title: Novel mutations in the GH gene (GH1) uncover putative splicing regulatory elements.
abstract: Mutations affecting exon 3 splicing are the main cause of autosomal dominant Isolated GH Deficiency II (IGHDII) by increasing the level of exon 3-skipped mRNA encoding the functionally inactive dominant-negative 17.5-kDa isoform. The exons and introns of the gene encoding GH (GH1) were screened for the presence of mutations in 103 sporadic isolated GH deficiency cases. Four different variations within exon 3 were identified in 3 patients. One carried c.261C>T (p.Pro87Pro) and c.272A>T (p.Glu91Val), the second c.255G>A (p.Pro85Pro) and c.261 C>T, and the third c.246G>C (p.Glu82Asp). All the variants were likely generated by gene conversion from an homologous gene in the GH1 cluster. In silico analysis predicted that positions c.255 and c.272 were included within 2 putative novel exon splicing enhancers (ESEs). Their effect on splicing was confirmed in vitro. Constructs bearing these 2 variants induced consistently higher levels both of transcript and protein corresponding to the 17.5-kDa isoform. When c.255 and c.272 were combined in cis with the c.261 variant, as in our patients, their effect was weaker. In conclusion, we identified 2 variations, c.255G>A and c.272A>T, located in 2 novel putative exon splicing enhancers and affecting GH1 splicing in vitro by increasing the production of alternatively spliced isoforms. The amount of aberrant isoforms is further regulated by the presence in cis of the c.261 variant. Thus, our results evidenced novel putative splicing regulatory elements within exon 3, confirming the crucial role of this exon in mRNA processing.
authors.affiliation: Laboratory of Genetics (D.B., S.M., I.F., P.M.-R., M.G.), Department of Health Sciences, University of Eastern Piedmont and Interdisciplinary Research Center of Autoimmune Diseases, and Unit of Pediatrics (A.P., G.E.W., S.B., F.P., G.B.), Department of Health Sciences, University of Eastern Piedmont, 28100 Novara, Italy.
authors.forename: Deepak
authors.initials: D
authors.lastname: Babu
authors.forename: Simona
authors.initials: S
authors.lastname: Mellone
authors.forename: Ileana
authors.initials: I
authors.lastname: Fusco
authors.forename: Antonella
authors.initials: A
authors.lastname: Petri
authors.forename: Gillian E
authors.initials: GE
authors.lastname: Walker
authors.forename: Simonetta
authors.initials: S
authors.lastname: Bellone
authors.forename: Flavia
authors.initials: F
authors.lastname: Prodam
authors.forename: Patricia
authors.initials: P
authors.lastname: Momigliano-Richiardi
authors.forename: Gianni
authors.initials: G
authors.lastname: Bona
authors.forename: Mara
authors.initials: M
authors.lastname: Giordano
mesh_terms: D000293:Adolescent; D017398:Alternative Splicing; D019943:Amino Acid Substitution; D002460:Cell Line; D002648:Child; D019295:Computational Biology; D004393:Dwarfism, Pituitary / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D005103:Expert Systems; D005785:Gene Conversion; D019382:Human Growth Hormone / Q000737:chemistry / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D010902:Pituitary Gland / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020218:Response Elements
chemical_list: D050505:Mutant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019382:Human Growth Hormone