The present invention relates to the “Stimulator of Interferon Genes” (STING) gene,
and to single nucleotide polymorphisms (SNPs) in the STING gene, as well as to the
STING protein and variants of STING protein resulting from the SNPs. The invention
5 particularly relates to methods and kits for distinguishing and detecting STING gene
SNPs (i.e. STING genotyping) and STING protein variants expressed by humans. The
present invention also relates to the uses of the methods and kits as a diagnostic tool to
assess the suitability of individuals for STING agonist or antagonist therapy, and also to
methods of treatment with a STING agonist or antagonist.
10
STING, also known as transmembrane protein 173, is an adaptor protein that binds
cyclic dinucleotides (CDN) or agonist compounds, leading to the activation of IκB
kinase (IKK) and TANK-binding kinase (TBK1), which when phosphorylated, activate
Nuclear Factor kappa-light-chain-enhancer of activated B cells (NFκB) and interferon
15 regulatory factor 3 (IRF3), respectively. These activated proteins translocate to the
nucleus and induce transcription of genes encoding type I interferons (IFN) and
inflammatory cytokines to promote an innate immune response against pathogens or
neoplasms [1].
20 Cancer immunotherapy modulates and leverages the host’s immune system to treat
cancer, and, over the past decade, there have been significant advances in the field of
cancer treatment. Numerous approaches have been explored to elicit or augment
anticancer innate immunity and/or adaptive immunity. Recently, activation of STING,
using STING agonists, has shown great potential to enhance antitumor immunity
25 through the induction of a variety of pro-inflammatory cytokines and chemokines,
including type I IFNs. A number of natural and synthetic STING agonists have been
discovered and developed, and tested in preclinical models and in the clinic for the
immunotherapy of various diseases, such as cancer and infectious diseases. For
example, cyclic dinucleotides (CDNs), such as cyclic dimeric guanosine monophosphate
30 (c-di-GMP), cyclic dimeric adenosine monophosphate (c-di-AMP), and cyclic GMPAMP (cGAMP), are a class of STING agonists that can elicit immune responses.
Conversely, STING antagonists have also become an area of therapeutic interest, as
they block overactive STING proteins in various autoimmune diseases. Recently, a
35 number of companies have developed various small molecule STING antagonists, such
as C-176 and H-151.
- 3 -
Analysis of single nucleotide polymorphism (SNP) data from the 1000 Genome Project
revealed that there are five major STING haplotypes that carry specific SNPs, resulting
in variations in the STING polypeptide sequence. The major STING variant is R232,
5 characterised by the presence of an arginine at the 232 position. This is the most
prevalent human STING haplotype with an allele frequency of 57.6%, and is therefore
considered to be the wild type. STING variant HAQ (R71H-G230A-R293Q), has an
allele frequency of 20.4% and contains three SNPs. This HAQ variant has a histidine at
the 71 position in place of an arginine, an alanine at the 230 position in place of glycine,
10 and a glutamine at the 293 position in place of arginine. STING variant H232 (R232H)
is characterised by a histidine at the 232 position in place of arginine, with an allele
frequency of 13.7%. STING haplotype AQ (G230A-R293Q), contains an alanine at the
230 position in place of glycine, and a glutamine at the 293 position in place of
arginine, with an allele frequency of 5.2%. Finally, STING variant Q (R293Q), contains
15 a glutamine at the 293 position in place of arginine, and has an allele frequency of 1.5%
[2].
Each of these STING protein variants differs in the production of IFNs upon activation.
For example, the natural STING ligand cGAMP is a weaker activator of the H232
20 variant. Therefore, it will be appreciated that the therapeutic application of a STING
agonist or an antagonist will be better directed if the identification of the STING alleles
present in a specific patient who is about to undergo STING agonist or antagonist
treatment is known.
25 Currently, there are three methods used for STING genotyping, namely Sanger
sequencing, Taqman-PCR, and FRET hybridisation probe based melt curve analysis.
Sanger sequencing requires highly advanced instrumentation, and is therefore a very
expensive method for detecting SNPs. In addition, the laboratory protocols and
interpretation of the results require significant dedicated hands-on operator time,
30 resulting in a long turnaround time of approximately 37 hours. TaqMan-PCR and FRET
hybridisation melt curve analysis are less time-consuming compared to Sanger
sequencing, with a total turnaround time of approximately 3 to 5 hours. However, the
initial cost of the instrumentation required for each of these methods is very high and is
not affordable by many laboratories. In addition, the FRET-based method also utilises
35 two sets of probes, adding to the overall cost of this technique.
- 4 -
Accordingly, there remains a significant need in the art for an improved method for
STING genotyping, i.e. for detecting the presence or absence of a genetic polymorphism
pattern in the STING gene of a subject. In particular, there is a need for a method that
provides a fast, cheap and convenient way by which the STING genotype of a patient
5 can be determined. Accordingly, this will lead to improved diagnostic tools for
assessing the suitability of STING agonist/antagonist therapy for patients suffering
from diseases, such as cancer, chronic inflammation associated with fibrosis or
autoinflammatory disease, or infectious diseases, such as those caused by viruses,
bacteria, parasites and fungi.
10
Thus, according to a first aspect of the invention, there is provided a method for
determining the genetic polymorphism pattern in the “Stimulator of Interferon Genes”
(STING) gene in a subject, the method comprising:
- amplifying, in a sample obtained from a subject, a sequence of genomic DNA
15 comprising a single nucleotide polymorphism (SNP) within the STING gene;
and
- performing restriction fragment length polymorphism (RFLP) pattern analysis
on the amplified DNA to determine the genetic polymorphism pattern in the
STING gene in the sample.
20
According to a second aspect, there is provided an apparatus for determining the
genetic polymorphism pattern in the “Stimulator of Interferon Genes” (STING) gene in
a subject, the apparatus comprising:
- means for amplifying, in a sample obtained from a subject, a sequence of
25 genomic DNA comprising a single nucleotide polymorphism (SNP) within the
STING gene; and
- means for performing restriction fragment length polymorphism (RFLP)
pattern analysis on the amplified DNA to determine the genetic polymorphism
pattern in the STING gene in the sample.
30
According to a third aspect, there is provided a method for determining the efficacy of a
treatment of a subject with a “Stimulator of Interferon Genes” (STING) agonist or a
STING antagonist, the method comprising:
- determining the genetic polymorphism pattern in the Stimulator of Interferon
35 Genes (STING) gene in a sample obtained from a subject using the method
according to the first aspect or the apparatus of the second aspect; and
- 5 -
- determining the suitability of the subject for STING agonist or antagonist
therapy based on the genetic polymorphism pattern in the STING gene.
In a fourth aspect of the invention, there is provided a diagnostic or prognostic tool for
5 assessing the suitability of a subject for “Stimulator of Interferon Genes” (STING)
agonist or antagonist therapy, comprising:
- determining the genetic polymorphism pattern in the Stimulator of Interferon
Genes (STING) gene in a sample obtained from a subject using the method of
the first aspect or the apparatus of the second aspect; and
10 - determining the suitability of the subject for STING agonist or antagonist
therapy based on the genetic polymorphism pattern.
According to a fifth aspect, there is provided a method of treating a subject suffering
from a disease characterised by underactive “Stimulator of Interferon Genes” (STING)
15 protein or underexpressed STING gene, the method comprising:
- amplifying, in a sample obtained from a subject, a sequence of genomic DNA
comprising a single nucleotide polymorphism (SNP) within the STING gene;
- performing restriction fragment length polymorphism (RFLP) pattern analysis
on the amplified DNA to determine the genetic polymorphism pattern in the
20 STING gene in the sample; and
- administering, or having administered, to the subject, a STING agonist, thereby
treating the disease.
According to a sixth aspect, there is provided a method of treating a subject suffering
25 from a disease characterised by overactive “Stimulator of Interferon Genes” (STING)
protein or overexpressed STING gene, the method comprising:
- amplifying, in a sample obtained from a subject, a sequence of genomic DNA
comprising a single nucleotide polymorphism (SNP) within the STING gene;
- performing restriction fragment length polymorphism (RFLP) pattern analysis
30 on the amplified DNA to determine the genetic polymorphism pattern in the
STING gene in the sample; and
- administering, or having administered, to the subject, a STING antagonist, thereby
treating the disease.
35 Advantageously, the methods and apparatus of the first and second aspects of the
invention enable the very quick and simple determination of the presence or absence of
- 6 -
certain SNPs in the STING gene and, accordingly, the specific STING alleles carried by
subjects using tissue or blood samples which can yield very accurate results within only
a few hours. The methods and apparatus are inexpensive and have minimal
requirements in terms of investment in instrumentation. In addition, genotyping can
5 be easily carried out by simple visualisation of restriction fragments by gel
electrophoresis, for which no specific software and little technical expertise is required.
Considering the number of patients being tested per day per institution, the methods of
the invention are both cost-effective and simple to perform. Therefore, the methods
provide a very fast and cheap way by which the STING genotype of a patient can be
10 determined. As such, the suitability of a patient for treatment with a STING agonist or
antagonist can be readily determined, as per the third and fourth aspects of the
invention. This means that the correct and most effective STING agonist or antagonist
is administered to the subject in subsequent therapy, according to the fifth and sixth
aspects of the invention.
15
The STING gene is located on chromosome 5 at position 31.2, and one embodiment of
the coding sequence of STING is known and readily accessible at
www.ncbi.nlm.nih.gov. The polymorphism pattern in the STING gene may comprise at
least one polymorphism or polymorphic region in the STING gene. The polymorphism
20 pattern in the STING gene comprises more than one, and preferably two,
polymorphisms or polymorphic regions in the STING gene. The polymorphism pattern
in the STING gene comprises three or more polymorphisms or polymorphic regions in
the STING gene.
25 The term “polymorphism” can refer to the co-existence, within a population, of more
than one form of a gene or portion thereof (e.g. an allelic variant). A portion of a gene of
which there are at least two different forms, i.e. two different nucleotide sequences, is
referred to as a “polymorphic region of a gene”. A specific genetic sequence at a
polymorphic region of a gene is known as an allele.
30
The term “allele” can refer to the different sequence variants found at different
polymorphic sites in DNA obtained from a subject. For example, each polymorphic
region of the STING gene has at least two different alleles. The sequence variants of
each allele may be single or multiple base changes, including without limitation
35 insertions, deletions, or substitutions, or may be a variable number of sequence
repeats. Thus, a polymorphic region may be a single nucleotide (i.e. a single nucleotide
- 7 -
polymorphism, or SNP), the identity of which differs in different alleles. In other
embodiments, a polymorphic region can also be several nucleotides long.
The term “genotype”, “allelic pattern” or “polymorphism pattern” can refer to the
5 identity of an allele or alleles at one or more polymorphic sites. A genotype, allelic
pattern or polymorphism pattern may consist of either a homozygous or heterozygous
state at one or more polymorphic sites.
There are currently known to be five major STING gene haplotypes that carry specific
10 SNPs, resulting in variations in the STING polypeptide sequence.
The term “haplotype” can refer to a set of genetic or DNA variants, such as SNPs, that
are usually inherited together, and these sets of haplotypes are usually located on one
chromosome. The alleles making up a haplotype can be located in different places on
15 the chromosome but that are inherited together.
Hence, the polymorphism pattern in the STING gene may comprise at least one SNP, at
least two SNPs, at least three SNPs, or at least four SNPs in the STING gene.
20 In a first embodiment, a STING variant is R232. In this embodiment, an individual has
an arginine at position 232. This is the most prevalent human STING variant with an
allele frequency of 57.6%, and is therefore considered to be the wild-type.
The SNP which encodes R232 is known as rs1131769. The alleles of STING rs1131769
25 SNP may be identified as (i) a G-allele, and (ii) an A-allele. Therefore, the method may
comprise detecting or determining the G-allele or the A-allele of the STING rs1131769
SNP.
The nucleotide sequence encoding one embodiment of human STING variant R232 is
30 referred to herein as SEQ ID No: 1, as follows:
GTTCATTTTTCACTCCTCCCTCCTAGGTCACACTTTTCAGAAAAAGAATCTGCATCCTGGAAACCAGAAG
AAAAATATGAGACGGGGAATCATCGTGTGATGTGTGTGCTGCCTTTGGCTGAGTGTGTGGAGTCCTGCTC
AGGTGTTAGGTACAGTGTGTTTGATCGTGGTGGCTTGAGGGGAACCCGCTGTTCAGAGCTGTGACTGCGG
35 CTGCACTCAGAGAAGCTGCCCTTGGCTGCTCGTAGCGCCGGGCCTTCTCTCCTCGTCATCATCCAGAGCA
GCCAGTGTCCGGGAGGCAGAAGATGCCCCACTCCAGCCTGCATCCATCCATCCCGTGTCCCAGGGGTCAC
GGGGCCCAGAAGGCAGCCTTGGTTCTGCTGAGTGCCTGCCTGGTGACCCTTTGGGGGCTAGGAGAGCCAC
- 8 -
CAGAGCACACTCTCCGGTACCTGGTGCTCCACCTAGCCTCCCTGCAGCTGGGACTGCTGTTAAACGGGGT
CTGCAGCCTGGCTGAGGAGCTGCGCCACATCCACTCCAGGTACCGGGGCAGCTACTGGAGGACTGTGCGG
GCCTGCCTGGGCTGCCCCCTCCGCCGTGGGGCCCTGTTGCTGCTGTCCATCTATTTCTACTACTCCCTCC
CAAATGCGGTCGGCCCGCCCTTCACTTGGATGCTTGCCCTCCTGGGCCTCTCGCAGGCACTGAACATCCT
5 CCTGGGCCTCAAGGGCCTGGCCCCAGCTGAGATCTCTGCAGTGTGTGAAAAAGGGAATTTCAACGTGGCC
CATGGGCTGGCATGGTCATATTACATCGGATATCTGCGGCTGATCCTGCCAGAGCTCCAGGCCCGGATTC
GAACTTACAATCAGCATTACAACAACCTGCTACGGGGTGCAGTGAGCCAGCGGCTGTATATTCTCCTCCC
ATTGGACTGTGGGGTGCCTGATAACCTGAGTATGGCTGACCCCAACATTCGCTTCCTGGATAAACTGCCC
CAGCAGACCGGTGACCGTGCTGGCATCAAGGATCGGGTTTACAGCAACAGCATCTATGAGCTTCTGGAGA
10 ACGGGCAGCGGGCGGGCACCTGTGTCCTGGAGTACGCCACCCCCTTGCAGACTTTGTTTGCCATGTCACA
ATACAGTCAAGCTGGCTTTAGCCGGGAGGATAGGCTTGAGCAGGCCAAACTCTTCTGCCGGACACTTGAG
GACATCCTGGCAGATGCCCCTGAGTCTCAGAACAACTGCCGCCTCATTGCCTACCAGGAACCTGCAGATG
ACAGCAGCTTCTCGCTGTCCCAGGAGGTTCTCCGGCACCTGCGGCAGGAGGAAAAGGAAGAGGTTACTGT
GGGCAGCTTGAAGACCTCAGCGGTGCCCAGTACCTCCACGATGTCCCAAGAGCCTGAGCTCCTCATCAGT
15 GGAATGGAAAAGCCCCTCCCTCTCCGCACGGATTTCTCTTGAGACCCAGGGTCACCAGGCCAGAGCCTCC
AGTGGTCTCCAAGCCTCTGGACTGGGGGCTCTCTTCAGTGGCTGAATGTCCAGCAGAGCTATTTCCTTCC
ACAGGGGGCCTTGCAGGGAAGGGTCCAGGACTTGACATCTTAAGATGCGTCTTGTCCCCTTGGGCCAGTC
ATTTCCCCTCTCTGAGCCTCGGTGTCTTCAACCTGTGAAATGGGATCATAATCACTGCCTTACCTCCCTC
ACGGTTGTTGTGAGGACTGAGTGTGTGGAAGTTTTTCATAAACTTTGGATGCTAGTGTACTTAGGGGGTG
20 TGCCAGGTGTCTTTCATGGGGCCTTCCAGACCCACTCCCCACCCTTCTCCCCTTCCTTTGCCCGGGGACG
CCGAACTCTCTCAATGGTATCAACAGGCTCCTTCGCCCTCTGGCTCCTGGTCATGTTCCATTATTGGGGA
GCCCCAGCAGAAGAATGGAGAGGAGGAGGAGGCTGAGTTTGGGGTATTGAATCCCCCGGCTCCCACCCTG
CAGCATCAAGGTTGCTATGGACTCTCCTGCCGGGCAACTCTTGCGTAATCATGACTATCTCTAGGATTCT
GGCACCACTTCCTTCCCTGGCCCCTTAAGCCTAGCTGTGTATCGGCACCCCCACCCCACTAGAGTACTCC
25 CTCTCACTTGCGGTTTCCTTATACTCCACCCCTTTCTCAACGGTCCTTTTTTAAAGCACATCTCAGATTA
[SEQ ID No: 1]
The amino acid sequence of one embodiment of human STING variant R232 (bold,
underlined) is referred to herein as SEQ ID No: 2, as follows:
30
MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEEL
RHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLA
PAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPD
NLSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFS
35 REDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSA
VPSTSTMSQEPELLISGMEKPLPLRTDFS
[SEQ ID No: 2]
Therefore, preferably the polymorphism pattern in the STING gene, which is
40 determined, corresponds to STING variant R232. Preferably, the STING variant R232
comprises or consists of the amino acid sequence substantially as set out in SEQ ID
- 9 -
No:2 and/or is encoded by a nucleic acid sequence substantially as set out in SEQ ID
No:1.
In a second embodiment, a STING variant is R71H-G230A-R293Q or “HAQ”, in which
5 three SNPs are present. This human STING variant has an allele frequency of 20.4%. In
this embodiment, a histidine replaces an arginine at position 71; an alanine replaces a
glycine at position 230; and a glutamine replaces an arginine at position 293.
The SNP which encodes R71H is known as rs11554776. The alleles of STING rs11554776
10 SNP may be identified as (i) a G-allele, and (ii) an A-allele. Therefore, the method may
comprise detecting or determining the G-allele or the A-allele of the STING rs11554776
SNP.
The SNP which encodes G230A is known as rs78233829. The alleles of STING
15 rs78233829 SNP may be identified as (i) a G-allele, and (ii) a C-allele. Therefore, the
method may comprise detecting or determining the G-allele or the C-allele of the
STING rs78233829 SNP.
The SNP which encodes R293Q is known as rs7380824. The alleles of STING
20 rs7380824 SNP may be identified as (i) a G-allele, and (ii) an A-allele. Therefore, the
method may comprise detecting or determining the G-allele or the A-allele of the
STING rs7380824 SNP.
The nucleotide sequence encoding one embodiment of human STING variant HAQ is
25 referred to herein as SEQ ID No: 3, as follows:
GTTCATTTTTCACTCCTCCCTCCTAGGTCACACTTTTCAGAAAAAGAATCTGCATCCTGGAAACCAGAAG
AAAAATATGAGACGGGGAATCATCGTGTGATGTGTGTGCTGCCTTTGGCTGAGTGTGTGGAGTCCTGCTC
AGGTGTTAGGTACAGTGTGTTTGATCGTGGTGGCTTGAGGGGAACCCGCTGTTCAGAGCTGTGACTGCGG
30 CTGCACTCAGAGAAGCTGCCCTTGGCTGCTCGTAGCGCCGGGCCTTCTCTCCTCGTCATCATCCAGAGCA
GCCAGTGTCCGGGAGGCAGAAGATGCCCCACTCCAGCCTGCATCCATCCATCCCGTGTCCCAGGGGTCAC
GGGGCCCAGAAGGCAGCCTTGGTTCTGCTGAGTGCCTGCCTGGTGACCCTTTGGGGGCTAGGAGAGCCAC
CAGAGCACACTCTCCGGTACCTGGTGCTCCACCTAGCCTCCCTGCAGCTGGGACTGCTGTTAAACGGGGT
CTGCAGCCTGGCTGAGGAGCTGCACCACATCCACTCCAGGTACCGGGGCAGCTACTGGAGGACTGTGCGG
35 GCCTGCCTGGGCTGCCCCCTCCGCCGTGGGGCCCTGTTGCTGCTGTCCATCTATTTCTACTACTCCCTCC
CAAATGCGGTCGGCCCGCCCTTCACTTGGATGCTTGCCCTCCTGGGCCTCTCGCAGGCACTGAACATCCT
CCTGGGCCTCAAGGGCCTGGCCCCAGCTGAGATCTCTGCAGTGTGTGAAAAAGGGAATTTCAACGTGGCC
CATGGGCTGGCATGGTCATATTACATCGGATATCTGCGGCTGATCCTGCCAGAGCTCCAGGCCCGGATTC
- 10 -
GAACTTACAATCAGCATTACAACAACCTGCTACGGGGTGCAGTGAGCCAGCGGCTGTATATTCTCCTCCC
ATTGGACTGTGGGGTGCCTGATAACCTGAGTATGGCTGACCCCAACATTCGCTTCCTGGATAAACTGCCC
CAGCAGACCGCTGACCGTGCTGGCATCAAGGATCGGGTTTACAGCAACAGCATCTATGAGCTTCTGGAGA
ACGGGCAGCGGGCGGGCACCTGTGTCCTGGAGTACGCCACCCCCTTGCAGACTTTGTTTGCCATGTCACA
5 ATACAGTCAAGCTGGCTTTAGCCGGGAGGATAGGCTTGAGCAGGCCAAACTCTTCTGCCAGACACTTGAG
GACATCCTGGCAGATGCCCCTGAGTCTCAGAACAACTGCCGCCTCATTGCCTACCAGGAACCTGCAGATG
ACAGCAGCTTCTCGCTGTCCCAGGAGGTTCTCCGGCACCTGCGGCAGGAGGAAAAGGAAGAGGTTACTGT
GGGCAGCTTGAAGACCTCAGCGGTGCCCAGTACCTCCACGATGTCCCAAGAGCCTGAGCTCCTCATCAGT
GGAATGGAAAAGCCCCTCCCTCTCCGCACGGATTTCTCTTGAGACCCAGGGTCACCAGGCCAGAGCCTCC
10 AGTGGTCTCCAAGCCTCTGGACTGGGGGCTCTCTTCAGTGGCTGAATGTCCAGCAGAGCTATTTCCTTCC
ACAGGGGGCCTTGCAGGGAAGGGTCCAGGACTTGACATCTTAAGATGCGTCTTGTCCCCTTGGGCCAGTC
ATTTCCCCTCTCTGAGCCTCGGTGTCTTCAACCTGTGAAATGGGATCATAATCACTGCCTTACCTCCCTC
ACGGTTGTTGTGAGGACTGAGTGTGTGGAAGTTTTTCATAAACTTTGGATGCTAGTGTACTTAGGGGGTG
TGCCAGGTGTCTTTCATGGGGCCTTCCAGACCCACTCCCCACCCTTCTCCCCTTCCTTTGCCCGGGGACG
15 CCGAACTCTCTCAATGGTATCAACAGGCTCCTTCGCCCTCTGGCTCCTGGTCATGTTCCATTATTGGGGA
GCCCCAGCAGAAGAATGGAGAGGAGGAGGAGGCTGAGTTTGGGGTATTGAATCCCCCGGCTCCCACCCTG
CAGCATCAAGGTTGCTATGGACTCTCCTGCCGGGCAACTCTTGCGTAATCATGACTATCTCTAGGATTCT
GGCACCACTTCCTTCCCTGGCCCCTTAAGCCTAGCTGTGTATCGGCACCCCCACCCCACTAGAGTACTCC
CTCTCACTTGCGGTTTCCTTATACTCCACCCCTTTCTCAACGGTCCTTTTTTAAAGCACATCTCAGATTA
20 [SEQ ID No: 3]
The amino acid sequence of one embodiment of human STING variant HAQ (bold,
underlined) is referred to herein as SEQ ID No: 4, as follows:
25 MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEEL
HHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLA
PAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPD
NLSMADPNIRFLDKLPQQTADRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFS
REDRLEQAKLFCQTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSA
30 VPSTSTMSQEPELLISGMEKPLPLRTDFS
[SEQ ID No: 4]
Therefore, preferably the polymorphism pattern in the STING gene, which is
determined, comprises a SNP which corresponds to STING variant R71H-G230A35 R293Q or “HAQ”. Preferably, the STING variant R71H-G230A-R293Q or “HAQ”
comprises or consists of the amino acid sequence substantially as set out in SEQ ID
No:4 and/or is encoded by a nucleic acid sequence substantially as set out in SEQ ID
No:3.
- 11 -
In a third embodiment, a STING variant is R232H or “H232”, in which one SNP is
present. This human STING variant has an allele frequency of 13.7%. In this
embodiment, a histidine replaces an arginine at position 232.
5 The nucleotide sequence encoding one embodiment of human STING variant H232 is
referred to herein as SEQ ID No: 5, as follows:
GTTCATTTTTCACTCCTCCCTCCTAGGTCACACTTTTCAGAAAAAGAATCTGCATCCTGGAAACCAGAAGAAAAATAT
GAGACGGGGAATCATCGTGTGATGTGTGTGCTGCCTTTGGCTGAGTGTGTGGAGTCCTGCTCAGGTGTTAGGTACAGT
10 GTGTTTGATCGTGGTGGCTTGAGGGGAACCCGCTGTTCAGAGCTGTGACTGCGGCTGCACTCAGAGAAGCTGCCCTTG
GCTGCTCGTAGCGCCGGGCCTTCTCTCCTCGTCATCATCCAGAGCAGCCAGTGTCCGGGAGGCAGAAGATGCCCCACT
CCAGCCTGCATCCATCCATCCCGTGTCCCAGGGGTCACGGGGCCCAGAAGGCAGCCTTGGTTCTGCTGAGTGCCTGCC
TGGTGACCCTTTGGGGGCTAGGAGAGCCACCAGAGCACACTCTCCGGTACCTGGTGCTCCACCTAGCCTCCCTGCAGC
TGGGACTGCTGTTAAACGGGGTCTGCAGCCTGGCTGAGGAGCTGCGCCACATCCACTCCAGGTACCGGGGCAGCTACT
15 GGAGGACTGTGCGGGCCTGCCTGGGCTGCCCCCTCCGCCGTGGGGCCCTGTTGCTGCTGTCCATCTATTTCTACTACT
CCCTCCCAAATGCGGTCGGCCCGCCCTTCACTTGGATGCTTGCCCTCCTGGGCCTCTCGCAGGCACTGAACATCCTCC
TGGGCCTCAAGGGCCTGGCCCCAGCTGAGATCTCTGCAGTGTGTGAAAAAGGGAATTTCAACGTGGCCCATGGGCTGG
CATGGTCATATTACATCGGATATCTGCGGCTGATCCTGCCAGAGCTCCAGGCCCGGATTCGAACTTACAATCAGCATT
ACAACAACCTGCTACGGGGTGCAGTGAGCCAGCGGCTGTATATTCTCCTCCCATTGGACTGTGGGGTGCCTGATAACC
20 TGAGTATGGCTGACCCCAACATTCGCTTCCTGGATAAACTGCCCCAGCAGACCGGTGACCATGCTGGCATCAAGGATC
GGGTTTACAGCAACAGCATCTATGAGCTTCTGGAGAACGGGCAGCGGGCGGGCACCTGTGTCCTGGAGTACGCCACCC
CCTTGCAGACTTTGTTTGCCATGTCACAATACAGTCAAGCTGGCTTTAGCCGGGAGGATAGGCTTGAGCAGGCCAAAC
TCTTCTGCCGGACACTTGAGGACATCCTGGCAGATGCCCCTGAGTCTCAGAACAACTGCCGCCTCATTGCCTACCAGG
AACCTGCAGATGACAGCAGCTTCTCGCTGTCCCAGGAGGTTCTCCGGCACCTGCGGCAGGAGGAAAAGGAAGAGGTTA
25 CTGTGGGCAGCTTGAAGACCTCAGCGGTGCCCAGTACCTCCACGATGTCCCAAGAGCCTGAGCTCCTCATCAGTGGAA
TGGAAAAGCCCCTCCCTCTCCGCACGGATTTCTCTTGAGACCCAGGGTCACCAGGCCAGAGCCTCCAGTGGTCTCCAA
GCCTCTGGACTGGGGGCTCTCTTCAGTGGCTGAATGTCCAGCAGAGCTATTTCCTTCCACAGGGGGCCTTGCAGGGAA
GGGTCCAGGACTTGACATCTTAAGATGCGTCTTGTCCCCTTGGGCCAGTCATTTCCCCTCTCTGAGCCTCGGTGTCTT
CAACCTGTGAAATGGGATCATAATCACTGCCTTACCTCCCTCACGGTTGTTGTGAGGACTGAGTGTGTGGAAGTTTTT
30 CATAAACTTTGGATGCTAGTGTACTTAGGGGGTGTGCCAGGTGTCTTTCATGGGGCCTTCCAGACCCACTCCCCACCC
TTCTCCCCTTCCTTTGCCCGGGGACGCCGAACTCTCTCAATGGTATCAACAGGCTCCTTCGCCCTCTGGCTCCTGGTC
ATGTTCCATTATTGGGGAGCCCCAGCAGAAGAATGGAGAGGAGGAGGAGGCTGAGTTTGGGGTATTGAATCCCCCGGC
TCCCACCCTGCAGCATCAAGGTTGCTATGGACTCTCCTGCCGGGCAACTCTTGCGTAATCATGACTATCTCTAGGATT
CTGGCACCACTTCCTTCCCTGGCCCCTTAAGCCTAGCTGTGTATCGGCACCCCCACCCCACTAGAGTACTCCCTCTCA
35 CTTGCGGTTTCCTTATACTCCACCCCTTTCTCAACGGTCCTTTTTTAAAGCACATCTCAGATTA
[SEQ ID NO: 5]
The amino acid sequence of one embodiment of human STING variant H232 (bold,
underlined) is referred to herein as SEQ ID No: 6, as follows:
40
- 12 -
MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEEL
RHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLA
PAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPD
NLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFS
5 REDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSA
VPSTSTMSQEPELLISGMEKPLPLRTDFS
[SEQ ID NO: 6]
Therefore, preferably the polymorphism pattern in the STING gene, which is
10 determined, comprises a SNP which corresponds to STING variant R232H or “H232”.
Preferably, the STING variant R232H or “H232” comprises or consists of the amino
acid sequence substantially as set out in SEQ ID No:6 and/or is encoded by a nucleic
acid sequence substantially as set out in SEQ ID No:5.
15 In a fourth embodiment, a STING variant is G230A-R293Q or “AQ”, in which two SNPs
are present. This human STING variant has an allele frequency of 5.2%. In this
embodiment, an alanine replaces a glycine at position 230; and a glutamine replaces an
arginine a SNP at position 293.
20 The SNP which encodes G230A is known as rs78233829. The alleles of STING
rs78233829 SNP may be identified as (i) a G-allele, and (ii) a C-allele. Therefore, the
method may comprise detecting or determining the G-allele or the C-allele of the
STING rs78233829 SNP.
25 The SNP which encodes R293Q is known as rs7380824. The alleles of STING
rs7380824 SNP may be identified as (i) a G-allele, and (ii) an A-allele. Therefore, the
method may comprise detecting or determining the G-allele or the A-allele of the
STING rs7380824 SNP.
30 The nucleotide sequence encoding one embodiment of human STING variant AQ is
referred to herein as SEQ ID No: 7, as follows:
GTTCATTTTTCACTCCTCCCTCCTAGGTCACACTTTTCAGAAAAAGAATCTGCATCCTGGAAACCAGAAG
AAAAATATGAGACGGGGAATCATCGTGTGATGTGTGTGCTGCCTTTGGCTGAGTGTGTGGAGTCCTGCTC
35 AGGTGTTAGGTACAGTGTGTTTGATCGTGGTGGCTTGAGGGGAACCCGCTGTTCAGAGCTGTGACTGCGG
CTGCACTCAGAGAAGCTGCCCTTGGCTGCTCGTAGCGCCGGGCCTTCTCTCCTCGTCATCATCCAGAGCA
GCCAGTGTCCGGGAGGCAGAAGATGCCCCACTCCAGCCTGCATCCATCCATCCCGTGTCCCAGGGGTCAC
GGGGCCCAGAAGGCAGCCTTGGTTCTGCTGAGTGCCTGCCTGGTGACCCTTTGGGGGCTAGGAGAGCCAC
- 13 -
CAGAGCACACTCTCCGGTACCTGGTGCTCCACCTAGCCTCCCTGCAGCTGGGACTGCTGTTAAACGGGGT
CTGCAGCCTGGCTGAGGAGCTGCGCCACATCCACTCCAGGTACCGGGGCAGCTACTGGAGGACTGTGCGG
GCCTGCCTGGGCTGCCCCCTCCGCCGTGGGGCCCTGTTGCTGCTGTCCATCTATTTCTACTACTCCCTCC
CAAATGCGGTCGGCCCGCCCTTCACTTGGATGCTTGCCCTCCTGGGCCTCTCGCAGGCACTGAACATCCT
5 CCTGGGCCTCAAGGGCCTGGCCCCAGCTGAGATCTCTGCAGTGTGTGAAAAAGGGAATTTCAACGTGGCC
CATGGGCTGGCATGGTCATATTACATCGGATATCTGCGGCTGATCCTGCCAGAGCTCCAGGCCCGGATTC
GAACTTACAATCAGCATTACAACAACCTGCTACGGGGTGCAGTGAGCCAGCGGCTGTATATTCTCCTCCC
ATTGGACTGTGGGGTGCCTGATAACCTGAGTATGGCTGACCCCAACATTCGCTTCCTGGATAAACTGCCC
CAGCAGACCGCTGACCGTGCTGGCATCAAGGATCGGGTTTACAGCAACAGCATCTATGAGCTTCTGGAGA
10 ACGGGCAGCGGGCGGGCACCTGTGTCCTGGAGTACGCCACCCCCTTGCAGACTTTGTTTGCCATGTCACA
ATACAGTCAAGCTGGCTTTAGCCGGGAGGATAGGCTTGAGCAGGCCAAACTCTTCTGCCAGACACTTGAG
GACATCCTGGCAGATGCCCCTGAGTCTCAGAACAACTGCCGCCTCATTGCCTACCAGGAACCTGCAGATG
ACAGCAGCTTCTCGCTGTCCCAGGAGGTTCTCCGGCACCTGCGGCAGGAGGAAAAGGAAGAGGTTACTGT
GGGCAGCTTGAAGACCTCAGCGGTGCCCAGTACCTCCACGATGTCCCAAGAGCCTGAGCTCCTCATCAGT
15 GGAATGGAAAAGCCCCTCCCTCTCCGCACGGATTTCTCTTGAGACCCAGGGTCACCAGGCCAGAGCCTCC
AGTGGTCTCCAAGCCTCTGGACTGGGGGCTCTCTTCAGTGGCTGAATGTCCAGCAGAGCTATTTCCTTCC
ACAGGGGGCCTTGCAGGGAAGGGTCCAGGACTTGACATCTTAAGATGCGTCTTGTCCCCTTGGGCCAGTC
ATTTCCCCTCTCTGAGCCTCGGTGTCTTCAACCTGTGAAATGGGATCATAATCACTGCCTTACCTCCCTC
ACGGTTGTTGTGAGGACTGAGTGTGTGGAAGTTTTTCATAAACTTTGGATGCTAGTGTACTTAGGGGGTG
20 TGCCAGGTGTCTTTCATGGGGCCTTCCAGACCCACTCCCCACCCTTCTCCCCTTCCTTTGCCCGGGGACG
CCGAACTCTCTCAATGGTATCAACAGGCTCCTTCGCCCTCTGGCTCCTGGTCATGTTCCATTATTGGGGA
GCCCCAGCAGAAGAATGGAGAGGAGGAGGAGGCTGAGTTTGGGGTATTGAATCCCCCGGCTCCCACCCTG
CAGCATCAAGGTTGCTATGGACTCTCCTGCCGGGCAACTCTTGCGTAATCATGACTATCTCTAGGATTCT
GGCACCACTTCCTTCCCTGGCCCCTTAAGCCTAGCTGTGTATCGGCACCCCCACCCCACTAGAGTACTCC
25 CTCTCACTTGCGGTTTCCTTATACTCCACCCCTTTCTCAACGGTCCTTTTTTAAAGCACATCTCAGATTA
[SEQ ID No: 7]
The amino acid sequence of one embodiment of human STING variant AQ (bold,
underlined) is referred to herein as SEQ ID No: 8, as follows:
30
MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEEL
RHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLA
PAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPD
NLSMADPNIRFLDKLPQQTADRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFS
35 REDRLEQAKLFCQTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSA
VPSTSTMSQEPELLISGMEKPLPLRTDFS
[SEQ ID No: 8]
Therefore, preferably the polymorphism pattern in the STING gene, which is
40 determined, comprises a SNP which corresponds to STING variant G230A-R293Q or
“AQ”. Preferably, the STING variant G230A-R293Q or “AQ” comprises or consists of
- 14 -
the amino acid sequence substantially as set out in SEQ ID No:8 and/or is encoded by a
nucleic acid sequence substantially as set out in SEQ ID No:7.
In a fifth embodiment, a STING variant is R293Q or “Q”, in which one SNP is present.
5 This human STING variant has an allele frequency of 1.5%. In this embodiment, a
glutamine replaces an arginine at position 293.
The SNP which encodes R293Q is known as rs7380824. The alleles of STING
rs7380824 SNP may be identified as (i) a G-allele, and (ii) an A-allele. Therefore, the
10 method may comprise detecting or determining the G-allele or the A-allele of the
STING rs7380824 SNP.
The nucleotide sequence encoding one embodiment of human STING variant Q is
referred to herein as SEQ ID No: 9, as follows:
15
GTTCATTTTTCACTCCTCCCTCCTAGGTCACACTTTTCAGAAAAAGAATCTGCATCCTGGAAACCAGAAG
AAAAATATGAGACGGGGAATCATCGTGTGATGTGTGTGCTGCCTTTGGCTGAGTGTGTGGAGTCCTGCTC
AGGTGTTAGGTACAGTGTGTTTGATCGTGGTGGCTTGAGGGGAACCCGCTGTTCAGAGCTGTGACTGCGG
CTGCACTCAGAGAAGCTGCCCTTGGCTGCTCGTAGCGCCGGGCCTTCTCTCCTCGTCATCATCCAGAGCA
20 GCCAGTGTCCGGGAGGCAGAAGATGCCCCACTCCAGCCTGCATCCATCCATCCCGTGTCCCAGGGGTCAC
GGGGCCCAGAAGGCAGCCTTGGTTCTGCTGAGTGCCTGCCTGGTGACCCTTTGGGGGCTAGGAGAGCCAC
CAGAGCACACTCTCCGGTACCTGGTGCTCCACCTAGCCTCCCTGCAGCTGGGACTGCTGTTAAACGGGGT
CTGCAGCCTGGCTGAGGAGCTGCGCCACATCCACTCCAGGTACCGGGGCAGCTACTGGAGGACTGTGCGG
GCCTGCCTGGGCTGCCCCCTCCGCCGTGGGGCCCTGTTGCTGCTGTCCATCTATTTCTACTACTCCCTCC
25 CAAATGCGGTCGGCCCGCCCTTCACTTGGATGCTTGCCCTCCTGGGCCTCTCGCAGGCACTGAACATCCT
CCTGGGCCTCAAGGGCCTGGCCCCAGCTGAGATCTCTGCAGTGTGTGAAAAAGGGAATTTCAACGTGGCC
CATGGGCTGGCATGGTCATATTACATCGGATATCTGCGGCTGATCCTGCCAGAGCTCCAGGCCCGGATTC
GAACTTACAATCAGCATTACAACAACCTGCTACGGGGTGCAGTGAGCCAGCGGCTGTATATTCTCCTCCC
ATTGGACTGTGGGGTGCCTGATAACCTGAGTATGGCTGACCCCAACATTCGCTTCCTGGATAAACTGCCC
30 CAGCAGACCGGTGACCGTGCTGGCATCAAGGATCGGGTTTACAGCAACAGCATCTATGAGCTTCTGGAGA
ACGGGCAGCGGGCGGGCACCTGTGTCCTGGAGTACGCCACCCCCTTGCAGACTTTGTTTGCCATGTCACA
ATACAGTCAAGCTGGCTTTAGCCGGGAGGATAGGCTTGAGCAGGCCAAACTCTTCTGCCAGACACTTGAG
GACATCCTGGCAGATGCCCCTGAGTCTCAGAACAACTGCCGCCTCATTGCCTACCAGGAACCTGCAGATG
ACAGCAGCTTCTCGCTGTCCCAGGAGGTTCTCCGGCACCTGCGGCAGGAGGAAAAGGAAGAGGTTACTGT
35 GGGCAGCTTGAAGACCTCAGCGGTGCCCAGTACCTCCACGATGTCCCAAGAGCCTGAGCTCCTCATCAGT
GGAATGGAAAAGCCCCTCCCTCTCCGCACGGATTTCTCTTGAGACCCAGGGTCACCAGGCCAGAGCCTCC
AGTGGTCTCCAAGCCTCTGGACTGGGGGCTCTCTTCAGTGGCTGAATGTCCAGCAGAGCTATTTCCTTCC
ACAGGGGGCCTTGCAGGGAAGGGTCCAGGACTTGACATCTTAAGATGCGTCTTGTCCCCTTGGGCCAGTC
ATTTCCCCTCTCTGAGCCTCGGTGTCTTCAACCTGTGAAATGGGATCATAATCACTGCCTTACCTCCCTC
40 ACGGTTGTTGTGAGGACTGAGTGTGTGGAAGTTTTTCATAAACTTTGGATGCTAGTGTACTTAGGGGGTG
- 15 -
TGCCAGGTGTCTTTCATGGGGCCTTCCAGACCCACTCCCCACCCTTCTCCCCTTCCTTTGCCCGGGGACG
CCGAACTCTCTCAATGGTATCAACAGGCTCCTTCGCCCTCTGGCTCCTGGTCATGTTCCATTATTGGGGA
GCCCCAGCAGAAGAATGGAGAGGAGGAGGAGGCTGAGTTTGGGGTATTGAATCCCCCGGCTCCCACCCTG
CAGCATCAAGGTTGCTATGGACTCTCCTGCCGGGCAACTCTTGCGTAATCATGACTATCTCTAGGATTCT
5 GGCACCACTTCCTTCCCTGGCCCCTTAAGCCTAGCTGTGTATCGGCACCCCCACCCCACTAGAGTACTCC
CTCTCACTTGCGGTTTCCTTATACTCCACCCCTTTCTCAACGGTCCTTTTTTAAAGCACATCTCAGATTA
[SEQ ID No: 9]
The amino acid sequence of one embodiment of human STING variant Q (bold,
10 underlined) is referred to herein as SEQ ID No: 10, as follows:
MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEEL
RHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLA
PAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPD
15 NLSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFS
REDRLEQAKLFCQTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSA
VPSTSTMSQEPELLISGMEKPLPLRTDFS
[SEQ ID No: 10]
Therefore, preferably the polymorphism pattern in the STING gene, which is
20 determined, comprises a SNP which corresponds to STING variant R293Q or “Q”.
Preferably, the STING variant R293Q or “Q” comprises or consists of the amino acid
sequence substantially as set out in SEQ ID No:2 and/or is encoded by a nucleic acid
sequence substantially as set out in SEQ ID No:1.
25 The sample is preferably a biological bodily sample taken from the test subject.
Determining the genetic polymorphism pattern in the STING gene in the sample is
therefore preferably carried out in vitro. The sample may comprise tissue, blood,
plasma, serum, spinal fluid, urine, sweat, saliva, sputum, tears, breast aspirate, prostate
fluid, seminal fluid, vaginal fluid, stool, cervical scraping, amniotic fluid, intraocular
30 fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin,
buccal scrapings, nails, bone marrow, cartilage, prions, bone powder, ear wax, or
combinations thereof. The sample may be a biopsy.
In another embodiment, the sample may be contained within the test subject, which
35 may be an experimental animal (e.g. a mouse or rat) or a human, wherein the method is
an in vivo based test. Alternatively, the sample may be an ex vivo sample or an in vitro
sample. Therefore, the cells being tested may be in a tissue sample (for ex vivo based
- 16 -
tests) or the cells may be grown in culture (an in vitro sample). Preferably, the
biological sample is an ex vivo sample.
The sample may be pretreated prior to being used in the invention (e.g. diluted,
5 concentrated, separated, partially purified, frozen etc.). Preferably, the sample is a
pretreated blood sample.
The sample may comprise blood, urine, or tissue. Most preferably, therefore, the
sample comprises a blood sample. The blood may be venous or arterial blood. The
10 apparatus may comprise a sample collection container for receiving the extracted
sample. Blood samples may be assayed immediately. Alternatively, the blood sample
may be stored at low temperatures, for example in a fridge or even frozen before the
assay is conducted. Detection of SNPs in the STING gene may be carried out on whole
blood. Preferably, however, the blood sample comprises blood serum. Preferably, the
15 blood sample comprises nucleated cells.
The blood may be further processed before the STING assay is performed, i.e.
determining the presence of SNPs in the STING gene. For instance, an anticoagulant,
such as citrate (such as sodium citrate), hirudin, heparin, PPACK, or sodium fluoride
20 may be added. Thus, the sample collection container may contain an anticoagulant in
order to prevent the blood sample from clotting. Alternatively, the blood sample may
be centrifuged or filtered to isolate the nucleated cell fraction of the blood or serum
fraction, which may be used for analysis. Hence, it is preferred that the presence of
SNPs in the STING gene is analysed or assayed in a nucleated cell fraction of the blood
25 or a blood serum sample. It is preferred that the presence of SNPs in the STING gene is
analysed in vitro from a blood serum sample or a nucleated cell fraction of the blood
taken from the subject.
Amplification techniques for amplifying the sequence of genomic DNA comprising
30 SNPs associated with STING are known to the skilled person and include, but are not
limited to, cloning, polymerase chain reaction (PCR), polymerase chain reaction of
specific alleles (PASA), polymerase chain ligation, nested polymerase chain reaction,
and the like.
35 Thus, the detecting step may comprise amplification of the sample, for example PCR
amplification. PCR involves amplifying DNA, preferably small amounts of DNA, to ease
- 17 -
subsequent detection of the genetic polymorphic patterns. Many variations of the basic
amplification protocol are well-known to those of skill in the art. PCR-based detection
means include multiplex amplification of a plurality of polymorphisms or markers,
simultaneously. For example, it is well-known to select PCR primers to generate PCR
5 products that do not overlap in size and which can be analysed simultaneously.
Alternatively, it is possible to amplify different markers with primers that are
differentially labelled and thus can each be differentially detected. Of course,
hybridization-based detection means allow the differential detection of multiple PCR
products in a sample. Other techniques are known to allow multiplex analysis of a
10 plurality of markers.
The skilled person would understand that the PCR reaction comprises a mixture of
reagents that are well-known in the art in performing the PCR reaction. For example,
the mixture may comprise a buffer, forward primer, reverse primer, template,
15 polymerase and water.
The PCR may be Quantitative (Q)-PCR, droplet-digital PCR and CrystalTM DigitalTM
PCR. All of these techniques are routine in molecular biology and known to those
skilled in the art.
20
Preferably, the activation of DNA polymerase and initial denaturation is carried out at a
temperature of between 94 and 98°C, between 95 and 98°C, between 96 and 98°C, or
between 97 and 98°C. Most preferably, the initial denaturation is carried out at a
temperature of 98°C. Preferably, the activation of DNA polymerase and initial
25 denaturation is carried out for a period of between 30 seconds and 5 minutes, or
between 1 and 4 minutes, or between 2 and 3 minutes. Most preferably, the initial
denaturation is carried out for a period of 2 minutes and 45 seconds.
Preferably, PCR is carried out for between 20 and 40 cycles, or between 25 and 35
30 cycles. Most preferably, PCR is carried out for 30 cycles. Preferably, the cycle includes
the following steps:
- denaturation;
- annealing; and
- extension.
35
- 18 -
Preferably, the denaturation step is carried out at a temperature of between 94 and
98°C, between 95 and 98°C, between 96 and 98°C, or between 97 and 98°C. Most
preferably, the denaturation step is carried out at a temperature of 98°C. Preferably,
the denaturation step is carried out for a period of between 20 seconds and 2 minutes,
5 between 20 seconds and 1 minute, or between 20 and 30 seconds. Most preferably, the
denaturation step is carried out for 30 seconds.
Preferably, the annealing step is carried out at a temperature of between 40 and 60°C,
or between 50 and 60°C. Most preferably, the annealing step is carried out at a
10 temperature of 60°C. Preferably, the annealing step is carried out for a period of
between 30 seconds and 2 minutes, or between 30 seconds and 1 minute. Most
preferably, the annealing step is carried out for 30 seconds.
Preferably, the extension step is carried out at a temperature of between 70 and 75°C,
15 between 71 and 74°C, or between 72 and 73°C. Most preferably, the extension step is
carried out at a temperature of 72°C. Preferably, the extension step is carried out for a
period of time between 30 seconds and 2 minutes, or between 30 seconds and 1 minute.
Most preferably, the extension step is carried out for 30 seconds.
20 Preferably, PCR includes a final extension step. Preferably, the final extension step is
carried out at a temperature of between 70 and 75°C, between 71 and 74°C, or between
72 and 73°C. Most preferably, the final extension is carried out at a temperature of
72°C. Preferably, the final extension is carried out for a period of time between 5 and 15
minutes, between 6 and 14 minutes, between 7 and 13 minutes, between 8 and 12
25 minutes, or between 9 and 11 minutes. Most preferably, the final extension is carried
out for a period of 10 minutes.
Preferably, PCR uses a pair of primers, a forward primer and a reverse primer. The
forward primer is designed so that it is complementary to a sequence of nucleotides
30 upstream of the sequence of interest, whilst the reverse primer is designed so that it is
complementary to a sequence of nucleotides downstream of the sequence of interest.
The STING nucleotide sequence to which the primer sequences are capable of
hybridizing to may be a DNA or RNA sequence. The RNA sequence may be a miRNA,
mRNA or siRNA.
35
- 19 -
The term “primer” designates, within the context of the present invention, a nucleotide
sequence of that can hybridize specifically to a target genetic sequence and serve to
initiate amplification. Primers of the invention may be a single-stranded nucleotide
sequence , with a length of between 10 and 50 nucleotides between 10 and 40
5 nucleotides, between 10 and 30 nucleotides, between 10 and 25 nucleotides between 11
and 50, between 11 and 40 nucleotides, between 11 and 30 nucleotides, between 11 and
25 nucleotides, between 13 and 50, between 13 and 40 nucleotides, between 13 and 30
nucleotides, between 13 and 25 nucleotides, between 14 and 50, between 14 and 40
nucleotides, between 14 and 30 nucleotides, between 14 and 25 nucleotides, between 15
10 and 50, between 15 and 40 nucleotides, between 15 and 30 nucleotides, between 15 and
25 nucleotides. Preferably, primers of the invention have a length of between 15 and 30
nucleotides. More preferably, primers of the invention have a length of between 15 and
25.
15 Preferably, primers are perfectly matched with the targeted sequence in the STING
nucleotide sequence, i.e. having 100% complementarity, allowing specific hybridization
thereto and substantially no hybridization to another region.
Preferably, the primers are designed to hybridize to sequences flanking the SNPs which
20 correspond to amino acid residues 71, 230, 232 and 293. This is because substitutions
at these residues correspond to the five major STING variants.
In an embodiment in which the SNP location corresponds to amino acid residue 71, the
forward primer is provided herein as follows:
25 GTCTGTTTTGTAGATCGAGAAATGG
[SEQ ID NO: 11]
Thus, in a preferred embodiment, the forward primer comprises or consists of a
nucleotide sequence substantially set out as SEQ ID No: 11, or a fragment or variant
thereof. Preferably, the forward primer is capable of hybridizing to a sequence
30 complementary to the nucleotide sequence as substantially set out in SEQ ID No: 11, or
a fragment or variant thereof, and amplifies, with its paired reverse primer, the target
sequence corresponding to amino acid residue 71.
In an embodiment in which the SNP location corresponds to amino acid residue 71, the
35 reverse primer is provided herein as follows:
AGAATGGTCATGGATTTCTTGG
- 20 -
[SEQ ID NO: 12]
Thus in a preferred embodiment, the reverse primer comprises or consists of a
nucleotide sequence substantially set out as SEQ ID No: 12, or a fragment or variant
thereof. Preferably, the reverse primer is capable of hybridizing to a sequence
5 complementary to the nucleotide sequence as substantially set out in SEQ ID No: 12, or
a fragment or variant thereof, and amplifies, with its paired forward primer, the target
sequence corresponding to amino acid residue 71. Preferably, the PCR product is 991
bp.
10 In an embodiment in which the SNP location corresponds to amino acid residue 230 or
232, the forward primer is provided herein as follows:
CAGCTAGGGACACTACAGCTCAGA
[SEQ ID NO: 13]
Thus in a preferred embodiment, the forward primer comprises or consists of a
15 nucleotide sequence substantially set out as SEQ ID No: 13, or a fragment or variant
thereof. Preferably, the forward primer is capable of hybridizing to a sequence
complementary to the nucleotide sequence as substantially set out in SEQ ID No: 13, or
a fragment or variant thereof, and amplifies, with its paired reverse primer, the target
sequence corresponding to amino acid residues 230 and 232.
20
In an embodiment in which the SNP location corresponds to amino acid residue 230 or
232, the reverse primer is provided herein as follows:
CTGGCCTCCTGTACAATGAGAGT
[SEQ ID NO: 14]
25 Thus in a preferred embodiment, the reverse primer comprises or consists of a
nucleotide sequence substantially set out as SEQ ID No: 14, or a fragment or variant
thereof. Preferably, the reverse primer is capable of hybridizing to a sequence
complementary to the nucleotide sequence as substantially set out in SEQ ID No: 14, or
a fragment or variant thereof, and amplifies, with its paired forward primer, the target
30 sequence corresponding to amino acid residues 230 and 232. Preferably, the PCR
product is 501 bp.
In an embodiment in which the SNP location corresponds to amino acid residue 293,
the forward primer is provided herein as follows:
35 CTCCATAGCCCCTTCTGACTCTT
[SEQ ID NO: 15]
- 21 -
Thus in a preferred embodiment, the forward primer comprises or consists of a
nucleotide sequence substantially set out as SEQ ID No: 15, or a fragment or variant
thereof. Preferably, the forward primer is capable of hybridizing to a sequence
complementary to the nucleotide sequence as substantially set out in SEQ ID No: 15, or
5 a fragment or variant thereof, and amplifies, with its paired reverse primer, the target
sequence corresponding to amino acid residue 293.
In an embodiment in which the SNP location corresponds to amino acid residue 293,
the reverse primer is provided herein as follows:
10 GGCTTAGTCTGGTCTTCCTCTTACC
[SEQ ID NO: 16]
Thus in a preferred embodiment, the reverse primer comprises or consists of a
nucleotide sequence substantially set out as SEQ ID No: 16, or a fragment or variant
thereof. Preferably, the reverse primer is capable of hybridizing to a sequence
15 complementary to the nucleotide sequence as substantially set out in SEQ ID No: 16, or
a fragment or variant thereof, and amplifies, with its paired forward primer, the target
sequence corresponding to amino acid residue 293. Preferably, the PCR product is 314
bp.
20 Preferably, the RFLP pattern analysis comprises subjecting the genomic DNA to
restriction enzyme digestion to produce at least one fragment; and subjecting the at
least one fragment to gel electrophoresis. The presence of SNPs in the STING gene will
result in fragments that display different migration profiles from the wild type fragment
patterns.
25
The skilled person would understand that the restriction enzyme digestion reaction
comprises a mixture of reagents that are well known in the art in performing the
reaction. For example, the mixture may comprise the PCR product, a buffer, a
restriction enzyme and water.
30
In one embodiment, the restriction enzyme is HhaI. Preferably, HhaI cuts the genomic
DNA at the position corresponding to amino acid residue 71.
In one embodiment, HpaII may cut the genomic DNA at the position corresponding to
35 amino acid residue 230. In another embodiment, the restriction enzyme is Eco91I.
- 22 -
Preferably, Eco91I cuts the genomic DNA at the position corresponding to amino acid
residue 230.
In another embodiment, the restriction enzyme is Hin1II. Preferably, Hin1II cuts the
5 genomic DNA at the position corresponding to amino acid residue 232.
In another embodiment, the restriction enzyme is HpaII. Preferably, HpaII cuts the
genomic DNA at the position corresponding to amino acid residue 293.
10 The five major STING haplotypes are characterised by the amino acid present at the 71,
230, 232 and 293 positions. Depending on the amino acid present at these positions,
and the restriction enzyme used, different length fragments are produced.
In an embodiment in which arginine is located at the 71 position, restriction enzyme
15 digestion produces a 750 bp fragment and a 241 bp fragment. In an embodiment in
which histidine is located at the 71 position, restriction enzyme digestion produces a
991 bp fragment.
In an embodiment in which glycine is located at the 230 position, restriction enzyme
20 digestion produces a 317 bp fragment and a 184 bp fragment. In an embodiment in
which alanine is present at the 230 position, restriction enzyme digestion produces a
501 bp fragment.
In an embodiment in which arginine is present at the 232 position, restriction enzyme
25 digestion produces a 501 bp fragment. In an embodiment in which histidine is present
at the 232 position, restriction enzyme digestion produces a 317 bp fragment and a 184
bp fragment.
In an embodiment in which an arginine is present at the 293 position, restriction
30 enzyme digestion produces a 162 bp fragment, a 116 bp fragment, and a 36 bp
fragment. In an embodiment in which glutamine is present at the 293 position,
restriction enzyme digestion produces a 198 bp fragment and a 116 bp fragment.
Preferably, the restriction enzyme digestion is carried out at a temperature of between
35 35 and 40°C, between 36 and 39°C, or between 37 and 38°C. Most preferably, the
restriction enzyme digestion is carried out at a temperature of 37°C.
- 23 -
Preferably, the restriction enzyme digestion is carried out for a period of between 5
minutes and 4 hours. More preferably, the restriction enzyme digestion is carried out
for a period of between 10 minutes and 3 hours, or15minutes and 2 hours. Most
5 preferably, the restriction enzyme digestion is carried out for a period of one hour.
In one embodiment, the fragment is electrophoresed on an agarose gel. Preferably, the
concentration of the agarose in the gel is between 0.5% and 4%, or between 1 and 3.5%.
Most preferably, the concentration of the agarose in the gel is between 2 and 3%.
10
Preferably, the fragment is electrophoresed on a polyacrylamide gel. Preferably, the
concentration of the polyacrylamide in the gel is between 5 and 10%. More preferably,
the concentration of the polyacrylamide in the gel is between 6 and 9%, or between 7
and 8%. Most preferably, the concentration of the polyacrylamide in the gel is 8%.
15 Preferably, the polyacrylamide gel is placed in 0.5x TBE buffer.
Preferably, the gel is incubated in ethidium bromide staining solution for between 20
seconds and 5 minutes, or between 30 seconds and 4 minutes, or between 40 seconds
and 3 minutes, or between 50 seconds and 2 minutes. Most preferably, the gel is
20 incubated in ethidium bromide staining solution for one minute. Preferably, the gel is
visualised using a UV gel documentation system.
The method of the fifth aspect may comprise administering, or having administered, to
the subject, a STING agonist to treat a disease selected from a group consisting of:
25 cancer, bacterial infection, viral infection, parasitic infection, fungal infection, immunemediated disorder, central nervous system disease, peripheral nervous system disease,
neurodegenerative disease, mood disorder, sleep disorder, cerebrovascular disease,
peripheral artery disease and cardiovascular disease. Most preferably, however, cancer
is treated with the STING agonist.
30
Examples of STING agonists include synthetic CDN STING agonists and smallmolecule STING agonists. For example, cyclic dinucleotides (CDNs), such as cyclic
dimeric guanosine monophosphate (c-di-GMP), cyclic dimeric adenosine
monophosphate (c-di-AMP), and cyclic GMP-AMP (cGAMP), are a class of STING
35 agonists that can elicit immune responses.
- 24 -
Examples of STING agonists also include small molecule modulators of STING, of
which more information can be found in WO2018/234805, WO2018/234807,
WO2019/243825, and WO2019/243823.
5 In an embodiment in which an individual has STING haplotype R232, HAQ, R232H,
AQ or Q, a STING agonist is administered.
It will be appreciated that an ‘agonist’, an ‘effector’ or an activator, as it relates to
STING, comprises a molecule, combination of molecules, or a complex, that stimulates
10 STING.
The method of the sixth aspect may comprise administering, or having administered, to
the subject, a STING antagonist to treat a disease selected from a group consisting of:
autoimmune disease, liver fibrosis, fatty liver disease, non-alcoholic steatohepatitis
15 (NASH), pulmonary fibrosis, lupus, sepsis, rheumatoid arthritis (RA), type I diabetes,
STING-associated vasculopathy with onset in infancy (SAVI), Aicardi-Goutieres
syndrome (AGS), familial chilblain lupus (FCL), systemic lupus erythematosus (SLE),
retinal vasculopathy, neuroinflammation, systemic inflammatory response syndrome,
pancreatitis, cardiovascular disease, non-alcoholic fatty liver disease, renal fibrosis,
20 stroke and age-related macular degeneration (AMD).
Examples of STING antagonists include small-molecule STING antagonists, such as C176 and H-151, and STING pathway antagonists.
25 Examples of STING antagonists also include small molecule modulators of STING.
It will be appreciated that an ‘antagonist’, as it relates to STING, comprises a molecule,
combination of molecules, or a complex, that inhibits, counteracts, downregulates,
and/or desensitizes STING. ‘Antagonist’ encompasses any reagent that inhibits a
30 constitutive activity of STING. A constitutive activity is one that is manifest in the
absence of a ligand/STING interaction. ‘Antagonist’ also encompasses any reagent that
inhibits or prevents a stimulated (or regulated) activity of STING.
The “subject” or “individual” may be a vertebrate, mammal, or domestic animal. Hence,
35 medicaments according to the invention may be used to treat any mammal, for example
- 25 -
livestock (e.g. a horse), pets, or may be used in other veterinary applications. Most
preferably, the subject is a human being.
It will be appreciated that the invention extends to any nucleic acid or peptide or
5 variant, derivative or analogue thereof, which comprises substantially the amino acid or
nucleic acid sequences of any of the sequences referred to herein, including variants or
fragments thereof. The terms “substantially the amino acid/nucleotide/peptide
sequence”, “variant” and “fragment”, can be a sequence that has at least 40% sequence
identity with the amino acid/nucleotide/peptide sequences of any one of the sequences
10 referred to herein, for example 40% identity with the sequence identified as SEQ ID
Nos: 1-16 and so on.
Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is
greater than 65%, more preferably greater than 70%, even more preferably greater than
15 75%, and still more preferably greater than 80% sequence identity to any of the
sequences referred to are also envisaged. Preferably, the amino
acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the
sequences referred to, more preferably at least 90% identity, even more preferably at
least 92% identity, even more preferably at least 95% identity, even more preferably at
20 least 97% identity, even more preferably at least 98% identity and, most preferably at
least 99% identity with any of the sequences referred to herein.
The skilled technician will appreciate how to calculate the percentage identity between
two amino acid/polynucleotide/polypeptide sequences. In order to calculate the
25 percentage identity between two amino acid/polynucleotide/polypeptide sequences, an
alignment of the two sequences must first be prepared, followed by calculation of the
sequence identity value. The percentage identity for two sequences may take different
values depending on:- (i) the method used to align the sequences, for example,
ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or
30 structural alignment from 3D comparison; and (ii) the parameters used by the
alignment method, for example, local vs global alignment, the pair-score matrix used
(e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and
constants.
35 Having made the alignment, there are many different ways of calculating percentage of
identity between the two sequences. For example, one may divide the number of
- 26 -
identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the
mean length of sequence; (iv) the number of non-gap positions; or (v) the number of
equivalenced positions excluding overhangs. Furthermore, it will be appreciated that
percentage of identity is also strongly length dependent. Therefore, the shorter a pair of
5 sequences is, the higher the sequence identity one may expect to occur by chance.
Hence, it will be appreciated that the accurate alignment of protein or DNA sequences
is a complex process. The popular multiple alignment program ClustalW (Thompson et
al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids
10 Research, 24, 4876-4882) is a preferred way for generating multiple alignments of
proteins or DNA in accordance with the invention. Suitable parameters for ClustalW
may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension
Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty =
10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein
15 alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that
it may be necessary to vary these and other parameters for optimal sequence alignment.
Preferably, calculation of percentage identities between two amino
acid/polynucleotide/polypeptide sequences may then be calculated from such an
20 alignment as (N/T)*100, where N is the number of positions at which the sequences
share an identical residue, and T is the total number of positions compared including
gaps and either including or excluding overhangs. Preferably, overhangs are included in
the calculation. Hence, a most preferred method for calculating percentage identity
between two sequences comprises (i) preparing a sequence alignment using the
25 ClustalW program using a suitable set of parameters, for example, as set out above; and
(ii) inserting the values of N and T into the following formula:- Sequence Identity =
(N/T)*100.
Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence
30 described herein could be varied or changed without substantially affecting the
sequence of the protein encoded thereby, to provide a functional variant thereof.
Suitable nucleotide variants are those having a sequence altered by the substitution of
different codons that encode the same amino acid within the sequence, thus producing
a silent (synonymous) change. Other suitable variants are those having homologous
35 nucleotide sequences but comprising all, or portions of, sequence, which are altered by
the substitution of different codons that encode an amino acid with a side chain of
- 27 -
similar biophysical properties to the amino acid it substitutes, to produce a
conservative change. For example small non-polar, hydrophobic amino acids include
glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar,
hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar
5 neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The
positively charged (basic) amino acids include lysine, arginine and histidine. The
negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will
therefore be appreciated which amino acids may be replaced with an amino acid having
similar biophysical properties, and the skilled technician will know the nucleotide
10 sequences encoding these amino acids.
All of the features described herein (including any accompanying claims, abstract and
drawings), and/or all of the steps of any method or process so disclosed, may be
combined with any of the above aspects in any combination, except combinations
15 where at least some of such features and/or steps are mutually exclusive.
For a better understanding of the invention, and to show how embodiments of the same
may be carried into effect, reference will now be made, by way of example, to the
accompanying Figure, in which:-
20 Figure 1 is a flow diagram illustrating one embodiment of a STING genotyping method
according to the invention;
Figure 2 shows SNP typing of human donor D1: STING R232/R232;
Figure 3 shows SNP typing of human donor D2: STING R232/HAQ;
Figure 4 shows SNP typing of human donor D3: STING H232/HAQ;
25 Figure 5 shows SNP typing of human donor D4: STING R232/H232;
Figure 6 shows SNP typing of human donor D5: STING HAQ/HAQ;
Figure 7 shows SNP typing of human donor D6: STING R232/H232;
Figure 8 shows SNP typing of human donor D7: STING H232/HAQ;
Figure 9 shows SNP typing of human donor D8: STING R232/R232;
30 Figure 10 shows SNP typing of human donor D9: STING R232/R232;
Figure 11 shows SNP typing of human donor D10: STING R232/R232;
Figure 12 shows SNP typing of human donor D11: STING R232/HAQ; and
Figure 13 shows SNP typing of human donor D12: STING R232/R232.
35 Examples
- 28 -
Referring to Figure 1, the inventors have designed a novel method for rapidly and
reliably identifying the presence or absence of mutations in the STING gene and,
accordingly, determining which STING alleles a subject carries using tissue or blood
samples. The method involves PCR amplification of a SNP-containing sequence of
5 genomic DNA obtained from a human subject. This is followed by RFLP analysis to
identify the subject’s STING variant according to the presence or absence of particular
SNPs.
Materials and methods
10
Collection of blood
500µl of fresh whole blood was collected from individual healthy donors in Vacuette
Na-Heparin coated vacuum tubes after obtaining signed consent (as per Institutional
Ethical Approval Committee guidelines).
15
Genomic DNA isolation and measurement
Commonly available commercial kits can be used for isolation of genomic DNA as per
the manufacturer’s protocol. Such kits are marketed by Qiagen (QIAamp DNA Blood
Mini Kit Cat #51104) and Promega (Wizard® Genomic DNA Purification Kit Cat
20 #A1120). In this example, the inventors used the QIAGEN kit. DNA was quantified and
measured by monitoring UV absorbance at 260nm using a spectrophotometer (Agilent
technologies Cary 60 UV-Vis).
PCR for each SNP position
25 PCR was carried out for each SNP position according to the below mentioned PCR
conditions and using the primer sets for each SNP amplification set out below.
a) Primer sets for each SNP amplification:
Primer sets for PCR of gDNA template
Primer_ID Primer SNP Sequence
Fwd71 SNP_71_Fwd GTCTGTTTTGTAGATCGAGAAATGG
[SEQ ID No: 11]
Rev71 SNP_71_Rev AGAATGGTCATGGATTTCTTGG
[SEQ ID No: 12]
Fwd230 SNP_230/232_Fwd CAGCTAGGGACACTACAGCTCAGA
[SEQ ID No: 13]
Rev230 SNP_230/232_Rev CTGGCCTCCTGTACAATGAGAGT
[SEQ ID No: 14]
- 29 -
Fwd293 SNP_293_Fwd CTCCATAGCCCCTTCTGACTCTT
[SEQ ID No: 15]
Rev293 SNP_293_Rev GGCTTAGTCTGGTCTTCCTCTTACC
[SEQ ID No: 16]
b) PCR reaction table for each SNP PCR:
Phusion Pol hSTING cloning PCR rxn using gDNA template
component Conc. Volume (ul) Final conc.
buffer 5x 4.0 1x
dNTPs 10mM 0.4 200uM
primer Fwd 10uM 1.0 0.5uM
primer Rev 10uM 1.0 0.5uM
Template 50-100ng 1.0
Phusion Pol 2U/ul 0.2 0.02U/ul
Water 12.4
Total 20.0
c) PCR cycling instructions:
5 Initial denaturation: 98°C for 2 min 45 seconds
Denaturation: 98°C for 30 seconds
Annealing: 60°C for 30 seconds
Extension: 72°C for 30 seconds
Cycles: 30 cycles
10 Final extension: 72°C for 10 minutes
Restriction enzyme digestion
The following reaction mix was prepared and incubated at 37°C for 1 hour. 5µl of the
digested sample was electrophoresed on 8% polyacrylamide gel in 0.5X TBE buffer for
15 resolution. The gel was incubated in ethidium bromide staining solution for a minute
and visualized using a UV gel documentation system.
Reagent Volume
PCR Product 5ul
FD Green 10x buffer 1ul
Restriction Enzyme 0.5ul (0.5U)
Water 3.5ul
Total 10ul
RFLP pattern analysis
- 30 -
Fragment Pattern expected in electrophoretic movement post digestion using gDNA
template:
PCR prod size
(bp)
Position
Restriction
Enzyme
Amino
Acid STING Variant Fragments
(bp)
991
71
HhaI
Arg(R71)
His (H71)
R232/H232
HAQ
750/ 241
991
501
230
Eco91I
Gly(G230)
Ala(A230)
R232/H232
HAQ/A230
317/184
501
501
232
Hin1II
Arg(R232)
His (H232)
R232/HAQ
H232
501
317/184
314
293
HpaII
Arg(R293)
Gln(Q293)
R232/H232
HAQ/Q293
162/116/36
198/116
Results
5 In this example, the inventors used their newly designed method to identify the
presence or absence of SNPs in the STING gene, and therefore determine the specific
STING alleles carried by twelve healthy human donors. They first collected fresh whole
blood from each individual healthy donor and then isolated and measured genomic
DNA from the samples. PCR amplification of a SNP containing sequence of genomic
10 DNA was then carried out to produce multiple copies of the DNA sample. The inventors
then used restriction enzyme digestion to digest the DNA pieces into smaller fragments.
Following this, the inventors carried out gel electrophoresis to separate the DNA
fragments produced according to their size. Finally, restriction fragment length
polymorphism (RFLP) pattern analysis was used to determine the specific STING
15 alleles carried in the genotype of each subject.
Using this newly designed method, the inventors were able to quickly and reliably
identify the SNPs present in the STING genes of healthy human donors, and therefore
determine the STING variant of the individual. The inventors designed the method to
20 identify SNPs that corresponded to the five major STING variants: R232, HAQ, H232,
AQ and Q. Therefore, the PCR primers were designed to hybridize to sequences
flanking the SNPs that correspond to amino acid residues 71, 230, 232 and 293, as
substitutions at these residues correspond to the five major STING variants.
25 As illustrated in Figures 2-13, the inventors then used RFLP pattern analysis based on
the electrophoretic movement of the DNA fragments to determine the STING variants
- 31 -
carried by each individual. For example, as illustrated in Figure 2, human donor D1
carries the STING alleles R232/R232. This is because RFLP pattern analysis of the PCR
products revealed the presence of arginine at position 71, glycine at position 230,
arginine at position 232 and arginine at position 293, based on the fragment lengths.
5 Accordingly, human donor D1 carries the STING alleles R232/R232. In comparison,
human donor D5 (Figure 6) carries the STING alleles HAQ/HAQ, since RFLP pattern
analysis of PCR products revealed the presence of histidine at position 71, alanine at
position 230, arginine at position 232 and glutamine at position 293. By identifying the
SNPs and determining the STING variant, this method is extremely informative in
10 revealing that both subjects would be suitable for treatment with a STING agonist.
Conclusions
In summary, therefore, the inventors have developed a novel method for determining
the specific STING alleles carried by individuals using tissue or blood samples. In
15 particular, this novel method is both rapid and reliable and can therefore yield accurate
results within a few hours. In addition, this novel method does not require the need for
expensive reagents and instruments. Therefore, considering the large number of
patients being tested, this method will be both cost effective and feasible to perform.
Accordingly, this method can therefore be used as a diagnostic tool to determine the
20 most effective STING agonist therapy for a patient based on their STING genotype.
References
1. Cell Reports 11, 1018–1030, May 19, 2015 (2015 Direct Activation of STING in the Tumor)
2. PLoS ONE 8(10): e77846. doi:10.1371/journal.pone.0077846, 2013 (2013 SNP of human STING)
25 3. The Journal of Immunology, 2017, 198: 000–000 (2016_The Common HAQ STING Is a Null
AlleleHuman)
4. Genes and Immunity (2011) 12, 263–269 (2011_Identification and characterization of a loss-offunction STING SNPs)

We Claim:

1. A method for determining the genetic polymorphism pattern in the “Stimulator
of Interferon Genes” (STING) gene in a subject, the method comprising:
5 - amplifying, in a sample obtained from a subject, a sequence of genomic DNA
comprising a single nucleotide polymorphism (SNP) within the STING gene;
and
- performing restriction fragment length polymorphism (RFLP) pattern analysis
on the amplified DNA to determine the genetic polymorphism pattern in the
10 STING gene in the sample.
2. A method for determining the efficacy of a treatment of a subject with a
“Stimulator of Interferon Genes” (STING) agonist or a STING antagonist, the method
comprising:
15 - determining the genetic polymorphism pattern in the Stimulator of Interferon
Genes (STING) gene in a sample obtained from a subject using the method
according to claim 1; and
- determining the suitability of the subject for STING agonist or antagonist
therapy based on the genetic polymorphism pattern in the STING gene.
20
3. The method according to either claim 1 or 2, wherein the polymorphism pattern
in the STING gene, which is determined, corresponds to STING variant R232,
optionally wherein the STING variant R232 comprises or consists of the amino acid
sequence substantially as set out in SEQ ID No:2 and/or is encoded by a nucleic acid
25 sequence substantially as set out in SEQ ID No:1.
4. The method according to either claim 1 or 2, wherein the polymorphism pattern
in the STING gene, which is determined, comprises a SNP which corresponds to STING
variant R71H-G230A-R293Q or “HAQ”, optionally wherein the STING variant R71H30 G230A-R293Q or “HAQ” comprises or consists of the amino acid sequence
substantially as set out in SEQ ID No:4 and/or is encoded by a nucleic acid sequence
substantially as set out in SEQ ID No:3.
5. The method according to either claim 1 or 2, wherein the polymorphism pattern
35 in the STING gene, which is determined, comprises a SNP which corresponds to STING
variant R232H or “H232”, optionally wherein the STING variant R232H or “H232”
- 33 -
comprises or consists of the amino acid sequence substantially as set out in SEQ ID
No:6 and/or is encoded by a nucleic acid sequence substantially as set out in SEQ ID
No:5.
5 6. The method according to either claim 1 or 2, wherein the polymorphism pattern
in the STING gene, which is determined, comprises a SNP which corresponds to STING
variant G230A-R293Q or “AQ”, optionally wherein the STING variant G230A-R293Q
or “AQ” comprises or consists of the amino acid sequence substantially as set out in
SEQ ID No:8 and/or is encoded by a nucleic acid sequence substantially as set out in
10 SEQ ID No:7.
7. The method according to either claim 1 or 2, wherein the polymorphism pattern
in the STING gene, which is determined, comprises a SNP which corresponds to STING
variant R293Q or “Q”, optionally wherein the STING variant R293Q or “Q” comprises
15 or consists of the amino acid sequence substantially as set out in SEQ ID No:2 and/or is
encoded by a nucleic acid sequence substantially as set out in SEQ ID No:1.
8. The method according to any preceding claim, wherein the amplification step
comprises PCR, optionally
20 wherein the PCR comprises an initial denaturation step carried out at a
temperature of between 94 and 98°C, or between 96 and 98°C, or between 97 and
98°C, and for a period of between 30 seconds and 5 minutes, or between 1 and 4
minutes, or between 2 and 3 minutes; and/or
wherein the PCR is carried out for between 20 and 40 cycles, or between 25 and
25 35 cycles, wherein a cycle includes a denaturation step, an annealing step and an
extension step.
9. The method according to claim 11, wherein the denaturation step is carried out
at a temperature of between 94 and 98°C, or between 95 and 98°C, or between 96 and
30 98°C, or between 97 and 98°C, and for a period of between 20 seconds and 2 minutes,
or between 20 seconds and 1 minute, or between 20 and 30 seconds; and/or
wherein the annealing step is carried out at a temperature of between 40 and
60°C, or between 50 and 60°C, and for a period of between 30 seconds and 2 minutes,
or between 30 seconds and 1 minute; and/or
- 34 -
wherein the extension step is carried out at a temperature of between 70 and
75°C, or between 71 and 74°C, or between 72 and 73°C, and for a period of between 30
seconds and 2 minutes, or between 30 seconds and 1 minute.
5 10. The method according to either claim 8 or 9, wherein the PCR includes a final
extension step carried out at a temperature of between 70 and 75°C, or between 71 and
74°C, or between 72 and 73°C, and for a period of between 5 and 15 minutes, or
between 6 and 14 minutes, or between 7 and 13 minutes, or between 8 and 12 minutes,
or between 9 and 11 minutes.
10
11. The method according to any one of claims 8 to 10, wherein the PCR uses a
forward primer and a reverse primer designed to hybridize to sequences flanking the
SNPs which correspond to amino acid residues 71, 230, 232 and 293 of the STING
variant.
15
12. The method according to claim 11, in which the SNP location corresponds to
amino acid residue 71, wherein the forward primer comprises a nucleotide sequence
substantially as set out in SEQ ID No: 11, or a variant or fragment thereof, and the
reverse primer comprises a nucleotide sequence substantially as set out in SEQ ID No:
20 12, or a variant or fragment thereof.
13. The method according to claim 11, in which the SNP location corresponds to
amino acid residue 230 or 232, wherein the forward primer comprises a nucleotide
sequence substantially as set out in SEQ ID No: 13, or a variant or fragment thereof,
25 and the reverse primer comprises a nucleotide sequence substantially as set out in SEQ
ID No: 14, or a variant or fragment thereof.
14. The method according to claim 11, in which the SNP location corresponds to
amino acid residue 293, wherein the forward primer comprises a nucleotide sequence
30 substantially as set out in SEQ ID No: 15, or a variant or fragment thereof, and the
reverse primer comprises a nucleotide sequence substantially as set out in SEQ ID No:
16, or a variant or fragment thereof.
15. The method according to any preceding claim, wherein the RFLP pattern
35 analysis comprises subjecting the genomic DNA to restriction enzyme digestion to
- 35 -
produce at least one fragment; and subjecting the at least one fragment to gel
electrophoresis.
16. The method according to claim 15, wherein the restriction enzyme digestion
5 reaction comprises the genomic DNA, a buffer, a restriction enzyme and water.
17. The method according to claim 16, wherein the restriction enzyme is HhaI and
cuts the genomic DNA at the position corresponding to amino acid residue 71.
10 18. The method according to claim 16, wherein the restriction enzyme is Eco91I and
cuts the genomic DNA at the position corresponding to amino acid residue 230.
19. The method according to claim 16, wherein the restriction enzyme is Hin1II and
cuts the genomic DNA at the position corresponding to amino acid residue 232.
15
20. The method according to claim 16, wherein the restriction enzyme is HpaII and
cuts the genomic DNA at the position corresponding to amino acid residue 293.
21. The method according to claim 17, wherein restriction enzyme digestion
20 produces a 750 bp fragment and a 241 bp fragment when an arginine is located at the 71
position, and/or a 991 bp fragment when a histidine is located at the 71 position.
22. The method according to claim 18, wherein restriction enzyme digestion
produces a 317 bp fragment and a 184 bp fragment when a glycine is located at the 230
25 position, and/or a 501 bp fragment when an alanine is present at the 230 position.
23. The method according to claim 19, wherein restriction enzyme digestion
produces a 501 bp fragment when an arginine is present at the 232 position, and/or a
317 bp fragment and a 184 bp fragment when a histidine is present at the 232 position.
30
24. The method according to claim 20, wherein restriction enzyme digestion
produces a 162 bp fragment, a 116 bp fragment, and a 36 bp fragment when an arginine
is present at the 293 position, and/or a 198 bp fragment and a 116 bp fragment when a
glutamine is present at the 293 position.
35
- 36 -
25. The method according to any one of claims 15 to 24, wherein the restriction
enzyme digestion is carried out at a temperature of between 35 and 40°C, or between
36 and 39°C, or between 37 and 38°C, and for a period of between 5 minutes and 4
hours, or between 10 minutes and 3 hours, or between 15 minutes and 2 hours.
5
26. The method according to any one of claims 15 to 25, wherein the gel
electrophoresis step comprises electrophoresing the fragment on a polyacrylamide or
agarose gel, wherein the concentration of the polyacrylamide in the gel is between 5 and
10%, or between 6 and 9%, or between 7 and 8% and the concentration of the agarose
10 in the gel is between 0.5 and 4%, or between 2 and 3%.
27. An apparatus for determining the genetic polymorphism pattern in the
“Stimulator of Interferon Genes” (STING) gene in a subject using the method of any
one of claims 1 to 26, the apparatus comprising:
15 - means for amplifying, in a sample obtained from a subject, a sequence of
genomic DNA comprising a single nucleotide polymorphism (SNP) within the
STING gene; and
- means for performing restriction fragment length polymorphism (RFLP)
pattern analysis on the amplified DNA to determine the genetic polymorphism
20 pattern in the STING gene in the sample.
28. A diagnostic or prognostic tool for assessing the suitability of a subject for
“Stimulator of Interferon Genes” (STING) agonist or antagonist therapy, comprising:
- determining the genetic polymorphism pattern in the Stimulator of Interferon
25 Genes (STING) in a sample obtained from a subject using the method of any
one of claims 1 to 26 or the apparatus of claim 27; and
- determining the suitability of the subject for STING agonist or antagonist
therapy based on the genetic polymorphism pattern.