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HTR4 gene structure and altered expression in the developing lung.

Hodge E, Nelson CP, Miller S, Billington CK, Stewart CE, Swan C, Malarstig A, Henry AP, Gowland C, Melén E, Hall IP, Sayers I - Respir. Res. (2013)

Bottom Line: Meta-analyses of genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) spanning the 5-hydroxytryptamine receptor 4 (5-HT₄R) gene (HTR4) associated with lung function.Radioligand binding experiments also indicated that HBEC and HASM cells did not express a significant 5-HT₄R population. 5' RACE in brain identified a novel N-terminal variant, containing an extended N-terminal sequence.Taken together, these data suggest a role for HTR4 in lung development, which may at least in part explain the genetic association with lung function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Respiratory Medicine, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.

ABSTRACT

Background: Meta-analyses of genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) spanning the 5-hydroxytryptamine receptor 4 (5-HT₄R) gene (HTR4) associated with lung function. The aims of this study were to i) investigate the expression profile of HTR4 in adult and fetal lung tissue and cultured airway cells, ii) further define HTR4 gene structure and iii) explore the potential functional implications of key SNPs using a bioinformatic approach.

Methods: Following reverse transcription (RT)-PCR in human brain, 5' rapid amplification of cDNA ends (5' RACE) was used to examine the exonic structure of HTR4 at the 5' end. Quantitative (Q)-PCR was used to quantify HTR4 mRNA expression in total RNA from cultured airway cells and whole lung tissue. Publically available gene microarray data on fetal samples of estimated gestational age 7-22 weeks were mined for HTR4 expression. Immunohistochemistry (IHC; in adult and fetal lung tissue) and a radioligand binding assay (in cultured airway cells) were used to analyze 5-HT₄R protein expression.

Results: IHC in adult lung, irrespective of the presence of chronic obstructive pulmonary disease (COPD), suggested low level expression of 5-HT₄R protein, which was most prominent in alveolar pneumocytes. There was evidence of differential 5-HT₄R protein levels during gestation in fetal lung, which was also evident in gene expression microarray data. HTR4 mRNA expression, assessed by Q-PCR, was <0.5% relative to brain in total adult lung tissue and in human airway smooth muscle (HASM) and bronchial epithelial cells (HBEC) derived from adult donors. Radioligand binding experiments also indicated that HBEC and HASM cells did not express a significant 5-HT₄R population. 5' RACE in brain identified a novel N-terminal variant, containing an extended N-terminal sequence. The functional significance of key HTR4 SNPs was investigated using the encyclopedia of DNA elements consortium (ENCODE) dataset. These analyses identified multiple alterations in regulatory motifs for transcription factors implicated in lung development, including Foxp1.

Conclusions: Taken together, these data suggest a role for HTR4 in lung development, which may at least in part explain the genetic association with lung function.

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Analysis of HTR4 gene structure and identification of a novel transcript variant in brain. 5′ RACE in cDNA derived from total brain tissue indicated expression of transcripts possessing exons 1-3, and also a novel transcript in which exons 1 and 2 are replaced by an alternate exon (a). The remaining sequence of the novel transcript aligns with the equivalent region of transcript b (b). The predicted protein structure of this novel variant has an elongated N-terminus and affects the position of an N-linked glycosylation site in this region (c).
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Figure 6: Analysis of HTR4 gene structure and identification of a novel transcript variant in brain. 5′ RACE in cDNA derived from total brain tissue indicated expression of transcripts possessing exons 1-3, and also a novel transcript in which exons 1 and 2 are replaced by an alternate exon (a). The remaining sequence of the novel transcript aligns with the equivalent region of transcript b (b). The predicted protein structure of this novel variant has an elongated N-terminus and affects the position of an N-linked glycosylation site in this region (c).

Mentions: 5′ RACE showed variation of HTR4 transcripts in cDNA derived from total brain tissue only; it was not possible to amplify HTR4 transcripts from total lung tissue, HASM, HBEC (undifferentiated and differentiated) and PBMC due to the extremely low abundance. Approximately half of the analyzed clones derived from brain cDNA showed the presence of exons 1–3, although exon 1 was truncated by 41bp at the 5′ end compared to the sequence reported by NCBI (Figure 6a). The remaining clones possessed a novel exon of 107bp, in place of exons 1 and 2. Two consecutive RT-PCR procedures in cDNA derived from total brain, i.e. using the amplicon from the first as a template in a second, and confirmation by sequencing showed that the exonic structure of the novel exon-containing variant was comparable to transcript b, since sequence alignment was of good quality for consecutive exons 3, 4, 5, 7 and 9 (Figure 6b). However, consecutive RT-PCR procedures failed to detect this novel transcript in total lung tissue or differentiated HBEC (data not shown). Using BLAST, we found that the sequence of the novel exon aligns with a region on the same genomic contig as the HTR4 gene (accession NG_029052.1). Using the same translational stop codon as that in transcript b, the novel transcript variant has an in-frame translational start codon (ATG) within this novel exon. The predicted protein structure of the novel transcript has an extended N-terminus (Figure 6c). Furthermore, the N-linked glycosylation site in the N-terminal region of transcript b is replaced in the novel transcript by an alternate site further upstream.


HTR4 gene structure and altered expression in the developing lung.

Hodge E, Nelson CP, Miller S, Billington CK, Stewart CE, Swan C, Malarstig A, Henry AP, Gowland C, Melén E, Hall IP, Sayers I - Respir. Res. (2013)

Analysis of HTR4 gene structure and identification of a novel transcript variant in brain. 5′ RACE in cDNA derived from total brain tissue indicated expression of transcripts possessing exons 1-3, and also a novel transcript in which exons 1 and 2 are replaced by an alternate exon (a). The remaining sequence of the novel transcript aligns with the equivalent region of transcript b (b). The predicted protein structure of this novel variant has an elongated N-terminus and affects the position of an N-linked glycosylation site in this region (c).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750317&req=5

Figure 6: Analysis of HTR4 gene structure and identification of a novel transcript variant in brain. 5′ RACE in cDNA derived from total brain tissue indicated expression of transcripts possessing exons 1-3, and also a novel transcript in which exons 1 and 2 are replaced by an alternate exon (a). The remaining sequence of the novel transcript aligns with the equivalent region of transcript b (b). The predicted protein structure of this novel variant has an elongated N-terminus and affects the position of an N-linked glycosylation site in this region (c).
Mentions: 5′ RACE showed variation of HTR4 transcripts in cDNA derived from total brain tissue only; it was not possible to amplify HTR4 transcripts from total lung tissue, HASM, HBEC (undifferentiated and differentiated) and PBMC due to the extremely low abundance. Approximately half of the analyzed clones derived from brain cDNA showed the presence of exons 1–3, although exon 1 was truncated by 41bp at the 5′ end compared to the sequence reported by NCBI (Figure 6a). The remaining clones possessed a novel exon of 107bp, in place of exons 1 and 2. Two consecutive RT-PCR procedures in cDNA derived from total brain, i.e. using the amplicon from the first as a template in a second, and confirmation by sequencing showed that the exonic structure of the novel exon-containing variant was comparable to transcript b, since sequence alignment was of good quality for consecutive exons 3, 4, 5, 7 and 9 (Figure 6b). However, consecutive RT-PCR procedures failed to detect this novel transcript in total lung tissue or differentiated HBEC (data not shown). Using BLAST, we found that the sequence of the novel exon aligns with a region on the same genomic contig as the HTR4 gene (accession NG_029052.1). Using the same translational stop codon as that in transcript b, the novel transcript variant has an in-frame translational start codon (ATG) within this novel exon. The predicted protein structure of the novel transcript has an extended N-terminus (Figure 6c). Furthermore, the N-linked glycosylation site in the N-terminal region of transcript b is replaced in the novel transcript by an alternate site further upstream.

Bottom Line: Meta-analyses of genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) spanning the 5-hydroxytryptamine receptor 4 (5-HT₄R) gene (HTR4) associated with lung function.Radioligand binding experiments also indicated that HBEC and HASM cells did not express a significant 5-HT₄R population. 5' RACE in brain identified a novel N-terminal variant, containing an extended N-terminal sequence.Taken together, these data suggest a role for HTR4 in lung development, which may at least in part explain the genetic association with lung function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Respiratory Medicine, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.

ABSTRACT

Background: Meta-analyses of genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) spanning the 5-hydroxytryptamine receptor 4 (5-HT₄R) gene (HTR4) associated with lung function. The aims of this study were to i) investigate the expression profile of HTR4 in adult and fetal lung tissue and cultured airway cells, ii) further define HTR4 gene structure and iii) explore the potential functional implications of key SNPs using a bioinformatic approach.

Methods: Following reverse transcription (RT)-PCR in human brain, 5' rapid amplification of cDNA ends (5' RACE) was used to examine the exonic structure of HTR4 at the 5' end. Quantitative (Q)-PCR was used to quantify HTR4 mRNA expression in total RNA from cultured airway cells and whole lung tissue. Publically available gene microarray data on fetal samples of estimated gestational age 7-22 weeks were mined for HTR4 expression. Immunohistochemistry (IHC; in adult and fetal lung tissue) and a radioligand binding assay (in cultured airway cells) were used to analyze 5-HT₄R protein expression.

Results: IHC in adult lung, irrespective of the presence of chronic obstructive pulmonary disease (COPD), suggested low level expression of 5-HT₄R protein, which was most prominent in alveolar pneumocytes. There was evidence of differential 5-HT₄R protein levels during gestation in fetal lung, which was also evident in gene expression microarray data. HTR4 mRNA expression, assessed by Q-PCR, was <0.5% relative to brain in total adult lung tissue and in human airway smooth muscle (HASM) and bronchial epithelial cells (HBEC) derived from adult donors. Radioligand binding experiments also indicated that HBEC and HASM cells did not express a significant 5-HT₄R population. 5' RACE in brain identified a novel N-terminal variant, containing an extended N-terminal sequence. The functional significance of key HTR4 SNPs was investigated using the encyclopedia of DNA elements consortium (ENCODE) dataset. These analyses identified multiple alterations in regulatory motifs for transcription factors implicated in lung development, including Foxp1.

Conclusions: Taken together, these data suggest a role for HTR4 in lung development, which may at least in part explain the genetic association with lung function.

Show MeSH
Related in: MedlinePlus