A Novel Familial PHP1B Variant With Incomplete Loss of Methylation at GNAS-A/B and Enhanced Methylation at GNAS-AS2

Abstract

Context: Pseudohypoparathyroidism type 1B (PHP1B), also referred to as inactivating PTH/PTHrP signaling disorder (iPPSD), is characterized by proximal renal tubular resistance to parathyroid hormone (PTH) leading to hypocalcemia, hyperphosphatemia, and elevated PTH values. Autosomal dominant PHP1B (AD-PHP1B) with loss of methylation at the maternal GNAS A/B:TSS-DMR (transcription start site-differentially methylated region) alone can be caused by maternal deletions involving STX16.

Objective: Characterize a previously not reported AD-PHP1B family with loss of methylation at GNAS A/B:TSS-DMR, but without evidence for a STX16 deletion on the maternal allele and assess GNAS-AS2:TSS-DMR methylation.

Methods: DNA from 24 patients and 10 controls were investigated. AD-PHP1B patients without STX16 deletion from a single family (n = 5), AD-PHP1B patients with STX16 deletion (n = 9), sporPHP1B (n = 10), unaffected controls (n = 10), patUPD20 (n = 1), and matUPD20 (n = 1). Methylation and copy number analyses were performed by pyrosequencing, methylation-sensitive multiplex ligation-dependent probe amplification, and multiplex ligation-dependent probe amplification.

Results: Molecular cloning of polymerase chain reaction-amplified, bisulfite-treated genomic DNA from healthy controls revealed evidence for 2 distinct GNAS-AS2:TSS-DMR subdomains, named AS2-1 and AS2-2, which showed 16.0 ± 2.3% and 31.0 ± 2.2% methylation, respectively. DNA from affected members of a previously not reported AD-PHP1B family without the known genetic defects revealed incomplete loss of methylation at GNAS A/B:TSS-DMR, normal methylation at the 3 well-established maternal and paternal DMRs, and, surprisingly, increased methylation at AS2-1 (32.9 ± 3.5%), but not at AS2-2 (30.5 ± 2.9%).

Conclusion: The distinct methylation changes at the novel GNAS-AS2:TSS-DMR will help characterize further different PHP1B/iPPSD3 variants and will guide the search for underlying genetic defects, which may provide novel insights into the mechanisms underlying GNAS methylation.

Keywords: GNAS; DMR; iPPSD; imprinting; pseudohypoparathyroidism.

Figure 1.

The GNAS locus (not to genomic scale). Mat, maternal chromosome; Pat, paternal chromosome; cen, centromere; tel, telomere; arrow, direction of transcription; pink rectangle, maternal transcript (NESP55); blue rectangles, paternal transcripts (A/B, XL and the GNAS-AS1 antisense); number in italic exons and 3′ untranslated region (UTR) of GNAS-AS1; gray rectangles, transcripts not expressed; black rectangles, transcripts expressed from both alleles; black stars, GNAS DMRs; GNAS A/B:TSS-DMR, GNAS-XL:EX1-DMR; GNAS-AS1:TSS-DMR (chr20:57,425,649-57,428,033; hg19); and GNAS-NESP:TSS-DMR; red star, GNAS-AS2:TSS-DMR (AS2-1: chr20: 57,427,685-57,427,748 and AS2-2: chr20:57,427,933-57,427,996; hg19); broken lines, splicing patterns; STX16 is a gene located approximately 220 kb upstream of the GNAS locus. A deletion of STX16-5-418nt and ST16-6-244nt probes by MS-MPLA is consistent with LOM at GNAS A/B:TSS-DMR for AD-PHP1B patients. A more detailed view of the GNAS-AS2:TSS-DMR: white circles CpG not methylated, black circles CpG methylated.

Figure 2.

Pedigree, laboratory results at the time of establishing the diagnosis and microsatellite markers of the iPPSD3/AD-PHP1B family (AD–), in which the recurrent 3-kb deletion of STX16 was excluded.

Figure 3.

Average methylation indexes of GNAS-DMRs. (A) GNAS AB:TSS-DMR, (B) GNAS-AS1:TSS-DMR, (C) GNAS-AS2:TSS-DMR, and (D) GNAS-AS2:TSS-DMR: [iUPD(20q)]pat and [iUPD(20q)]mat patients. AD–, PHP1B patients without STX16 deletion (pyrosequencing for 3 patients [208/II-2, 208/II-3, and 208/III-1]); AD+, PHP1B patients with STX16 deletion (n = 9), sporPHP1B (n = 10), controls (n = 10), patUPD20 (n = 1), matUPD20 (n = 1).

Figure 4.

GNAS-AS2:TSS-DMR methylation indexes determined by pyrosequencing after subcloning. black circles, controls; green squares, AD– patients; red triangles, AD+ patients; and blue triangles, sporPHP1B patients.