Targeted long-read sequencing reveals allele-specific methylation profiles at the GNAS locus in...

Patients with pseudohypoparathyroidism (PHP) show parathyroid hormone resistance due to reduced Gsα expression. Loss-of-function variants in maternal Gsα-coding exons cause PHP type 1A (PHP1A), and methylation defects in GNAS-differentially methylated regions (DMRs) at the GNAS locus cause PHP type 1B (PHP1B). The GNAS-AS1:TSS-DMR (AS1-DMR), GNAS-AS2:TSS-DMR (AS2-DMR), GNAS-XL:Ex1-DMR (XL-DMR), and GNAS-A/B:TSS-DMR (A/B-DMR) are maternally methylated. The GNAS-NESP:TSS-DMR (NESP-DMR) is paternally methylated. STX16 functions as a long-range enhancer for NESP55 and XL. PHP1B cases commonly exhibit hypomethylated A/B-DMR and various methylation patterns in other GNAS-DMRs; however, the regulatory mechanisms for methylation patterns in GNAS-DMRs remain unclear. We evaluated allele-specific methylation levels for all CpGs within the GNAS locus together with structural abnormalities using targeted long-read sequencing (T-LRS) in a PHP1A case with a deletion affecting the 3' side A/B-DMR to GNAS exon 1, eight PHP1B cases (three familial cases [maternal STX16 deletion, maternal GNAS duplication, and retrotransposon insertion on the maternal allele], a sporadic case with STX16 deletion, and four epimutation cases [two typical and two atypical methylation patterns]), and an individual with paternal GNAS duplication showing normal phenotype. The PHP1A case showed normal methylation at the intact A/B-DMR, narrowing the critical region for the establishment and/or maintenance of the maternal methylation imprint in the A/B-DMR. The case with STX16 deletion and the case with maternal duplication causing elongation of the distance between STX16 and the AS2-DMR showed hypomethylated AS2-DMR, suggesting the incomplete enhancer effect of STX16 on the AS2-DMR. Furthermore, we detected the precise region of the AS2-DMR. The case with retrotransposon insertion (chr20:58,863,683) showed markedly hypomethylated A/B-DMR, plus mild hypomethylation of the AS1-DMR, AS2-DMR, and XL-DMR, undetected by conventional methylation analyses. Sporadic cases exhibited mosaic methylation abnormalities in all GNAS-DMRs, indicating that abnormal methylation occurred during early embryogenesis. T-LRS is a powerful tool for clarifying methylation pattern regulation in GNAS-DMRs. (300/300).