Blood dna methylation biomarker diagnostic test for anxiety and depressive disorders

Abstract

A method for diagnosing or giving a prognosis for anxious temperament or trait-like anxiety in a human or non-human primate subject comprising the steps of (a) obtaining DNA from a blood or saliva sample from the subject and (b) quantifying methylation in a set of differentially methylated regions (DMRs) selected from SEQ ID NOs: 1-32, 34-44, 46-53, and 55-74, GCTCA, CGCACCG, AGAGGCAG, and AGCTCG or DMR-associated genes selected from DIP2C, GRB10, INPP5A, C17ORF97, PDXK, CACNA2D4, TRAPPC9, CRTC1, MEGF6, HIVEP3, OPCML, PITPNM2, ZFPM1, RAP1GAP2, NFATC1, RNF126, FSTL3, GNAS, SH3BP2, NEURL1B, MAD1L1, HSPA12B, IGF2, PEG10, PEG3, SLC16A3, SYTL1, and ZIM2, wherein a significant change methylation indicates the present of anxious temperament or trait-like anxiety, wherein the change is relative to DNA from a second human or non-human primate who does not have anxious temperament or trait-like anxiety. Also disclosed is a biomarker panel of DMR and DMR-associated genes for the diagnosis or prognosis of anxious temperament or trait-like anxiety.

1 - 26 . (canceled) 
     
     
         27 . A method of amplifying one or more differentially methylated region (DMR) associated genes comprising the steps of:
 (a) providing a reaction mixture comprising bisulfite modified target DNA from a subject and at least one pair of primers designed to amplify at least one DMR-associated gene selected from the group consisting of DIP2C, GRB10, INPP5A, GNAS, PDXK, TRAPPC9, C17ORF97, CACNA2D4, CRTC1, MEGF6, HIVEP3, OPCML, PITPNM2, ZFPM1, RAP1GAP2, NFATC1, RNF126, FSTL3, SH3BP2, NEURL1B, MAD1L1, HSPA12B, IGF2, PEG10, PEG3, SLC16A3, SYTL1, ZIM2, BRD3, DDX50, DUSP8, EHMT1, HCN2, IL17D, MICAL3, NACC2, PKD1, and VWA1, wherein the primer pair comprises a first and a second primer that are complementary to the DMR-associated gene;   (b) heating the reaction mixture to a first predetermined temperature for a first predetermined time;   (c) cooling the reaction mixture to a second predetermined temperature for a second predetermined time under conditions to allow the first and second primers to hybridize with their complementary sequences on the target DNA; and   (d) repeating steps (b) and (c) wherein an amplified target DNA sample is formed.   
     
     
         28 . The method of  claim 27 , wherein the reaction mixture additionally comprises a polymerase and a plurality of free nucleotides comprising adenine, thymine, cytosine, and guanine. 
     
     
         29 . The method of  claim 27 , wherein the reaction mixture additionally comprises a reaction buffer and MgCl 2 . 
     
     
         30 . The method of  claim 27 , wherein the primers are specific for a DMR selected from the group consisting of SEQ ID NOs:1-75. 
     
     
         31 . The method of  claim 27 , wherein at least one of the primers in the primer pair is biotinylated. 
     
     
         32 . The method of  claim 27 , wherein the target DNA is isolated from a blood sample or a saliva sample from the subject. 
     
     
         33 . The method of  claim 27 , wherein the subject is a human or non-human primate. 
     
     
         34 . The method of  claim 27 , wherein step (a) comprises providing at least two reaction mixtures, each of the at least two reaction mixtures comprising the bisulfite modified target DNA from the subject and at least one pair of primers designed to amplify at least one different DMR-associated gene. 
     
     
         35 . The method of  claim 27 , wherein step (a) comprises providing at least three reaction mixtures, each of the at least three reaction mixtures comprising the bisulfite modified target DNA from the subject and at least one pair of primers designed to amplify at least one different DMR-associated gene. 
     
     
         36 . The method of  claim 27 , wherein step (a) comprises providing at least four reaction mixtures, each of the at least four reaction mixtures comprising the bisulfite modified target DNA from the subject and at least one pair of primers designed to amplify at least one different DMR-associated gene. 
     
     
         37 . The method of  claim 27 , wherein step (a) comprises providing at least five reaction mixtures, each of the at least five reaction mixtures comprising the bisulfite modified target DNA from the subject and at least one pair of primers designed to amplify at least one different DMR-associated gene. 
     
     
         38 . The method of  claim 27 , wherein step (a) comprises providing at least 10 reaction mixtures, each of the at least 10 reaction mixtures comprising the bisulfite modified target DNA from the subject and at least one pair of primers designed to amplify at least one different DMR-associated gene. 
     
     
         39 . The method of  claim 27 , wherein step (a) comprises providing at least 15 reaction mixtures, each of the at least 15 reaction mixtures comprising the bisulfite modified target DNA from the subject and at least one pair of primers designed to amplify at least one different DMR-associated gene. 
     
     
         40 . The method of  claim 27 , wherein step (a) comprises providing at least 20 reaction mixtures, each of the at least 20 reaction mixtures comprising the bisulfite modified target DNA from the subject and at least one pair of primers designed to amplify at least one different DMR-associated gene. 
     
     
         41 . A method for estimating risk of anxious temperament in a subject comprising the steps of:
 (a) isolating and bisulfite modifying target DNA from a blood or saliva sample from the subject;   (b) quantifying methylation in at least one of DMR-associated genes DIP2C, GRB10, INPP5A, GNAS, PDXK, TRAPPC9, C17ORF97, CACNA2D4, CRTC1, MEGF6, HIVEP3, OPCML, PITPNM2, ZFPM1, RAP1GAP2, NFATC1, RNF126, FSTL3, SH3BP2, NEURL1B, MAD1L1, HSPA12B, IGF2, PEG10, PEG3, SLC16A3, SYTL1, ZIM2, BRD3, DDX50, DUSP8, EHMT1, HCN2, IL17D, MICAL3, NACC2, PKD1, and VWA1 in target DNA from the subject by contacting the bisulfite modified target DNA with a primer pair directed to at least one of DMR-associated genes under conditions suitable for amplification of the DMR-associated gene to obtain methylation quantification therein; and   (c) transforming the methylation quantification of the DMR-associated gene into an estimation of risk of anxious temperament, wherein a change in methylation of at least 10% compared to methylation in the same DMR-gene from a subject unaffected by anxious temperament indicates a risk of anxious temperament in the subject.   
     
     
         42 . A biomarker panel comprising probes specific to at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or at least 35 of DIP2C, GRB10, INPP5A, GNAS, PDXK, TRAPPC9, C17ORF97, CACNA2D4, CRTC1, MEGF6, HIVEP3, OPCML, PITPNM2, ZFPM1, RAP1GAP2, NFATC1, RNF126, FSTL3, SH3BP2, NEURL1B, MAD1L1, HSPA12B, IGF2, PEG10, PEG3, SLC16A3, SYTL1, ZIM2, BRD3, DDX50, DUSP8, EHMT1, HCN2, IL17D, MICAL3, NACC2, PKD1, and VWA1.