Method for determining the level of risk of severe covid-19 disease in a patient

Abstract

A method for assaying the presence of two genetic polymorphisms whose occurrence significantly modifies the course of COVID-19 disease is disclosed. The genetic polymorphisms, namely rs143334143 locus and at the rs74956615. This method represents a new tool in the public health protection against COVID-19 disease.

Description

[0001] Method for determining the level of risk of severe COVID-19 disease in a patient

[0002] The invention relates to the fields of genetic diagnostics and public health, and in particular to a method for determining the level of risk of severe COVID-19 disease in a patient, comprising testing for the presence of two genetic polymorphisms whose presence significantly modifies the course of COVID-19 disease. The method of the invention provides a new tool in the public health protection against COVID-19 disease. COVID-19 disease, caused by the SARS-C0V2 virus, has taken a huge toll during the 2019-2021 pandemic due to insufficient intensive care units, the lack of effective triage, primary prophylaxis and appropriate drugs. Currently, the availability of primary prophylaxis in the form of protective vaccination allows for a reduction in the number of cases and their severity, but an additional tool assessing the individual genetic susceptibility of individuals to severe disease could be helpful at the triage stage of symptomatic patients and could also be a tool to assist in typing individuals who should be subjected to stricter epidemiological surveillance in the form of more frequent, regular vaccination.

[0003] The aim of the invention is to provide a method suitable for testing predisposition to severe COVID-19, in particular to identify individuals belonging to a group at increased risk of severe COVID-19. A patient at increased risk of severe COVID-19 is considered to be a person who has at least about 1.5-2 times the risk of severe COVID-19 found in the general population. In such individuals, strict application of primary prophylaxis is advisable: protective vaccination and following the Ministry of Health's anti-epidemic recommendations.

[0004] Essence of the invention

[0005] The subject of the invention is a method for determining the level of risk of severe COVID-19 disease in a patient, characterised in that the allele configuration at the rs143334143 locus and at the rs74956615 locus is assessed in a DNA sample taken from the patient, whereby:

[0006] - in the case where the GG allele configuration is found at the rs143334143 locus and the AT or AA allele configuration is found at the rs74956615 locus, the subject shall be considered to be at increased risk of severe COVID-19, - in the case where the AA or AG allele configuration is found at the rs143334143 locus and the TT allele configuration is found at the rs74956615 locus, the subject shall be considered to be at reduced risk of severe COVID-19, and

[0007] - in the case where one of the other configurations is found, the subject shall be considered to be at the average population risk of severe COVID-19.

[0008] Detailed description of the invention

[0009] A study of the genetic profiles of Caucasian patients with a different course of COVID- 19 was carried out. The following groups of patients with a different course of COVID- 19 participated in the study: a group of patients who were severely affected by COVID- 19, i.e. required hospitalisation, a group of patients who were symptomatic but did not require hospitalisation, asymptomatic or did not become infected despite being with a COVID-19 patient. A total of 340 patients participated in the study. DNA was extracted from peripheral blood and full whole-genome sequencing (WGS) was performed.

[0010] Surprisingly, for some polymorphism configurations at the rs143334143 locus and at the rs74956615 locus a statistically significant correlation with a different course of COVID-19 than the course observed in the general population was observed. These observations are summarised in Table 1 below.

[0011] Table 1. Results of the association between different allele configurations at the rs143334143 locus and at the rs74956615 locus and the course of COVID-19, in particular the risk of a severe course of COVID-19

[0012] Surprisingly, carriers of the GG allele configuration at the rs143334143 locus and the AT or AA allele configuration at the rs74956615 locus were found to have an increased risk of severe SARS-CoV-2 virus infection (HR>=1 .4).

[0013] At the same time, surprisingly, carriers of the AA or AG allele configuration at the rs143334143 locus and the TT allele configuration at the rs74956615 locus were found to have a reduced (HR<1 ) risk of severe COVID-19.

[0014] Also surprisingly, the remaining allele configurations were found to have a risk that is to be considered at the population-based risk.

[0015] The above observations should not be applied to non-Caucasian populations due to the very rare occurrence of the A allele in rs74956615.

[0016] Subsequently, in order to allow an easy assessment of the allele configuration at the rs143334143 locus and at the rs74956615 locus, a set of primers and probes was designed to allow for the identification and amplification of the flanking regions of the rs143334143 and rs74956615 polymorphisms by real-time PCR (qPCR).

[0017] The resulting assay allows for genotyping by PCR, especially real-time PCR (qPCR), in at most two sealed tubes using specially designed TaqMan-type molecular probes. In an embodiment of the invention, the TaqMan probes have been optimally labelled with two different universal fluorescent dyes with different fluorescence light emission spectra, which makes it possible to perform the assays in most commercially available PCR reaction devices and not only in devices designed for the assays of a particular manufacturer. Preferably, the use of MGB-type probes with increased affinity to DNA excludes the risk of genotyping error, while increasing the specific light emission of the probe.

[0018] In a preferred embodiment, the method is based on the use of endpoint genotyping technology with qPCR for two polymorphisms with a significant impact on population resistance to COVID-19: rs143334143 and rs74956615. Two TaqMan MGB probes are used in a multiplex reaction to discriminate between the alleles. One probe is fully complementary to the allele sequence of the frequent variant (A), the other to the sequence of the rare variant (B). Each probe is labelled at the 5’ end with a distinguishing fluorochrome - in the implementation of the invention this is FAM or VIC - and at the 3’ end with a quencher, for example NFQ. In the elongation phase of the PCR reaction, the perfectly matched probe is cleaved by Taq DNA polymerase, which has 5’— >3’ exonuclease activity. The fluorochrome is released, resulting in fluorescence emission. A probe that is not a perfect match will only be cleaved by the polymerase to a small extent. Therefore, the fluorochrome will be released to a much lesser extent.

[0019] The generated fluorescent signal (VIC or FAM) is read in the last cycle of the PCR reaction (endpoint) and directly indicates the allele configuration (AA, AB, BB) without the need for long and labour-intensive post-PCR steps that increase the risk of contamination.

[0020] The qPCR endpoint genotyping technology is characterised by simplicity of use and ease of interpretation of the results obtained, high throughput (up to 192 samples at a time), and the possibility of performing with generally available laboratory equipment (qPCR thermocycler) using DNA of different quality collected from both swab and peripheral blood. The assay for both polymorphisms is performed under the same reaction conditions and can be performed simultaneously, which is important when using the assay for screening large populations.

[0021] The subject of the invention in an embodiment is illustrated in Figures 1 and 2, where the results obtained in an exemplary implementation of the assay using the real-time PCR reaction for the rs143334143 polymorphism, using a homozygous GG control sample, a heterozygous GA control sample, a homozygous AA control sample and a non-template control (NTC) sample are shown. Detection channels of 465 nm and 540 nm were used. Fig. 1 shows the PCR reaction kinetics with differences in the fluorescence profiles for the probes used, while Fig. 2 shows the interpretation of the data derived from the ratio of fluorescence levels for the two probes, which are indicative of a given genotype.

[0022] The subject of the invention in an embodiment is illustrated in Figures 3 and 4, where the results obtained in an exemplary implementation of the assay using the real-time PCR reaction for the rs74956615 polymorphism, using a homozygous TT control sample, a heterozygous TA control sample, a homozygous AA control sample and a non-template control (NTC) sample are shown. Detection channels of 465 nm and 540 nm were used. Fig. 3 shows the PCR reaction kinetics with differences in the fluorescence profiles for the probes used, while Fig. 4 shows the interpretation of the data derived from the ratio of fluorescence levels for the two probes, which are indicative of a given genotype.

[0023] An exemplary implementation of the invention is given below.

[0024] Embodiment of the invention

[0025] In the present implementation, genotyping for the rs143334143 and rs74956615 polymorphisms in a test sample from a subject against control samples containing each of the 3 genotypes and a non-template control (NTC) is performed according to the following protocol.

[0026] DNA from subjects was isolated from peripheral blood, oral epithelial swab or salivabased kit (spit kit), provided that a minimum of 50 ng of genomic DNA was isolated.

[0027] For each assay, three control samples for each genotype and one non-template control (NTC) sample were preferably used. In the present implementation, the control samples of the genotypes preferably indicate the correctness of the reaction performed (correct performance of the reagents, correct thermal profile of the instrument, no inhibition of the reaction). The NTC control sample allows for the verification that the reagent eluate has not been contaminated with biological material of human origin or with the amplified PCR product during the preparation of the amplification reaction. If a positive signal is detected for the NTC control sample in at least one of the two detection channels, this indicates that reagent contamination has occurred.

[0028] 1 . The sequences of the primers, probes and controls used according to the invention: a) oligonucleotide sequences of the rs143334143 polymorphism primer: sequences SEQ ID NO. 1 and 2, b) oligonucleotide sequences of the rs74956615 polymorphism primer: sequence SEQ ID NO. 3 and 4, c) oligonucleotide sequences of the probes: sequence SEQ ID NO. 5, 6, 7, 8.

[0029] 2. The concentrations of primers and probes used according to the invention: a) Final concentrations of the primer and probe concentrate for the rs143334143 polymorphism: 100 pM b) Final concentrations of primer and probe concentrate for the rs74956615 polymorphism: 100 pM Appropriate amounts of primers and probes were prepared at the concentrations given above. All components were mixed by vortex and pelleted before mixture preparation. Mixtures of the probe and primer concentrates were then prepared and aliquoted in appropriate amounts into tubes.

[0030] 3. Positive controls used according to the invention: a) Positive controls for the rs143334143 polymorphism: b) Positive controls for the rs74956615 polymorphism:

[0031] 4. Description of the genotyping kit

[0032] The mastermix used in the assay contains Taq polymerase blocked with a monoclonal antibody and other components required for the real-time PCR reaction. Mastermix is stable at 4 degrees C, making it easy to transport and store prior to use. The kit also includes: 2 probe and primer mixes,

[0033] • 2 sets of 3 positive control samples for each genotype

[0034] • deionised PCR-grade water.

[0035] All assay components should be stored at +4°C in a dark place until first use. After dilution of primer concentrates, they should be transferred to -20°C. Reagents should be transported at +4°C, reducing delivery costs by eliminating the need to freeze the kit.

[0036] 5. Assay protocol: a) Preparation of DNA samples

[0037] • The concentration of DNA for the assay should be in the range of 5-50 ng / pL.

[0038] • For more concentrated samples, dilute with water (included in the kit) to a concentration of 5-50 ng / pL. b) Reagent preparation

[0039] • Before using the assay for the first time, add 247.5 pL of DEPC water (supplied) to each of the reagents PC1 and PC2 to obtain working concentrations.

[0040] • Mix the reagents thoroughly by pipetting or vortexing.

[0041] • After mixing, centrifuge the reagents briefly to prevent droplets from settling on the cap or walls. c) PCR reaction preparation

[0042] • The assay consists of 2 PCR reactions, separately with reagents MX, PC1 , K1A, K1 B, K1C (rs143334143 assay) and separately with reagents MX, PC2, K2A, K2B, K2C (rs74956615 assay).

[0043] Reaction preparation method:

[0044] • Insert the prepared reactions, pipetted into a 96-well plate, into a PCR thermocycler. It is possible to perform a joint reaction for both mixtures 1 and 2 on one reaction plate, provided that up to 48 reactions are prepared.

[0045] The PCR reaction profile is identical for both polymorphisms:

[0046] 6. Genotyping

[0047] • Genotype determination for each polymorphism is based on the 3 genotypespecific reaction controls included in the kit.

[0048] • The controls must be included in the assay each time, as must the non-template control (NTC). These controls determine the clusters around which the signals for the test samples will cluster.

[0049] The genotypes in the reaction controls are shown in the table below.

[0050] 7. Genotype determination for the rs143334143 polymorphism

[0051] Homozygotes of the dominant allele (GG) and the K1A control are grouped in the top left corner (green), heterozygotes (GA) and the K1 B control in the middle of the diagram (red), homozygotes of the rarer allele (AA) and the K1C control at the bottom right (blue). Reactions with no or insufficient DNA matrix are grouped near the bottom left corner (Figure 2).

[0052] 8. Genotype determination for the rs74956615 polymorphism

[0053] Homozygotes of the dominant allele (TT) and the K2A control group in the upper left corner (green), heterozygotes (TA) and the K2B control in the middle of the diagram (red), homozygotes of the rarer allele (AA) and the K2C control at the bottom right (blue). Reactions with no or insufficient DNA matrix are grouped near the bottom left corner (Figure 4).

[0054] 9. Interpretation of assay results

[0055] The assay is considered to have been performed correctly when:

[0056] • the matrix-free control does not demonstrate PCR amplification and

[0057] • all control samples (K1A, K1 B, K1C, K2A, K2B, K2C) demonstrate the correct genotype according to the assay leaflet and

[0058] • the results for the test sample can be assigned to one of the 3 genotypes described in points 7 to 8.

[0059] If the assay result is inconclusive, it must be performed again using a new DNA sample. The correlations revealed in Table 1 should be used to determine the risk level for severe COVID-19.

[0060] Specifically, in the case where the GG allele configuration is found at the rs143334143 locus and the AT or AA allele configuration is found at the rs74956615 locus, the subject shall be considered to be at an increased risk of severe COVID-19.

[0061] On the other hand, in the case where the AA or AG allele configuration is found at the rs143334143 locus and the TT allele configuration is found at the rs74956615 locus, the subject shall be considered to be at a reduced risk of severe COVID-19.

[0062] Furthermore, in the case where one of the other configurations is found, the subject shall be considered to be at the average population risk of severe COVID-19.

[0063] The present embodiment is not a limiting example of the scope of the invention. The assay has been validated for thermocyclers: Roche LightCycler480, Cobas Z480 and Bio-Rad CFX96.

[0064] After designing the assay and developing the qPCR reaction conditions, the performance of the assay was confirmed in a validation study using 50 DNA samples. Endpoint genotyping by qPCR was performed on previously sequenced clinical samples, pre-screened by whole-genome sequencing (WGS), and obtained 100% genotype concordance with the orthogonal method. Sequence listing

[0065] Sequence SEQ ID NO. 1

[0066] Primer description: rs143334143_F

[0067] Sequence: 5‘-CGGGCCTT GAAGGAATCAGA-3’

[0068] Sequence SEQ ID NO. 2

[0069] Primer description: rs143334143_R

[0070] Sequence: 5‘-CCTCCCGTGAAGATACAGAGACT-3’

[0071] Sequence SEQ ID NO. 3

[0072] Primer description: rs74956615_F

[0073] Sequence: 5‘-GGCGAAACCCTGTCTCTACT-3’

[0074] Sequence SEQ ID NO. 4

[0075] Primer description: rs74956615_R

[0076] Sequence: 5‘-CCCTTGTTTTCTCCCAGTTCC-3’

[0077] Sequence SEQ ID NO. 5

[0078] Probe description: rs143334143_A

[0079] Sequence: 5‘-VIC- AAGTCAGTTGTCAAAGTT-NFQ-3’.

[0080] Sequence SEQ ID NO. 6

[0081] Probe description: rs143334143_B

[0082] Sequence: 5‘-6FAM- AGTCAGTTGACAAAGTT-NFQ-3’

[0083] Sequence SEQ ID NO. 7

[0084] Probe description: rs74956615_A

[0085] Sequence: 5‘-VIC- TGAGCTGAGACTGTGCCATT-NFQ-3’.

[0086] Sequence no. SEQ ID NO. 8

[0087] Probe description: rs74956615_B

[0088] Sequence: 5‘-6FAM- TGAGCCGAGACTGTGCCATT-NFQ-3’

Claims

Claims1. A method for determining the level of risk of severe COVID-19 disease in a patient, characterised in that the allele configuration at the rs143334143 locus and at the rs74956615 locus is assessed in a DNA sample taken from the patient, whereby a polymerase chain reaction is performed using at least one pair of primers selected from oligonucleotide sequences: SEQ ID NO. 1 , 2, 3 or 4, whereby:- in the case where the GG allele configuration is found at the rs143334143 locus and the AT or AA allele configuration is found at the rs74956615 locus, the subject shall be considered to be at increased risk of severe COVID-19,- in the case where the AA or AG allele configuration is found at the rs143334143 locus and the TT allele configuration is found at the rs74956615 locus, the subject shall be considered to be at reduced risk of severe COVID-19, and- in the case where one of the other configurations is found, the subject shall be considered to be at the average population risk of severe COVID-19.

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