Polymorphic variation and transcriptional regulation of muscarinic receptor genes in human airway cells
Muscarinic M2 and M3 receptors are important for the development of airway
hyperresponsiveness in asthma. While there is currently no evidence to suggest altered
function of M3 receptors in airway disease, a dysfunctionality of M2 receptors has been
reported to occur in asthmatic patients. Current studies suggest that this
dysfunctionality may be caused by allosteric modulation induced by endogenous
mediators released by pro-inflammatory cells in inflamed airways. However, it is not
known whether genetic variation in the coding or transcriptional regulatory regions of
the gene contributes to this observed dysfunction.
In order to identify potential genetic contribution, the following approaches were used:
(a) the muscarinic M2 and M3 receptor genes and flanking sequences were screened for
polymorphic variation in asthmatic individuals and controls, using single stranded
conformation polymorphism analysis and direct sequencing; (b) the promoter region
and 5'UTR arrangement of the muscarinic M2 receptor gene were identified, using a
5'RACE approach on cDNA obtained from human airway smooth muscle (HASM)
cells; (c) regions of major transcriptional regulatory activity were identified using
transient transfection reporter gene assays; (d) the major regulatory region was
screened for polymorphic genetic variation, using an approach which involved cloning
and sequencing; (e) identified promoter polymorphic variants were studied in a
reporter gene assay system in order to identify the polymorphism contribution to
transcriptional regulation and (t) transmission disequilibrium testing was performed in
asthmatic families in order to identify whether overtransmission of any promoter
variants occurs to asthmatic offspring.
This study identified two rare degenerate single nucleotide polymorphisms in the
coding region of the muscarinic M2 receptor gene at positions 976 (A~C) and 1197
(T~C) (c.f. ATG). In addition, a common polymorphism was identified within the
3'UTR (1696T~A) (allelic frequency 65%) and an extra 'A' was present at position
1793 in all samples investigated by sequencing or RFLP analysis, suggesting that there
is a missing base in the published NCBI sequence M16404. TDT analysis suggested
that the 1696T ~A SNP is not linked to asthma.
The muscarinic M2 receptor coding sequence is preceded by a splice site located 46bp
upstream of the ATG start codon. Five exons with alternative splicing patterns are
present upstream of this splice site, separated by introns ranging from 87bp to 145.4kb.
Three major regions of transcriptional initiation were identified (TSS 1, TSS2, TSS3),
with multiple transcriptional start sites (TSSs) clustered around each region. There is
evidence for the gene being under the control of a TAT A-less promoter with Sp 1,
GATA and AP binding sites. Polymorphism screening revealed a O.5kb hypervariable
region located 648bp upstream of the most 5' TSS, a multiallelic (CA) tandem repeat
96bp downstream of the most 5' TSS and a common C~A SNP located 136bp
upstream of the most 5' TSS. Functional studies in primary HASM cells and the
BEAS-2B cell line, demonstrated the highest promoter activity to be upstream of the
TSS3 cluster, with potential repressor elements operating in a cell-type dependent
manner, located upstream of ISSl. Based on the frequency of 5'RACE products
obtained, transcription initiation occurs most frequently from the TSS3 cluster, and
transcription factor consensus sequence mapping shows the region upstream of TSS3
to be the richest in Spl, AP and GATA sites.
Seven variants of the CA repeat have been identified, and have been shown to
influence reporter gene transcriptional regulation in HASM and BEAS-2B cells. The
three low expressing variants ~(CA)I, ~(CA)3 and ~(CA)8 showed positive TDT
results in asthmatic families, suggesting that these polymorphisms may contribute to
the expression of the asthma phenotype.
This project has identified a high conserved status of the human muscarinic M2 and M3
gene coding sequences, and concludes that the observed genetic variation within these
genes is unlikely to contribute to phenotypic variation. Specific CA repeat promoter
variants, have however been shown to alter transcriptional regulation in transiently
transfected cell culture models, and to be linked to the asthma phenotype in patients.
These variants may therefore contribute to the development of the disease status or to
its severity. Subsequent studies may identify further roles of these polymorphisms in
determining treatment responses.