Treatment of septicemia with PCSK9 and LDLR modulators

Abstract

To provide an in vitro method for assessing the risk of developing sepsis, systemic inflammatory response syndrome (SIRS), septic shock, or multiple organ dysfunction syndrome (MODS) in a subject. Also provided are pharmaceutical compositions for the treatment of sepsis, SIRS, septic shock or MODS in a subject.SOLUTION: Determining or having determined the presence or absence of i) a PCSK9 variant nucleic acid sequence encoding a Pro-protein convertase subtilisin / kexin type 9 (Arg46Leu) PCSK9; and ii) a low-density-lipoprotein receptor (LDLR) variant nucleic acid sequence comprising a single nucleotide polymorphism rs6511720 in a biological sample obtained from the subject, wherein the subject is a PCSK9 reference and being a LDLR reference indicates that the subject has an increased risk of developing SIRS / MODS.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Reference to sequence listing This application contains a Sequence Listing which has been submitted electronically as a text file of 340 kilobytes in size, designated 18923805302SEQ, generated on August 24, 2021. The Sequence Listing is hereby incorporated by reference.

[0002] The present disclosure relates generally to the treatment of subjects with sepsis, systemic inflammatory response syndrome (SIRS), septic shock, and / or multiple organ dysfunction syndrome (MODS) with proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors and / or low-density lipoprotein receptor (LDLR) agonists, methods for identifying subjects at increased risk for developing sepsis, SIRS, septic shock, and / or MODS, and methods for detecting variant nucleic acid molecules and variant polypeptides of PCSK9 and / or LDLR. [Background technology]

[0003] Sepsis is a systemic inflammatory response to infection that can lead to organ failure and death. The annual incidence of sepsis exceeds 48 million cases, accounting for approximately 20% of deaths worldwide (Non-Patent Document 1). Bacterial cell wall components (including lipopolysaccharide (LPS) and lipoteichoic acid (LTA) for Gram-negative and Gram-positive bacteria, respectively) are important mediators of sepsis and septic shock. In healthy individuals, LPS / LTA are incorporated into lipoprotein particles (including LDL and HDL) and removed from plasma via the LDL receptor (LDLR) in hepatocytes as a primary step in the sequestration of pathogen toxins from the circulation. Generally, sepsis is characterized by two or more measures of a systemic inflammatory response, plus a known or suspected infection. Severe sepsis is sepsis accompanied by acute organ dysfunction. Sepsis can progress to severe sepsis when, in addition to the symptoms of sepsis, there are signs of organ dysfunction, such as difficulty breathing (lung problems), low or no urine output (kidneys), abnormal liver tests (liver), and altered mental status (brain). Nearly all patients with severe sepsis require treatment in an intensive care unit (ICU). Septic shock is the most severe level and is diagnosed when a subject's blood pressure drops to dangerous levels.

[0004] Systemic inflammatory response syndrome (SIRS) is the body's enhanced defense response to harmful stressors (infection, trauma, surgery, acute inflammation, ischemia or reperfusion, or malignancy, to name a few) to localize and then eliminate endogenous or exogenous causes of injury. It involves the release of acute-phase reactants that are direct mediators of a wide range of autonomic, endocrine, hematologic, and immunological changes in the subject. A dysregulated cytokine storm has the capacity to trigger a massive inflammatory cascade that can lead to reversible or irreversible end-organ dysfunction and death.

[0005] During sepsis, direct tissue toxicity caused by systemic hypotension, impaired microcirculatory perfusion, and inflammatory immune responses can contribute to organ failure. Failure of two or more vital organ systems is termed multiple organ dysfunction syndrome (MODS) and resembles a critical condition associated with high morbidity and mortality. Importantly, no specific therapeutic strategies exist to effectively prevent the development of MODS during sepsis.

[0006] Proprotein convertase subtilisin / kexin type 9 is an enzyme (encoded by PCSK9) that binds LDLR and prevents its recycling to the cell surface. It has been observed that the addition of exogenous PCSK9 protein reduced LPS / LTA uptake into cells in vitro (Non-Patent Documents 2, 3, 4). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Rudd et al.,Lancet,2020,395,200-2011 [Non-patent document 2] Boyd,Inn.Immunol.,2016 [Non-patent document 3] Grin,Sci.Rep.,2018 [Non-patent document 4] Leung,Sci.Rep.,2019 Summary of the Invention

[0008] The present disclosure provides methods of treating a subject with sepsis or severe sepsis, comprising administering a PCSK9 inhibitor and / or an LDLR agonist to a subject in need thereof.

[0009] The present disclosure also provides a method of treating a subject having SIRS, comprising administering a PCSK9 inhibitor and / or an LDLR agonist to a subject in need thereof.

[0010] The present disclosure also provides a method of treating a subject having septic shock, comprising administering a PCSK9 inhibitor and / or an LDLR agonist to a subject in need thereof.

[0011] The present disclosure also provides a method of treating a subject with MODS, comprising administering a PCSK9 inhibitor and / or an LDLR agonist to a subject in need thereof.

[0012] The present disclosure provides a method of treating a subject with a therapeutic agent that treats or inhibits sepsis, SIRS, septic shock, and / or MODS, wherein the subject is suffering from sepsis, SIRS, septic shock, and / or MODS, comprising determining whether the subject has i) a PCSK9 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of PCSL9; and / or ii) an LDLR variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of LDLR by obtaining or having obtained a biological sample from the subject; and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype that includes i) a PCSK9 variant nucleic acid molecule; and / or ii) an LDLR variant nucleic acid molecule; and if the subject is a PCSK9 reference and an LDLR reference, then the subject is suffering from sepsis, SIRS, septic shock, and / or MODS. administering or continuously administering to the subject a therapeutic agent treating or inhibiting S at a standard dosage, and administering a PCSK9 inhibitor and / or LDLR agonist to the subject; if the subject is an LDLR reference and heterozygous for a PCSK9 variant nucleic acid molecule, then administering or continuously administering to the subject a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at an amount equal to or less than the standard dosage, and administering a PCSK9 inhibitor and / or LDLR agonist to the subject; if the subject is a PCSK9 reference and heterozygous for an LDLR variant nucleic acid molecule, then administering or continuously administering to the subject a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at an amount equal to or less than the standard dosage, and administering a PCSK9 inhibitor and / or LDLR agonist to the subject;If the subject is an LDLR reference and homozygous for the PCSK9 variant nucleic acid molecule, then a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is also administered or continuously administered to the subject in an amount equal to or less than the standard dosage, and administering an LDLR agonist to the subject; if the subject is a PCSK9 reference and homozygous for the LDLR variant nucleic acid molecule, then a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is also administered or continuously administered to the subject in an amount equal to or less than the standard dosage, and administering a PCSK9 inhibitor to the subject; if the subject is heterozygous for both the PCSK9 variant nucleic acid molecule and the LDLR variant nucleic acid molecule, then a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is also administered or continuously administered to the subject in an amount equal to or less than the standard dosage, and administering a PCSK9 inhibitor to the subject. Also provided is a method comprising the steps of administering or continuously administering to the subject a therapeutic agent treating or inhibiting MODS at an amount equal to or less than a standard dosage, and administering to the subject a PCSK9 inhibitor and / or an LDLR agonist; if the subject is homozygous for both the PCSK9 variant nucleic acid molecule and the LDLR variant nucleic acid molecule, also administering to the subject a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at an amount equal to or less than a standard dosage; and indicating that the presence of a genotype having both a PCSK9 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of PCSL9 and an LDLR variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of LDLR indicates that the subject has a reduced risk of developing sepsis, SIRS, septic shock, and / or MODS.

[0013] The present disclosure also provides a method for identifying a subject at risk of developing sepsis, SIRS, septic shock, and / or MODS, comprising determining or having determined the presence or absence of a PCSK9 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of PCSL9 and / or an LDLR variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of LDLR in a biological sample obtained from the subject; if the subject is a PCSK9 reference and an LDLR reference, the subject has an increased risk of developing sepsis, SIRS, septic shock, and / or MODS; and if the subject is heterozygous or homozygous for the LDLR variant nucleic acid molecule and / or is heterozygous or homozygous for the PCSK9 variant nucleic acid molecule, the subject has a reduced risk of developing sepsis, SIRS, septic shock, and / or MODS.

[0014] The present disclosure provides a method for detecting a PCSK9 variant nucleic acid molecule in a subject, comprising assaying a sample obtained from the subject to determine whether the nucleic acid molecule in the sample is a genomic nucleic acid molecule comprising a nucleotide sequence comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or its complement; an mRNA molecule having a nucleotide sequence comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, or its complement; an mRNA molecule having a nucleotide sequence comprising a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or its complement; or an mRNA molecule having a nucleotide sequence comprising a uracil at a position corresponding to position 137 set forth in SEQ ID NO:19, or its complement. A molecule, or its complement; or a cDNA molecule produced from the mRNA molecule, wherein the cDNA molecule has a nucleotide sequence that includes a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a cDNA molecule produced from the mRNA molecule, wherein the cDNA molecule has a nucleotide sequence that includes a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a cDNA molecule produced from the mRNA molecule, wherein the cDNA molecule has a nucleotide sequence that includes a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement.

[0015] The present disclosure also provides a method for detecting an LDLR variant nucleic acid molecule in a subject, comprising assaying a sample obtained from the subject to determine whether the nucleic acid molecule in the sample is a genomic nucleic acid molecule comprising a nucleotide sequence comprising a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof.

[0016] The present disclosure also provides a method for detecting the presence of a PCSK9 Arg46Leu polypeptide, comprising performing an assay on a sample obtained from a subject to determine whether the PCSK9 protein in the sample comprises a leucine at a position corresponding to position 46 set forth in SEQ ID NO:42.

[0017] The present disclosure relates to a genomic nucleic acid molecule having a nucleotide sequence encoding a proprotein convertase subtilisin / kexin type 9 (PCSK9) polypeptide, the nucleotide sequence comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and / or a genomic nucleic acid molecule having a nucleotide sequence encoding a low density lipoprotein receptor (LDLR) polypeptide, the nucleotide sequence comprising a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, or a complement thereof; an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or a complement thereof; or a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 217 set forth in SEQ ID NO:19, or a complement thereof. or a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or a complement thereof; or a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or a complement thereof.

[0018] The present disclosure relates to a) a subject that is a reference for a PCSK9 genomic nucleic acid molecule and / or an LDLR genomic nucleic acid molecule, a PCSK9 mRNA molecule and / or an LDLR mRNA molecule, or a PCSK9 cDNA molecule and / or an LDLR cDNA molecule; or b) a subject that is heterozygous for i) a genomic nucleic acid molecule having a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2; and / or ii) a genomic nucleic acid molecule having a nucleotide sequence encoding an LDLR polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4; an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, or its complement; an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or its complement; or a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 137 set forth in SEQ ID NO:19. Also provided are proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors and / or low-density lipoprotein receptor (LDLR) agonists for use in treating sepsis, systemic inflammatory response syndrome (SIRS), septic shock, and / or multiple organ dysfunction syndrome (MODS) in a subject having an mRNA molecule having the nucleotide sequence of:

[0019] The accompanying figures, which are incorporated by reference and constitute a part of this specification, illustrate several features of the present disclosure.

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0021] [Figure 1]

[0033] Figure 1 shows summary data for meta-analysis of PCSK9 p.R46L and 28-day mortality across all studied cohorts in severe sepsis. [Figure 2-1] Genetically predicted reduction in LDL levels and sepsis mortality based on genotype group is shown (calculated as follows: N_Alt_allele_rs11591147 × LDL_weighting_rs11591147 + N_Alt_allele_rs6511720 × LDL_weighting_rs6511720; SNP weighting was estimated jointly using linear regression (covariates: age and sex) in UKB500K with levels measured on the LDL raw scale (mg / dL); weighting was −11.7 for PCSK9 variants and −5.9 for LDLR variants). [Figure 2-2] Genetically predicted reduction in LDL levels and sepsis mortality based on genotype group is shown (calculated as follows: N_Alt_allele_rs11591147 × LDL_weighting_rs11591147 + N_Alt_allele_rs6511720 × LDL_weighting_rs6511720; SNP weighting was estimated jointly using linear regression (covariates: age and sex) in UKB500K with levels measured on the LDL raw scale (mg / dL); weighting was −11.7 for PCSK9 variants and −5.9 for LDLR variants). [Figure 3-1]Genetically predicted reduction in LDL levels and sepsis mortality based on 10 mg / dL group is shown. [Figure 3-2] Genetically predicted reduction in LDL levels and sepsis mortality based on 10 mg / dL group is shown. DETAILED DESCRIPTION OF THE INVENTION

[0022] Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0023] Unless expressly stated otherwise, it is in no way intended that any method or aspect described herein be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically states in the claim or description that the steps are to be limited to a particular order, no order is intended to be implied in any way. This is true for all potentially imprecise criteria for interpretation, including questions of logic regarding the arrangement of steps or operational flow, the plain meaning derived from grammatical construction or punctuation, or the number or type of aspects described herein.

[0024] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0025] As used herein, the term "about" means that the recited numerical value is an approximation and that small variations would not significantly affect the practice of the disclosed embodiments. When numerical values ​​are used, unless otherwise indicated by context, the term "about" means that the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments.

[0026] As used herein, the term "comprising" may be replaced with "consisting of" or "consisting essentially of," as desired, in certain embodiments.

[0027] As used herein, the term "isolated," with respect to a nucleic acid molecule or polypeptide, means that the nucleic acid molecule or polypeptide is in a condition other than its native environment (e.g., away from blood and / or animal tissue). In some embodiments, an isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides (particularly other nucleic acid molecules, polypeptides of animal origin). In some embodiments, the nucleic acid molecule or polypeptide can be in a highly purified form (i.e., greater than 95% pure, or greater than 99% pure). When used in this context, the term "isolated" does not exclude the presence of alternative physical forms (such as dimers, or alternatively phosphorylated or derivatized forms) of the same nucleic acid molecule or polypeptide.

[0028] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" include polymeric forms of nucleotides of any length, including DNA and / or RNA, which may be single-stranded, double-stranded, or multi-stranded. A reference to one strand of a nucleic acid also refers to its complement.

[0029] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs, etc.), pet animals (e.g., dogs, cats, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), and non-human primates (e.g., apes and monkeys, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a patient under the care of a physician.

[0030] Rare variants in the PCSK9 gene and the LDLR gene associated with a reduced risk of developing sepsis, SIRS, septic shock, and / or MODS in a subject have been identified according to the present disclosure. For example, a genetic mutation that changes the guanine nucleotide at position 427 of the human PCSK9 reference (see SEQ ID NO: 1) to thymine and / or a genetic mutation that changes the guanine nucleotide at position 2,269 of the human LDLR reference (see SEQ ID NO: 3) to thymine has been observed to indicate that a person with such a mutation may have a reduced risk of developing sepsis, severe sepsis, SIRS, septic shock, and / or MODS. Such variants in the PCSK9 gene and the LDLR gene are believed to have no known association with sepsis, severe sepsis, SIRS, septic shock, and / or MODS. In summary, the genetic analysis described herein unexpectedly indicates that the PCSK9 gene and the LDLR gene, and in particular variants in the PCSK9 gene and the LDLR gene, are associated with a reduced risk of developing sepsis, severe sepsis, SIRS, septic shock, and / or MODS. Thus, a subject who is a PCSK9 reference and / or a LDLR reference has an increased risk of developing sepsis, SIRS, septic shock, and / or MODS, and the subject can be treated so that sepsis, severe sepsis, SIRS, septic shock, and / or MODS are prevented, the symptoms are reduced, and / or the onset of the symptoms is inhibited. Accordingly, the present disclosure provides methods that utilize the identification of such variants in a subject to identify or stratify the risk of developing sepsis, severe sepsis, SIRS, septic shock, and / or MODS in such a subject, or to diagnose a subject as having an increased risk of developing sepsis, severe sepsis, SIRS, septic shock, and / or MODS, so that at-risk subjects or subjects with active disease can be treated accordingly.

[0031] For the purposes of this disclosure, any particular subject can be classified as having any one of the following PCSK9 genotypes and LDLR genotypes: i) a PCSK9 reference and an LDLR reference; ii) a heterozygote or homozygote for a PCSK9 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of PCSK9 and an LDLR reference; iii) a heterozygote or homozygote for an LDLR variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of LDLR and a PCSK9 reference; or iii) a heterozygote or homozygote for both a PCSK9 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of PCSK9 and an LDLR variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of LDLR. A subject is a PCSK9 reference and / or an LDLR reference if the subject has no copies of the PCSK9 variant nucleic acid molecule and / or the LDLR variant nucleic acid molecule. A subject is a heterozygote for a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule if the subject has a single copy of the PCSK9 variant nucleic acid molecule and / or the LDLR variant nucleic acid molecule. As used herein, a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule is any PCSK9 nucleic acid molecule and / or any LDLR nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) having a genetic variation described herein that encodes a polypeptide with partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. A subject with a PCSK9 polypeptide with partial loss of function (or predicted partial loss of function) is hypomorphic for PCSK9. A PCSK9 variant nucleic acid molecule can be any nucleic acid molecule encoding a PCSK9 Arg46Leu polypeptide. If a subject has two copies of a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule, the subject is homozygous for the PCSK9 variant nucleic acid molecule and / or the LDLR variant nucleic acid molecule.

[0032] For subjects who are genotyped or determined to be PCSK9 reference and LDLR reference, such subjects have an increased risk of developing sepsis, severe sepsis, SIRS, septic shock, and / or MODS. For subjects who are genotyped or determined to be heterozygous for a PCSK9 variant nucleic acid molecule and / or heterozygous for an LDLR variant nucleic acid molecule, such subjects can be treated with a PCSK9 inhibitor and / or an agent that reduces LDL (such as an LDLR agonist).

[0033] In any of the embodiments described herein, the PCSK9 variant nucleic acid molecule and / or the LDLR variant nucleic acid molecule can be any PCSK9 nucleic acid molecule and / or any LDLR nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, a cDNA molecule, etc.) that encodes a polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. For example, the PCSK9 variant nucleic acid molecule can be any nucleic acid molecule that encodes a PCSK9 Arg46Leu polypeptide.

[0034] In any of the embodiments described herein, the predicted loss-of-function polypeptide of PCSK9 can be any PCSK9 polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function. In any of the embodiments described herein, the predicted loss-of-function polypeptide of PCSK9 can be any of the PCSK9 polypeptides described herein (including, for example, a PCSK9 Arg46Leu polypeptide).

[0035] In any of the embodiments described herein, the subject may have sepsis, severe sepsis, SIRS, septic shock, and / or MODS. In any of the embodiments described herein, the subject may have sepsis. In any of the embodiments described herein, the subject may have severe sepsis. In any of the embodiments described herein, the subject may have SIRS. In any of the embodiments described herein, the subject may have septic shock. In any of the embodiments described herein, the subject may have MODS.

[0036] Symptoms of sepsis include, but are not limited to, fever, diarrhea, decreased blood pressure, vascular leakage, and / or disseminated blood clotting in various organs. Underlying dysfunctions include, but are not limited to, arterial hypotension, metabolic acidosis, decreased systemic vascular resistance, tachypnea, organ dysfunction, and sepsis.

[0037] Symptoms of SIRS include, but are not limited to, any two or more of the following: 1) body temperature >38°C or <36°C; 2) heart rate >90 beats / min; 3) respiratory rate >20 breaths / min or PaCO2 <32 mmHg; and 4) white blood cell count >12,000 / μL or <4000 / μL or band neutrophil ratio >10%.

[0038] Symptoms of septic shock include, but are not limited to, a decrease in blood pressure to dangerous levels in a subject with sepsis.

[0039] Symptoms of MODS include, but are not limited to, failure of two or more vital organ systems.

[0040] The present disclosure provides methods of treating a subject having sepsis, SIRS, septic shock, and / or MODS, comprising administering to the subject a PCSK9 inhibitor and / or an LDLR agonist.

[0041] The present disclosure also provides a method of treating a subject having sepsis, comprising administering to the subject a PCSK9 inhibitor and / or an LDLR agonist.

[0042] The present disclosure also provides a method of treating a subject having SIRS, the method comprising administering to the subject a PCSK9 inhibitor and / or an LDLR agonist.

[0043] The present disclosure also provides a method of treating a subject having septic shock, comprising administering to the subject a PCSK9 inhibitor and / or an LDLR agonist.

[0044] The present disclosure also provides a method of treating a subject with MODS, the method comprising administering to the subject a PCSK9 inhibitor and / or an LDLR agonist.

[0045] In some embodiments, the PCSK9 inhibitor and / or LDLR agonist comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNA (siRNA), and short hairpin RNA (shRNA). Such antisense molecules can be designed to target any region of a PCSK9 nucleic acid molecule and / or an LDLR nucleic acid molecule (such as an mRNA molecule). In some embodiments, the antisense molecule, siRNA, or shRNA hybridizes to a sequence within a PCSK9 and / or LDLR genomic nucleic acid molecule or mRNA molecule and reduces the expression of a PCSK9 polypeptide and / or an LDLR polypeptide in cells of a subject. In some embodiments, the PCSK9 inhibitor and / or LDLR agonist comprises an antisense molecule that hybridizes to a PCSK9 and / or LDLR genomic nucleic acid molecule or mRNA molecule and reduces the expression of a PCSK9 polypeptide and / or an LDLR polypeptide in cells of a subject. In some embodiments, the PCSK9 inhibitor and / or LDLR agonist comprises an siRNA molecule that hybridizes to a PCSK9 and / or LDLR genomic nucleic acid molecule or mRNA molecule and reduces expression of a PCSK9 polypeptide and / or an LDLR polypeptide in cells in a subject. In some embodiments, the PCSK9 inhibitor and / or LDLR agonist comprises an shRNA molecule that hybridizes to a PCSK9 and / or LDLR genomic nucleic acid molecule or mRNA molecule and reduces expression of a PCSK9 polypeptide and / or an LDLR polypeptide in cells in a subject.

[0046] In some embodiments, the PCSK9 inhibitor and / or LDLR agonist comprises a nuclease agent that induces one or more nicks or double-strand breaks at the recognition sequence(s), or a DNA-binding protein that binds to the recognition sequence within the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule. The recognition sequence may be located within the coding region of the PCSK9 gene and / or the LDLR gene or within a regulatory region that affects gene expression. The recognition sequence of the DNA-binding protein or nuclease agent may be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein-coding region. The recognition sequence may include or be adjacent to the start codon of the PCSK9 gene and / or the LDLR gene. For example, the recognition sequence may be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents can be used, each targeting a nuclease recognition sequence that includes or is adjacent to the start codon. As another example, two nuclease agents (one targeting a nuclease recognition sequence that includes or is adjacent to the start codon and one targeting a nuclease recognition sequence that includes or is adjacent to the stop codon) can be used, and cleavage by the nuclease agents can result in the deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-stranded break in the desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA-binding protein that binds to the desired recognition sequence can be used in the methods and compositions disclosed herein.

[0047] Nuclease agents and DNA-binding proteins suitable for use herein include, but are not limited to, zinc finger proteins or zinc finger nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALENs), or clustered regularly interspersed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary, for example, about 30-36 bp for zinc finger proteins or ZFN pairs, about 15-18 bp for each ZFN, about 36 bp for TALE proteins or TALENs, and about 20 bp for CRISPR / Cas guide RNAs.

[0048] In some embodiments, a CRISPR / Cas system can be used to modify a PCSK9 genomic nucleic acid molecule and / or an LDLR genomic nucleic acid molecule in a cell. The methods and compositions disclosed herein can use the CRISPR-Cas system by utilizing a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-directed cleavage of a PCSK9 nucleic acid molecule and / or an LDLR nucleic acid molecule.

[0049] Cas proteins generally contain at least one RNA recognition or binding domain capable of interacting with a gRNA. Cas proteins may also contain a nuclease domain (e.g., a DNase domain or an RNase domain), a DNA-binding domain, a helicase domain, a protein-protein interaction domain, a dimerization domain, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 protein and wild-type Cpf1 protein (e.g., FnCpf1). Cas proteins may have full cleavage activity to generate double-strand breaks in PCSK9 genomic nucleic acid molecules and / or LDLR genomic nucleic acid molecules, or may be nickases that generate single-strand breaks in PCSK9 genomic nucleic acid molecules and / or LDLR genomic nucleic acid molecules. Additional examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), and Cse Cas proteins include, but are not limited to, CasC, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, as well as homologs or modified versions thereof. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, Cas proteins can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Cas proteins can be provided in any form. For example, the Cas protein can be provided in the form of a protein (such as a Cas protein complexed with a gRNA).Alternatively, the Cas protein may be provided in the form of a nucleic acid molecule (such as RNA or DNA) that encodes the Cas protein.

[0050] In some embodiments, targeted genetic modification of a PCSK9 genomic nucleic acid molecule and / or an LDLR genomic nucleic acid molecule can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize to one or more gRNA recognition sequences within a target genomic locus in the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located within the region of SEQ ID NO: 1 and / or SEQ ID NO: 3. The gRNA recognition sequence can also include or be adjacent to a position corresponding to position 427 set forth in SEQ ID NO: 1 or position 2,269 set forth in SEQ ID NO: 3. For example, the gRNA recognition sequence can be located about 1,000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides from a position corresponding to position 427 set forth in SEQ ID NO: 1 or position 2,269 set forth in SEQ ID NO: 3. The gRNA recognition sequence may include or be adjacent to the start codon of the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule or the stop codon of the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule. For example, the gRNA recognition sequence may be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start or stop codon.

[0051] The gRNA recognition sequence within the target genomic locus in the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence (a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease). A canonical PAM is the sequence 5'-NGG-3' (where "N" is any nucleobase and is followed by two guanine ("G") nucleobases). The gRNA can transport Cas9 to any location in the genome for gene editing, but editing cannot occur at any site other than the site where Cas9 recognizes the PAM. In addition, 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells. Generally, the PAM is approximately 2-6 nucleotides downstream of the DNA sequence targeted by the gRNA. The PAM can be adjacent to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM on the 3' end. In some embodiments, the gRNA recognition sequence can be flanked on the 5' end by a PAM. For example, the cleavage site of the Cas protein can be about 1 to about 10, about 2 to about 5, or 3 base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when Cas9 from S. pyogenes or a closely related Cas9 is used), the PAM sequence of the non-complementary strand can be 5'-NGG-3' (where N is any DNA nucleotide and is immediately 3' to the gRNA recognition sequence on the non-complementary strand of the target DNA). Therefore, the PAM sequence of the complementary strand will be 5'-CCN-3' (where N is any DNA nucleotide and is immediately 5' to the gRNA recognition sequence on the non-complementary strand of the target DNA).

[0052] A gRNA is an RNA molecule that binds to a Cas protein and targets the Cas protein to a specific location within a PCSK9 genomic nucleic acid molecule and / or an LDLR genomic nucleic acid molecule. Exemplary gRNAs are gRNAs that are effective in directing a Cas enzyme to bind to or cleave a PCSK9 genomic nucleic acid molecule and / or an LDLR genomic nucleic acid molecule, and the gRNA comprises a DNA-targeting segment that hybridizes to a gRNA recognition sequence within a PCSK9 genomic nucleic acid molecule and / or an LDLR genomic nucleic acid molecule that includes or is adjacent to a position corresponding to position 427 set forth in SEQ ID NO:1 or position 2,269 set forth in SEQ ID NO:3. For example, a gRNA can be selected such that it hybridizes to a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from a position corresponding to position 427 set forth in SEQ ID NO: 1 or position 2,269 set forth in SEQ ID NO: 3. Other exemplary gRNAs comprise a DNA-targeting segment that hybridizes to a gRNA recognition sequence present in a PCSK9 genomic nucleic acid molecule and / or an LDLR genomic nucleic acid molecule that includes or is adjacent to a start or stop codon. For example, a gRNA can be selected so that it hybridizes to a gRNA recognition sequence located about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the start codon or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the stop codon. Suitable gRNAs can contain about 17 to about 25 nucleotides, about 17 to about 23 nucleotides, about 18 to about 22 nucleotides, or about 19 to about 21 nucleotides. In some embodiments, a gRNA can contain 20 nucleotides.

[0053] Examples of suitable gRNA recognition sequences located within the PCSK9 reference gene are shown in Table 1 as SEQ ID NOs: 43-68. Examples of suitable gRNA recognition sequences located within the LDLR reference gene are shown in Table 1 as SEQ ID NOs: 69-89. [Table 1-1] [Table 1-2]

[0054] The Cas protein and gRNA form a complex, and the Cas protein cleaves the target PCSK9 genomic nucleic acid molecule and / or the target LDLR genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at a site within or outside the nucleic acid sequence present in the target PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule to which the DNA-targeting segment of the gRNA binds. For example, formation of a CRISPR complex (comprising a gRNA hybridized to a gRNA recognition sequence and complexed with a Cas protein) can result in cleavage of one or both strands within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the nucleic acid sequence present in the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule to which the DNA-targeting segment of the gRNA binds.

[0055] Such methods may result in PCSK9 genomic nucleic acid molecules and / or LDLR genomic nucleic acid molecules in which, for example, the region of SEQ ID NO: 1 or SEQ ID NO: 3 is disrupted, the start codon is disrupted, the stop codon is disrupted, or the coding sequence is disrupted or deleted. Optionally, the cell may be further contacted with one or more additional gRNAs that hybridize to additional gRNA recognition sequences within the target genomic locus in the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (e.g., a second gRNA that hybridizes to a second gRNA recognition sequence), cleavage by the Cas protein may generate two or more double-strand breaks or two or more single-strand breaks.

[0056] In some embodiments, the PCSK9 inhibitor comprises a small molecule or an antibody. In some embodiments, the PCSK9 inhibitor is alirocumab or evolocumab, or a combination thereof. In some embodiments, the PCSK9 inhibitor is monoclonal antibody SAR236553 / REGN727 or AMG145, or a combination thereof. In some embodiments, the PCSK9 inhibitor is lupane peptide, resveratrol, lycopene, or eugenol, or any combination thereof.

[0057] In some embodiments, LDLR agonist comprises small molecule or antibody.In some embodiments, LDLR agonist is one of the PCSK9 inhibitors described herein.In some embodiments, LDLR agonist is lupane peptide, resveratrol, lycopene, suramin or puromycin, or any combination thereof.

[0058] In some embodiments, the method of treatment further comprises detecting the presence or absence of a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule in a biological sample from the subject. As used throughout this disclosure, a "PCSK9 variant nucleic acid molecule" is any PCSK9 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule, etc.) that encodes a PCSK9 polypeptide having partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function.

[0059] The present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits sepsis, SIRS, septic shock, and / or MODS. In some embodiments, the subject is suffering from sepsis, SIRS, septic shock, and / or MODS. In some embodiments, the subject is suffering from sepsis. In some embodiments, the subject is suffering from SIRS. In some embodiments, the subject is suffering from septic shock. In some embodiments, the subject is suffering from MODS. In some embodiments, the method includes determining whether the subject has a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule by obtaining or having obtained a biological sample from the subject and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype that includes a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule.

[0060] In some embodiments, where the subject is a PCSK9 reference and an LDLR reference, a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is administered to the subject at a standard dosage or administered continuously, and a PCSK9 inhibitor and / or LDLR agonist is administered to the subject.

[0061] In some embodiments, when a subject is an LDLR reference and heterozygous for a PCSK9 variant nucleic acid molecule, a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is administered or administered continuously in an amount equal to or less than the standard dosage, and a PCSK9 inhibitor and / or LDLR agonist is administered to the subject.

[0062] In some embodiments, when a subject is a PCSK9 reference and heterozygous for an LDLR variant nucleic acid molecule, a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is administered or administered continuously in an amount equal to or less than the standard dosage, and a PCSK9 inhibitor and / or LDLR agonist is administered to the subject.

[0063] In some embodiments, when a subject is an LDLR reference and homozygous for a PCSK9 variant nucleic acid molecule, a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is administered at or continuously administered in an amount equal to or less than the standard dosage, and an LDLR agonist is administered to the subject.

[0064] In some embodiments, when a subject is a PCSK9 reference and homozygous for an LDLR variant nucleic acid molecule, a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is administered at or continuously administered in an amount equal to or less than the standard dosage, and a PCSK9 inhibitor is administered to the subject.

[0065] In some embodiments, when a subject is heterozygous for both a PCSK9 variant nucleic acid molecule and an LDLR variant nucleic acid molecule, a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is administered at or below the standard dosage or is administered continuously, and a PCSK9 inhibitor and / or LDLR agonist is administered to the subject.

[0066] In some embodiments, when a subject is homozygous for both a PCSK9 variant nucleic acid molecule and an LDLR variant nucleic acid molecule, a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is administered at or continuously administered at a dosage that is the same as or less than a standard dosage.

[0067] The presence of a genotype having a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule indicates that the subject has a reduced risk of developing sepsis, SIRS, septic shock, and / or MODS. In some embodiments, the subject is a PCSK9 reference and an LDLR reference. In some embodiments, the subject is a heterozygote or homozygote for the PCSK9 variant nucleic acid molecule and an LDLR reference. In some embodiments, the subject is a heterozygote or homozygote for the LDLR variant nucleic acid molecule and a PCSK9 reference. In some embodiments, the subject is a heterozygote or homozygote for the LDLR variant nucleic acid molecule and a PCSK9 heterozygote or homozygote for the PCSK9 variant nucleic acid molecule.

[0068] Detecting the presence or absence of a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule in a biological sample from a subject and / or determining whether a subject has a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from the subject.

[0069] In some embodiments, if the subject is a PCSK9 reference and / or an LDLR reference, the subject is also administered a standard dosage of a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS. In some embodiments, if the subject is heterozygous or homozygous for a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule, the subject is also administered a standard dosage or less of a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS.

[0070] In some embodiments, the treatment method further comprises detecting the presence or absence of a predicted loss-of-function polypeptide of PCSK9 in a biological sample from the subject. In some embodiments, if the subject does not have a predicted loss-of-function polypeptide of PCSK9, the subject is also administered a standard dosage of a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS. In some embodiments, if the subject has a predicted loss-of-function polypeptide of PCSK9, the subject is also administered a dosage of a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS that is the same as or less than the standard dosage.

[0071] The present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits sepsis, SIRS, septic shock, and / or MODS. In some embodiments, the subject is suffering from sepsis, SIRS, septic shock, and / or MODS. In some embodiments, the subject is suffering from sepsis. In some embodiments, the subject is suffering from SIRS. In some embodiments, the subject is suffering from septic shock. In some embodiments, the subject is suffering from MODS. In some embodiments, the method includes obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine whether the subject has a predicted loss-of-function polypeptide of PCSK9. If the subject does not have a predicted loss-of-function polypeptide of PCSK9, the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is administered to the subject at a standard dosage or continuously administered, and a PCSK9 inhibitor and / or LDLR agonist is administered to the subject. If the subject has a predicted loss-of-function polypeptide of PCSK9, the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is administered to the subject at an amount equal to or less than the standard dosage or continuously administered, and a PCSK9 inhibitor and / or LDLR agonist is administered to the subject. The presence of a predicted loss-of-function polypeptide of PCSK9 indicates that the subject has a reduced risk of developing sepsis, SIRS, septic shock, and / or MODS. In some embodiments, the subject has a predicted loss-of-function polypeptide of PCSK9. In some embodiments, the subject does not have a predicted loss-of-function polypeptide of PCSK9.

[0072] Detecting the presence or absence of a predicted loss-of-function polypeptide of PCSK9 in a biological sample from a subject and / or determining whether a subject has a predicted loss-of-function polypeptide of PCSK9 can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the polypeptide can be present in a cell obtained from the subject.

[0073] Examples of therapeutic agents and therapies for treating or inhibiting sepsis, severe sepsis, SIRS, septic shock, and / or MODS may depend on the affected organ(s), the severity of the condition, the time of diagnosis, and the underlying cause(s) (e.g., infection, trauma, etc.). Early treatments include antibiotics and intravenous fluids, and high-flow oxygen. Treatments may also include administering red blood cells, vasoconstrictors (e.g., norepinephrine in cases of hypotensive septic shock), and, in some cases, steroids. Surgical intervention may be used to control the source of infection (e.g., drainage of pus from an abscess). Further sepsis management includes monitoring organ function to identify affected organs and systems. Additional therapies may then be implemented to specifically target the affected organs. In cases of severe sepsis, particularly those resulting in MODS, treatment is typically largely limited to supportive care (i.e., hemodynamic protection) and respiration. In addition, several classes of therapeutic agents are used in clinical sepsis management, including recombinant activated protein C, TLR4 antagonists, C5a antagonists, C1 inhibitors, endotoxin removal devices, caspase inhibitors, estrogen receptor-β, and statins, among others (reviewed in Shukla et al., Br. J. Pharmacol., 2014, 171, 5011-5031).

[0074] In some embodiments, the dose of a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% (i.e., less than the standard dose) for a subject who is heterozygous for a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule compared to a subject who is a PCSK9 reference and / or an LDLR reference (which may receive a standard dose). In some embodiments, the dose of a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS may be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. Additionally, the dose of a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS in a subject who is heterozygous for a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule may be administered less frequently compared to a subject who is a PCSK9 reference and / or an LDLR reference.

[0075] The administration of the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS, and / or PCSK9 inhibitor and / or LDLR agonist can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months.The repeated administration can be the same dose or different doses.The administration can be repeated 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more.For example, according to a certain dosing regimen, the subject can receive a long-term therapy (for example, 6 months, 1 year, or more).

[0076] Administration of therapeutic agents for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS, and / or PCSK9 inhibitors and / or LDLR agonists can occur by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially harmful to the recipient thereof.

[0077] The terms "treat," "treating," and "treatment," as well as "prevent," "preventing," and "prevention," as used herein, refer to eliciting a desired biological response (e.g., a therapeutic effect and a prophylactic effect, respectively). In some embodiments, a therapeutic effect is a reduction / reduction in sepsis, SIRS, septic shock, and / or MODS (i.e., pathology), a reduction / reduction in the severity of sepsis, SIRS, and septic shock, and / or MODS (e.g., a reduction or inhibition of the onset of sepsis, SIRS, septic shock, and / or MODS), a reduction / reduction in symptoms and pathology-related effects, a delay in the onset of symptoms and pathology-related effects, a reduction in the severity of symptoms of pathology-related effects, a reduction in the severity of acute episodes, a reduction in the number of symptoms and pathology-related effects, a reduction in the severity of symptoms and pathology-related effects, a reduction in the severity of acute episodes, a reduction in the number of symptoms and pathology-related effects, a reduction in the severity of symptoms and pathology-related effects, a reduction in the severity of acute episodes, a reduction in the number of symptoms and pathology-related effects, a reduction in the severity of symptoms and pathology-related effects, a reduction in the severity of acute episodes, a reduction in the number of symptoms and pathology-related effects, a reduction in the severity of acute episodes ... the effects of the agent or composition comprising the agent include one or more of: reducing the latency of the effects of sepsis, SIRS, septic shock, and / or MODS, ameliorating symptoms and pathology-related effects, reducing secondary symptoms, reducing secondary infections, preventing recurrence of sepsis, SIRS, septic shock, and / or MODS, reducing the number or frequency of recurrent episodes, increasing the latency between symptomatic episodes, increasing the time to sustained progression, promoting remission, inducing remission, increasing remission, rapid recovery, or increasing the effectiveness of or reducing resistance to alternative therapies and / or increasing the survival time of the affected host animal after administration of the agent or composition comprising the agent. A prophylactic effect can include complete or partial avoidance / inhibition or delay (e.g., complete or partial avoidance / inhibition or delay, etc.) of the onset / progression of sepsis, SIRS, septic shock, and / or MODS, as well as increasing the survival time of the affected host animal after administration of the therapeutic protocol. Treatment of sepsis, SIRS, septic shock, and / or MODS encompasses treatment of subjects already diagnosed with any form of sepsis, SIRS, septic shock, and / or MODS at any clinical stage or presentation, delaying the onset or development or progression or worsening of symptoms or signs of sepsis, SIRS, septic shock, and / or MODS, and / or preventing and / or reducing the severity of sepsis, SIRS, septic shock, and / or MODS.

[0078] As used herein, the phrase "in need thereof" means that an "individual," "subject," or "patient" has been identified as having need for a particular method, prevention, or treatment. In some embodiments, identification can be diagnosis by any means. An "individual," "subject," or "patient" can be in need of any of the methods, preventions, and treatments described herein.

[0079] The present disclosure also provides methods for identifying a subject at increased risk of developing sepsis, SIRS, septic shock, and / or MODS. In some embodiments, the methods identify a subject at increased risk of developing sepsis. In some embodiments, the methods identify a subject at increased risk of developing SIRS. In some embodiments, the methods identify a subject at increased risk of developing septic shock. In some embodiments, the methods identify a subject at increased risk of developing MODS. In some embodiments, the methods include determining or having determined the presence or absence of a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) in a biological sample obtained from the subject. If the subject lacks a PCSK9 variant nucleic acid molecule and an LDLR variant nucleic acid molecule (i.e., the subject is classified by genotype as a PCSK9 reference and an LDLR reference), then the subject has an increased risk of developing sepsis, SIRS, septic shock, and / or MODS. If the subject has a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule (i.e., the subject is heterozygous or homozygous for the PCSK9 variant nucleic acid molecule and / or the LDLR variant nucleic acid molecule), then the subject has a reduced risk of developing sepsis, SIRS, septic shock, and / or MODS.

[0080] Having a single copy of a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule is more protective against developing sepsis, SIRS, septic shock, and / or MODS than not having a copy of a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule. Without intending to be limited to any particular theory or mechanism of action, it is believed that a single copy of a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule (i.e., heterozygous for a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule) protects a subject from developing sepsis, SIRS, septic shock, and / or MODS, and it is also believed that having two copies of a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule (i.e., homozygous for a predicted loss-of-function PCSK9 variant and / or a predicted loss-of-function LDLR variant) may be more protective against developing sepsis, SIRS, septic shock, and / or MODS than a single-copy subject. Thus, in some embodiments, a single copy of a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule may not be completely protective, but instead may partially or incompletely protect a subject from developing sepsis, SIRS, septic shock, and / or MODS. Without wishing to be bound by any particular theory, there may be additional factors or molecules involved in the development of sepsis, SIRS, septic shock, and / or MODS that are still present in a subject having a single copy of a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule, thus resulting in less than complete protection from developing sepsis, septic shock, and / or MODS.

[0081] Determining whether a subject has a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule in a biological sample from the subject and / or determining whether a subject has a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from the subject.

[0082] In some embodiments, when a subject is identified as having an increased risk of developing sepsis, SIRS, septic shock, and / or MODS, the subject is further treated with a therapeutic agent that treats or inhibits sepsis, SIRS, septic shock, and / or MODS, and / or a PCSK9 inhibitor and / or an LDLR agonist, as described herein. For example, when a subject is a PCSK9 reference and / or an LDLR reference and therefore has an increased risk of developing sepsis, SIRS, septic shock, and / or MODS, the subject is administered a PCSK9 inhibitor and / or an LDLR agonist. In some embodiments, such a subject is also administered a therapeutic agent that treats or inhibits sepsis, SIRS, septic shock, and / or MODS. In some embodiments, if the subject is heterozygous for a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule, the subject is administered a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at a dosage equal to or less than the standard dosage, and also is administered a PCSK9 inhibitor and / or an LDLR agonist.

[0083] In some embodiments, the subject is a PCSK9 reference and an LDLR reference. In some embodiments, the subject is a heterozygote or homozygote for a PCSK9 variant nucleic acid molecule and an LDLR reference. In some embodiments, the subject is a heterozygote or homozygote for an LDLR variant nucleic acid molecule and a PCSK9 reference. In some embodiments, the subject is a heterozygote or homozygote for an LDLR variant nucleic acid molecule and a PCSK9 heterozygote or homozygote for a PCSK9 variant nucleic acid molecule.

[0084] The present disclosure also provides methods for detecting the presence or absence of PCSK9 variant genomic nucleic acid molecules and / or LDLR variant genomic nucleic acid molecules in a biological sample from a subject, and / or PCSK9 variant mRNA molecules in a biological sample from a subject, and / or PCSK9 variant cDNA molecules produced from mRNA molecules in a biological sample from a subject. It is understood that gene sequences within a population and mRNA molecules encoded by such genes may vary due to polymorphisms (such as single nucleotide polymorphisms). The sequences provided herein for PCSK9 and / or LDLR variant genomic nucleic acid molecules, PCSK9 variant mRNA molecules, and PCSK9 variant cDNA molecules are merely exemplary sequences. Other sequences for PCSK9 and / or LDLR variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules are also possible.

[0085] A biological sample can be derived from any cell, tissue, or biological fluid from a subject. A biological sample can include any clinically significant tissue, such as a bone marrow sample, tumor biopsy, fine needle aspirate, or a sample of body fluid (such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine). In some cases, the sample includes a buccal swab. The biological sample used in the methods disclosed herein can vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. Depending on the assay used, the biological sample can be processed differently. For example, when detecting any PCSK9 and / or any LDLR variant nucleic acid molecule, preliminary processing designed to isolate or enrich the biological sample for genomic DNA can be used. Various techniques can be used for this purpose. When detecting the level of any PCSK9 and / or any LDLR variant mRNA molecule, different techniques can be used to enrich the biological sample containing mRNA molecules. A variety of methods can be used to detect the presence or level of mRNA molecules or the presence of particular variant genomic DNA loci.

[0086] In some embodiments, detecting a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule in a subject comprises assaying or genotyping a biological sample obtained from the subject to determine whether a PCSK9 genomic nucleic acid molecule and / or an LDLR genomic nucleic acid molecule in the biological sample, and / or a PCSK9 mRNA molecule in the biological sample, and / or a PCSK9 cDNA molecule produced from an mRNA molecule in the biological sample contains one or more variations that cause or are predicted to cause a loss of function (partial or complete).

[0087] In some embodiments, methods for detecting the presence or absence of a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule produced from an mRNA molecule, etc.) in a subject include performing an assay on a biological sample obtained from the subject, wherein the assay determines whether a nucleic acid molecule in the biological sample contains a particular nucleotide sequence.

[0088] In some embodiments, the PCSK9 nucleotide sequence comprises a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2 (for a genomic nucleic acid molecule); a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or a uracil at a position corresponding to position 137 set forth in SEQ ID NO:19 (for an mRNA molecule); or a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, a thymine at a position corresponding to position 217 set forth in SEQ ID NO:33, or a thymine at a position corresponding to position 137 set forth in SEQ ID NO:34 (for a cDNA molecule obtained from an mRNA molecule).

[0089] In some embodiments, the PCSK9 genomic nucleotide sequence comprises a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or its complement.

[0090] In some embodiments, the PCSK9 mRNA nucleotide sequence comprises a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or its complement; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or its complement; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or its complement.

[0091] In some embodiments, the PCSK9 cDNA nucleotide sequence comprises a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement.

[0092] In some embodiments, the LDLR nucleotide sequence comprises a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO: 4 (for a genomic nucleic acid molecule). In some embodiments, the LDLR genomic nucleotide sequence comprises a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO: 4, or a complement thereof.

[0093] In some embodiments, the biological sample comprises cells or cell lysates. Such a method can further comprise, for example, obtaining a biological sample comprising a PCSK9 genomic nucleic acid molecule or mRNA molecule and / or a LDLR genomic nucleic acid molecule from a subject, and if it is mRNA, optionally reverse transcribing the mRNA into cDNA. Such an assay can comprise, for example, determining the identity of these positions of a specific PCSK9 nucleic acid molecule and / or LDLR nucleic acid molecule. In some embodiments, the method is an in vitro method.

[0094] In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a PCSK9 genomic nucleic acid molecule and / or an LDLR genomic nucleic acid molecule, a PCSK9 mRNA molecule, or a PCSK9 cDNA molecule produced from the mRNA molecule in the biological sample, wherein the sequenced portion comprises one or more variations described herein.

[0095] In some embodiments, the determining, detecting, or sequence analyzing step is performed using: i) at least a portion of the nucleotide sequence of a PCSK9 genomic nucleic acid molecule and / or an LDLR genomic nucleic acid molecule in the biological sample, the portion being sequenced including a position corresponding to position 427 set forth in SEQ ID NO:2, or its complement; or a position corresponding to position 2,269 set forth in SEQ ID NO:4, or its complement; ii) at least a portion of the nucleotide sequence of a PCSK9 mRNA molecule in the biological sample, the portion being sequenced including a position corresponding to position 428 set forth in SEQ ID NO:17, or its complement; a position corresponding to position 217 set forth in SEQ ID NO:18, or its complement; or a position corresponding to position 137 set forth in SEQ ID NO:19, or its complement; and / or iii) a PCSK9 mRNA molecule produced from mRNA in the biological sample. The method includes sequencing at least a portion of the nucleotide sequence of the cDNA molecule, the portion being sequenced including a position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement. If the sequenced portion of the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule in the biological sample contains a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2 or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, respectively; or if the sequenced portion of the PCSK9 mRNA molecule in the biological sample contains a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, position 217 set forth in SEQ ID NO:18, or position 137 set forth in SEQ ID NO:19; or if the sequenced portion of the PCSK9 cDNA molecule in the biological sample contains a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; then the PCSK9 nucleic acid molecule and / or the LDLR nucleic acid molecule in the biological sample is a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule.

[0096] In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 427 set forth in SEQ ID NO:2, or its complement; or a position corresponding to position 2,269 set forth in SEQ ID NO:4, or its complement. If the sequenced portion of the PCSK9 nucleic acid molecule and / or the LDLR nucleic acid molecule in the biological sample comprises a thymine at the position corresponding to position 427 set forth in SEQ ID NO:2 or a thymine at the position corresponding to position 2,269 set forth in SEQ ID NO:4, respectively, then the PCSK9 nucleic acid molecule and / or the LDLR nucleic acid molecule in the biological sample is a PCSK9 variant nucleic acid molecule and / or an LDLR variant nucleic acid molecule.

[0097] In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a PCSK9 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 428 set forth in SEQ ID NO: 17, or its complement; a position corresponding to position 217 set forth in SEQ ID NO: 18, or its complement; or a position corresponding to position 137 set forth in SEQ ID NO: 19, or its complement. If the sequenced portion of the PCSK9 nucleic acid molecule in the biological sample comprises uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19, then the PCSK9 nucleic acid molecule in the biological sample is a PCSK9 variant nucleic acid molecule.

[0098] In some embodiments, the determining, detecting, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a PCSK9 cDNA molecule produced from an mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement. If the sequenced portion of the PCSK9 nucleic acid molecule in the biological sample comprises a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, position 217 set forth in SEQ ID NO: 33, or position 137 set forth in SEQ ID NO: 34, then the PCSK9 nucleic acid molecule in the biological sample is a PCSK9 variant nucleic acid molecule.

[0099] In some embodiments, the determining, detecting, or sequence analyzing step comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PCSK9 genomic nucleic acid molecule adjacent to a position corresponding to position 427 set forth in SEQ ID NO:2 or an LDLR genomic nucleic acid molecule adjacent to a position corresponding to position 2,269 set forth in SEQ ID NO:4; a PCSK9 mRNA molecule adjacent to a position corresponding to position 428 set forth in SEQ ID NO:17, position 217 set forth in SEQ ID NO:18, or position 137 set forth in SEQ ID NO:19; and / or a PCSK9 cDNA molecule adjacent to a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; and b) contacting the biological sample with a primer that hybridizes to at least a portion of the nucleotide sequence of a PCSK9 genomic nucleic acid molecule corresponding to position 427 set forth in SEQ ID NO:2 or an LDLR genomic nucleic acid molecule corresponding to position 2,269 set forth in SEQ ID NO:4; a PCSK9 mRNA molecule adjacent to a position corresponding to position 428 set forth in SEQ ID NO:17, position 217 set forth in SEQ ID NO:18, or position 137 set forth in SEQ ID NO:19. mRNA molecule; and / or extending a primer through a position in the nucleotide sequence of a PCSK9 cDNA molecule corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; and c) determining whether the extension product of the primer comprises: i) a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4; ii) a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, position 217 set forth in SEQ ID NO:18, or position 137 set forth in SEQ ID NO:19; and / or iii) a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34.

[0100] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule adjacent to a position corresponding to position 427 set forth in SEQ ID NO:2 or position 2,269 set forth in SEQ ID NO:4, respectively; b) extending the primer through at least the position in the nucleotide sequence of the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule corresponding to position 427 set forth in SEQ ID NO:2 or position 2,269 set forth in SEQ ID NO:4, respectively; and c) determining whether the extension product of the primer comprises a thymine at the position corresponding to position 427 set forth in SEQ ID NO:2 or position 2,269 set forth in SEQ ID NO:4.

[0101] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PCSK9 mRNA molecule proximate to a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; b) extending the primer through at least a position in the nucleotide sequence of the PCSK9 mRNA molecule corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; and c) determining whether the extension product of the primer comprises uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19.

[0102] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a PCSK9 cDNA molecule proximate to a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; b) extending the primer through at least the position in the nucleotide sequence of the PCSK9 cDNA molecule corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; and c) determining whether the extension product of the primer comprises a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34.

[0103] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) contacting the biological sample with i) a first primer that hybridizes to a portion of the nucleotide sequence of a PCSK9 genomic nucleic acid molecule adjacent to a position corresponding to position 427 set forth in SEQ ID NO:2, and ii) a second primer that hybridizes to a portion of the nucleotide sequence of an LDLR genomic nucleic acid molecule adjacent to position 2,269 set forth in SEQ ID NO:4; b) extending the first primer through at least the position in the nucleotide sequence of the PCSK9 genomic nucleic acid molecule corresponding to position 427 set forth in SEQ ID NO:2; and extending the second primer through at least the position in the nucleotide sequence of the LDLR genomic nucleic acid molecule corresponding to position 2,269 set forth in SEQ ID NO:4; and c) determining whether an extension product of the first primer comprises a thymine at the position corresponding to position 427 set forth in SEQ ID NO:2; and determining whether an extension product of the second primer comprises a thymine at the position corresponding to position 2,269 set forth in SEQ ID NO:4.

[0104] In some embodiments, the assay involves sequencing the entire nucleic acid molecule. In some embodiments, only the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule is analyzed. In some embodiments, only the PCSK9 mRNA molecule is analyzed. In some embodiments, only the PCSK9 cDNA molecule obtained from the mRNA molecule is analyzed.

[0105] In some embodiments, the determining, detecting, or sequence analyzing step is performed by detecting a) i) at least a portion of a genomic nucleic acid molecule encoding a PCSK9 polypeptide and / or an LDLR polypeptide, the amplified portion comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; ii) at least a portion of a PCSK9 mRNA molecule encoding a PCSK9 polypeptide, the amplified portion comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, or a complement thereof; a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or a complement thereof; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO:19, or a complement thereof; or iii) a PCSK9 mRNA molecule encoding a PCSK9 polypeptide, the amplified portion comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; a) amplifying at least a portion of a cDNA molecule, the amplified portion comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, or its complement; a thymine at a position corresponding to position 217 set forth in SEQ ID NO:33, or its complement; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO:34, or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) comparing the labeled nucleic acid molecule with a mutation-specific probe comprising: i) a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or its complement; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or its complement; a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or its complement; and / or iii) a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement;d) detecting the detectable label;

[0106] In some embodiments, the determining step, detecting step, or sequence analysis includes: a) amplifying at least a portion of a nucleic acid molecule encoding a PCSK9 polypeptide and / or an LDLR polypeptide, the portion to be amplified comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or its complement; or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or its complement; or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or its complement; and d) detecting the detectable label.

[0107] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a PCSK9 polypeptide, the amplified portion comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; and d) detecting the detectable label.

[0108] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a PCSK9 polypeptide, the amplified portion comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement; and d) detecting the detectable label.

[0109] In some embodiments, the nucleic acid molecule is mRNA and the determining step further comprises reverse transcribing the mRNA into cDNA prior to the amplifying step.

[0110] In some embodiments, the determining step, detecting step, or sequence analysis involves detecting nucleic acid molecules in a biological sample with a mutation-specific probe comprising a detectable label, the probe identifying: i) a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or its complement; an amplified nucleic acid molecule comprising: a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or its complement; ii) a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, or its complement; a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or its complement; or a uracil at a position corresponding to position 1 set forth in SEQ ID NO:19, or its complement. and / or iii) a thymine, or its complement, at a position corresponding to position 428 set forth in SEQ ID NO: 32; a thymine, or its complement, at a position corresponding to position 217 set forth in SEQ ID NO: 33; or a thymine, or its complement, at a position corresponding to position 137 set forth in SEQ ID NO: 34; and / or iv) a mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified cDNA molecule that comprises a uracil at a position corresponding to position 37 set forth in SEQ ID NO: 32, or its complement; a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement; and detecting the detectable label.

[0111] In some embodiments, the determining step, detecting step, or sequence analysis comprises contacting a genomic nucleic acid molecule in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified PCSK9 nucleic acid molecule and / or an LDLR nucleic acid molecule comprising a thymine, or its complement, at a position corresponding to position 427 set forth in SEQ ID NO:2, or a thymine, or its complement, at a position corresponding to position 2,269 set forth in SEQ ID NO:4, respectively; and detecting the detectable label.

[0112] In some embodiments, the determining step, detecting step, or sequence analysis comprises contacting mRNA molecules in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or its complement; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or its complement; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or its complement; and detecting the detectable label.

[0113] In some embodiments, the determining step, detecting step, or sequence analysis comprises contacting a cDNA molecule in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement; and detecting the detectable label.

[0114] In some embodiments, the determining step, detecting step, or sequence analysis comprises: contacting a genomic nucleic acid molecule in the biological sample with a first mutation-specific probe comprising a first detectable label, the first mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or its complement; and contacting a nucleic acid molecule in the biological sample with a second mutation-specific probe comprising a second detectable label, the second mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or its complement; and detecting the first detectable label and the second detectable label.

[0115] Mutation-specific polymerase chain reaction techniques can be used to detect mutations (such as SNPs in nucleic acid sequences). Mutation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0116] In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is present in a cell obtained from the subject.

[0117] In some embodiments, the assay comprises contacting the biological sample with a primer or probe (such as a mutation-specific primer or a mutation-specific probe) that specifically hybridizes under stringent conditions to the PCSK9 variant genomic sequence, variant mRNA sequence, or variant cDNA sequence, and / or the LDLR variant genomic sequence, and does not specifically hybridize to the corresponding PCSK9 reference sequence and / or LDLR reference sequence, and determining whether hybridization occurs.

[0118] In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay also comprises reverse transcribing mRNA into cDNA, such as by reverse transcriptase polymerase chain reaction (RT-PCR).

[0119] In some embodiments, the methods utilize probes and primers of sufficient nucleotide length to bind to target nucleotide sequences and specifically detect and / or identify polynucleotides comprising PCSK9 variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules, and / or LDLR variant genomic nucleic acid molecules. The operator can determine the hybridization or reaction conditions to achieve this result. The nucleotide length can be any length sufficient for use in the selected detection method, including any of the assays described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under high stringency hybridization conditions. The probes and primers can have complete nucleotide sequence identity to consecutive nucleotides within the target nucleotide sequence, although probes that differ from the target nucleotide sequence and retain the ability to specifically detect and / or identify the target nucleotide sequence can be designed by conventional methods. The probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity to the nucleotide sequence of the target nucleic acid molecule.

[0120] In some embodiments, to determine whether a PCSK9 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or its complement, and / or an LDLR nucleic acid molecule (genomic nucleic acid molecule), or its complement in a biological sample contains a nucleotide sequence comprising: i) a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2 or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4 (genomic nucleic acid molecule); ii) a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or a uracil at a position corresponding to position 137 set forth in SEQ ID NO:19 (mRNA molecule); or iii) a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, a thymine at a position corresponding to position 217 set forth in SEQ ID NO:33, or a thymine at a position corresponding to position 137 set forth in SEQ ID NO:34 (cDNA molecule), the biological sample is subjected to the following steps: i) a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2 or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4; ii) a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or a uracil at a position corresponding to position 137 set forth in SEQ ID NO:19 (mRNA molecule); or iii) a first primer derived from 5'-flanking sequence adjacent to i) a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2 or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4; ii) a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or a uracil at a position corresponding to position 137 set forth in SEQ ID NO:19; or iii) a first primer derived from 5'-flanking sequence adjacent to i) a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, a thymine at a position corresponding to position 217 set forth in SEQ ID NO:33, or a thymine at a position corresponding to position 137 set forth in SEQ ID NO:34; and i) a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2 or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4; ii) a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or a uracil at a position corresponding to position 137 set forth in SEQ ID NO:19; or iii) a second primer derived from 3'-flanking sequence adjacent to a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, a thymine at a position corresponding to position 217 set forth in SEQ ID NO:33, or a thymine at a position corresponding to position 137 set forth in SEQ ID NO:34;The amplification method can be performed using a primer pair comprising: i) a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4; ii) a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or a uracil at a position corresponding to position 137 set forth in SEQ ID NO:19; or iii) a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, a thymine at a position corresponding to position 217 set forth in SEQ ID NO:33, or a thymine at a position corresponding to position 137 set forth in SEQ ID NO:34. In some embodiments, the amplicon can range in length from the primer pair plus one nucleotide base pair to any length of an amplicon producible by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to approximately 20,000 nucleotide base pairs. Optionally, the primer pair comprises: i) a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4; ii) a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or a uracil at a position corresponding to position 137 set forth in SEQ ID NO:19; or iii) a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, a thymine at a position corresponding to position 217 set forth in SEQ ID NO:33, or a thymine at a position corresponding to position 137 set forth in SEQ ID NO:34; and i) a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4; ii) a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, or a uracil at a position corresponding to position 137 set forth in SEQ ID NO:19;or iii) flanked by a region comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides on each side of a position comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34;

[0121] Similar amplicons can be generated from mRNA and / or cDNA sequences. PCR primer pairs can be derived from known sequences, for example, by using computer programs designed for this purpose (such as the PCR Primer Analysis Tool in Vector NTI Version 10 (Informax Inc., Bethesda, Md.); PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer3 (Version 0.4.0.COPYRGT, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.)). Additionally, sequences can be visually scanned and primers manually identified using known guidelines.

[0122] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods include nucleic acid hybridization methods other than sequencing, including using labeled primers or probes on purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, the target nucleic acid molecule can be amplified prior to or simultaneously with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0123] In hybridization techniques, stringent conditions can be used to ensure that a probe or primer specifically hybridizes to its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably more highly than other non-target sequences (at least 2-fold above background, at least 3-fold, at least 4-fold, or more, including 10-fold above background). In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably more highly than other nucleotide sequences by at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably more highly than other nucleotide sequences by at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably more highly than other nucleotide sequences by at least 4-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than other nucleotide sequences than 10-fold above background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0124] Suitable stringency conditions that promote DNA hybridization (e.g., 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by a 2× SSC wash at 50° C.) are known or can be found in *Current Protocols in Molecular Biology*, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection are those in which the salt concentration is less than about 1.5 M NaCl at pH 7.0-8.3. + ions, typically about 0.01 to 1.0 M Na + The conditions for hybridization will be ionic concentrations (or other salts) and temperatures of at least about 30°C for short probes (e.g., 10-50 nucleotides) and at least about 60°C for longer probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents (e.g., formamide). Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash period will be at least long enough to reach equilibrium.

[0125] The present disclosure also provides a method for detecting the presence of predicted loss-of-function polypeptide of PCSK9, comprising: carrying out an assay on a biological sample obtained from a subject to determine whether the PCSK9 polypeptide in the subject contains one or more variations that cause the polypeptide to have loss-of-function (partial or complete) or predicted loss-of-function (partial or complete).The predicted loss-of-function polypeptide of PCSK9 can be any of the PCSK9 variant polypeptides described herein.In some embodiments, the method detects the presence of PCSK9Arg46Leu variant polypeptide.

[0126] In some embodiments, the method includes performing an assay on a sample obtained from the subject to determine whether a PCSK9 polypeptide in the sample comprises a leucine at a position corresponding to position 46 set forth in SEQ ID NO:42.

[0127] In some embodiments, the detecting step comprises sequencing at least a portion of the polypeptide that includes a position corresponding to position 46 set forth in SEQ ID NO:42 or SEQ ID NO:35.

[0128] In some embodiments, the detecting step comprises an immunoassay to detect the presence of a polypeptide comprising a position corresponding to position 46 set forth in SEQ ID NO:42 or SEQ ID NO:35.

[0129] In some embodiments, the subject has an increased risk of developing sepsis, SIRS, septic shock, and / or MODS when the subject does not have a predicted loss-of-function polypeptide of PCSK9, hi some embodiments, the subject has a decreased risk of developing sepsis, SIRS, septic shock, and / or MODS when the subject has a predicted loss-of-function polypeptide of PCSK9.

[0130] The present disclosure also provides isolated nucleic acid molecules that hybridize to PCSK9 variant genomic nucleic acid molecules and / or LDLR variant genomic nucleic acid molecules, PCSK9 variant mRNA molecules, and / or PCSK9 variant cDNA molecules (such as any of the genomic variant nucleic acid molecules, mRNA variant molecules, and cDNA variant molecules disclosed herein). In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of a PCSK9 nucleic acid molecule and / or LDLR nucleic acid molecule comprising: i) position 427 set forth in SEQ ID NO:2, or positions 2,269 set forth in SEQ ID NO:4; ii) position 428 set forth in SEQ ID NO:17, position 217 set forth in SEQ ID NO:18, or position 137 set forth in SEQ ID NO:19; or iii) position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34.

[0131] In some embodiments, such isolated nucleic acid molecules have a nucleotide sequence of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55 , at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 10 to 35, about 10 to 30, about 10 to 25, about 12 to 30, about 12 to 28, about 12 to 24, about 15 to 30, about 15 to 25, about 18 to 30, about 18 to 25, about 18 to 24, or about 18 to 22 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 18 to 30 nucleotides.In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

[0132] In some embodiments, such isolated nucleic acid molecules hybridize under stringent conditions to PCSK9 variant nucleic acid molecules (such as genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules) and / or LDLR variant nucleic acid molecules (such as genomic nucleic acid molecules). Such nucleic acid molecules can be used, for example, as probes, primers, mutation-specific probes, or mutation-specific primers as described or exemplified herein, including, but not limited to, primers, probes, antisense RNA, shRNA, and siRNA, each of which is described in more detail elsewhere herein and can be used in any of the methods described herein.

[0133] In some embodiments, the isolated nucleic acid molecule hybridizes to at least about 15 contiguous nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a PCSK9 variant genomic nucleic acid molecule and / or an LDLR variant genomic nucleic acid molecule, a PCSK9 variant mRNA molecule, and / or a PCSK9 variant cDNA molecule. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to 100 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to 35 nucleotides.

[0134] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, and the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence complementary to a portion of the nucleotide sequence encoding a PCSK9 polypeptide and / or an LDLR polypeptide, the portion comprising: i) a position corresponding to position 247 set forth in SEQ ID NO: 17, or its complement; or a position corresponding to position 2,269 set forth in SEQ ID NO: 4, or its complement; ii) a position corresponding to position 428 set forth in SEQ ID NO: 17, or its complement; a position corresponding to position 217 set forth in SEQ ID NO: 18, or its complement; or a position corresponding to position 137 set forth in SEQ ID NO: 19, or its complement; or iii) a position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement. In some embodiments, the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence comprising: i) positions corresponding to positions 426-428 set forth in SEQ ID NO:2, or their complements; ii) positions corresponding to positions 427-429 set forth in SEQ ID NO:17, or their complements; positions corresponding to positions 216-218 set forth in SEQ ID NO:18, or their complements; or positions corresponding to positions 136-138 set forth in SEQ ID NO:19, or their complements; and / or iii) positions corresponding to positions 427-429 set forth in SEQ ID NO:32, or their complements; positions corresponding to positions 216-218 set forth in SEQ ID NO:33, or their complements; or positions corresponding to positions 136-138 set forth in SEQ ID NO:34, or their complements.

[0135] In some embodiments, the mutation-specific probe and the mutation-specific primer comprise DNA. In some embodiments, the mutation-specific probe and the mutation-specific primer comprise RNA.

[0136] In some embodiments, the probes and primers described herein (including mutation-specific probes and mutation-specific primers) have nucleotide sequences that specifically hybridize to any of the nucleic acid molecules disclosed herein, or their complements. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0137] In some embodiments, primers (including mutation-specific primers) can be used in second-generation sequencing or high-throughput sequencing. In some instances, primers (including mutation-specific primers) can be modified. In particular, primers can contain various modifications used in different steps of, for example, massively parallel signature sequencing (MPSS), polony sequencing, and 454 pyrosequencing. Modified primers can be used in multiple steps of the process, including biotinylated primers in the cloning step and fluorescently labeled primers used in the bead loading and detection steps. Polony sequencing is generally performed using paired-end tag libraries, where each molecule of DNA template is approximately 135 bp in length. Biotinylated primers are used in the bead loading step and emulsion PCR. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. Adapters can contain 5'-biotin tags for immobilization of DNA libraries onto streptavidin-coated beads.

[0138] The probes and primers described herein can be used to detect nucleotide variations within any of the PCSK9 variant genomic nucleic acid molecules and / or LDLR variant genomic nucleic acid molecules, PCSK9 variant mRNA molecules, and / or PCSK9 variant cDNA molecules disclosed herein. The primers described herein can be used to amplify any of the PCSK9 variant genomic nucleic acid molecules and / or LDLR variant genomic nucleic acid molecules, PCSK9 variant mRNA molecules, or any of the PCSK9 variant cDNA molecules described herein, or any fragment thereof.

[0139] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3'-ends of the primers hybridizes to a guanine (rather than a thymine) at a position corresponding to position 427 set forth in SEQ ID NO: 1 in a specific PCSK9 genomic nucleic acid molecule, then the presence of an amplified fragment will indicate the presence of a PCSK9 reference genomic nucleic acid molecule. Conversely, if one of the 3'-ends of the primers hybridizes to a thymine (rather than a guanine) at a position corresponding to position 427 set forth in SEQ ID NO: 2 in a specific PCSK9 genomic nucleic acid molecule, then the presence of an amplified fragment will indicate the presence of a PCSK9 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 427 set forth in SEQ ID NO: 2 may be at the 3'-end of the primer. In addition, if one of the 3'-ends of the primers hybridizes to a guanine (rather than a uracil) at a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19 in a particular PCSK9 mRNA molecule, then the presence of an amplified fragment will indicate the presence of a PCSK9 reference mRNA molecule. Conversely, if one of the 3'-ends of the primers hybridizes to a uracil (rather than a guanine) at a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19 in a particular PCSK9 mRNA molecule, then the presence of an amplified fragment will indicate the presence of a PCSK9 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19 may be at the 3'-end of the primer.In addition, if one of the 3'-ends of the primers hybridizes to a guanine (rather than a thymine) at a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34 in a particular PCSK9 cDNA molecule, then the presence of an amplified fragment will indicate the presence of a PCSK9 reference cDNA molecule. Conversely, if one of the 3'-ends of the primers hybridizes to a thymine (rather than a guanine) at a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34 in a particular PCSK9 cDNA molecule, then the presence of an amplified fragment will indicate the presence of a PCSK9 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the thymine at the position corresponding to position 428 set forth in SEQ ID NO:32, the thymine at the position corresponding to position 217 set forth in SEQ ID NO:33, or the thymine at the position corresponding to position 137 set forth in SEQ ID NO:34 may be at the 3'-end of the primer.

[0140] "Specifically hybridize," in the context of this disclosure, means that a probe or primer (e.g., a mutation-specific probe or mutation-specific primer, etc.) does not hybridize to a nucleic acid sequence encoding a PCSK9 reference genomic nucleic acid molecule and / or an LDLR reference genomic nucleic acid molecule, a PCSK9 reference mRNA molecule, and / or a PCSK9 reference cDNA molecule.

[0141] In some embodiments, the probe (e.g., mutation-specific probe, etc.) comprises a label. In some embodiments, the label is a fluorescent label, a radioisotope label, or biotin.

[0142] The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are bound. A solid support is a solid-phase substrate or support to which a molecule (such as any of the probes disclosed herein) can be associated. A form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which multiple different probes are coupled in an array, grid, or other organized pattern. A form for a solid-state substrate is a microtiter dish (such as a standard 96-well type). In some embodiments, a multi-well glass slide, usually containing one array per well, can be used.

[0143] The present disclosure also provides a molecular complex comprising or consisting of any of the PCSK9 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or a complement thereof, and any of the mutation-specific primers or mutation-specific probes described herein. The present disclosure also provides a molecular complex comprising or consisting of any of the LDLR nucleic acid molecules (genomic nucleic acid molecules) described herein, or a complement thereof, and any of the mutation-specific primers or mutation-specific probes described herein. In some embodiments, the PCSK9 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule), or a complement thereof, and / or the LDLR nucleic acid molecule (genomic nucleic acid molecule), or a complement thereof, in the molecular complex is single-stranded. In some embodiments, the PCSK9 nucleic acid molecule and / or the LDLR nucleic acid molecule is any of the genomic nucleic acid molecules described herein. In some embodiments, the PCSK9 nucleic acid molecule is any of the mRNA molecules described herein. In some embodiments, the PCSK9 nucleic acid molecule is any of the cDNA molecules described herein. In some embodiments, the molecular complex comprises or consists of any of the PCSK9 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the mutation-specific primers described herein. In some embodiments, the molecular complex comprises or consists of any of the LDLR nucleic acid molecules (genomic nucleic acid molecules) described herein, or their complements, and any of the mutation-specific primers described herein. In some embodiments, the molecular complex comprises or consists of any of the PCSK9 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the mutation-specific probes described herein.In some embodiments, the molecular complex comprises or consists of any of the LDLR nucleic acid molecules (genomic nucleic acid molecules) described herein, or their complements, and any of the mutation-specific probes described herein.

[0144] In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe hybridized to a genomic nucleic acid molecule comprising a nucleotide sequence encoding a PCSK9 polypeptide, wherein the mutation-specific primer or the mutation-specific probe hybridizes to a thymine at a position corresponding to position 427 set forth in SEQ ID NO: 2, or its complement. In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe hybridized to a genomic nucleic acid molecule comprising a nucleotide sequence encoding an LDLR polypeptide, wherein the mutation-specific primer or the mutation-specific probe hybridizes to a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO: 4, or its complement.

[0145] In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe that hybridizes to the CTT codon at positions corresponding to positions 426-428 set forth in SEQ ID NO:2.

[0146] In some embodiments, the molecular complex comprises or consists of a genomic nucleic acid molecule comprising SEQ ID NO: 2. In some embodiments, the molecular complex comprises or consists of a genomic nucleic acid molecule comprising SEQ ID NO: 4.

[0147] In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe hybridized to a PCSK9 mRNA molecule comprising a nucleotide sequence encoding a PCSK9 polypeptide, wherein the mutation-specific primer or the mutation-specific probe hybridizes to a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or its complement; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or its complement; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or its complement.

[0148] In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe that hybridizes to a CUU codon at a position corresponding to positions 427-429 set forth in SEQ ID NO:17, positions 216-218 set forth in SEQ ID NO:18, or positions 136-138 set forth in SEQ ID NO:19.

[0149] In some embodiments, the molecular complex comprises or consists of a PCSK9 mRNA molecule comprising SEQ ID NO: 17. In some embodiments, the molecular complex comprises or consists of an mRNA molecule comprising SEQ ID NO: 18. In some embodiments, the molecular complex comprises or consists of an mRNA molecule comprising SEQ ID NO: 19.

[0150] In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe hybridized to a PCSK9 cDNA molecule comprising a nucleotide sequence encoding a PCSK9 polypeptide, wherein the mutation-specific primer or the mutation-specific probe hybridizes to a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement.

[0151] In some embodiments, the molecular complex comprises or consists of a mutation-specific primer or a mutation-specific probe that hybridizes to the CTT codon at a position corresponding to positions 427-429 set forth in SEQ ID NO:32, positions 216-218 set forth in SEQ ID NO:33, or positions 136-138 set forth in SEQ ID NO:34.

[0152] In some embodiments, the molecular complex comprises or consists of a PCSK9 cDNA molecule comprising SEQ ID NO: 32. In some embodiments, the molecular complex comprises or consists of a cDNA molecule comprising SEQ ID NO: 33. In some embodiments, the molecular complex comprises or consists of a DNA molecule comprising SEQ ID NO: 34.

[0153] In some embodiments, the molecular complex comprises a mutation-specific probe or a mutation-specific primer comprising a label. In some embodiments, the label is a fluorescent label, a radioisotope label, or biotin. In some embodiments, the molecular complex further comprises a non-human polymerase.

[0154] The nucleotide sequence of the PCSK9 reference genomic nucleic acid molecule is set forth in SEQ ID NO: 1. With reference to SEQ ID NO: 1, position 427 is a guanine.

[0155] A variant genomic nucleic acid molecule of PCSK9 exists in which the guanine at position 427 is replaced by a thymine. The nucleotide sequence of this PCSK9 variant genomic nucleic acid molecule is set forth in SEQ ID NO:2.

[0156] The nucleotide sequence of the LDLR reference genomic nucleic acid molecule is shown in SEQ ID NO: 3. With reference to SEQ ID NO: 3, position 2,269 is a guanine.

[0157] A variant genomic nucleic acid molecule of LDLR exists in which the guanine at position 2,269 is replaced by a thymine. The nucleotide sequence of this LDLR variant genomic nucleic acid molecule is set forth in SEQ ID NO:4.

[0158] The nucleotide sequence of the first PCSK9 reference mRNA molecule is set forth in SEQ ID NO: 5. With reference to SEQ ID NO: 5, position 428 is guanine. The nucleotide sequence of the second PCSK9 reference mRNA molecule is set forth in SEQ ID NO: 6. With reference to SEQ ID NO: 6, position 217 is guanine. The nucleotide sequence of the third PCSK9 reference mRNA molecule is set forth in SEQ ID NO: 7. With reference to SEQ ID NO: 7, position 137 is guanine.

[0159] A first PCSK9 variant mRNA molecule exists in which the guanine at position 428 (with reference to SEQ ID NO: 5) is replaced by uracil. The nucleotide sequence of this PCSK9 variant mRNA molecule is set forth in SEQ ID NO: 17. The first PCSK9 variant mRNA molecule comprises a CUU codon at positions 427-429 set forth in SEQ ID NO: 17.

[0160] A second PCSK9 variant mRNA molecule exists in which the guanine at position 217 (with reference to SEQ ID NO: 6) is replaced by uracil. The nucleotide sequence of this PCSK9 variant mRNA molecule is set forth in SEQ ID NO: 18. The second PCSK9 variant mRNA molecule comprises a CUU codon at positions 216-218 as set forth in SEQ ID NO: 18.

[0161] A third PCSK9 variant mRNA molecule exists in which the guanine at position 137 (with reference to SEQ ID NO: 7) is replaced by uracil. The nucleotide sequence of this PCSK9 variant mRNA molecule is set forth in SEQ ID NO: 19. The third PCSK9 variant mRNA molecule comprises a CUU codon at positions 136-138 as set forth in SEQ ID NO: 19.

[0162] The nucleotide sequence of the first PCSK9 reference cDNA molecule is set forth in SEQ ID NO:20. With reference to SEQ ID NO:20, position 428 is guanine. The nucleotide sequence of the second PCSK9 reference cDNA molecule is set forth in SEQ ID NO:21. With reference to SEQ ID NO:21, position 217 is guanine. The nucleotide sequence of the third PCSK9 reference cDNA molecule is set forth in SEQ ID NO:22. With reference to SEQ ID NO:22, position 137 is guanine.

[0163] A first PCSK9 variant cDNA molecule exists in which the guanine at position 428 (with reference to SEQ ID NO:20) is replaced by a thymine. The nucleotide sequence of this PCSK9 variant cDNA molecule is set forth in SEQ ID NO:32. The first PCSK9 variant cDNA molecule comprises a CTT codon at positions 427-429 as set forth in SEQ ID NO:32.

[0164] A second PCSK9 variant cDNA molecule exists in which the guanine at position 217 (with reference to SEQ ID NO:21) is replaced by a thymine. The nucleotide sequence of this PCSK9 variant cDNA molecule is set forth in SEQ ID NO:33. The second PCSK9 variant cDNA molecule comprises a CTT codon at positions 216-218 as set forth in SEQ ID NO:33.

[0165] A third PCSK9 variant cDNA molecule exists in which the guanine at position 137 (with reference to SEQ ID NO:22) is replaced by a thymine. The nucleotide sequence of this PCSK9 variant cDNA molecule is set forth in SEQ ID NO:34. The third PCSK9 variant cDNA molecule contains a CTT codon at positions 136-138 as set forth in SEQ ID NO:34.

[0166] Genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be from any organism. For example, genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be human or orthologs from other organisms (such as non-human mammals, rodents, mice, or rats). It is understood that gene sequences within a population can vary due to polymorphisms (such as single nucleotide polymorphisms). The examples provided herein are only illustrative sequences. Other sequences are also possible.

[0167] Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triple helix-forming molecules, and external guide sequences. Functional polynucleotides can act as effectors, inhibitors, regulators, and stimulators of the specific activity possessed by target molecules, or functional polynucleotides can possess de novo activity independent of other molecules.

[0168] The isolated nucleic acid molecules disclosed herein can comprise RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to a heterologous nucleic acid sequence (e.g., in a vector) or a heterologous label. For example, the isolated nucleic acid molecules disclosed herein can be within a vector comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence, or can be an exogenous donor sequence comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to a heterologous label. The label can be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, enzyme, or fluorophore quencher). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioisotope labels, dyes, stains, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme (in which case enzyme-dependent secondary signal generation occurs). The term "label" can also refer to a "tag" or hapten that can selectively bind to a conjugated molecule such that the conjugated molecule can subsequently be added with a substrate to generate a detectable signal. For example, biotin can be used as a tag to bind to the tag along with an avidin or streptavidin conjugate of horseradish peroxidase (HRP), and a colorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorogenic substrate can be used to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3xFLAG, 6xHis or polyhistidine, glutathione-S-transferase (GST), maltose-binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes, and their colorimetric, fluorogenic, and chemiluminescent substrates, as well as other labels.

[0169] The disclosed nucleic acid molecules can include, for example, nucleotides, or non-natural or modified nucleotides (such as nucleotide analogs or nucleotide substitutes). Such nucleotides include nucleotides containing modified bases, sugars, or phosphate groups, or nucleotides incorporating non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0170] The nucleic acid molecules disclosed herein may also contain one or more nucleotide analogs or substitutions. A nucleotide analog is a nucleotide containing a modification to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as different purine or pyrimidine bases (e.g., pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl, etc.). Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine, and 6-azothymine, These include, but are not limited to, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo (such as 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0171] Nucleotide analogs can also contain modifications to the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural and synthetic modifications of ribose and deoxyribose. Sugar modifications include the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl (where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1-10 Alkyl or C 2-10 Alkenyl, and C 2-10Exemplary 2' sugar modifications include, but are not limited to, -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON[(CH2) n Other modifications at the 2' position include, but are not limited to, C 1-10 Examples of suitable sugars include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in 2'-5'-linked oligonucleotides, and the 5' position of 5'-terminal nucleotides. Modified sugars can also include those containing modifications at the bridging ring oxygen (e.g., CH and S). Nucleotide sugar analogs can also have sugar mimetics (e.g., cyclobutyl moieties) in place of the pentofuranosyl sugar.

[0172] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. These phosphate linkages or modified phosphate linkages between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages can contain opposite polarities (such as 3'-5' to 5'-3' or 2'-5' to 5'-2'). Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0173] The present disclosure also provides vectors comprising any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vector can be a viral or non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or cosmid (e.g., a circular double-stranded DNA into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses (e.g., cauliflower mosaic virus and tobacco mosaic virus), yeast artificial chromosomes (YACs), Epstein-Barr (EBV)-derived episomes, and other expression vectors known in the art.

[0174] Desirable regulatory sequences for mammalian host cell expression can include, for example, viral elements that direct high-level polypeptide expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., the CMV promoter / enhancer), simian virus 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyoma-derived promoters and / or enhancers, as well as strong mammalian promoters (e.g., the native immunoglobulin promoter and actin promoter). Methods for expressing polypeptides in bacterial or fungal cells (e.g., yeast cells) are also well known. The promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally-regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter).

[0175] The percent identity (or percent complementarity) between specific stretches of nucleotide sequences within a nucleic acid molecule or amino acid sequences within a polypeptide can be determined using the BLAST program (Basic Local Alignment Search Tool) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Science, Inc.) with default settings using the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). Group, University Research Park, Madison Wis.) When percent sequence identity is referred to herein, a higher percentage of sequence identity is preferred over a lower one.

[0176] The present disclosure also provides compositions comprising any one or more of the isolated nucleic acid molecules, genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules disclosed herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cocrystals, and lipid microtubules. The carrier may comprise a buffered salt solution, PBS, HBSS, etc.

[0177] As used herein, the phrase "corresponding to," or grammatical variations thereof, when used in the context of numbering a particular nucleotide or sequence or position of a nucleotide, refers to the numbering of a defined reference sequence when a particular nucleotide or nucleotide sequence is compared to a reference sequence (e.g., SEQ ID NO:1 (genomic PCSK9), SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 (PCSK9 mRNA), or SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22 (PCSK9 cDNA), etc.). In other words, the number of a residue (e.g., a nucleotide or amino acid, etc.) or the position of a residue (e.g., a nucleotide or amino acid, etc.) in a particular polymer is specified with respect to the reference sequence, rather than by the actual number position of the residue within the particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence can be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps are present, the numbering of the residues in a particular nucleotide or nucleotide sequence is done with respect to the reference sequence to which it is aligned.

[0178] For example, a nucleic acid molecule comprising a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, means that when the nucleotide sequence of a PCSK9 genomic nucleic acid molecule is aligned to the sequence of SEQ ID NO:2, the PCSK9 sequence has a thymine residue at a position corresponding to position 427 of SEQ ID NO:2. The same applies to an mRNA molecule comprising a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, and a cDNA molecule comprising a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32. In other words, these terms refer to a nucleic acid molecule encoding a PCSK9 polypeptide, wherein the genomic nucleic acid molecule has a nucleotide sequence that includes a thymine residue homologous to the thymine residue at position 427 of SEQ ID NO:2 (or the mRNA molecule has a nucleotide sequence that includes a uracil residue homologous to the uracil residue at position 428 of SEQ ID NO:17, or the cDNA molecule has a nucleotide sequence that includes a thymine residue homologous to the thymine residue at position 428 of SEQ ID NO:32).

[0179] As described herein, the position within the PCSK9 genomic nucleic acid molecule and / or the LDLR genomic nucleic acid molecule corresponding to position 427 set forth in SEQ ID NO:2 or position 2,269 set forth in SEQ ID NO:4, respectively, can be identified, for example, by performing sequence alignment between the nucleotide sequence of a particular PCSK9 nucleic acid molecule and / or LDLR nucleic acid molecule and the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4, respectively. There are various computational algorithms that can be used to perform sequence alignment, for example, to identify the nucleotide position corresponding to position 427 in SEQ ID NO:2 or position 2,269 in SEQ ID NO:4. For example, sequence alignment can be performed by using the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res., 1997, 25, 3389-3402) or CLUSTALW software (Sievers and Higgins, Methods Mol. Biol., 2014, 1079, 105-116). However, sequences can also be aligned manually.

[0180] The amino acid sequence of the PCSK9 reference polypeptide is set forth in SEQ ID NO: 35. With reference to SEQ ID NO: 35, the PCSK9 reference polypeptide is 692 amino acids in length. With reference to SEQ ID NO: 35, position 46 is an arginine.

[0181] A PCSK9 variant polypeptide exists (Arg46Leu or R46L), the amino acid sequence of which is set forth in SEQ ID NO: 42. With reference to SEQ ID NO: 42, the PCSK9 variant polypeptide is also 692 amino acids in length. With reference to SEQ ID NO: 42, position 46 is a leucine.

[0182] The amino acid sequence of the first LDLR reference polypeptide is set forth in SEQ ID NO:36. With reference to SEQ ID NO:36, the LDLR reference polypeptide is 860 amino acids in length. The amino acid sequence of the second LDLR reference polypeptide is set forth in SEQ ID NO:37. With reference to SEQ ID NO:37, the LDLR reference polypeptide is 682 amino acids in length. The amino acid sequence of the third LDLR reference polypeptide is set forth in SEQ ID NO:38. With reference to SEQ ID NO:38, the LDLR reference polypeptide is 692 amino acids in length. The amino acid sequence of the fourth LDLR reference polypeptide is set forth in SEQ ID NO:39. With reference to SEQ ID NO:39, the LDLR reference polypeptide is 819 amino acids in length. The amino acid sequence of the fifth LDLR reference polypeptide is set forth in SEQ ID NO:40. With reference to SEQ ID NO:40, the LDLR reference polypeptide is 858 amino acids in length. The amino acid sequence of the sixth LDLR reference polypeptide is set forth in SEQ ID NO:41. With reference to SEQ ID NO:41, the LDLR reference polypeptide is 739 amino acids in length.

[0183] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids. The nucleotide sequences follow the standard convention of starting at the 5'-end of the sequence and proceeding forward to the 3'-end (i.e., left to right on each line). Only one strand of each nucleotide sequence is shown, but any reference to the presented strand is understood to encompass the complementary strand. The amino acid sequences follow the standard convention of starting at the amino-terminus of the sequence and proceeding forward to the carboxy-terminus (i.e., left to right on each line).

[0184] The present disclosure also provides a therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS for use in treating sepsis, SIRS, septic shock, and / or MODS in a subject (or for use in preparing a medicament for treating sepsis, SIRS and septic shock, and / or MODS), wherein the subject has any of the PCSK9 genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules described herein, and / or has any of the LDLR genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules described herein. The therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS may be any of the therapeutic agents for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS described herein.In some embodiments, the subject is diagnosed with i) a genomic nucleic acid molecule having a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and / or a genomic nucleic acid molecule having a nucleotide sequence encoding an LDLR9 polypeptide, wherein the nucleotide sequence comprises a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; ii) an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 428 set forth in SEQ ID NO:17, or a complement thereof; or an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence comprises a uracil at a position corresponding to position 217 set forth in SEQ ID NO:18, if or a complement thereof; or an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence includes a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; or iii) a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence includes a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or a complement thereof; a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence includes a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or a complement thereof; or a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, wherein the nucleotide sequence includes a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or a complement thereof.

[0185] The present disclosure also provides a PCSK9 inhibitor and / or an LDLR agonist for use in treating sepsis, systemic inflammatory response syndrome (SIRS), septic shock, and / or multiple organ dysfunction syndrome (MODS) in a subject. In some embodiments, the subject is administered a PCSK9 genomic nucleic acid molecule and / or an LDLR genomic nucleic acid molecule, PCSK9 In some embodiments, the subject is a mRNA molecule and / or an LDLR mRNA molecule, or a PCSK9 cDNA molecule and / or an LDLR cDNA molecule. In some embodiments, the subject is a PCSK9 gene encoding a genomic nucleic acid molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a thymine at position 427 set forth in SEQ ID NO:2; and / or a genomic nucleic acid molecule having a nucleotide sequence encoding a LDLR polypeptide, the nucleotide sequence comprising a thymine at position 2,269 set forth in SEQ ID NO:4; an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a uracil at position 428 set forth in SEQ ID NO:17, or a complement thereof; an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a uracil at position 217 set forth in SEQ ID NO:18, or a complement thereof; or a PCSK9 gene encoding a genomic nucleic acid molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a uracil at position 217 set forth in SEQ ID NO:19, or a complement thereof; The molecule is heterozygous for an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence including a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or its complement; or a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence including a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence including a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence including a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement.

[0186] In some embodiments, the PCSK9 inhibitor is an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to PCSK9 mRNA. In some embodiments, the PCSK9 inhibitor comprises a Cas protein and a gRNA that hybridizes to a gRNA recognition sequence within a PCSK9 genomic nucleic acid molecule. In some embodiments, the Cas protein is Cas9 or Cpf1. In some embodiments, the gRNA recognition sequence includes or is adjacent to position 427 set forth in SEQ ID NO:1. In some embodiments, the gRNA recognition sequence is located about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides from the position corresponding to position 427 set forth in SEQ ID NO:1. In some embodiments, the PAM sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence. In some embodiments, the gRNA comprises about 17 to about 23 nucleotides. In some embodiments, the gRNA recognition sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 43-68.

[0187] In some embodiments, the LDLR agonist is an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to an LDLR mRNA molecule. In some embodiments, the LDLR agonist comprises a Cas protein and a gRNA that hybridizes to a gRNA recognition sequence within an LDLR genomic nucleic acid molecule. In some embodiments, the Cas protein is Cas9 or Cpf1. In some embodiments, the gRNA recognition sequence includes or is adjacent to position 2,269 set forth in SEQ ID NO:3. In some embodiments, the gRNA recognition sequence is located about 1,000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides from the position corresponding to position 2,269 set forth in SEQ ID NO:3. In some embodiments, the PAM sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence. In some embodiments, the gRNA comprises about 17 to about 23 nucleotides. In some embodiments, the gRNA recognition sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 69-89.

[0188] All patent documents, websites, other publications, accession numbers, and the like, cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. If different versions of a sequence are associated with accession numbers at different times, the version associated with the accession number as of the effective filing date of this application is intended. Effective filing date means the earlier of the actual filing date or the filing date of a priority application, and the accession number, if applicable, is referenced. Similarly, if different versions of a publication, website, or the like are published at different times, the most recently published version as of the effective filing date of this application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure may be used in combination with any other feature, step, element, embodiment, or aspect unless specifically indicated otherwise. While the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0189] The following examples are provided to describe the embodiments in more detail. They are intended to illustrate, not limit, the claimed embodiments. The following examples provide one of ordinary skill in the art with a disclosure and description of how the compounds, compositions, articles, devices, and / or methods described herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in ° C. or is ambient temperature, and pressure is at or near atmospheric.

[0190] "Example" Example 1: Human genetics of PCSK9 and sepsis mortality To assess whether loss of function in PCSK9 and / or LDLR is associated with improved sepsis survival, we investigated the association between loss of function in PCSK9 and 28-day mortality due to severe sepsis (as defined by ICD10 codes) using specific genetic data from the GHS and UKB cohorts. Additionally, summary data were collected and a meta-analysis of 28-day mortality due to severe sepsis (clinically defined or defined by ICD10 codes for suspected / confirmed infection and acute organ failure) was performed across nine cohorts (totaling 3,228 fatal and 10,732 surviving severe sepsis cases). Cohorts with summary data included in the meta-analysis included GHS, Gen-Sep, Vanderbilt, SPH, VASST, UKB, NTNU, Estonia, and Copenhagen (see Figure 1).

[0191] The meta-analysis focused on PCSK9 p.Arg46Leu (rs11591147, AAF 0.012), a well-characterized missense variant that acts as a loss-of-function variant with a high effect on lowering LDL (in UKB: reference carriers: 138.23 mg / dL, heterozygote carriers: 126.8 mg / dL, homozygote carriers: 103.8 mg / dL (Cohen, N. Eng. J. Med., 2005); in UKB p-value = 4.9E-324) and a significant reduction in the odds of coronary artery disease (UKB: odds ratio = 0.79, p = 6.4E-14). Additionally, we also tested an LDLR intronic variant (rs6511720, AAF 0.12) with a significant effect on LDL-lowering (-5.9 mg / dL per allele (p = 3.9E-324)) and significantly reduced odds of CAD (odds ratio = 0.90, p = 6.2E-13) in the GHS. Meta-analysis results for the association between PCSK9 p.Arg46Leu and 28-day mortality due to severe sepsis showed an odds ratio = 0.83 (0.65, 1.06) and p = 0.13, consistent with the association of PCSK9 p.Arg46Leu with reduced odds of mortality due to severe sepsis.

[0192] Furthermore, the combined effect of PCSK9 and LDLR loss on 28-day sepsis mortality was analyzed. From this analysis, the combined effect of sepsis mortality for carriers of both PCSK9 p.Arg46Leu and LDLR rs6511720 was found to be odds ratio = 0.33 (0.08, 1.32) and p = 0.12 (see Figures 2 and 3), consistent with a reduced odds of death due to sepsis. Similar combined effects in repeated analyses were observed in Estonia and HUNT. In summary, an association between PCSK9 p.R46L and reduced mortality due to severe sepsis was observed.

[0193] Various modifications of the subject matter described, and those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Each reference cited in this application (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, GeneBank accession numbers, and the like) is incorporated herein by reference in its entirety and for all purposes.

Claims

1. 1. A method of treating a subject having sepsis, comprising administering a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor and / or a low-density lipoprotein receptor (LDLR) agonist to the subject in need thereof.

2. 10. The method of claim 1, wherein the sepsis is severe sepsis.

3. 1. A method of treating a subject having systemic inflammatory response syndrome (SIRS), comprising administering to the subject in need thereof a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor and / or a low-density lipoprotein receptor (LDLR) agonist.

4. A method of treating a subject having septic shock, comprising administering a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor and / or a low-density lipoprotein receptor (LDLR) agonist to the subject in need thereof.

5. 1. A method of treating a subject having multiple organ dysfunction syndrome (MODS), comprising administering a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor and / or a low-density lipoprotein receptor (LDLR) agonist to the subject in need thereof.

6. 6. The method of any one of claims 1 to 5, wherein the PCSK9 inhibitor comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes to PCSK9 mRNA.

7. The method of any one of claims 1 to 6, wherein the LDLR agonist comprises an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to LDLR mRNA.

8. The method of any one of claims 1 to 5, wherein the PCSK9 inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence in a PCSK9 genomic nucleic acid molecule.

9. The method of any one of claims 1 to 6, wherein the LDLR agonist comprises a Cas protein and a gRNA that hybridizes to a gRNA recognition sequence within an LDLR genomic nucleic acid molecule.

10. 10. The method of claim 8 or claim 9, wherein the Cas protein is Cas9 or Cpf1.

11. 11. The method of any one of claims 8 to 10, wherein the gRNA recognition sequence comprises or is adjacent to a position corresponding to position 427 set forth in SEQ ID NO:1 or position 2,269 set forth in SEQ ID NO:

3.

12. 12. The method of any one of claims 8 to 11, wherein the gRNA recognition sequence is located about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides from a position corresponding to position 427 set forth in SEQ ID NO:1 or position 2,269 set forth in SEQ ID NO:

3.

13. 13. The method of any one of claims 8 to 12, wherein a protospacer adjacent motif (PAM) sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence.

14. 14. The method of any one of claims 8 to 13, wherein the gRNA comprises from about 17 to about 23 nucleotides.

15. 15. The method of any one of claims 8 to 14, wherein the gRNA recognition sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 43-68 or any one of SEQ ID NOs: 69-89.

16. Detecting the presence or absence of a PCSK9 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of PCSK9 in a biological sample from the subject; and / or detecting the presence or absence of an LDLR variant nucleic acid molecule encoding a predicted loss-of-function LDLR polypeptide in a biological sample from said subject; The method of any one of claims 1 to 15, further comprising:

17. If the subject is a PCSK9 reference and an LDLR reference, then a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is also administered or administered continuously to the subject at a standard dosage, and the PCSK9 inhibitor and / or the LDLR agonist are administered to the subject; If the subject is an LDLR reference and heterozygous for a PCSK9 variant nucleic acid molecule, then the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is also administered or continuously administered to the subject in an amount equal to or less than a standard dosage, and the PCSK9 inhibitor and / or the LDLR agonist is administered to the subject; If the subject is a PCSK9 reference and heterozygous for an LDLR variant nucleic acid molecule, then the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is also administered or continuously administered to the subject in an amount equal to or less than a standard dosage, and the PCSK9 inhibitor and / or the LDLR agonist are administered to the subject; If the subject is an LDLR reference and homozygous for a PCSK9 variant nucleic acid molecule, then the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is also administered or continuously administered to the subject in an amount equal to or less than a standard dosage, and the LDLR agonist is administered to the subject; If the subject is a PCSK9 reference and homozygous for an LDLR variant nucleic acid molecule, then the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is also administered or continuously administered to the subject in an amount equal to or less than a standard dosage, and the PCSK9 inhibitor is administered to the subject; if the subject is heterozygous for both a PCSK9 variant nucleic acid molecule and an LDLR variant nucleic acid molecule, then the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is also administered or continuously administered to the subject in an amount equal to or less than a standard dosage, and the PCSK9 inhibitor and / or the LDLR agonist are administered to the subject; If the subject is homozygous for both a PCSK9 variant nucleic acid molecule and an LDLR variant nucleic acid molecule, the subject is also administered a therapeutic agent that treats or inhibits sepsis, SIRS, septic shock, and / or MODS at a dosage that is the same as or less than a standard dosage.

17. The method of claim 16.

18. 18. The method of claim 16 or 17, wherein the PCSK9 variant nucleic acid molecule is a nucleic acid molecule encoding an Arg46Leu polypeptide.

19. the PCSK9 variant nucleic acid molecule a genomic nucleic acid molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2; an mRNA molecule having a nucleotide sequence comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; or a cDNA molecule produced from an mRNA molecule, said cDNA molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; 19. The method of claim 18, wherein:

20. 19. The method of claim 18, wherein the LDLR variant nucleic acid molecule is a genomic nucleic acid molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:

4.

21. The method of any one of claims 16 to 20, wherein the detecting step is carried out in vitro.

22. the detecting step comprises: i) sequencing at least a portion of the nucleotide sequence of the PCSK9 genomic nucleic acid molecule in the biological sample, the sequenced portion comprising a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and ii) sequencing at least a portion of the nucleotide sequence of the LDLR genomic nucleic acid molecule in the biological sample, the sequenced portion comprising a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; If the sequenced portion of the PCSK9 genomic nucleic acid molecule in the biological sample contains a thymine at the position corresponding to position 427 set forth in SEQ ID NO:2, then the PCSK9 genomic nucleic acid molecule in the biological sample is a PCSK9 variant genomic nucleic acid molecule; If the sequenced portion of the LDLR genomic nucleic acid molecule in the biological sample contains a thymine at the position corresponding to position 2,269 set forth in SEQ ID NO:4, then the LDLR genomic nucleic acid molecule in the biological sample is an LDLR variant genomic nucleic acid molecule. The method according to any one of claims 16 to 21.

23. the detecting step comprises sequencing at least a portion of the nucleotide sequence of the PCSK9 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; If the sequenced portion of the PCSK9 mRNA molecule in the biological sample contains uracil at the position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19, then the PCSK9 mRNA molecule in the biological sample is a PCSK9 variant mRNA molecule; The method according to any one of claims 16 to 21.

24. the detecting step comprises sequencing at least a portion of the nucleotide sequence of the PCSK9 cDNA molecule produced from an mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 428 set forth in SEQ ID NO:32, or a complement thereof; a position corresponding to position 217 set forth in SEQ ID NO:33, or a complement thereof; or a position corresponding to position 137 set forth in SEQ ID NO:34, or a complement thereof; If the sequenced portion of the PCSK9 cDNA molecule produced from the mRNA in the biological sample contains a thymine at the position corresponding to position 428 set forth in SEQ ID NO: 32, position 217 set forth in SEQ ID NO: 33, or position 137 set forth in SEQ ID NO: 34, then the PCSK9 cDNA molecule produced from the mRNA molecule in the biological sample is a PCSK9 variant cDNA molecule; The method according to any one of claims 16 to 21.

25. the detecting step a) contacting the biological sample with i) a first primer that hybridizes to a portion of the nucleotide sequence of the PCSK9 genomic nucleic acid molecule adjacent to a position corresponding to position 427 set forth in SEQ ID NO:2, and ii) a second primer that hybridizes to a portion of the nucleotide sequence of the LDLR genomic nucleic acid molecule adjacent to position 2,269 set forth in SEQ ID NO:4; b) extending the first primer through the position of the nucleotide sequence of the PCSK9 genomic nucleic acid molecule corresponding to at least position 427 set forth in SEQ ID NO:2; and extending the second primer through the position of the nucleotide sequence of the LDLR genomic nucleic acid molecule corresponding to at least position 2,269 set forth in SEQ ID NO:4; c) determining whether the extension product of the first primer contains a thymine at the position corresponding to position 427 of SEQ ID NO:2; and determining whether the extension product of the second primer contains a thymine at the position corresponding to position 2,269 of SEQ ID NO:4; The method according to any one of claims 16 to 21, comprising:

26. the detecting step a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the PCSK9 mRNA molecule adjacent to a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; b) extending the primer through the position of the nucleotide sequence of the PCSK9 mRNA molecule corresponding to at least position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; c) determining whether the extension product of the primer comprises uracil at the position corresponding to position 428 set forth in SEQ ID NO: 17, uracil at the position corresponding to position 217 set forth in SEQ ID NO: 18, or uracil at the position corresponding to position 137 set forth in SEQ ID NO: 19; The method according to any one of claims 16 to 21, comprising:

27. the detecting step a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the PCSK9 cDNA molecule adjacent to a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; b) extending the primer through the position of the nucleotide sequence of the PCSK9 cDNA molecule corresponding to at least position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; c) determining whether the extension product of the primer contains a thymine at the position corresponding to position 428 set forth in SEQ ID NO:32, a thymine at the position corresponding to position 217 set forth in SEQ ID NO:33, or a thymine at the position corresponding to position 137 set forth in SEQ ID NO:34; The method according to any one of claims 16 to 21, comprising:

28. The method of any one of claims 22 to 27, wherein the detecting step comprises sequencing the entire nucleic acid molecule.

29. the detecting step a) amplifying at least a portion of the genomic nucleic acid molecule encoding the PCSK9 polypeptide, said portion comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and amplifying at least a portion of the genomic nucleic acid molecule encoding the LDLR polypeptide, said portion comprising a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising: i) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule containing a thymine, or a complement thereof, at the position corresponding to position 427 set forth in SEQ ID NO:2; and ii) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule containing a thymine, or a complement thereof, at the position corresponding to position 2,269 set forth in SEQ ID NO:4; d) detecting the detectable label; The method according to any one of claims 16 to 21, comprising:

30. the detecting step a) amplifying at least a portion of the mRNA molecule encoding the PCSK9 polypeptide, the portion comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule containing a uracil at the position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at the position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at the position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; d) detecting the detectable label; The method according to any one of claims 16 to 21, comprising:

31. the detecting step a) amplifying at least a portion of the cDNA molecule encoding the PCSK9 polypeptide, the portion comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, or a complement thereof; a thymine at a position corresponding to position 217 set forth in SEQ ID NO:33, or a complement thereof; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO:34, or a complement thereof; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule comprising a thymine at the position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a thymine at the position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a thymine at the position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement; d) detecting the detectable label; The method according to any one of claims 16 to 21, comprising:

32. 32. The method of claim 31 , wherein the nucleic acid molecules in the sample are mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step.

33. the detecting step contacting the genomic nucleic acid molecule in the biological sample with: i) a first mutation-specific probe comprising a first detectable label, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule that contains a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and ii) a second mutation-specific probe comprising a second detectable label, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule that contains a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; detecting the first detectable label and the second detectable label; The method according to any one of claims 16 to 21, comprising:

34. the detecting step contacting the mRNA molecule in the biological sample with a mutation-specific probe comprising a detectable label, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; detecting the detectable label; The method according to any one of claims 16 to 21, comprising:

35. the detecting step contacting the cDNA molecules produced from mRNA molecules in the biological sample with a mutation-specific probe comprising a detectable label, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or a complement thereof; a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or a complement thereof; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or a complement thereof; detecting the detectable label; The method according to any one of claims 16 to 21, comprising:

36. 1. A method of treating a subject with a therapeutic agent that treats or inhibits sepsis, SIRS, septic shock, and / or MODS, wherein the subject has sepsis, SIRS, septic shock, and / or MODS, comprising: determining whether the subject has i) a PCSK9 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of PCSL9; and / or ii) an LDLR variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of LDLR; obtaining or having obtained a biological sample from said subject; and performing or having performed a sequence analysis on the biological sample to determine whether the subject has a genotype that includes: i) the PCSK9 variant nucleic acid molecule; and / or ii) the LDLR variant nucleic acid molecule; determining the If the subject is a PCSK9 reference and an LDLR reference, then the therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS is also administered or administered continuously to the subject at a standard dosage, and a PCSK9 inhibitor and / or an LDLR agonist is administered to the subject; If the subject is an LDLR reference and heterozygous for the PCSK9 variant nucleic acid molecule, then administering or continuously administering to the subject the therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at an amount equal to or less than a standard dosage, and administering to the subject a PCSK9 inhibitor and / or an LDLR agonist; If the subject is a PCSK9 reference and heterozygous for the LDLR variant nucleic acid molecule, then administering or continuously administering to the subject the therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at an amount equal to or less than a standard dosage, and administering to the subject a PCSK9 inhibitor and / or an LDLR agonist; If the subject is an LDLR reference and homozygous for the PCSK9 variant nucleic acid molecule, then administering or continuously administering to the subject the therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at an amount equal to or less than a standard dosage, and administering to the subject an LDLR agonist; If the subject is a PCSK9 reference and homozygous for the LDLR variant nucleic acid molecule, then administering or continuously administering to the subject the therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at an amount equal to or less than a standard dosage, and administering to the subject a PCSK9 inhibitor; if the subject is heterozygous for both the PCSK9 variant nucleic acid molecule and the LDLR variant nucleic acid molecule, then administering or continuously administering to the subject the therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at an amount equal to or less than a standard dosage, and administering to the subject a PCSK9 inhibitor and / or an LDLR agonist; If the subject is homozygous for both the PCSK9 variant nucleic acid molecule and the LDLR variant nucleic acid molecule, then also administering to the subject the therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS in an amount equal to or less than a standard dosage; indicating that the presence of a genotype having both the PCSK9 variant nucleic acid molecule and the LDLR variant nucleic acid molecule indicates that the subject has a reduced risk of developing sepsis, SIRS, septic shock, and / or MODS; The method comprising:

37. 37. The method of claim 36, wherein the subject is a PCSK9 reference, a LDLR reference, and the subject is administered with a standard dosage or continuously administered with the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS, and is administered with the PCSK9 inhibitor and / or the LDLR agonist.

38. 37. The method of claim 36, wherein the subject is LDLR reference, and is heterozygous for the PCSK9 variant nucleic acid molecule, and the subject is administered or continuously administered the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock and / or MODS at the same amount as or less than the standard dosage, and is administered the PCSK9 inhibitor and / or the LDLR agonist.

39. 37. The method of claim 36, wherein the subject is a PCSK9 reference and is heterozygous for the LDLR variant nucleic acid molecule, and the subject is administered or continuously administered the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock and / or MODS at the same or less than the standard dosage, and is administered the PCSK9 inhibitor and / or the LDLR agonist.

40. 37. The method of claim 36, wherein the subject is LDLR reference and is homozygous for the PCSK9 variant nucleic acid molecule, and the subject is administered or continuously administered the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock and / or MODS at the same or less than the standard dosage, and is administered an LDLR agonist.

41. 37. The method of claim 36, wherein the subject is a PCSK9 reference and is homozygous for the LDLR variant nucleic acid molecule, and the subject is administered or continuously administered the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock and / or MODS at the same or less than the standard dosage, and is administered a PCSK9 inhibitor.

42. 37. The method of claim 36, wherein the subject is heterozygous for both the PCSK9 variant nucleic acid molecule and the LDLR variant nucleic acid molecule, and the subject is administered or continuously administered the therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at an amount equal to or less than a standard dosage, and is administered the PCSK9 inhibitor and / or the LDLR agonist.

43. The method of any one of claims 36 to 42, wherein the PCSK9 variant nucleic acid molecule is a nucleic acid molecule encoding the Arg46Leu polypeptide.

44. the PCSK9 variant nucleic acid molecule a genomic nucleic acid molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2; an mRNA molecule having a nucleotide sequence comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; or a cDNA molecule produced from an mRNA molecule, said cDNA molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; 44. The method of claim 43, wherein:

45. 45. The method of any one of claims 36 to 44, wherein the LDLR variant nucleic acid molecule is a genomic nucleic acid molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:

4.

46. The sequence analysis comprises: i) sequencing at least a portion of the nucleotide sequence of the PCSK9 genomic nucleic acid molecule in the biological sample, the sequenced portion comprising a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and ii) sequencing at least a portion of the nucleotide sequence of the LDLR genomic nucleic acid molecule in the biological sample, the sequenced portion comprising a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; If the sequenced portion of the PCSK9 genomic nucleic acid molecule in the biological sample contains a thymine at the position corresponding to position 427 set forth in SEQ ID NO:2, then the PCSK9 genomic nucleic acid molecule in the biological sample is a predicted loss-of-function variant genomic nucleic acid molecule of PCSK9; If the sequenced portion of the LDLR genomic nucleic acid molecule in the biological sample contains a thymine at the position corresponding to position 2,269 set forth in SEQ ID NO:4, then the LDLR genomic nucleic acid molecule in the biological sample is a predicted loss-of-function variant genomic nucleic acid molecule of LDLR.

46. ​​The method according to any one of claims 36 to 45.

47. The sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the PCSK9 mRNA molecule in the biological sample, the portion to be sequenced comprising a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; position 217 set forth in SEQ ID NO: 18, or a complement thereof; or position 137 set forth in SEQ ID NO: 19, or a complement thereof; If the sequenced portion of the PCSK9 mRNA molecule in the biological sample contains uracil at the position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19, then the PCSK9 mRNA molecule in the biological sample is a PCSK9 variant mRNA molecule; 46. ​​The method according to any one of claims 36 to 45.

48. The sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the PCSK9 cDNA molecule produced from the mRNA molecule in the biological sample, the portion comprising: a position corresponding to position 428 set forth in SEQ ID NO: 32, or a complement thereof; a position corresponding to position 217 set forth in SEQ ID NO: 33, or a complement thereof; or a position corresponding to position 137 set forth in SEQ ID NO: 34, or a complement thereof; If the sequenced portion of the PCSK9 cDNA molecule produced from the mRNA in the biological sample contains a thymine at the position corresponding to position 428 set forth in SEQ ID NO: 32, position 217 set forth in SEQ ID NO: 33, or position 137 set forth in SEQ ID NO: 34, then the PCSK9 cDNA molecule produced from the mRNA molecule in the biological sample is a PCSK9 variant cDNA molecule; 46. ​​The method according to any one of claims 36 to 45.

49. wherein the sequence analysis a) contacting the biological sample with i) a first primer that hybridizes to a portion of the nucleotide sequence of the PCSK9 genomic nucleic acid molecule adjacent to a position corresponding to position 427 set forth in SEQ ID NO:2, and ii) a second primer that hybridizes to a portion of the nucleotide sequence of the LDLR genomic nucleic acid molecule adjacent to position 2,269 set forth in SEQ ID NO:4; b) extending the first primer through the position of the nucleotide sequence of the PCSK9 genomic nucleic acid molecule corresponding to at least position 427 set forth in SEQ ID NO:2; and extending the second primer through the position of the nucleotide sequence of the LDLR genomic nucleic acid molecule corresponding to at least position 2,269 set forth in SEQ ID NO:4; c) determining whether the extension product of the first primer contains a thymine at the position corresponding to position 427 of SEQ ID NO:2; and determining whether the extension product of the second primer contains a thymine at the position corresponding to position 2,269 of SEQ ID NO:4; The method of any one of claims 36 to 45, comprising:

50. wherein the sequence analysis a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the PCSK9 mRNA molecule adjacent to a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; b) extending the primer through the position of the nucleotide sequence of the PCSK9 mRNA molecule corresponding to at least position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; c) determining whether the extension product of the primer contains uracil at the position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; and The method of any one of claims 36 to 45, comprising:

51. wherein the sequence analysis a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the PCSK9 cDNA molecule adjacent to a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; b) extending the primer through the position of the nucleotide sequence of the PCSK9 cDNA molecule corresponding to at least position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; c) determining whether the extension product of the primer contains a thymine at the position corresponding to position 428 set forth in SEQ ID NO: 32, position 217 set forth in SEQ ID NO: 33, or position 137 set forth in SEQ ID NO: 34; and The method of any one of claims 36 to 45, comprising:

52. 52. The method of any one of claims 46 to 51, wherein the sequence analysis comprises sequencing the entire nucleic acid molecule.

53. wherein the sequence analysis a) amplifying at least a portion of the nucleic acid molecule encoding the PCSK9 polypeptide, said portion comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and amplifying at least a portion of the nucleic acid molecule encoding the LDLR polypeptide, said portion comprising a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising: i) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule containing a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and ii) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule containing a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; d) detecting the detectable label; The method of any one of claims 36 to 45, comprising:

54. wherein the sequence analysis a) amplifying at least a portion of the mRNA molecule encoding the PCSK9 polypeptide, the portion comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule containing a uracil at the position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at the position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at the position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; d) detecting the detectable label; The method of any one of claims 36 to 45, comprising:

55. wherein the sequence analysis a) amplifying at least a portion of the cDNA molecule encoding the PCSK9 polypeptide, the portion comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, or a complement thereof; a thymine at a position corresponding to position 217 set forth in SEQ ID NO:33, or a complement thereof; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO:34, or a complement thereof; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule comprising a thymine at the position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a thymine at the position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a thymine at the position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement; d) detecting the detectable label; The method of any one of claims 36 to 45, comprising:

56. 56. The method of claim 55, wherein the nucleic acid molecules in the sample are mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step.

57. wherein the sequence analysis contacting the genomic nucleic acid molecule in the biological sample with ii) a first mutation-specific probe comprising a first detectable label, the first mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and ii) a second mutation-specific probe comprising a second detectable label, the second mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; detecting the first detectable label and the second detectable label; The method of any one of claims 36 to 45, comprising:

58. wherein the sequence analysis contacting the mRNA molecule in the biological sample with a mutation-specific probe comprising a detectable label, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; detecting the detectable label; The method of any one of claims 36 to 45, comprising:

59. wherein the sequence analysis contacting the cDNA molecules produced from mRNA molecules in the biological sample with a mutation-specific probe comprising a detectable label, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or a complement thereof; a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or a complement thereof; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or a complement thereof; detecting the detectable label; The method of any one of claims 36 to 45, comprising:

60. 46. ​​The method of any one of claims 36 to 45, wherein the nucleic acid molecule is present in a cell obtained from the subject.

61. The method of any one of claims 36 to 60, wherein the PCSK9 inhibitor comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes to PCSK9 mRNA, and the LDLR agonist comprises an antisense nucleic acid molecule, a siRNA, or a shRNA that hybridizes to LDLR mRNA.

62. The method according to any one of claims 36 to 60, wherein the PCSK9 inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence in a PCSK9 genomic nucleic acid molecule, and the LDLR agonist comprises a Cas protein and a gRNA that hybridizes to a gRNA recognition sequence in a LDLR genomic nucleic acid molecule.

63. 63. The method of claim 62, wherein the Cas protein is Cas9 or Cpf1.

64. The method of claim 62 or claim 63, wherein the PCSK9 gRNA recognition sequence comprises or is adjacent to the position corresponding to position 427 set forth in SEQ ID NO: 1, and the LDLR gRNA recognition sequence comprises or is adjacent to the position corresponding to position 2,269 set forth in SEQ ID NO:

3.

65. 64. The method of claim 62 or claim 63, wherein the PCSK9 gRNA recognition sequence is located at about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10 or about 5 nucleotides from the position corresponding to position 427 as set forth in SEQ ID NO:1, and the LDLR gRNA recognition sequence is located at about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10 or about 5 nucleotides from the position corresponding to position 2,269 as set forth in SEQ ID NO:

3.

66. 64. The method of Claim 62 or Claim 63, wherein a protospacer adjacent motif (PAM) sequence is about 2 to 6 nucleotides downstream of the gRNA recognition sequence.

67. 67. The method of any one of claims 62-66, wherein the gRNA comprises from about 17 to about 23 nucleotides.

68. The method of any one of claims 62 to 67, wherein the PCSK9 gRNA recognition sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 43 to 68.

69. The method of any one of claims 62 to 67, wherein the LDLR gRNA recognition sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 69 to 89.

70. 1. A method for identifying a subject at risk for developing sepsis, SIRS, septic shock, and / or MODS, comprising: determining or having determined the presence or absence of a PCSK9 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of PCSL9 and / or an LDLR variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of LDLR in a biological sample obtained from said subject; When the subject is a PCSK9 reference and an LDLR reference, the subject has an increased risk of developing sepsis, SIRS, septic shock, and / or MODS; when the subject is heterozygous or homozygous for an LDLR variant nucleic acid molecule and / or is heterozygous or homozygous for a PCSK9 variant nucleic acid molecule, the subject has a reduced risk of developing sepsis, SIRS, septic shock, and / or MODS; The method comprising:

71. 71. The method of claim 70, wherein the PCSK9 variant nucleic acid molecule is a nucleic acid molecule encoding the Arg46Leu polypeptide.

72. the PCSK9 variant nucleic acid molecule a genomic nucleic acid molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2; an mRNA molecule having a nucleotide sequence comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; or a cDNA molecule produced from an mRNA molecule, said cDNA molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; 71. The method of claim 70, wherein:

73. 72. The method of claim 71, wherein the LDLR variant nucleic acid molecule is a genomic nucleic acid molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:

4.

74. 74. The method of any one of claims 70 to 73, wherein the determining step is carried out in vitro.

75. the determining step comprises: i) sequencing at least a portion of the nucleotide sequence of the PCSK9 genomic nucleic acid molecule in the biological sample, the sequenced portion comprising a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and ii) sequencing at least a portion of the nucleotide sequence of the LDLR genomic nucleic acid molecule in the biological sample, the sequenced portion comprising a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; If the sequenced portion of the PCSK9 genomic nucleic acid molecule in the biological sample contains a thymine at the position corresponding to position 427 set forth in SEQ ID NO:2, then the PCSK9 genomic nucleic acid molecule in the biological sample is a PCSK9 variant genomic nucleic acid molecule; if the sequenced portion of the LDLR genomic nucleic acid molecule in the biological sample contains a thymine at the position corresponding to position 2,269 set forth in SEQ ID NO:4, then the LDLR genomic nucleic acid molecule in the biological sample is an LDLR variant genomic nucleic acid molecule; 75. The method of any one of claims 70 to 74.

76. The determining step comprises sequencing at least a portion of the nucleotide sequence of the PCSK9 mRNA molecule in the biological sample, the portion to be sequenced comprising a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; If the sequenced portion of the PCSK9 mRNA molecule in the biological sample contains uracil at the position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19, then the PCSK9 mRNA molecule in the biological sample is a PCSK9 variant mRNA molecule; 75. The method of any one of claims 70 to 74.

77. The determining step comprises sequencing at least a portion of the nucleotide sequence of the PCSK9 cDNA molecule produced from the mRNA molecule in the biological sample, the portion to be sequenced comprising a position corresponding to position 428 set forth in SEQ ID NO: 32, or a complement thereof; a position corresponding to position 217 set forth in SEQ ID NO: 33, or a complement thereof; or a position corresponding to position 137 set forth in SEQ ID NO: 34, or a complement thereof; If the sequenced portion of the PCSK9 cDNA molecule produced from the mRNA in the biological sample contains a thymine at the position corresponding to position 428 set forth in SEQ ID NO: 32, position 217 set forth in SEQ ID NO: 33, or position 137 set forth in SEQ ID NO: 34, then the PCSK9 cDNA molecule produced from the mRNA molecule in the biological sample is a PCSK9 variant cDNA molecule; 75. The method of any one of claims 70 to 74.

78. the determining step: a) contacting the biological sample with i) a first primer that hybridizes to a portion of the nucleotide sequence of the PCSK9 genomic nucleic acid molecule adjacent to a position corresponding to position 427 set forth in SEQ ID NO:2, and ii) a second primer that hybridizes to a portion of the nucleotide sequence of the LDLR genomic nucleic acid molecule adjacent to position 2,269 set forth in SEQ ID NO:4; b) extending the first primer through the position of the nucleotide sequence of the PCSK9 genomic nucleic acid molecule corresponding to at least position 427 set forth in SEQ ID NO:2; and extending the second primer through the position of the nucleotide sequence of the LDLR genomic nucleic acid molecule corresponding to at least position 2,269 set forth in SEQ ID NO:4; c) determining whether the extension product of the first primer contains a thymine at the position corresponding to position 427 of SEQ ID NO:2; and determining whether the extension product of the second primer contains a thymine at the position corresponding to position 2,269 of SEQ ID NO:4; 75. The method of any one of claims 70 to 74, comprising:

79. the determining step: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the PCSK9 mRNA molecule adjacent to a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; b) extending the primer through the position of the nucleotide sequence of the PCSK9 mRNA molecule corresponding to at least position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; c) determining whether the extension product of the primer contains uracil at the position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; and 75. The method of any one of claims 70 to 74, comprising:

80. the determining step: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the PCSK9 cDNA molecule adjacent to a position corresponding to position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; b) extending the primer through the position of the nucleotide sequence of the PCSK9 cDNA molecule corresponding to at least position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; c) determining whether the extension product of the primer contains a thymine at the position corresponding to position 428 set forth in SEQ ID NO: 32, position 217 set forth in SEQ ID NO: 33, or position 137 set forth in SEQ ID NO: 34; and 75. The method of any one of claims 70 to 74, comprising:

81. 81. The method of any one of claims 75 to 80, wherein the determining step comprises sequencing the entire nucleic acid molecule.

82. the determining step: a) amplifying at least a portion of the genomic nucleic acid molecule encoding the PCSK9 polypeptide, said portion comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and amplifying at least a portion of the genomic nucleic acid molecule encoding the LDLR polypeptide, said portion comprising a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising: i) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule containing a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and ii) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule containing a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; d) detecting the detectable label; 75. The method of any one of claims 70 to 74, comprising:

83. the determining step: a) amplifying at least a portion of the mRNA molecule encoding the PCSK9 polypeptide, the portion comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule containing a uracil at the position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at the position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at the position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; d) detecting the detectable label; 75. The method of any one of claims 70 to 74, comprising:

84. the determining step: a) amplifying at least a portion of the cDNA molecule encoding the PCSK9 polypeptide, the portion comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO:32, or a complement thereof; a thymine at a position corresponding to position 217 set forth in SEQ ID NO:33, or a complement thereof; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO:34, or a complement thereof; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule comprising a thymine at the position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a thymine at the position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a thymine at the position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement; d) detecting the detectable label; 75. The method of any one of claims 70 to 74, comprising:

85. 85. The method of Claim 84, wherein the nucleic acid molecules in the sample are mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step.

86. the detecting step contacting the genomic nucleic acid molecule in the biological sample with a first mutation-specific probe comprising a first detectable label, the first mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising a thymine, or a complement thereof, at a position corresponding to position 427 set forth in SEQ ID NO:2; and contacting the nucleic acid molecule in the biological sample with a second mutation-specific probe comprising a second detectable label, the second mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising a thymine, or a complement thereof, at a position corresponding to position 2,269 set forth in SEQ ID NO:4; detecting the first detectable label and the second detectable label; 75. The method of any one of claims 70 to 74, comprising:

87. the detecting step contacting the mRNA molecule in the biological sample with a mutation-specific probe comprising a detectable label, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; detecting the detectable label; 75. The method of any one of claims 70 to 74, comprising:

88. the detecting step contacting the cDNA molecules produced from mRNA molecules in the biological sample with a mutation-specific probe comprising a detectable label, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or a complement thereof; a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or a complement thereof; or a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or a complement thereof; detecting the detectable label; 75. The method of any one of claims 70 to 74, comprising:

89. If the subject is a PCSK9 reference and an LDLR reference, then administering or continuously administering to the subject a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at a standard dosage, and administering to the subject a PCSK9 inhibitor and / or an LDLR agonist; If the subject is an LDLR reference and heterozygous for the PCSK9 variant nucleic acid molecule, then administer or continuously administer to the subject a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at an amount equal to or less than a standard dosage, and administer to the subject a PCSK9 inhibitor and / or an LDLR agonist; If the subject is a PCSK9 reference and heterozygous for the LDLR variant nucleic acid molecule, then administer or continuously administer to the subject a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS in an amount equal to or less than a standard dosage, and administer to the subject a PCSK9 inhibitor and / or an LDLR agonist; If the subject is an LDLR reference and homozygous for the PCSK9 variant nucleic acid molecule, then administering or continuously administering to the subject a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS in an amount equal to or less than a standard dosage, and administering to the subject an LDLR agonist; If the subject is a PCSK9 reference and homozygous for the LDLR variant nucleic acid molecule, then administering or continuously administering to the subject a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS in an amount equal to or less than a standard dosage, and administering to the subject a PCSK9 inhibitor; If the subject is heterozygous for both the PCSK9 variant nucleic acid molecule and the LDLR variant nucleic acid molecule, then administering or continuously administering to the subject a therapeutic agent treating or inhibiting sepsis, SIRS, septic shock, and / or MODS in an amount equal to or less than a standard dosage, and administering to the subject a PCSK9 inhibitor and / or an LDLR agonist; If the subject is homozygous for both the PCSK9 variant nucleic acid molecule and the LDLR variant nucleic acid molecule, then administering a therapeutic agent for treating or inhibiting sepsis, SIRS, septic shock, and / or MODS at a dosage equal to or less than a standard dosage; 89. The method of any one of claims 70 to 88.

90. 1. A method for detecting a proprotein convertase subtilisin / kexin type 9 (PCSK9) variant nucleic acid molecule in a subject, comprising assaying a sample obtained from the subject to detect a nucleic acid molecule in the sample that: a genomic nucleic acid molecule comprising a nucleotide sequence comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; an mRNA molecule having a nucleotide sequence that includes uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; an mRNA molecule having a nucleotide sequence that includes uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or an mRNA molecule having a nucleotide sequence that includes uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; or a cDNA molecule produced from an mRNA molecule, said cDNA molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or a complement thereof; said cDNA molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or a complement thereof; or said cDNA molecule having a nucleotide sequence comprising a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or a complement thereof; determining whether

91. 1. A method for detecting a low-density lipoprotein receptor (LDLR) variant nucleic acid molecule in a subject, the method comprising assaying a sample obtained from the subject to determine whether a nucleic acid molecule in the sample is a genomic nucleic acid molecule comprising a nucleotide sequence comprising a thymine at position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof.

92. 92. The method of claim 90 or claim 91, wherein the method is an in vitro method.

93. 91. The method of claim 90, wherein the assay comprises sequencing at least a portion of the nucleic acid molecule, the sequenced portion comprising a thymine at the position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof.

94. 92. The method of claim 91, wherein the assay comprises sequencing at least a portion of the nucleic acid molecule, the portion to be sequenced comprising a thymine at the position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof.

95. 91. The method of claim 90, wherein the assay comprises sequencing at least a portion of the mRNA molecule, the portion comprising uracil, or a complement thereof, at the position corresponding to position 428 set forth in SEQ ID NO: 17; uracil, or a complement thereof, at the position corresponding to position 217 set forth in SEQ ID NO: 18; or uracil, or a complement thereof, at the position corresponding to position 137 set forth in SEQ ID NO:

19.

96. 92. The method of claim 91, wherein the assay comprises sequencing at least a portion of the cDNA molecule, the portion comprising a thymine at the position corresponding to position 428 set forth in SEQ ID NO: 32, or a complement thereof; a thymine at the position corresponding to position 217 set forth in SEQ ID NO: 33, or a complement thereof; or a thymine at the position corresponding to position 137 set forth in SEQ ID NO: 34, or a complement thereof.

97. wherein the assay a) contacting the sample with a primer that hybridizes to a portion of the nucleotide sequence of the PCSK9 genomic nucleic acid molecule adjacent to the position corresponding to position 427 set forth in SEQ ID NO:2; b) extending the primer through the position of the nucleotide sequence of the PSCK9 genomic nucleic acid molecule corresponding to at least position 427 set forth in SEQ ID NO:2; c) determining whether the extension product of the primer contains a thymine at the position corresponding to position 427 set forth in SEQ ID NO:2; 91. The method of claim 90, comprising:

98. wherein the assay a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the LDLR genomic nucleic acid molecule adjacent to the position corresponding to position 2,269 set forth in SEQ ID NO:4; b) extending the primer through the position of the nucleotide sequence of the LDLR genomic nucleic acid molecule corresponding to at least position 2,269 set forth in SEQ ID NO:4; c) determining whether the extension product of the primer contains a thymine at the position corresponding to position 2,269 set forth in SEQ ID NO:4; 92. The method of claim 91, comprising:

99. wherein the assay a) isolating the sample from the PCSK9 fragment adjacent to a position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; contacting the mRNA molecule with a primer that hybridizes to a portion of the nucleotide sequence of the mRNA molecule; b) extending the primer through the position of the nucleotide sequence of the PCSK9 mRNA molecule corresponding to at least position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; c) determining whether the extension product of the primer contains uracil at the position corresponding to position 428 set forth in SEQ ID NO: 17, position 217 set forth in SEQ ID NO: 18, or position 137 set forth in SEQ ID NO: 19; and 91. The method of claim 90, comprising:

100. wherein the assay a) isolating the sample from the PCSK9 fragment adjacent to a position corresponding to position 428 set forth in SEQ ID NO: 32, position 217 set forth in SEQ ID NO: 33, or position 137 set forth in SEQ ID NO: 34; contacting the cDNA molecule with a primer that hybridizes to a portion of said nucleotide sequence; b) extending the primer through the position of the nucleotide sequence of the PCSK9 cDNA molecule corresponding to at least position 428 set forth in SEQ ID NO:32, position 217 set forth in SEQ ID NO:33, or position 137 set forth in SEQ ID NO:34; c) determining whether the extension product of the primer contains a thymine at the position corresponding to position 428 set forth in SEQ ID NO: 32, position 217 set forth in SEQ ID NO: 33, or position 137 set forth in SEQ ID NO: 34; and 91. The method of claim 90, comprising:

101. 101. The method of any one of claims 93 to 100, wherein the assay comprises sequencing the entire nucleic acid molecule.

102. wherein the assay a) amplifying at least a portion of said genomic nucleic acid molecule encoding said PCSK polypeptide, said portion comprising a thymine at said position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule that contains a thymine at the position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; d) detecting the detectable label; 91. The method of claim 90, comprising:

103. wherein the assay a) amplifying at least a portion of the nucleic acid molecule encoding the LDLR polypeptide, said portion comprising a thymine at said position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule that contains a thymine at the position corresponding to position 2,269 set forth in SEQ ID NO:4, or its complement; d) detecting the detectable label; 92. The method of claim 91, comprising:

104. wherein the assay a) amplifying at least a portion of the mRNA molecule encoding the PCSK9 polypeptide, the portion comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule containing a uracil at the position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at the position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at the position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; d) detecting the detectable label; 91. The method of claim 90, comprising:

105. wherein the assay a) amplifying at least a portion of the cDNA molecule encoding the PCSK9 polypeptide, the portion comprising a thymine at the position corresponding to position 428 set forth in SEQ ID NO:32, or a complement thereof; a thymine at the position corresponding to position 217 set forth in SEQ ID NO:33, or a complement thereof; or a thymine at the position corresponding to position 137 set forth in SEQ ID NO:34, or a complement thereof; b) labeling the amplified nucleic acid molecules with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule comprising a thymine at the position corresponding to position 428 set forth in SEQ ID NO: 32, or its complement; a thymine at the position corresponding to position 217 set forth in SEQ ID NO: 33, or its complement; or a thymine at the position corresponding to position 137 set forth in SEQ ID NO: 34, or its complement; d) detecting the detectable label; 91. The method of claim 90, comprising:

106. 106. The method of claim 105, wherein the nucleic acid molecules in the sample are mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step.

107. wherein the assay contacting the genomic nucleic acid molecule in the biological sample with a mutation-specific probe comprising a detectable label, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule that contains a thymine at the position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; detecting the detectable label; 91. The method of claim 90, comprising:

108. wherein the assay contacting a genomic nucleic acid molecule in a biological sample with a mutation-specific probe comprising a detectable label, said mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to said nucleotide sequence of said amplified nucleic acid molecule comprising a thymine at said position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; detecting the detectable label; 92. The method of claim 91, comprising:

109. wherein the assay contacting the mRNA molecule with a mutation-specific probe comprising a detectable label, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of the amplified nucleic acid molecule comprising: a uracil at the position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a uracil at the position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a uracil at the position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; detecting the detectable label; 91. The method of claim 90, comprising:

110. wherein the assay contacting the cDNA molecules produced from mRNA molecules in the biological sample with a mutation-specific probe comprising a detectable label, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the amplified nucleic acid molecule comprising a thymine at the position corresponding to position 428 set forth in SEQ ID NO:32, or a complement thereof; a thymine at the position corresponding to position 217 set forth in SEQ ID NO:33, or a complement thereof; or a thymine at the position corresponding to position 137 set forth in SEQ ID NO:34, or a complement thereof; detecting the detectable label; 91. The method of claim 90, comprising:

111. 111. The method of any one of claims 90 to 110, wherein the nucleic acid molecule is present in a cell obtained from the subject.

112. A method for detecting the presence of a proprotein convertase subtilisin / kexin type 9 (PCSK9) Arg46Leu variant polypeptide, comprising performing an assay on a sample obtained from a subject to determine whether the PCSK9 protein in the sample contains a leucine at a position corresponding to position 46 set forth in SEQ ID NO:

42.

113. 113. The method of claim 112, wherein the assay comprises sequencing the polypeptide.

114. 113. The method of claim 112, wherein the assay is an immunoassay.

115. a genomic nucleic acid molecule having a nucleotide sequence encoding a proprotein convertase subtilisin / kexin type 9 (PCSK9) polypeptide, said nucleotide sequence comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2, or a complement thereof; and / or a genomic nucleic acid molecule having a nucleotide sequence encoding a low-density lipoprotein receptor (LDLR) polypeptide, said nucleotide sequence comprising a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4, or a complement thereof; an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, said nucleotide sequence comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, said nucleotide sequence comprising a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, said nucleotide sequence comprising a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; or a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or a complement thereof; a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or a complement thereof; or a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or a complement thereof; A therapeutic agent that treats or inhibits sepsis, systemic inflammatory response syndrome (SIRS), septic shock, and / or multiple organ dysfunction syndrome (MODS) for use in treating sepsis, SIRS, septic shock, and / or MODS in a subject having the disease.

116. a) a subject that is a reference for a PCSK9 genomic nucleic acid molecule and / or a LDLR genomic nucleic acid molecule, a PCSK9 mRNA molecule and / or a LDLR mRNA molecule, or a PCSK9 cDNA molecule and / or a LDLR cDNA molecule; or b) a subject who is heterozygous for i) a genomic nucleic acid molecule having a nucleotide sequence encoding a PCSK9 polypeptide, said nucleotide sequence comprising a thymine at a position corresponding to position 427 set forth in SEQ ID NO:2; and / or ii) a genomic nucleic acid molecule having a nucleotide sequence encoding an LDLR polypeptide, said nucleotide sequence comprising a thymine at a position corresponding to position 2,269 set forth in SEQ ID NO:4; a subject that is an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 428 set forth in SEQ ID NO: 17, or a complement thereof; a mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 217 set forth in SEQ ID NO: 18, or a complement thereof; or a subject that is an mRNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a uracil at a position corresponding to position 137 set forth in SEQ ID NO: 19, or a complement thereof; or a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a thymine at a position corresponding to position 428 set forth in SEQ ID NO: 32, or a complement thereof; a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a thymine at a position corresponding to position 217 set forth in SEQ ID NO: 33, or a complement thereof; or a cDNA molecule having a nucleotide sequence encoding a PCSK9 polypeptide, the nucleotide sequence comprising a thymine at a position corresponding to position 137 set forth in SEQ ID NO: 34, or a complement thereof.

1. A proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor and / or low-density lipoprotein receptor (LDLR) agonist for use in the treatment of sepsis, systemic inflammatory response syndrome (SIRS), septic shock, and / or multiple organ dysfunction syndrome (MODS) in

117. 117. The PCSK9 inhibitor of claim 116, which is an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes to PCSK9 mRNA.

118. 117. The PCSK9 inhibitor of claim 116, comprising a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within a PCSK9 genomic nucleic acid molecule.

119. 119. The PCSK9 inhibitor of claim 118, wherein the Cas protein is Cas9 or Cpf1.

120. The PCSK9 inhibitor of claim 118 or claim 119, wherein the gRNA recognition sequence comprises or is adjacent to position 427 set forth in SEQ ID NO:

1.

121. The PCSK9 inhibitor of claim 120 or claim 121, wherein said gRNA recognition sequence is located at about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10 or about 5 nucleotides from the position corresponding to position 427 of SEQ ID NO:

1.

122. 119. The LDLR agonist of claim 118, which is an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to an LDLR mRNA molecule.

123. The LDLR agonist of claim 116, comprising a Cas protein and a gRNA that hybridizes to a gRNA recognition sequence within an LDLR genomic nucleic acid molecule.

124. The LDLR agonist of claim 123, wherein the Cas protein is Cas9 or Cpf1.

125. The LDLR agonist of claim 123 or claim 124, wherein the gRNA recognition sequence includes or is adjacent to position 2,269 set forth in SEQ ID NO:

3.

126. The LDLR agonist of claim 123 or claim 124, wherein the gRNA recognition sequence is located at about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides from the position corresponding to position 2,269 of SEQ ID NO:

3.

127. 127. The PCSK9 inhibitor or LDLR agonist of any one of claims 118-126, wherein a protospacer adjacent motif (PAM) sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence.

128. 128. The PCSK9 inhibitor or LDLR agonist of any one of claims 118-127, wherein the gRNA comprises from about 17 to about 23 nucleotides.

129. The PCSK9 inhibitor of any one of claims 118 to 121, wherein the gRNA recognition sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 43 to 68.

130. The LDLR agonist according to any one of claims 122 to 126, wherein the gRNA recognition sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 69 to 89.

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