Discordance between genetic and epigenetic defects in pseudohypoparathyroidism type 1b revealed by inconsistent loss of maternal imprinting at GNAS1

Abstract

Although the molecular basis of pseudohypoparathyroidism type 1b (PHP type 1b) remains unknown, a defect in imprinting at the GNAS1 locus has been suggested by the consistent finding of paternal-specific patterns of DNA methylation on maternally inherited GNAS1 alleles. To characterize the relationship between the genetic and epigenetic defects in PHP type 1b, we analyzed allelic expression and methylation of CpG islands within exon 1A of GNAS1 in patients with sporadic PHP type 1b and in affected and unaffected individuals from five multigenerational kindreds with PHP type 1b. All subjects with resistance to parathyroid hormone (PTH) showed loss of methylation of the exon 1A region on the maternal GNAS1 allele and/or biallelic expression of exon 1A-containing transcripts, consistent with an imprinting defect. Paternal transmission of the disease-associated haplotype was associated with normal patterns of GNAS1 methylation and PTH responsiveness. We found that affected and unaffected siblings in one kindred had inherited the same GNAS1 allele from their affected mother, evidence for dissociation between the genetic and epigenetic GNAS1 defects. The absence of the epigenetic defect in subjects who have inherited a defective maternal GNAS1 allele suggests that the genetic mutation may be incompletely penetrant, and it indicates that the epigenetic defect, not the genetic mutation, leads to renal resistance to PTH in PHP type 1b.

Figures

Figure 1

Exon 1A methylation status revealed by restriction digestion of genomic DNA. Genomic DNA was digested with PstI alone or PstI plus NgoMIV, which cuts only unmethylated sequences. Digests were transferred to nylon membranes and hybridized to a radiolabeled genomic DNA probe corresponding to exon 1A of GNAS1. A representative autoradiogram (top) indicates results of individuals from family R labeled with the pedigree number corresponding to figure 4. Bottom, positions of the recognition sites for the two restriction enzymes with respect to exon 1A (striped box), along with the corresponding position of the 900-bp probe. Digestion of DNA from unaffected individuals with PstI generates a single, 2.8-kb fragment from both alleles, whereas digestion with both PstI and NgoMIV yields a 2.8-kb fragment from the maternal (methylated) allele and fragments of 1.4 kb, 1.2 kb, and 90 bp (not shown) from the paternal (unmethylated) allele. Digestion of DNA from subjects with PHP type 1b with PstI yields a single fragment of 2.8-kb, but after digestion with PstI plus NgoM IV there is complete loss of the 2.8-kb fragment, indicating that both alleles are unmethylated within the DMR of exon 1A (see text).

Figure 2

Bisulfite sequencing of exon 1A of GNAS1. Autoradiograms showing the nucleotide sequence of a portion of GNAS1 exon 1A after bisulfite treatment of genomic DNA in unaffected subjects, a PHP1b carrier (*), and subjects with PHP1b from family R, identified with pedigree numbers corresponding to figure 4. PCR products were directly sequenced, to avoid quantitative errors that might arise because of different cloning efficiencies (see text), and thus represent sequences derived from maternal and paternal alleles. The sequence ladder from the unaffected and carrier subject shows both Cs and Ts at multiple positions, corresponding to cytosines within CpGs that were methylated (maternal allele) or unmethylated (paternal allele), respectively. By contrast, the sequence ladder from the subjects with PHP type 1b shows only Ts, indicating that cytosines on both maternal and paternal alleles were unmethylated.

Figure 3

RT-PCR expression analysis of GNAS1 exon 1A. Total RNA from fresh peripheral blood leukocytes or transformed lymphoblasts was reverse transcribed and amplified using an exon 1A–specific upstream primer and an exon 6 downstream primer. Direct sequencing of the RT-PCR products is shown for subjects who are heterozygous for a common T/C polymorphism in exon 5. The normal individuals show uniallelic expression of exon 1A transcripts, as indicated by either the C or T (arrow in the sequence ladder) at the polymorphic nucleotide site, indicating expression from only the paternal allele. By contrast, the subject with PHP type 1b shows both the C and T nucleotides, indicating biallelic expression of exon 1A.

Figure 4

Family R pedigree. Unaffected individuals are indicated by an open circle (female) or square (male). An individual carrying the defective PHP type 1b allele is indicated by a blackened upper left quadrant of the symbol; PTH resistance is indicated by a blackened lower right quadrant; normal imprinting is indicated by a blackened lower left quadrant; loss of maternal imprinting is indicated by a blackened upper right quadrant. NT = not tested. Imprinting of exon 1A was determined by restriction endonuclease digestion using methylation sensitive enzymes or by bisulfite sequencing (see text).