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These laboratory abnormalities are caused by maternal loss-of-methylation (LOM) at GNAS exon A\/B, which reduces in cis expression of the stimulatory G protein \u03b1-subunit (Gs\u03b1). Paternal Gs\u03b1 expression in proximal renal tubules is silenced through unknown mechanisms, hence LOM at exon A\/B reduces further Gs\u03b1 protein in this kidney portion, leading to PTH resistance. In a previously reported PHP1B family, affected members showed variable LOM at exon A\/B, yet no genetic defect was found by whole-genome sequencing despite linkage to GNAS. Using targeted long-read sequencing (T-LRS), we discovered an approximately 2800-bp maternally inherited retrotransposon insertion nearly 1200\u2009bp downstream of exon XL not found in public databases or in 13,675 DNA samples analyzed by short-read whole-genome sequencing. T-LRS data furthermore confirmed normal methylation at exons XL, AS, and NESP and showed that LOM comprising exon A\/B is broader than previously thought. The retrotransposon most likely causes the observed epigenetic defect by impairing function of a maternally derived NESP transcript, consistent with findings in mice lacking full-length NESP mRNA and in PHP1B patients with deletion of exon NESP and adjacent intronic sequences. In addition to demonstrating that T-LRS is an effective strategy for identifying a small disease-causing variant that abolishes or severely reduces exon A\/B methylation, our data demonstrate that this sequencing technology has major advantages for simultaneously identifying structural defects and altered methylation. \u00a9 2022 American Society for Bone and Mineral Research (ASBMR).<\/jats:p>","DOI":"10.1002\/jbmr.4647","type":"journal-article","created":{"date-parts":[[2022,7,10]],"date-time":"2022-07-10T21:59:40Z","timestamp":1657490380000},"page":"1711-1719","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["Targeted Long-Read Sequencing Identifies a Retrotransposon Insertion as a Cause of Altered GNAS Exon A\/B Methylation in a Family With Autosomal Dominant Pseudohypoparathyroidism Type 1b (PHP1B)"],"prefix":"10.1093","volume":"37","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6096-8601","authenticated-orcid":false,"given":"Danny E","family":"Miller","sequence":"first","affiliation":[{"name":"Department of Genome Sciences University of Washington School of Medicine Seattle WA USA"},{"name":"Division of Genetic Medicine, Department of Pediatrics University of Washington and Seattle Children's Hospital Seattle WA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick","family":"Hanna","sequence":"additional","affiliation":[{"name":"Endocrine Unit Massachusetts General Hospital and Harvard Medical School Boston MA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miranda","family":"Galey","sequence":"additional","affiliation":[{"name":"Department of Genome Sciences University of Washington School of Medicine Seattle WA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monica","family":"Reyes","sequence":"additional","affiliation":[{"name":"Endocrine Unit Massachusetts General Hospital and Harvard Medical School Boston MA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3455-002X","authenticated-orcid":false,"given":"Agn\u00e8s","family":"Linglart","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Saclay, Inserm, Physiologie et Physiopathologie Endocrinienne Bic\u00eatre Paris Saclay Hospital Le Kremlin Bic\u00eatre France"},{"name":"AP-HP, Endocrinology and Diabetology for Children Bic\u00eatre Paris Saclay Hospital Le Kremlin Bic\u00eatre France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Evan E","family":"Eichler","sequence":"additional","affiliation":[{"name":"Brotman Baty Institute for Precision Medicine University of Washington Seattle WA USA"},{"name":"Howard Hughes Medical Institute 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