US 20180371542A1 (19 ) United States (12 ) Patent Application Publication (10 ) Pub. No. : US 2018 /0371542 A1 Anton (43 ) Pub . Date : Dec . 27 , 2018 (54 ) RECEPTOR BLOCKADE IN ( 52 ) U . S. CI. TREATING ALCOHOL USE DISORDERS CPC ...... C12Q 1/ 6876 ( 2013. 01 ) ; A61K 31/ 485 (71 ) Applicant: MUSC Foundation for Research ( 2013 .01 ) ; C12Q 2600 / 106 ( 2013 .01 ) ; C12Q Development, Charleston , SC ( US ) 2600 / 156 ( 2013 .01 ) ; A61P 25 / 32 (2018 .01 ) ( 72 ) Inventor: Raymond F . Anton , Charleston (US ) (57 ) ABSTRACT (73 ) Assignee : MUSC Foundation for Research Development, Charleston , SC (US ) Provided are methods for treating alcohol use disorders using antagonists . In some embodiments , the (21 ) Appl. No. : 16 /019 ,091 presently disclosed methods include assaying nucleic acid from a subject regarding the subject' s genotype with respect ( 22 ) Filed : Jun . 26 , 2018 to the COMT and OPRM1 genes and administering or not Related U .S . Application Data administering an opioid to the subject on (60 ) Provisional application No . 62 /525 , 123 , filed on Jun . the basis therefore . Also provided are methods for detecting 26 , 2017 . susceptibility to an opioid receptor antagonist therapy for disorders associated with opioid receptor activity and meth Publication Classification ods for identifying and treating human subjects having (51 ) Int. Ci. susceptibility to opioid receptor antagonist therapies for C12Q 1 /6876 ( 2006 .01 ) A61K 31/ 485 (2006 . 01) disorders associated with opioid receptor activity . A61P 25 /32 ( 2006 . 01 ) Specification includes a Sequence Listing . Patent Application Publication Dec. 27 , 2018 Sheet 1 of 7 US 2018 /0371542 A1

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OPIOID RECEPTOR BLOCKADE IN naltrexone ) that remove these inconsistencies and methods TREATING ALCOHOL USE DISORDERS of detecting susceptibility to opioid receptor antagonist treatment. CROSS -REFERENCE TO RELATED APPLICATION SUMMARY [ 0001] This application claims the benefit of U . S . Provi- [0006 ] This Summary lists several embodiments of the sional Patent Application Ser . No . 62 /525 , 123 , filed Jun . 26 , presently disclosed subject matter, and in many cases, lists 2017 , the disclosure of which is incorporated herein by variations and permutations of these embodiments . This reference in its entirety . Summary is merely exemplary of the numerous and varied embodiments . Mention of one or more representative fea GRANT STATEMENT tures of a given embodiment is likewise exemplary . Such an [0002 ] This invention was made with government support embodiment can typically exist with or without the feature under R01AA017633 and P50 AA010761, and ( s ) mentioned ; likewise , those features can be applied to K05AA017435 awarded by the National Institute on Alco other embodiments of the presently disclosed subjectmatter , hol Abuse and Alcoholism . The government has certain whether listed in this Summary or not. To avoid excessive rights in the invention . repetition , this Summary does not list or suggest all possible combinations of such features. TECHNICAL FIELD 10007 ] In some embodiments , the presently disclosed sub ject matter relates to methods and compositions related to 10003 ] The presently disclosed subject matter relates to the use of opioid receptor antagonists in the treatment of treating disorders associated with opioid receptor activity , psychiatric, mental, and /or neurological disorders . particularly to methods for treating disorders associated with [0008 ] In some embodiments , the presently disclosed opioid receptor activity including but not limited to alcohol methods relate to treating psychiatric , mental, and / or neu use disorders using opioid receptor antagonists , and also to rological disorders ( such as , for example , alcohol use dis methods for detecting susceptibility to opioid receptor order (AUD )) . In some embodiments , the presently dis antagonist therapy for disorders associated with opioid closed methods comprise assaying the nucleic acid from a receptor activity. subject for the genotype of the dopamine (DA ) -catabolizing enzyme catechol- O -methyltransferase (COMT ) and the BACKGROUND genotype of the opioid mu receptor gene (OPRM1 ) , wherein [0004 ] It is well documented that naltrexone is efficacious when one or two alleles encoding a methionine at amino acid in the treatment of alcohol dependence and has been residue 158 of COMT and two alleles encoding an aspara approved by the U . S . Food and Drug Administration for this gine at amino acid residue 40 of OPRM1 is detected or indication since 1994 . Nevertheless, the effect is moderate at wherein two alleles encoding valine at amino acid residue best , and it is recognized that not all individuals with an 158 of COMT and at least one allele encoding an aspartic alcohol use disorder (AUD ) respond to it. Genetic differ acid at amino acid residue 40 of OPRM1 is detected , an ences have been suggested as one factor thatmight influence opioid receptor antagonist is administered to the subject ; and both response to alcohol and the ability of naltrexone to wherein when one or two alleles encoding a methionine at modify this response . There have been a number of animal amino acid residue 158 of COMT and at least one allele and human clinical laboratory studies, suggesting that a encoding an aspartic acid at amino acid residue 40 of single nucleotide polymorphism ( SNP ) in the mu opioid OPRM1 is detected or wherein two alleles encoding valine gene (OPRM1 ) ( A118G ) leading to a missense asparagine to at amino acid residue 158 of COMT and two alleles encod aspartic acid amino acid substitution at position 40 ing an asparagine at amino acid residue 40 of OPRM1 is ( Asn40Asp ; rs1799971) can lead to differences in alcohol detected , an opioid receptor antagonist is not administered to effects and response to naltrexone. It has been shown that the subject. In some embodiments , the genotype of COMT some of this enhanced alcohol response and drinking behav is assayed prior to administering the opioid receptor antago ior in rodents engineered to have a homologous SNP is nist or wherein the genotype of COMT is assayed after because of increased dopamine release in the nucleus opioid receptor antagonist therapy has commenced , and accumbens, which in general is thought to be a signature of wherein when one or two alleles encoding a methionine at reinforcement and addiction . Of interest, naltrexone has amino acid residue 158 of COMT and at least one allele been shown in animals to both reduce nucleus accumbens encoding an aspartic acid at amino acid residue 40 of dopamine release and to reduce drinking in rodent and OPRM1 is detected or wherein two alleles encoding valine nonhuman primate drinking models. Also of interest, a at amino acid residue 158 of COMT and two alleles encod homologous SNP to that found in humans in the mu opioid ing an asparagine at amino acid residue 40 of OPRM1 is receptor gene occurring naturally in non - human primates detected , the opioid receptor antagonist therapy is discon also appears to confer both sensitivity to alcohol response tinued . In some embodiments , the opioid receptor antagonist and response to naltrexone in the reduction of alcohol effects is a naltrexone . In some embodiments , the genotype of the and consumption . This finding parallels data in human COMT and OPRM1 polymorphisms are detected by a clinical laboratory studies and clinical trials where naltrex nucleic acid amplification process followed by sequencing one appears to exert a stronger effect on those individuals or gel electrophoresis or direct sequencing . with the Asp40 OPRM1 (A118G ) SNP. However, this find [ 0009 ] In some embodiments, the presently disclosed ing is not universal as several reports suggest that it may not methods further comprise assaying the nucleic acid from a be as salient. subject for the genotype of the variable number tandem [ 0005 ] What is needed are new methods of treating AUD repeats ( VNTR ) polymorphism in the dopamine transporter with opioid receptor antagonists (such as, for example gene DAT1/ SLC6A3 , wherein when one or two alleles for 9 US 2018 /0371542 A1 Dec. 27 , 2018

tandem repeats and an asparagine at amino acid residue 40 encoding a valine at an amino acid position corresponding to of OPRM1 is detected or wherein two alleles for 10 tandem amino acid residue 158 of the COMT gene product of SEQ repeats and an aspartic acid at amino acid residue 40 of ID NO : 8 or two alleles encoding a dopamine transporter OPRM1 is detected , an opioid receptor antagonist is admin DAT1/ SLC6A3 10 -repeat variable number tandem repeat istered to the subject ; and wherein when one or two alleles (VNTR ) . In some embodiments , the disorder associated with for 9 tandem repeats and an aspartic acid at amino acid opioid receptor activity is an alcohol use disorder (AUD ) . In residue 40 of OPRM1 is detected or wherein two alleles for some embodiments , at least one of the one or more geno 10 tandem repeats and an asparagine at amino acid residue typing assays is performed prior to administering the opioid 40 of OPRM1 is detected , an opioid receptor antagonist is receptor antagonist therapy to the subject. In some embodi not administered to the subject . ments , at least one of the one or more genotyping assays are [0010 ] In some embodiments , the presently disclosed sub performed after administering the opioid receptor antagonist jectmatter also provides methods for detecting susceptibility therapy to the subject , and further wherein the opioid to an opioid receptor antagonist therapy for alcohol use receptor antagonist therapy is discontinued if the subject has disorder. In some embodiments , the presently disclosed at least one allele encoding an aspartic acid at an amino acid methods comprise obtaining a tissue sample from a subject, position corresponding to amino acid residue 40 of the assaying nucleic acid from the tissue sample from the OPRM1 gene product of SEQ ID NO : 12 and at least one subject for the genotype encoding the amino acid substitu allele encoding a methionine at an amino acid position tion at residue 158 of the DA - catabolizing enzyme catechol corresponding to amino acid residue 158 of the COMT gene O -methyltransferase (COMT ) and the genotype encoding an product of SEQ ID NO : 8 or at least one allele for a amino acid substitution at residue 40 of the opioid mu DAT1 /SLC6A3 9- repeat variable number tandem repeat receptor gene (OPRM1 ) , wherein when one or two alleles (VNTR ) ; or the subject is homozygous for an asparagine at encode for a methionine at amino acid residue 158 of COMT an amino acid position corresponding to amino acid residue and a genome that encodes for an asparagine at amino acid 40 the of OPRM1 gene product of SEQ ID NO : 12 and is residue 40 of OPRM1 or wherein two alleles encoding homozygous for a valine at an amino acid position corre valine at amino acid residue 158 of COMT and the genome sponding to amino acid residue 158 of the COMT gene encodes for an aspartic acid at amino acid residue 40 of product of SEQ ID NO : 8 or is homozygous for a DAT1 / OPRM1 indicates that the subject is susceptible to dopamine SLC6A3 10 - repeat variable number tandem repeat ( VNTR ) . modulator therapy . In some embodiments , the opioid receptor antagonist [0011 ] In some embodiments of the presently disclosed therapy comprises administering an effective amount of methods , the opioid receptor antagonist is a naltrexone. naltrexone to the subject. In some embodiments , at least one [0012 ] In some embodiments of the presently disclosed of the one or more genotyping assays comprises a nucleic methods, the genotype of the OPRM1 and COMT polymor acid amplification process followed by sequencing or gel phisms are detected by a nucleic acid amplification process electrophoresis of an amplification product produced followed by sequencing or gel electrophoresis or direct thereby . sequencing. [0015 ] Thus, it is an object of the presently disclosed [0013 ] In some embodiments , the presently disclosed subject matter to provide methods for treating psychiatric , methods further comprise assaying the nucleic acid from a mental, and /or neurological disorders associated with opioid subject for the genotype of the variable number tandem receptor activity ( such as, for example , alcohol use disorder repeats (VNTR ) polymorphism in the dopamine transporter ( AUD ) ) using opioid receptor antagonists . gene DAT1 / SLC6A3 . [0016 ] An object of the presently disclosed subject matter [0014 ] The presently disclosed subjectmatter also provide having been stated hereinabove , and which is achieved in in some embodiments methods for treating subjects with whole or in part by the compositions and methods disclosed disorders associated with opioid receptor activity . In some herein , other objects will become evident as the description embodiments , the methods comprising performing or having proceeds when taken in connection with the accompanying performed one or more genotyping assays on nucleic acids Figures as best described herein below . isolated from the subject to determine the subject' s genotype with respect to a first gene and a second gene , wherein the BRIEF DESCRIPTION OF THE FIGURES first gene is an opioid mu receptor (OPRM1 ) gene and the second gene is selected from the group consisting of a [0017 ] A more complete understanding of the presently dopamine (DA ) - catabolizing enzyme catechol- O -methyl disclosed subject matter can be obtained by reference to the transferase (COMT ) gene and a dopamine transporter accompanying Figures, when considered in conjunction DAT1/ SLC6A3 gene; and administering an opioid receptor with the subsequent Detailed Description . The embodiments antagonist therapy to the subject when the subject' s geno illustrated in the Figures are intended to be exemplary only , type has two alleles encoding an asparagine at an amino acid and should not be construed as limiting the presently dis position corresponding to amino acid residue 40 the of closed subject matter to the illustrated embodiments . The OPRM1 gene product of SEQ ID NO : 12 and has at least one accompanying Figures , which are incorporated in and con allele encoding a methionine at an amino acid position stitute a part of this disclosure , thus illustrate several corresponding to amino acid residue 158 of the COMT gene embodiments and together with the description taken in its product of SEQ ID NO : 8 or at least one allele encoding a entirety are illustrative of the disclosed compositions and dopamine transporter DAT1/ SLC6A3 9 - repeat variable methods . number tandem repeat (VNTR ) ; or the subject ' s genotype [ 0018 ] FIG . 1 is a flow chart showing consolidated stan has at least one allele encoding an aspartic acid at an amino dards of reporting Clinical Trials Diagram . Legend : Those acid position corresponding to amino acid residue 40 the of with valid data had more than one week of drinking data OPRM1 gene product of SEQ ID NO : 12 and has two alleles reported post -randomization . US 2018 /0371542 A1 Dec. 27 , 2018

[0019 ] FIGS . 2A and 2B are a series of graphs showing the [0025 ] SEQ ID NO : 4 is the sequence of the 40 -base -pair percent heavy drinking days % HDD ) over the 4 study variable number tandem repeat (VNTR ) polymorphism . It months. % HDD was analyzed in mixed model of medica corresponds to the insertion / deletion ( indel) variation single tion (naltrexone ( triangles ) vs . placebo ( open circles ) ) x nucleotide polymorphism (SNP ) rs28363170 of the human OPRM1 allelexCOMT (COMT158 Met Carrier vs . Val/ Val DAT1/ SLC6A3 VNTR , where the 9 - repeat (9R ) allele has homozygotes ) xtime ( study weeks ). FIG . 2A shows the nine consecutive repeats of SEQ ID NO : 4 and the 10 -re results for OPRM1 Asn / Asn homozygotes , with the top peated ( 10R ) allele has ten consecutive repeats of SEQ ID panel being COMT158 Met Carriers and the bottom panel NO : 4 . being COMT158 Val/ Val homozygotes . FIG . 2B shows the [ 0026 ] SEQ ID NO : 5 is the nucleotide sequence of the results for OPRM1 Asp carriers , with the top panel being single nucleotide polymorphism (SNP ) rs1799971 of the COMT158 Met Carriers and the bottom panel being human OPRM1 gene . The polymorphism is located at COMT158 Val / Val homozygotes . Statistically for Medica nucleotide 26 of SEQ ID NO : 5 (this position corresponds to tionxOPRM1xCOMT is ( F = 4 .25 , p = 0 .041 ) . nucleotide position 561 of SEQ ID NO : 11) , wherein the [ 0020 ] FIG . 3 is a bar graph showing the percent heavy nucleotide at this position is in some embodiments an drinking days (% HDD ) over the 4 months of the study. % adenine and is in some embodiments a guanine . With respect HDD was analyzed as univariate mixed model ofmedication to the exemplary OPRM1 amino acid sequence of SEQ ID ( naltrexone , black squares vs . placebo , gray squares ) x NO : 12 , amino acid position 40 is shown as an asparagine OPRM1 allele (at least one Asp40 allele , right two pairs of residue as SEQ ID NO : 11 shows position 561 to be an bars vs . Asn /Asn homozygotes , left two pairs of bars ) x adenine , but when the nucleotide at position 561 of SEQ ID COMT (at least one COMT Met158 allele , first and third bar NO : 11 is a guanine, the amino acid at the amino acid pairs vs. Val/ Val homozygotes, second and fourth bar pairs ) x position that corresponds to residue number 40 of SEQ ID time ( 4 months ). Values for treatment response are given for NO : 12 would be an aspartic acid . each gene pair by group . Significant treatment [0027 ] SEQ ID NO : 6 is the nucleotide sequence of the response is obtained only for those who are OPRM1 Asn ( A single nucleotide polymorphism ( SNP ) rs4680 of the human allele ) homozygotes who are also COMT 158 Met ( allele ) COMT gene. The polymorphism is located at nucleotide 26 carriers ( p = 0 . 03 ) and for those who are OPRM1 Asp40 (G of SEQ ID NO : 6 (corresponds to nucleotide 721 of SEQ ID allele ) carriers who are COMT 158 Val/ Val ( A / A allele ) NO : 7 ) , wherein the nucleotide at this position is in some homozygotes (p = 0 .05 ). Statistically , p = 0 . 065 for Medica embodiments a guanine and is in some embodiments an tionxOPRM1xCOMTXF = 3 .47 . adenine . With respect to the exemplary COMT amino acid [0021 ] FIGS. 4A and 4B are a series of graphs showing the sequence of SEQ ID NO : 8 , amino acid position 158 is percent heavy drinking days ( % HDD ) over the 4 study shown as a valine residue as SEQ ID NO : 7 shows position months. % HDD was analyzed in mixed model of medica 721 to be a guanine , but when the nucleotide at position 721 tion ( naltrexone ( triangles ) vs . placebo ( open circles ) ) x of SEQ ID NO : 7 is an adenine , the amino acid at the amino OPRM1 allelexDAT1 VNTR allele (9R carriers vs. 10R acid position that corresponds to residue number 158 of SEQ homozygotes )xtime ( study weeks) . FIG . 4A shows the ID NO : 8 is a methionine . results for OPRM1 Asn / Asn homozygotes , with the top [0028 ] SEQ ID NOs : 7 and 8 are nucleotide and amino panel being DAT1 VNTR 9R carriers and the bottom panel acid sequences , respectively , of exemplary human COMT being DAT1 VNTR 10R homozygotes . FIG . 4B shows the gene products . The nucleotide sequence corresponds to results for OPRM1 Asp carriers , with the top panel being Accession No . NM _ 000754 . 3 in the GENBANK® biose DAT1 VNTR R carriers and the bottom panel being DAT1 quence database , and the amino acid sequence corresponds VNTR 10R homozygotes. MedicationxOPRM1XDAT by to Accession No. NP _ 000745 . 1 in the GENBANK® biose time ( F = 3 .63 p = 0 .015 ) quence database . [0022 ] FIG . 5 is a bar graph showing the number of Drinks [0029 ] SEQ ID NOs : 9 and 10 are nucleotide and amino Per Drinking Day (DPDD ) in the end (month 4 ) of the trial. acid sequences , respectively , of exemplary human DAT1/ % HDD was analyzed in mixed model ofmedication (nal SLC6A3 gene products . The nucleotide sequence corre trexone, black squares vs . placebo , gray squares ) xOPRM1 sponds to Accession No . NM _ 001044 . 4 in the GENBANK® allele (at least one Asp40 allele , third and fourth pairs ofbars biosequence database , and the amino acid sequence corre vs . Asn /Asn homozygotes , first and second pairs of bars ) x sponds to Accession No. NP _ 001035 . 1 in the GENBANK® DAT1 VNTR genotype (at least one 9R allele , first and third biosequence database . bar pairs vs . 10R homozygotes, second and fourth bar (0030 ] SEQ ID NOs: 11 and 12 are nucleotide and amino pairs )xtime ( 4 months ) . Statistically, p = 0 .017 for Medica acid sequences, respectively , of exemplary human OPRM1 tionxOPRM1XDAT1 VNTRXF = 5 . 98 . gene products . The nucleotide sequence corresponds to Accession No . NM _ 000914 . 4 in the GENBANK® biose BRIEF DESCRIPTION OF THE SEQUENCE quence database , and the amino acid sequence corresponds to Accession No . NP _ 000905 . 3 in the GENBANK® biose LISTING quence database. There are numerous different transcription [0023 ] SEQ ID NOs: 1 and 2 are the nucleotide sequences and /or splice variants of the human OPRM1 gene , all of of an exemplary oligonucleotide pair that together can be which are encompassed within the presently disclosed sub employed for determining the number of DAT1 /SLC6A3 ject matter. As such , SEQ ID NOs: 11 and 12 are intended VNTR repeats in a nucleic acid sample . to be exemplary only and not limiting . [ 0024 ] SEQ ID NO : 3 is the nucleotide sequence of an [0031 ] As is known in the art, in some embodiments exemplary oligonucleotide that can be employed with SEQ multiple gene products can be generated from a particular ID NO : 2 for determining the number of DAT1 /SLC6A3 genetic locus, for example by alternative transcriptional VNTR repeats in a nucleic acid sample . initiation sites, alternative splicing , etc . It is understood that US 2018 /0371542 A1 Dec. 27 , 2018 the GENBANK® Accession Nos. presented herein are value . When such a range is expressed , in some embodi meant to be exemplary only, and other gene products for ments the range includes from the one particular value which the nucleotide and / or amino acid sequences are not and /or to the other particular value . Similarly , when values explicitly disclosed herein are also intended to be encom are expressed as approximations, such as but not limited to passed by the names of the corresponding genes. Thus , for by use of the antecedent “ about ," it will be understood that example , transcript variants of the sequences in the the particular value forms an exemplary non - limiting Sequence Listing are also included with the definitions of embodiment. It will be further understood that the endpoints the genes described herein , as are the amino acid variants of each of the ranges are significant both in relation to the encoded thereby . other endpoint, and independently of the other endpoint . [0041 ] It is also understood that there are a number of DETAILED DESCRIPTION values disclosed herein , and that each value is also herein [0032 ] Before the present compounds , compositions , disclosed as " about" that particular value in addition to the articles, devices , and / or methods are disclosed and value itself. For example , if the value “ 10 ” is disclosed , then described , it is to be understood that they are not limited to “ about 10 ” is also disclosed . It is also understood that when specific synthetic methods or specific recombinant biotech a value is disclosed that “ less than or equal to ” the value , nology methods unless otherwise specified , or to particular “ greater than or equal to the value ” and possible ranges reagents unless otherwise specified , as such may , of course , between values are also disclosed , as appropriately under vary . It is also to be understood that the terminology used stood by the skilled artisan . For example , if the value “ 10 ” herein is for the purpose of describing particular embodi is disclosed , “ less than or equal to 10 ” as well as " greater ments only and is not intended to be limiting. than or equal to 10 ” are also disclosed . It is also understood [0033 ] Headings are included herein for reference and to that throughout the disclosure , data are provided in a number aid in locating certain sections. These headings are not of different formats , and that these data represent in some intended to limit the scope of the concepts described therein embodiments endpoints and starting points , and ranges for under , and these concepts can have applicability in other any combination of the data points . By way of example and sections throughout the entire description . not limitation , if a particular data point “ 10 ” and a particular data point “ 15 ” are disclosed , it is understood that greater I . Definitions than , greater than or equal to , less than , less than or equal to , and equal to 10 and 15 are considered disclosed as well as [ 0034 ] The terminology used herein is for the purpose of between 10 and 15 . It is also understood that each unit describing particular embodiments only and is not intended between two particular units are also disclosed . For to be limiting of the presently disclosed subject matter. example , if 10 and 15 are disclosed , then 11 , 12 , 13 , and 14 [0035 ] While the following terms are believed to be well are also disclosed . understood by one of ordinary skill in the art, the following [0042 ] Unless otherwise indicated , all numbers expressing definitions are set forth to facilitate explanation of the quantities of components , reaction conditions , and so forth presently disclosed subject matter. used in the specification and claims are to be understood as [0036 ] All technical and scientific terms used herein , being modified in all instances by the term “ about” . The term unless otherwise defined below , are intended to have the “ about” , as used herein when referring to a measurable value same meaning as commonly understood by one of ordinary such as an amount of mass, weight, time, volume, concen skill in the art . References to techniques employed herein are tration , or percentage , is meant to encompass variations of in intended to refer to the techniques as commonly understood some embodiments + 20 % , in some embodiments + 10 % , in in the art , including variations on those techniques or some embodiments + 5 % , in some embodiments + 1 % , in substitutions of equivalent techniques that would be appar some embodiments + 0 . 5 % , and in some embodiments ent to one of skill in the art . While the following terms are + 0 . 1 % from the specified amount, as such variations are believed to be well understood by one of ordinary skill in the appropriate to perform the disclosed methods and / or employ art , the following definitions are set forth to facilitate expla the disclosed compositions . Accordingly , unless indicated to nation of the presently disclosed subject matter . the contrary , the numerical parameters set forth in this [ 0037 ] In describing the presently disclosed subject mat specification and attached claims are approximations that ter , it will be understood that a number of techniques and can vary depending upon the desired properties sought to be steps are disclosed . Each of these has individual benefit and each can also be used in conjunction with one or more , or in obtained by the presently disclosed subject matter . some cases all, of the other disclosed techniques. [ 0043 ] As used herein , the term “ and / or” when used in the [0038 ] Accordingly , for the sake of clarity , this description context of a list of entities, refers to the entities being present will refrain from repeating every possible combination of singly or in combination . Thus, for example , the phrase “ A , the individual steps in an unnecessary fashion . Nevertheless, B , C , and / or D ” includes A , B , C , and D individually , but the specification and claims should be read with the under also includes any and all combinations and subcombinations standing that such combinations are entirely within the of A , B , C , and D . scope of the presently disclosed and claimed subjectmatter . [0044 ] In this disclosure and in the claims which follow , [0039 ] As used in the specification and the appended reference will be made to a number of terms and phrases claims, the singular forms “ a ," " an ” , and “ the ” include plural which shall be defined to have the following meanings: referents unless the context clearly dictates otherwise . Thus , [0045 ] " Optional” or “ optionally ” means that the subse for example , reference to " a pharmaceutical carrier” quently described event or circumstance may or may not includes mixtures of two or more such carriers, and the like . occur, and that the description includes instances where said 10040 ) Ranges can be expressed herein as from “ about” event or circumstance occurs and instances where it does one particular value , and /or to “ about” another particular not. US 2018 /0371542 A1 Dec . 27 , 2018

[ 0046 ] “ Probes ” are molecules capable of interacting with antagonists , inverse , and mixed / antago a target nucleic acid , typically in a sequence specific manner , nists . As used herein , whether a given agent acts as an for example through hybridization . The hybridization of antagonist or an agonist can depend on the disorder for nucleic acids is well understood in the art and discussed which use of the antagonist or agonist is desired . herein . Typically , a probe can be made from any combina [0054 ] An exemplary disorder associated with opioid tion of nucleotides or nucleotide derivatives or analogs receptor activity is alcohol use disorder ( AUD ) , a chronic available in the art . relapsing brain disease characterized by compulsive alcohol [0047 ] “ Primers ” are a subset of probes which are capable use , loss of control over alcohol intake, and a negative of supporting some type of enzymatic manipulation and emotional state when not using . An estimated 16 million which can hybridize with a target nucleic acid such that the people have been diagnosed as having AUD in the United enzymatic manipulation can occur. A primer can be made States alone . To be diagnosed with AUD , individuals must from any combination of nucleotides and /or nucleotide meet at least two of the criteria outlined in the Diagnostic derivatives and / or analogs available in the art which do not and Statistical Manual ofMental Disorders (DSM ) including interfere with the enzymatic manipulation . amount or duration of consumption , inability to reduce or [0048 ] Throughout this disclosure, various publications stop drinking , time spent drinking or recovering , craving, are referenced . The disclosures of these publications are interference of drinking on work , school, or family , main hereby incorporated by reference into this application in taining consumption despite problems resulting from con their entireties in order to more fully describe the state of the sumption , reducing activities to place more emphasis on art to which this pertains. The references disclosed are also consumption , increased risk behavior while consuming or individually and specifically incorporated by reference intoxicated , continued consumption despite feelings of herein for the material contained in them that is discussed in depression or anxiety, increased average consumption over the sentence in which the reference is relied upon . the past year, and presence of withdrawal symptoms. II . Disorders Associated with Opioid Receptor Activities 10055 ] Treatment for AUD can comprise counseling , [0049 ] In some embodiments , the presently disclosed sub behavioral modification , and pharmacological intervention . ject matter provides methods for treating subjects with Currently , three drugs , Naltrexone, Acamprosate , and Dis disorders associated with opioid receptor activity , detecting ulfiram are approved for treating alcohol use disorder. susceptibility of subject to treatment with an opioid receptor [0056 ] There have been reports that functional genetic antagonist for disorders associated with opioid receptor differences in several dopamine system genes , including but activity , and identifying and treating subjects ( e . g . , human not limited to the OPRM1 gene , alter reward -based brain subjects ) having susceptibility to opioid receptor agonist mechanisms. Dopamine (DA ) signaling regulates many and / or antagonist therapies for disorders associated with aspects of AUD . Alcohol cues and intravenous alcohol opioid receptor activity in the subjects . self -administration both increase DA release in the human [ 0050 ] As used herein , the phrase " disorder associated ventral striatum ( VS ) . Individuals with AUD , relative to with opioid receptor activity ” refers to any disease , disorder, controls , display enhanced alcohol- induced but blunted or condition at least one symptom of which can be improved amphetamine - induced VS DA release , and , unlike controls , or treated by administering to a subject in need thereof an demonstrate no association between striatal DA release and opioid receptor antagonist or an opioid receptor agonist, prefrontal glucose metabolism , suggesting impaired cortical depending on whether the symptom results from undesirably modulation of VS DA signaling . high opioid receptor activity or undesirably low opioid [0057 ] Because it has been speculated that dopamine receptor activity . Exemplary disorders associated with opi tone/ release might underlie the stimulant response to alcohol oid receptor activity include , but are not limited to alcohol observed in heavy drinkers and early - stage alcoholics and use disorders ( AUD ) , addiction and /or abuse , whether because naltrexone reduces ventral striatal dopamine output intentional or unintentional; depression , anxiety , compulsive in rodents and also reduces alcohol- induced stimulation in disorders , and pain . man , it is likely that there might be a salient interaction [0051 ] As used herein , the phrases “ ” and between the opiate and dopamine systems that might be “ opioid receptor antagonist ” refer to any agent that inhibits genetically based and that can be modified by naltrexone signaling through an opioid receptor either directly or indi and / or other opioid antagonists . For instance, people who rectly. Exemplary opioid receptor antagonists include nal are carriers of the DAT1 9 -repeat VNTR might be more trexone, , , and . See also likely to have elevated dopamine levels in nucleus accum Niciu & Arias , 2013 . Other agents that have opioid receptor bens after alcohol consumption or cue - presentation and antagonist activities include , but are not limited to nalor therefore be more likely to respond to naltrexone second phine , dinicotinate , , , ary to its ability to decrease alcohol or cue - induced dop and . amine release . Similarly , certain alleles of the COMT rs4680 [0052 ] As used herein , the phrase " opioid agonist ” and SNP (also known as Val158Met) might be more likely to “ opioid receptor agonist " refer to any agent that enhances or have elevated dopamine levels . The COMT Met allele has augments signaling through an opioid receptor either been associated with reduced catechol- O -methyltransferase directly or indirectly. Exemplary opioid receptor agonists efficiency, likely increasing extrasynaptic DA accumulation , include , buprenorphine , , hydroco and also with heightened DA receptor sensitivity among done , and its derivatives, , , mor AUD individuals . phine, and . [0058 ] In some embodiments , disclosed herein are meth [0053 ] As is known in the art, some antagonists and ods of treating a psychiatric , mental, and /or neurological agonists have overlapping activities such that under different disorder (such as, for example , AUD ) comprising assaying circumstances they can act as either antagonists or agonists . a nucleic acid from a subject with respect to the genotype of Exemplary such agents include partial agonists , competitive the dopamine (DA ) - catabolizing enzyme catechol- O - meth US 2018 /0371542 A1 Dec . 27 , 2018 yltransferase (COMT ) and the genotype of the opioid mu a psychiatric , mental, and / or neurological disorder ( such as, receptor gene (OPRM1 ) , wherein when one or two alleles 1019for example , alcohol use disorder (AUD ) ) , wherein the encoding a methionine at amino acid residue 158 of COMT genotype of COMT is assayed after opioid receptor antago and two copies of an allele encoding an asparagine at amino nist therapy has commenced , and wherein when one or two acid residue 40 of OPRM1 is detected or wherein two alleles alleles encoding a methionine at amino acid residue 158 of encoding valine at amino acid residue 158 of COMT and at COMT and at least one allele encoding an aspartic acid at least one allele encoding an aspartic acid at amino acid amino acid residue 40 of OPRM1 is detected or wherein two residue 40 of OPRM1 is detected , an opioid receptor antago alleles encoding valine at amino acid residue 158 of COMT nist is administered to the subject; and wherein when one or and two alleles encoding an asparagine at amino acid residue two alleles encoding a methionine at amino acid residue 158 40 of OPRM1 is detected , the opioid receptor antagonist of COMT and at least one allele encoding an aspartic acid therapy is discontinued . at amino acid residue 40 of OPRM1 is detected or wherein [0062 ] It is understood and herein provided that the dis two alleles encoding valine at amino acid residue 158 of closed methods of treating, methods of detecting the sus COMT and two alleles encoding an asparagine at amino acid ceptibility to opioid receptor antagonist therapy and kits are residue 40 of OPRM1 is detected , an opioid receptor antago not limited to alcohol use disorder, but can be used for any nist is not administered to the subject . psychiatric , mental, and / or neurological disorder where opi [0059 ] It is understood and herein provided that the detec oid receptor modulation can have an effect on the treatment tion genotype COMT and OPRM1 can also be used to detect of the subject . For example , the psychiatric , mental , and /or susceptibility to opioid receptor antagonist therapy . Accord neurological disorder can comprise schizophrenia , bipolar ingly , disclosed herein are methods of detecting susceptibil disorder , depression , and chemical addition (including , but ity to an opioid receptor antagonist therapy for alcohol use not limited to cocaine addiction , opioid addiction , amphet disorder comprising obtaining a tissue sample from a sub amine ( including methamphetamine ) addiction , nicotine ject , assaying nucleic acid from the tissue sample from the addiction , prescription drug addiction , and alcohol use dis subject for the genotype encoding the amino acid substitu order ) . tion at residue 158 of the DA -catabolizing enzyme catechol [ 0063 ] The disclosed methods of detection and treatment O -methyltransferase (COMT ) and the genotype encoding an assay for nucleic acid polymorphisms of the COMT and amino acid substitution at residue 40 of the opioid mu OPRM1 and / or COMT, the VNTR of DAT1 , and OPRM1. receptor gene (OPRM1 ) , wherein when one or two alleles Such polymorphisms can be detected using any method encode for a methionine at amino acid residue 158 of COMT known in the art for detection of nucleic acids . and a genome that encodes for an asparagine at amino acid residue 40 of OPRM1 or wherein two alleles encoding III. DNA Detection and Quantification valine at amino acid residue 158 of COMT and the genome 10064 ] As indicated throughout, the methods disclosed encodes for an aspartic acid at amino acid residue 40 of herein relate to the detection of nucleic acid variation in the OPRM1 indicates that the subject is susceptible to opioid form of, for example, the alleles for the polymorphism receptor antagonist therapy. encoding the amino acid at residue 158 of COMT, the allele [0060 ] As used in the methods of treating a psychiatric , for the polymorphism encoding the amino acid at residue 40 mental, and /or neurological disorder ( such as, for example , ofOPRM1 , and the allele for the number of variable number alcohol use disorder ( AUD ) ) or methods of detecting sus tandem repeats (VNTR ) of DAT1 . For these latter expres ceptibility to opioid receptor antagonist treatment disclosed sion level detections , the methods comprise detecting either herein , a opioid receptor antagonist as used in the disclosed the abundance or presence ofmRNA , or both . Alternatively , methods can comprise any antibody , biologic , small mol detection can be directed to the abundance or presence of ecule, and / or nucleic acid modulators ( including , but not DNA , for example , cDNA . Thus, disclosed herein are meth limited to small interfering RNAs ( siRNAs ) , small hairpin ods of treating a psychiatric , mental, and / or neurological RNAs ( shRNAs ), antisense molecules , zinc finger nucle disorder (such as, for example , alcohol use disorder (AUD ) ) ases , meganucleases, TAL ( TALE ) nucleases, triplexes, comprising assaying the nucleic acid from a subject for the modified triplexes ). Such opioid receptor antagonists can genotype of the dopamine (DA ) - catabolizing enzyme cat include but are not limited to naloxone , naltrexone , nalme echol- O -methyltransferase (COMT ) and the genotype of the fene , , nalorphine , nalorphine dinicotinate , opioid mu receptor gene (OPRM1 ) , wherein when one or levallorphan , samidorphan , nalodeine , , methyln two alleles encoding a methionine at amino acid residue 158 altrexone , , 6ß -naltrexol , , , of COMT and two alleles encoding an asparagine at amino methylsamidorphan , and . acid residue 40 of OPRM1 is detected or wherein two alleles 10061] In some embodiments , it is understood and herein encoding valine at amino acid residue 158 of COMT and at provided that the disclosed treatment methods can be least one allele encoding an aspartic acid at amino acid applied prior to any opioid receptor antagonist therapy residue 40 of OPRM1 is detected , an opioid receptor antago and / or as a modification of an opioid receptor antagonist nist is administered to the subject; and wherein when one or therapy . Thus , in some embodiments , disclosed herein are two alleles encoding a methionine at amino acid residue 158 methods of treating a psychiatric , mental, and / or neurologi of COMT and at least one allele encoding an aspartic acid cal disorder ( such as , for example , alcohol use disorder at amino acid residue 40 of OPRM1 is detected or wherein ( AUD ) ) comprising assaying the nucleic acid from a subject two alleles encoding valine at amino acid residue 158 of for the genotype of the dopamine (DA ) -catabolizing enzyme COMT and two alleles encoding an asparagine at amino acid catechol - O -methyltransferase (COMT ) and the genotype of residue 40 of OPRM1 is detected , an opioid receptor antago the opioid mu receptor gene (OPRM1 ) , wherein the geno nist is not administered to the subject; wherein the genotype type of COMT is assayed prior to administering the opioid of the COMT and OPRM1 polymorphisms ( and the geno receptor antagonist. Also disclosed are methods of treating type of the variable number tandem repeats (VNTR ) poly US 2018 /0371542 A1 Dec. 27 , 2018 morphism in the dopamine transporter gene DAT1 /SLC6A3 spots . Microarrays require specialized robotics and/ or imag when included ) are detected by a nucleic acid amplification ing equipment that generally are not commercially available process followed by sequencing or gel electrophoresis or by as a complete system . Terminologies that have been used in direct sequencing. Also disclosed herein are methods of the literature to describe this technology include , but not detecting susceptibility to an opioid receptor antagonist limited to : biochip , DNA chip , DNA microarray , therapy for alcohol use disorder comprising obtaining a GENECHIP® (Affymetrix , Inc which refers to its high tissue sample from a subject, assaying nucleic acid from the density , oligonucleotide- based DNA arrays) , and gene array . tissue sample from the subject for the genotype encoding the [0068 ] DNA microarrays , or DNA chips are fabricated by amino acid substitution at residue 158 of the DA -cataboliz high -speed robotics , generally on glass or nylon substrates, ing enzyme catechol- O -methyltransferase ( COMT) and the for which probes with known identity are used to determine genotype encoding an amino acid substitution at residue 40 complementary binding , thus allowing massively parallel of the opioid mu receptor gene (OPRM1 ) , wherein when one gene expression and gene discovery studies . An experiment or two alleles encode for a methionine at amino acid residue with a single DNA chip can provide information on thou 158 of COMT and a genome that encodes for an asparagine sands of genes simultaneously . It is herein contemplated that at amino acid residue 40 of OPRM1 or wherein two alleles the disclosed microarrays can be used to monitor gene encoding valine at amino acid residue 158 of COMT and the expression , disease diagnosis , gene discovery , drug discov genome encodes for an aspartic acid at amino acid residue 40 of OPRM1 indicates that the subject is susceptible to ery ( pharmacogenomics) , and toxicological research or toxi dopamine modulator therapy ; wherein the genotype of the cogenomics . COMT and OPRM1 polymorphisms (and the genotype of [0069 ] There are two variants of the DNA microarray the variable number tandem repeats (VNTR ) polymorphism technology , in terms of the property of arrayed DNA in the dopamine transporter gene DAT1 /SLC6A3 when sequence with known identity . Type I microarrays comprise included ) are detected by a nucleic acid amplification pro a probe cDNA ( typically about 500 - 5 ,000 bases long ) that is cess followed by sequencing or gel electrophoresis or by immobilized to a solid surface such as glass using robot direct sequencing . spotting and exposed to a set of targets either separately or [ 0065 ] A number of widely used procedures exist for in a mixture . This method is traditionally referred to as DNA detecting and determining the abundance of a particular microarray . With Type I microarrays, localized multiple DNA in a sample . For example , the technology of PCR copies of one or more polynucleotide sequences , preferably permits amplification and subsequent detection of minute copies of a single polynucleotide sequence are immobilized quantities of a target nucleic acid . Details of PCR are well on a plurality of defined regions of the substrate ' s surface . described in the art, including , for example, U . S . Pat. Nos. A polynucleotide refers to a chain of nucleotides ranging 4 ,683 , 195 to Mullis et al. , 4 ,683 ,202 to Mullis , and 4 , 965 , from 5 to 10 ,000 nucleotides . These immobilized copies of 188 to Mullis et al. Generally, oligonucleotide primers are a polynucleotide sequence are suitable for use as probes in annealed to the denatured strands of a target nucleic acid , hybridization experiments . and primer extension products are formed by the polymer [0070 ] To prepare beads coated with immobilized probes , ization of deoxynucleoside triphosphates by a polymerase. A beads are immersed in a solution containing the desired typical PCR method involves repetitive cycles of template probe sequence and then immobilized on the beads by nucleic acid denaturation , primer annealing and extension of covalent or noncovalent means. Alternatively, when the the annealed primers by the action of a thermostable poly probes are immobilized on rods, a given probe can be merase . The process results in exponential amplification of spotted at defined regions of the rod . Typical dispensers the target nucleic acid , and thus allows the detection of include a micropipette delivering solution to the substrate targets existing in very low concentrations in a sample . It is with a robotic system to control the position of the micropi understood and herein contemplated that there are variant pette with respect to the substrate . There can be a multiplic PCR methods known in the art that may also be utilized in ity of dispensers so that reagents can be delivered to the the disclosed methods, for example , Quantitative PCR reaction regions simultaneously . In one embodiment , a ( QPCR ) ; microarrays , real- time PCT ; hot start PCR ; nested microarray is formed by using ink - jet technology based on PCR ; allele - specific PCR ; digital droplet PCR (ddPCR ) , the piezoelectric effect, whereby a narrow tube containing a digital droplet quantitative PCR (ddQPCR ) , and Touchdown liquid of interest, such as oligonucleotide synthesis reagents , PCR . is encircled by an adapter. An electric charge sent across the [0066 ] III. A . Microarrays adapter causes the adapter to expand at a different rate than [0067 ] An array is an orderly arrangement of samples, the tube and forces a small drop of liquid onto a substrate . providing a medium for matching known and unknown [ 0071 ] Tissue samples may be any sample containing DNA samples based on base- pairing rules and automating polynucleotides (polynucleotide targets ) of interest and the process of identifying the unknowns . An array experi obtained from any bodily fluid ( blood, urine, saliva , phlegm , ment can make use of common assay systems such as gastric juices , etc . ) , cultured cells, biopsies, or other tissue microplates or standard blotting membranes , and can be preparations . DNA or RNA can be isolated from the sample created by hand or make use of robotics to deposit the according to any of a number of methods well known to sample . In general , arrays are described as macroarrays or those of skill in the art . In one embodiment, total RNA is microarrays , the difference being the size of the sample isolated using the TRIzol total RNA isolation reagent ( Life spots . Macroarrays contain sample spot sizes of about 300 Technologies, Inc. , Rockville , Md. ) and RNA is isolated microns or larger and can be easily imaged by existing gel using oligo d ( T ) column chromatography or glass beads . and blot scanners . The sample spot sizes in microarray can After hybridization and processing, the hybridization signals be 300 microns or less , but typically less than 200 microns obtained should reflect accurately the amounts of control in diameter and these arrays usually contains thousands of target polynucleotide added to the sample . US 2018 /0371542 A1 Dec. 27 , 2018

[0072 ] The plurality of defined regions on the substrate [0077 ] In a differential hybridization experiment, poly can be arranged in a variety of formats . For example , the nucleotide targets from two or more different biological regions may be arranged perpendicular or in parallel to the samples are labeled with two or more different fluorescent length of the casing . Furthermore , the targets do not have to labels with different emission wavelengths . Fluorescent sig be directly bound to the substrate , but rather can be bound nals are detected separately with different photomultipliers to the substrate through a linker group . The linker groups set to detect specific wavelengths . The relative abundances/ may typically vary from about 6 to 50 atoms long. Linker expression levels of the target polynucleotides in two or groups include ethylene glycol oligomers , diamines , diacids more samples is obtained . Typically , microarray fluores and the like . Reactive groups on the substrate surface react cence intensities can be normalized to take into account with one of the terminal portions of the linker to bind the variations in hybridization intensities when more than one linker to the substrate . The other terminal portion of the microarray is used under similar test conditions . In one linker is then functionalized for binding the probes . embodiment, individual polynucleotide probe / target com [0073 ] Sample polynucleotides may be labeled with one or plex hybridization intensities are normalized using the inten more labeling moieties to allow for detection of hybridized sities derived from internal normalization controls contained probe /target polynucleotide complexes. The labeling moi on each microarray . eties can include compositions that can be detected by [ 0078 ] Type II microarrays comprise an array of oligo spectroscopic , photochemical , biochemical , bioelectronic , nucleotides (typically about 20 to about 80 -mer oligos ) or immunochemical , electrical, optical or chemical means . The peptide nucleic acid ( PNA ) probes that is synthesized either labeling moieties include radioisotopes , such as 32P , 33P or in situ (on - chip ) or by conventional synthesis followed by 35S , chemiluminescent compounds, labeled binding pro on -chip immobilization . The array is exposed to labeled teins, heavy metal atoms, spectroscopic markers , such as sample DNA , hybridized , and the identity / abundance of fluorescent markers and dyes , magnetic labels , linked complementary sequences are determined . This method , enzymes ,mass spectrometry tags , spin labels , electron trans " historically ” called DNA chips, was developed at Affyme fer donors and acceptors, biotin , and the like . trix , Inc ., which sells its photolithographically fabricated [0074 ] Labeling can be carried out during an amplification products under the GENECHIP® trademark . reaction , such as polymerase chain reaction and in vitro or [ 0079 ] The basic concept behind the use of Type II arrays in vivo transcription reactions. Alternatively , the labeling for gene expression is simple : labeled cDNA or CRNA moiety can be incorporated after hybridization once a probe targets derived from the mRNA of an experimental sample target complex his formed . In one embodiment, biotin is first are hybridized to nucleic acid probes attached to the solid incorporated during an amplification step as described support . By monitoring the amount of label associated with above . After the hybridization reaction , unbound nucleic each DNA location , it is possible to infer the abundance of acids are rinsed away so that the only biotin remaining each mRNA species represented . Although hybridization has bound to the substrate is that attached to target polynucle been used for decades to detect and quantify nucleic acids , otides that are hybridized to the polynucleotide probes . the combination of theminiaturization of the technology and Then , an avidin - conjugated fluorophore , such as avidin the large and growing amounts of sequence information , phycoerythrin , that binds with high affinity to biotin is have enormously expanded the scale at which gene expres added . [ 0075 ] Hybridization causes a polynucleotide probe and a sion can be studied . complementary target to form a stable duplex through base [ 0080 ] Microarray manufacturing can begin with a 5 - inch pairing . Hybridization methods are well known to those square quartz wafer . Initially the quartz is washed to ensure skilled in the art Stringent conditions for hybridization can uniform hydroxylation across its surface . Because quartz is be defined by salt concentration , temperature , and other naturally hydroxylated , it provides an excellent substrate for chemicals and conditions . Varying additional parameters , the attachment of chemicals , such as linker molecules, that such as hybridization time, the concentration of detergent are later used to position the probes on the arrays. ( , SDS ) or solvent ( formamide ) , and [0081 ] The wafer is placed in a bath of silane, which reacts the inclusion or exclusion of carrier DNA , are well known with the hydroxyl groups of the quartz , and forms a matrix to those skilled in the art . Additional variations on these of covalently linked molecules . The distance between these conditions will be readily apparent to those skilled in the art . silane molecules determines the probes ' packing density , [0076 ] Methods for detecting complex formation are well allowing arrays to hold over 500 ,000 probe locations , or known to those skilled in the art. In one embodiment, the features, within a mere 1 . 28 square centimeters . Each of polynucleotide probes are labeled with a fluorescent label these features harbors millions of identical DNA molecules . and measurement of levels and patterns of complex forma The silane film provides a uniform hydroxyl density to tion is accomplished by fluorescence microscopy, preferably initiate probe assembly. Linker molecules , attached to the confocal fluorescence microscopy . An argon ion laser silane matrix , provide a surface that may be spatially acti excites the fluorescent label, emissions are directed to a vated by light. photomultiplier and the amount of emitted light detected and [ 0082 ] Probe synthesis occurs in parallel, resulting in the quantitated . The detected signal should be proportional to addition of an A , C , T , or G nucleotide to multiple growing the amount of probe / target polynucleotide complex at each chains simultaneously . To define which oligonucleotide position of the microarray . The fluorescence microscope can chains will receive a nucleotide in each step , photolitho be associated with a computer- driven scanner device to graphic masks, carrying 18 to 20 square micron windows generate a quantitative two - dimensional image of hybrid that correspond to the dimensions of individual features , are ization intensities . The scanned image is examined to deter placed over the coated wafer. The windows are distributed mine the abundance / expression level of each hybridized over the mask based on the desired sequence of each probe . target polynucleotide . When ultraviolet light is shone over the mask in the first step US 2018 /0371542 A1 Dec. 27 , 2018 of synthesis , the exposed linkers become deprotected and the dynamic range , the more accurate the quantitation . When are available for nucleotide coupling . combined with RT- PCR , a real - time RT- PCR reaction 10083 ) Once the desired features have been activated , a reduces the time needed for measuring the amount of solution containing a single type of deoxynucleotide with a amplicon by providing for the visualization of the amplicon removable protection group is flushed over the wafer 's as the amplification process is progressing. surface . The nucleotide attaches to the activated linkers , [0091 ] The real- time PCR system is based on the detection initiating the synthesis process . and quantitation of a fluorescent reporter . This signal 10084 ] Although each position in the sequence of an increases in direct proportion to the amount of PCR product oligonucleotide can be occupied by 1 of 4 nucleotides , in a reaction . By recording the amount of fluorescence resulting in an apparent need for 25x4 , or 100 , different emission at each cycle, it is possible to monitor the PCR masks per wafer , the synthesis process can be designed to reaction during exponential phase where the first significant significantly reduce this requirement. Algorithms that help increase in the amount of PCR product correlates to the minimize mask usage calculate how to best coordinate probe initial amount of target template . The higher the starting growth by adjusting synthesis rates of individual probes and copy number of the nucleic acid target, the sooner a signifi identifying situations when the same mask can be used cant increase in fluorescence is observed . A significant multiple times . increase in fluorescence above the baseline value measured [0085 ) Some of the key elements of selection and design during the 3 - 15 cycles can indicate the detection of accu are common to the production of all microarrays , regardless mulated PCR product . of their intended application . Strategies to optimize probe [0092 ] A fixed fluorescence threshold is set significantly hybridization , for example, are invariably included in the above the baseline that can be altered by the operator. The process of probe selection . Hybridization under particular parameter C , ( threshold cycle ) is defined as the cycle pH , salt , and temperature conditions can be optimized by number at which the fluorescence emission exceeds the fixed taking into accountmelting temperatures and using empiri threshold . cal rules that correlate with desired hybridization behaviors . [0093 ] There are three main fluorescence -monitoring sys [0086 ] To obtain a complete picture of a gene ' s activity , tems for DNA amplification : ( 1 ) hydrolysis probes ; ( 2 ) some probes are selected from regions shared by multiple hybridizing probes ; and ( 3 ) DNA -binding agents . Hydroly splice or polyadenylation variants . In other cases, unique sis probes include TAQMAN® brand probes , molecular probes that distinguish between variants are favored . Inter beacons and scorpions . They use the fluorogenic 5 ' exonu probe distance is also factored into the selection process . clease activity of Taq polymerase to measure the amount of [0087 ] A different set of strategies is used to select probes target sequences in cDNA samples. for genotyping arrays that rely on multiple probes to inter [0094 ] TAQMAN® brand probes are oligonucleotides rogate individual nucleotides in a sequence . The identity of longer than the primers (20 - 30 bases long with a Tm value a target base can be deduced using four identical probes that of 10° C . higher ) that contain a fluorescent dye usually on vary only in the target position , each containing one of the the 5 ' base , and a quenching dye ( usually TAMRA ) typically four possible bases . on the 3 ' base. When irradiated , the excited fluorescent dye [0088 ] Alternatively , the presence of a consensus transfers energy to the nearby quenching dye molecule sequence can be tested using one or two probes representing rather than fluorescing ( this is called FRET = Förster or specific alleles . To genotype heterozygous or genetically fluorescence resonance energy transfer ) . Thus , the close mixed samples, arrays with many probes can be created to proximity of the reporter and quencher prevents emission of provide redundant information , resulting in unequivocal any fluorescence while the probe is intact. TAQMAN® genotyping . In addition , generic probes can be used in some brand probes are designed to anneal to an internal region of applications to maximize flexibility . Some probe arrays , for a PCR product. When the polymerase replicates a template example , allow the separation and analysis of individual on which a TAQMAN® brand probe is bound , its 5 ' exo reaction products from complex mixtures , such as those nuclease activity cleaves the probe . This ends the activity of used in some protocols to identify single nucleotide poly quencher (no FRET) and the reporter dye starts to emit morphisms (SNPs ). fluorescence which increases in each cycle proportional to [0089 ] III. B . Real- Time PCR the rate of probe cleavage . Accumulation of PCR products [0090 ] Real -time PCR monitors the fluorescence emitted is detected by monitoring the increase in fluorescence of the during the reaction as an indicator of amplicon production reporter dye ( note that primers are not labelled ) . TAQ during each PCR cycle ( i. e ., in real time ) as opposed to the MAN® brand assay uses universal thermal cycling param endpoint detection . The real - time progress of the reaction eters and PCR reaction conditions . Because the cleavage can be viewed in some systems. Real- time PCR does not occurs only if the probe hybridizes to the target, the origin detect the size of the amplicon and thus does not allow the of the detected fluorescence is specific amplification . The differentiation between DNA and cDNA amplification ; how process of hybridization and cleavage does not interfere with ever, it is not influenced by non - specific amplification unless the exponential accumulation of the product. One specific SYBR Green is used . Real- time PCR quantitation eliminates requirement for fluorogenic probes is that there be no G at post - PCR processing of PCR products . This helps to the 5 ' end . A ‘ G ’ adjacent to the reporter dye can quench increase throughput and reduce the chances of carryover reporter fluorescence even after cleavage . contamination . Real- time PCR also offers a wide dynamic 10095 ) Molecular beacons also contain fluorescent (FAM , range of up to 107- fold . Dynamic range of any assay TAMRA , TET , ROX ) and quenching dyes ( typically DAB determines how much target concentration can vary and still CYL ) at either end but they are designed to adopt a hairpin be quantified . A wide dynamic range means that a wide structure while free in solution to bring the fluorescent dye range of ratios of target and normalizer can be assayed with and the quencher in close proximity for FRET to occur. They equal sensitivity and specificity . It follows that the broader have two arms with complementary sequences that form a US 2018 /0371542 A1 Dec . 27 , 2018 very stable hybrid or stem . The close proximity of the real amplification (rather than signal drift ) , there has to be an reporter and the quencher in this hairpin configuration inflection point. This is the point on the growth curve when suppresses reporter fluorescence . When the beacon hybrid the log - linear phase begins. It also represents the greatest izes to the target during the annealing step , the reporter dye rate of change along the growth curve . (Signal drift is is separated from the quencher and the reporter fluoresces characterized by gradual increase or decrease in fluores ( FRET does not occur ) . Molecular beacons remain intact cence without amplification of the product. ) The important during PCR and must rebind to target every cycle for parameter for quantitation is the Cr. The higher the initial fluorescence emission . This will correlate to the amount of amount of genomic DNA , the sooner accumulated product PCR product available. All real- time PCR chemistries allow is detected in the PCR process, and the lower the C , value . detection ofmultiple DNA species (multiplexing ) by design The threshold should be placed above any baseline activity ing each probe /beacon with a spectrally unique fluor / quench and within the exponential increase phase (which looks pair as long as the platform is suitable for melting curve linear in the log transformation ) . Some software allows analysis if SYBR green is used . By multiplexing, the target determination of the cycle threshold (CT ) by a mathematical ( s ) and endogenous control can be amplified in single tube . analysis of the growth curve . This provides better run - to -run [0096 ] With Scorpion probes, sequence - specific priming reproducibility . AC value of 40 means no amplification and and PCR product detection is achieved using a single this value cannot be included in the calculations. Besides oligonucleotide . The Scorpion probe maintains a stem - loop being used for quantitation , the C7 value can be used for configuration in the unhybridized state . The fluorophore is qualitative analysis as a pass/ fail measure . 00991 Multiplex TAQMAN® brand assays can be per to the 3 ' end . The 3 ' portion of the stem also contains formed using multiple dyes with distinct emission wave sequence that is complementary to the extension product of lengths . Available dyes for this purpose are FAM , TET, VIC the primer. This sequence is linked to the 5 ' end of a specific and JOE ( the most expensive ) . TAMRA is reserved as the primer via a non - amplifiable monomer . After extension of quencher on the probe and ROX as the passive reference . the Scorpion primer, the specific probe sequence is able to For best results , the combination of FAM ( target ) and VIC bind to its complement within the extended amplicon thus ( endogenous control) is recommended ( they have the largest opening up the hairpin loop . This prevents the fluorescence difference in emission maximum ) whereas JOE and VIC from being quenched and a signal is observed . should not be combined . It is important that if the dye layer [0097 ] Another alternative is the double - stranded DNA has not been chosen correctly , the machine will still read the binding dye chemistry , which quantitates the amplicon pro other dye 's spectrum . For example , both VIC and FAM emit duction ( including non -specific amplification and primer fluorescence in a similar range to each other and when doing dimer complex ) by the use of a non - sequence specific fluorescent intercalating agent ( SYBR - green I or ethidium case ofmultiplexing , the spectral compensation for the post bromide ) . It does not bind to ssDNA . SYBR green is a run analysis should be turned on ( on ABI 7700 : Instrument / fluorogenic minor groove binding dye that exhibits little Diagnostics/ Advanced Options/ Miscellaneous ) . Activating fluorescence when in solution but emits a strong fluorescent spectral compensation improves dye spectral resolution . signal upon binding to double - stranded DNA . Disadvan tages of SYBR green - based real- time PCR include the [0100 ) III .C . Nested PCR requirement for extensive optimization . Furthermore , non [0101 ] The disclosed methods can further utilize nested specific amplifications require follow -up assays (melting PCR . Nested PCR increases the specificity of DNA ampli point curve or dissociation analysis ) for amplicon identifi fication , by reducing background due to non - specific ampli cation . The method has been used in HFE -C282Y genotyp fication of DNA . Two sets of primers are being used in two ing . Another controllable problem is that longer amplicons successive PCRs . In the first reaction , one pair of primers is create a stronger signal ( if combined with other factors , this used to generate DNA products , which besides the intended may cause CDC camera saturation , see below ). Normally target, may still consist of non - specifically amplified DNA SYBR green is used in singleplex reactions , however when fragments . The product( s ) are then used in a second PCR coupled with melting point analysis , it can be used for with a set of primers whose binding sites are completely or multiplex reactions . partially different from and located 3 ' of each of the primers 0098 ] The threshold cycle or the C , value is the cycle at used in the first reaction . Nested PCR is often more suc which a significant increase in ARn is first detected ( for cessful in specifically amplifying long DNA fragments than definition of ARn , see below ). The threshold cycle is when conventional PCR , but it requires more detailed knowledge the system begins to detect the increase in the signal of the target sequences. associated with an exponential growth of PCR product 10102 ] III. D . Primers and Probes during the log -linear phase . This phase provides the most useful information about the reaction (certainly more impor [0103 ] As used herein , “ primers ” are a subset of probes tant than the end- point ) . The slope of the log - linear phase is a reflection of the amplification efficiency . The efficiency manipulation and which can hybridize with a target nucleic (Eff ) of the reaction can be calculated by the formula : acid such that the enzymatic manipulation can occur. A Eff = 10 (- 1/ slope ) - 1 . The efficiency of the PCR should be primer can be made from any combination of nucleotides or 90 - 100 % ( 3 . 6 > slope > 3 . 1 ) . A number of variables can affect nucleotide derivatives or analogs available in the art which the efficiency of the PCR . These factors include length of the do not interfere with the enzymatic manipulation . amplicon , secondary structure and primer quality . Although [0104 ] As used herein , “ probes ” are molecules capable of valid data can be obtained that fall outside of the efficiency interacting with a target nucleic acid , typically in a sequence range, the qRT- PCR should be further optimized or alterna specific manner , for example through hybridization . The tive amplicons designed . For the slope to be an indicator of hybridization of nucleic acids is well understood in the art US 2018 /0371542 A1 Dec. 27 , 2018 and discussed herein . Typically , a probe can be made from nucleic acid of interest. The size of the product can be such any combination of nucleotides or nucleotide derivatives or that the size can be accurately determined to within 3 , or 2 analogs available in the art . or 1 nucleotides . [ 0105 ] Disclosed are compositions including primers and [0109 ] In certain embodiments the product can be , for probes , which are capable of interacting with the disclosed example , at least 20 , 21, 22 , 23, 24 , 25 , 26 , 27, 28 , 29 , 30 , COMT, OPRM1, and /or DAT1 nucleic acids or its comple 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , ment. In certain embodiments the primers are used to 47 , 48 , 49 , 50 , 51, 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61, 62 , support nucleic acid extension reactions , nucleic acid rep 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , lication reactions, and/ or nucleic acid amplification reac 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91, 92 , 93 , 94 , tions. Typically , the primers will be capable of being 95 , 96 , 97 , 98 , 99 , 100 , 125 , 150 , 175 , 200 , 225, 250 , 275 , extended in a sequence specific manner . Extension of a 300 , 325 , 350 , 375 , 400 , 425 , 450 , 475 , 500 , 550 , 600 , 650 , primer in a sequence specific manner includes any methods 700 , 750 , 800 , 850 , 900 , 950 , 1000 , 1250 , 1500 , 1750 , 2000 , wherein the sequence and /or composition of the nucleic acid 2250 , 2500 , 2750 , 3000 , 3500 , or 4000 nucleotides long . molecule to which the primer is hybridized or otherwise [0110 ] In other embodiments the product can be , for associated directs or influences the composition or sequence example , less than or equal to 20 , 21 , 22, 23 , 24 , 25 , 26 , 27 , of the product produced by the extension of the primer. 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , Extension of the primer in a sequence specific manner 44 , 45 , 46 , 47, 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , therefore includes , but is not limited to , PCR , DNA sequenc 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , ing , DNA extension , DNA polymerization , RNA transcrip 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , tion , or reverse transcription . Techniques and conditions that 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 125 , 150 , 175 , 200 , 225 , amplify the primer in a sequence specific manner are dis 250 , 275 , 300 , 325 , 350 , 375 , 400 , 425 , 450 , 475 , 500 , 550 , closed . In certain embodiments the primers are used for the 600 , 650 , 700 , 750 , 800 , 850 , 900 , 950 , 1000 , 1250 , 1500 , DNA amplification reactions , such as PCR or direct 1750 , 2000 , 2250 , 2500 , 2750 , 3000 , 3500 , or 4000 nucleo sequencing . It is understood that in certain embodiments the tides long . primers can also be extended using non -enzymatic tech 10111 ] Thus , it is understood and herein provided that the niques , where for example , the nucleotides or oligonucle disclosed RT- PCR and PCR reactions that comprise a por otides used to extend the primer are modified such that they tion of the disclosed methods or performed using the dis will chemically react to extend the primer in a sequence closed kits require forward and reverse primers to form a specific manner. Typically , the disclosed primers hybridize primer pair. Herein disclosed , the forward and reverse with the disclosed nucleic acids or region of the nucleic primer pair in the disclosed methods and kits can be SEQ ID acids or they hybridize with the complement of the nucleic NO : 1 or SEQ ID NO : 3 ; and SEQ ID NO : 2 . acids or complement of a region of the nucleic acids . As an [0112 ] It is understood and herein contemplated that there example of the use of primers , one or more primers can be are situations where it may be advantageous to utilize more used to create extension products from and templated by a than one primer pair to detect the presence of a fusion , first nucleic acid . truncation , or over expression mutation . Such RT -PCR or [0106 ] The size of the primers or probes for interaction PCR reactions can be conducted separately , or in a single with the nucleic acids can be any size that supports the reaction . When multiple primer pairs are placed into a single desired enzymatic manipulation of the primer, such as DNA reaction , this is referred to as “ multiplex PCR .” For amplification or the simple hybridization of the probe or example , the reaction can comprise a first COMT and / or primer . A typical primer or probe would be at least 6 , 7 , 8 , OPRM1 forward and reverse primer pair , as well as , second 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , COMT and / or OPRM1 forward and reverse primer. In some 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , instances , the second forward and reverse primer can be 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49, 50 , 51 , 52 , 53 , 54 , 55 , 56 , internal ( i. e ., nested ) to the first COMT and /or OPRM1 57 , 58 , 59 , 60 , 61, 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , forward and reverse primer. 73 , 74 , 75 , 76 , 77, 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , (0113 ]. Thus , disclosed herein in some embodiments are 89 , 90 , 91, 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99, 100 , 125, 150 , 175 , methods of detecting a susceptibility to opioid antagonist 200 , 225 , 250 , 275 , 300 , 325 , 350 , 375 , 400 , 425 , 450 , 475 , therapy or treating a psychiatric , mental, and /or neurological 500 , 550 , 600 , 650 , 700 , 750 , 800 , 850 , 900 , 950 , 1000 , disorder (such as, for example, alcohol use disorder ( AUD ) ) , 1250 , 1500 , 1750 , 2000, 2250 , 2500 , 2750 , 3000 , 3500 , or assaying the nucleic acid from a subject for the genotype of 4000 nucleotides long . the dopamine (DA ) - catabolizing enzyme catechol - O -meth [0107 ] In other embodiments a primer or probe can be less yltransferase ( COMT) and the genotype of the opioid mu than or equal to 6 , 7 , 8 , 9 , 10 , 11 , 12 13 , 14 , 15 , 16 , 17 , 18 , receptor gene (OPRM1 ) further comprising assaying DNA 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , and RNA in a tissue sample for the number genotype of 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , alleles coding for the number of VNTR repeats comprising 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65, 66 , wherein the PCR reaction a primer pair capable of specifi 67 , 68, 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , cally hybridizing to one or more DAT1 sequences for 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , example , 5 ' - TGTGGTGTAGGGAACGGCCTGAG - 3 ' 99 , 100 , 125 , 150 , 175 , 200 , 225 , 250, 275 , 300 , 325 , 350 , (SEQ ID NO : 1 ) and 5 -CTTCCTGGAGGTCACGGCT 375 , 400 , 425 , 450 , 475 , 500 , 550 , 600 , 650 , 700 , 750 , 800 , CAAGG - 3 ' ( SEQ ID NO : 2 ) . An alternative forward primer 850 , 900 , 950 , 1000 , 1250 , 1500 , 1750 , 2000 , 2250 , 2500 , is 5 '- TGCGGTGTAGGGAACGGCCTGAG - 3 ' (SEQ ID 2750 , 3000 , 3500 , or 4000 nucleotides long. NO : 3 ) . [0108 ] The primers for the nucleic acid of interest typi [0114 ] III .E . Fluorescent Change Probes and Primers cally will be used to produce extension products and / or other 0115 ] Fluorescent change probes and fluorescent change replicated or amplified products that contain a region of the primers refer to all probes and primers that involve a change US 2018 /0371542 A1 Dec. 27 , 2018 in fluorescence intensity or wavelength based on a change in donormoiety such that , when the acceptor and donor are in the form or conformation of the probe or primer and nucleic proximity (when the probes are hybridized to a target acid to be detected , assayed or replicated . Examples of sequence ), fluorescence resonance energy transfer from the fluorescent change probes and primers include molecular donor to the acceptor causes the acceptor to fluoresce . beacons, Amplifluors, FRET probes , cleavable FRET Fluorescent activated probes are typically pairs of probes probes, TAQMAN® brand probes, scorpion primers , fluo designed to hybridize to adjacent sequences such that the rescent triplex oligos including but not limited to triplex acceptor and donor are brought into proximity . Fluorescent molecular beacons or triplex FRET probes, fluorescent activated probes can also be single probes containing both a water- soluble conjugated polymers , PNA probes and QPNA donor and acceptor where , when the probe is not hybridized probes . to a target sequence , the donor and acceptor are not in [0116 ] Fluorescent change probes and primers can be proximity but where the donor and acceptor are brought into classified according to their structure and /or function . Fluo proximity when the probe hybridized to a target sequence . rescent change probes include hairpin quenched probes , This can be accomplished , for example , by placing the donor cleavage quenched probes , cleavage activated probes , and and acceptor on opposite ends of the probe and placing fluorescent activated probes . Fluorescent change primers target complement sequences at each end of the probe where include stem quenched primers and hairpin quenched prim the target complement sequences are complementary to ers . adjacent sequences in a target sequence. If the donor moiety [0117 ] Hairpin quenched probes are probes that when not of a fluorescent activated probe is itself a fluorescent label, bound to a target sequence form a hairpin structure (and , it can release energy as fluorescence ( typically at a different typically , a loop ) that brings a fluorescent label and a wavelength than the fluorescence of the acceptor ) when not quenching moiety into proximity such that fluorescence in proximity to an acceptor (that is , when the probes are not from the label is quenched . When the probe binds to a target hybridized to the target sequence ). When the probes hybrid sequence , the stem is disrupted , the quenching moiety is no ize to a target sequence , the overall effect would then be a longer in proximity to the fluorescent label and fluorescence reduction of donor fluorescence and an increase in acceptor increases . Examples of hairpin quenched probes are molecu fluorescence . FRET probes are an example of fluorescent lar beacons, fluorescent triplex oligos , triplex molecular activated probes. beacons, triplex FRET probes, and QPNA probes . [0121 ] Stem quenched primers are primers that when not [ 0118 ] Cleavage activated probes are probes where fluo hybridized to a complementary sequence form a stem struc rescence is increased by cleavage of the probe . Cleavage ture ( either an intramolecular stem structure or an intermo activated probes can include a fluorescent label and a lecular stem structure ) that brings a fluorescent label and a quenching moiety in proximity such that fluorescence from quenching moiety into proximity such that fluorescence the label is quenched . When the probe is clipped or digested from the label is quenched . When the primer binds to a ( typically by the 5 '- 3 ' exonuclease activity of a polymerase complementary sequence, the stem is disrupted , the quench during amplification ), the quenching moiety is no longer in ing moiety is no longer in proximity to the fluorescent label proximity to the fluorescent label and fluorescence and fluorescence increases. In the disclosed method , stem increases . TAQMAN® brand probes are an example of quenched primers are used as primers for nucleic acid cleavage activated probes . synthesis and thus become incorporated into the synthesized [0119 ] Cleavage quenched probes are probes where fluo or amplified nucleic acid . Examples of stem quenched rescence is decreased or altered by cleavage of the probe . primers are peptide nucleic acid quenched primers and Cleavage quenched probes can include an acceptor fluores hairpin quenched primers . cent label and a donor moiety such that , when the acceptor and donor are in proximity , fluorescence resonance energy [0122 ] Peptide nucleic acid quenched primers are primers transfer from the donor to the acceptor causes the acceptor associated with a peptide nucleic acid quencher or a peptide to fluoresce . The probes are thus fluorescent, for example , nucleic acid fluor to form a stem structure. The primer when hybridized to a target sequence . When the probe is contains a fluorescent label or a quenching moiety and is clipped or digested ( typically by the 5 '- 3' exonuclease activ associated with either a peptide nucleic acid quencher or a ity of a polymerase during amplification ), the donor moiety peptide nucleic acid fluor, respectively . This puts the fluo is no longer in proximity to the acceptor fluorescent label rescent label in proximity to the quenching moiety . When and fluorescence from the acceptor decreases . If the donor the primer is replicated , the peptide nucleic acid is displaced , moiety is itself a fluorescent label, it can release energy as thus allowing the fluorescent label to produce a fluorescent fluorescence ( typically at a different wavelength than the signal. fluorescence of the acceptor ) when not in proximity to an [0123 ] Hairpin quenched primers are primers that when acceptor. The overall effect would then be a reduction of not hybridized to a complementary sequence form a hairpin acceptor fluorescence and an increase in donor fluorescence . structure ( and , typically , a loop ) that brings a fluorescent Donor fluorescence in the case of cleavage quenched probes label and a quenching moiety into proximity such that is equivalent to fluorescence generated by cleavage activated fluorescence from the label is quenched . When the primer probes with the acceptor being the quenching moiety and the binds to a complementary sequence , the stem is disrupted , donor being the fluorescent label. Cleavable FRET ( fluores the quenching moiety is no longer in proximity to the cence resonance energy transfer ) probes are an example of fluorescent label and fluorescence increases. Hairpin cleavage quenched probes. quenched primers are typically used as primers for nucleic [0120 ] Fluorescent activated probes are probes or pairs of acid synthesis and thus become incorporated into the syn probes where fluorescence is increased or altered by hybrid thesized or amplified nucleic acid . Examples of hairpin ization of the probe to a target sequence . Fluorescent acti quenched primers are Amplifluor primers and scorpion vated probes can include an acceptor fluorescent label and a primers . US 2018 /0371542 A1 Dec . 27 , 2018

[0124 ] Cleavage activated primers are similar to cleavage erythrin B , Polyazaindacene Pontochrome Blue Black , Por activated probes except that they are primers that are incor phyrin , Primuline , Procion Yellow , Pyronine , Pyronine B , porated into replicated strands and are then subsequently Pyrozal Brilliant Flavin 7GF, Quinacrine Mustard , Rhod cleaved . amine 123 , Rhodamine 5 GLD , Rhodamine 6G , Rhodamine [0125 ] III . F . Labels B , Rhodamine B 200 , Rhodamine B Extra , Rhodamine BB , [0126 ] To aid in detection and quantitation of nucleic acids Rhodamine BG , Rhodamine WT, Serotonin , Sevron Bril produced using the disclosed methods, labels can be directly liant Red 2B , Sevron Brilliant Red 4G , Sevron Brilliant Red incorporated into nucleotides and nucleic acids or can be B , Sevron Orange , Sevron Yellow L , SITS (Primuline ) , coupled to detection molecules such as probes and primers . SITS (Stilbene Isothiosulphonic acid ) , Stilbene, Snarf 1 , As used herein , a label is any molecule that can be associated sulpho Rhodamine B Can C , Sulpho Rhodamine G Extra , with a nucleotide or nucleic acid , directly or indirectly , and Tetracycline, Thiazine Red R , Thioflavin S , Thioflavin TCN , which results in a measurable , detectable signal, either Thioflavin 5 , Thiolyte , Thiozol Orange , Tinopol CBS , True directly or indirectly . Many such labels for incorporation Blue , Ultralite , Uranine B , Uvitex SFC , Xylene Orange , and into nucleotides and nucleic acids or coupling to nucleic acid XRITC . probes are known to those of skill in the art . Examples of [0128 ] The absorption and emission maxima, respectively , labels suitable for use in the disclosed method are radioac for some of these fluors are : FITC (490 nm ; 520 nm ) , Cy3 tive isotopes, fluorescent molecules, phosphorescent mol (554 nm ; 568 nm ) , Cy3 . 5 (581 nm ; 588 nm ) , Cy5 (652 nm : ecules, enzymes, antibodies, and ligands. Fluorescent labels , 672 nm ) , Cy5 . 5 (682 nm ; 703 nm ) and Cy7 (755 nm ; 778 especially in the context of fluorescent change probes and nm ), thus allowing their simultaneous detection . Other primers , are useful for real - time detection of amplification . examples of fluorescein dyes include 6 - carboxyfluorescein [0127 ] Examples of suitable fluorescent labels include ( 6 - FAM ) , 2 , 4 ', 1 , 4 , - tetrachlorofluorescein ( TET) , 2 ' , 4 ', 5 ', 7 ' , fluorescein isothiocyanate (FITC ) , 5 , 6 -carboxymethyl fluo 1 , 4 -hexachlorofluorescein (HEX ), 2 ', 7 ' - dimethoxy - 4 ', 5 - di rescein , Texas red , nitrobenz- 2 -oxa - 1 , 3 -diazol - 4 - yl (NBD ) , chloro - 6 - carboxyrhodamine (JOE ) , 2 - chloro - 5 - fluoro - 7 ', 8 ' coumarin , dansyl chloride , rhodamine , amino -methyl cou fused phenyl - 1 , 4 - dichloro - 6 -carboxyfluorescein (NED ), and marin (AMCA ) , Eosin , Erythrosin , BODIPY® , CASCADE 2 ' - chloro - 7 '- phenyl- 1 , 4 -dichloro - 6 - carboxyfluorescein BLUE® , OREGON GREEN® , pyrene , lissamine , xan (VIC ) . Fluorescent labels can be obtained from a variety of thenes , acridines , oxazines , phycoerythrin , macrocyclic che commercial sources, including Amersham Pharmacia Bio lates of lanthanide ions such as quantum dyeTM , fluorescent tech , Piscataway , N . J . , United States of America ; Molecular energy transfer dyes, such as thiazole orange - ethidium het Probes , Eugene , Oreg . , and Research Organics, Cleveland , erodimer , and the cyanine dyes Cy3 , Cy3 . 5 , Cy5 , Cy5 . 5 and Ohio ., United States of America . Cy7 . Examples of other specific fluorescent labels include [0129 ] Additional labels of interest include those that 3 -Hydroxypyrene 5 ,8 , 10 - Tri Sulfonic acid , 5 -Hydroxy provide for signal only when the probe with which they are Tryptamine ( 5 -HT ) , Acid Fuchsin , Alizarin Complexon , associated is specifically bound to a target molecule, where Alizarin Red , Allophycocyanin , Aminocoumarin , Anthroyl such labels include : “ molecular beacons " as described in Stearate , Astrazon Brilliant Red 4G , Astrazon Orange R , Tyagi & Kramer, 1996 and European Patent Publication EP Astrazon Red 6B , Astrazon Yellow 7 GLL , Atabrine, 0 070 685 B1. Other labels of interest include those Auramine, Aurophosphine , Aurophosphine G , BAO 9 described in U . S . Pat. No . 5 , 563 , 037 which is incorporated (Bisaminophenyloxadiazole ), BCECF , Berberine Sulphate , herein by reference . Bisbenzamide , Blancophor FFG Solution , Blancophor SV , [0130 ] Labeled nucleotides are a form of label that can be Bodipy F1, Brilliant Sulphoflavin FF , Calcien Blue, Calcium directly incorporated into the amplification products during Green , Calcofluor RW Solution , Calcofluor White, Calco synthesis . Examples of labels that can be incorporated into phor White ABT Solution , Calcophor White Standard Solu amplified nucleic acids include nucleotide analogs such as tion , Carbostyryl , Cascade Yellow , Catecholamine, China BrdUrd , aminoallyldeoxyuridine , 5 -methylcytosine , bro crine , Coriphosphine 0 , Coumarin -Phalloidin , CY3. 1 8 , mouridine , and nucleotides modified with biotin or with CY5 .1 8 , CY7 , Dans ( 1 -Dimethyl Amino Naphaline 5 suitable haptens such as digoxygenin . Suitable fluorescence Sulphonic Acid ), Dansa (Diamino Naphtyl Sulphonic Acid ) , labeled nucleotides are Fluorescein - isothiocyanate - dUTP , Dansyl NH - CH3, Diamino Phenyl Oxydiazole (DAO ) , Cyanine - 3 - dUTP and Cyanine - 5 - dUTP. One example of a Dimethylamino - 5 - Sulphonic acid , Dipyrrometheneboron nucleotide analog label for DNA is BrdUrd (bromodeoxyu Difluoride , Diphenyl Brilliant Flavine 7GFF , Dopamine , ridine, BrdUrd , BrdU , BUdR , Sigma - Aldrich Co ) . Other Erythrosin ITC , Euchrysin , FIF (Formaldehyde Induced examples of nucleotide analogs for incorporation of label Fluorescence ) , Flazo Orange , Fluo 3 , Fluorescamine, Fura into DNA are AA - DUTP (aminoallyl - deoxyuridine triphos 2 , Genacryl Brilliant Red B , Genacryl Brilliant Yellow phate , Sigma - Aldrich Co . ) , and 5 -methyl - dCTP (Roche 10GF, Genacryl Pink 3G , Genacryl Yellow 5GF, Gloxalic Molecular Biochemicals ) . One example of a nucleotide Acid , Granular Blue , Haematoporphyrin , Indo - 1, Intrawhite analog for incorporation of label into RNA is biotin - 16 -UTP Cf Liquid , Leucophor PAF , Leucophor SF , Leucophor WS , (biotin - 16 - uridine - 5 '- triphosphate , Roche Molecular Bio Lissamine Rhodamine B200 (RD200 ) , Lucifer Yellow CH , chemicals ) . Fluorescein , Cy3 , and Cy5 can be linked to Lucifer Yellow VS . Magdala Red , Marina Blue , Maxilon DUTP for direct labeling . Cy3 . 5 and Cy7 are available as Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF , avidin or anti -digoxygenin conjugates for secondary detec MPS (Methyl Green Pyronine Stilbene ) , Mithramycin , NBD tion of biotin - or digoxygenin - labeled probes. Amine, Nitrobenzoxadidole , Noradrenaline, Nuclear Fast [0131 ] Labels that are incorporated into amplified nucleic Red , Nuclear Yellow , Nylosan Brilliant Flavin E8G , Oxadi acid , such as biotin , can be subsequently detected using azole , Pacific Blue , Pararosaniline ( Feulgen ) , Phorwite AR sensitive methods well -known in the art. For example , biotin Solution , Phorwite BKL , Phorwite Rev , Phorwite RPA , can be detected using streptavidin - alkaline phosphatase con Phosphine 3R , Phthalocyanine, Phycoerythrin R , Phyco jugate ( Tropix , Inc. ), which is bound to the biotin and US 2018 /0371542 A1 Dec. 27 , 2018 14 subsequently detected by chemiluminescence of suitable method made it susceptible to sequence - specific bias or loss substrates ( for example , chemiluminescent substrate CSPD : of specific sequences. Polony sequencing, combined an in disodium , 3 - ( 4 -methoxyspiro - [ 1 , 2 , - dioxetane - 3 - 2 - ( 5 vitro paired - tag library with emulsion PCR , an automated chloro tricyclo [ 3 .3 .1 . 13 .7 ] decane ) -4 - yl ) phenyl phosphate ; microscope, and ligation -based sequencing chemistry to Tropix , Inc. ) . Labels can also be enzymes, such as alkaline sequence an E . coli genome at an accuracy of > 99 . 9999 % phosphatase , soybean peroxidase , horseradish peroxidase and a cost approximately 1/ 10 that of Sanger sequencing . and polymerases , that can be detected , for example , with [0135 ] A parallelized version of pyrosequencing , the chemical signal amplification or by using a substrate to the method amplifies DNA inside water droplets in an oil enzyme which produces light (for example, a chemilumi solution ( emulsion PCR ), with each droplet containing a nescent 1 , 2 -dioxetane substrate ) or fluorescent signal. single DNA template attached to a single primer - coated bead [0132 ] Molecules that combine two ormore of these labels that then forms a clonal colony . The sequencing machine are also considered labels . Any of the known labels can be contains many picolitre - volume wells each containing a used with the disclosed probes , tags , and method to label and single bead and sequencing enzymes . Pyrosequencing uses detect nucleic acid amplified using the disclosed method . luciferase to generate light for detection of the individual Methods for detecting and measuring signals generated by nucleotides added to the nascent DNA , and the combined labels are also known to those of skill in the art . For data are used to generate sequence read - outs. This technol example, radioactive isotopes can be detected by scintilla ogy provides intermediate read length and price per base tion counting or direct visualization ; fluorescent molecules compared to Sanger sequencing on one end and Solexa and can be detected with fluorescent spectrophotometers ; phos SOLID on the other. phorescent molecules can be detected with a spectropho [0136 ] A sequencing technology based on reversible dye tometer or directly visualized with a camera ; enzymes can terminators . DNA molecules are first attached to primers on be detected by detection or visualization of the product of a a slide and amplified so that local clonal colonies are reaction catalyzed by the enzyme; antibodies can be detected formed . Four types of reversible terminator bases (RT - bases ) by detecting a secondary label coupled to the antibody . As are added , and non - incorporated nucleotides are washed used herein , detection molecules are molecules which inter away . Unlike pyrosequencing , the DNA can only be act with amplified nucleic acid and to which one or more extended one nucleotide at a time. A camera takes images of labels are coupled . the fluorescently labeled nucleotides , then the dye along [0133 ] The disclosed methods can use any sequencing with the terminal 3' blocker is chemically removed from the technique known in the art including, but not limited to DNA , allowing the next cycle . methods disclosed by Sanger ( dideoxy method ) , Maxam Gilbert ( chemical cleavage ) , automated sequencing ( for [0137 ] SOLID technology employs sequencing by liga example ABI systems) and next generation sequencing . tion . Here , a pool of all possible oligonucleotides of a fixed From a technical perspective High - throughput or Next Gen length are labeled according to the sequenced position . eration Sequencing (NGS ) represents an attractive option for Oligonucleotides are annealed and ligated ; the preferential detecting the somatic mutations within a gene . Unlike PCR , ligation by DNA ligase for matching sequences results in a microarrays , high -resolution melting and mass spectrom signal informative of the nucleotide at that position . Before etry, which all indirectly infer sequence content, NGS sequencing , the DNA is amplified by emulsion PCR . The directly ascertains the identity of each base and the order in resulting bead , each containing only copies of the same which they fall within a gene. The newest platforms on the DNA molecule , are deposited on a glass slide . The result is market have the capacity to cover an exonic region 10 ,000 sequences of quantities and lengths comparable to Illumina times over ,meaning the content of each base position in the sequencing . sequence is measured thousands of different times . This high [0138 ] Ion semiconductor sequencing is based on using level of coverage ensures that the consensus sequence is standard sequencing chemistry , but with a novel , semicon extremely accurate and enables the detection of rare variants ductor based detection system . This method of sequencing is within a heterogeneous sample . For example , in a sample based on the detection of hydrogen ions that are released extracted from FFPE tissue, relevant mutations are only during the polymerization of DNA , as opposed to the optical present at a frequency of 1 % with the wild -type allele methods used in other sequencing systems. A micro well comprising the remainder . When this sample is sequenced at containing a template DNA strand to be sequenced is 10 ,000x coverage , then even the rare allele , comprising only flooded with a single type of nucleotide . If the introduced 1 % of the sample , is uniquely measured 100 times over. nucleotide is complementary to the leading template nucleo Thus , NGS can provide reliably accurate results with very tide it is incorporated into the growing complementary high sensitivity , making it ideal for clinical diagnostic test strand . This causes the release of a hydrogen ion that triggers ing of FFPEs and other mixed samples . a hypersensitive ion sensor, which indicates that a reaction [0134 ] Examples of Next Generation Sequencing tech has occurred . If homopolymer repeats are present in the niques include, but are not limited to Massively Parallel template sequence multiple nucleotides will be incorporated Signature Sequencing (MPSS ) , Polony sequencing , pyrose in a single cycle . This leads to a corresponding number of quencing, Reversible dye - terminator sequencing , SOLID released hydrogens and a proportionally higher electronic sequencing, Ion semiconductor sequencing , DNA nanoball signal. sequencing, Helioscope single molecule sequencing , Single [ 0139 ] DNA nanoball sequencing is a type of high molecule real time (SMRT ) sequencing , Single molecule throughput sequencing technology used to determine the real time (RNAP ) sequencing, and Nanopore DNA sequenc entire genomic sequence of an organism . The method uses ing . MPSS was a bead -based method that used a complex rolling circle replication to amplify small fragments of approach of adapter ligation followed by adapter decoding , genomic DNA into DNA nanoballs . Unchained sequencing reading the sequence in increments of four nucleotides ; this by ligation is then used to determine the nucleotide US 2018 /0371542 A1 Dec . 27 , 2018 15 sequence. This method of DNA sequencing allows large regions and require excessive amounts of time to run indi numbers of DNA nanoballs to be sequenced per run . vidual nucleotides across growing DNA strands . The SBS 10140 ] Helicos ' s single -molecule sequencing uses DNA NGS platform uses a direct sequencing approach to produce fragments with added polyA tail adapters , which are a sequencing strategy with very a high precision , rapid pace attached to the flow cell surface . The next steps involve and low cost . extension - based sequencing with cyclic washes of the flow [0150 ] SBS sequencing is initialized by fragmenting of the cell with fluorescently labeled nucleotides (one nucleotide template DNA into fragments , amplification , annealing of type at a time, as with the Sanger method ) . The reads are DNA sequencing primers , and finally affixing as a high performed by the Helioscope sequencer . density array of spots onto a glass chip . The array of DNA [0141 ] SMRT sequencing is based on the sequencing by fragments are sequenced by extending each fragment with synthesis approach . The DNA is synthesized in zero -mode modified nucleotides containing cleavable chemical moi wave - guides (ZMWs ) small well - like containers with the eties linked to fluorescent dyes capable of discriminating all capturing tools located at the bottom of the well. The four possible nucleotides . The array is scanned continuously sequencing is performed with use of unmodified polymerase by a high - resolution electronic camera (Measure ) to deter ( attached to the ZMW bottom ) and fluorescently labeled mine the fluorescent intensity of each base ( A , C , G or T ) nucleotides flowing freely in the solution . that was newly incorporated into the extended DNA frag [ 0142 ] The wells are constructed in a way that only the ment. After the incorporation of each modified base the array fluorescence occurring by the bottom of the well is detected . is exposed to cleavage chemistry to break off the fluorescent The fluorescent label is detached from the nucleotide at its dye and end cap allowing additional bases to be added . The incorporation into the DNA strand , leaving an unmodified process is then repeated until the fragment is completely DNA strand . sequenced or maximal read length has been achieved . [0143 ] Single molecule real time sequencing based on RNA polymerase (RNAP ) , which is attached to a polysty IV . Nucleic Acids rene bead , with distal end of sequenced DNA is attached to [0151 ] There are a variety of molecules disclosed herein another bead , with both beads being placed in optical traps . that are nucleic acid based , including for example the RNAP motion during transcription brings the beads in closer nucleic acids that encode, for example primers that hybridize and their relative distance changes , which can then be to COMT and /or OPRM1. The disclosed nucleic acids are recorded at a single nucleotide resolution . The sequence is made up of for example , nucleotides, nucleotide analogs , or deduced based on the four readouts with lowered concen nucleotide substitutes . Non - limiting examples of these and trations of each of the four nucleotide types (similarly to other molecules are discussed herein . It is understood that Sangers method ) . for example , when a vector is expressed in a cell , that the [ 0144 ] Nanopore sequencing is based on the readout of expressed mRNA will typically be made up of A , C , G , and electrical signal occurring at nucleotides passing by alpha U . Likewise , it is understood that if , for example , an hemolysin pores covalently bound with cyclodextrin . The antisense molecule is introduced into a cell or cell environ DNA passing through the nanopore changes its ion current. ment through for example exogenous delivery , it is advan This change is dependent on the shape, size and length of the tageous that the antisense molecule be made up of nucleo DNA sequence . Each type of the nucleotide blocks the ion tide analogs that reduce the degradation of the antisense flow through the pore for a different period of time. molecule in the cellular environment. [0145 ] VisiGen Biotechnologies uses a specially engi [0152 ] III . A . Nucleotides and Related Molecules neered DNA polymerase. This polymerase acts as a sensor [0153 ] A nucleotide is a molecule that contains a base having incorporated a donor fluorescent dye by its active moiety , a sugarmoiety and a phosphate moiety . Nucleotides center. This donor dye acts by FRET ( fluorescent resonant can be linked together through their phosphate moieties and energy transfer ) , inducing fluorescence of differently labeled sugar moieties creating an internucleoside linkage . The base nucleotides . This approach allows reads performed at the moiety of a nucleotide can be adenin - 9 - yl ( A ) , cytosin - 1 - yl speed at which polymerase incorporates nucleotides into the ( C ) , guanin - 9 - yl ( G ) , uracil - 1 - yl ( U ) , and thymin - 1 - yl ( T ) . sequence ( several hundred per second ). The nucleotide The sugar moiety of a nucleotide is a ribose or a deoxyri fluorochrome is released after the incorporation into the bose . The phosphate moiety of a nucleotide is pentavalent DNA strand . phosphate . A non - limiting example of a nucleotide would be [0146 ] Sequencing by hybridization is a non -enzymatic 3 '- AMP (3 '- adenosine monophosphate ) or 5 -GMP (5 ' method that uses a DNA microarray . A single pool of DNA guanosine monophosphate ) . There are many varieties of whose sequence is to be determined is fluorescently labeled these types of molecules available in the art and available and hybridized to an array containing known sequences. herein . Strong hybridization signals from a given spot on the array [0154 ] A nucleotide analog is a nucleotide which contains identify its sequence in the DNA being sequenced . some type of modification to either the base , sugar, or [0147 ] Mass spectrometry may be used to determine mass phosphate moieties . Modifications to nucleotides are well differences between DNA fragments produced in chain known in the art and would include for example , 5 -meth termination reactions. ylcytosine ( 5 -me - C ) , 5 -hydroxymethyl cytosine , xanthine, [0148 ] Another NGS approach is sequencing by synthesis hypoxanthine, and 2 - aminoadenine as well as modifications (SBS ) technology which is capable of overcoming the at the sugar or phosphate moieties . There are many varieties limitations of existing pyrosequencing based NGS plat - of these types of molecules available in the art and available forms. herein . [0149 ] Such technologies rely on complex enzymatic cas [0155 ] Nucleotide substitutes are molecules having simi cades for read out, are unreliable for the accurate determi- lar functional properties to nucleotides, but which do not nation of the number of nucleotides in homopolymeric contain a phosphate moiety , such as peptide nucleic acid US 2018 /0371542 A1 Dec. 27 , 2018

(PNA ) . Nucleotide substitutes are molecules that will rec tion of the probe or primer . A typical primer or probe would ognize nucleic acids in a Watson - Crick or Hoogsteen man be at least 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , ner, but which are linked together through a moiety other 20 , 21, 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , than a phosphate moiety. Nucleotide substitutes are able to 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49, 50 , 51 , conform to a double helix type structure when interacting 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , with the appropriate target nucleic acid . There are many 68, 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79, 80 , 81, 82 , 83 , varieties of these types of molecules available in the art and 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , available herein . 100 , 125 , 150 , 175 , 200 , 225 , 250 , 275 , 300 , 325 , 350 , 375 . [0156 ] It is also possible to link other types ofmolecules 400 , 425 , 450 , 475 , 500 , 550 , 600 , 650 , 700 , 750 , 800 , 850 , ( conjugates ) to nucleotides or nucleotide analogs to enhance 900 , 950 , 1000 , 1250 , 1500 , 1750 , 2000 , 2250 , 2500 , 2750 , for example , cellular uptake . Conjugates can be chemically 3000 , 3500 , or 4000 nucleotides long . linked to the nucleotide or nucleotide analogs. Such conju 10162 ] In other embodiments a primer or probe can be less gates include but are not limited to lipid moieties such as a than or equal to 6 , 7 , 8 , 9 , 10 , 11 , 12 13 , 14 , 15 , 16 , 17 , 18 , cholesterol moiety . ( Letsinger et al. , 1989 ) . There are many 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31, 32 , 33 , 34 , varieties of these types ofmolecules available in the art and 35 , 36 , 37 , 38 , 39 , 40 , 41, 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , available herein . 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59, 60 , 61, 62 , 63 , 64 , 65 , 66 , 10157 ] A Watson -Crick interaction is at least one interac 67 , 68 , 69, 70 , 71, 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , tion with the Watson - Crick face of a nucleotide, nucleotide 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , analog , or nucleotide substitute . The Watson -Crick face of a 99 , 100 , 125 , 150 , 175 , 200 , 225 , 250 , 275, 300 , 325 , 350 , nucleotide , nucleotide analog , or nucleotide substitute 375 , 400 , 425 , 450 , 475 , 500 , 550 , 600 , 650 , 700 , 750 , 800 , includes the C2 , N1, and C positions of a purine based 850 , 900 , 950 , 1000 , 1250 , 1500 , 1750 , 2000 , 2250 , 2500 , nucleotide , nucleotide analog , or nucleotide substitute and 2750 , 3000 , 3500 , or 4000 nucleotides long . the C2 , N3, C4 positions of a pyrimidine based nucleotide , 0163 ] The primers for the COMT, OPRM1 and / or DAT1 nucleotide analog, or nucleotide substitute . gene typically will be used to produce an amplified DNA [0158 ] A Hoogsteen interaction is the interaction that takes product that contains a region of the COMT, OPRM1 and / or place on the Hoogsteen face of a nucleotide or nucleotide DAT1 gene or the complete gene . In general, typically the analog , which is exposed in the major groove of duplex size of the product will be such that the size can be DNA . The Hoogsteen face includes the N7 position and accurately determined to within 3 , or 2 or 1 nucleotides . reactive groups (NH , or O ) at the C6 position of purine 0164 ] In certain embodiments this product is at least 20 , nucleotides . 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31, 32 , 33 , 34 , 35 , 36 , [0159 ] IV .B . Primers and Probes 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51, 52 , [0160 ] Disclosed are compositions including primers and 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61, 62 , 63 , 64 , 65 , 66 , 67 , 68 , probes , which are capable of interacting with the disclosed 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81, 82 , 83 , 84 , nucleic acids, such as the COMT primers , OPRM1 primers, 85 , 86 , 87 , 88 , 89 , 90 , 91, 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , and /or DAT1 primers (SEQ ID NO : 1 or SEQ ID NO : 3 ; and 125 , 150 , 175 , 200 , 225 , 250 , 275 , 300 , 325 , 350 , 375 , 400 , SEQ ID NO : 2 ) as disclosed herein . In certain embodiments 425 , 450 , 475 , 500 , 550 , 600 , 650 , 700 , 750 , 800 , 850 , 900 , the primers are used to support DNA amplification reactions . 950 , 1000 , 1250 , 1500 , 1750 , 2000 , 2250 , 2500 , 2750 , 3000 , Typically , the primers will be capable of being extended in 3500 , or 4000 nucleotides long . a sequence specific manner. Extension of a primer in a 10165 ]. In other embodiments the product is less than or sequence specific manner includes any methods wherein the equal to 20 , 21, 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32, 33 , sequence and /or composition of the nucleic acid molecule to 34 , 35 , 36 , 37 , 38, 39 , 40 , 41, 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , which the primer is hybridized or otherwise associated 50 , 51, 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , directs or influences the composition or sequence of the 66 , 67 , 68 , 69, 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81, product produced by the extension of the primer . Extension 82, 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91, 92 , 93 , 94 , 95 , 96 , 97 , of the primer in a sequence specific manner therefore 98, 99 , 100 , 125 , 150 , 175 , 200 , 225 , 250 , 275 , 300 , 325 , includes , but is not limited to , PCR , DNA sequencing , DNA 350 , 375 , 400 , 425 , 450 , 475 , 500 , 550 , 600 , 650 , 700 , 750 , extension , DNA polymerization , RNA transcription , or 800 , 850 , 900 , 950 , 1000 , 1250 , 1500 , 1750 , 2000 , 2250 , reverse transcription . Techniques and conditions that 2500 , 2750 , 3000 , 3500 , or 4000 nucleotides long . amplify the primer in a sequence specific manner are pre ferred . In certain embodiments the primers are used for the V . Kits DNA amplification reactions , such as PCR or direct [0166 ] Disclosed herein are kits that are drawn to reagents sequencing . It is understood that in certain embodiments the that can be used in practicing the methods disclosed herein . primers can also be extended using non -enzymatic tech The kits can include any reagent or combination of reagent niques , where for example, the nucleotides or oligonucle discussed herein or that would be understood to be required otides used to extend the primer are modified such that they or beneficial in the practice of the disclosed methods . For will chemically react to extend the primer in a sequence example , the kits could include primers to perform the specific manner. Typically , the disclosed primers hybridize amplification reactions discussed in certain embodiments of with the disclosed nucleic acids or region of the nucleic the methods , as well as the buffers and enzymes required to acids or they hybridize with the complement of the nucleic use the primers as intended . For example , disclosed is a kit acids or complement of a region of the nucleic acids. for treating a psychiatric , mental, and / or neurological dis [0161 ] The size of the primers or probes for interaction order ( such as, for example , alcohol use disorder (AUD ) ) or with the nucleic acids in certain embodiments can be any detecting susceptibility to opioid receptor antagonist treat size that supports the desired enzymatic manipulation of the ment disclosed herein , comprising the primers that specifi primer, such as DNA amplification or the simple hybridiza cally hybridize to COMT (more preferably are able to US 2018 /0371542 A1 Dec. 27 , 2018 17 specifically hybridize to COMT and able to amplify the types (DAT1 9 - repeat or COMTMet carriers ) . These results nucleic acids encoding amino acid 158 ) and OPRM1 (more could lead to more personized treatment approaches. preferably are able to specifically hybridize to OPRM1 and 0172 ) The following Examples provide results from an able to amplify the nucleic acids encoding amino acid 40 ) . expanded analysis of a single site prospective study in which In some embodiments , the disclosed kits for treating a individuals with DSM -IV alcohol dependence were pro psychiatric , mental, and / or neurological disorder ( such as , spectively genotyped for their OPRM1 Al 18G status ( G - al for example , alcohol use disorder ( AUD ) ) can also comprise lele carriers vs . A - allele homozygotes ) and then randomized an opioid receptor antagonist ( such as, for example naltrex to receive naltrexone or placebo (Schacht et al. , 2017 ) . In a one ) . pre - planned analysis , these same individuals were also genotyped for the DAT1 /SLC6A3 VNTR and COMT EXAMPLES Val158Met SNP to evaluate the epistatic effects of these genotypes with OPRM1 A118G genotype on naltrexone [0167 ] The following EXAMPLES are put forth so as to efficacy . provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compo Materials and Methods for the Examples sitions , articles, devices and / or methods claimed herein are made and evaluated , and are intended to be purely exem [0173 ] Trial Design and Participants . The study was a plary and are not intended to limit the disclosure. Efforts 16 -week randomized clinical trial ( clinicaltrials . gov have been made to ensure accuracy with respect to numbers # NCT00920829 ) of naltrexone vs . placebo with initial strati ( e. g ., amounts , temperature , etc. ), but some errors and fication by OPRM1 A118G genotype (A -allele homozygotes deviations should be accounted for . Unless indicated other versus G - allele carriers ) . Participants were between 18 - 70 wise , parts are parts by weight , temperature is in ° C . or is years of age , met DSM - IV criteria for alcohol dependence at ambient temperature , and pressure is at or near atmo by SCID interview ( First et al. , Structured Clinical Interview spheric for DSM - IV Axis I Disorders Patient Edition . SCID - 1 / P , Version 2 .0 ed . New York , NY : New York State Psychiatric Institute ; 1997 ) , could not be of African American descent Introduction to the Examples by self -report ( due to low frequency of G alleles ) , con sumed , on average , at least 5 drinks per day prior to Opioid and Dopamine Genes Interact to Predict screening , and were required to be abstinent at least 4 days Naltrexone Response in an Alcohol Use Disorder prior to randomization . Participants did not meet DSM - IV Clinical Trial criteria for dependence for any other drug but nicotine . Cocaine or marijuana use prior to screening was allowable , [ 0168 ] While the opiate antagonist , naltrexone , is but no psychoactive drug could be evident in the urine at approved for Alcohol Use Disorder (AUD ) , not everyone screening . Participants were not taking psychotropic medi benefits . These Examples evaluated whether the OPRM1 cations other than antidepressants ( stable dose for onemonth SNP rs1799971 interacts with the dopamine transport gene required ) and did notmeet criteria for current major depres DAT1 /SLC6A3 VNTR rs28363170 or the catechol- o -meth sion , bipolar disorder, psychoses, PTSD , or eating disorders . yltransferase ( COMT) gene SNP rs4680 in predicting nal They had to be medically stable (not having liver enzymes , trexone response . ALT and AST, more than 3 times normal ). Females were [0169 ] DSM - IV alcohol dependent individuals were ran required to use a reliable form of contraception or be domly assigned to naltrexone ( 50 mg/ day ) or placebo based post -menopausal , and were not pregnant or nursing. on their OPRM1 A118G genotype (75 G allele carriers and [0174 ] Recruitment. The study was approved by the Insti 77 A allele homozygotes ) who were also genotyped for tutional Review Board (IRB ) at the Medical University of DAT1 VNTR ( 9 vs 10 repeats ) or COMT 158 SNP (Val / Val South Carolina (Charleston , S . C . , United States of America ) ( A , A alleles ) vs. Met (at least one G allele ) carriers ). Heavy and participants signed informed consent before formal drinking days ( % HDD ) were evaluated over 16 weeks and assessment and genotyping. Participants , recruited primarily at the end of treatment. Effect sizes ( d ) for naltrexone by community advertisement, weren ' t currently receiving response were calculated based on genotypes . other alcohol treatment. After genotyping (blind to subjects [ 0170 ] Naltrexone , relative to placebo , significantly and study staff ) , individuals with at least one G allele and reduced % HDD among OPRM1 G carriers who also had approximately 1 /3 of A - allele homozygotes ( temporally DAT1 10 / 10 ( p = 0 . 021 ; d = 0 .72 ) or COMT Val/ Val genotypes proximal to G allele carriers ) were offered participation . ( p = 0 .05 ; d = 0 .80 ) , and to a similar degree in those OPRM1 Subsequently , pre - planned genotyping for the DAT1 VNTR A homozygotes who were also DAT1 9 -repeat carriers and COMT Val158Met SNP was conducted to form the (p = 0 .09 ; d = 0 .70 ) or COMT met carriers (p = 0 .03 ; d = 0 .63 ) . combined gene groups for this evaluation ( see FIG . 1 , Other genotype combinations did not respond differentially CONSORT Diagram ) . to naltrexone treatment. Smoking status did not materially [0175 ] Medication . After at least 4 days of abstinence , affect the pharmacogenetic findings. participants were randomized to receive naltrexone ( 25 mg [0171 ] While not wishing to be bound by any particular for 2 days , then 50 mg thereafter ) or identical placebo theory of operation , AUD individuals with more opioid ( distributed in labeled blister packs) for 16 weeks. responsive genotypes (OPRM1 G carriers ) might respond 0176 ] Assessment and Medical Management (MM ) . better to naltrexone because they have normal/ less dopamine Medical management (MM ), a manualized intervention tone promoting genotypes (DAT1 10 / 10 or COMT Val/ Val ) (Pettinati et al. , 2005 ) designed and used previously ( Anton while those with less responsive opioid responsive geno et al ., 2006b ), and also utilized in previous naltrexone types (OPRM1 A homozygotes ) might respond better to pharmacogenetic studies (Oslin et al . , 2003 ; Anton et al. , naltrexone because they have dopamine enhancing pheno 2008 ) was administered . During each MM session ( con US 2018 /0371542 A1 Dec. 27 , 2018 ducted on weeks 1 , 2 , 3 , 4 , 6 , 8 , 10 , 12 , 16 ) medication heavy drinking days in the COMBINE Study ( Anton et al. , adverse effects using the SAFTEE ( Johnson et al. , 2005 ) 2008 ) . The sample size for this exploratory epistatic evalu were collected , compliance reviewed and motivated . Par ation was limited by that collected for the original OPRM1 ticipants were encouraged , but not required , to attend Alco gene evaluation ( Schacht et al. , 2017 ) . holics Anonymous meetings . [0182 ] Randomization . Participating subjects were [ 01771 Assessment. Multiple assessments were adminis entered into a pre -constructed randomization program based tered prior to randomization , including : SCID - IV , Alcohol on OPRM1 genotype ( strata ) with the following URN Dependence Scale ( ADS ; Skinner & Allen , 1982 ) , Obses randomization variables : sex , smoking status , alcohol use sive Compulsive Drinking Scale (OCDS ; Anton et al. , disorder family history , antidepressant, and recent cocaine 1996 ) , Form - 90 (modified time- line followback method for use . The program assigned them to a study medication group documenting alcohol consumption ; Tonigan et al . , 1997 ) , based on a priori determination . All subjects , assessment and Drinker Inventory of Consequences (DrInC ) , the Clinical treatment staff , were blind to both genotype and medication Institute Withdrawal Assessment for Alcohol- Revised assignment. Since DAT1 and COMT genotyping were done ( Forcehimes et al. , 2007 ) , and baseline physical complaints . later they did not serve as randomization variables . Lab tests included a health screen , liver function tests , pregnancy test ( females ) and alcohol use markers gamma 10183 ] Statistical Analysis . Chi- Square or ANOVA were glutamyltransferase (GGT ) and carbohydrate - deficient used to evaluate differences in baseline demographic and alcohol use data between the 2 combinations of 8 treatment transferrin (Litten et al ., 2010 ; Helander et al. , 2010 ; Anton groups : medication (naltrexone vs . placebo )xOPRM1 geno et al. , 2006a ) . type ( A - allele homozygotes vs. G - allele carriers ) xeither [ 0178 ] During each study visit , the calendar based time DAT1 genotype [ 10R homozygotes vs . 9R carriers ] or line follow -back ( Sobell & Sobell , 2000 ) was used to assess COMT genotype ( Val allele homozygotes vs. Met allele daily drinking . Study drop -outs were paid ( $ 50 ) to return at carriers ; see Table 1 ) . Two baseline predictors of percent week 16 for drinking assessment. heavy drinking days during the trial, " current employment " [0179 ] Outcome Measures. The primary a priori defined and “ time since last drink prior to randomization ” , were used outcome measure was percent heavy drinking days ( 5 or as covariates in the intent- to - treat (ITT ) analysis (all subjects more standard drinks per day for men and 4 or more for with at least 1 week of drinking data ) using a linear mixed women ) per month , over the course of treatment. model (SPSS ver. 22 , — Linear Mixed , IBM , Armonk , N . Y ., [ 0180 ] Genetic and Biological Tests /Assays . The Clinical United States of America ) with an unstructured variancel Neurobiology Lab (CNL ) at Medical University of South covariance matrix focusing on 4 bins of monthly percent Carolina (MUSC ; Raymond Anton , Director) extracted heavy drinking data with medication group , time , OPRM1 genomic DNA from peripheral blood mononuclear cells genotype, and either DAT1 or COMT genotypes as factors. using a commercial DNA extraction kit /procedure ( Qiagen Based on previous data (Anton et al. , 2008 and Schacht et Inc ., Valencia , Calif ., United States of America ). OPRM1 al. , 2017 ), the initial protocol stipulated that the last month A118G ( Asn40 vs . Asp40 alleles ) and COMT 158 ( Val vs . of treatment would be analyzed independently as contrasts Met alleles ) were determined by a 5' nuclease genotyping within the mixed model . Effect sizes of naltrexone compared assay ( commercially available under the registered trade to placebo were calculated for each gene combination for mark TAQMAN® ) using primers and allele -specific probes comparative / illustrative purposes. Since nicotine use was ( Catalogue No . C _ 8950074 - 1 ; Applied Biosystems, Foster found to affect naltrexone response ( Schacht et al. , 2017 ; City , Calif. , United States of America . Samples previously Anton et al. , 2018 ) , a sensitivity analysis was performed to genotyped in the laboratory of Dr. David Goldman (NIAAA evaluate the effect of smoking as a factor in the main ITT Intramural) and in the CNL were used as controls ( 3 each per analysis . genotype/ assay ). The DAT1 VNTR was assessed after PCR amplification by specific primers 5 '- TGTGGTG TAGGGAACGGCCTGAG -3 ' (SEQ ID NO : 1 ) and 5 '- CT Example 1 TCCTGGAGGTCACGGCTCAAGG - 3 ' (SEQ ID NO : 2 ) ( Thermo Fisher Scientific , Waltham , Mass ., United States of Randomization and Baseline Characteristics America ). Amplified samples were electrophoresed on 2 . 0 % agarose gels and visualized with ethidium bromide under [ 0184 ] Of those initially screened , 358 individuals con ultraviolet light. Previously identified and sequenced con sented to participate , and 152 were randomized with equal trols for each VNTR length ( 9 or 10 repeats ) were amplified distribution between OPRM1 gene groups ( see FIG . 1 ) . in each assay and two raters compared them with subject Since 6 did not have valid outcome data , there were 146 samples to call genotypes . Four subjects who carried an evaluable individuals . Of those, 40 terminated the study 11 -repeat allele were reclassified as 10 carriers for this early (similar across all gene /medication groups in numbers analysis . For each polymorphism , genotypes were in Hardy and reasons ) , but full 16 -week drinking data was available Weinberg equilibrium and consistent with published fre on 126 ( range 84 - 91 % across gene /medication groups) . quencies among individuals of European American descent Determined by pill count, 82 % of subjects took at least 80 % (Kang et al. , 1999 ; Palmatier et al. , 1999 ) . RA supervised of study medication , again similar across all groups. quality control in a blinded fashion . % CDT was measured [0185 ] A 2 OPRM1 genex2 allele ( independently for with a reference HPLC assay (Helander et al. , 2010 ), other DAT1 and COMT alleles )x2 medication group ANOVA blood chemistries including GGT were measured with an showed there were no statistical differences between geno auto -analyzer . type /medication groups across a range of demographic and [0181 ] Sample Size Estimates . Sample size estimates for drinking variables , including sex , age, nicotine use , antide the original study were guided by power analysis based on pressant and cocaine use , baseline drinking , biomarker, and the magnitude of the OPRM1 by medication interaction on severity measures ( Table 1 ) . US 2018 /0371542 A1 Dec. 27 , 2018 19

Example 2 p = 0 .005 ). For the OPRM1xCOMT interaction , only those OPRM1 G -allele carriers , who also had COMT Val/ Val Drinking Outcome Measures genotypes , were significantly more responsive to naltrexone [0186 ] The main ITT analysis on percent heavy drinking than placebo ( F = 7 . 3 , 1 , 123 , p = 0 . 008 ) . As seen , while effect days ( HDD % ) included the evaluable participants in each sizes remained large for the gene/ medication groups dis group for the combination ofOPRM1XCOMT (see FIGS . 2 cussed above, OPRM1 G - allele carriers who were also either and 3 ) or OPRM1XDAT1 ( see FIGS . 4 and 5 ) . DAT1 10R homozygotes or COMT Val/ Val homozygotes [0187 ] As shown in FIG . 2A , COMT158Met carriers (i . e ., benefitted the most from naltrexone , both during the course those subjects whose genomes had one or two COMT of the study and at the end of treatment . rs4680 alleles that encoded methionine at amino acid 158 of SEQ ID NO : 8 ; also referred to herein as the “ COMT A [0191 ] Given our previous findings that nicotine use! allele” ) responded better to naltrexone when their genomes smoking status might influence naltrexone response also included two copies of the OPRM1 asparagine allele (Schacht et al. , 2017 ; Anton et al. , 2018 ) and despite similar (i . e . , the allele that has an adenine at nucleotide position 561 nicotine use between all gene and medication groups (see of SEO ID NO : 11 ; also referred to herein as the " OPRM1 Table 1 ) , a sensitivity analysis , whereby nicotine -use was A allele ” or the “ OPRM1- Asn40 allele ” ) . However, if the entered as a covariate into the ITT analyses , found that the subject had at least one copy of the OPRM1 aspartic acid pharmacogenetic results were not materially changed . allele (i . e ., the allele that has a guanine at nucleotide position 561 of SEQ ID NO : 11 ; also referred to herein as the TABLE 1 “ OPRM1 G allele ” or the “ OPRM1-Asp40 allele ” ), those subjects who were homozygous for the COMT158 Val allele Demographic , Alcohol Use , and Severity Measures ( i. e ., the allele that has a guanine at nucleotide position 721 of SEQ ID NO : 7 ; also referred to herein as the “ COMT G allele” ) responded better to naltrexone (see FIG . 2B ). FIG . OPRM1x OPRM1x 3 also shows that the response of COMT158 Met carriers | Total DAT1 COMT whose genomes also included two copies of the OPRM1 Sample Statistica Statistica asparagine allele was statistically significantly different Characteristic ( n = 146 ) P value P value from placebo ( p = 0 .03 ) and the response of COMT158 Val/ Val homozygotes whose genomes also included at least Demographics one copy of the OPRM1 aspartic acid allele was statistically significantly different from placebo ( p = 0 .05 ) Age , y (mean (SD )) 49. 3 ( 10 . 1 ) 0 . 88 0 . 10 [0188 ] The interactions among OPRM1 and DAT1 VNTR Sex , M , No. (% ) 101 (69 . 2 ) 0 .88 0 . 97 genotypes was also examined . As shown in FIG . 4A , those Married , No. ( % ) 94 ( 64 .4 ) 0 . 33 0 . 12 subjects who were homozygous for the OPRM1 asparagine Employed , No. ( % ) 114 (78 . 1 ) 0 . 16 0 . 74 allele responded better to naltrexone when their genomes Education s 12 years , No . ( % ) 24 ( 16. 4 ) 0 .55 0 . 72 also included two copies of the DAT1 VNTR 9R allele (i . e . , those subjects whose genomes had one or two copies of the Current Nicotine Use , No. ( % ) 57 ( 39 .0 ) 0 . 78 0 . 77 DAT1 VNTR 9 - repeat allele ). However, if the subject had at Cocaine Use , No. ( % ) 19 (13 . 0 ) 0 .75 0 . 49 least one copy of the OPRM1 aspartic acid allele , those Antidepressant use, No. ( % ) 48 (32 . 9 ) 0 .72 0 . 83 subjects who were homozygous for the DAT1 VNTR 10 - re Alcohol use and severity peat allele ( i . e . , those subjects whose genomes had two indicators , mean ( SD ) copies of the DAT1 VNTR 10 - repeat allele ) responded better to naltrexone ( see FIG . 4B ) . FIG . 5 also shows that the Drinking days ( % ) 85 . 3 ( 19 . 3 ) 0 .88 0 . 22 response of DAT1 VNTR 9R carriers whose genomes also Heavy drinking days ( % ) 79. 7 ( 22. 3 ) 0 . 34 0 . 28 included two copies of the OPRM1 asparagine allele was Drinks per drinking days 11. 2 (4 . 8 ) 0 .84 0 . 44 statistically significantly different from placebo ( p = 0 . 02 ) Drinks per day 9 . 6 ( 5 .0 ) 0 . 88 0 . 39 and the response of DAT1 VNTR 1OR homozygotes whose Days from last drink to 0 . 13 0 . 44 genomes also included at least one copy of the OPRM1 6 . 9 ( 4 . 4 ) aspartic acid allele was statistically significantly different randomization from placebo ( p = 0 .003 ) ADS score 15 .4 (6 .4 ) 0 . 96 0 . 25 [0189 ] In order to compare genotype by medication dif OCDS scored 25 . 6 ( 8 . 1 ) 0 . 89 0 . 58 ferences across the spectrum of genotypes , and to illustrate Drinking consequences No . ( % ) 41 .4 ( 18 .6 ) 0 . 54 0 .23 the clinical significance of the pharmacogenetic interactions, GGT > 63 IU /L 46 (31 . 5 ) 0 . 62 0 . 32 the univariate analyses in each genotype group and the effect dCDT s 1 . 7 % No . ( % ) 80 (56 ) 0 . 37 0 . 59 size of naltrexone over placebo , for the full 16 weeks of treatment as well as during the last 4 weeks of treatment , is Test of differences across medication groups and two genes. ANOVA testing linear given in Table 2 . variables and Chi Square testing categorical variables . [0190 ] Planned simple effects analyses showed that at the Drinking calculated during the 30 days prior to screening. 16 -week ( end of study ) time point, differences between the Alcohol Dependence Scale groups were generally in the same direction as the full study dobsessive Compulsive Drinking Scale interval ( see Table 2 ) . However , for the OPRM1XDAT1 Drinker Inventory of Consequences interaction , only those OPRM1 G - allele carriers, who also had the DAT1 10 / 10 genotype , were significantly more Disialo - Carbohydrate Deficient Transferrin - biomarker of heavy drinking responsive to naltrexone than placebo ( F = 8 . 17 , 1 , 119 , US 2018 /0371542 A1 Dec . 27 , 2018 20

TABLE 2 TAQMAN® PCR ) on a one to one basis in a blinded fashion . In an exploratory fashion DAT1 VNTR (using Significance and Effect Sizes of the Interaction of Carious OPRM1 primers from ABI, PCR , and chromatographic separation ) and DAT or COMT Genes and Naltrexone Response on Percent Heavy Drinking Days over the Course of the Trial ( 16 weeks) and status was explored in these participants . Smoking ( 40 % ) , at the End of the Study ( last 4 weeks ) antidepressant use (33 % ), and sex (30 % Female ) were equally distributed across groups . Nine sessions of Medical Treatment Effects Management (wks . 1 , 2 , 3 , 4 , 6 , 8 , 10 , 12, 16 ) were provided and drinking assessed by TLFB during the 16 -week treat All 4 months Last Month ment. Percent heavy drinking days ( % HDD ) over the 4 Effect Effect study months were analyzed in mixed model of medication Genotypes N p value * Sizes* * p value* Size* * (ntx vs . plac . )xOPRM1 allele (at least one Asp40 allele vs . OPRM1" DAT Asn / Asn homozygotes) < DAT1 ( at least one 9 VNTR vs . 10 / 10 homozygotes ) xtime ( study month ) (FIG . 2 ) . Drinks AA 9 Carr. 26 0 .085 0 . 70 0 .25 0 .46 per Drinking Day (DPDD ) in the end (month 4 ) of the trial A / A 10 / 10 47 0 . 4 0 . 25 0 . 82 0 . 07 G Carriers 9 Carr. 0 .62 - 0 . 17 0 . 5 - 0 . 24 was also explored (FIG . 5 ) . G Carriers 10 /10 0 .021 0 . 72 0 . 005 0 .89 [0195 ] The overall study demographics and drinking char acteristics are given in Table 1 of 146 evaluable individuals , OPRM1 COMTd n = 73 were OPRM1 Asp40 carriers and 73 Asn40 homozy A / A Met Carr . 51 0 .03 0 .63 0 .27 0 . 32 gotes. DAT1 9 carriers (n = 59 ) and 10 /10 homozygotes AA Val / Val 22 0 .78 - 0 . 12 0 .94 - 0 .03 ( n = 87) were not significantly different across OPRM1 and G Carriers Met Carr. WNA 0 .69 0 . 11 0 . 92 0 . 03 medication groups ( group size range 12 - 24 ) . There were no G Carriers Val /Val 20 0 .05 0 . 80 0 .008 1 . 09 significant differences in any baseline demographic and * Univariate analysis testing the interaction ofmedication group (naltrexone x placebo ) in drinking variables across the 8 study groups. each genetic combination . * * Effect size if for naltrexone over placebo in reduction of percent heavy drinking days [0196 ] Of 146 evaluable individuals n = 73 were OPRM1 for G at the 118 position of the OPRM1 gene coding for Asn40 or Asp40 respectively Asp40 carriers and 73 Asn40 homozygotes . DAT1 9 carriers bNumber of 9 or 10 forty base pair repeats of the DAT1 gene and 10 / 10 homozygotes were not significantly different Carr. : carriers across OPRM1 and medication groups ( group size range dVal or Met allele at the 158 position on the COMT gene 12 - 24 ) . Study completers (73 % ) and complete drinking data (86 % ) did not differ across groups . There was a significant Discussion of the Examples four -way interaction ( p = 0 .015 ) such that those Asp40 car riers who also had DAT1 10 / 10 genotypes and those Asn40 [ 0192 ] While great emphasis has been placed on the genotypes that who were DAT1 9 carriers all had less % OPRM1 Asp40 predicting naltrexone response , results have HDD over the course of the study when on naltrexone been inconsistent. One reason is that OPRM1 Asp40 might compared to placebo ( FIGS . 2 and 5 ) . This was not modified be interacting with other genetic variants (epistasis ). One by sex , age , antidepressant use or smoking status . The same important possibility is the functional VNTR variant in the pharmacogenetic relationships held when percent days dopamine transporter coding (DAT1 ) gene . DAT1 VNTR OR abstinent ( p = 0 .022 ) , drinks per day ( p = 0 . 051) and drinks per carriers to have lower DAT function leading to more syn drinking day ( p = 0 .024 ) were similarly analyzed . aptic dopamine availability underlying more reward and cue [0197 ] Alcohol dependent individuals (DSM IV ) with a sensitivity . As shown herein OPRM1 Asn40 /DAT1 9R car mean 80 % heavy drinking days (HDD ) were randomized to riers had less drinking on naltrexone that was also observed naltrexone ( 50 mg) or placebo, based on their OPRM1 allele in OPRM1 Asp40 /DAT1 10 / 10 homozygotes ( i . e . , Asp40 / status ( Asp40 vs . Asn40 homozygotes — TAQMAN® brand DAT 10 /10 and Asn40 /DAT 9 variants did best on naltrex PCR ) on a one to one basis in a blinded fashion . In an one) . Overall, this data indicates that genetically based exploratory fashion COMT (using primers from ABI, PCR , dopamine system variation / tone can influence how opiate and chromatographic separation ) status was explored in systemsmight respond and interact with medication . DAT 9 these participants . Smoking ( 40 % ) , antidepressant use carriers putatively have lower DAT transport function and ( 33 % ) , and sex ( 30 % Female ) were equally distributed likely more synaptic dopamine . Accordingly , Asp40 carriers across groups. Nine sessions of MedicalManagement (wks . only respond to opioid receptor blockade if they have 1 , 2 , 3 , 4 , 6 , 8, 10 , 12 , 16 ) were provided and drinking normal synaptic dopamine (DAT 10 / 10 carriers) . Con assessed by TLFB during the 16 -week treatment. Percent versely, Asn40 homozygotes only respond to opioid receptor heavy drinking days % HDD ) over the 4 study months were blockade if they have a downstream excess of synaptic analyzed in mixed model of medication ( naltrexone vs . dopamine (DAT 9 carriers ) . These results indicate that an placebo ) OPRM1 allele ( at least one Asp40 allele vs. Asn / epistatic balance of the opioid and dopamine systems exists Asn homozygotes )xCOMT (at least one Met158 allele vs . and should be taken into account in AUD in regard to Val /Val homozygotes )xtime ( study month ). pharmacogenetic treatment response and personalized medi [0198 ]. There was a significant four- way interaction ( p = 0 . cine initiatives. 015 ) such that those Asp40 carriers who also had Val /Val158 [ 0193] As shown herein , OPRM1 Asn40 /Met158 carriers genotypes and those Asn40 genotypes that who were had less drinking on naltrexone that was also observed in Met158 carriers all had less % HDD over the course of the OPRM1 Asp40 / COMT Val/ Val158 carriers study when on naltrexone compared to placebo (FIGS . 3 , 4A [0194 ] 146 Caucasian alcohol dependent individuals and 4B , and 5 ). This was not modified by sex , age , antide (DSM IV ) with a mean 80 % heavy drinking days (HDD ) pressant use or smoking status . The same pharmacogenetic were randomized to naltrexone (50 mg) or placebo , based on relationships held when percent days abstinent, drinks per their OPRM1 allele status (Asp40 vs . Asn40 homozygotes — day , and drinks per drinking day, were similarly analyzed . US 2018 /0371542 A1 Dec. 27 , 2018

[0199 ] These Examples had high internal validity , being [0204 ] Given the original hypothesis and sample size conducted at one site with the same staff providing MM available , individuals could not be genotyped and subse throughout the study , and using a priori OPRM1 genotyping quently randomized based on two / three genes, so some and selection . Data collection was performed blind to all systematic selection and treatment assignment bias cannot genotypes and medication group assignment. Individuals of be ruled out. Also , sample size limitation did not allow valid clinical interest ( some taking antidepressants and some analysis of 4 -way interactions between all three genotypes having cocaine use ) were included , and equally distributed and medication response . Despite these limitations, the across study groups. The analyses of DAT1 and COMT findings are underpinned by a well - executed clinical trial. genotypes were hypothesis driven , and pre - planned , with the Further, given the moderate expense of genotyping, clini genotyping done prior to any OPRM1 outcome analyses . cians might consider providing this service to AUD patients [0200 ] In our initial report (Schacht et al. , 2017 ) , while who might want to enhance their expectations of response to there was a significant main effect of naltrexone over pla naltrexone and other opioid antagonist . cebo overall , OPRM1 A118G status was not predictive of response in the ITT analysis (but did have some predictive REFERENCES value in the most adherent individuals ) . In the current [ 0205 ] All references listed in the instant disclosure , expanded analysis of the epistatic interaction of the OPRM1 including but not limited to all patents, patent applications 118 genotypes with several dopamine genes, very strong and and publications thereof , scientific journal articles , and significantmedication by genotype interactions emerged . As database entries ( including but not limited to GENBANK® the results show , those OPRM1 G allele carriers who were biosequence database entries and including all annotations also DAT1 10 , 10 or COMT Val /Val homozygotes responded available therein ) are incorporated herein by reference in remarkably well to naltrexone compared to placebo ( effect their entireties to the extent that they supplement, explain , sizes of 0 . 7 and 0 . 8 ) . In addition , those OPRM1 AA provide a background for, and / or teach methodology , tech homozygotes who were either DAT1 9R - carriers or COMT niques, and / or compositions employed herein . The discus met -carriers also responded better to naltrexone than pla - sion of the references is intended merely to summarize the cebo ( effect sizes 0 .70 and 0 .63 respectively ) . These effect assertions made by their authors . No admission is made that sizes are much greater than the small to moderate effect sizes any reference (or a portion of any reference ) is relevant prior of naltrexone efficacy in unselected AUD individuals across art. Applicants reserve the right to challenge the accuracy many trials (Srisurapanont & Jarusuraisin , 2005 ; Maisel et and pertinence of any cited reference . al. , 2013 ) , and may explain the inconsistent results reported [ 0206 ] Aarts et al . ( 2010 ) Neuropsychopharmacology in past naltrexone/ OPRM1 A118G pharmacogenetic studies . 35 (9 ) : 1943 - 1951 . In this study, individuals with OPRM1 G allele who were [0207 ] Anton & Youngblood ( 2006 ) Alcohol Clin Exp Res DAT1 9R - carriers or COMT met -carriers or who were 30 (11 ) : 1878 - 1883. OPRM1 AA homozygotes who were DAT1 10 , 10 or COMT [0208 ] Anton et al. ( 1996 ) Arch Gen Psychiatry 53 : 225 Val/ Val homozygotes were, on average, naltrexone non 231 . responders . [0209 ] Anton et al . (2006a ) Alcohol Clin Exp Res 30 ( 11 ): [0201 ] This pattern of epistatic interaction suggests that 1878 - 1883 . AUD individuals who are OPRM1 G carriers ( those with [0210 ] Anton et al . (2006b ) JAMA 295 ( 17 ): 2003 -2017 . putative increased endogenous opioid tone ) might need to 10211 ] Anton et al . ( 2008 ) Arch Gen Psychiatry 65 ( 2 ) : 135 144 . also have low /normal dopamine- tone , since genotypes nor [0212 ] Anton et al . 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( 2009) Proc Natl Acad Sci USA a way to unravel the complex effects of alcohol on brain 106 ( 2 ) :617 -622 . transmitter systems. The fact that response to a targeted (and [0219 ] European Patent Publication EP 0 070 685 B1. pharmacologically specific ) medication like naltrexone [0220 First et al. ( 1997 ) DSM - IV Axis I Disorders could be differentially impacted by these interactions is both Patient Edition . SCID - 1/ P , Version 2 . 0 ed . New York State novel and noteworthy . It also suggests that multiple genes Psychiatric Institute , New York , N . Y ., United States of might need to be examined to provide the precision that America . personalized treatment of AUD might require , not a surprise [0221 ] Forbes et al . (2009 ) Mol Psychiatry 14 ( 1 ) :60 - 70 . given the biological and pharmacological response hetero [0222 ] Forcehimes et al. (2007 ) Addict Behav 32 ( 8 ) : 1699 geneity of the disorder . 1704 . [ 0203] The impact of nicotine -use / smoking on the phar [0223 ] Fuke et al. (2001 ) Pharmacogenomics J 1 ( 2 ) : 152 macogenetic findings was analyzed . It was found that nico 156 . tine -use / smoking did not materially impact the results pre [0224 ] Goldman et al . (2005 ) J Stud Alcohol Suppl. 2005 sented here . However , future larger studies might further ( 15 ): 56 -64 ; discussion 33 . explore more subtle interactions of nicotine - use and geno [0225 ] Gonzales & Weiss ( 1998 ) J Neurosci 18 (24 ): type in AUD treatments . 10663 - 10671. US 2018 /0371542 A1 Dec. 27 , 2018

[ 0226 ] Grant et al. ( 2013) Eur Neuropsychopharmacol [0247 ] Pettinati et al. ( 2005 ) J Stud Alcohol Suppl 66 ( 15) : 23 ( 11 ) : 1587 - 1596 . 170 - 178 ; discussion 68 -69 . [ 0227] Grant et al. ( 2016 ) JAMA Psychiatry 73 ( 1 ) : 39 - 47 . [0248 ] Ramchandani et al. ( 2011) Mol Psychiatry 16 ( 8 ): [ 0228 ] Greenfield et al. ( 2014 ) J Stud Alcohol Drugs 809 - 817 . 75 ( 2 ) :319 - 327 . [0249 ] Rehm et al. ( 2014 ) Alcohol Clin Exp Res 38 ( 4 ) : [0229 ] Hasin et al. (2007 ) Arch Gen Psychiatry 64 (7 ): 830 1068 - 1077 . 842 . [0250 ] Schacht ( 2016 ) Pharmacogenomics J 16 ( 5 ): 430 [0230 ] Heinz et al. ( 2000 ) Neuropsychopharmacology 438 . 22 ( 2 ) : 133 - 139 . [0251 ] Schacht et al. (2013 ) Neuropsychopharmacology [0231 ] Helander et al. ( 2010 ) Clin Chem Lab Med 48 ( 11 ): 38 ( 3 ) :414 -422 . 1585 - 1592 . [ 0252 ] Schacht et al. ( 2017 ) Neuropsychopharmacology [ 0232 ] Job et al. (2007 ) Biol Psychiatry 62 (6 ): 627 -634 . 42 ( 13 ) : 2640 - 2653 . [ 0233] Johnson et al . ( 2005 ) J Stud Alcohol Suppl Suppl [0253 ] Skinner & Allen ( 1982 ) J Abnorm Psychol 91 ( 3 ) : 15 (15 ) : 157 - 167; discussion 140 . [ 0234 ] Jones et al. ( 2015 ) Alcohol Clin Exp Res 39 ( 3 ) : 199 - 209 . 391 - 402 . [0254 ] Sobell & Sobell ( 2000 ) Alcohol Timeline Follow [0235 ] Kang et al. ( 1999 ) Biol Psychiatry 46 ( 2 ) :151 - 160 . back ( TLFB ) . Handbook of Psychiatric Measures. Ameri [0236 ] Lachman (2008 ) Neuropsychopharmacology can Psychiatric Association Publishing . Washington , 33 (13 ): 3027 - 3029. D . C ., United States of America pages 477 -479 . [0237 ] Lachman et al. ( 1996 ) Pharmacogenetics 6 ( 3 ): 243 [0255 ] Srisurapanont & Jarusuraisin ( 2005 ) Int J Neurop 250 . sychopharmacol 8 ( 2 ): 267 - 280 . [0238 ] Letsinger et al. (1989 ) Proc Natl Acad SciUSA [0256 ] Stewart et al . (2009 ) Am J Hypertens 22 ( 1 ) : 87 - 91 . 86 :6553 -6556 . [ 0257 ] Sullivan et al. ( 1989 ) Br J Addict 84 ( 11 ): 1353 [ 0239] Litten et al. ( 2010 ) Alcohol Clin Exp Res 34 ( 6 ) : 1357 . 955 - 967. [0258 ] Tonigan et al. ( 1997 ) J Stud Alcohol 58 ( 4 ): 358 [0240 ] Maisel et al. ( 2013 ) Addiction 108( 2) :275 -293 . 364 . [0241 ] Mark et al. (2009 ) Drug Alcohol Depend 99 ( 1 -3 ): [ 0259 ] Tyagi & Kramer ( 1996 ) Nature Biotechnology 345 - 349 . 14 : 303 - 308 . [0242 ] McCane et al . (2018 ) Alcohol 67: 15 - 22 . [0260 ] Zindel & Kranzler (2014 ) J Stud Alcohol Drugs [ 0243 ] Niciu & Arias ( 2013 ) CNS Drugs 27 ( 10 ): 777 -787 . Suppl 75 Suppl 17 : 79 -88 . [0244 ] Oslin et al. (2003 ) Neuropsychopharmacology [0261 ] It will be understood that various details of the 28 : 1546 - 1552. presently disclosed subject matter may be changed without [ 0245 ) Oslin et al. ( 2015 ) JAMA Psychiatry 72 ( 5 ): 430 departing from the scope of the presently disclosed subject 437 . matter. Furthermore, the foregoing description is for the [0246 ] Palmatier et al . (1999 ) Biol Psychiatry 46 (4 ) :557 purpose of illustration only , and not for the purpose of 567. limitation .

SEQUENCE LISTING

< 160 > NUMBER OF SEQ ID NOS : 12 < 210 > SEQ ID NO 1 < 211 > LENGTH : 23 < 212 > TYPE : DNA 3 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Artificially synthesized oligonucleotide primer

< 400 > SEQUENCE : 1 tgtggtgtag ggaacggcct gag 23

< 210 > SEQ ID NO 2 < 211 > LENGTH : 24 < 212 > TYPE : DNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Artificially synthesized oligonucleotide primer < 400 > SEQUENCE : 2 cttcctggag gtcacggctc aagg 24

< 210 > SEQ ID NO 3 < 211 > LENGTH : 23 < 212 > TYPE : DNA US 2018 /0371542 A1 Dec . 27 , 2018 23

- continued < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : Artificially synthesized oligonucleotide primer < 400 > SEQUENCE : 3 tgcggtgtag ggaacggcct gag 23

< 210 > SEO ID NO 4 < 211 > LENGTH : 40 < 212 > TYPE : DNA < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 4 actggagcgt gtactacccc aggacgcatg cagggccccc 40

< 210 > SEQ ID NO 5 < 211 > LENGTH : 51 < 212 > TYPE : DNA < 213 > ORGANISM : Homo sapiens 20 > FEATURE : 1 > NAME / KEY : misc _ feature < 222 > LOCATION : ( 26 ) . . ( 26 ) < 223 > OTHER INFORMATION : r = a org < 400 > SEQUENCE : 5 ggtcaacttg tcccacttag atggcracct gtccgaccca tgcggtccga a 51

< 210 > SEQ ID NO 6 < 211 > LENGTH : 51 < 212 > TYPE : DNA < 213 > ORGANISM : Homo sapiens < 220 > FEATURE : V 1 > NAME / KEY : misc _ feature V 22 > LOCATION : ( 26 ) . . ( 26 ) < 223 > OTHER INFORMATION : r = a org < 400 > SEQUENCE : 6 ccagcggatg gtggatttcg ctggcrtgaa ggacaaggtg tgcatgcctg a 51

< 210 > SEO ID NO 7 < 211 > LENGTH : 2304 < 212 > TYPE : DNA < 213 > ORGANISM : Homo sapiens < 220 > FEATURE : 21 > NAME / KEY : CDS 22 > LOCATION : ( 250 ) . . ( 1065 ) < 400 > SEQUENCE : 7 cggcctgcgt ccgccaccgg aagcgccctc ctaatccccg cagcgccacc gccattgccg 60 ccatcgtcgt ggggcttctg gggcagctag ggctgcccgc cgcgctgcct gcgccggacc 120 ggggcgggtc cagtcccggg cgggccgtcg cgggagagaa ataacatctg ctttgctgcc 180 gagctcagag gagaccccag acccctcccg cagccagagg gctggagcct gctcagaggt 240 gctttgaag atg ccg gag gcc ccg cct ctg ctg ttg gca gct gtg ttg ctg 291 Met Pro Glu Ala Pro Pro Leu Leu Leu Ala Ala Val Leu Leu 10 ggc ctg gtg ctg ctg gtg gtg ctg ctg ctg ctt ctg agg cac tgg ggc 339 Gly Leu Val Leu Leu Val Val Leu Leu Leu Leu Leu Arg His Trp Gly 15 20 25 30 tgg ggc ctg tgc ctt atc ggc tgg aac gag ttc atc ctg cag ccc atc 387 Trp Gly Leu Cys Leu Ile Gly Trp Asn Glu Phe Ile Leu Gin Pro Ile 35 40 45 US 2018 /0371542 A1 Dec . 27 , 2018 24

- continued cac aac ctg ctc atg ggt gac acc aag gag cag cgc atc ctg aac cac 435 His Asn Leu Leu Met Gly Asp Thr Lys Glu Gin Arg Ile Leu Asn His 50 55 60 gtg ctg cag cat gcg gag ccc ggg aac gca cag agc gtg ctg gag gcc 483 Val Leu Gln His Ala Glu Pro Gly Asn Ala Gln Ser Val Leu Glu Ala 65 70 75 att gac acc tac tgc gag cag aag gag tgg gcc atg aac gtg ggc gac 531 Ile Asp Thr Tyr Cys Glu Gin Lys Glu Trp Ala Met Asn Val Gly Asp 80 85 90 aag aaa ggc aag atc gtg gac gcc gtg att cag gag cac cag ccc tcc 579 Lys Lys Gly Lys Ile Val Asp Ala Val Ile Gin Glu His Gin Pro Ser 95 100 105 110 gtg ctg ctg gag ctg ggg gcc tac tgt ggc tac tca gct gtg cgc atg 627 Val Leu Leu Glu Leu Gly Ala Tyr Cys Gly Tyr Ser Ala Val Arg Met 115 120 125 gcc cgc ctg ctg tca cca ggg gcg agg ctc atc acc atc gag atc aac 675 Ala Arg Leu Leu Ser Pro Gly Ala Arg Leu Ile Thr Ile Glu Ile Asn 130 135 140 ccc gac tgt gcc gcc atc acc cag cgg atg gtg gat ttc gct ggc gtg 723 Pro Asp Cys Ala Ala Ile Thr Gin Arg Met Val Asp Phe Ala Gly Val 145 150 155 aag gac aag gtc acc ctt gtg gtt gga gcg tcc cag gac atc atc ccc 771 Lys Asp Lys Val Thr Leu Val Val Gly Ala Ser Gin Asp Ile Ile Pro 160 165 170 cag ctg aag aag aag tat gat gtg gac aca ctg gac atg gtc ttc ctc 819 Gin Leu Lys Lys Lys Tyr Asp Val Asp Thr Leu Asp Met Val Phe Leu 175 180 185 190 gac cac tgg aag gac cgg tac ctg ccg gac acg ctt ctc ttg gag gaa 867 Asp His Trp Lys Asp Arg Tyr Leu Pro Asp Thr Leu Leu Leu Glu Glu 195 200 205 tgt ggc ctg ctg cgg aag ggg aca gtg cta ctg gct gac aac gtg atc 915 Cys Gly Leu Leu Arg Lys Gly Thr Val Leu Leu Ala Asp Asn Val Ile 210 215 220 tgc cca ggt gcg cca gac ttc cta gca cac gtg cgc ggg agc agc tgc 963 Cys Pro Gly Ala Pro Asp Phe Leu Ala His Val Arg Gly Ser Ser Cys 225 230 235 ttt gag tgc aca cac tac caa tog ttc ctg gaa tac agg gag gtg gtg 1011 Phe Glu Cys Thr His Tyr Gin Ser Phe Leu Glu Tyr Arg Glu Val Val 240 245 250 gac ggc ctg gag aag gcc atc tac aag ggc cca ggc agc gaa gca ggg 1059 Asp Gly Leu Glu Lys Ala Ile Tyr Lys Gly Pro Gly Ser Glu Ala Gly 255 260 265 270 CCC tga ctgccccccc ggcccccctc tcgggctctc tcacccagcc tggtactgaa 1115 Pro ggtgccagac gtgctcctgc tgaccttctg cggctccggg ctgtgtccta aatgcaaagc 1175 acacctcggc cgaggcctgc gccctgacat gctaacctct ctgaactgca acactggatt 1235 gttctttttt aagactcaat catgacttct ttactaacac tggctagcta tattatctta 1295 tatactaata tcatgtttta aaaatataaa atagaaatta agaatctaaa tatttagata 1355 taactcgact tagtacatcc ttctcaactg ccattcccct gctgcccttg acttgggcac 1415 caaacattca aagctcccct tgacggacgc taacgctaag ggcggggccc ctagctggct 1475 gggttctggg tggcacgcct ggcccactgg cctcccagcc acagtggtgc agaggtcago 1535 cctcctgcag ctaggccagg ggcacctgtt agccccatgg ggacgactgc cggcctggga 1595 US 2018 /0371542 A1 Dec . 27 , 2018 25

- continued aacgaagagg agtcagccag cattcacacc tttctgacca agcaggcgct ggggacaggt 1655 ggaccccgca gcagcaccag cccctctggg ccccatgtgg cacagagtgg aagcatctcc 1715 ttccctactc cccactgggc cttgcttaca gaagaggcaa tggctcagac cagctcccgc 1775 atccctgtag ttgcctccct ggcccatgag tgaggatgca gtgctggttt ctgcccacct 1835 acacctagag ctgtccccat ctcctccaag gggtcagact gctagccacc tcagaggctc 1895 caagggccca gttcccaggc ccaggacagg aatcaaccct gtgctagctg agttcacctg 1955 caccgagacc agcccctagc caagattcta ctcctgggct caaggcctgg ctagccccca 2015 gccagcccac tcctatggat agacagacca gtgagcccaa gtggacaagt ttggggccac 2075 ccagggacca gaaacagagc ctctgcagga cacagcagat gggcacctgg gaccacctcc 2135 acccagggcc ctgccccaga cgcgcagagg cccgacacaa gggagaagcc agccacttgt 2195 gccagacctg agtggcagaa agcaaaaagt tcctttgctg ctttaatttt taaattttct 2255 tacaaaaatt taggtgttta ccaatagtct tattttggct tatttttaa 2304

< 210 > SEQ ID NO 8 < 211 > LENGTH : 271 212 > TYPE : PRT < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 8 Met Pro Glu Ala Pro Pro Leu Leu Leu Ala Ala Val Leu Leu Gly Leu Met Pro Glu Ala pro Pro Leu Leu Leu Ala Ala val Leu Leu 15Gly Leu Val Leu Leu Val Val Leu Leu Leu Leu Leu Arg His Trp Gly Trp Gly 25 Leu Cys Leu Ile Gly Trp Asn Glu Phe Ile Leu Gin Pro Ile His Asn Leu Leu Met Gly Asp Thr Lys Glu Gin Arg Ile Leu Asn His Val Leu 50

Gln His Ala Glu Pro Gly Asn Ala Gln Ser Val Leu Glu Ala Ile Asp Thr Tyr cys Glu Gin Lys Glu Trp Ala Met Asn Val Gly Asp Lys Lys 95 Gly Lys Ile Val Asp Ala Val Ile Gin Glu His Gin Pro Ser Val Leu 105 Leu Glu Leu Gly Ala Tyr Cys Gly Tyr Ser Ala Val Arg Met Ala Arg

Leu Leu Ser Pro Gly Ala Arg Leu Ile Thr Ile Glu Ile Asn Pro Asp 130 Cys Ala Ala Ile Thr Gin Arg Met Val Asp Phe Ala Gly Val Lys Asp

Lys Val Thr Leu Val Val Gly Ala Ser Gin Asp Ile Ile Pro Gln Leu 175 Lys Lys Lys Tyr Asp Val Asp Thr Leu Asp Met Val Phe Leu Asp His 185 Trp Lys Asp Arg Tyr Leu Pro Asp Thr Leu Leu Leu Glu Glu Cys Gly

Leu Leu Arg Lys Gly Thr Val Leu Leu Ala Asp Asn Val Ile Cys Pro 210 Gly Ala Pro Asp Phe Leu Ala His Val Arg Gly Ser Ser Cys Phe Glu 240 US 2018 /0371542 A1 Dec . 27 , 2018

- continued

Cys Thr His Tyr Gin Ser Phe Leu Glu Tyr Arg Glu Val Val Asp Gly 245 250 255 Leu Glu Lys Ala Ile Tyr Lys Gly Pro Gly Ser Glu Ala Gly Pro 260 265 270

< 210 > SEQ ID NO 9 < 211 > LENGTH : 3952 < 212 > TYPE : DNA < 213 > ORGANISM : Homo sapiens < 220 > FEATURE : < 221 > NAME / KEY : CDS < 222 > LOCATION : ( 127 ) . . ( 1989 ) < 400 > SEQUENCE : 9 cgctgcggag cgggagggga ggcttcgcgg aacgctctcg gcgccaggac tcgcgtgcaa 60 agcccaggcc cgggcggcca gaccaagagg gaagaagcac agaattcctc aactcccagt 120 gtgccc atg agt aag agc aaa tgc tcc gtg gga ctc atg tct tcc gtg 168 Met Ser Lys Ser Lys Cys Ser Val Gly Leu Met Ser Ser Val 10 gtg gcc ccg gct aag gag ccc aat gcc gtg ggc ccg aag gag gtg gag 216 Val Ala Pro Ala Lys Glu Pro Asn Ala Val Gly Pro Lys Glu Val Glu 15 20 25 30 ctc atc ctt gtc aag gag cag aac gga gtg cag ctc acc agc tcc acc 264 Leu Ile Leu Val Lys Glu Gin Asn Gly Val Gin Leu Thr Ser Ser Thr 35 40 45 ctc acc aac ccg cgg cag agc ccc gtg gag gcc cag gat cgg gag acc 312 Leu Thr Asn Pro Arg Gin Ser Pro Val Glu Ala Gin Asp Arg Glu Thr 50 55 60 tgg ggc aag aag atc gac ttt ctc ctg tcc gtc att ggc ttt gct gtg 360 Trp Gly Lys Lys Ile Asp Phe Leu Leu Ser Val Ile Gly Phe Ala Val 65 70 75 gac ctg gcc aac gtc tgg cgg ttc ccc tac ctg tgc tac aaa aat ggt 408 Asp Leu Ala Asn Val Trp Arg Phe Pro Tyr Leu Cys Tyr Lys Asn Gly 80 85 90 ggc ggt gcc ttc ctg gtc ccc tac ctg ctc ttc atg gtc att gct ggg 456 Gly Gly Ala Phe Leu Val Pro Tyr Leu Leu Phe Met Val Ile Ala Gly 95 100 105 110 atg cca ctt ttc tac atg gag ctg gcc ctc ggc cag ttc aac agg gaa 504 Met Pro Leu Phe Tyr Met Glu Leu Ala Leu Gly Gin Phe Asn Arg Glu 115 120 125 ggg gcc gct ggt gtc tgg aag atc toc ccc ata ctg aaa ggt gtg ggc 552 Gly Ala Ala Gly Val Trp Lys Ile Cys Pro Ile Leu Lys Gly Val Gly 130 135 140 ttc acg gtc atc ctc atc tca ctg tat gtc ggc ttc ttc tac aac gtc 600 Phe Thr Val Ile Leu Ile Ser Leu Tyr Val Gly Phe Phe Tyr Asn Val 145 150 155 atc atc gcc tgg gcg ctg cac tat ctc ttc tcc tcc ttc acc acg gag 648 Ile Ile Ala Trp Ala Leu His Tyr Leu Phe Ser Ser Phe Thr Thr Glu 160 165 170 ctc ccc tgg atc cac tgc aac aac tcc tgg aac agc ccc aac tgc tcg 696 Leu Pro Trp Ile His Cys Asn Asn Ser Trp Asn Ser Pro Asn cys Ser 175 180 185 190 gat gcc cat cct ggt gac tcc agt gga gac agc tcg ggc ctc aac gac 744 Asp Ala His Pro Gly Asp Ser Ser Gly Asp Ser Ser Gly Leu Asn Asp 195 200 205 act ttt ggg acc aca cct gct gcc gag tac ttt gaa cgt ggc gtg ctg 792 Thr Phe Gly Thr Thr Pro Ala Ala Glu Tyr Phe Glu Arg Gly Val Leu US 2018 /0371542 A1 Dec . 27 , 2018 27

- continued 210 215 220 cac ctc cac cag agc cat ggc atc gac gac ctg ggg cct ccg cgg tgg 840 His Leu His Gin Ser His Gly Ile Asp Asp Leu Gly Pro Pro Arg Trp 225 230 235 cag ctc aca gcc tgc ctg gtg ctg gtc atc gtg ctg ctc tac ttc ago 888 Gin Leu Thr Ala Cys Leu Val Leu Val Ile Val Leu Leu Tyr Phe Ser 240 245 250 ctc tgg aag ggc gtg aag acc tca ggg aag gtg gta tgg atc aca gcc 936 Leu Trp Lys Gly Val Lys Thr Ser Gly Lys Val Val Trp Ile Thr Ala 255 260 265 270 acc atg cca tac gtg gtc ctc act gcc ctg ctc ctg cgt ggg gtc acc 984 Thr Met Pro Tyr Val Val Leu Thr Ala Leu Leu Leu Arq Gly Val Thr 75 280 285 ctc cct gga gcc ata gac ggc atc aga gca tac ctg agc gtt gac ttc 1032 Leu Pro Gly Ala Ile Asp Gly Ile Arg Ala Tyr Leu Ser Val Asp Phe 290 295 300 tac cgg ctc tgc gag gcg tct gtt tgg att gac gcg gcc acc cag gtg 1080 Tyr Arg Leu Cys Glu Ala Ser Val Trp Ile Asp Ala Ala Thr Gin Val 305 310 315 tgc ttc tcc ctg ggc gtg ggg ttc ggg gtg ctg atc gcc ttc tcc ago 1128 Cys Phe Ser Leu Gly Val Gly Phe Gly Val Leu Ile Ala Phe Ser Ser 320 325 330 tac aac aag ttc acc aac aac tgc tac agg gac gcg att gtc acc acc 1176 Tyr Asn Lys Phe Thr Asn Asn Cys Tyr Arg Asp Ala Ile Val Thr Thr 335 340 345 350 tcc atc aac tcc ctg acg agc ttc tcc tcc ggc ttc gtc gtc ttc tcc 1224 Ser Ile Asn Ser Leu Thr Ser Phe Ser Ser Gly Phe Val Val Phe Ser 355 360 365 ttc ctg ggg tac atg gca cag aag cac agt gtg ccc atc ggg gac gtg 1272 Phe Leu Gly Tyr Met Ala Gin Lys His Ser Val Pro Ile Gly Asp Val 370 375 380 gcc aag gac ggg cca ggg ctg atc ttc atc atc tac ccg gaa gcc atc 1320 Ala Lys Asp Gly Pro Gly Leu Ile Phe Ile Ile Tyr Pro Glu Ala Ile 385 390 395 gcc acg ctc cct ctg toc tca gcc tgg gcc gtg gtc ttc ttc atc atg 1368 Ala Thr Leu Pro Leu Ser Ser Ala Trp Ala Val Val Phe Phe Ile Met 400 405 410 ctg ctc acc ctg ggt atc gac agc gcc atg ggt ggt atg gag tca gtg 1416 Leu Leu Thr Leu Gly Ile Asp Ser Ala Met Gly Gly Met Glu Ser Val 415 20 425 430 atc acc ggg ctc atc gat gag ttc cag ctg ctg cac aga cac cgt gag 1464 Ile Thr Gly Leu Ile Asp Glu Phe Gln Leu Leu His Arg His Arg Glu 435 440 445 ctc ttc acg ctc ttc atc gtc ctg gcg acc ttc ctc ctg tcc ctg ttc 1512 Leu Phe Thr Leu Phe Ile Val Leu Ala Thr Phe Leu Leu Ser Leu Phe 450 455 460 tgc gtc acc aac ggt ggc atc tac gtc ttc acg ctc ctg gac cat ttt 1560 Cys Val Thr Asn Gly Gly Ile Tyr Val Phe Thr Leu Leu Asp His Phe 465 470 475 gca gcc ggc acg tcc atc ctc ttt gga gtg ctc atc gaa gcc atc gga 1608 Ala Ala Gly Thr Ser Ile Leu Phe Gly Val Leu Ile Glu Ala Ile Gly 480 485 490 gtg gcc tgg ttc tat ggt gtt ggg cag ttc agc gac gac atc cag cag 1656 Val Ala Trp Phe Tyr Gly Val Gly Gln Phe Ser Asp Asp Ile Gin Gin 495 500 505 510 atg acc ggg cag cgg ccc agc ctg tac tgg cgg ctg tgc tgg aag ctg 1704 Met Thr Gly Gin Arg Pro Ser Leu Tyr Trp Arg Leu Cys Trp Lys Leu US 2018 /0371542 A1 Dec . 27 , 2018 28

- continued 515 520 525 gtc agc ccc tgc ttt ctc ctg ttc gtg gtc gtg gtc agc att gtg acc 1752 Val Ser Pro Cys Phe Leu Leu Phe Val Val Val Val Ser Ile Val Thr 530 535 540 ttc aga ccc ccc cac tac gga gcc tac atc ttc ccc gac tgg gcc aac 1800 Phe Arg Pro Pro His Tyr Gly Ala Tyr Ile Phe Pro Asp Trp Ala Asn 545 550 555 gcg ctg ggc tgg gtc atc gcc aca tcc tcc atg gcc atg gtg ccc atc 1848 Ala Leu Gly Trp Val Ile Ala Thr Ser Ser Met Ala Met Val Pro Ile 560 565 570 tat gcg gcc tac aag ttc tgc agc ctg cct ggg toc ttt cga gag aaa 1896 Tyr Ala Ala Tyr Lys Phe Cys Ser Leu Pro Gly Ser Phe Arg Glu Lys 575 580 585 590 ctg gcc tac gcc att gca ccc gag aag gac cgt gag ctg gtg gac aga 1944 Leu Ala Tyr Ala Ile Ala Pro Glu Lys Asp Arg Glu Leu Val Asp Arg 595 600 605 ggg gag gtg cgc cag ttc acg ctc cgc cac tgg ctc aag gtg tag 1989 Gly Glu Val Arg Gin Phe Thr Leu Arg His Trp Leu Lys Val 610 615 620 agggagcaga gacgaagacc ccaggaagtc atcctgcaat gggagagaca cgaacaaacc 2049 aaggaaatct aagtttcgag agaaaggagg gcaacttcta ctcttcaacc tctactgaaa 2109 acacaaacaa caaagcagaa gactcctctc ttctgactgt ttacaccttt ccgtgccggg 2169 agcgcacctc gccgtgtctt gtgttgctgt aataacgacg tagatctgtg cagcgaggtc 2229 caccccgttg ttgtccctgc agggcagaaa aacgtctaac ttcatgctgt ctgtgtgagg 2289 ctccctccct ccctgctccc tgctcccggc tctgaggctg ccccaggggc actgtgttct 2349 caggcgggga tcacgatcct tgtagacgca cctgctgaga atccccgtgc tcacagtago 2409 ttcctagacc atttactttg cccatattaa aaagccaagt gtcctgcttg gtttagctgt 2469 gcagaaggtg aaatggagga aaccacaaat tcatgcaaag tcctttcccg atgcgtggct 2529 cccagcagag gccgtaaatt gagcgttcag ttgacacatt gcacacacag tctgttcaga 2589 ggcattggag gatgggggtc ctggtatgtc tcaccaggaa attctgttta tgttcttgca 2649 gcagagagaa ataaaactcc ttgaaaccag ctcaggctac tgccactcag gcagcctgtg 2709 ggtccttgcg gtgtagggaa cggcctgaga ggagcgtgtc ctatccccgg acgcatgcag 2769 ggcccccaca ggagcgtgtc ctatccccgg acgcatgcag ggcccccaca ggagcatgtc 2829 ctatccctgg acgcatgcag ggcccccaca ggagcgtgta ctaccccaga acgcatgcag 2889 ggcccccaca ggagcgtgta ctaccccagg acgcatgcag ggcccccact ggagcgtgta 2949 ctaccccagg acgcatgcag ggcccccaca ggagcgtgtc ctatccccgg accggacgca 3009 tgcagggccc ccacaggagc gtgtactacc ccaggacgca tgcagggccc ccacaggagc 3069 gtgtactacc ccaggatgca tgcagggccc ccacaggagc gtgtactacc ccaggacgca 3129 tgcagggccc ccatgcaggc agcctgcaga ccacactctg cctggccttg agccgtgacc 3189 tccaggaagg gaccccactg gaattttatt tctctcaggt gcgtgccaca tcaataacaa 3249 cagtttttat gtttgcgaat ggctttttaa aatcatattt acctgtgaat caaaacaaat 3309 tcaagaatgc agtatccgcg agcctgcttg ctgatattgc agtttttgtt tacaagaata 3369 attagcaata ctgagtgaag gatgttggcc aaaagctgct ttccatggca cactgccctc3429 toccactgac aggaaagtgg atgccatagt ttgaattcat gcctcaagtc ggtgggcctg 3489 US 2018 /0371542 A1 Dec . 27 , 2018

- continued cctacgtgct gcccgagggc aggggccgtg cagggccagt catggctgtc ccctgcaagt 3549 ggacgtgggc tccagggact ggagtgtaat gctcggtggg agccgtcagc ctgtgaactg 3609 ccaggcagct gcagttagca cagaggatgg cttccccatt gccttctggg gagggacaca 3669 gaggacggct tccccatcgc cttctggccg ctgcagtcag cacagagagc ggcttcccca 3729 ttgccttctg gggagggaca cagaggacag cttccccatc gccttctggc tgctgcagtc 3789 agcacagaga gcggcttccc catcgccttc tggggagggg ctccgtgtag caacccaggt 3849 gttgtccgtg tctgttgacc aatctctatt cagcatcgtg tgggtcccta agcacaataa 3909 aagacateca caatggaaaa actgcaaaaa aaaaaaaaaa aaa 3952

< 210 > SEQ ID NO 10 < 211 > LENGTH : 620 < 212 > TYPE : PRT < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 10 Met Ser Lys Ser Lys Cys Ser Val Gly Leu Met Ser Ser Val Val Ala 15 Pro Ala Lys Glu Pro Asn Ala Val Gly Pro Lys Glu Val Glu Leu Ile Ala Lys Glu Pro Asn Ala val 30 Leu Val Lys Glu Gln Asn Gly Val Gln Leu Thr Ser Ser Thr Leu Thr 35 Asn Pro Arg Gin Ser Pro Val Glu Ala Gin Asp Arg Glu Thr Trp Gly Lys Lys Ile Asp Phe Leu Leu Ser Val Ile Gly Phe Ala Val Asp Leu 70 75 Ala Asn Val Trp Arg Phe Pro Tyr Leu Cys Tyr Lys Asn Gly Gly Gly

Ala Phe Leu Val Pro Tyr Leu Leu Phe Met Val Ile Ala Gly Met Pro 110 Leu Phe Tyr Met Glu Leu Ala Leu Gly Gin Phe Asn Arg Glu Gly Ala 115 Ala Gly Val Trp Lys Ile Cys Pro Ile Leu Lys Gly Val Gly Phe Thr

Val Ile Leu Ile Ser Leu Tyr Val Gly Phe Phe Tyr Asn Val Ile Ile 150 155

Ala Trp Ala Leu His Tyr Leu Phe Ser Ser Phe Thr Thr Glu Leu Pro

Trp Ile His Cys Asn Asn Ser Trp Asn Ser Pro Asn cys Ser Asp Ala 190 His Pro Gly Asp Ser Ser Gly Asp Ser Ser Gly Leu Asn Asp Thr Phe 195 Gly Thr Thr Pro Ala Ala Glu Tyr Phe Glu Arg Gly Val Leu His Leu

His Gin Ser His Gly Ile Asp Asp Leu Gly Pro Pro Arg Trp Gin Leu 230 235 240 Thr Ala Cys Leu Val Leu Val Ile Val Leu Leu Tyr Phe Ser Leu Trp

Lys Gly Val Lys Thr Ser Gly Lys Val Val Trp Ile Thr Ala Thr Met 270

Pro Tyr Val Val Leu Thr Ala Leu Leu Leu Arg y Val Thr Leu Pro US 2018 /0371542 A1 Dec . 27 , 2018 30

- continued 275 280 285 Gly Ala Ile Asp Gly Ile Arg Ala Tyr Leu Ser Val Asp Phe Tyr Arg

Leu Cys Glu Ala Ser Val Trp Ile Asp Ala Ala Thr Gln Val Cys Phe 305 Ser Leu Gly Val Gly Phe Gly Val Leu Ile Ala Phe Ser Ser Tyr Asn Lys Phe Thr Asn Asn Cys Tyr Arg Asp Ala Ile Val Thr Thr Ser Ile 350 Asn Ser Leu Thr Ser Phe Ser Ser Gly Phe Val Val Phe Ser Phe Leu 355 360 365 Gly Tyr Met Ala Gln Lys His Ser Val Pro Ile Gly Asp Val Ala Lys

Asp Gly Pro Gly Leu Ile Phe Ile Ile Tyr Pro Glu Ala Ile Ala Thr 385 Leu Pro Leu Ser Ser Ala Trp Ala Val Val Phe Phe Ile Met Leu Leu

Thr Leu Gly Ile Asp Ser Ala Met Gly Gly Met Glu Ser Val Ile Thr 430 Gly Leu Ile Asp Glu Phe Gin Leu Leu His Arg His Arg Glu Leu Phe 435 440 445 Thr Leu Phe Ile Val Leu Ala Thr Phe Leu Leu Ser Leu Phe Cys Val

Thr Asn Gly Gly Ile Tyr Val Phe Thr Leu Leu Asp His Phe Ala Ala 465 Gly Thr Ser Ile Leu Phe Gly Val Leu Ile Glu Ala Ile Gly Val Ala Trp Phe Tyr Gly Val Gly Gin Phe Ser Asp Asp Ile Gin Gin Met Thr 510 Gly Gin Arg Pro Ser Leu Tyr Trp Arg Leu Cys Trp Lys Leu Val Ser 515 520 525 Pro Cys Phe Leu Leu Phe Val Val Val Val Ser Ile Val Thr Phe Arg

Pro Pro His Tyr Gly Ala Tyr Ile Phe Pro Asp Trp Ala Asn Ala Leu 545 Gly Trp Val Ile Ala Thr Ser Ser Met Ala Met Val Pro Ile Tyr Ala Ala Tyr Lys Phe Cys Ser Leu Pro Gly Ser Phe Arg Glu Lys Leu Ala 590 Tyr Ala Ile Ala Pro Glu Lys Asp Arg Glu Leu Val Asp Arg Gly Glu 595 600 605 Val Arg Gin Phe Thr Leu Arg His Trp Leu Lys Val 610 620

< 210 > SEQ ID NO 11 < 211 > LENGTH : 15279 < 212 > TYPE : DNA < 213 > ORGANISM : Homo sapiens < 220 > FEATURE : < 221 > NAME /KEY : CDS < 222 > LOCATION : (444 ) . . ( 1646 ) < 400 > SEQUENCE : 11 US 2018 /0371542 A1 Dec . 27 , 2018 31

- continued gctaccaaag actaactcta tctctctccc caacccttct ctccatctcc ctcctttaga 60 tgtgtttgca cagaagagtg cccagtgaag agacctactc cttggatcgc tttgcgcaaa 120 atccacccct tttccctcct ccctcccttc cagcctccga atcccgcatg goccacgctc 180 ccctcctgca gcggtgcggg gcaggtgatg agcctctgtg aactactaag gtgggagggg 240 gctatacgca gaggagaatg tcagatgctc agctcggtcc cctccgcctg acgctcctct 300 ctgtctcagc caggactggt ttctgtaaga aacagcagga gotgtggcag cggcgaaagg 360 aagcggctga ggcgcttgga acccgaaaag tctcggtgct cctggctacc tcgcacagcg 420 gtgcccgccc ggccgtcagt acc atg gac agc agc gct gcc ccc acg aac gcc 473 Met Asp Ser Ser Ala Ala Pro Thr Asn Ala 10 agc aat tgc act gat gcc ttg gcg tac tca agt tgc tcc cca gca ccc 521 Ser Asn Cys Thr Asp Ala Leu Ala Tyr Ser Ser Cys Ser Pro Ala Pro 15 20 25 agc ccc ggt tcc tgg gtc aac ttg tcc cac tta gat ggc aac ctg tcc 569 Ser Pro Gly Ser Trp Val Asn Leu Ser His Leu Asp Gly Asn Leu Ser 35 40 gac cca tgc ggt ccg aac cgc acc gac ctg ggc ggg aga gac agc ctg 617 Asp Pro Cys Gly Pro Asn Arg Thr Asp Leu Gly Gly Arg Asp Ser Leu 45 50 55 tgc cct ccg acc ggc agt ccc tcc atg atc acg gcc atc acg atc atg 665 Cys Pro Pro Thr Gly Ser Pro Ser Met Ile Thr Ala Ile Thr Ile Met 60 65 70 gcc ctc tac tcc atc gtg tgc gtg gtg ggg ctc ttc gga aac ttc ctg 713 Ala Leu Tyr Ser Ile Val Cys Val Val Gly Leu Phe Gly Asn Phe Leu 75 80 85 90 gtc atg tat gtg att gtc aga tac acc aag atg aag act gcc acc aac 761 Val Met Tyr Val Ile Val Arg Tyr Thr Lys Met Lys Thr Ala Thr Asn 95 100 105 atc tac att ttc aac ctt gct ctg gca gat gcc tta gcc acc agt acc 809 Ile Tyr Ile Phe Asn Leu Ala Leu Ala Asp Ala Leu Ala Thr Ser Thr 110 115 120 ctg ccc ttc cag agt gtg aat tac cta atg gga aca tgg cca ttt gga 857 Leu Pro Phe Gln Ser Val Asn Tyr Leu Met Gly Thr Trp Pro Phe Gly 125 130 135 acc atc ctt tgc aag ata gtg atc tcc ata gat tac tat aac atg ttc 905 Thr Ile Leu Cys Lys Ile Val Ile Ser Ile Asp Tyr Tyr Asn Met Phe 140 145 150 acc agc ata ttc acc ctc tgc acc atg agt gtt gat cgatac att gca 953 Thr Ser Ile Phe Thr Leu Cys Thr Met Ser Val Asp Ara Tyr Ile Ala 155 160 165 170 gtc toc cac cct gtc aag gcc tta gat ttc cgt act ccc cga aat gcc 1001 Val Cys His Pro Val Lys Ala Leu Asp Phe Arg Thr Pro Arg Asn Ala 175 180 185 aaa att atc aat gtc tgc aac tgg atc ctc tct tca gcc att ggt ctt 1049 Lys Ile Ile Asn Val Cys Asn Trp Ile Leu Ser Ser Ala Ile Gly Leu 190 195 200 cct gta atg ttc atg gct aca aca aaa tac agg caa ggt tcc ata gat 1097 Pro Val Met Phe Met Ala Thr Thr Lys Tyr Arg Gin Gly Ser Ile Asp 205 210 215 tot aca cta aca ttc tct cat cca acc tgg tac tgg gaa aac ctg ctg 1145 Cys Thr Leu Thr Phe Ser His Pro Thr Trp Tyr Trp Glu Asn Leu Leu 220 225 230 aag atc tgt gtt ttc atc ttc gcc ttc att atg cca gtg ctc atc att 1193 Lys Ile Cys Val Phe Ile Phe Ala Phe Ile Met Pro Val Leu Ile Ile US 2018 /0371542 A1 Dec . 27 , 2018 32

- continued 235 240 245 250 acc gtg tgc tat gga ctg atg atc ttg cgc ctc aag agt gtc cgc atg 1241 Thr Val Cys Tyr Gly Leu Met Ile Leu Arg Leu Lys Ser Val Arg Met 255 260 265 ctc tct ggc tcc aaa gaa aag gac agg aat ctt cga agg atc acc agg 1289 Leu Ser Gly Ser Lys Glu Lys Asp Arg Asn Leu Arg Arg Ile Thr Arg 270 275 280 atg gtg ctg gtg gtg gtg gct gtg ttc atc gtc tgc tgg act ccc att 1337 Met Val Leu Val Val Val Ala Val Phe Ile Val Cys Trp Thr Pro Ile 285 290 295 cac att tac gtc atc att aaa gcc ttg gtt aca atc cca gaa act acg 1385 His Ile Tyr Val Ile Ile Lys Ala Leu Val Thr Ile Pro Glu Thr Thr 300 305 310 ttc cag act gtt tct tgg cac ttc tgc att gct cta ggt tac aca aac 1433 Phe Gin Thr Val Ser Trp His Phe Cys Ile Ala Leu Gly Tyr Thr Asn 315 320 325 330 agc tgc ctc aac cca gtc ctt tat gca ttt ctg gat gaa aac ttc aaa 1481 Ser Cys Leu Asn Pro Val Leu Tyr Ala Phe Leu Asp Glu Asn Phe Lys 335 340 345 cga tgc ttc aga gag ttc tgt atc cca acc tct tcc aac att gag caa 1529 Arg Cys Phe Arg Glu Phe Cys Ile Pro Thr Ser Ser Asn Ile Glu Gln 350 355 360 caa aac tcc act cga att cgt cag aac act aga gac cac ccc tcc acg 1577 Gln Asn Ser Thr Arg Ile Arg Gln Asn Thr Arg Asp His Pro Ser Thr 365 370 375 gcc aat aca gtg gat aga act aat cat cag cta gaa aat ctg gaa gca 1625 Ala Asn Thr Val Asp Arg Thr Asn His Gin Leu Glu Asn Leu Glu Ala 380 385 390 gaa act gct ccg ttg ccc taa cagggtctca toccattccg accttcacca 1676 Glu Thr Ala Pro Leu Pro 395 400 agcttagaag ccaccatgta tgtggaagca ggttgcttca agaatgtgta ggaggctcta 1736 attctctagg aaagtgcctg cttttaggtc atccaacctc tttcctctct ggccactctg 1796 ctctgcacat tagagggaca gccaaaagta agtggagcat ttggaaggaa aggaatatac 1856 cacaccgagg agtccagttt gtgcaagaca cccagtggaa ccaaaaccca tcgtggtatg 1916 tgaattgaag tcatcataaa aggtgaccct tctgtctgta agattttatt ttcaagcaaa 1976 tatttatgac ctcaacaaag aagaaccatc ttttgttaag ttcaccgtag taacacataa 2036 agtaaatgct acctctgatc aaagcacctt gaatggaagg tccgagtctt tttagtgttt 2096 tgcaagggaa tgaatccatt attctatttt agacttttaa cttcacctta aaattagcat 2156 ctggctaagg catcattttc acctccattt cttggttttg tattgtttaa aaaaataara 2016 tctctttcat ctagctccat aattgcaagg gaagagatta gcatgaaagg taatctgaaa 2276 cacagtcatg tgtcagctgt agaaaggttg attctcatgc actgcaaata cttccaaaga 2336 gtcatcatgg gggatttttc attcttaggc tttcagtggt ttgttcctca gttttaaatg 2396 tgcaattttc ttgctcctat ttaagtgttc acaaaaggta atagtcaatg agctcatcac 2456 ttcatccatg caggaagtca agcattaaaa tgtactcttt atttctcact ggtttctcca 2516 tactgcaggc tccccacata ttattttctt tttttaactc agctcagaat ccttatgcct 2576 tttgaatcag tgtgataaaa tgacagatgt ttttgttgat toccagaaca gtttaaactt 2636 tttttaaaca ataaatccaa taaacata at ctcaagacag actcatatga ccatacagaa 2696 US 2018 /0371542 A1 Dec . 27 , 2018 33

- continued aagtgtgtca tctatttttc aatcctgtgt agttacttgg atgtgaaata ttaacaatgg 2756 cccaaaaata ttttcctqaa qattgtqttt atataatgtcatcaccaata ttttgatott 2816 tttgatctct gotaaatgtc aagatttcct ttgtaaagtg togatcttct aagtagtttc 2876 tttaagacaa ttctccgctt taactgattt tctttgttgt gaaacacagt agagatttgg 2936 caatcaacca ttttacttga tctaggtaga cagccaagtc agatggccca tgcctagaag 2996 ctctccattt tgaacttttg tcagcattga ttaaaagaat caaatacctt gtagttatct 3056 atgatgatac aagtaaaaaa ctaggctgct gacttctgag tattcctgag cctacaattg 3116 taaaattgta gactccattg taaaatttgt attttttcat caatctgaca aggcacaaat 3176 atgtgccaga tatacaaaag caatgtttct agaaaacagc tatcatggat cagaataact 3236 gaatttactc tcagatctat tggctatagt tatgtggact caacccacgt atccagtaga 3296 tgggaaaaaa caaaagccaa aataagtttt ttagtgtttc cttctgatga agtttcatgt 3356 ttgcttgtaa taatctccat ttctcaaata ttatgttcca taatagacat acattatgtt 3416 taatttttta tattttctga caaaagtaac taaaactagg accttaaaaa gatttagaat 3476 gttaaataag tgtactaggg tgtatatatt tacatatata cactactaga gcttccaaaa 3536 gtaaaatgga taattcaaac agaacacaat gtaatatttg tatgtaaata actgaggagg 3596 aaaatccatg cttttcatgg getaggatgg tttctcccaa gagatgacat agtattgctt 3656 ttgctcatca ggctgtttct cagcaatcat tgtttctgct taataccagc tcctagtacg 3716 aattatctgg catgttgaga gcaactttgt cttcaagtag gacctgatct atctttttcc 3776 acaaatgtca tgtgtgtgaa caagtttctt ccatgtcatc tttgagactc tacacagaat 3836 acacaataaa gggagttatt tttaaaataa gacattctaa agtaaataat aaata aggtc3896 attgtcaacg tttttcattc aaaaccattt tttaacgtaa atttgctaga accaccttcc 3956 aattccaagg caaggagaga cattacaacc ctgactcaac tggatgggct aaggtttctg 4016 ataaaatctg aagataaaga aaatggaata ttctgcttttttcttccttc taatttcacc 4076 cttgcctaag gatgagattt cttcccaggt tggtatccca gaaatgcaga ctgtagctat 4136 ggggcggaag ctttgtttct ttacctgatc acttgctgtg gaaattctag cttattgtgt 4196 tccaagtagt tagtggtttt tctccttcag tctccactgt tattttgctc cttcatccct 4256 ctttccttgc caatcattag aaaggaaaga agaggaaaga gactcgctgg agcactggtg 4316 agtctctagg accctgctat cctatcccaa cagggctgtc agacggagaa ctcctaatgt 4376 ggccatttga aacacttctc aacattgaaa tagacagttg aagttttaaa ataacctctt 4436 ctaagacacg gctatgagta ggtaagagag cattcattcc cttcaataat atgactgtgt 4496 tgataaaact gataaccatt cacttgcaaa tgttattatt gaataagtct cacttagctc 4556 atttaatatt acccaaaaga tgctaacaaa ttctgtttcc cacattgtca cagcatgccc 4616 tttacatctc aggatccagg caaaagttga aattcagaaa catagatatg aaatgtaaga 4676 tacaaagaaa acacctctgc aaagattccg accacattta tcaaaaagtc cccaaagcat 4736 tcaaaatctt tacttaagtc aagtctattt atacgtttaa aagctaaaaa caagatcttt 4796 ttggtaatgc ttcaattaaa tgttttatct aaatatgtgg aaatgataag atgcgtactg 4856 catctctcat ataata aaga taatcagttt tatacaqaca tattctccat ctactqacaa 4916 ttaccaagat aagatggcac tagaaatttc tccagcttac gocatgaata ctgcagaagc 4976 US 2018 /0371542 A1 Dec . 27 , 2018 34

- continued tgatactatc cgttgtggtt ttacaaattc tagagggttc tagccaaagc aacctaagaa 5036 taggacatgg tagcttaagt ttttcagctt cttaactggc cacacacaca caagttgtgt 5096 ttgtacaatt cttgaggtca atcagaacca aaaaatctgt tgctggaaga aatattatcc 5156 tcttcataga aatatccacc agcagaaaat tggtttctca aggaatccct actgcccttg 5216 tagaaacato aagattcttg cctggattct caacataagt ctttactcac aggcctattg 5276 cttggtttca gaagagtgag aacatgaaag ttcataaatg cctgggccac tgcaactcta 5336 accactgtgt ttcccccagt ttgatatggt tcaggataca tagtcataga acagggcatg 5396 cagattgtat ttaagaccac tgcaagtaag gtctaaggca aaagtaaatt aatgagtcca 5456 actctggggcatccatttaa gagccattta atccatttaa ttacaaataa tttcaaccca 5516 tagtcagtgt tcttcactgt cttcaaaaat ataaaaagta caaggaattg tgtacattac 5576 aaactgctcc agaaacaaaa ccaaatgtgg atagctttgt gagctgcagt gtgtggcaaa 5636 tgttcaacct tttgttatge aatatcacccatatcaaata ccattcttaa agcagtagac 5696 agatgagtca agttcaattt aatgcaaaca atattactgt gttctaagcg cttctgttac 5756 togaaagggg tctgatccag accccaaaag agggttcttg gacctcatgc aagaaagaat 5816 tcaggagtaa agtgaaagtg aaatgggtca ggatcaatgg tctcattgtc taaggtgtta 00v tccgagctcg ttgtctcaca accaagaaaa ttaaggagca tggacacaaa gggtgaggtt 5936 ggagcaaaag tttaataagc aaaagaggaa agctctctgc agcagagacg ggagcccaag 5996 tgggttgctg tttttacagc tgaatccaaa agcttttata agaaactcct ctcatctctg 6056 cagctatttg agtaacttct cttatctgaa aagctgtctg tacaactgcc tctatctatg 6116 cagctatggg gtgtctctag gcgagcacaa agcatagctt ctcttgttgt ataattgtgg 6176 gtttgtttta agtaagccac tttcctccct gcaagttccc acggagcaga aaaaaggagg 6236 aaactttttc ctgggagccc actaatcaca cagtgaacaa aaggettcta tgctgggcct 6296 tactttcta a cagtgcagca gttatagtct gagttttctccaggctgctc catttttgcc 6356 tgtagctatg atttttcagg cagcctgctt ctccgaggac tagtcttagc tgtttaccta 6416 actgattggt ccttttcttc tccctgaaaa gcaagtttat taagaaagca aaggaataaa 6476 gaatggctac tccataggca gcgtagcccc aagggctgct ggttggctat ttttgtggtt 6536 atttcttgat tatatgctaa acaaggggtg gattattcat gggttttctg ggaaaggagt 6596 gggcaattcc cagaactgag ggttcttccc cttttaagac cgcatagggc aacttcctga 6656 cattgccatg gcatttgtaa actgtcgtgg tactggtggg aatgtttttt agcatgctaa 6716 tocaatataa ttagtgtata atgagtagtg aggacaacca gaggtaactt tcattgccat 6776 cttggtttta gtggggtttg gccggcttct ttaccacatc cttttatcag taagatcttc 6836 gtgacctgta ccttgtgcca acctcctatc tcttcctgtg acttggaatg cctaacctcc 6896 tgggaatgca goccagtagg tctcagcctt attttaccca gcctctattc aagatggagt 6956 cgctctggtt caaacacctc tgacacttga attacaaata taaggaccat tgacactgag 7016 attttaaggg aggaaaaaca gattgacagt ggactaaagg tacttttgta gcaaggtact 7076 ttccacacaa tattgaataa atgcagtgta tacattttta aaggaatttt aagagctcgg 7136 aaatcattaa aattaagttt atcaattttt gaaagcatct tctgactcaa atatatgaaa 7196 agattattct agaccttagg cagatattca gagaaagatc agttttctat gggttttttc 7256 US 2018 /0371542 A1 Dec . 27 , 2018 35

- continued catcatgcat tttatacaaa tttaatatac tttttcaact cactttgcat ttcctgttac 7316 cttgtactga gaacttcatt aaaacctgca cttgaaattg cacttaaact ttacaaaatc 7376 acagaagaag ttgttttcca taggtgtggg gtgggatgtt agagctctag acattaattc 7436 ctaggaatcg catacctacg gaaaacagtc ctctgacctc ctgtgacaca tggggtgagc 7496 ccttcctttt tgtctcaatc tcaaaaaaca gtaagtcttt aatccatttg catcaaaaag 7556 tacttcatat gotcgaaact aaagtaaaac tttattgaaa tacattaata tgctcctgga 7616 acatactact gggtctcaga cagtgcagaa gctttattgt ttatttggga agagcaaggt 7676 aagaatcaag tagaaatgat aaagggcaag gaaaaaagat gaaagcttac tcatattaac 7736 cattctacca ttggaattat ttgccaacac accttgctgc tacttagaga aagtagttca 7796 cctcactacc ttttatccaa agaaattatc totaaaagca ctcagacatt ttgtagagca 7856 agtagcaatc tattcaaagt tgtaaaggtt ctgtagaatc tctcagacca ggtacaggac 7916 ctaccaaagg ccacagccaa gcacaagcag ctacattaga cagttttaca gctctgtatt 7976 tcaggaaaac ttctgtcctg tgggagcaat acaaaactat accagttttt tgttagtgta 8036 aaaattgcCC aatattaacc agagcagcccaaaatatttc agggtaagaa tagatatatt 8096 tatatttttc aqatqatata tcttcatttt cqattttqaa aqaacaqtaa tactaattat 8156 atcccatgta aggggctact gacaattttg atggtacctg aatttgcctc tatcatgcat 8216 ctcaatgatt tgttgtcatc caaagctatt tcatgaatca aatatcgttt tctacctgcc 8276 ccacaactgt gtacataaaa cctaaacctc tgaagcaata aacctcttcc attacacagg 8336 tttagattca gagttttctt gottaagttc caactaaaag tattacattc ttagcataag 8396 tatactcata aagaaaaata agtatttgtt ttaggtttta gagagagagc acagagtccc 8456 tttgagacag tggggaaaat tcatcttcat attgtcacat gcactgtaat aggaatgttt 8516 agcaaaaaaa accttccaga gaaaggtggt ttccaatatt acctacaact tcctttgcaa 8576 tttgattttt gaaaggacct aaaagttgaa aacaggctat cacatcccat ttgctttaaa 8636 gtctcttaaa cttacgctct ttcgcttcaa atgcataaat gttttattta agtttgcatt 8696 gcccactaag gctagacatt tttttttttt tttttttttt tttttttttg agacagagtc 8756 tcgctctgtc gcccaggctg gagtacagtg gcgggatctc ggctcactgc aagctccgcc 8816 tcccgggttc acgccattct cctgcctcag cctcccaagt agctgggact acaggcgccc 8876 gccactacgc ccggctaatt ttttgtattt ttagtagaga cggggtttca ccgttttagc 8936 cgggatgatc togatctcct gacctcgtga tccgcccgcc tcggcctccc aaagtgctgg 8996 gattacaggc gtgagccacc gcgcccggcc cggctagaca ttttttgata aattcacagg 9056 gttacaaaat accaaacgga aatgagataa gtggtataaa ccacagaaga tataggagaa 9116 gagaaaaaaa aaagaggaaa taaagaagac aactcttttc ctaagagtct gggtaaaatt . 9176 gaacatagcc atattcactg aacaacatga gtgagcttca ttaatttaag cacagcaaaa 9236 ctgctttaat taacaagacc agagagaagg gagaggagac tacatttgtg tgacctaatg 9296 gttgtgattt cactgtccaa gaggacaaag acaaagaaat tctgggaagg agaacaacaa 9356 ttatattccc ccatttcaag aagggcagaa gtgtcccaac actacccaat atttgcaaaa 9416 ttcaaatgtc tcataggctc ttcttccctg gttccctcag gagctgggtt tctgggttgc 9476 agaagtgctt ttcatattct gtatctggtt gtggtggcaa tgtcaccacc ctacactget 9536 US 2018 /0371542 A1 Dec . 27 , 2018 36

- continued gtgacaccga aacaaccaag cctagaatca gctggtgcct cttttcatct gcagggtaga 9596 tttggcttcc atggttgttc actgctctgt gttaggaagg ctcagtgaca ggtgtacagc 9656 cttcagtaat gcctcaaagg ttctccaagc agaggtaaac atgtgggtcc tgctggtgac 9716 atattagact tcttactttc cccaaataaa aaagtgcctg ctgggcgcgg tggctcacgc 9776 ctgtaattcc agcactttgg gaggccgagg cgggcggaac acaaggtcag gagatcaaga 9836 ccatcctggc caatatggta aaaccccatctctactaaaa atacaaa att aggaaggcgt 9896 ggtggtgcac gcctgtaatc ccagctagtc gggaggctga ggcaggagaa ttgottgaac 9956 tggggaggcg gaagttgcag tgagccaaga tcgcagcatt gcactccagc ctgggcaaca 10016 gaatgagatt gtctcaaaaa aaaaaaaaag tgccacatgc catgctatgt gcccaaagtt 10076 tccttcacac aacacagcct tgagatgcag tattaaattc tacacttttc ctaccatagt 10136 gatacatgtg gottttcttt gctgtgttct gagatgtcat gotttgaaat cagtggccat 10196 tatcatctaa ggattccgcc agagacttcc aaaagaagag gtctcattta taaagtgaat 10256 ttgaataaaa tgaccagtta ggtgttttca gaaacaccta tgccctactt gcctactctt 10316 caagggttta ggggcttagg gggaggtttt gtttgggttt tttgttgctg ttgottgttt 10376 atttgtttgt tgtgtttaag acgttttact tgtccctgaa atgtttgtca tcacacagat 10436 acacgctcag gataagaact accagactag attaggaggt ccacaccacc aattgagatg 10496 tacctgtgct catgacttga cattgtggtg ggccggctac aaccctcccc acccctcgct 10556 ttcactaaat aacaactctc ttctctccat cattttgact tagagccagt cagaattcaa 10616 tctccaatat cctgactagc acaagaaatc cataggttga ttcttgttct cctgcatctc 10676 tgcaggtggc aaacctgatt cctaatgcct gttcctgcct ctgcaggggt tcattcagaa 10736 aacaggaaaa taacaaaggc ttcctgtaat tctctttggc tgtaatacaa tttgttcccg 10796 tctgccccca ggctcaccca gtgctctgtc tcagtggtaa gotgtaactg atcactgctg 10856 tattaactca a aactcattt qctttatqqa aattcatqac cottatttct caaqqqacca 10916 gagaaaacca ataggcctac tccccagctg agtactttcc atgcaagcta ccgcatctgt 10976 ataaattagg ttcaaataac agagtatttc caggatttat aaattcagta ttacaaatag 11036 taatctggca agtgttctca ggatcccctt gctacctgta attcaatcat ataacttctg 11096 aatgggctgg gggaaaataa caataagaaa aactggtgtt tacctgaaga tctgcccagt 11156 gatttgtgtg ttttcttaat aaactttacc cacttattaa aagaataaaa tgaaggtgga 11216 gttaattctg actacgggat tcctttttca cttttataat gaactccttc cttctaacta 11276 aatcttatca taaqcaaato tatqcaccaa attatttaqt acaattccta ataacaqeta 11336 aaggaccatt tatttgaagc aatgttcacc atagcaaaat tccagtgaag totaagaact 11396 gggacagtcc gttgaggatc cttgtgccag gatgtatgtt gccccatgaa tgtgcacatg 11456 catattaaaa tatqqqcacc tcttttaatt cttttttttc tcataataaq tttqaaactc 11516 acagtaggaa attgagagat caatttggtt actgttttat cattgatcct gaagacagtt 11576 gaagcaatca tactggttgt tctcgaacta gctggtttcc cagagacagc tggagactga 11636 gcacataaag acatcattga ggaaaaaggc taccttgtac ctcatggaga gotgaaggtc 11696 tgataaatgg gaactgccag gtaatagcta tgctatttct gacataaatt taaaaactag 11756 tattgtttct tctagctctg tttttgcata gtgcacagag atctttgtaa aaaacaggaa 11816 US 2018 /0371542 A1 Dec . 27 , 2018 37

- continued attaatgtta aattggatct ataaacataa gtcaatttgg ctctattatg tcaaaagaga 11876 ataqqagttt taacttatat ctqtqtttta ttaatatttt qaagtatagg aacctcatag 11936 tgtagcagga tgagccacag acaaaacctc tcagacaccg agttgtagaa ggaagggctt 11996 tattcagctg ggagcatcga ccagctactg tctcaaaatc caagctccct gagtacacaa 12056 tttctgtccc ttttaagggc tcacaacact agatttcaca tgaaagggtc gtgattgatt 12116 tgagcaagca aggggtatgt gacaggggct gcatgcaccg gtggtctggg aggaacagaa 12176 caggacaggg agttcttcta tacaatagag aacagaacaa tgttcttcta tacaatgtaa 12236 ggaatctatg aataacatcg gcttctaaat cataagttga tttttaacta ctgggtttag 12296 gccaggcggg cccaggcctg gtttcgggcc tggcgctgag ctgcctgtat ttggttttac 12356 ttccttgttg tttttactga atatgaaaca atataaaaca atgtgagagg gtctttctct 12416 cctctcaatg tcaacatcat atatgattgg agacttccac ataattgagt tttagtgccc 12476 actgttacag aaaatcataa tggaaaaact aaaatgtcaa taataatttc agatgtgtat 12536 tttagttctc ataagaacat ctacattcat ttgaaaaata gttctatatc tattcttgaa 12596 acatatttct ttagttcaag gtctgatgag ctcccagact gttggggatt gctctacagc 12656 tgctcaagct tttgaagact cctgtgattt ttttaaatgg ctggtttggt tgaagtttct 12716 cttatcagtc aggcactttg cattttaagc gtactttacc accgacacoc tcccccccca 12776 gcacacacac acacacacac acacacacac acacaacata gtgaaatgga CCcgtgggaa 12836 ttatatgata gttgtaatca aaataaaatg caatcaatac taaaatacaa tttaccaaag 12896 gottacctcc ttatttgaaa ctccagcatc aataatttac ttgcactctt gttatttaca 12956 tttgtactct ggaagtaaac ttaaaatgaa aattagaatt tgctttcaat tatactatct 13016 ctatctaaat cttaatttga aatttaaatt attttqtctc tacccaaacc atcqatttca 13076 tggaaatgtt taaattttct tttttttttt tttttttgat ggagtctcac tctgtcgccc 13136 aggctggagt gcagtggctc aatcttggct cactgcaacc tctgcctccc gggttcacac 13196 cattctcctg cttcagcctc ctgagtagct gggactacag gtgcccgcca caacacctgg 13256 ctaatttttt gtatttttag tagagatggg gtttcaccac gttagccagg atggtttcga 13316 tctcctgacc tcgtgatctg cctgcctcgg cctcccaaag tgttgggatt acaggtgtga 13376 gccactgcgc ctggccagaa atgtttaaaatttcataaat cttgaatcca taaaattcaa 13436 tqctaccatt tataatttaa tactcctacc ataaaaattt ctgtattaqa cataqctata 13496 tgaatatttg cctata aatccccatcaatt taataggaat taagttagaa atactagtat 13556 atatattccc tttatatact aattgtatat ccatataaaa gcattagtac cattatatga 13616 aagtatatat gccattccat aaaaatatat ctaccaatat aaatagaata tataaaggga 13676 atatatatat accaatataa atqaqaattt atattqacac atacatttcc attattttaa 13736 tgggaattaa agaaaaaatg cctgttttca ctaagtcatc cttcccctgg caatacattt 13796 cctgaacttt tacatactta aatagccagt tatgaaaatg taaaacaatg agtgatcttg 13856 ttgttttcat tttattatgt tatatgaaaa aaagaaacct tgtaagtgca gatttatttt 13916 aaaaaattga aaaactactc tgtcaaacat agcaaatagg aaagttaaaa aaaagtaagt 13976 ctaaagatta ggtgctctgt cacttgtcat tacatttttt tataacatta atgacacatt 14036 ttttgctatc caaatataat ttccactaga aaagaaaaga tgtcctccat gttttaaatt 14096 US 2018 /0371542 A1 Dec . 27 , 2018 38

- continued gcatttgaaa ttcaggtaac taataaccct tggttttaaa agatacatat aatgtttccg 14156 taggaaaaaa atcaaaatat tgtaaagaat ?tgagtaaat gagagcctct catggttgat 14216 taagttcaga cattttaaac agactcctgc ccacaaacta tttttcctct ccaggaataa 14276 gaatggcaac tgaattgttc cttctttatt ctatagcttt aagtcaaacc taacataagc 14336 aatcaaccct tccacccatt gtcctctttc tagctgctta tattcctgag totgaaagaa 14396 gggcagaatt aattogtttc cttgacagtg ttgggggtga aataaaagat agacccctgc 14456 tgctctgcac gtagattcag tttgtatgcc agggtgacat tttaatttac agtagtccag 14516 acacctaaac aggacataga aatgtcaact ggtcatatta aaataaaaaa gtatagaata 14576 gtttctgttt tacggatttt gtatccaagt aaatqaaaac acaatcacct ttcaaaacat 14636 ggtatgaaat ccacagttgt aacagtgagc acaacatgct ttctgtgtgt gtttttaagc 14696 tctttcccca catctgcttt tcaaaaatat ttgcctggaa acctgtattt cgcctataga 14756 gacaaataca tatattgttt gttgtgataa tgcacaaaaa ggaatgttaa aaaaaaacac 14816 acaacattgt tttccttttg atggtctggg agtttttcta taagtttttg gttttttttt 14876 ttttttctct tcattagtgt gttagttcca tcatcatgtc tgtttactat tgaaaaataa 14936 atatcttcat ttaaagtaga gacaaagcta ctatttcaca tttccaggta ggacaggatg 14996 atcagatgca gctttcaaaa gaactacctg ctaaatcata atggtctcat gtgcagatca 15056 aaatgtcaac tagtatatca aacagtgagcaattcactaa attattttta cattactcct 15116 atttgactgt ttcagatagt tgtatataaa aaatataaat ttttgcattg tattgttctt 15176 gatgtaccad catacataaa catattaggc tgtattgtaa gtttcctgca tgtaatatgt 15236 aatgtgtaat tatatgtgat aaataaaacc taaaactgat aca 15279

< 210 > SEQ ID NO 12 < 211 > LENGTH : 400 < 212 > TYPE : PRT < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 12 Met Asp Ser Ser Ala Ala Pro Thr Asn Ala Ser Asn Cys Thr Asp Ala

Leu Ala Tyr Ser Ser Cys Ser Pro Ala Pro Ser Pro Gly Ser Trp Val 25 30 Asn Leu Ser His Leu Asp Gly Asn Leu Ser Asp Pro Cys Gly Pro Asn 40 Arg Thr Asp Leu Gly Gly Arg Asp Ser Leu Cys Pro Pro Thr Gly Ser

Pro Ser Met Ile Thr Ala Ile Thr Ile Met Ala Leu Tyr Ser Ile Val

Cys Val Val Gly Leu Phe Gly Asn Phe Leu Val Met Tyr Val Ile Val

Arg Tyr Thr Lys Met Lys Thr Ala Thr Asn Ile Tyr Ile Phe Asn Leu 105 110

Ala Leu Ala Asp Ala Leu Ala Thr Ser Thr Leu Pro Phe Gln Ser Val 120 Asn Tyr Leu Met Gly Thr Trp Pro Phe Gly Thr Ile Leu Cys Lys Ile

Val Ile Ser Ile Asp Tyr Tyr Asn Met Phe Thr Ser Ile Phe Thr Leu US 2018 /0371542 A1 Dec . 27 , 2018 39

- continued 145 150 155 160 Cys Thr Met Ser Val Asp Arg Tyr Ile Ala Val Cys His Pro Val Lys 170 175 Ala Leu Asp Phe Arg Thr Pro Arg Asn Ala Lys Ile Ile Asn Val Cys Asn Trp Ile Leu Ser Ser Ala Ile Gly Leu Pro Val Met Phe Met Ala 195 200 205 Thr Thr Lys Tyr Arg Gin Gly Ser Ile Asp Cys Thr Leu Thr Phe Ser

His Pro Thr Trp Tyr Trp Glu Asn Leu Leu Lys Ile Cys Val Phe Ile 225 230 235 Phe Ala Phe Ile Met Pro Val Leu Ile Ile Thr Val Cys Tyr Gly Leu 250 255 Met Ile Leu Arg Leu Lys Ser Val Arg Met Leu Ser Gly Ser Lys Glu

Lys Asp Arg Asn Leu Arg Arg Ile Thr Arg Met Val Leu Val Val Val 275 280 285 Ala Val Phe Ile Val Cys Trp Thr Pro Ile His Ile Tyr Val Ile Ile

Lys Ala Leu Val Thr Ile Pro Glu Thr Thr Phe Gln Thr Val Ser Trp 305 310 315 His Phe Cys Ile Ala Leu Gly Tyr Thr Asn Ser Cys Leu Asn Pro Val 330 335 Leu Tyr Ala Phe Leu Asp Glu Asn Phe Lys Arg Cys Phe Arg Glu Phe

Cys Ile Pro Thr Ser Ser Asn Ile Glu Gin Gin Asn Ser Thr Arg Ile 355 360 365 Arg Gln Asn Thr Arg Asp HisHis Pro SerSer Thr AlaAla Asn Thr Val Asp Arq

Thr Asn His Gin Leu Glu Asn Leu Glu Ala Glu Thr Ala Pro Leu Pro 385 390 395 400

What is claimed is : and has at least one allele encoding an aspartic acid 1 . A method for treating a subject with a disorder asso at an amino acid position corresponding to amino ciated with opioid receptor activity , the method comprising : acid residue 40 of the OPRM1 gene product of SEQ ( a ) performing or having performed one or more geno ID NO : 12 ; or typing assays on nucleic acids isolated from the subject ( iii ) the subject has at least one allele encoding a VNTR with respect to an opioid mu receptor (OPRM1 ) gene 9 - repeat and has two alleles encoding an asparagine product and also a dopamine (DA ) - catabolizing at an amino acid position corresponding to amino enzyme catechol- O -methyltransferase ( COMT) gene acid residue 40 the of OPRM1 gene product of SEQ product and / or a variable number tandem repeat ID NO : 12 ; or (VNTR ) polymorphism in the dopamine transporter ( iv ) the subject is homozygous for a VNTR 10 - repeat gene DAT1 /SLC6A3 ; and and has at least one allele encoding an aspartic acid (b ) administering an opioid receptor antagonist therapy to at an amino acid position corresponding to amino the subject, wherein : acid residue 40 of the OPRM1 gene product of SEQ ( i ) the subject has at least one allele encoding a methio nine at an amino acid position corresponding to ID NO : 12 . amino acid residue 158 of the COMT gene product 2 . The method of claim 1 , wherein the disorder associated of SEQ ID NO : 8 and has two alleles encoding an with opioid receptor activity is an alcohol use disorder asparagine at an amino acid position corresponding (AUD ) . to amino acid residue 40 the ofOPRM1 gene product 3 . The method of claim 1 , wherein at least one of the one of SEO ID NO : 12 ; or or more genotyping assays is performed prior to adminis ( ii ) the subject is homozygous for a valine at an amino tering the opioid receptor antagonist therapy to the subject . acid position corresponding to amino acid residue 4 . The method of claim 1 , wherein at least one of the one 158 of the COMT gene product of SEQ ID NO : 8 or more genotyping assays is performed after administering US 2018 /0371542 A1 Dec . 27 , 2018 40 the opioid receptor antagonist therapy to the subject, and ing to amino acid residue 40 the of OPRM1 gene further wherein the opioid receptor antagonist therapy is product of SEQ ID NO : 12 ; or discontinued if: ( d ) the subject is homozygous for a VNTR 10 -repeat ( v ) the subject has at least one allele encoding a methio and has at least one allele encoding an aspartic nine at an amino acid position corresponding to amino acid at an amino acid position corresponding to acid residue 158 of the COMT gene product of SEQ ID amino acid residue 40 of the OPRM1 gene product NO : 8 and has at least one allele encoding an aspartic of SEQ ID NO : 12 ; and acid at an amino acid position corresponding to amino ( ii ) the subject is not susceptible to an opioid receptor acid residue 40 of the OPRM1 gene product of SEQ ID antagonist if: NO : 12 ; or ( e ) the subject has at least one allele encoding a ( vi) the subject is homozygous for a valine at an amino methionine at an amino acid position correspond acid position corresponding to amino acid residue 158 ing to amino acid residue 158 of the COMT gene of the COMT gene product of SEQ ID NO : 8 and is product of SEQ ID NO : 8 and has at least one homozygous for an asparagine at an amino acid posi allele encoding an aspartic acid at an amino acid tion corresponding to amino acid residue 40 the of position corresponding to amino acid residue 40 OPRM1 gene product of SEQ ID NO : 12 ; or of the OPRM1 gene product of SEQ ID NO : 12 ; ( vi) the subject has at least one allele encoding a VNTR or 9 - repeat and has at least one allele encoding an aspartic ( f ) the subject is homozygous for a valine at an acid at an amino acid position corresponding to amino amino acid position corresponding to amino acid acid residue 40 the of OPRM1 gene product of SEQ ID residue 158 of the COMT gene product of SEQ ID NO : 12 ; or NO : 8 and is homozygous for asparagine at an ( viii ) the subject is homozygous for a VNTR 10 -repeat amino acid position corresponding to amino acid and is homozygous for an asparagine at an amino acid residue 40 the of OPRM1 gene product of SEQ ID position corresponding to amino acid residue 40 of the OPRM1 gene product of SEQ ID NO : 12 . NO : 12 ; or 5 . The method of claim 1 , wherein the opioid receptor ( g) the subject has at least one allele encoding a antagonist is naltrexone . VNTR 9 - repeat and has at least one allele encod 6 . The method of claim 1 , wherein the genotyping assay ing an aspartic acid at an amino acid position comprises a nucleic acid amplification process followed by corresponding to amino acid residue 40 the of sequencing or gel electrophoresis of an amplification prod OPRM1 gene product of SEQ ID NO : 12 ; or uct produced thereby . ( h ) the subject is homozygous for a VNTR 10 -repeat 7 . A method for detecting susceptibility of a subject to an and is homozygous for an asparagine at an amino opioid receptor antagonist for a disorder associated with acid position corresponding to amino acid residue opioid receptor activity in the subject, the method compris 40 of the OPRM1 gene product of SEQ ID NO : ing : 12 . ( a ) obtaining a biological sample from the subject; and 8 . The method of claim 7 , wherein the opioid receptor ( b ) performing or having performed one or more geno antagonist is naltrexone . typing assays on the biological sample from the subject 9 . The method of claim 7 , wherein at least one of the one with respect to an opioid mu receptor (OPRM1 ) gene or more genotyping assays comprises a nucleic acid ampli product and also a dopamine (DA ) - catabolizing fication process followed by sequencing or gel electropho enzyme catechol- O -methyltransferase (COMT ) gene resis of an amplification product produced thereby . product and /or a variable number tandem repeat 10 . A method for identifying and treating a human subject (VNTR ) polymorphism in the dopamine transporter having susceptibility to an opioid receptor antagonist gene DAT1/ SLC6A3 ; therapy for a disorder associated with opioid receptor activ wherein : ity in the subject , the method comprising: ( i) the subject is susceptible to an opioid receptor ( a ) obtaining a nucleic acid sample from a human subject ; antagonist if: ( b ) performing or having performed on the nucleic acid ( a ) the subject has at least one allele encoding a sample one or more genotyping assays to determine methionine at an amino acid position correspond genotypes of the subject with respect to an opioid mu ing to amino acid residue 158 of the COMT gene receptor (OPRM1 ) gene product and either or both of product of SEO ID NO : 8 and has two alleles a dopamine (DA ) -catabolizing enzyme catechol - O encoding an asparagine at an amino acid position methyltransferase (COMT ) gene product and a variable corresponding to amino acid residue 40 the of number tandem repeat (VNTR ) polymorphism in the OPRM1 gene product of SEQ ID NO : 12 ; or dopamine transporter gene DAT1/ SLC6A3 ; and ( b ) the subject is homozygous for a valine at an (c ) administering an opioid receptor antagonist to the amino acid position corresponding to amino acid subject if the genotypes determined indicate that the residue 158 of the COMT gene product of SEQ ID subject: NO : 8 and has at least one allele encoding an ( i) has at least one allele encoding a methionine at an aspartic acid at an amino acid position correspond amino acid position corresponding to amino acid ing to amino acid residue 40 of the OPRM1 gene residue 158 of the COMT gene product of SEQ ID product of SEQ ID NO : 12 ; or NO : 8 and has two alleles encoding an asparagine at ( c ) the subject has at least one allele encoding a an amino acid position corresponding to amino acid VNTR 9 - repeat and has two alleles encoding an residue 40 the of OPRM1 gene product of SEQ ID asparagine at an amino acid position correspond NO : 12 ; or US 2018 /0371542 A1 Dec. 27 , 2018

( b ) is homozygous for a valine at an amino acid amine (DA )- catabolizing enzyme catechol- O -methyl position corresponding to amino acid residue 158 of transferase ( COMT) gene and a dopamine transporter the COMT gene product of SEQ ID NO : 8 and has DAT1/ SLC6A3 gene; and at least one allele encoding an aspartic acid at an ( b ) administering an opioid receptor antagonist therapy to amino acid position corresponding to amino acid the subject when : residue 40 of the OPRM1 gene product of SEQ ID ( i) the subject' s genotype has two alleles encoding an NO : 12 ; or asparagine at an amino acid position corresponding ( c ) has at least one allele encoding a VNTR 9 - repeat to amino acid residue 40 the of OPRM1gene product and has two alleles encoding an asparagine at an of SEQ ID NO : 12 and has at least one allele amino acid position corresponding to amino acid encoding a methionine at an amino acid position residue 40 the of OPRM1 gene product of SEQ ID corresponding to amino acid residue 158 of the NO : 12 ; or COMT gene product of SEQ ID NO : 8 or at least one ( d ) is homozygous for a VNTR 10 - repeat and has at allele encoding a dopamine transporter DAT1/ least one allele encoding an aspartic acid at an amino SLC6A3 9 -repeat variable number tandem repeat acid position corresponding to amino acid residue 40 (VNTR ) ; or of the OPRM1 gene product of SEQ ID NO : 12 . ( ii ) the subject ' s genotype has at least one allele encod 11 . The method of claim 10 , wherein the genotypes are ing an aspartic acid at an amino acid position cor determined prior to administering the opioid receptor responding to amino acid residue 40 the of OPRM1 antagonist. gene product of SEQ ID NO : 12 and has two alleles 12 . The method of claim 11 , wherein the genotypes are encoding a valine at an amino acid position corre determined after an opioid receptor antagonist therapy has sponding to amino acid residue 158 of the COMT commenced and the opioid receptor antagonist therapy is gene product of SEQ ID NO : 8 or two alleles discontinued if: encoding a dopamine transporter DAT1 /SLC6A3 ( e ) the subject has at least one allele encoding a methio 10 - repeat variable number tandem repeat (VNTR ) . nine at an amino acid position corresponding to amino 16 . The method of claim 15 , wherein the disorder asso acid residue 158 of the COMT gene product of SEQ ID ciated with opioid receptor activity is an alcohol use disorder NO : 8 and has at least one allele encoding an aspartic (AUD ) . acid at an amino acid position corresponding to amino 17 . The method of claim 15 , wherein at least one of the acid residue 40 of the OPRM1 gene product of SEQ ID one or more genotyping assays is performed prior to admin NO : 12 ; or istering the opioid receptor antagonist therapy to the subject. ( f ) the subject is homozygous for a valine at an amino acid 18 . The method of claim 15 , wherein at least one of the position corresponding to amino acid residue 158 of the one or more genotyping assays are performed after admin COMT gene product of SEQ ID NO : 8 and has two istering the opioid receptor antagonist therapy to the subject, alleles encoding an asparagine at an amino acid posi and further wherein the opioid receptor antagonist therapy is tion corresponding to amino acid residue 40 the of discontinued if : OPRM1 gene product of SEQ ID NO : 12 ; or ( iii ) the subject has at least one allele encoding an aspartic ( g ) the subject has at least one allele encoding a VNTR acid at an amino acid position corresponding to amino 9 - repeat and has at least one allele encoding an aspartic acid residue 40 of the OPRM1 gene product of SEQ ID acid at an amino acid position corresponding to amino NO : 12 and at least one allele encoding a methionine at acid residue 40 the of OPRM1 gene product of SEQ ID an amino acid position corresponding to amino acid NO : 12 ; or residue 158 of the COMT gene product of SEQ ID NO : ( h ) the subject is homozygous for a VNTR 10 - repeat and 8 or at least one allele for a DAT1/ SLC6A3 9 - repeat has two alleles encoding an asparagine at an amino acid variable number tandem repeat ( VNTR ); or position corresponding to amino acid residue 40 of the ( iv ) the subject is homozygous for an asparagine at an OPRM1 gene product of SEQ ID NO : 12 . amino acid position corresponding to amino acid resi 13 . The method of claim 11, wherein the opioid receptor due 40 the of OPRM1 gene product of SEO ID NO : 12 antagonist is naltrexone. and is homozygous for a valine at an amino acid 14 . The method of claim 11, wherein at least one of the one or more genotyping assays comprises a nucleic acid position corresponding to amino acid residue 158 of the amplification process followed by sequencing or gel elec COMT gene product of SEQ ID NO : 8 or is homozy trophoresis of an amplification product produced thereby. gous for a DAT1 / SLC6A3 10 - repeat variable number 15 . A method for treating a subject with a disorder tandem repeat ( VNTR ) . associated with opioid receptor activity , the method com 19. The method of claim 15 , wherein the opioid receptor prising : antagonist therapy comprises administering an effective ( a ) performing or having performed one or more geno amount of naltrexone to the subject. typing assays on nucleic acids isolated from the subject 20 . The method of claim 15 , wherein at least one of the to determine the subject' s genotype with respect to a one or more genotyping assays comprises a nucleic acid first gene and a second gene , wherein the first gene is amplification process followed by sequencing or gel elec an opioid mu receptor (OPRM1 ) gene and the second trophoresis of an amplification product produced thereby. gene is selected from the group consisting of a dop