Blood biomarkers and diagnostic methods for small vessel diseases

Abstract

The present subject matter provides, inter alia compositions, systems, kits and methods for diagnosing and treating small vessel diseases (SDVDs).

What is claimed is: 
     
         1 . A method for diagnosing a small vessel disease (SVD) in a subject comprising
 (a) assaying the level of (i) Neurogenic Locus Notch Homolog Protein 3 (NOTCH3), collagen18α1/endostatin, High-Temperature Requirement A Serine Peptidase 1 (HTRA1), and/or insulin-like growth factor binding protein 1 (IGFBP-1), (ii) a protein-protein complex comprising NOTCH3 bound to collagen18α1/endostatin, HTRA1, IGFBP-1, and/or NOTCH3, in a test sample from said subject; and   (b) diagnosing the subject with the SVD if
 (i) the level of collagen18α1/endostatin, IGFBP-1, and/or HTRA1 is elevated in the test sample compared to a normal control, and/or 
 (ii) the level of collagen18α1/endostatin or NOTCH3 is reduced in the test sample compared to the normal control, and/or 
 (iii) the protein-protein complex comprising NOTCH3 bound to collagen18α1/endostatin, HTRA1, IGFBP-1, and/or NOTCH3 is detected in the test sample. 
   
     
     
         2 . The method of  claim 1 , wherein NOTCH3 is a NOTCH3 extracellular domain (N3ECD) protein comprising the amino acid sequence of SEQ ID NO: 5, and wherein assaying the level of NOTCH3 comprises contacting the N3ECD protein with an anti-N3ECD antibody. 
     
     
         3 . The method of  claim 1 , wherein (i) assaying the level of NOTCH3 comprises detecting the level of a NOTCH3 protein comprising the amino acid sequence of SEQ ID NO: 3 or 5; (ii) assaying the level of collagen18α1/endostatin comprises detecting the level of a collagen18α1/endostatin protein comprising the amino acid sequence of SEQ ID NO: 1; (iii) assaying the level of HTRA1 comprises detecting the level of a HTRA1 protein having the amino acid sequence of SEQ ID NO: 9; and/or (iv) assaying the level of IGFBP-1 comprises detecting the level of an IGFBP-1 protein comprising the amino acid sequence of SEQ ID NO: 7. 
     
     
         4 . The method of  claim 2 , wherein (i) assaying the level of collagen18α1/endostatin comprises contacting the collagen18α1/endostatin protein with an anti-collagen18α1/endostatin antibody; (ii) assaying the level of IGFBP-1 comprises contacting the IGFBP-1 protein with an anti-IGFBP-1 antibody; (iii) assaying the level of HTRA1 comprises contacting the HTRA1 protein with an anti-HTRA1 antibody; and/or (iv) assaying the level of NOTCH3 comprises contacting the NOTCH3 protein with an anti-NOTCH3 antibody. 
     
     
         5 . The method of  claim 1 , wherein (i) assaying the level of collagen18α1/endostatin comprises detecting the level of collagen18α1/endostatin mRNA; (ii) assaying the level of HTRA1 comprises detecting the level of HTRA1 mRNA; (iii) assaying the level of IGFBP-1 comprises detecting the level of IGFBP-1 mRNA; and/or (iv) assaying the level of NOTCH3 comprises detecting the level of NOTCH3 mRNA. 
     
     
         6 . The method of  claim 5 , wherein (i) detecting the level of collagen18α1/endostatin mRNA comprises contacting the collagen18α1/endostatin mRNA with a probe or a primer that is complementary to SEQ ID NO: 2; (ii) detecting the level of HTRA1 mRNA comprises contacting the HTRA1 mRNA with a probe or a primer that is complementary to SEQ ID NO: 10; (iii) detecting the level of IGFBP-1 mRNA comprises contacting the IGFBP-1 mRNA with a probe or a primer that is complementary to SEQ ID NO: 8; and/or (iv) detecting the level of NOTCH3 mRNA comprises contacting the NOTCH3 mRNA with a probe or a primer that is complementary to SEQ ID NO: 4. 
     
     
         7 . The method of  claim 1 , wherein said subject is diagnosed with the SVD if (i) the level of collagen18α1/endostatin, IGFBP-1, and/or HTRA1 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 5-50%, 50-75%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold higher in said test sample compared to a normal control; and/or (ii) the level of collagen18α1/endostatin or NOTCH3 reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, 5-50%, 50-75%, or 75-100% in said test sample compared to a normal control. 
     
     
         8 . A method for identifying whether a subject is at risk of developing a SVD comprising
 (a) assaying the level of (i) collagen18α1/endostatin, IGFBP-1, HTRA1, and/or NOTCH3, and/or (ii) a protein-protein complex comprising NOTCH3 bound to collagen18α1/endostatin, HTRA1, IGFBP-1, and/or NOTCH3 in a test sample from said subject; and   (b) identifying the subject as at risk of developing the SVD if
 (i) the level of collagen18α1/endostatin, IGFBP-1, and/or HTRA1 is elevated in the test sample compared to a normal control, and/or 
 (ii) the level of collagen18α1/endostatin or NOTCH3 is reduced in the test sample compared to the normal control, and/or 
 (iii) the protein-protein complex comprising NOTCH3 bound to collagen18α1/endostatin, HTRA1, IGFBP-1, and/or NOTCH3 is detected in the test sample. 
   
     
     
         9 . The method of  claim 8 , wherein said subject is identified as at risk of developing the SVD if (i) the level of collagen18 α1/endostatin, IGFBP-1, and/or HTRA1 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 5-50%, 50-75%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold higher in said test sample compared to a normal control; and/or (ii) the level of collagen18 α1/endostatin or NOTCH3 reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, 5-50%, 50-75%, or 75-100%, in said test sample compared to a normal control. 
     
     
         10 . The method of  claim 8 , further comprising directing the subject to obtain (i) additional screening or an additional diagnostic test for the SVD if the subject is identified as at risk of developing the SVD; or (ii) treatment to reduce, delay, or prevent the onset or progression of the SVD. 
     
     
         11 . A method for monitoring whether a SVD is progressing in a subject who has been diagnosed with the SVD, comprising periodically determining the level of collagen18 α1/endostatin, IGFBP-1, HTRA1, NOTCH3, and/or a protein-protein complex comprising NOTCH3 bound to collagen18 α1/endostatin, HTRA1, IGFBP-1, and/or NOTCH3 in said subject, and
 (1) identifying the SVD as worsening if (i) the level of collagen18 α1/endostatin, IGFBP-1, HTRA1, and/or the complex increases over time, and/or (ii) the level of collagen18 α1/endostatin or NOTCH3 decreases over time; 
 (2) identifying the SVD as improving if (i) the level of collagen18 α1/endostatin, IGFBP-1, HTRA1, and/or the complex decreases over time, and/or (ii) the level of collagen18 α1/endostatin or NOTCH3 increases over time; or 
 (3) identifying the SVD as neither worsening nor improving if the level of collagen18 α1/endostatin, IGFBP-1, HTRA1, NOTCH3, and/or the complex remains the same or about the same over time, 
 wherein determining the level of collagen18 α1/endostatin, IGFBP-1, HTRA1, and/or NOTCH3 comprises 
 assaying the level of collagen18 α1/endostatin, IGFBP-1, HTRA1, NOTCH3, and/or the complex in a test sample from the subject. 
 
     
     
         12 . The method of  claim 11 , wherein the level of collagen18 α1/endostatin, IGFBP-1, NOTCH3, HTRA1, and/or the complex is determined at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times and/or at least once every 1, 2, 3, or 4 weeks; at least once every 1, 2, 3, 4, 5, or 6 weeks; or at least once every 1, 2, 3, 4, or 5 years. 
     
     
         13 . A method for identifying a prognosis for a subject who has been diagnosed with a SVD comprising
 (a) assaying the level of collagen18α1/endostatin, IGFBP-1, HTRA1, NOTCH3, and/or a protein-protein complex comprising NOTCH3 bound to collagen18α1/endostatin, HTRA1, IGFBP-1, and/or NOTCH3 in a test sample from the subject; and   (b) comparing the level of collagen18α1/endostatin, IGFBP-1, HTRA1, NOTCH3, and/or the complex determined in (a) to a value in a database to identify the subject's risk of suffering from a symptom of SVD.   
     
     
         14 . The method of  claim 13 , wherein said database contains (i) collagen18 α1/endostatin, IGFBP-1, HTRA1, NOTCH3, and/or complex level values from subjects who have suffered from said symptom of SVD; or (ii) absolute or relative risk values calculated based on collagen18 α1/endostatin, IGFBP-1, HTRA1, NOTCH3, and/or complex level values from subjects who have suffered from said symptom of SVD. 
     
     
         15 . The method of  claim 14 , wherein said absolute or relative risk values comprise mean or median level values calculated using collagen18 α1/endostatin, IGFBP-1, HTRA1, NOTCH3, complex level values from subjects who have suffered from said symptom of SVD. 
     
     
         16 . A method of prophylaxis for a SVD, and administering to a subject a compound that is used to treat the SVD if the subject has been identified as at risk of suffering from SVD according to the method of  claim 8 , wherein said compound wherein said compound comprises a an anti-angiogenic compound, an anti-vascular endothelial growth factor (VEGF) compound, an anti-platelet compound, an antihypertensive compound, a platelet antiaggregant, a cholesterol-lowering compound, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, nimodipine, propentofylline, posatirelin, bevacizumab, ranibizumab, pegaptanib, aflibercept, nicardipine, verteporfin, anecortave acetate, triamcinolone acetonide, aspirin, dipyridamole, ticlopidine, a thrombolytic agent, an anticoagulant, or clopidogrel. 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 17 , wherein the SVD is cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and the compound is not a thrombolytic agent. 
     
     
         19 .- 20 . (canceled) 
     
     
         21 . The method of  claim 1 , wherein the SVD comprises Age-Related Macular Degeneration (AMD). 
     
     
         22 . The method of  claim 1 , wherein the level of collagen18α1/endostatin, IGFBP-1, HTRA1, and/or NOTCH3 is the level of collagen18α1/endostatin and HTRA1. 
     
     
         23 . The method of  claim 1 , which does not comprise assaying and/or detecting the HTRA1. 
     
     
         24 . The method of  claim 1 , wherein NOTCH3 is an extracellular portion of NOTCH3. 
     
     
         25 . The method of  claim 1 , wherein the extracellular portion of NOTCH3 is the NOTCH3 extracellular domain (N3ECD). 
     
     
         26 . The method of  claim 1 , wherein said test sample comprises a bodily fluid from said subject. 
     
     
         27 . The method of  claim 1 , wherein said bodily fluid comprises whole blood, a component of whole blood, plasma, serum, saliva, tears, vitreous, cerebrospinal fluid, sweat, cerebrospinal fluid, or urine. 
     
     
         28 . The method of  claim 1 , wherein said test sample is other than a tissue biopsy. 
     
     
         29 . The method of  claim 1 , wherein the subject does not contain a mutated HTRA1 gene. 
     
     
         30 . The method of  claim 1 , which does not comprise assaying and/or detecting the level of Fas ligand (FasL). 
     
     
         31 . The method of  claim 1 , wherein said SVD comprises cerebral SVD. 
     
     
         32 . The method of  claim 1 , wherein said SVD comprises a stroke, vascular cognitive impairment, dementia, nephropathy, retinopathy, or neuropathy. 
     
     
         33 . The method of  claim 1 , wherein said SVD comprises cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), age-related macular degeneration (AMD), CADASIL, NOTCH3 loss of function-associated SVD, nephropathy, microangiopathy, heart failure, Alagille syndrome, familial tetralogy of Fallot, patent ductus arteriosus, a cerebral cavernous malformation, pulmonary arterial hypertension, or diabetic retinopathy. 
     
     
         34 . The method of  claim 1 , wherein said SVD comprises AMD and said AMD is dry AMD or wet AMD. 
     
     
         35 . The method of  claim 1 , wherein said subject has diabetes. 
     
     
         36 . The method of  claim 1 , wherein assaying the level of collagen18α1/endostatin, IGFBP-1, HTRA1, and/or NOTCH3 comprises contacting said collagen18α1/endostatin, IGFBP-1, HTRA1, and/or NOTCH3 with an collagen18α1/endostatin-specific, IGFBP-1-specific HTRA1-specific and/or NOTCH3-specific binding agent. 
     
     
         37 . The method of  claim 36 , wherein said binding agent comprises an antibody or a fragment thereof, a polypeptide or a fragment thereof, or a nucleic acid. 
     
     
         38 . The method of  claim 37 , wherein said binding agent comprises an antibody or a fragment thereof. 
     
     
         39 . The method of  claim 38 , wherein said antibody comprises an anti-collagen18α1/endostatin antibody, an anti-IGFBP-1 antibody, an anti-HTRA1 antibody or an anti-NOTCH3 antibody. 
     
     
         40 . The method of  claim 38 , wherein said antibody or fragment thereof is attached to a solid support. 
     
     
         41 . The method of  claim 1 , wherein said assaying comprises an enzyme immunoassay (EIA). 
     
     
         42 . The method of  claim 1 , wherein said assaying comprises an enzyme-linked immunosorbent assay (ELISA), a Western blot, a mass spectrometry assay, a radioimmunoassay, or a fluoroimmunoassay. 
     
     
         43 . The method of  claim 1 , wherein said assaying comprises high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), protein immunoprecipitation, immunoelectrophoresis, or protein immunostaining. 
     
     
         44 . The method of  claim 37 , wherein said nucleic acid comprises a probe or a primer that is complementary to mRNA that encodes collagen18α1/endostatin, IGFBP-1, HTRA1, and/or NOTCH3. 
     
     
         45 . The method of  claim 1 , wherein said assaying comprises a polymerase chain reaction (PCR). 
     
     
         46 . The method of  claim 1 , wherein said assaying comprises quantitative reverse transcription PCR, microarray analysis, RNA sequencing, Northern analysis, a Nuclease Protection Assay, or in situ hybridization. 
     
     
         47 .- 67 . (canceled) 
     
     
         68 . The method according to  claim 1 , further comprising administering to a subject a compound that is used to treat the SVD, wherein said compound comprises a an anti-angiogenic compound, an anti-vascular endothelial growth factor (VEGF) compound, an anti-platelet compound, an antihypertensive compound, a platelet antiaggregant, a cholesterol-lowering compound, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, nimodipine, propentofylline, posatirelin, bevacizumab, ranibizumab, pegaptanib, aflibercept, nicardipine, verteporfin, anecortave acetate, triamcinolone acetonide, aspirin, dipyridamole, ticlopidine, a thrombolytic agent, an anticoagulant, or clopidogrel. 
     
     
         69 . (canceled)