Analysis of GNAS1 and Overlapping Transcripts Identifies the Parental Origin of Mutations in Patients with Sporadic Albright Hereditary Osteodystrophy.

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Analysis of GNAS1 and Overlapping Transcripts Identifies the Parental Origin of Mutations in Patients with Sporadic Albright Hereditary Osteodystrophy and Reveals a Model System in Which to Observe the Effects of Splicing Mutations on Translated and Untranslated Messenger RNA Sarah J. Rickard, Louise C. Wilson The American Journal of Human Genetics Volume 72, Issue 4, Pages 961-974 (April 2003) DOI: 10.1086/374566 Copyright © 2003 The American Society of Human Genetics Terms and Conditions

Figure 1 Schematic representation of the GNAS1 cluster, including exons 1–13 of GNAS1 and upstream imprinted exons NESP55, XLαs, and exon 1A. a, Summary of imprinting. Arrows above and below depict paternally and maternally expressed transcripts, respectively. The transcripts for Gsα and XLαs have ORFs starting in their unique 5′ exons and ending in exon 13. Exons 2–13 of GNAS1 lie within the 3′ UTR of NESP55. Exon 1A is probably not translated. b, Location of primers used to amplify the Gsα, NESP55, and exon 1A transcripts (not to scale). The American Journal of Human Genetics 2003 72, 961-974DOI: (10.1086/374566) Copyright © 2003 The American Society of Human Genetics Terms and Conditions

Figure 2 Sequence data from patient AHO-33. A heterozygous IVS4:−11A→G mutation was present in genomic DNA. Gsα cDNA amplification products showed only normal transcript, owing to NMD of the mutant transcript, whereas amplification of the NESP55 transcript revealed retention of intron 4. The base substitution is depicted in boldface. Intronic and exonic sequences are given in lowercase and uppercase, respectively. Exon 4 sequence is underlined. The American Journal of Human Genetics 2003 72, 961-974DOI: (10.1086/374566) Copyright © 2003 The American Society of Human Genetics Terms and Conditions

Figure 3 Sequence data showing genomic DNA and NESP55 and exon 1A cDNA products. a, Patient AHO-10. The heterozygous c.565-568delGACT deletion is present in genomic DNA, as denoted by the inverted triangle. Only deleted sequence is present in the NESP55 transcript, and only normal sequence is present in exon 1A, indicating that the GNAS1 mutation is maternally derived. b, Patient AHO-11. The heterozygous c.432+1G→A intron 5 splice-site mutation is present in genomic DNA. In NESP55 cDNA, normal exon 5 splicing has occurred. In exon 1A cDNA, skipping of exon 5 has occurred, confirming that the GNAS1 mutation is paternally derived. Exonic sequence is given in uppercase, and intronic sequence is given in lowercase. Exons 4, 5, and 6 sequences are given in boldfaced, normal, and underlined text, respectively. The American Journal of Human Genetics 2003 72, 961-974DOI: (10.1086/374566) Copyright © 2003 The American Society of Human Genetics Terms and Conditions

Figure 4 Imprinting of GNAS1 and overlapping transcripts in normal lymphoblast cell lines. The transcripts were amplified using an upstream primer in the unique exon and a downstream primer in exon 10 of GNAS1. Because of alternative splicing of exon 3 and multiple FokI restriction-enzyme cut sites, nested amplification from exons 4–6 was performed on each transcript prior to FokI digestion. The FokI − allele (c.393T) gave a 195-bp product, and the FokI + allele (c.393C) gave two products, of 122 and 73 bp (only the 122-bp product was visualized). An electropherogram demonstrating the fragments from one cell line (sized on an ABI 377 sequencer) is shown. a, Monoallelic expression of exon 1A, with only the + allele being present. b, Biallelic expression of Gsα, with − and + alleles being present. c, Monoallelic expression of NESP55 (opposite to exon 1A), with only the − allele being present. The American Journal of Human Genetics 2003 72, 961-974DOI: (10.1086/374566) Copyright © 2003 The American Society of Human Genetics Terms and Conditions

Figure 5 Effect of the c.586-1G→A mutation (patient AHO-15). The G→A substitution obliterates the normal AG acceptor site in intron 7 (underlined) and causes NMD of the mutant Gsα transcipt. Analysis of the NESP55 transcript reveals that exon skipping does not occur. Instead, the novel splice site created 1 nt downstream is used, resulting in deletion of a single G nucleotide with a frameshift and premature termination in exon 8. Consensus splice-acceptor sequence is defined as NCAGG (Shapiro and Senapathy 1987). Intronic and exonic sequences are given in lowercase and uppercase, respectively. The American Journal of Human Genetics 2003 72, 961-974DOI: (10.1086/374566) Copyright © 2003 The American Society of Human Genetics Terms and Conditions

Figure 6 Splice-recognition sequences within GNAS1 intron 4. The putative branch site GCTAAAT (underlined) is disrupted by the IVS4:−11A→G mutation (arrow). A second possible upstream branch site, CCCTGAC, is also shown (dotted underline). The diamond symbol denotes the conserved adenine nucleotide within the branch sites. The unusually short polypyrimidine tract CATTTC is given in italics. Exon 5 sequence is given in uppercase. The American Journal of Human Genetics 2003 72, 961-974DOI: (10.1086/374566) Copyright © 2003 The American Society of Human Genetics Terms and Conditions