Sporadic pseudohypoparathyroidism type 1B in monozygotic twins: insights into the pathogenesis of methylation defects

Context: Sporadic pseudohypoparathyroidism type 1B (sporPHP1B) is an
imprinting disease without a defined genetic cause, characterized by broad
methylation changes in differentially methylated regions (DMRs) of the
GNAS gene. Objective: This work aims to provide insights into the
causative event leading to the GNAS methylation defects through
comprehensive molecular genetic analyses of a pair of female monozygotic
twins concordant for sporPHP1B who were conceived naturally i.e., without
assisted reproductive techniques. Methods: Using the leukocyte genome of
the twins and family members, we performed targeted bisulfite sequencing,
methylation-sensitive restriction enzyme (MSRE)-qPCR, whole-genome
sequencing (WGS), high-density SNP array, and Sanger sequencing. Results:
Methylation analyses by targeted bisulfite sequencing and MSRE-qPCR
revealed almost complete loss of methylation at the GNAS AS, XL, and A/B
DMRs and gain of methylation at the NESP55 DMR in the twins, but not in
other family members. Except for the GNAS locus, we did not find apparent
methylation defects at other imprinted genome loci of the twins. WGS, SNP
array, and Sanger sequencing did not detect the previously described
genetic defects associated with familial PHP1B. Sanger sequencing also
ruled out any novel genetic alterations in the entire NESP55/AS region.
However, the analysis of 28 consecutive SNPs could not exclude the
possibility of paternal heterodisomy in a span of 22 kb comprising exon
NESP55 and AS exon 5. Conclusion: Our comprehensive analysis of a pair of
monozygotic twins with sporPHP1B ruled out all previously described
genetic causes. Twin concordance indicates that the causative event was an
imprinting error earlier than the timing of monozygotic twinning.