. This cohort consisted of 146 hospitalized patients,while the rest represented less severe symptoms orasymptomatic cases. In total, 2217 controls were selectedfrom LGDB, representing the group of the general popu-lation and disease-specific biobank participants from theLatvian population with available genome-wide genotyp-ing data. This study was approved by the Central MedicalEthics Committee of Latvia (No. 01-29.1.2/928). The meanage of cases and controls were 47.3 [standard deviation(SD) = 17.8] and 54.9 (SD = 13.1) years old, respectively.The proportion of females was 64.5% in the case group and62.0% in the controls. After the genotype quality controland imputation using the TOPMed r2 imputation server, weperformed an association study with sex and age ascovariates. Association was performed with PLINK 1.9(Chang et al. 2015) according to the parameters defined inSAIGE software (Zhou et al. 2020), adding the first 20principal components (PCs) to control for populationstratification.We selected a total of nine significantly associated SNPsreported in the GWAS catalog from two previously pub-lished studies analyzing the association between severeCOVID-19 cases and population controls (Pairo-Castineiraet al. 2021; The Severe COVID-19 GWAS Group 2020).Table 1 summarizes the list of the selected SNPs. No sig-nificant deviation in allele frequencies (AF) was foundbetween the control group and the global AFs from the1000G Phase 3 reference set (The 1000 Genomes ProjectConsortium et al. 2015). Out of the nine selected variants,we identified three significantly associated LZTFL1 gene& Janis Klovinsklovins@biomed.lu.lv1 Latvian Biomedical Research and Study Centre, Riga 1067,Latvia2 Riga Stradins University, Riga 1007, Latvia123Virologica Sinica (2021) 36:1241–1244 www.virosin.orghttps://doi.org/10.1007/s12250-021-00448-x www.springer.com/12250(0123456789().,-volV)(0123456789().,-volV)polymorphisms rs71325088 [Bonferroni adjusted P-value(Padj) = 0.007, odds ratio (OR) = 1.46 95% confidenceinterval (95% CI) 1.17–1.81], rs11385942 (Padj = 0.005,OR = 1.47 95% CI 1.18–1.820) and rs73064425 (Padj-= 0.007, OR = 1.45 95% CI 1.17–1.80) after applying theBonferroni correction for multiple testing (Table 1). Weobserved a high degree of linkage disequilibrium (LD)between all three SNPs reflected by almost identical fre-quencies in case and control groups. These variants werealso the most significant (Phet = 7.2 9 10-25) in theworldwide meta-analysis of individuals with the SARS-CoV-2 infection, hospitalization, and critical illness (TheCOVID-19 Host Genetics Initiative and Ganna 2021). Allof these variants are believed to represent the region to be aremnant of Neanderthal gene pool introgression into themodern human population (Zeberg and Pääbo 2020).LZFTL1 gene has been implicated in ciliogenesis andintracellular trafficking of ciliary proteins, probablyimpacting airway epithelial cell function (Promchan andNatarajan 2020; Shelton et al. 2021). Leading polymor-phism rs11385942 is located at 618 bp upstream and670 bp downstream from LZTFL exons as well as 1800 bpupstream and downstream from CTCF and transcriptionfactor binding sites (GTEx Consortium 2018).Even though the primary analysis was performed on thecase group that included all patients regardless of severity,there was an obvious bias toward the inclusion of symp-tomatic patients as SARS-CoV-2 was more likely to betested in these patients than asymptomatic cases. We alsotested the association of the selected SNPs with diseaseseverity in our study group using hospitalized patients ascases against the same group of controls (Table 1). Allthree SNPs from the 3p21.31 region displayed a strongerassociation in the frame of this comparison (Padj-= 0.00006, OR = 2.14 95% CI 1.54–2.99) and 4.2 timeshigher homozygote prevalence for rs11385942, supportingthe notion that this locus increased the risk for respiratorysymptoms. Studies that account for participants’ exposureto the infection are needed to answer this question. How-ever, the other SNPs included in the study did not reachstatistical significance. The OR for almost all SNPs (ex-cluding rs6489867) was similar to those published previ-ously (Pairo-Castineira et al. 2021; The Severe COVID-19GWAS Group 2020) (Table 1). Probably larger samplesize is needed to replicate other loci. It should be noted thatthe 3p.21.31 locus is the only replicated locus out of all 28COVID-19 variants released in GWAS catalog to date,representing variants above P = 5 9 10-8 level.We also explored the 500 kb regions around thers11385942 to evaluate the association of other SNPs withthe main trait in our study. In total nine 3p21.31 locuspolymorphisms, rs2191031, rs3774641, rs72893671,rs35896106, rs13071258, rs34668658, rs17763742,Table1AssociationoftheselectedsinglenucleotidepolymorphismsandSARS-CoV-2infectionChrSNPIDRiskalleleMappedgenesCOVID-19patients(n=475)vs.controls(n=2217)HospitalizedCOVID-19patients(n=146)vs.controls(n=2217)P/PadjAF(case/ctrl)OR(95%CI)P/PadjAF(case/ctrl)OR(95%CI)GWAScatalogOR(95%CI)3rs71325088CSLC6A20,LZTFL10.0007/0.0070.13/0.091.46(1.17–1.81)0.00001/0.00010.16/0.092.10(1.50–2.92)1.9a(1.73–2.0)3rs11385942ALZTFL10.0005/0.0050.13/0.091.47(1.18–1.82)0.000006/0.000060.16/0.092.14(1.54–2.99)2.11b(1.70–2.61)3rs73064425TAC099782.3,LZTFL10.0008/0.0070.13/0.091.45(1.17–1.80)0.00001/0.000090.16/0.092.13(1.52–2.97)1.7aNR6rs143334143ACCHCR10.7/10.09/0.090.94(0.73–1.22)0.4/10.10/0.091.18(0.78–1.80)1.3a(1.27–1.48)9rs657152AABO0.1/0.8910.47/0.441.13(0.98–1.31)0.1/10.48/0.441.22(0.95–1.55)1.32b(1.20–1.47)12rs6489867COAS1,AC004551.10.9/10.30/0.310.99(0.85–1.16)0.7/10.29/0.310.96(0.73–1.25)1.2a(1.14–1.25)19rs2109069ADPP90.01/0.1150.30/0.271.22(1.04–1.44)0.1/10.30/0.271.22(0.94–1.59)1.2a(1.19–1.31)19rs11085727TTYK20.03/0.2350.31/0.281.20(1.02–1.40)0.1/10.32/0.281.23(0.95–1.60)1.2a(1.18–1.31)21rs13050728TAP000295.1,IFNAR20.6/10.39/0.381.04(0.90–1.21)0.06/10.43/0.381.28(0.99–1.64)1.2a(1.16–1.28)BoldrepresentsthesignificantP-valuesafterBonferronicorrectionaFromPairo-Castineiraetal.2021bFromTheSevereCOVID-19GWASGroup2020.BoldrepresentthesignificantP-values.Chr,Chromosomenumber;SNP,Singlenucleotidepolymorphism;AF,Allelefrequency;Padj,BonferroniadjustedP-value;ctrl,controls;OR,Oddsratio;CI,Confidenceinterval;NR,notrecorded1242 Virologica Sinica123rs17763537 and rs73062389 displayed lower P-value inour cohort compared to the leading rs11385942, with thestrongest association estimated for rs2191031(P = 0.00005, OR = 1.40 95% CI 1.19–1.64) located in theLZTFL1 gene (Fig. 1). Among these newly discoveredvariants, rs2191031, rs3774641 are located in a promoterflank and rs73062389 in CTCF region, while rs35896106overlaps enhancer of SLC6A20 gene. The prevalence ofrs2191031 is 2.3 times higher in our population comparedto replicated polymorphisms. To date, no function orclinical relevance has been ascribed for this variant; how-ever, rs2191031 is equivalently associated with differentialexpression of the LZTFL1 gene in the esophagus mucosa(P = 10-5, normalized effect size = - 0.22) (GTExConsortium 2018) and multiple other tissues pointing tosimilar etiology as replicated variants (Shelton et al.2021). However, it is important to note that none of thesenine variants have a lower P-value than rs11385942 in acohort of hospitalized COVID-19 patients.Using the retrospectively collected samples from apopulation-based biobank has some limitations. We cannotassess the exposure to SARS-CoV-2 for this group ofpeople and match that with the case group, as we do nothave the information about the possible rate of infectionamong this group. However, such a design does notincrease type I error, and this study did not aim to findgenetic factors facilitating protection against SARS-CoV-2infection, in which such a design would not be appropriate.Another shortcoming was that most patients included inthis study were recruited in December 2020, and extensivephenotype data, including follow-ups, were not yet avail-able for analysis. Such data would allow to elaborate theinteraction between different clinical data and genotypeand include the development of post-COVID-19 compli-cations as an essential phenotype for association study.In conclusion, we demonstrate supportive evidence forthe involvement of human 3p21.31 locus in the patho-physiology of COVID-19 disease using an independentcohort of patients and controls from the Latvian population.It highlights the importance of this genomic region forgenetic risk estimation in relation to SARS-CoV-2 infec-tion and the robustness of proper genetic associationstudies for replication purposes. Notably, the results pre-sented here provide a preliminary indication of variantswith possible functional effects and calls for further studiesexploring the validation of these variants.Acknowledgements This study was funded by the Ministry of Edu-cation and Science, Republic of Latvia, project ‘‘Establishment ofCOVID-19 related biobank and integrated platform for research datain Latvia’’, project No. VPP-COVID-2020/1-0016. We acknowledgeThe Boris and Inara Teterev Foundation for support to Riga StradinsUniversity inpatient sample collection. The authors acknowledge theLatvian Biomedical Research and Study Centre and the GenomeDatabase of the Latvian Population for providing the infrastructure,biological material, and data.Fig. 1 Manhattan plot of 1 Mb region at 3p21.31 locus. rs11385942variant is emphasized with a diamond shape and set as a reference forLD estimation with other SNPs in the region. Rs codes for three SNPsselected for replication are depicted in dark blue, while rs codes forother SNPs displaying lower P-value in our association study are inlight blue. The dotted line represents the common genome-widesignificance threshold. LD, linkage disequilibrium; SNP, singlenucleotide polymorphism.R. Rescenko et al.: LZTFL1 Gene Locus is Associated with Increased COVID-19 Risk 1243123Compliance with Ethical StandardsConflict of interest The authors declare that they have no conflict ofinterest.Animal and Human Rights Statement This study was approved by theCentral Medical Ethics Committee of Latvia (No. 01–29.1.2/928).ReferencesChang CC, Chow CC, Tellier LC, Vattikuti S, Purcell SM, Lee JJ(2015) Second-generation PLINK: rising to the challenge oflarger and richer datasets. GigaScience 4:7COVID-19 Host Genetics Initiative (2021) Mapping the humangenetic architecture of COVID-19. 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Replication of LZTFL1 Gene Region as a Susceptibility Locus for COVID-19 in Latvian Population

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Replication of LZTFL1 Gene Region as a Susceptibility Locus for COVID-19 in Latvian Population

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