Patent application title: Genetic variations associated with psychiatric disorders
Inventors:
Thomas Bourgeron (Paris, FR)
Jonas Melke (Goteborg, SE)
Hany Goubran Botros (Le Pre Saint Gervais, FR)
Jean-Marie Launay (Argenteuil, FR)
Marion Leboyer (Paris, FR)
Christopher Gillberg (Goteborg, SE)
Pauline Chaste (Paris, FR)
Richard Delorme (Paris, FR)
Assignees:
INSTITUT PASTEUR
IPC8 Class: AA61K3845FI
USPC Class:
424 945
Class name: Drug, bio-affecting and body treating compositions enzyme or coenzyme containing transferases (2. ), lyase (4.), isomerase (5.), ligase (6.)
Publication date: 2009-10-22
Patent application number: 20090263368
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Patent application title: Genetic variations associated with psychiatric disorders
Inventors:
Marion Leboyer
Christopher Gillberg
Thomas Bourgeron
Jonas Melke
Hany Goubran Botros
Jean-Marie Launay
Pauline Chaste
Richard Delorme
Agents:
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, L.L.P.
Assignees:
Institut Pasteur
Origin: ALEXANDRIA, VA US
IPC8 Class: AA61K3845FI
USPC Class:
424 945
Patent application number: 20090263368
Abstract:
The present invention relates to a method for determining likelihood of an
individual of developing a psychiatric disorder. More particularly, the
method of the invention is based on the identification of genetic
variations in genes involved in the modulation of melatonin and their
consequences on such pathway for the determination of whether an
individual is susceptible of being afflicted by a psychiatric disorder
such as autism spectrum disorders (ASD), Deficits and Hyper Activity
Disorder (ADHD) and anorexia.Claims:
1. A method for determining likelihood of an individual of developing a
psychiatric disorder, comprising:identifying at least one genetic
variation in a gene involved in the modulation of melatonin
and/orassaying the mRNA transcript level of said gene and/or,assaying the
enzymatic activity of an enzyme involved in the melatonin biosynthesis
pathway in a sample of said individual, and/orassaying the melatonin
level,whereby identification of a genetic variation and/or decrease in
the enzymatic activity and/or a decrease in the transcript level of said
gene and/or a decrease in melatonin level as compared to a
psychiatric-free individual, is indicative of a likelihood that said
individual develops a psychiatric disorder.
2. The method according to claim 1, wherein said gene is ASMT gene.
3. The method according to claim 2, wherein said genetic variation is a point mutation.
4. The method according to claim 3, wherein said point mutation is a mutation at position 17, 81, 210, 306 or 326 as defined by the position in SEQ ID NO:1.
5. The method according to claim 4, wherein said point mutation is a N17K mutation.
6. The method according to claim 4, wherein said point mutation is a K81E mutation.
7. The method according to claim 4, wherein said point mutation is a R210H mutation.
8. The method according to claim 4, wherein said point mutation is a G306A mutation.
9. The method according to claim 4, wherein said point mutation is a L326F mutation.
10. The method according to claim 2, wherein said genetic variation is a splice site mutation.
11. The method according to claim 10, wherein the splice site mutation is IVS5+2T>C.
12. The method according to claim 2, wherein said genetic variation is located in a single nucleotide polymorphism (SNP) of said ASMT gene.
13. The method according to claim 12, wherein said SNP is Rs5989681 or Rs6588809.
14. The method according to claim 3, wherein said point mutation is a mutation at position 17, 81, 228 or 298 as defined by the position in SEQ ID NO:2.
15. The method according to claim 3, wherein said point mutation is a mutation at position 17, 81, 231 or 251 as defined by the position in SEQ ID NO:3.
16. The method according to claim 1, wherein said gene is AA.NAT gene.
17. The method according to claim 16, wherein said genetic variation is a point mutation.
18. The method according to claim 17, wherein said point mutation is a mutation at position 3, 13, 62, 157 or 163 as defined by the position in SEQ ID NO:4.
19. The method according to claim 18, wherein said point mutation is a T3M mutation.
20. The method according to claim 18, wherein said point mutation is a A13S mutation.
21. The method according to claim 18, wherein said point mutation is a V62I mutation.
22. The method according to claim 18, wherein said point mutation is a A157V mutation.
23. The method according to claim 18, wherein said point mutation is a A163V mutation.
24. The method according to claim 1, wherein said enzyme consists of HIOMT (Hydroxyindole-O-methyltransferase) or functional derivatives thereof.
25. The method according to claim 1, wherein said gene is a gene coding for a melatonin receptor.
26-42. (canceled)
43. The method according to claim 25, wherein the melanine receptor consists of MTNR1A or MTNR1B.
44. The method according to claim 25, wherein said genetic variation is a point mutation.
45. The method according to claim 44, wherein said point mutation is a Y170X mutation in the MTNR1A receptor as defined by the position in SEQ ID NO: 5.
46. The method according to claim 1, wherein said psychiatric disorder is selected from the group consisting of autism spectrum disorders (ASD), Attention Deficits and Hyper Activity Disorder (ADHD) and anorexia.
47. A composition for treating and/or preventing a psychiatric disorder in an individual having a defective gene involved in the modulation of melatonin, comprising an acceptable carrier and a therapeutically effective amount of a molecule promoting melatonin modulation.
48. The composition according to claim 47, wherein said molecule is a polypeptide.
49. The composition according to claim 48, wherein said polypeptide is an enzyme.
50. The composition according to claim 49, wherein said enzyme is HIOMT (Hydroxyindole-O-methyltransferase) enzyme or functional derivatives thereof.
51. A method for treating and/or preventing a psychiatric disorder in an individual having a defective gene involved in the modulation of melatonin comprising the administration in said individual of a composition as defined in claim 47.
52. The method according to claim 51, wherein said psychiatric disorder is selected from the group consisting of autism spectrum disorders (ASD), Attention Deficits and Hyper Activity Disorder (ADHD) and anorexia.
53. A method for treating and/or preventing ASD in an individual comprising the administration in said individual of a composition comprising melatonin or a functional derivative thereof.
54. An isolated polynucleotide encoding a polypeptide or functional derivative thereof, wherein said polypeptide or functional derivative thereof comprises a point mutation in the amino acid sequence as defined in SEQ ID NO:1.
55. An isolated polynucleotide encoding a polypeptide or functional derivative thereof, wherein said polypeptide or functional derivative thereof comprises a point mutation in the amino acid sequence as defined in SEQ ID NO:4.
56. An isolated polynucleotide encoding a polypeptide or functional derivative thereof, wherein said polypeptide or functional derivative thereof comprises a point mutation in the amino acid sequence as defined in SEQ ID NO:5.
57. An isolated polynucleotide comprising a regulatory sequence of the ASMT gene, wherein said regulatory sequence comprises an insertion/deletion mutation compared to the wild type regulatory sequence of said ASMT gene.
58. An isolated polynucleotide comprising a regulatory sequence of the AANAT gene, wherein said regulatory sequence comprises an insertion/deletion mutation compared to the wild type regulatory sequence of said AANAT gene.
59. The isolated polynucleotide according to claim 57, wherein said regulatory sequence consists of a promoter.
60. An isolated polypeptide encoded by a polynucleotide as defined in claim 54.
61. The method according to claim 43, wherein said genetic variation is a point mutation.
62. The isolated polynucleotide according to claim 58, wherein said regulatory sequence consists of a promoter.
63. An isolated polypeptide encoded by a polynucleotide as defined in claim 55.
64. An isolated polypeptide encoded by a polynucleotide as defined in claim 56.
Description:
FIELD OF THE INVENTION
[0001]The present invention relates to methods for determining likelihood of an individual of developing a psychiatric disorder. More particularly, the method of the invention is based on the identification of genetic variations in genes involved in the modulation of melatonin and their consequences on such pathway for the determination of whether an individual is susceptible of being afflicted by a psychiatric disorder such as autism, Deficits and Hyper Activity Disorder (ADHD) and anorexia and for the treatment of such a psychiatric disorder.
BRIEF DESCRIPTION OF THE PRIOR ART
Autism Spectrum Disorders (ASD) and Attention-Deficit/Hyperactivity Disorder (ADHD)
[0002]ASD and ADHD are among the most predominant psychiatric syndromes in children and seem to be strongly influenced by genes 1-4. ASD is characterised by impairments in communication skills, social interaction and restricted, repetitive and stereotyped patterns of behaviour 5. ASD includes autistic disorder (classical autism), disintegrative disorder, pervasive development disorder not otherwise specified (PDD-NOS) and Asperger syndrome. The recurrence risk of autism in sib-ships is approximately 45 times greater than in the general population and twin studies have documented a higher concordance rate in monozygotic (60%-91%) than in dizygotic twins (0%-6%)1. Furthermore, approximately 10% of individuals with autism have chromosomal rearrangements (e.g. duplication of 15q) or mutations in genes associated with other syndromes such as FMR1 (Fragile X syndrome), TSC1 and TSC2 (tuberous sclerosis) or NF1 (Neurofibromatosis).
[0003]ADHD is the most commonly diagnosed behavioural disorder in childhood affecting a relatively stable rate of 8-12% of all young children and likely represents an extreme of normal behaviour 6. It is a condition characterized by behavioural symptoms of inattention and/or hyperactivity-impulsivity, with onset in childhood 5. Such symptoms include restlessness, difficulty with organizing tasks, distractibility, forgetfulness, difficulty awaiting turns, and frequent interrupting. ADHD significantly impacts learning in school-age children and leads to impaired functioning throughout the life span. Family, twin, and adoption studies provided compelling evidence that genes play a strong role in mediating susceptibility to ADHD 3,4.
[0004]Despite the clear clinical boundaries between these two disorders, there are behavioural, cognitive, and neurobiological deficits that suggest some degree of phenotypic overlap such as maladaptive social functioning and executive function deficits7,8. Furthermore, there is a significant subgroup of children with ASD and symptoms of ADHD who respond to stimulant medication9, suggesting that common neurobiological mediators may be present in a subset of cases. Shared susceptibility genes for ASD and ADHD were also suggested from the results of genetic studies, which pointed at the same chromosomal regions using linkage analyses (e.g. 16p13 and 17p11)10 or chromosomal rearrangements (e.g. Xp22.3)11,12 13.
ASMT (Acetyl Serotonin Methyl Transferase) Gene and AA NAT (Arylalkylamine Acetyltransferase) Gene.
[0005]ASMT gene is located on the pseudo autosomal region 1 (PAR1), common to the X and the Y chromosome 14 ASMT encodes the enzyme HIOMT (Hydroxyindole-O-methyltransferase; EC 2.1.1.4), which catalyses the last step of melatonin biosynthesis. Melatonin (N-acetyl-5-methoxytryptamine) is the major secretory product of the pineal gland. It is produced through the conversion of tryptophan, first to serotonin, then N-acetylserotonin, and finally to melatonin15 16. The synthesis of melatonin exhibits a pronounced circadian rhythm: its concentration in the body is typically lower during the day and reaches a maximal level at night in the darkness15. The regulation of melatonin production in the pineal gland involves especially norepinephrine (NE) couples to beta-adrenergic receptors17. Melatonin has been functionally linked to the regulation of circadian and seasonal rhythms, immune function, and is a powerful free radical scavenger and antioxidant 15,18. Nevertheless, the consequence of the clock and calendar information that the melatonin cycle imparts to the organism is still not fully understood.
[0006]The enzyme AA.NAT is located before ASMT in the biosynthesis pathway and transforms the serotonin into N. acetyl serotonin.
[0007]A typical circadian rhythms are frequently observed in individuals with neuropsychiatric disorders and numerous studies have reported abnormal synthesis of melatonin or benefit from treatment with melatonin 19-21 22-25 26. WO 02/076452 and WO 2004/028532 describe the use of melatonin in the treatment of ADHD. However, neither of these studies could discriminate between a cause or a consequence of the disorders.
The Melatonin Receptors
[0008]In humans, two distinct melatonin receptors MTNR1A and MTNR1B have been reported so far. Both types of receptors were identified in a wide variety of tissues with different expression profiles. A third melatonin related receptor GPR50 was very recently identified. Although this receptor is structurally related to the melatonin receptors, with a 45% homology at the amino acid levels, it does not bind to melatonin. Nevertheless, the heterodimer MTNR1A/GPR50 abolishes high-affinity agonist binding and G protein coupling to the MTNR1A.
[0009]Activation of the MTNR1A leads to inhibition of forskolin stimulated cAMP formation, PKA activity, and phosphorylation of the cAMP-responsive element binding protein, a transcription factor. Activation of the MTNR1A also increases phosphorylation of mitogen-activated protein kinase and MEK1-2 and ERK1/2 probably leading to induction of synthesis of filamentous structures in non-neuronal tissues. Melatonin induction of filamentous structures in non-neuronal cells is dependent on expression of the human MTNR1A melatonin receptor. MTNR1A is coupled to parallel signal transduction pathways and regulates functional responses of melatonin in ion channels. Similar to the second messenger pathways of the MTNR1A receptor, activation of the MTNR1B also inhibits forskolin stimulated cAMP formation. Additionally coupling to this receptor can also lead to inhibition of cGMP formation.
[0010]Deletion of the large C-terminal tail of GPR50 suppresses the inhibitory effect of GPR50 on MT1 without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT1. This effect appears to be specific for the GPR50/MT1 heterodimer since it was not observed for heterodimers with the closely related MT2. In mammals this receptor has been detected in various brain structures and peripheral tissues.
[0011]In humans, MTNR1A and MTNR1B were found in the hippocampus, throughout the cerebellar cortex in distinct cell populations. MTNR1A is localized in various areas related to dopaminergic behaviors, including Brodmann area 10 (i.e. prefrontal cortex), putamen, substantia nigra, amydala, and hippocampus. Melatonin may exert inhibitory effects, especially in the night, where melatonin levels are high.
[0012]There is thus a need for new tools marker associated with susceptibility to psychiatric disorders. Consequently, on the bases of both genetics and biochemical data, the inventors of the present invention propose that mutations in the ASMT gene cause an absence or a decrease of melatonin and confer an increased risk to neuropsychiatric disorders such as ASD and ADHD. The inventors thus demonstrate that some genetic variations in genes involved in melatonin biosynthesis cause an absence or a decrease of melatonin during childhood and adulthood and confer a risk to psychiatric disorders such as autism, ADHD and anorexia and thus provide new diagnostic and treatment methods with regards to such psychiatric disorders.
SUMMARY OF THE INVENTION
[0013]One aspect of the invention is to provide a method for determining likelihood of an individual of developing a psychiatric disorder, comprising: [0014]identifying at least one genetic variation in a gene involved in the modulation of melatonin and/or [0015]assaying the mRNA transcript level of said gene and/or, [0016]assaying the enzymatic activity of an enzyme involved in the melatonin biosynthesis pathway in a sample of said individual, and/or [0017]assaying the melatonin level,whereby identification of a genetic variation and/or decrease in the enzymatic activity and/or a decrease in the transcript level of said gene and/or a decrease in melatonin level as compared to a psychiatric-free individual, is indicative of a likelihood that said individual develops a psychiatric disorder.
[0018]Another aspect of the invention concerns a composition for treating and/or preventing a psychiatric disorder in an individual having a defective gene involved in the modulation of melatonin, comprising an acceptable carrier and a therapeutically effective amount of a molecule promoting melatonin biosynthesis.
[0019]A further aspect of the invention is to provide a method for treating and/or preventing a psychiatric disorder in an individual having a defective gene involved in the modulation of melatonin comprising the administration in said individual of a composition of the invention.
[0020]The invention also provides the use of a composition comprising an acceptable carrier and a therapeutically effective amount of a molecule promoting melatonin biosynthesis, for the preparation of a medicament for treating and/or preventing a psychiatric disorder in an individual having a defective gene involved in the modulation of melatonin.
[0021]Yet, another aspect of the invention is to provide a method for treating and/or preventing ASD in an individual comprising the administration in said individual of a composition comprising melatonin or a functional derivative thereof.
[0022]The invention also provides the use of a composition comprising melatonin or a functional derivative thereof for the preparation of a medicament for treating and/or preventing ASD in an individual.
[0023]Yet a further aspect of the invention is to provide an isolated polynucleotide encoding a polypeptide or functional derivative thereof, characterized in that said polypeptide or functional derivative thereof comprises a point mutation in the amino acid sequence as defined in SEQ ID NO: 1, 4, 5 or 6.
[0024]A further aspect of the invention is to provide an isolated polynucleotide comprising a regulatory sequence of the ASMT gene or of the AANAT gene, characterized in that said regulatory sequence comprises an insertion/deletion mutation compared to the wild type regulate sequence of said ASMT gene or of said AANAT.
[0025]Another aspect of the invention concerns an isolated polypeptide encoded by a polynucleotide of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0026]FIG. 1: Linkage and association of ASMT with ASD and ADHD. a. Linkage analysis of the PAR1 using 71 families with ASD. b. Linkage disequilibrium map of the ASMT gene. c. Association study of the ASMT gene in ASD and ADHD.
[0027]FIG. 2: Analysis of the ASMT mRNA in BLCL from ASD and controls. a. relative proportion of ASMT isoforms. Insert. ASMT isoforms from a control (C5), two ASD (A39 and A40), and cDNA from pineal gland. b. Relative abundance of ASMT correlated with rs4446909 genotype. c. Relative abundance of ASMT correlated with rs5989681 genotype. d. Promoter B sequence of the ASMT gene (SEQ ID NO:27 and SEQ ID NO:28). Rare and frequent variations identified in ASD are indicated in red and purple, respectively.
[0028]FIG. 3: Biochemical analyses in blood and BLCL from ASD, parents and controls. a. Serotonin concentration in the blood. b. ASMT activity in blood platelets. c. Melatonin concentration in the blood. d. ASMT activity in BLCL. C1 control age-matched for parents; C2 control age-matched for ASD.
[0029]FIG. 4: ASMT transcript level and activity in individuals with ASD and controls. ASD with GG and CG genotype at rs5989681 are in red and orange circle, respectively. Controls with GG and CG genotype at rs5989681 are in green and blue square, respectively. Individuals with low medium ASMT isoform and non-synonymous mutations are indicated by double lines and arrows, respectively.
[0030]FIG. 5: Position and segregation of the ASMT mutations in families with ASD, ADHD and anorexia.
[0031]FIG. 6: Melatonin concentration in the boy with ASD and their parents during the night. 0 is 20h30.
[0032]FIG. 7: Amino acid sequence of the HIOMT enzyme alternatively spliced with exon 6 and 7 (SEQ ID NO: 1; NCBI accession number AAA75290).
[0033]FIG. 8: Amino acid sequence of the HIOMT enzyme alternatively spliced with exon 7 and lacking exon 6 (SEQ ID NO: 2; NCBI accession number AAA75291).
[0034]FIG. 9: Amino acid sequence of the HIOMT enzyme lacking exon 6 and 7 (SEQ ID NO: 3; NCBI accession number AAA75289).
[0035]FIG. 10: Amino acid sequence of the AA.NAT enzyme (SEQ ID NO: 4; NCBI accession number NM--001088).
[0036]FIG. 11: A. Localisation of the variations within the MTNR1A/MTNR1B receptors. B. Family with ADHD and the STOP mutation of MTNR1A. The variations changing amino acids highly or less conserved during evolution are indicated in red and orange respectively. The amino acids, which directly bind to melatonin are indicated in blue.
[0037]FIG. 12: Amino acid sequence of the melatonin MTNR1A receptor (SEQ ID NO: 5; NCBI accession number P48039).
[0038]FIG. 13: Amino acid sequence of the melatonin MTNR1B receptor (SEQ ID NO: 6; NCBI accession number AAS00461).
DETAILED DESCRIPTION OF THE INVENTION
[0039]The present invention relates to methods for determining likelihood of an individual of developing a psychiatric disorder. The present invention further relates to compositions and methods for treating and/or preventing a psychiatric disorder such as autism, Deficits and Hyper Activity Disorder (ADHD) and anorexia.
1. Method of Diagnosis
[0040]According to an embodiment, the present invention provides a method for determining likelihood of an individual of developing a psychiatric disorder.
[0041]More specifically, the method of the invention is preferably achieved by identifying at least one genetic variation in a gene involved in the modulation of melatonin or in its mRNA transcript or by assaying the mRNA transcript level of said gene, or by assaying melatonin level.
[0042]As used herein, the expression "gene involved in the modulation of melatonin" refers to genes involved in the melatonin biosynthesis pathway and genes involved in the signalisation induced by the melatonin, such as its receptors.
[0043]Preferred susceptibility genes for psychiatric disorders contemplated by the method of the invention are ASMT (Acetyl serotonin methyl transferase) and AA-NAT (Arylalkylamine N. acetyl transferase) genes and genes that code for the melatonin receptors, such as the melatonin receptors MTNR1A and MTNR1B.
[0044]As used herein, the expression "genetic variation of a gene" refers to any variation that may occur within the DNA sequence of the genes contemplated by the present invention. Such a DNA sequence comprises, but is not limited to, regulatory sequences such as promoters, introns, exons, and coding sequences. By the term "genetic variation", it is meant any variation that could interfere with the transcription and/or translation of a specific gene. Genetic variation may be recombination events or mutations such as substitution/deletion/insertion events like point and splice site mutations.
[0045]With respect to the ASMT gene, preferred genetic variations that the method of the invention is interested in, are the following preferred point mutations located at position 17, 81, 210, 306 or 326 as defined by the position in SEQ ID NO:1 (FIG. 7). More preferably the point mutation is selected from the group consisting of a N17K mutation, a K81E mutation, a R210H mutation, a G306A mutation and a L326F mutation. According to other preferred genetic variations regarding the ASMT gene concern point mutations located at position 17, 81, 228 or 298 as defined by the position in SEQ ID NO:2 (FIG. 8) or 17, 81, 231 or 251 as defined by the position in SEQ ID NO:3 (FIG. 9).
[0046]Another preferred genetic variation regarding the ASMT gene is a splice site mutation consisting of IVS5+2T>C.
[0047]Another preferred genetic variation regarding the ASMT gene is a mutation in a single nucleotide polymorphism (SNP) such as Rs5989681 or Rs6588809.
[0048]With respect to the AA.NAT gene, preferred genetic variations that the method of the invention is interested in, are the following preferred point mutations located at position 3, 13, 62, 157 or 163 as defined by the position in SEQ ID NO:4 (FIG. 10). More preferably the point mutation is selected from the group consisting of a T3M mutation, a A13S mutation, a V62I mutation, a A157V mutation and a A163V mutation.
[0049]With respect to a melatonin receptor encoding gene, preferred genetic variations that the method of the invention is interested in are those that occur preferably the genes coding for the melatonin receptors, such as MTNR1A (SEQ ID NO: 5) and MTNR1B (SEQ ID NO: 6). A preferred point mutation is a Y170X mutation in the MTNR1A protein as defined by the position in SEQ ID NO: 5 (FIG. 12).
[0050]According to another preferred embodiment, the method of the invention can be achieved by assaying the mRNA transcript level of a gene involved in the modulation of melatonin, such as those involved in its biosynthesis.
[0051]According to another preferred embodiment, the method of the invention can be achieved by assaying in a sample of the individual, the enzymatic activity of an enzyme involved in the melatonin biosynthesis pathway. Such an enzyme is preferably the HIOMT (Hydroxyindole-O-methyltransferase) enzyme. It will be understood that any suitable assay method known to one skilled in the art, such as the one described in Chanut et al.28 and Finocchiaro et al.29, is within the scope of the present invention.
[0052]According to another preferred embodiment, the method of the invention can be achieved by assaying in a sample of the individual, the melatonin level. Methods for assaying melatonin levels are known by one skilled in the art (see Tordjman et al.25, Chanut et al.28 and Finocchiaro et al.29).
[0053]As used herein, the term "sample" refers to a variety of sample types obtained from an individual and can be used in accordance with the method of the invention. The definition encompasses blood, saliva, urine and other samples of biological origin.
[0054]As one skilled in that art may appreciate in view of the above, identification of a genetic variation and/or decrease in the enzymatic activity of said enzyme as compared to a psychiatric-free individual and/or a decrease in the transcript level of said gene, is indicative of a higher likelihood that the individual develops a psychiatric disorder.
2. Composition and Method of Use
[0055]According to another embodiment, the present invention concerns a composition for treating and/or preventing a psychiatric disorder in an individual having a defective gene involved in the modulation of melatonin. By the term "preventing" it refers to a process by which a psychiatric disorder is obstructed or delayed, whereas the term "treating" is intended, for the purposes of this invention, that the symptoms of the psychiatric disorder be ameliorated or completely eliminated.
[0056]As used herein, the term "defective gene" refers to a gene involved in the modulation of melatonin, such as those involved in its biosynthesis pathway, which undergoes genetic variation(s) that impairs or completely stops the production and/or the activity of the protein, such as an enzyme coded by the gene, thus interfering with the biosynthesis of melatonin.
[0057]More particularly, the composition of the invention comprises an acceptable carrier and a therapeutically effective amount of a molecule, preferably a polypeptide, such as an enzyme or functional derivatives thereof, promoting melatonin biosynthesis. Preferably, the enzyme is HIOMT (Hydroxyindole-O-methyltransferase) enzyme or functional derivatives thereof. A "functional derivative" of an enzyme, as is generally understood and used herein, refers to a protein/peptide sequence or fragment thereof or peptidomimetic thereof that possesses a functional biological activity that is substantially similar to the biological activity of the whole enzyme.
[0058]As used herein, the expression "an acceptable carrier" means a vehicle for containing the enzyme, preferably HIOMT, or functional derivatives thereof present in the composition of the invention that can be administered to an individual without adverse effects. Suitable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions. Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.
[0059]According to another embodiment, the present invention provides a method for treating and/or preventing a psychiatric disorder in an individual having a defective gene involved in the modulation of melatonin. In one embodiment, the method of the invention comprises the step of administrating in the individual a composition as defined above.
[0060]The amount of enzyme, preferably HIOMT, or functional derivatives thereof, present in the composition of the invention is preferably a therapeutically effective amount. A therapeutically effective amount of enzyme or functional derivatives thereof present in the composition of the invention is the amount necessary to allow the same to perform its role in the melatonin biosynthesis pathway without causing overly negative effects in the individual to which the composition is administered. The exact amount of enzyme or functional derivatives thereof present in the composition of the invention to be used and the composition to be administered will vary according to factors such as the type of psychiatric disorder being treated, the mode of administration, as well as the other ingredients in the composition.
[0061]The composition of the invention may be given to an individual through various routes of administration. For instance, the composition may be administered in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents. They may be given parenterally, for example intravenously, intramuscularly or sub-cutaneously by injection, by infusion or per os. Suitable dosages will vary, depending upon factors such as the amount of each of the components in the composition, the desired effect (short or long term), the route of administration, the age and the weight of the individual to be treated. Any other methods well known in the art may be used for administering the composition of the invention.
[0062]In another embodiment, the method for treating and/or preventing ASD in an individual comprises the step of administering in the individual a composition comprising melatonin or a functional derivative thereof.
3. Polynucleotides and Polypeptides of the Invention
[0063]A further embodiment of the invention concerns an isolated polynucleotide encoding a polypeptide or functional derivative thereof, characterized in that said polypeptide or functional derivative thereof comprises a point mutation in the amino acid sequence as defined in SEQ ID NO: 1, 4, 5 or 6.
[0064]Related aspects of the invention concern an isolated polynucleotide comprising a regulatory sequence of the ASMT gene or of the AANAT gene. The regulatory sequence comprises an insertion/deletion mutation compared to the wild type regulatory sequence of said ASMT or AANAT gene. Preferably, the regulatory sequence consists of a promotor.
[0065]According to another embodiment of the invention, there is provided an isolated polypeptide encoded by a polynucleotide as defined above.
[0066]As used herein, the term "isolated" is meant to describe a polynucleotide or a polypeptide that is in an environment different from that in which the polynucleotide or the polypeptide naturally occurs.
[0067]The present invention will be more readily understood by referring to the following example. This example is illustrative of the wide range of applicability of the present invention and is not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described.
Example 1
Mutations in the ASMT and AA-NAT Gene Decrease Melatonin Synthesis and Confer Susceptibility to Psychiatric Disorders
[0068]During the search of susceptibility factors to autism spectrum disorders (ASD) or Attention Deficits and Hyper Activity Disorder (ADHD), the inventors identified the ASMT (Acetyl serotonin methyl transferase) gene and the AA.NAT gene as a susceptibility gene for neuropsychiatric disorders. This gene is located on the pseudo autosomal region 1 (PAR1), common to the X and the Y chromosome14. ASMT encodes the enzyme HIOMT (Hydroxyindole-O-methyltransferase; EC 2.1.1.4), which catalyses the last step of melatonin biosynthesis15,16.
[0069]Polymorphisms located in the promoter or the coding sequence and rare mutations affecting the splicing and the protein function are associated with a decrease of ASMT transcript level and/or activity. In individuals carrying these genetic variations, the decrease in ASMT activity leads to a decrease in melatonin concentration. Melatonin (N-acetyl-5-methoxytryptamine) is the major secretory product of the pineal gland. The synthesis of melatonin exhibits a pronounced circadian rhythm: its concentrations in the body are typically lower during the day and reach maximal levels at night in the darkness.
Materials and Methods
Mutation Screening and Genotyping
[0070]Both genotyping and screening for mutation was performed by direct sequencing. Coding regions including the exon/intron boundaries, the 5' and 3' untranslated regions, and the regulatory regions of the ASMT gene were amplified by PCR using HotstarTaq (Qiagen). For primers and PCR conditions, see table 8. Sequencing of PCR products was performed using the BigDye Terminator Cycle Sequencing Kit (V3.1, Applied Biosystems). Samples were then subjected to electrophoresis using an ABI PRISM genetic analyzer (Applied Biosystems). ABI electropherogram data were imported and analyzed for variation using the Genalys software (Author: Masazum Takahashi).
RT-PCR and Quantitative RT-PCR
[0071]For analysis of ASMT transcripts from the investigated subjects, RNA was isolated from the generated lymphoblastoid cell lines using the NucleoSpin® RNA II kit (MACHEREY-NAGEL). Quantification of total RNA was performed by measurement of absorbance at OD260 on a Biophotometer (Eppendorf). Oligo(dT) primed cDNA was prepared from 5 μg of this RNA using superscript II (Invitrogen) according to the manufacturers instructions in a reaction volume of 40 μl. For control of DNA contamination, a parallel tube without reverse transcriptase (RT negative control) was included in the RT reactions. All cDNA samples were tested on an agarose gel (3%) using a GAPDH PCR (for primers, see table 8), followed by a dilution with 40 to 80 μl dH20 (DEPC) in order to homogenize samples for real-time PCR analysis. The cDNA was used directly in TaqMan assays using the ABI PRISM 7500 Sequence Detection System (PE Biosystems). Each reaction was performed in triplicate and the cDNA was added to each reaction (25 μL) containing 1.25 μL of the assay and 12.5 μL of the TaqMan Universal PCR Master Mix (Applied Biosystems). Study samples were run in duplicate or triplicate on 96-well optical PCR plates (ABgene). Quantification of ASMT mRNA was performed using commercially available Assays-on-Demand. Two different assays were used, one covering the boundary between exon 1B and exon 2 (Hs00946625_m1), and one covering the boundary between exon 8 and exon 9 (Hs00187839_m1). Relative values of expression were determined for each sample using the standard curve method (ABI user's manual), and these values were normalized to the Ct values of GAPDH, a standard "housekeeping" control gene, using the glyceraldehyde phosphate dehydrogenase assay Hs99999905_m1. For ASMT and GAPDH, the thresholds were set at 0.2 and 0.25, respectively, which was within the linear region of the semi-log plot in all assays.
Results
Linkage Between the Pseudo Autosomal Region 1 and ASD
[0072]The Pseudo Autosomal Region 1 (PAR1) is only 2.7 Mb of DNA located on the tip of the short arms of the sex chromosomes and containing fifteen genes. A fine mapping of the PAR1 using four microsatellites markers (DXYS233, DXYS234, DXYS228 and DXYS229) was conducted on 52 families with at least two children with ASD. The inventors observed an excess of allelic sharing (Genehunter NPL=2.12; P=0.014 and ASPEX mlod=1.53). Within this region, the inventors focused their study on the ASMT gene using a combination of polymorphisms located in the promoters and intron 3 and adding 22 new families (FIG. 1a, Table 1). Consistent with the initial fine mapping results, the inventors observed an increased of ASMT allelic sharing in affected sib pairs (Genehunter NPL=2.95; P=0.0014 and ASPEX mlod=2.29; 0.0012). When families with only males affected sib pairs are used in the analysis, the linkage is dramatically increased (Genehunter NPL=4.8; P=4.86×10-7 and ASPEX mlod=5.92; P=1.81×10-7). However, in linkage studies of PAR1, the excess of paternal sharing should be taken with great care since this region is not fully independent from the sex chromosome segregation. This distortion effect specific to the paternal meiosis could account for the excess of sharing in male-male ASP and of non-sharing in male-female ASP. Nevertheless, the significant excess of maternal allelic sharing of ASMT in affected sib pairs (ASPEX maternal mlod=1.9; P=0.003) prompt the inventors to analyse the genetic variability of the ASMT gene in ASD compared to the control population.
Genetic Variability of the ASMT Gene in ASD, ADHD and Controls
[0073]To determine whether frequent variations in ASMT significantly associate with ASD, the inventors genotyped one insertion/deletion located in the promoter A and twelve Single Nucleotide Polymorphisms (SNPs) in 275 ASD, 95 ADHD and 187 geographically-matched controls. The genotype region covers 41.3 kb, including the coding exons and the two promoters (FIG. 1b). The ASMT gene can be divided in five haplotype blocks of high inter-marker linkage disequilibrium (D'>0.8). Comparison of allelic, and genotype frequencies are presented in FIG. 1c and table 2. Overall, a significant association was observed between ASD and one SNP rs4446909 located in the promoter B (P=0.028). When the French and the Scandinavian samples are studied separately, a trend for association with rs4446909 is observed in the French sample (P=0.078) and a significant association with a close marker rs5989681 is present in the Scandinavian sample (P=0.047). The haplotype analysis indicated that one haplotype ACGC (including three SNPs in the promoter and one in the 5'UTR) was significantly less frequent in ASD (P=0.012) (Table 3). When populations are studied separately, although not significant, this haplotype was less frequent in both the French (P=0.057) and the Scandinavian ASD population (P=0.067) compared to their related control groups. In contrast, the GGGC haplotype could represent an "at risk haplotype" since it was more frequently observed in ASD (P=0.097). The same SNPs were genotyped in 95 Swedish individuals with ADHD, but no significant association was found. However, when individuals with ASD or ADHD were pooled and compared with controls, the association was more significant (rs4446909 P=0.015, ACGC, P=0.0077; GGGC, P=0.042).
Correlation Between ASMT Genotype and Transcript Level
[0074]The ASMT gene contains two promoters (A and B) and two alternative spliced exons (Exon 6 and 7) (FIG. 2a). Promoter A was previously shown to be exclusive to retina 27. Consistent with this, the inventors could not amplify ASMT transcripts originated from promoter A using human B lymphoblastoid cell lines (BLCL) or pineal gland cDNA. In contrast, the inventors could detect the three known alternative isoforms derived from promoter B (FIG. 2b insert). The long isoform contains all exons including exon 6, which is a Long Interspersed Nuclear Element (LINE). The medium isoform does not contain the LINE insertion and codes for the isoform homologous to the ASMT protein of other species. The shortest isoform does not contain the LINE insertion and exon 7. Only the medium isoform is supposed to be functional since the insertion of exon 6 or the deletion of exon 7 greatly modify the O-methylase domain of ASMT. Using fluorescent RT-PCR, the inventors could ascertain the relative proportion of the three ASMT isoforms. Overall, there was no significant difference in the abundance of the three isoforms between ASD and controls (FIG. 2b). However, in several individuals (13/48 patients and 4/23 controls), the relative percentage of the medium isoform was relatively low (<20%). For example, in the individual ASD49, the long ASMT isoform including the LINE was repetitively more abundant than the medium isoform (FIG. 2b insert). Interestingly, one non synonymous SNP rs6588809 (R190W), located in exon6 within the LINE element, was associated with the level of LINE insertion (P=0.004). In average, individuals homozygotes for the C allele had 3.6 fold more insertion of the LINE in the ASMT transcript compared to individuals homozygotes for the T allele. This variation rs6588809 is probably located in one exonic splicing enhancer (ESE), a sequence which binds to splicing factors such as SR proteins. Despite, its functional consequence on the relative abundance of ASMT isoforms, no association was observed between rs6588809 and ASD.
[0075]To quantify the level of ASMT transcripts, the TaqMan technology and two independent probes E1 CE2 and E8E9 located in the 5' and the 3' end of the mRNA respectively were used. No significant difference in ASMT mRNA level was observed between our sample of 60 ASD and 35 control individuals (FIG. 2c). In both samples, the ASMT transcript level was heterogenous ranging from 0.0019 to 1.39 compared to GAPDH mRNA level. However, when the ASMT genotypes and transcript levels were compared, the inventors could observe a very significant association between the transcript level and two SNPs rs4446909 (5' Probe P=0.0009; 3' probe P=0.06) and rs5989681 (5' probe P=0.000057; 3'probe P=0.02). These SNPs are distant from 109 bp, in high linkage disequilibrium (D'=0.94) and located in promoter B (FIG. 2d). The SNP rs4446909 is situated at -207 bp from the transcription site in a CCCAC box and six nucleotides downstream a CAAT/10 mer box also present in the promoter A (FIG. 2d). SNP rs5989681 is located at -97 bp from the transcription site in a putative binding site for the transcription factor NF-kappaB. Individuals homozygote for the G allele of both SNPs had less ASMT transcripts compared to heterozygotes individuals. Two individuals with a rs4446909 G/G genotype and a rs5989681 C/G genotype had a high transcript level, suggesting that the C allele of rs5989681 could be responsible for the high transcript level. Interestingly, these two SNPs (rs4446909 and rs5989681) were the one associated with ASD. The G alleles of both SNPs were more frequent in ASD and ADHD and were associated to a low ASMT transcript level. To explore further the relation between the ASMT gene and the susceptibility to these disorders, the inventors measured the enzyme activity in families with ASD and controls.
ASMT Activity in ASD and Control Individuals.
[0076]The ASMT activity was first investigated in the blood platelets of ASD, their parents and control individuals. As already reported in other samples, the inventors could observe an increase of serotonin concentration in the blood of ASD (5HT 1.0±0.65 μM; P=1.46×10-7) and their parents (5HT 0.80±0.24 μM; P=1.85×10-10) compared to controls (5HT: 0.42±0.22 μM; FIG. 3a). Remarkably, the inventors also observed a strong decrease of ASMT activity for almost all of the individuals with ASD (ASMT blood 0.77±0.47 pmoles/109 platelets/30 min P=1.15×10-10) and their parents (1.18±0.87 pmoles/109 platelets/30 min P=0.00045) compared to control (ASMT blood 1.811±0.68 pmoles/109 platelets/30 min; FIG. 3b). In parents, the ASMT activity was significantly decreased in the mothers (P=0.016), but not in the fathers (P=0.80) compared to sex- and age-matched controls. The decrease in ASMT was accompanied by a significant decrease of blood melatonin concentration in ASD (ML 0.07±0.04 nM; P=5.2×10-10) and their parents (ML 0.09±0.04 nM; P=2.04×10-6) compared to controls (ML 0.14±0.04 nM) (FIG. 3C). In parents, the melatonin concentration was significantly lower in mother (P=3.35×10-5) and in the fathers (P=0.007). To replicate these results, BLCLs from 51 individuals with ASD (11 already analysed in the blood sample) and 33 new independent controls were analysed (FIG. 3d). Consistent, with the results obtained using the blood platelets, the level of ASMT activity was dramatically decreased in ASD (ASMT BLCL 3.29±2.5 pmoles/mg prot/30 min) compared to controls (ASMT BLCL 7.97±2.5 pmoles/mg prot/30 min; P=7.2×10-7).
[0077]The inventors next plotted the relative quantity of ASMT mRNA against the level of the enzyme activity (FIG. 4). In the control sample, the inventors could not detect a significant correlation between the RNA amount and the enzyme activity, suggesting that the level of ASMT transcript is not a strong limiting factor for enzyme activity in BLCL. In the ASD sample, the ASMT activity was decreased in almost all of the individuals, independently of their transcript level. For several individuals with ASD, the inventors cannot exclude that the low ASMT activity could be the direct consequence of a very low ASMT transcript level and/or a specific strong decrease in the medium isoform. Nevertheless, these results indicated that the global decrease of ASMT activity observed in ASD could not be explained solely by a decrease in ASMT mRNA. To further investigate this enzymatic deficiency, we screen the ASMT gene for mutation.
Mutation Screening of the ASMT Gene in ASD, ADHD and Controls
[0078]All exons of the ASMT gene, including the two promoters and alternatively spliced exons were directly sequenced in individuals with ASD (n=275) or ADHD (n=103). Several genetic variants were identified including a splice site mutation (IVS5+2T>C), five rare non-synonymous variations (N17K, K81E, R210H, G306A, L326F) and two synonymous variation (N167N, Q205Q). The location, the segregation and the frequency of these rare variations are indicated in FIG. 5 and table 4. The splice site mutation (IVS5+2T>C) was present in two families with ASD and one with ADHD, but never observed in 411 controls. In one patient carrying the splice site mutation (IVS5+2T>C), the inventors could detect additional transcripts isoforms compared to controls, which originate from a donor splice site located 31 bp in intron 5 (FIG. 5). This abnormal spliced transcripts leads to different C-terminal protein sequences after amino acid G188 and premature truncation of all ASMT isoforms (FIG. 5). In ASD family 1, the mother transmitted the mutation to her son, who was homozygote for the G alleles of rs4446909 and rs5989681. In ASD family 2, the father transmitted the splice site mutation to his two sons with autism. The mother had previously a first child with autism but with a milder phenotype and from a different father. The haplotype analysis showed that the mother has transmitted the same ASMT GGGT promoter haplotype to all her affected sons. In the ADHD family 1, the mother carrying the splice site mutation had history of neuropsychiatric disorders including attention and impulsivity problems. She transmitted the mutation to her son with ADHD and to her daughter with anorexia, but not to her unaffected daughter. All individuals carried the haplotype. The N17K variation was observed in two ASD families (family 3 and 4) with parents originated from Asiatic countries. In family 3 and 4, the mother transmitted the mutation. In ASD family 5, the mother transmitted the non-synonymous variation K81E. In ASD family 6 from Norway, the father transmitted a variation G306A to his two sons with autism. The variation L326F was identified in two independent families with ASD and one with ADHD. In family 7 and 8, the mutations were transmitted by the mother and the father, respectively. In ADHD family 2, the son with ADHD has a 326 mutation and there is a daughter with anorexia. None of these non-synonymous variations were observed in controls except L326F, which was present in 3/400 controls. To ascertain the functional effects of these rare variants, the inventors measured the ASMT enzyme activity and the melatonin concentration in the carriers.
Biochemical and Clinical Characterisation of the Individuals Carrying Rare Variations of the ASMT Gene.
[0079]Biochemical analyses of the blood platelets indicate that individuals carrying the splice site mutation (IVS5+2T>C) or the L326F mutation have low HIOMT activity, a decrease of melatonin and a relatively high concentration of 5-HT compared to controls (Table 5). Consistent with the results obtained on the blood samples, ASMT activity in BLCL was found reduced in all individuals carrying the (IVS5+2T>C) and the L326F mutations. In these individuals, the melatonin concentration was also found decreased compared to controls. To further explore the ASMT deficit in vivo, the inventors investigated family 1 carrying the splice site mutation (FIG. 6). The father does not carry the mutation and showed a normal increase of melatonin during the night. In contrast, both carriers of the ASMT splice mutation, the unaffected mother and the son with ASD, showed no increase of melatonin during the night. Despite this absence of melatonin, no obvious difference in the sleep pattern was observed compared to controls.
Mutations of AANAT in Individuals with ASD and ADHD
[0080]In the melatonin biosynthesis pathway, the enzyme AANAT is located before the ASMT and transform the serotonin into N-acetyl serotonin. Mutations in the AANAT gene were screened and five rare non-synonymous variations (T3M, A13S, V621, A157V, A163V) were indetified in individuals with ASD and ADHD. These variations were not observed in 200 controls.
Example 2
Mutations in the Melatonin Receptors MTNR1A and MTNR1B Decrease Melatonin Binding and Confer Susceptibility to Psychiatric Disorders
[0081]During the search of susceptibility factors to autism or Attention Deficits and Hyper Activity Disorder (ADHD), the inventors identified genetic mutations in the ASMT (Acetyl serotonin methyl transferase) gene (see Example 1). In this present example, the inventors report genetic variations in the melatonin receptors including a stop mutation in a patient with ADHD, which is absent from all controls tested (n=150). In addition, the inventors identified an ASMT splice site mutation in a patient with OCD. These results confirm the results of Example 1 showing that the melatonin pathway is associated to psychiatric disorder.
Results
[0082]The inventors investigated whether variations in MTNR1A and MTNR1B were associated with ASD and ADHD, by directly sequencing all exons and the promoters of individuals with ASD (n=78-130) or ADHD (n=79-102). Several exonic mutations were identified (Table 9, FIG. 11A), including a stop mutation in a patient with ADHD, caused by a point mutation at position 170 (see SEQ ID NO: 5), namely a Y170X mutation. This woman has a son suffering from ADHD with autistic traits, but no DNA is available for this patient.
[0083]On the basis of the results shown in Example 1, the inventors have now evidenced that the melatonin receptor MTNR1A can also be mutated in individuals with ADHD. These results confirm that abnormal melatonin pathway is a risk factor for neuropsychiatric disorders.
General Conclusions
[0084]Melatonin is synthesised in two steps from the neurotransmitter serotonin. The HIOMT enzyme catalyses the final reaction transforming N-acetylserotonin into melatonin. This hormone is mainly produced in the pineal gland but also in other tissues such as retina or lymphocytes. It is secreted during the circadian cycle with high level during the night and low level during the day. The physiological role of melatonin is still unclear but it plays crucial roles correlated to the circadian or seasonal rhythms. Furthermore, melatonin was shown to have anti-oxidative or anti-aging properties as well as effects on hormonal autocrine/paracrine functions, on the immune system, on the modulation of neurotransmitter release.
[0085]Sleep problems are frequently reported in patients with neuro-psychiatric disorders. In addition, an increase concentration of serotonin in individuals with autism as well as in there relatives is one of the most replicated finding in autism. However, despite numerous genetic studies on the serotonin transporters or receptors, the reason why there is high level of serotonin was never elucidated. The outcomes of the present invention are extremely important for the diagnostic and therapy of autism and ADHD for at least three major reasons:
A. The biochemical analysis of the melatonin biosynthesis is the first biological marker associated with susceptibility to neuropsychiatric disorders (autism, ADHD and OCD). This assay could be used as a screening test since concentration of melatonin could be quantified with a simple saliva sample and the HIOMT enzymatic activity can be measured using a blood sample.B. In the case of a decrease melatonin concentration is identified in one patient, the supplementation with exogenous melatonin is feasible and could correct the genetic deficit.C. The identification of these mutations should permit to find others susceptibility factors having roles in the same biological pathway.
TABLE-US-00001 TABLE 1 ASMT allelic sharing in ASD sib-pairs families Affected IBD status Parental sharing % (IBD1:IBD0)* sib pairs 0 M1 P1 2 P value Maternal P value Paternal P value Male-Male 3 4 5 15 0.0033 71 (20:8) 0.023 75 (24:8) 0.0047 Male-Female 8 8 1 5 0.11 60 (15:10) 0.31 31 (6:18) 0.014 Female-Female 1 0 1 1 -- 33 (1:2) -- 75 (3:1) -- All 12 12 7 21 0.049 64 (36:20) 0.033 62 (33:27) 0.44 *Thirteen additional families. informative for only one of the parent. were included in the parental sharing compare to the IBD status.
TABLE-US-00002 TABLE 2 Association study of ASMT frequent variations identified in ASD, ADHD and controls. ASD Geno- ASD C FR P ASD ADHD C ASD P All ASD + ALL P P SNP Localisation type FR FR value SWE SWE SWE P value value ASD ADHD C value value EIA 1757953 A 0.644 0.600 0.52 0.637 nd 0.581 0.46 0.32 A:A 20 12 17 nd 14 37 26 A:G 27 30 0.24 17 nd 22 0.64 44 52 0.28 G:G 3 3 6 nd 7 11 10 rs4446909 1977610 A 0.234 0.309 0.078 0.212 0.222 0.280 0.14 0.21 0.028 0.017 A:A 8 6 4 1 9 12 13 15 A:G 57 38 0.17 28 33 33 0.32 0.056 85 118 71 0.09 0.039 G:G 91 37 53 45 49 144 189 86 rs5989681 1777719 C 0.338 0.358 0.65 0.230 0.265 0.324 0.047 0.23 0.21 0.1 C:C 17 9 5 2 10 22 24 19 C:G 72 40 0.84 30 30 39 0.15 0.089 107 141 79 0.44 0.24 G:G 68 32 52 40 42 120 160 74 EIBC 1777747 A 0.101 0.062 0.14 0.098 0.086 0.121 0.48 0.29 0.74 0.85 A:A 3 1 1 1 0 4 5 1 A:G 26 8 0.35 15 12 22 0.32 0.18 41 53 30 0.62 0.62 G:G 129 72 71 68 69 200 268 141 rs6644635 1777821 C 0.661 0.648 0.77 0.622 0.619 0.682 0.85 0.74 0.81 0.79 C:C 68 32 32 32 52 100 64 C:T 7 11 0.81 43 45 51 0.6 0.92 161 0.46 0.64 T:T 17 8 11 7 8 28 116 rs 588802 1785774 C 0.593 0.568 0.62 0.425 0.476 0.491 0.29 0.82 0.96 0.66 C:C 58 19 20 20 11 78 98 30 C:T 62 37 0.13 34 40 30 0.096 0.58 96 136 67 0.018 0.023 T:T 30 10 33 24 12 63 87 22 rs5948991 178616 C 0.163 0.210 0.34 0.141 0.000 0.118 0.61 nd 0.78 nd C:C 2 5 1 nd 3 3 8 C:T 13 27 0.65 9 nd 16 0.73 nd 22 43 0.9 nd T:T 37 56 29 nd 74 66 130 rs6588809 1795429 C 0.491 0.512 0.65 0.474 0.433 0.453 0.69 0.7 0.93 0.76 C:C 45 19 22 17 16 67 84 35 C:T 75 44 0.28 47 44 46 0.74 0.79 122 166 90 0.27 0.29 T:T 48 17 27 29 24 75 104 41 I6A 1793531 A 0.494 0.425 0.15 0.484 0.494 0.494 0.83 0.99 0.39 0.36 A:A 39 15 25 24 19 84 88 34 A:G 88 38 0.29 43 41 27 0.41 0.48 131 172 85 0.65 0.55 G:G 41 27 28 25 20 69 94 47 rs7471973 1795551 C 0.814 0.787 0.48 0.802 0.833 0.831 0.47 0.96 0.99 0.83 C:C 109 48 63 63 57 172 235 105 C:T 54 30 0.72 28 24 29 0.091 0.16 82 106 59 0.27 0.19 T:T 4 2 5 3 0 9 12 2 rs4521942 1795844 G 0.913 0.930 0.49 0949 0.944 0.944 0.83 0.98 0.54 0.7 G:G 139 80 90 80 80 229 309 160 G:T 27 13 0.67 8 8 10 0.5 0.54 35 43 23 0.49 0.46 T:T 1 0 1 1 0 2 3 0 rs4933063 1799231 C 0.910 0.914 0.86 0.872 0.930 0.896 0.48 0.2 0.67 1 C:C 147 79 75 80 73 222 302 152 C:T 28 12 0.67 21 13 17 0.77 0.36 49 62 29 0.84 0.83 T:T 2 2 2 0 1 4 4 3 rs11346829 1799288 A 0.116 0.086 0.28 0.097 0.102 0.099 0.95 0.92 0.41 0.44 A:A 2 0 2 2 2 4 6 2 A:G 37 16 0.44 15 15 14 0.99 0.99 52 67 30 0.72 0.74 G:G 138 77 81 76 75 219 295 152 indicates data missing or illegible when filed
TABLE-US-00003 TABLE 3 Haplotypic association using the four frequent polymorphisms located in the promoter B and 5'UT5 of ASMT French sample (%) Scandinavian sample (%) Combined sample P ASD ADHD ASD ASD + ADHD ASD Control values ASD ADHD Controls P value Pvalue P value P value GGGC 0.308 0.279 0.51 0.396 0.374 0.313 0.099 0.058 0.096 0.042 ACGC 0.225 0.304 0.057 0.185 0.214 0.267 0.067 0.24 0.012 0.0077 GGGT 0.242 0.306 0.13 0.272 0.267 0.241 0.54 0.90 0.43 0.47 GCGC 0.112 0.052 0.022 0.017 0.051 0.037 0.26 0.82 0.086 0.17 GGAT 0.092 0.054 0.14 0.098 0.078 0.108 0.75 0.25 0.68 0.76 GCGT 0 0 -- 0.013 0 0.013 0.99 -- -- -- AGGC 0 0 -- 0.018 0 0.011 0.59 -- 0.32 --
TABLE-US-00004 TABLE 4 ASMT Rare variations identified in ASD, ADHD and controls. French Swedish Swedish French Swedish th Africa Exon/Intron Variant Nucleotide* ASD ASD ADHD Control Control Control All controls 5' UTR C/T 0|136 0|82 0|103 0|84 1|90 Nd 1/174 E1A A/g 2|136 0|82 0|103 0|84 0|90 Nd 0/174 E1A G/C 2|136 0|82 0|103 0|84 0|90 Nd 0/174 E1A A/T 2|136 0|82 0|103 0|84 0|90 Nd 0/174 E1A A/g 2|136 0|82 0|103 0|84 0|90 Nd 0/174 E1A C/T 1|136 0|82 0|103 0|84 0|90 Nd 0/174 E1A C/A 0|136 1|82 0|103 0|84 0|90 Nd 0/174 E1B N17K C232A 1|140 1|80 0|103 0|79 0|92 Nd 0/171 E1B A/T 1|140 0|80 0|103 0|79 0|92 Nd 0/171 E1B C/T 2|140 0|80 0|103 0|79 0|92 Nd 0/171 E1B C/T 1|140 1|80 0|103 0|79 0|92 Nd 0/171 E1B C/A 0|140 1|80 0|103 0|79 0|92 Nd 0/171 E2 K81E A422G 1|150 0|86 0|103 0|67 0|53 Nd 0/120 INTRON 2 C/A 1|150 0|86 0|103 0|67 0|53 Nd 0/120 INTRON 2 G/A 3|150 0|86 0|103 0|67 0|53 Nd 0/120 INTRON 4 C/T 1|174 0|97 0|103 0|94 0|93 Nd 0/187 E5 N167N 0|182 1|102 0|103 0|94 0|145 0|172 0/411 INTRON 5 IVS5 + 2T > C T/C 2|182 0|102 1|102 0|94 0|145 0|172 0/411 INTRON 6 G/A 1|168 0|95 0|103 0|80 0|87 Nd 0/167 E7 D238G A894G 0|166 0|97 0|103 0|94 1|97 Nd 1/191 E7 K247R A921G 0|166 0|97 0|103 1|94 0|97 Nd 1/191 INTRON 7 C/T 0|166 0|97 0|103 0|94 1|97 Nd 1/191 E8 P271L G994T 0|176 0|96 0|103 0|94 1|93 Nd 1/187 E8 C301S G1083C 0|176 0|96 0|103 1|94 0|93 Nd 1/187 E9 G306A G1098C 0|186 1|101 0|103 0|88 0|145 0|95 0/328 R319Q G1137A 0|186 0|101 0|103 1|88 0|145 0|95 0/328 L326F C1157T 1|186 1|101 1|103 0|88 2|145 1|95 3/328 *reference sequence Genebank NM004043 indicates data missing or illegible when filed
TABLE-US-00005 TABLE 5 Biochemical measurement in blood and BLCL from families carrying rare variations Blood sample BLCL HIOMT HIOMT Axe I ASMT 5-HT (pmoles/109 ML (pmoles/ ML Diagnosis genotype (μM) platelets/30 min) (nM) mg proi/30 min) (pM) Father Nd +/+ 1.14 0.96 0.12 -- Mother Nd +/(IVS5 + 2T > 1.18 0.39 0.05 -- C) Proband 1 Autism +/(IVS5 + 2T > 0.89 0.22 0.04 <0.01 <0.02 C) Father Nd +/+ 0.90 0.88 0.09 -- -- Mother Nd +/L326F 0.96 <0.06 <0.02 -- -- Proband 2 Autism +/L326F 0.80 <0.06 <0.02 <0.01 <0.02 Controls 0.12-1.17 0.86-3.36 0.1-0.26 3.3-13.4 2.3-10 range (n = 47)
TABLE-US-00006 TABLE 6 Genetic variations of the AANAT gene identified in individuals with ASD, ADHD and controls. ASD ADHD Controls Localisation* (n = 287) (n = 96) (n = 165) Promoter TG 169:179 170:164 G 179:169 180:140 5'UTR Intron 1 Exon2 C242T 3 0 (T3M) G271T 1 0 (A13S) Intron 2 GA Exon3 G418A 1 1 0 (V62I) Intron 3 Exon 4 TC (H122H) 1 0 0 CT (R128R) 1 0 0 C689T 0 1 0 (A152V) CT (A156A) 0 1 0 C704T 1 0 0 (A157V) C722T 1 1 0 (A163V) GA (G177D) 0 0 1 CA (I181I) 1 0 0 3'UTR CT GA *reference sequence Genebank NM_001088
TABLE-US-00007 TABLE 7 Allelic and haplotypic association using two AANAT frequent promoter polymorphisms Swedish Feench ADHD and n = 92 Swedish Major Allele French Swedish ASD P Frequence P Controls controls N = 192 value P value value n = 95 N = 95 Single rs G 0.51 0.54 T 0.57 0.45 G 0.512 T 0.53 rs G 0.51 0.21 G 0.57 0.45 G 0.596 G 0.53 Haplo- type Rs-rs TG 0.433 0.17 0.52 0.68 0.481 0.499 GC 0.426 0.89 0.38 0.27 0.411 0.438 GG 0.086 0.42 0.05 0.405 0.101 0.032 TC 0.055 0.038 0.05 0.405 0 0.032
TABLE-US-00008 TABLE 8 PCR and sequencing primers for Human ASMT Size PCR Ta Seq EXON Size (exon) Primers (bp) (° C.) comment primer 1A 78 ASMT1AF (SEQ ID NO: 7): 875 67 5% DMSO Fwd and GAGCGATTCTTCTGCCTCAGC (1m elong) rev ASMT1AR (SEQ ID NO: 8): TCTGCGCACACTCCCAGGTG 1B/C 136 ASMT1BF (SEQ ID NO: 9): 820 67 5% DMSO Fwd and coding: 69 GAGGCAGGAGAATCGCTTGAA (1m elong) rev ASMT1BR (SEQ ID NO: 10): GGCTACATCGTGGGTGTACGTC 2 175 ASMT2F (SEQ ID NO: 11): 552 58 Rev TGGTGCAATCTCATTTGACTCTG ASMT2R (SEQ ID NO: 12): GGGTTCATGCCATTCTCCTG 3 130 ASMT3F1 (SEQ ID NO: 13): 950 64 5% DMSO Fwd and CAGCTGTACAAGGCAAGAGGA (1m elong) rev ASMT3R2 (SEQ ID NO: 14): CTTTCACCTCCTCCACTGCCA 4 69 ASMT4F (SEQ ID NO; 15); 283 55 Rev GCCTGGGCTACAGAGCTGAAA ASMT4R (SEQ ID NO: 16): CTCCTGGGTTGTGCCATTTG 5 119 ASMT5F (SEQ ID NO: 17): 331 64 Fwd CCTGTGGGGTATAGCTCCGTTC ASMT5R (SEQ ID NO; 18): CGCACATGTCAAAGCATCAGA 6 84 ASMT6F (SEQ ID NO: 19): 342 64 Fwd AGCTTGCAGTGAGCGGAAATC ASMT6R (SEQ ID NO: 20): GCACCCATCGACTCGTCATTT 7 141 ASMT7F(SEQ ID NO: 21): 352 64 Rev TGGGTTGGACCCTTCATGAGT ASMT7R (SEQ ID NO: 22): GTGTTTCCGGGAGTGAGAGGA 8 123 ASMT8F (SEQ ID NO: 23): 338 64 Fwd AGCCTGGAAGACCTGGGAAAG ASMT8R (SEQ ID NO: 24): CCTGTGGGATGATTTCAGTGC 9 279 ASMT9F (SEQ ID NO: 25): 506 64 Fwd coding: 212 GGTGCCCTGACTGTCCTCTGA ASMT9R (SEQ ID NO: 26): CCATCAGCGTGGTCCTCAGTA All PCRs performed with Qiagen HotstarTaq with the temperature profile: 15 min at 95° 35 cycles of: 30s at 95°. 30s at Ta. 30s-1 min at 72° followed by a final extension step of 10 min at 72°.
TABLE-US-00009 TABLE 9 Variations identified in ASD, ADHD and controls MTNR1A MTNR1B Rares Rares variants ASD ADHD Controls variants ASD ADHD Co Gly166 Glu 4/79 1/87 5/100 Gly24Glu 8/78 8/62 2/78 HO 1/62 HO Tyr170X 0/79 1/79 0/100 Arg 231 His 2/130 2/99 Ile189Ile* 2/79 8/87 5/100 Lys 243 Arg 8/130 3/102 2/87 HO Ile 212Thr 2/79 0/87 0/100 Arg330 Gln 1/130 0/102 Ala266Val 2/79 4/87 4/100 Arg308Arg 3/79 5/87 2/100 1/79 HO Thr315Thr 5/79 5/87 2/100 1/79 HO Lys334Asn 1/79 2/87 2/100 *P = 0.05 between ADHD and Controls; HO: homozygotes; N/A: not available indicates data missing or illegible when filed
REFERENCES
[0086]1. Folstein, S. E. & Rosen-Sheidley, B. Genetics of autism: complex aetiology for a heterogeneous disorder. Nat Rev Genet 2, 943-955 (2001). [0087]2. Veenstra-VanderWeele, J. & Cook, E. H. Molecular genetics of autism spectrum disorder. Mol Psychiatry 9, 819-832 (2004). [0088]3. Bobb, A. J., Castellanos, F. X., Addington, A. M. & Rapoport, J. L. Molecular genetic studies of ADHD: 1991 to 2004. Am J Med Genet B Neuropsychiatr Genet 132, 109-25 (2005). [0089]4. Faraone, S. V. et al. Molecular genetics of attention-deficit/hyperactivity disorder. Biol Psychiatry 57, 1313-23 (2005). [0090]5. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, 4th Ed. (American Psychiatric Press, Washington D.C., 1994). [0091]6. Biederman, J. & Faraone, S. V. Attention-deficit hyperactivity disorder. Lancet 366, 237-48 (2005). [0092]7. Smalley, S. L. Genetic influences in childhood-onset psychiatric disorders: autism and attention-deficit/hyperactivity disorder. American Journal of Human Genetics 60, 1276-82. (1997). [0093]8. Ozonoff, S. & Jensen, J. Brief report: specific executive function profiles in three neurodevelopmental disorders. J Autism Dev Disord 29, 171-7 (1999). [0094]9. Handen, B. L., Johnson, C. R. & Lubetsky, M. Efficacy of methylphenidate among children with autism and symptoms of attention-deficit hyperactivity disorder. J Autism Dev Disord 30, 245-55 (2000). [0095]10. Smalley, S. L. et al. Genetic linkage of attention-deficit/hyperactivity disorder on chromosome 16p13, in a region implicated in autism. Am J Hum Genet 71, 959-63 (2002). [0096]11. Thomas, N. S. et al. Xp deletions associated with autism in three females. Human Genetics 104, 43-48 (1999). [0097]12. Boycott, K. M. et al. A familial contiguous gene deletion syndrome al Xp22.3 characterized by severe learning disabilities and ADHD. Am J Med Genet A 122, 139-47 (2003). [0098]13. Doherty, M. J. et al. An Xp; Yq translocation causing a novel contiguous gene syndrome in brothers with generalized epilepsy, ichthyosis, and attention deficits. Epilepsia 44, 1529-35 (2003). [0099]14. Yi, H., Donohue, S. J., Klein, D. C. & McBride, O. W. Localization of the hydroxyindole-O-methyltransferase gene to the pseudoautosomal region: implications for mapping of psychiatric disorders. Hum Mol Genet 2, 127-31 (1993). [0100]15. Reiter, R. J. Melatonin: clinical relevance. Best Pract Res Clin Endocrinol Metab 17, 273-85 (2003). [0101]16. Simonneaux, V. & Ribelayga, C. Generation of the melatonin endocrine message in mammals: a review of the complex regulation of melatonin synthesis by norepinephrine, peptides, and other pineal transmitters. Pharmacol Rev 55, 325-95 (2003). [0102]17. Reiter, R. J. Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr Rev 12, 151-80 (1991). [0103]18. Nelson, R. J. & Drazen, D. L. Melatonin mediates seasonal changes in immune function. Ann N Y Acad Sci 917, 404-15 (2000). [0104]19. Nir, I. et al. Brief report: circadian melatonin, thyroid-stimulating hormone, prolactin, and cortisol levels in serum of young adults with autism. J Autism Dev Disord 25, 641-54 (1995). [0105]20. Lord, C. What is melatonin? Is it a useful treatment for sleep problems in autism? J Autism Dev Disord 28, 345-6 (1998). [0106]21. Hayashi, E. Effect of melatonin on sleep-wake rhythm: the sleep diary of an autistic male. Psychiatry Clin Neurosci 54, 383-4 (2000). [0107]22. Kulman, G. et al. Evidence of pineal endocrine hypofunction in autistic children. Neuroendocrinol Lett 21, 31-34 (2000). [0108]23. Pacchierotti, C., Iapichino, S., Bossini, L., Pieraccini, F. & Castrogiovanni, P. Melatonin in psychiatric disorders: a review on the melatonin involvement in psychiatry. Front Neuroendocrinol 22, 18-32 (2001). [0109]24. Yun, A. J., Bazar, K. A. & Lee, P. Y. Pineal attrition, loss of cognitive plasticity, and onset of puberty during the teen years: is it a modern maladaptation exposed by evolutionary displacement? Med Hypotheses 63, 939-50 (2004). [0110]25. Tordjman, S., Anderson, G. M., Pichard, N., Charbuy, H. & Touitou, Y. Nocturnal excretion of 6-sulphatoxymelatonin in children and adolescents with autistic disorder. Biol Psychiatry 57, 134-8 (2005). [0111]26. Ishizaki, A., Sugama, M. & Takeuchi, N. [Usefulness of melatonin for developmental sleep and emotional/behavior disorders--studies of melatonin trial on 50 patients with developmental disorders]. No To Hattatsu 31, 428-37 (1999). [0112]27. Rodriguez, I. R., Mazuruk, K., Schoen, T. J. & Chader, G. J. Structural analysis of the human hydroxyindole-O-methyltransferase gene. Presence of two distinct promoters. J Biol Chem 269, 31969-77 (1994). [0113]28. Chanut E, Nguyen-Legros J, Versaux-Botteri C, Trouvin J H, Launay J M. Determination of melatonin in rat pineal, plasma and retina by high-performance liquid chromatography with electrochemical detection. J Chromatogr B Biomed Sci Appl. 1998 May 8; 709(1):11-8 [0114]29. Finocchiaro L M, Nahmod V E, Launay J M. Melatonin biosynthesis and metabolism in peripheral blood mononuclear leucocytes. Biochem J. 1991 Dec. 15; 280 (Pt 3):727-31.
Sequence CWU
1
361373PRTHomo sapiens 1Met Gly Ser Ser Glu Asp Gln Ala Tyr Arg Leu Leu Asn
Asp Tyr Ala1 5 10 15Asn
Gly Phe Met Val Ser Gln Val Leu Phe Ala Ala Cys Glu Leu Gly 20
25 30Val Phe Asp Leu Leu Ala Glu Ala
Pro Gly Pro Leu Asp Val Ala Ala 35 40
45Val Ala Ala Gly Val Arg Ala Ser Ala His Gly Thr Glu Leu Leu Leu
50 55 60Asp Ile Cys Val Ser Leu Lys Leu
Leu Lys Val Glu Thr Arg Gly Gly65 70 75
80Lys Ala Phe Tyr Arg Asn Thr Glu Leu Ser Ser Asp Tyr
Leu Thr Thr 85 90 95Val
Ser Pro Thr Ser Gln Cys Ser Met Leu Lys Tyr Met Gly Arg Thr
100 105 110Ser Tyr Arg Cys Trp Gly His
Leu Ala Asp Ala Val Arg Glu Gly Arg 115 120
125Asn Gln Tyr Leu Glu Thr Phe Gly Val Pro Ala Glu Glu Leu Phe
Thr 130 135 140Ala Ile Tyr Arg Ser Glu
Gly Glu Arg Leu Gln Phe Met Gln Ala Leu145 150
155 160Gln Glu Val Trp Ser Val Asn Gly Arg Ser Val
Leu Thr Ala Phe Asp 165 170
175Leu Ser Val Phe Pro Leu Met Cys Asp Leu Gly Gly Thr Arg Ile Lys
180 185 190Leu Glu Thr Ile Ile Leu
Ser Lys Leu Ser Gln Gly Gln Lys Thr Lys 195 200
205His Arg Val Phe Ser Leu Ile Gly Gly Ala Gly Ala Leu Ala
Lys Glu 210 215 220Cys Met Ser Leu Tyr
Pro Gly Cys Lys Ile Thr Val Phe Asp Ile Pro225 230
235 240Glu Val Val Trp Thr Ala Lys Gln His Phe
Ser Phe Gln Glu Glu Glu 245 250
255Gln Ile Asp Phe Gln Glu Gly Asp Phe Phe Lys Asp Pro Leu Pro Glu
260 265 270Ala Asp Leu Tyr Ile
Leu Ala Arg Val Leu His Asp Trp Ala Asp Gly 275
280 285Lys Cys Ser His Leu Leu Glu Arg Ile Tyr His Thr
Cys Lys Pro Gly 290 295 300Gly Gly Ile
Leu Val Ile Glu Ser Leu Leu Asp Glu Asp Arg Arg Gly305
310 315 320Pro Leu Leu Thr Gln Leu Tyr
Ser Leu Asn Met Leu Val Gln Thr Glu 325
330 335Gly Gln Glu Arg Thr Pro Thr His Tyr His Met Leu
Leu Ser Ser Ala 340 345 350Gly
Phe Arg Asp Phe Gln Phe Lys Lys Thr Gly Ala Ile Tyr Asp Ala 355
360 365Ile Leu Ala Arg Lys 3702345PRTHomo
sapiens 2Met Gly Ser Ser Glu Asp Gln Ala Tyr Arg Leu Leu Asn Asp Tyr Ala1
5 10 15Asn Gly Phe Met
Val Ser Gln Val Leu Phe Ala Ala Cys Glu Leu Gly 20
25 30Val Phe Asp Leu Leu Ala Glu Ala Pro Gly Pro
Leu Asp Val Ala Ala 35 40 45Val
Ala Ala Gly Val Arg Ala Ser Ala His Gly Thr Glu Leu Leu Leu 50
55 60Asp Ile Cys Val Ser Leu Lys Leu Leu Lys
Val Glu Thr Arg Gly Gly65 70 75
80Lys Ala Phe Tyr Arg Asn Thr Glu Leu Ser Ser Asp Tyr Leu Thr
Thr 85 90 95Val Ser Pro
Thr Ser Gln Cys Ser Met Leu Lys Tyr Met Gly Arg Thr 100
105 110Ser Tyr Arg Cys Trp Gly His Leu Ala Asp
Ala Val Arg Glu Gly Arg 115 120
125Asn Gln Tyr Leu Glu Thr Phe Gly Val Pro Ala Glu Glu Leu Phe Thr 130
135 140Ala Ile Tyr Arg Ser Glu Gly Glu
Arg Leu Gln Phe Met Gln Ala Leu145 150
155 160Gln Glu Val Trp Ser Val Asn Gly Arg Ser Val Leu
Thr Ala Phe Asp 165 170
175Leu Ser Val Phe Pro Leu Met Cys Asp Leu Gly Gly Gly Ala Gly Ala
180 185 190Leu Ala Lys Glu Cys Met
Ser Leu Tyr Pro Gly Cys Lys Ile Thr Val 195 200
205Phe Asp Ile Pro Glu Val Val Trp Thr Ala Lys Gln His Phe
Ser Phe 210 215 220Gln Glu Glu Glu Gln
Ile Asp Phe Gln Glu Gly Asp Phe Phe Lys Asp225 230
235 240Pro Leu Pro Glu Ala Asp Leu Tyr Ile Leu
Ala Arg Val Leu His Asp 245 250
255Trp Ala Asp Gly Lys Cys Ser His Leu Leu Glu Arg Ile Tyr His Thr
260 265 270Cys Lys Pro Gly Gly
Gly Ile Leu Val Ile Glu Ser Leu Leu Asp Glu 275
280 285Asp Arg Arg Gly Pro Leu Leu Thr Gln Leu Tyr Ser
Leu Asn Met Leu 290 295 300Val Gln Thr
Glu Gly Gln Glu Arg Thr Pro Thr His Tyr His Met Leu305
310 315 320Leu Ser Ser Ala Gly Phe Arg
Asp Phe Gln Phe Lys Lys Thr Gly Ala 325
330 335Ile Tyr Asp Ala Ile Leu Ala Arg Lys 340
3453298PRTHomo sapiens 3Met Gly Ser Ser Glu Asp Gln Ala
Tyr Arg Leu Leu Asn Asp Tyr Ala1 5 10
15Asn Gly Phe Met Val Ser Gln Val Leu Phe Ala Ala Cys Glu
Leu Gly 20 25 30Val Phe Asp
Leu Leu Ala Glu Ala Pro Gly Pro Leu Asp Val Ala Ala 35
40 45Val Ala Ala Gly Val Arg Ala Ser Ala His Gly
Thr Glu Leu Leu Leu 50 55 60Asp Ile
Cys Val Ser Leu Lys Leu Leu Lys Val Glu Thr Arg Gly Gly65
70 75 80Lys Ala Phe Tyr Arg Asn Thr
Glu Leu Ser Ser Asp Tyr Leu Thr Thr 85 90
95Val Ser Pro Thr Ser Gln Cys Ser Met Leu Lys Tyr Met
Gly Arg Thr 100 105 110Ser Tyr
Arg Cys Trp Gly His Leu Ala Asp Ala Val Arg Glu Gly Arg 115
120 125Asn Gln Tyr Leu Glu Thr Phe Gly Val Pro
Ala Glu Glu Leu Phe Thr 130 135 140Ala
Ile Tyr Arg Ser Glu Gly Glu Arg Leu Gln Phe Met Gln Ala Leu145
150 155 160Gln Glu Val Trp Ser Val
Asn Gly Arg Ser Val Leu Thr Ala Phe Asp 165
170 175Leu Ser Val Phe Pro Leu Met Cys Asp Leu Gly Gly
Asp Phe Phe Lys 180 185 190Asp
Pro Leu Pro Glu Ala Asp Leu Tyr Ile Leu Ala Arg Val Leu His 195
200 205Asp Trp Ala Asp Gly Lys Cys Ser His
Leu Leu Glu Arg Ile Tyr His 210 215
220Thr Cys Lys Pro Gly Gly Gly Ile Leu Val Ile Glu Ser Leu Leu Asp225
230 235 240Glu Asp Arg Arg
Gly Pro Leu Leu Thr Gln Leu Tyr Ser Leu Asn Met 245
250 255Leu Val Gln Thr Glu Gly Gln Glu Arg Thr
Pro Thr His Tyr His Met 260 265
270Leu Leu Ser Ser Ala Gly Phe Arg Asp Phe Gln Phe Lys Lys Thr Gly
275 280 285Ala Ile Tyr Asp Ala Ile Leu
Ala Arg Lys 290 2954207PRTHomo sapiens 4Met Ser Thr
Gln Ser Thr His Pro Leu Lys Pro Glu Ala Pro Arg Leu1 5
10 15Pro Pro Gly Ile Pro Glu Ser Pro Ser
Cys Gln Arg Arg His Thr Leu 20 25
30Pro Ala Ser Glu Phe Arg Cys Leu Thr Pro Glu Asp Ala Val Ser Ala
35 40 45Phe Glu Ile Glu Arg Glu Ala
Phe Ile Ser Val Leu Gly Val Cys Pro 50 55
60Leu Tyr Leu Asp Glu Ile Arg His Phe Leu Thr Leu Cys Pro Glu Leu65
70 75 80Ser Leu Gly Trp
Phe Glu Glu Gly Cys Leu Val Ala Phe Ile Ile Gly 85
90 95Ser Leu Trp Asp Lys Glu Arg Leu Met Gln
Glu Ser Leu Thr Leu His 100 105
110Arg Ser Gly Gly His Ile Ala His Leu His Val Leu Ala Val His Arg
115 120 125Ala Phe Arg Gln Gln Gly Arg
Gly Pro Ile Leu Leu Trp Arg Tyr Leu 130 135
140His His Leu Gly Ser Gln Pro Ala Val Arg Arg Ala Ala Leu Met
Cys145 150 155 160Glu Asp
Ala Leu Val Pro Phe Tyr Glu Arg Phe Ser Phe His Ala Val
165 170 175Gly Pro Cys Ala Ile Thr Val
Gly Ser Leu Thr Phe Met Glu Leu His 180 185
190Cys Ser Leu Arg Gly His Pro Phe Leu Arg Arg Asn Ser Gly
Cys 195 200 2055350PRTHomo sapiens
5Met Gln Gly Asn Gly Ser Ala Leu Pro Asn Ala Ser Gln Pro Val Leu1
5 10 15Arg Gly Asp Gly Ala Arg
Pro Ser Trp Leu Ala Ser Ala Leu Ala Cys 20 25
30Val Leu Ile Phe Thr Ile Val Val Asp Ile Leu Gly Asn
Leu Leu Val 35 40 45Ile Leu Ser
Val Tyr Arg Asn Lys Lys Leu Arg Asn Ala Gly Asn Ile 50
55 60\Phe Val Val Ser Leu Ala Val Ala Asp Leu Val Val
Ala Ile Tyr Pro65 70 75
80Tyr Pro Leu Val Leu Met Ser Ile Phe Asn Asn Gly Trp Asn Leu Gly
85 90 95Tyr Leu His Cys Gln Val
Ser Gly Phe Leu Met Gly Leu Ser Val Ile 100
105 110Gly Ser Ile Phe Asn Ile Thr Gly Ile Ala Ile Asn
Arg Tyr Cys Tyr 115 120 125Ile Cys
His Ser Leu Lys Tyr Asp Lys Leu Tyr Ser Ser Lys Asn Ser 130
135 140Leu Cys Tyr Val Leu Leu Ile Trp Leu Leu Thr
Leu Ala Ala Val Leu145 150 155
160Pro Asn Leu Arg Ala Gly Thr Leu Gln Tyr Asp Pro Arg Ile Tyr Ser
165 170 175Cys Thr Phe Ala
Gln Ser Val Ser Ser Ala Tyr Thr Ile Ala Val Val 180
185 190Val Phe His Phe Leu Val Pro Met Ile Ile Val
Ile Phe Cys Tyr Leu 195 200 205Arg
Ile Trp Ile Leu Val Leu Gln Val Arg Gln Arg Val Lys Pro Asp 210
215 220Arg Lys Pro Lys Leu Lys Pro Gln Asp Phe
Arg Asn Phe Val Thr Met225 230 235
240Phe Val Val Phe Val Leu Phe Ala Ile Cys Trp Ala Pro Leu Asn
Phe 245 250 255Ile Gly Leu
Ala Val Ala Ser Asp Pro Ala Ser Met Val Pro Arg Ile 260
265 270Pro Glu Trp Leu Phe Val Ala Ser Tyr Tyr
Met Ala Tyr Phe Asn Ser 275 280
285Cys Leu Asn Ala Ile Ile Tyr Gly Leu Leu Asn Gln Asn Phe Arg Lys 290
295 300Glu Tyr Arg Arg Ile Ile Val Ser
Leu Cys Thr Ala Arg Val Phe Phe305 310
315 320Val Asp Ser Ser Asn Asp Val Ala Asp Arg Val Lys
Trp Lys Pro Ser 325 330
335Pro Leu Met Thr Asn Asn Asn Val Val Lys Val Asp Ser Val 340
345 3506362PRTHomo sapiens 6Met Ser Glu
Asn Gly Ser Phe Ala Asn Cys Cys Glu Ala Gly Gly Trp1 5
10 15Ala Val Arg Pro Gly Trp Ser Gly Ala
Gly Ser Ala Arg Pro Ser Arg 20 25
30Thr Pro Arg Pro Pro Trp Val Ala Pro Ala Leu Ser Ala Val Leu Ile
35 40 45Val Thr Thr Ala Val Asp Val
Val Gly Asn Leu Leu Val Ile Leu Ser 50 55
60Val Leu Arg Asn Arg Lys Leu Arg Asn Ala Gly Asn Leu Phe Leu Val65
70 75 80Ser Leu Ala Leu
Ala Asp Leu Val Val Ala Phe Tyr Pro Tyr Pro Leu 85
90 95Ile Leu Val Ala Ile Phe Tyr Asp Gly Trp
Ala Leu Gly Glu Glu His 100 105
110Cys Lys Ala Ser Ala Phe Val Met Gly Leu Ser Val Ile Gly Ser Val
115 120 125Phe Asn Ile Thr Ala Ile Ala
Ile Asn Arg Tyr Cys Tyr Ile Cys His 130 135
140Ser Met Ala Tyr His Arg Ile Tyr Arg Arg Trp His Thr Pro Leu
His145 150 155 160Ile Cys
Leu Ile Trp Leu Leu Thr Val Val Ala Leu Leu Pro Asn Phe
165 170 175Phe Val Gly Ser Leu Glu Tyr
Asp Pro Arg Ile Tyr Ser Cys Thr Phe 180 185
190Ile Gln Thr Ala Ser Thr Gln Tyr Thr Ala Ala Val Val Val
Ile His 195 200 205Phe Leu Leu Pro
Ile Ala Val Val Ser Phe Cys Tyr Leu Arg Ile Trp 210
215 220Val Leu Val Leu Gln Ala Arg Arg Lys Ala Lys Pro
Glu Ser Arg Leu225 230 235
240Cys Leu Lys Pro Ser Asp Leu Arg Ser Phe Leu Thr Met Phe Val Val
245 250 255Phe Val Ile Phe Ala
Ile Cys Trp Ala Pro Leu Asn Cys Ile Gly Leu 260
265 270Ala Val Ala Ile Asn Pro Gln Glu Met Ala Pro Gln
Ile Pro Glu Gly 275 280 285Leu Phe
Val Thr Ser Tyr Leu Leu Ala Tyr Phe Asn Ser Cys Leu Asn 290
295 300Ala Ile Val Tyr Gly Leu Leu Asn Gln Asn Phe
Arg Arg Glu Tyr Lys305 310 315
320Arg Ile Leu Leu Ala Leu Trp Asn Pro Arg His Cys Ile Gln Asp Ala
325 330 335Ser Lys Gly Ser
His Ala Glu Gly Leu Gln Ser Pro Ala Pro Pro Ile 340
345 350Ile Gly Val Gln His Gln Ala Asp Ala Leu
355 360721DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 7gagcgattct tctgcctcag c
21820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8tctgcgcaca ctcccaggtg
20921DNAArtificial SequenceDescription of Artificial Sequence Synthetic
primer 9gaggcaggag aatcgcttga a
211022DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10ggctacatcg tgggtgtacg tc
221123DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 11tggtgcaatc tcatttgact ctg
231220DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 12gggttcatgc cattctcctg
201321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
13cagctgtaca aggcaagagg a
211421DNAArtificial SequenceDescription of Artificial Sequence Synthetic
primer 14ctttcacctc ctccactgcc a
211521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 15gcctgggcta cagagctgaa a
211620DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 16ctcctgggtt gtgccatttg
201722DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 17cctgtggggt atagctccgt tc
221821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18cgcacatgtc aaagcatcag a
211921DNAArtificial SequenceDescription of Artificial Sequence Synthetic
primer 19agcttgcagt gagcggaaat c
212021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 20gcacccatcg actcgtcatt t
212121DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 21tgggttggac ccttcatgag t
212221DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 22gtgtttccgg gagtgagagg a
212321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
23agcctggaag acctgggaaa g
212421DNAArtificial SequenceDescription of Artificial Sequence Synthetic
primer 24cctgtgggat gatttcagtg c
212521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25ggtgccctga ctgtcctctg a
212621DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 26ccatcagcgt ggtcctcagt a
2127430DNAHomo sapiensCDS(359)..(427)
27caggacctgc tcaatccata agacgattaa tgagtgctaa tgatgggtga gcatattggt
60ttgggatcag ttctacagct gatgagtcaa gggctcgggg gggcctgatg cagactattt
120tagggctgcg gaggggcgcc cagaaagcca gggaatcgcc acacgctttg tgatccccta
180atacggttaa tgcaaatttc tagagtgatc cgtctttgtt tgagtactgg gcaggcagca
240gggagagtca ggcagcagct gtgagcgggt ggctcttccc caccttgcca gcaggctctg
300tgctccttga agcaagcgct ccagaggctc cggaagccac ggctggattg gagacaag
358atg gga tcc tca gag gac cag gcc tat cgc ctc ctt aat gac tac gcc
406Met Gly Ser Ser Glu Asp Gln Ala Tyr Arg Leu Leu Asn Asp Tyr Ala1
5 10 15aac ggc ttc atg gtg tcc
cag gta 430Asn Gly Phe Met Val Ser
Gln 202823PRTHomo sapiens 28Met Gly Ser Ser Glu Asp Gln Ala
Tyr Arg Leu Leu Asn Asp Tyr Ala1 5 10
15Asn Gly Phe Met Val Ser Gln 202959DNAHomo
sapiens 29ttcccactta tgtgtgacct tggtggyaag tacccctcac cactaatacc
tcggaggtg 593065PRTHomo sapiens 30Trp Ala Asp Gly Lys Cys Ser His
Leu Leu Glu Arg Ile Tyr His Thr1 5 10
15Cys Lys Pro Gly Gly Ala Ile Leu Val Ile Glu Ser Leu Leu
Asp Glu 20 25 30Asp Arg Arg
Gly Pro Leu Leu Thr Gln Phe Tyr Ser Leu Asn Met Leu 35
40 45Val Gln Thr Glu Gly Gln Glu Arg Thr Pro Thr
His Tyr His Met Leu 50 55
60Leu653165PRTMacaca sp. 31Trp Ala Asp Gly Lys Cys Ser His Leu Leu Glu
Arg Val Tyr His Thr1 5 10
15Cys Lys Pro Gly Gly Gly Ile Leu Val Ile Glu Ser Leu Leu Asp Glu
20 25 30Asp Arg Arg Gly Pro Leu Leu
Thr Gln Leu Tyr Ser Leu Asn Met Leu 35 40
45Val Gln Thr Glu Gly Gln Glu Arg Thr Pro Thr His Tyr His Met
Leu 50 55 60Leu653265PRTBovine sp.
32Trp Thr Asp Ala Lys Cys Ser His Leu Leu Gln Arg Val Tyr Arg Ala1
5 10 15Cys Arg Thr Gly Gly Gly
Ile Leu Val Ile Glu Ser Leu Leu Asp Thr 20 25
30Asp Gly Arg Gly Pro Leu Thr Thr Leu Leu Tyr Ser Leu
Asn Met Leu 35 40 45Val Gln Thr
Glu Gly Arg Glu Arg Thr Pro Ala Glu Tyr Arg Ala Leu 50
55 60Leu653365PRTRattus sp. 33Trp Ala Asp Gly Ala Cys
Val Glu Leu Leu Gly Arg Leu His Arg Ala1 5
10 15Cys Thr Ser Ser Gly Ala Leu Leu Leu Val Glu Ala
Val Leu Ala Lys 20 25 30Gly
Gly Ala Gly Pro Leu Arg Ser Leu Leu Leu Ser Leu Asn Met Met 35
40 45Leu Gln Ala Glu Gly Trp Glu Arg Gln
Ala Ser Asp Tyr Arg Asn Leu 50 55
60Ala653465PRTGallus sp. 34Trp Asp Asp Lys Lys Cys Arg Gln Leu Leu Ala
Glu Val Tyr Lys Ala1 5 10
15Cys Arg Pro Gly Gly Gly Val Leu Leu Val Glu Ser Leu Leu Ser Glu
20 25 30Asp Arg Ser Gly Pro Val Glu
Thr Gln Leu Tyr Ser Leu Asn Met Leu 35 40
45Val Gln Thr Glu Gly Lys Glu Arg Thr Ala Val Glu Tyr Ser Glu
Leu 50 55 60Leu653562PRTDanio sp.
35Trp Ser Asp Glu Lys Leu His Val Leu Leu Ser Lys Leu Cys Lys Met1
5 10 15Trp Thr Pro Gly Cys Gly
Leu Leu Val Ser Glu Ile Leu Leu Asp Glu 20 25
30Glu Arg Arg Arg Pro Ser Arg Ala Leu Leu Gln Ala Leu
Ser Met Thr 35 40 45Glu Gly Lys
Gln Arg Ser Thr Ala Glu Tyr Ser Ala Leu Leu 50 55
603665PRTFugu sp. 36Trp Thr Asp Gln Arg Cys Leu Glu Leu Leu
Arg Arg Val His Gly Ala1 5 10
15Cys Arg Pro Gly Gly Ser Val Leu Leu Val Glu Ala Leu Leu Tyr Glu
20 25 30Asp Gly Ser Gly Pro Leu
Thr Ala Gln Leu Tyr Ser Leu Asn Met Leu 35 40
45Val Gln Thr Glu Gly Arg Glu Arg Ser Ala Ala Gln Tyr Ala
Ala Leu 50 55 60Leu65
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