ESPE2023 Rapid Free Communications Bone, Growth Plate and Mineral Metabolism (6 abstracts)
Familial pseudohypoparathyroidism type IB associated with an SVA retrotransposon insertion in the GNAS locus.
Masayo Kagami 1 , Sayaka Kawashima 1,2 , Akiko Yuno 3 , Shinichiro Sano 1,4 , Akie Nakamura 1,5 , Keisuke Ishiwata 6 , Tomoyuki Kawasaki 7 , Kazuyoshi Hosomichi 8 , Kazuhiko Nakabayashi 6 , Hidenori Akustu 7 , Hirotomo Saitsu 9 , Maki Fukami 1 , Takeshi Usui 10,11 & Tsutomu Ogata 1,9,12
1Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan. 2Department of Pediatrics, Tohoku University Graduate School of Medicine, Sendai, Japan. 3Department of Endocrinology and Metabolism, Kin-ikyo Chuo Hospital, Sapporo, Japan. 4Department of Endocrinology and Metabolism, Shizuoka Children’s Hospital, Shizuoka, Japan. 5Department of Pediatrics, Hokkaido University School of Medicine, Sapporo, Japan. 6Department of Maternal Fetal Biology, National Research Institute for Child Health and Development, Tokyo, Japan. 7Department of Reproductive Medicine, National Research Institute for Child Health and Development, Tokyo, Japan. 8Department of Bioinformatics and Genomics, Graduate School of Advanced Preventive Medical Sciences, Kanazawa University, Kanazawa, Japan. 9Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Japan. 10Research Support Center, Shizuoka General Hospital, Shizuoka, Japan. 11Shizuoka Graduate University of Public Health, Shizuoka, Japan. 12Department of Pediatrics, Hamamatsu Medical Center, Hamamatsu, Japan
Loss of methylation (LOM) at GNAS-A/B:TSS-differentially methylated regions (DMRs) in the GNAS locus is observed in pseudohypoparathyroidism type 1B (PHP1B). Many PHP1B cases are sporadic, but autosomal dominant-PHP1B has a deletion involving NESP55 expressed from the maternal allele or STX16 located upstream of the GNAS locus on the maternal allele. We report the possible first familial PHP1B cases with retrotransposon insertion in the GNAS locus on the maternal allele. To our knowledge, they are the possible first cases with imprinting disorders caused by retrotransposon insertion. The two sibling cases experienced tetany and/or cramps from school-age and had hypocalcemia and an increased serum intact PTH level together with overweight, round face, and normal intellectual levels. Methylation analysis for DMRs in the GNAS locus showed only LOM of the GNAS-A/B:TSS-DMR. Copy number abnormalities at STX16 and the GNAS locus were not detected by array comparative genomic hybridization. Whole-genome sequencing and Sanger sequencing revealed an approximately 1000-bp SVA retrotransposon insertion upstream of the first exon of A/B on the GNAS locus in these siblings. To detect the parental origin of retrotransposon insertion, we conducted long PCR for a region encompassing the retrotransposon insertion using gDNA treated with methylation sensitive restriction endonuclease in these patients. Junction between reference sequence and inserted retrotransposon sequence was detected on the allele having the methylated GNAS-XL:Ex1-TSS-DMR, namely, the maternally inherited allele. Whole-genome methylome analysis by Enzymatic Methyl-Seq in the siblings showed normal methylation status in the region surrounding the insertion site and mild LOM of the GNAS-A/B:TSS-DMR. We conducted transcriptome analysis using mRNA from skin fibroblasts and induced pluripotent stem cells (iPSCs) derived from the siblings and detected no aberrant NESP55 transcripts. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis in skin fibroblasts showed increased A/B expression in the patients and no NESP55 expression, even in a control. qRT-PCR analysis in iPSCs showed decreased NESP55 expression with normal methylation status of the GNAS-NESP:TSS-DMR in the patients. The retrotransposon insertion in the siblings likely caused decreased NESP55 expression that could lead to increased A/B expression via LOM of the GNAS-A/B:TSS-DMR, subsequent reduced Gsα expression, and finally, PHP1B development.
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