Methylation defect in imprinted genes detected in patients with an Albright's hereditary osteodystrophy like phenotype and platelet Gs hypofunction

Abstract

Background: Pseudohypoparathyroidism (PHP) indicates a group of heterogeneous disorders whose common feature is represented by impaired signaling of hormones that activate Gsalpha, encoded by the imprinted GNAS gene. PHP-Ib patients have isolated Parathormone (PTH) resistance and GNAS epigenetic defects while PHP-Ia cases present with hormone resistance and characteristic features jointly termed as Albright's Hereditary Osteodystrophy (AHO) due to maternally inherited GNAS mutations or similar epigenetic defects as found for PHP-Ib. Pseudopseudohypoparathyroidism (PPHP) patients with an AHO phenotype and no hormone resistance and progressive osseous heteroplasia (POH) cases have inactivating paternally inherited GNAS mutations.

Methodology/principal findings: We here describe 17 subjects with an AHO-like phenotype that could be compatible with having PPHP but none of them carried Gsalpha mutations. Functional platelet studies however showed an obvious Gs hypofunction in the 13 patients that were available for testing. Methylation for the three differentially methylated GNAS regions was quantified via the Sequenom EpiTYPER. Patients showed significant hypermethylation of the XL amplicon compared to controls (36 ± 3 vs. 29 ± 3%; p<0.001); a pattern that is reversed to XL hypomethylation found in PHPIb. Interestingly, XL hypermethylation was associated with reduced XLalphaS protein levels in the patients' platelets. Methylation for NESP and ExonA/B was significantly different for some but not all patients, though most patients have site-specific CpG methylation abnormalities in these amplicons. Since some AHO features are present in other imprinting disorders, the methylation of IGF2, H19, SNURF and GRB10 was quantified. Surprisingly, significant IGF2 hypermethylation (20 ± 10 vs. 14 ± 7%; p<0.05) and SNURF hypomethylation (23 ± 6 vs. 32 6%; p<0.001) was found in patients vs. controls, while H19 and GRB10 methylation was normal.

Conclusion/significance: In conclusion, this is the first report of methylation defects including GNAS in patients with an AHO-like phenotype without endocrinological abnormalities. Additional studies are still needed to correlate the methylation defect with the clinical phenotype.

Figure 1. Overall GNAS, IGF2, H19, SNURF and GRB10 methylation in AHO-like patients.

Dot plot representation of overall methylation values (averages expressed as % of methylation) for NESP, XL, Exon A/B (A), IGF2, H19 (B), SNURF and GRB10 (C) in AHO-like patients (indicated as ‘PPHP’) vs. the control population (indicated as ‘crls’). Individuals with significant hyper- or hypomethylation (patients 1 to 5) in the NESP, Exon A/B, H19 and GRB10 are indicated as follow: patient 1 =  red, 2 =  green, 3 =  blue, 4 =  brown, 5 =  yellow. Medians are displayed as black lines. ** p<0.01 and * p<0.05, two-tailed unpaired T-test.

Figure 2. GNAS, IGF2, H19, SNURF and GRB10 methylation at single CpG sites for AHO-like patients.

Single CpG site methylation values rapresentations for all patients studied via Sequenom EpiTYPER mass-array for NESP, XL, exon A/B (A), IGF2, H19 (B), SNURF and GRB10 (C) amplicons. % of methylation are reported as mean of three replicates from at least two separate plates and two independent DNA bisulphite treatment. White include the normal methylation values that are within the mean +/− SD value of the indicated number of normal controls. Values that are significantly hyper- or hypomethylated are depicted as red or green diagonal striped rectangles, respectively (Z-test, p<0.05). Red or green rectangles indicate methylation values that are outside the SD values but are not yet significant, indicative for a trend towards hyper or hypomethylation, respectively. Grey rectangles are CpG values that failed in the analysis. The mean (AVG) and Standard Deviation (SD) for each CpG in the controls are shown in the last rows. The last column in white shows the overall degree of methylation for the complete amplicon for each patient and the mean and SD for the controls. * Z-test, p<0.05.

Figure 3. XLalphaS and Gsalpha expression in platelets from AHO-like patients.

XLalphaS, CAP1 and Gsalpha expression in AHO-like platelets. A. Immunoblot analysis of XLalphas, CAP1 and Gsalpha protein in platelet lysates from XL hypermethylated AHO-like patients 12, 13, 10, 14, 15, 16 and 3 controls and B. correspondent densitometric scanning of XLalphaS protein in platelet lysates from AHO-like patients with XL hypermethylation (patients 6 to 16) and 5 controls (Controls). Results are expressed as percentage of controls (taken as 100%). Mean as well as SD are depicted as black horizontal and vertical lines, respectively. *, p value<0.05, two-tailed unpaired T-test.