Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.
Department of Pediatrics, Tohoku University Graduate School of Medicine, Sendai, Japan.
J Bone Miner Res. 2022 Oct;37(10):1850-1859. doi: 10.1002/jbmr.4652. Epub 2022 Aug 17.
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 parathyroid hormone (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. 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. © 2022 American Society for Bone and Mineral Research (ASBMR).
GNAS 基因座的 GNAS-A/B:TSS-差异甲基化区域（DMR）中的甲基化丢失（LOM）在假性甲状旁腺功能减退症 1B 型（PHP1B）中观察到。许多 PHP1B 病例是散发性的，但常染色体显性遗传的 PHP1B 具有涉及从母本等位基因表达的 NESP55 或位于母本等位基因上 GNAS 基因座上游的 STX16 的缺失。我们报告了可能的首例母本等位基因 GNAS 基因座逆转录转座子插入的家族性 PHP1B 病例。据我们所知，它们是可能的首例由逆转录转座子插入引起的印迹障碍病例。这两个同胞病例从上学时就经历了抽搐和/或痉挛，伴有低钙血症和血清完整甲状旁腺激素（PTH）水平升高，同时伴有超重、圆脸和正常智力水平。对 GNAS 基因座 DMR 的甲基化分析仅显示 GNAS-A/B:TSS-DMR 的 LOM。通过 arrayCGH 未检测到 STX16 和 GNAS 基因座的拷贝数异常。全基因组测序和 Sanger 测序揭示了这些同胞中 GNAS 基因座第一外显子上游约 1000bp 的 SVA 逆转录转座子插入。通过对同胞进行的 Enzymatic Methyl-Seq 全基因组甲基组分析显示，插入位点周围区域的甲基化状态正常，GNAS-A/B:TSS-DMR 出现轻度 LOM。我们使用来自皮肤成纤维细胞和诱导多能干细胞（iPSC）的 mRNA 进行了转录组分析，这些细胞是从同胞中获得的，并检测到没有异常的 NESP55 转录本。皮肤成纤维细胞的定量逆转录酶 PCR（qRT-PCR）分析显示，患者的 A/B 表达增加，而 NESP55 表达不存在，即使在对照中也是如此。在 iPSC 中的 qRT-PCR 分析显示，NESP55 表达降低，而患者的 GNAS-NESP:TSS-DMR 甲基化状态正常。同胞中的逆转录转座子插入可能导致 NESP55 表达降低，从而通过 GNAS-A/B:TSS-DMR 的 LOM 导致 A/B 表达增加，随后 Gsα 表达减少，最终导致 PHP1B 发育。 © 2022 美国骨骼与矿物质研究协会（ASBMR）。
