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. 2009 Mar 15;18(6):1037-51.
doi: 10.1093/hmg/ddn439. Epub 2008 Dec 22.

Expansion of the Human Mu-Opioid Receptor Gene Architecture: Novel Functional Variants

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Free PMC article

Expansion of the Human Mu-Opioid Receptor Gene Architecture: Novel Functional Variants

Svetlana A Shabalina et al. Hum Mol Genet. .
Free PMC article

Abstract

The mu-opioid receptor (OPRM1) is the principal receptor target for both endogenous and exogenous opioid analgesics. There are substantial individual differences in human responses to painful stimuli and to opiate drugs that are attributed to genetic variations in OPRM1. In searching for new functional variants, we employed comparative genome analysis and obtained evidence for the existence of an expanded human OPRM1 gene locus with new promoters, alternative exons and regulatory elements. Examination of polymorphisms within the human OPRM1 gene locus identified strong association between single nucleotide polymorphism (SNP) rs563649 and individual variations in pain perception. SNP rs563649 is located within a structurally conserved internal ribosome entry site (IRES) in the 5'-UTR of a novel exon 13-containing OPRM1 isoforms (MOR-1K) and affects both mRNA levels and translation efficiency of these variants. Furthermore, rs563649 exhibits very strong linkage disequilibrium throughout the entire OPRM1 gene locus and thus affects the functional contribution of the corresponding haplotype that includes other functional OPRM1 SNPs. Our results provide evidence for an essential role for MOR-1K isoforms in nociceptive signaling and suggest that genetic variations in alternative OPRM1 isoforms may contribute to individual differences in opiate responses.

Figures

Figure 1.
Figure 1.
The structure of the human and mouse OPRM1 gene. (A) Conventional structures of the mouse (upper panel) and human (middle panel) OPRM1 genes are shown in accordance with the NCBI database, UCSC genome browser and published data (43,76,77). Our version of the structure of the human OPRM1 gene (lower panel) is based on multispecies genome alignments created by OWEN (36) and comparative genomes analysis. Exons and introns are shown by vertical and horizontal boxes, respectively. Shaded boxes represent constitutive exons. Maximal sizes of human exons (for lower panel) are shown in parentheses (nt): exon 11 (206), exon 1 (580), exon T (117), exon 14 (105), exon 13 (1200), exon 2 (353), exon 3 (521), exon R (488), exon Y (109), exon 16 (314), exon X (1271), exon 17 (128), exon 5 (1013), exon 4 (304), exon 18 (412), exon 6 (124), exon 7 (89), exon 9 (393). (B) Alignment of human (1), chimpanzee (2), macaca (3), rat (4) and mouse (5) exon 13 and its conserved vicinity regions. Splicing boundaries are indicated by vertical bars. Enhancers of splicing (39) near exon–intron boundaries are marked in yellow. SNP rs563649 is highlighted in red. Conserved nucleotides for all species are marked by stars. Structurally conserved IRES in the human sequence is marked by green. Predicted uORFs in the human sequence are marked by arrows.
Figure 2.
Figure 2.
Expression pattern of exon 13 containing OPRM1 gene splice variant in human and mouse. (A) The schematic diagram illustrates relative positions of PCR primers designed to amplify the new alternative MOR-1K variants in mouse and human. The arrow indicates the relative position of SNP rs563649. (B) RT–PCR was performed on total RNA samples from the human brain regions known to express OPRM1 with hU2 and hL5 primers specific for exons 13 and exon 2, respectively. The exon 13 containing OPRM1 gene splice variant MOR-1K was detected in CNS but not in peripheral leukocytes even after a secondary PCR round with nested PCR primers. The PCR product size was 1229 nt, which was three times longer than the predicted 385 nt based on homology with the mouse genome. (C) RT–PCR analysis of mouse spinal cord with primer pairs mU2-mL3 and mU2-mL1 yielded PCR products of predicted size. A longer mouse isoform orthologous to human exon 13 was below the level of detection even by secondary PCR with the nested PCR primers mU3-mL3, mU3-mL1 or mU3-mL2. (D) The schematic diagram illustrates the exonic composition and relative positions of PCR primers designed to amplify the major MOR-1 variant and the newly identified alternative MOR-1K variant. The arrows indicate relative positions of translation initiation start codons and stop codons. (E) The predicted protein structure of MOR-1 and MOR-1K isoforms. Translation of the MOR-1K variants results in a 6 transmembrane domain (6TM) receptor, truncated at the N-terminus. (F) RT–PCR results demonstrate the relative expression pattern of human MOR-1 (primers hU1-L3) and MOR-1K (primers hU5-L3) variants. GAP3DH was used as a control for cDNA loading. All major PCR products shown in this figure were sequenced and aligned with human or mouse genomes.
Figure 3.
Figure 3.
Allelic variants of structurally conserved IRES in exon 13 of human OPRM1. (A) The local stem-loop structure associated with putative IRES within the 5′-UTR of OPRM-1K isoform. The major allele C of SNP rs563649 is shown in red. Translation start codon of downstream uORF is shown in blue. (B) A cloning of putative IRES allelic variants into secreted alkaline phoshatase (SEAP) reporter vector and associated translation detection experiments. (C and D) The expression of IRES-SEAP constructs transiently transfected into human neuroblastoma BE2C cells. Relative mRNA (C) and SEAP activity (D) levels were measured 8, 24 and 48 h after transfection. Reporter construct with the T allele showed significantly higher SEAP activity than reporter construct with the C allele, although mRNA levels of construct with the C allele were significantly higher than those with the T allele (P < 0.05, n = 4), suggesting higher translation activity of the IRES T allelic variant.

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