ESPE Abstracts

Background: Pseudohypoparathyroidism type 1a (PHP1a) is characterized by Albright hereditary osteodystrophy (AHO) and multi-hormone resistance, most commonly to parathyroid hormone (PTH) and thyroid-stimulating hormone. This rare disorder is caused by inactivating mutations involving exons 1–13 of the imprinted GNAS gene that encodes the alpha-subunit of the stimulatory G protein (Gαs). Due to paternal imprinting of Gαs transcripts, GNAS mutations on the maternal allele cause PHP1a while similar mutations on the paternal GNAS allele cause pseudopseudohypoparathyroidism (PPHP) characterized by the physical findings of AHO but without hormone resistance. More than 200 point mutations and small coding insertions/deletions (indels) have been described in PHP1a, but small intragenic deletions are rarely reported due to limitations of Sanger and whole exome sequence analysis.

Objective and hypotheses: To identify the underlying genetic defects for three patients with clinical diagnosis of PHP1a/PPHP, negative GNAS sequencing, and normal SNP array.

Method: Whole genome sequencing (WGS) was applied to two patients with clinical diagnosis of PHP1a to search for genomic structural variations and Sanger was performed to confirm and define their boundaries. Pyrosequencing was used to determine percent methylation at CpGs in the exon A/B differentially methylated region (DMR).

Results: We identified two novel small genomic deletions, supported by different bioinformatic algorithms. For family 1, an 1,438-bp deletion (chr20:g.57,465,324-57,466,761) that involves partial exon 1 was identified in the proband with clinical diagnosis of PHP1a and slightly reduced methylation level (34%) at exon A/B. Subsequently Sanger confirmed the deletion is inherited from mother who is affected with PPHP. This deletion starts 923-bp up-stream of the deletion described by Reyes et al., Bone 2017 (chr20:g.57,466,247-57,468,263) in a PHP1a patient with normal (50%) methylation at exon A/B, suggesting this 923 nucleotides could potentially highlight genetic elements important for normal establishment and/or maintenance of imprinting at exon A/B. WGS, followed by Sanger, revealed another novel small deletion (chr20:57,467,470-57,473,862) that includes exon 2 of GNAS in the second PHP1a patient with 43% methylation at exon A/B.

Conclusion: Our results demonstrate WGS can detect relatively small deletions beyond Microarray resolution and could be used as a single test to capture nearly all known genetic variations. The first deletion identified herein in a patient with slightly reduced methylation level at exon A/B further refines a region that may be critical for normal imprinting of the exon A/B DMR.