Methods for the diagnosis, risk assessment, and monitoring of autism spectrum disorders

Abstract

Methods for the diagnosis, risk assessment, and monitoring of Autism Spectrum Disorder (ASD) are disclosed. More specifically the present invention relates to the measurement of small molecules (metabolites) in human plasma that are found to have different abundances between persons with a clinical manifestation of ASD and subjects not expressing symptoms of ASD. Further, this invention relates to the monitoring of putative therapeutic strategies designed to ameliorate the biochemical abnormalities associated with ASD.

1 . A method for diagnosing a human subject's Autism Spectrum Disorder (ASD) health state or change in ASD health state or identifying a human subject's risk of ASD, the method comprising the steps of:
 a) analyzing at least one blood sample from said subject by high resolution mass spectrometry to obtain accurate mass intensity data for ionized metabolites;   b) comparing the accurate mass intensity data to corresponding data obtained from one or more than one reference blood sample to identify an increase or decrease in accurate mass intensity; and   c) using said increase or decrease in accurate mass intensity for diagnosing said subject's ASD health state or change in ASD health state or identifying the risk of ASD in said subject,   wherein the accurate mass intensity is measured, in Daltons, at one or more of the following hydrogen and electron adjusted accurate masses, or neutral accurate masses ±5 ppm,   wherein the hydrogen and electron adjusted accurate masses or neutral accurate masses at which intensity is increased in ASD are: 174.1408, 218.2034, 242.2033, 246.2345, 249.8832, 256.2189, 258.2346, 302.2219, 328.2402, 329.2436, 362.0842, 558.4652, 592.4705, 594.4849, 594.4858, 595.4887, 596.5017, 596.5018, 597.5053, 613.338, 622.4949, 724.5244, 728.5573, 747.5203, 749.5371, 750.5406, 766.4787, 766.5359, 775.5516, 776.5559, 777.5689, 779.4864, 791.5471, 792.494, 792.5522, 793.4944, 805.5608, 806.5089, 807.5133, 819.5794, 820.5267, 826.5555, 826.5563, 827.544, 828.5476, 834.5398, 837.5888, 841.5387, 851.5681, 851.5694, 852.5713, 852.5719, 856.6691, 858.6834, 858.6842, 859.6879, 876.7233, 879.5982, 879.5992, 904.7514, 905.7564, 950.7566, and 962.7618, and   wherein the hydrogen and electron adjusted accurate masses or neutral accurate masses at which intensity is decreased in ASD are: 878.7575, 550.4964, 551.4998, 893.7762, 906.7790, 865.7508, 753.5273, 894.7838, 549.4840, 866.7550, 548.4807, 760.5813, 604.5431, 946.8169, 892.7708, 605.5462, 860.7753, 863.7358, 920.8001, 759.5781, 919.7934, 562.4959, 782.5645, 576.5117, 876.7429, and 577.5154.   
     
     
         2 . The method of  claim 1 , wherein said method comprises monitoring an ASD therapy in the human subject, and wherein said increase or decrease in accurate mass intensity is used in step (c) to determine whether the therapy is improving the biochemical state of the subject. 
     
     
         3 . The method of  claim 2 , wherein the therapy is a carnitine therapy. 
     
     
         4 . The method of  claim 1 , wherein the accurate mass intensities are ionized metabolites. 
     
     
         5 . The method of  claim 1 , further comprising
 analyzing at least one blood sample from said patient by mass spectrometry to obtain accurate mass intensity data for one or more than one internal control metabolite; and   calculating a ratio for each of the accurate mass intensities obtained in step (a) to the accurate mass intensities obtained for the one or more than one internal control metabolite;   wherein the comparing step (b) comprises comparing each ratio to one or more corresponding ratios obtained for one or more than one reference blood sample.   
     
     
         6 . The method of  claim 5 , wherein the internal control metabolite is cholic acid. 
     
     
         7 . The method of  claim 1 , wherein the determination of the ASD health state or change in ASD health state comprises the determination of: the presence or absence of ASD, a biochemical ASD phenotype of the subject, an elevated risk of ASD, or a positive, negative, or nil effect of an ASD therapeutic strategy on the subject's underlying biochemical ASD phenotype. 
     
     
         8 . The method of  claim 7 , wherein the biochemical ASD phenotype is characterized as any one of:
 a) elevated levels of saturated or monounsaturated very long chain fatty acid (VLCFA)-containing ethanolamine phospholipids;   b) elevated levels of docosahexaenoic acid (22:6, DHA)-containing ethanolamine phospholipids;   c) elevated levels of polyunsaturated VLCFA-containing ethanolamine phospholipids;   d) decreased levels of 18:3, 20:3, 22:3, or 24:3-containing ethanolamine phospholipids; and   e) combinations thereof.   
     
     
         9 . The method of  claim 1 , wherein the blood sample is whole blood, plasma, serum, or a subfraction of whole blood. 
     
     
         10 . The method of  claim 1 , wherein the accurate mass intensity data is obtained using a mass spectrometer selected from the group consisting of: a Fourier transform ion cyclotron resonance, time of flight, orbitrap, quadrupole and triple quadrupole mass spectrometer. 
     
     
         11 . The method of  claim 1 , wherein a liquid/liquid extraction is performed on the blood samples whereby non-polar metabolites are dissolved in an organic solvent and polar metabolites are dissolved in an aqueous solvent. 
     
     
         12 . The method of  claim 11 , wherein the metabolites are ionized by positive or negative electrospray ionization, positive or negative atmospheric pressure chemical ionization, or a combination thereof. 
     
     
         13 . The method of  claim 1 , wherein the reference sample is taken from a non-ASD subject, one or more ASD subjects not on a therapeutic regimen, or from the human subject at a pre-therapy stage or at an earlier-therapy stage. 
     
     
         14 . The method of  claim 1 , wherein the intensity is measured at or ±1 ppm of the hydrogen and electron adjusted accurate mass, or neutral accurate mass. 
     
     
         15 . A method for diagnosing a human subject's Autism Spectrum Disorder (ASD) health state or change in ASD health state or identifying a human subject's risk of ASD, the method comprising the steps of:
 a) analyzing at least one blood sample from said subject using an analytic device or system comprising a mass spectrometer to obtain quantifying data for one or more than one metabolite marker;   b) comparing the quantifying data for said one or more than one metabolite marker to corresponding data obtained from one or more than one reference blood sample to identify an increase or decrease in the level of said one or more than one metabolite marker in said blood sample; and   c) using said increase or decrease in the level of said one or more than one metabolite marker to diagnose the human subject's ASD health state or change in ASD health state, or to identify the risk of ASD in said subject,   wherein the one or more than one metabolite marker comprises one or more than one molecule selected from the group consisting of ethanolamine phospholipids; docosahexaenoic acid (DHA)-containing phospholipids; DHA precursor-containing phospholipids; catabolic products of DHA beta-oxidation-containing phospholipids; polyunsaturated very long chain fatty acids (VLCFA)-containing phospholipids; and combinations thereof, and   wherein the human subject is diagnosed with ASD based on having: elevated levels of ethanolamine phospholipids containing saturated or monounsaturated VLCFA, docosahexaenoic acid (22:6, DHA), VLCFA DHA precursor (24:5, 24:6), catabolic products of DHA beta-oxidation (20:6), or polyunsaturated VLCFA; decreased levels of ethanolamine phospholipids containing 18:3, 20:3, 22:3, 24:3 fatty acids; or combinations thereof.   
     
     
         16 . The method of  claim 15 , wherein said method comprises monitoring an ASD therapy in the human subject, and wherein said increase or decrease in the level of said one or more than one metabolite marker is used in step (c) to determine whether the therapy is improving the biochemical state of the subject. 
     
     
         17 . The method of  claim 16 , wherein the therapy is a carnitine therapy. 
     
     
         18 . The method of  claim 15 , wherein the one or more than one metabolite marker is selected from the group consisting of: PtdEtn 16:0/18:0, PtdEtn 16:0/20:0, PtdEtn 16:0/22:0, PtdEtn 16:0/24:0, PtdEtn 16:0/26:0, PtdEtn 16:0/28:0, PtdEtn 16:0/30:0, PtdEtn 16:0/32:0, PtdEtn 16:0/34:0, PtdEtn 16:0/36:0, PtdEtn 16:0/38:0, PtdEtn 16:0/40:0, PtdEtn 18:0/18:0, PtdEtn 18:0/20:0, PtdEtn 18:0/22:0, PtdEtn 18:0/24:0, PtdEtn 18:0/26:0, PtdEtn 18:0/28:0, PtdEtn 18:0/30:0, PtdEtn 18:0/32:0, PtdEtn 18:0/34:0, PtdEtn 18:0/36:0, PtdEtn 18:0/38:0, PtdEtn 18:0/40:0, PtdEtn 16:0/18:1, PtdEtn 16:0/20:1, PtdEtn 16:0/22:1, PtdEtn 16:0/24:1, PtdEtn 16:0/26:1, PtdEtn 16:0/28:1, PtdEtn 16:0/30:1, PtdEtn 16:0/32:1, PtdEtn 16:0/34:1 PtdEtn 16:0/36:1, PtdEtn 16:0/38:1, PtdEtn 16:0/40:1, PtdEtn 18:0/18:1, PtdEtn 18:0/20:1, PtdEtn 18:0/22:1, PtdEtn 18:0/24:1, PtdEtn 18:0/26:1, PtdEtn 18:0/28:1, PtdEtn 18:0/30:1, PtdEtn 18:0/32:1, PtdEtn 18:0/34:1, PtdEtn 18:0/36:1, PtdEtn 18:0/38:1, PtdEtn 18:0/40:1, PtdEtn 16:0/18:2, PtdEtn 16:0/20:2, PtdEtn 16:0/22:2, PtdEtn 16:0/24:2, PtdEtn 16:0/26:2, PtdEtn 16:0/28:2, PtdEtn 16:0/30:2, PtdEtn 16:0/32:2, PtdEtn 16:0/34:2, PtdEtn 16:0/36:2, PtdEtn 16:0/38:2, PtdEtn 16:0/40:2, PtdEtn 18:0/18:2, PtdEtn 18:0/20:2, PtdEtn 18:0/22:2, PtdEtn 18:0/24:2, PtdEtn 18:0/26:2, PtdEtn 18:0/28:2, PtdEtn 18:0/30:2, PtdEtn 18:0/32:2, PtdEtn 18:0/34:2, PtdEtn 18:0/36:2, PtdEtn 18:0/38:2, PtdEtn 18:0/40:2, PtdEtn 16:0/18:3, PtdEtn 16:0/20:3, PtdEtn 16:0/22:3, PtdEtn 16:0/24:3, PtdEtn 16:0/26:3, PtdEtn 16:0/28:3, PtdEtn 16:0/30:3, PtdEtn 16:0/32:3, PtdEtn 16:0/34:3, PtdEtn 16:0/36:3, PtdEtn 16:0/38:3, PtdEtn 16:0/40:3, PtdEtn 18:0/18:3, PtdEtn 18:0/20:3, PtdEtn 18:0/22:3, PtdEtn 18:0/24:3, PtdEtn 18:0/26:3, PtdEtn 18:0/28:3, PtdEtn 18:0/30:3, PtdEtn 18:0/32:3, PtdEtn 18:0/34:3, PtdEtn 18:0/36:3, PtdEtn 18:0/38:3, PtdEtn 18:0/40:3, PtdEtn 16:0/20:4, PtdEtn 16:0/22:4, PtdEtn 16:0/24:4, PtdEtn 16:0/26:4, PtdEtn 16:0/28:4, PtdEtn 16:0/30:4, PtdEtn 16:0/32:4, PtdEtn 16:0/34:4, PtdEtn 16:0/36:4, PtdEtn 16:0/38:4, PtdEtn 16:0/40:4, PtdEtn 18:0/20:4, PtdEtn 18:0/22:4, PtdEtn 18:0/24:4, PtdEtn 18:0/26:4, PtdEtn 18:0/28:4, PtdEtn 18:0/30:4, PtdEtn 18:0/32:4, PtdEtn 18:0/34:4, PtdEtn 18:0/36:4, PtdEtn 18:0/38:4, PtdEtn 18:0/40:4, PtdEtn 16:0/20:5, PtdEtn 16:0/22:5, PtdEtn 16:0/24:5, PtdEtn 16:0/26:5, PtdEtn 16:0/28:5, PtdEtn 16:0/30:5, PtdEtn 16:0/32:5, PtdEtn 16:0/34:5, PtdEtn 16:0/36:5, PtdEtn 16:0/38:5, PtdEtn 16:0/40:5, PtdEtn 18:0/20:5, PtdEtn 18:0/22:5, PtdEtn 18:0/24:5, PtdEtn 18:0/26:5, PtdEtn 18:0/28:5, PtdEtn 18:0/30:5, PtdEtn 18:0/32:5, PtdEtn 18:0/34:5, PtdEtn 18:0/36:5, PtdEtn 18:0/38:5, PtdEtn 18:0/40:5, PtdEtn 16:0/20:6, PtdEtn 16:0/22:6, PtdEtn 16:0/24:6, PtdEtn 16:0/26:6, PtdEtn 16:0/28:6, PtdEtn 16:0/30:6, PtdEtn 16:0/32:6, PtdEtn 16:0/34:6, PtdEtn 16:0/36:6, PtdEtn 16:0/38:6, PtdEtn 16:0/40:6, PtdEtn 18:0/20:6, PtdEtn 18:0/22:6, PtdEtn 18:0/24:6, PtdEtn 18:0/26:6, PtdEtn 18:0/28:6, PtdEtn 18:0/30:6, PtdEtn 18:0/32:6, PtdEtn 18:0/34:6, PtdEtn 18:0/36:6, PtdEtn 18:0/38:6, PtdEtn 18:0/40:6, PlsEtn 16:0/18:1, PlsEtn 16:0/18:2, PlsEtn 16:0/18:3, PlsEtn 16:0/20:4, PlsEtn 16:0/22:6, PlsEtn 18:0/18:1, PlsEtn 18:0/18:2, PlsEtn 18:0/18:3, PlsEtn 18:0/20:4, PlsEtn 18:0/22:6, PlsEtn 18:1/18:1, PlsEtn 18:1/18:2, PlsEtn 18:1/18:3, PlsEtn 18:1/20:4, PlsEtn 18:1/22:6 and combinations thereof. 
     
     
         19 . The method of  claim 18 , wherein the human subject is diagnosed with ASD based on a statistically significant (p<0.05) increase or decrease in the level of said one or more than one metabolite marker relative to the corresponding data of the reference blood sample from a non-ASD subject. 
     
     
         20 . The method of  claim 15 , wherein the blood samples are analyzed by MS/MS transition. 
     
     
         21 . The method of  claim 20 , wherein the one or more than one metabolite marker is characterized by a MS/MS transition selected from the group consisting of: 718.5/255.2, 746.6/255.2, 774.6/255.2, 802.6/255.2, 830.7/255.2, 858.7/255.2, 886.7/255.2, 914.8/255.2, 942.8/255.2, 970.8/255.2, 998.9/255.2, 1026.9/255.2, 746.6/283.2, 774.6/283.2, 802.6/283.2, 830.7/283.2, 858.7/283.2, 886.7/283.2, 914.8/283.2, 942.8/283.2, 970.8/283.2, 998.9/283.2, 1026.9/283.2, 1054.9/283.2, 716.5/255.2, 744.6/255.2, 772.6/255.2, 800.6/255.2, 828.6/255.2, 856.7/255.2, 884.7/255.2, 912.7/255.2, 940.8//255.2, 968.8/255.2, 996.8/255.2, 1024.9/255.2, 744.6/283.2, 772.6/283.2, 800.6/283.2, 828.6/283.2, 856.7/283.2, 884.7/283.2, 912.7/283.2, 940.8/283.2, 968.8/283.2, 996.8/283.2, 1024.9/283.2, 1052.9/283.2, 714.5/255.2, 742.5/255.2, 770.6/255.2, 798.6/255.2, 826.6/255.2, 854.7/255.2, 882.7/255.2, 910.7/255.2, 938.8/255.2, 966.8/255.2, 994.8/255.2, 1022.9/255.2, 742.5/283.2, 770.6/283.2, 798.6/283.2, 826.6/283.2, 854.7/283.2, 882.7/283.2, 910.7/283.2, 938.8/283.2, 966.8/283.2, 994.8/283.2, 1022.9/283.2, 1050.9/283.2, 712.5/255.2, 740.5/255.2, 768.6/255.2, 796.6/255.2, 824.6/255.2, 852.6/255.2, 880.7/255.2, 908.7/255.2, 936.7/255.2, 964.8/255.2, 992.8/255.2, 1020.8/255.2, 740.5/283.2, 768.6/283.2, 796.6/283.2, 824.6/283.2, 852.6/283.2, 880.7/283.2, 908.7/283.2, 936.7/283.2, 964.8/283.2, 992.8/283.2, 1020.8/283.2, 1048.9/283.2, 738.5/255.2, 766.5/255.2, 794.6/255.2, 822.6/255.2, 850.6/255.2, 878.7/255.2, 906.7/255.2, 934.7/255.2, 962.8/255.2, 990.8/255.2, 1018.8/255.2, 766.5/283.2, 794.6/283.2, 822.6/283.2, 850.6/283.2, 878.7/283.2, 906.7/283.2, 934.7/283.2, 962.8/283.2, 990.8/283.2, 1018.8/283.2, 1046.9/283.2, 736.5/255.2, 764.5/255.2, 792.6/255.2, 820.6/255.2, 848.6/255.2, 876.6/255.2, 904.7/255.2, 932.7/255.2, 960.7/255.2, 988.8/255.2, 1016.8/255.2, 764.5/283.2, 792.6/283.2, 820.6/283.2, 848.6/283.2, 876.6/283.2, 904.7/283.2, 932.7/283.2, 960.7/283.2, 988.8/283.2, 1016.8/283.2, 1044.8/283.2, 734.5/255.2, 762.5/255.2, 790.5/255.2, 818.6/255.2, 846.6/255.2, 874.6/255.2, 902.7/255.2, 930.7/255.2, 958.7/255.2, 986.8/255.2, 1014.8/255.2, 762.5/283.2, 790.5/283.2, 818.6/283.2, 846.6/283.2, 874.6/283.2, 902.7/283.2, 930.7/283.2, 958.7/283.2, 986.8/283.2, 1014.8/283.2, 1042.8/283.2, 700.5/281.2, 698.5/279.2, 696.5/277.2, 722.5/303.2, 746.5/327.2, 728.5/281.2, 726.5/279.2, 724.5/277.2, 750.5/303.2, 774.5/327.2, 726.5/281.2, 724.5/279.2, 722.5/277.2, 748.5/303.2, 772.5/327.2, and combinations thereof. 
     
     
         22 . The method of  claim 21 , wherein the human subject is diagnosed with ASD based on a statistically significant (p<0.05) increase or decrease in the level of said one or more than one metabolite marker relative to the corresponding data of the reference blood sample from a non-ASD subject. 
     
     
         23 . The method of  claim 15 , wherein the analytic device or system further comprises a chromatography column, and the blood samples are analyzed by liquid chromatography (LC) and MS/MS transition. 
     
     
         24 . The method of  claim 15 , further comprising:
 analyzing at least one blood sample from said subject to obtain quantifying data for one or more than one internal control metabolite; and   obtaining a ratio for each of the levels of said one or more than one metabolite marker to the level obtained for the one or more than one internal control metabolite;   wherein the comparing step (b) comprises comparing each ratio to one or more corresponding ratios obtained for the one or more than one reference blood sample.   
     
     
         25 . The method of  claim 24 , wherein the internal control metabolite is cholic acid. 
     
     
         26 . The method of  claim 15 , wherein the human subject is diagnosed with ASD based on having either: elevated levels of ethanolamine phospholipids containing saturated or monounsaturated very long chain fatty acids (VLCFA); docosahexaenoic acid (22:6, DHA); VLCFA DHA precursor (24:5, 24:6); catabolic products of DHA beta-oxidation (20:6), polyunsaturated VLCFA; decreased levels of ethanolamine phospholipids containing 18:3, 20:3, 22:3, 24:3 fatty acids or combinations thereof. 
     
     
         27 . The method of  claim 15 , wherein the determination of the ASD health state or change in ASD health state comprises the determination of: the presence or absence of ASD, the biochemical ASD phenotype of the subject, an elevated risk of ASD, or a positive, negative, or nil effect of an ASD therapeutic strategy on the subject's underlying biochemical ASD phenotype. 
     
     
         28 . The method of  claim 27 , wherein ASD is detected by measuring any one of the following biochemical ASD phenotypes:
 a) elevated levels of saturated or monounsaturated very long chain fatty acid (VLCFA)-containing ethanolamine phospholipids;   b) elevated levels of docosahexaenoic acid (22:6, DHA)-containing ethanolamine phospholipids;   c) elevated levels of polyunsaturated VLCFA-containing ethanolamine phospholipids;   d) decreased levels of 18:3, 20:3, 22:3, or 24:3-containing ethanolamine phospholipids; and   e) combinations thereof.   
     
     
         29 . The method of  claim 15 , wherein the blood sample is whole blood, plasma, serum, or a subfraction of whole blood. 
     
     
         30 . The method of  claim 15 , wherein the mass spectrometer of the analytic device or system is selected from the group consisting of: a Fourier transform ion cyclotron resonance, time of flight, orbitrap, quadrupole and triple quadrupole mass spectrometer. 
     
     
         31 . The method of  claim 15 , wherein a liquid/liquid extraction is performed on the blood samples whereby non-polar metabolites are dissolved in an organic solvent and polar metabolites are dissolved in an aqueous solvent. 
     
     
         32 . The method of  claim 31 , wherein the analytic device or system analyzes the extracted samples by positive or negative electrospray ionization, positive or negative atmospheric pressure chemical ionization, or a combination thereof. 
     
     
         33 . The method of  claim 15 , wherein the reference blood sample is taken from a non-ASD subject, one or more ASD subjects not on a therapeutic regimen, or from the human subject at a pre-therapy stage or at an earlier-therapy stage. 
     
     
         34 . The method of  claim 15 , wherein the analytic device or system analyzes the level of said one or more than one metabolite marker in said sample using a colorimetric chemical assay, an antibody-based enzyme-linked immunosorbant assay (ELISA), a dipstick chemical assay, or mass spectrometry.