Intragenic GNAS deletion involving exon A/B in pseudohypoparathyroidism type 1A resulting in an apparent loss of exon A/B methylation: potential for misdiagnosis of pseudohypoparathyroidism type 1B

Abstract

Context: Several endocrine diseases that share resistance to PTH are grouped under the term pseudohypoparathyroidism (PHP). Patients with PHP type Ia show additional hormone resistance, defective erythrocyte G(s)alpha activity, and dysmorphic features termed Albright's hereditary osteodystrophy (AHO). Patients with PHP-Ib show less diverse hormone resistance and normal G(s)alpha activity; AHO features are typically absent in PHP-Ib. Mutations affecting G(s)alpha coding exons of GNAS and epigenetic alterations in the same gene are associated with PHP-Ia and -Ib, respectively. The epigenetic GNAS changes in familial PHP-Ib are caused by microdeletions near or within GNAS but without involving G(s)alpha coding exons.

Objective: We sought to identify the molecular defect in a patient who was diagnosed with PHP-Ia based on clinical presentation (hormone resistance and AHO) but displayed the molecular features typically associated with PHP-Ib (loss of methylation at exon A/B) without previously described genetic mutations.

Methods: Microsatellite typing, comparative genome hybridization, and allelic dosage were performed for proband and her parents.

Results: Comparative genome hybridization revealed a deletion of 30,431 bp extending from the intronic region between exons XL and A/B to intron 5. The same mutation was also demonstrated, by PCR, in the patient's mother, but polymorphism and allele dosage analyses indicated that she had this mutation in a mosaic manner.

Conclusion: We discovered a novel multiexonic GNAS deletion transmitted to our patient from her mother who is mosaic for this mutation. The deletion led to different phenotypic manifestations in the two generation and appeared, in the patient, as loss of GNAS imprinting.

Figure 1

aCGH profile at chromosome and gene level. The figure shows chromosome 20 ideogram and the aCGH profile of the patient, with the lost probes in the gray area; also under the ideogram is a gene view of the deleted region.

Figure 2

A, Agarose electrophoresis showing the PCR of the deleted region (294 bp) and the internal PCR control. The first lane corresponds to the weight marker; lane 2, PCR amplification of genomic DNA of the patient; lane 3, PCR amplification of genomic DNA of the mother; lane 4, PCR amplification of genomic DNA of the father. The loss of the region at GNAS locus in the patient and her mother is evident. B, Sequencing of the deletion, with the sense sequence on the top and the reverse on the bottom with the dotted line marking the point where deletion occurred. C, Plain text of the sequence with the used primers underlined. In black is represented the upstream sequence and in red the downstream one.

Figure 3

Results of allele dosage analyses for the rs7121 SNP. The height of the columns represents the dosage of the respective allele in the genomic DNA for the patient and the mean of the controls (sd is included for allele C because data were normalized for allele T). Diminished dosage is observed for the patient’s allele C. Data are representative of two independent experiments with similar results.

Figure 4

Electropherogram of the semiquantitative amplification of exon A/B comprising the pentanucleotide repeat polymorphism. A, PCR amplification of the heterozygous father; B, PCR amplification of the heterozygous mother; C, PCR amplification of the mother’s genomic DNA after the overnight digestion with HpaII enzyme. Note that the amplification of the short allele (allele with two repeats) relative to the long allele (allele with three repeats) is less intense in the mother than the father. This short allele is the only one amplified after HpaII digestion, indicating that the deletion is present in the maternally inherited chromosome in the mother. The numbers under peaks represent the size of the amplicon (top) and the height of the peak (bottom).