High-throughput Molecular Analysis of Pseudohypoparathyroidism 1b Patients Reveals Novel Genetic and Epigenetic Defects

Abstract

Context: Patients with pseudohypoparathyroidism type 1b (PHP1b) show disordered imprinting of the maternal GNAS allele or paternal uniparental disomy (UPD). Genetic deletions in STX16 or in upstream exons of GNAS are present in many familial but not sporadic cases.

Objective: Characterization of epigenetic and genetic defects in patients with PHP1b.

Design and patients: DNA from 84 subjects, including 26 subjects with sporadic PHP1b, 27 affected subjects and 17 unaffected and/or obligate gene carriers from 12 PHP1b families, 11 healthy individuals, and 3 subjects with PHP1a was subjected to quantitative pyrosequencing of GNAS differentially methylated regions (DMRs), microarray analysis, and microsatellite haplotype analysis.

Setting: Academic medical center.

Main outcome measurements: Molecular pathology of PHP1b.

Results: Healthy subjects, unaffected family members and obligate carriers of paternal PHP1b alleles, and subjects with PHP1a showed normal methylation of all DMRs. All PHP1b subjects showed loss of methylation (LOM) at the exon A/B DMR. Affected members of 9 PHP1b kindreds showed LOM only at the exon A/B DMR, which was associated with a 3-kb deletion of STX16 exons 4 through 6 in 7 families and a novel deletion of STX16 and adjacent NEPEPL1 in 1 family. A novel NESP deletion was found in 1 of 2 other families with more extensive methylation defects. One sporadic PHP1b had UPD of 20q, 2 had 3-kb STX16 deletions, and 5 had apparent epigenetic mosaicism.

Conclusions: We found diverse patterns of defective methylation and identified novel or previously known mutations in 9 of 12 PHP1b families.

Keywords: GNAS; epigenetics; imprinting; parathyroid hormone; pseudohypoparathyroidism.

Figure 1.

GNAS locus and differentially methylated regions (DMRs). The figure shows the organization of the GNAS locus and relevant genes (not drawn to scale) with solid black circles denoting methylation of DMRs on the maternal (M) or paternal (P) alleles. Solid arrows show the direction of transcription from promoters in all tissues; the dotted arrow denotes transcription of G_sα from the paternal allele in only some tissues (see text). Nucleotides sequences (GRCh37; hg19) in GNAS (20q13.32) that were analyzed by pyrosequencing were in the DMRs for NESP55 (ADS471; 20:g.57,415,807-57,415,853), XL (ADS470; 20:g.57,429,235-57,429,362), and exon A/B (ADS464; 20:g.57,464,773-57,464,927). The STX16 gene is located approximately 220kb centromeric of the GNAS locus.

Figure 2.

Array CGH and pedigree of patient F11-4 with a novel deletion of NESP. (A) The aCGH map for patient F11-1, corresponding to the region of chromosome 20q containing NESP, XL, and AS exons. The novel deletion of NESP is shown with breakpoints as indicated. (B) A 4-generation pedigree for family 11. The kindred designation and methylation pattern are indicated beneath each patient who was evaluated. Males are denoted by squares and females are denoted by circles.

Figure 3.

Heatmap of the results of the pyrosequencing analysis at the 3 DMRs. Each row represents a subject, with PHP1b patients denoted as sporadic (S) or familial (F, with kindred number); the diagnosis and mutation when known are also shown (ND, not detected). Each column represents a CpG site that was assessed for percent methylation; the color indicates the degree of methylation (see legend on figure) and the actual percentage methylation is indicated in each box. The sections are divided into the DMRs for NESP, for XL and for A/B. The results are clustered in groups, as denoted in the rightmost column, that are described more fully in Table 2 and the text. The key for the heatmap colors is shown at the bottom of the figure.

Figure 4.

Array CGH of patients with 3-kb STX16 deletion. Exons 3 through 7 of the STX16 gene are shown with positional coordinates in the upper panel, with the deleted exons indicated in red. The middle panel shows the aCGH results for patient F4-2 with exons indicated as solid boxes. This array is representative of patients with the common 3-kb deletion, with the deletion and breakpoints indicated. The lower panel shows the aCGH for patient F7-1, which demonstrates a novel 3-kb deletion and breakpoints.

Figure 5.

Sanger sequencing electropherograms from patients with deletion of STX16 exons 4-6. Nucleotide sequences are shown above each electropherogram from patients with PHP1b (denoted on left) who had a 3-kb deletion in STX16 encompassing exons 4-6. Note T/C difference (box) indicating that subject F7-1 has breakpoints that differ (20:57,243,740-C or 57,246,717-T) from the other subjects. See Fig. 3 and text.

Figure 6.

Array CGH of a patient F1-1 with a novel deletion of STX16-NPEPL1. The upper panel shows the results of aCGH covering the region of chromosome 20q from STX16 through GNAS, with results for patient F1-1 showing the novel 205-kb deletion and breakpoints indicated on the map. The lower panel shows a sequencing electropherogram, with nucleotides above the graphic, that depicts a complex 206-kb deletion of the STX16 and NPEPL1 genes. Sanger sequencing reveals 2 adjacent deletions, 20:57,151,892-57,289,110 and 20:57289120-57358140, which are separated by 9 bp.

Figure 7.

Summary of deletions affecting STX16 in patients with PHP1b. Breakpoint structures of deletions disrupting STX16 in PHP1b patients. The common intragenic deletions that affect exons 2-4 and 4-6 are shown as black lines directly above the STX16 gene. The previously reported deletions of 87.5 kb from Yang et al (35) and 24.6 kb from Elli et al (34) are shown below the novel 206-kb deletion identified in this study.

Figure 8.

Summary of deletions affecting NESP in patients with PHP1b. Breakpoint structures of deletions disrupting NESP in PHP1b patients. The region of chromosome 20q that contains STX16 through GNAS is depicted above an inset that shows the NESP-AS2 region (not drawn to scale) with genomic coordinates for each exon. The 7 previously described deletions are shown above the novel deletion that was identified in family 11 that removes the entire NESP exon. The methylation patterns for the 4 or 3 DMRs analyzed in previous studies and this study are shown to the right.