Genomic sequence of the human &mgr;-opioid receptor gene and...

Details Genomic sequence of the human &mgr;-opioid receptor gene and... Genomic sequence of the human &mgr;-opioid receptor gene and...

1999-09-27

2003-03-25

Fredman, Jeffrey (Department: 1637)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S022100

Reexamination Certificate

active

06538120

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to the genomic sequence of the human &mgr;-opioid receptor gene and its variants, polymorphisms and mutations and their use.
As is known, the human &mgr;-opioid receptor controls pain perception, ‘reward’ mechanisms and further important physiological functions. It is the highly specific target for morphine, the classical painkiller in contemporary medicine. In addition, it is target for further medically important analgesics, anesthetics and therapeutics such as e.g. methadone and fentanyl and commonly used addictive substances such as e.g. heroin and methadone. A number of (patho)physiological, biochemical, pharmacological findings, and observations from &mgr;-opioid receptor knock out mice demonstrate that the &mgr; opioid receptor gene is playing a major role in analgesia and anesthesia, and in the development and maintenance of addictions (dependence on opiates, alcoholism and other forms of dependence). Therefore, variants in the regulating, coding and intronic regions of this gene may contribute to genetic risk for addictions. In addition, such variants may affect the responsiveness of this receptor to endogenous and exogenous receptor ligands.
Addictions are common diseases of international dimension and cause, in general, scarcely comprehensible grave economic damages to the amount of billions up to trillions, not to speak of the deleterious psychosocial consequences for the individual, his family and society.
The genomic sequence of the human &mgr; opioid receptor gene is not known. So far only the cDNA of the &mgr; opioid receptor gene has been described; the first cDNA of a &mgr; opioid receptor was cloned by means of probes targeted against conserved regions of the &dgr; opioid receptor from a rat cDNA gene bank by Chen et al. (Molecular cloning and functional expression of a mu opioid receptor from rat brain. Mol. Pharmacol. 44, 8-12, 1993), the first human MOR cDNA by Wang et al. (Mu opiate receptor: cDNA cloning and expression. Proc. Natl. Acad. Sci. U.S.A. 90, 10230-10234, 1993). The only promoter sequence so far known was cloned from a mouse gene bank (Min. et al., Genomic structure and analysis of promoter sequence of a mouse &mgr; opioid receptor gene. Proc. Natl. Acad. Sci. U.S.A. 91, 9081-9085,1994).
Actual findings obtained from ‘knockout’ mice with an interrupted &mgr; opioid receptor gene show clearly that the analgesic and ‘reward’ inducing and dependence inducing effects of morphine are brought about specifically through the &mgr;-opioid receptor subtype, yet not through &dgr;- and &kgr; opioid receptor subtypes. Accordingly, the &mgr; opioid receptor is mandatory for morphine action in vivo (Matthes et al., Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the &mgr; opioid receptor gene. Nature 383, 819-823, 1996). A series of pharmacological and other studies have shown that the &mgr;opioid receptor expressed in brain is of key importance for the development of tolerance, addiction and analgesia (Reisine, Neurotransmitter Receptors V., Neuropharmacology 34, 463-472, 1995). In this context, endorphins come into consideration as endogenous ligands. Exogenous &mgr; opioid receptor ligands such as morphine, codeine, methadone and fentanyl have been used for a long time as analgesics and therapeutics in clinics.
As is known, the clinical use of opiates causes undesired side effects such as breathing disturbances, miosis, nausea and vomiting, sedation, depression and dependence.
It is the objective of the invention to identify and provide the genomic sequence of the human &mgr; opioid receptor which may be used as basis for the development of specific and efficient analgesics, anesthetics and therapeutics for addiction, in particular for developing e.g. analgesics not having addictive side effects, or for developing diagnostic kits.
According to the invention it was possible to identify and provide the genomic sequence of the human &mgr; opioid receptor gene and of variants, polymorphisms and mutants in specific populations.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The genomic DNA sequence of the human &mgr; opioid receptor amplified and sequenced according to the invention consists of
1) a promoter region (incl. 5′ regulatory region) SEQ ID no. 1 of a length of altogether 2412 bp.
The promoter is cloned according to methods known per se by amplifying the five various genomic DNA regions covering the human &mgr; opioid receptor promoter region.
2) Intron 2 the genomic DNA sequence of which is between nucleotides 855 and 856 of the cDNA sequence (or between nucleotides 643 and 644 related to A of the translation starting point) of a length of 773 bp according to SEQ ID no. 2.
3) the 5′ region of intron 1 the genomic DNA sequence of which is behind nucleotide 502 of the cDNA sequence (or behind nucleotide 290 related to the translation starting point) of a length of 383 bp according to SEQ ID no. 3; and the 3′ region the genomic DNA sequence of which is in front of nucleotide 503 of the cDNA sequence (or in front of nucleotide 291 related to the translation starting point) of a length of 538 bp according to SEQ ID no. 4.
4) the 5′ region of intron 3 the genomic DNA sequence of which is behind nucleotide 1376 of the cDNA sequence (or behind nucleotide 1164 related to the translation starting point) of a length of 300 bp according to SEQ ID no. 5; and the 3′ region the genomic DNA sequence of which is in front of nucleotide 1377 of the cDNA sequence (or in front of nucleotide 1165 related to the translation starting point) of a length of 400 bP according to SEQ ID no. 6.
Sequencing of the intron is carried out analogously to sequencing of promoters.
In addition, there was detected that the human cDNA sequence already known consisting of 2162 bp is defective in the first 16 nucleotides. According to the invention the cDNA has SEQ ID no. 7.
The genomic sequence of the human &mgr; opioid receptor gene is represented in illustrations 1a and 1b in a survey.
Four different transcription starting positions were identified in positions 212, 329, 371 and 421 bp upstream of the translation star site (ATG).
Variants, polymorphisms and mutants in specific populations are marked by base exchange. According to the invention these are base exchanges in up to 100 nucleotide positions, preferably up to 37 base exchanges are identified. In the cDNA region, in particular, preferably up to 20 base exchanges are identified, most preferably up to 11 base exchanges.
In a scheme according to the invention base exchanges take place in the following positions of the promoter (=RG=5′ regulatory region), of the exons (=cDNA sequence) and of the introns:
Exchange of
Exchange of
amino acids/
Region
Name
nucleotides
Splice variant
position
cDNA
−1793/4T→A
T→A at
RG
−1793/4
−1768ins22
Insertion of
RG
22 bp behind
−1768
−1699insT
Insertion of T
RG
behind −1699
−1595T→C
T→C at −1595
RG
−1565T→C
T→C at −1565
RG
−1469T→C
T→C at −1469
RG
−1320A→G
A→G at −1320
RG
−1255A→T
A→T at −1255
RG
−1236A→G
A→G at −1236
RG
−1171A→G
A→G at −1171
RG
−1045A→G
A→G at −1045
RG
 −995C→A
C→A at −995
RG
 −692G→C
G→C at −692
RG
 −665del3
Deletion of
RG
3 bp from
−665 to −663
 −554G→A
G→A at −554
RG
 −488G→T
G→T at −488
RG
 −254A→C
A→C at −254
RG
 −236A→G
A→G at −236
RG
 −172G→T
G→T at −172
Exon 1
41
 −133C→T
C→T at −133
Exon 1
80
 −111C→T
C→T at −111
Exon 1
102
 −38C→A
C→A at −38
Exon 1
175
A6V(C→T)
C→T at 17
Ala→Val at 6
Exon 1
229
N40D(A&r

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