Molecular Definition of Pseudohypoparathyroidism

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Pseudohypoparathyroidism type Ib (PHP-lb) is characterized by hypocalcemia and hyperphosphatemia due to renal resistance towards parathyroid hormone (PTH). Autosomal dominant PHP-lb (AD-PHP-lb) was mapped to chromosome 20q13.3, shows paternal imprinting, and loss of GNAS exon A/B methylation. Over the past funding period, we identified two different microdeletions about 220 kb up-stream of exon A/B, which remove STX16 exons 2-4 or 4-6. The 4.4-kb deletion (S7X76del2-4) was identified in one AD-PHP-lb kindred; the 3-kb deletion (STX76del4-6) was found in >30 unrelated AD-PHP-lb kindreds. We identified also two similar GNAS deletions, which remove exon NESP55 and antisense exons 3 and 4; both lead to loss of all maternal methylation imprints. It is uncertain how the deletions contribute to methylation changes, and how these reduce Gsa expression in proximal renal tubules. Interestingly, all non-familial, sporadic cases of PHP-lb (sporPHP-lb) do not carry any of the known deletions, yet these patients show methylation changes that usually affect all differentially methylated GNAS regions. It is conceivable that these patients have a de novo deletion within GNAS, or that they have an autosomal recessive form of PHP-lb. For the next funding period, we plan to expand our mutational analysis of the GNAS locus to identify novel microdeletions/mutations in sporPHP-lb (Aim 1), which is expected to provide additional information as to which genomic region is required for establishing or maintaining the GNAS methylation imprints; these efforts may identify control elements involved in regulating DMA methylation. We have furthermore collected DNA from one family with an autosomal recessive PHP-lb form (AR-PHP-lb) and will search for other families with two or more siblings affected by PHP-lb, who sjiow broad GNAS methylation changes, yet lack STX16 or GNAS mutations (Aim 2). Genetic linkage studies may then help identify of a novel gene involved in the regulation of GNAS methylation. In Aim 3, we plan to determine how mice lacking exon Nesp55 and adjacent regions (Nesp55del/AS3-4del) on the maternal, but not the paternal allele, develop PTH-resistance. We plan to determine whether primary or clonal proximal tubular cells with maternal, but not paternal, Nesp55del/AS3-4del show reduced/absent Gsa expression. These cells will then be analyzed through microarray experiments to determine which proteins are involved in Gnas methylation and the silencing of Gsa expression in proximal renal tubules and possibly other tissues/cell types. Aim 4 will determine how S7X76del4-6 or STX76del2-4 regulate methylation at human exon A/B, and where the equivalent regulatory region is located in the mouse. These studies are expected to provide additional insights into the mechanisms regulating A/B methylation and thus silencing of Gsa expression from the maternal allele.