title: HTR4 gene structure and altered expression in the developing lung.
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.
authors.affiliation: Division of Respiratory Medicine, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.
authors.forename: Emily
authors.initials: E
authors.lastname: Hodge
authors.forename: Carl P
authors.initials: CP
authors.lastname: Nelson
authors.forename: Suzanne
authors.initials: S
authors.lastname: Miller
authors.forename: Charlotte K
authors.initials: CK
authors.lastname: Billington
authors.forename: Ceri E
authors.initials: CE
authors.lastname: Stewart
authors.forename: Caroline
authors.initials: C
authors.lastname: Swan
authors.forename: Anders
authors.initials: A
authors.lastname: Malarstig
authors.forename: Amanda P
authors.initials: AP
authors.lastname: Henry
authors.forename: Catherine
authors.initials: C
authors.lastname: Gowland
authors.forename: Erik
authors.initials: E
authors.lastname: Melén
authors.forename: Ian P
authors.initials: IP
authors.lastname: Hall
authors.forename: Ian
authors.initials: I
authors.lastname: Sayers
mesh_terms: D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D006801:Humans; D008168:Lung / Q000196:embryology* / Q000502:physiology*; D008297:Male; D011985:Receptors, Serotonin / Q000235:genetics*
chemical_list: D011985:Receptors, Serotonin; C104425:serotonin 7 receptor
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