Methylation analysis of GNAS1. (a) The normal allele-specific methylation and expression patterns of the four known first exons of GNAS1, which splice onto exon 2 to produce transcripts encoding NESP55, XLαs, a transcript of unknown function (exon 1A), and G_sα (exon 1). Horizontal arrows indicate transcriptionally active promoters. The imprinting of NESP55 and XLαs have been defined previously (8, 9, 11). Exon 1 is probably paternally imprinted in some tissues, indicated by the dashed arrow. NESP55 protein is unrelated to G_sα, and its entire coding region is located within its first exon. In contrast, XLαs and G_sα proteins have identical COOH-terminal domains (encoded by exons 2–13), while their unique NH₂-terminal domains are encoded within their respective first exons. Exon 1A does not have a translational start site, and its transcripts are likely to be untranslated. The imprinting of the exon 1A region has been defined previously in mice (10). (b) Southern analyses of leukocyte genomic DNA from a normal subject (N), PHPIB patients 1, 3, and 7, the mother of patient 1 (1M), and the father of patient 1 (1F), using genomic DNA probes from the NESP55 (left panel; ref. 9), XLαs (middle panel; ref. 8), and exon 1A (right panel) regions. Above are the relevant restriction maps depicting each upstream exon as a black box and the position of the probes below. Patient 1 is abnormally methylated in all three regions, patient 3 is abnormally methylated only in the exon 1A region, and patient 7 is abnormally methylated in the NESP55 and exon 1A, but not the XLαs, regions. Bg, BglII; S, SacII; F, FspI, N, NgoMIV; A, AscI; Bs, BssHII; P, PstI; Mat, maternal allele; Pat, paternal allele.