1. Development of a Diagnostic Service for Pseudohypoparathyroidism type 1b Jennifer Greatwood

2. Pseudohypoparathyroidism (PHP) Introduction- Pseudohypoparathyroidism Screening strategy Results Conclusion

3. Pseudohypoparathyroidism (PHP) is characterised by hypocalcaemia, hyperphosphataemia and elevated levels of serum parathyroid hormone (PTH). Besides PTH resistance, affected individuals may show distinctive but variable features. These clinical findings are termed Albright’s hereditary osteodystrophy (AHO). Pseudohypoparathyroidism (PHP) short stature, obesity, short limbs round face, mental retardation Brachydactyly- hands/feet Characteristic ‘dimpling’ replacing the knuckles

4. PHP variants PHP type 1aPPHP Pseudopseudohypoparathyroidism PHP type 1b PHP (PTH resistance) + AHO phenotype AHO phenotype PHP (PTH resistance)

5. PTH is synthesised by the parathyroid glands and regulates calcium and phosphorous concentrations in extracellular fluid by acting on target organs. In PHP, the biochemical characteristics are caused by end–organ resistance to PTH rather than deficiency of PTH. Parathyroid hormone The PTH normally mediates its actions via a Gs  -coupled receptor. In PHP hormone resistance is due to a deficiency of the Gs  subunit.

6. GNAS locus GNAS is a complex imprinted locus on 20q13. Encodes the Gs  subunit, which is generated from the most downstream promoter (exon 1). Gs  is imprinted in a tissue-specific manner, being expressed primarily from the maternal allele in certain hormone responsive tissues, such as the renal proximal tubules. PHP type-1b is associated with epimutations at the GNAS locus on chromosome 20q13.

7. GNAS locus & PHP-1b Sporadic PHP-1b Variable GNAS imprinting defects that may involve the upstream DMRs NESP55 & NESPAS, in addition to GNAS exon 1A. This epigenotype can result from: - maternally inherited microdeletions in NESP55 - paternal UPD of 20q or - an epimutation affecting the GNAS locus. Familial PHP-1b LOMM at GNAS exon 1A. This epigenotype has been associated with maternally inherited microdeletions in STX16 gene.

8. Screening strategy for PHP- type 1b Bisulphite treatment of DNA Methylation-specific PCR to examine the methylation status of the three DMRs: GNAS exon 1A, NESP55 and NESP-AS/XL  S. Methylation-specific PCR to examine the methylation status of the three DMRs: GNAS exon 1A, NESP55 and NESP-AS/XL  S. Microdeletion analysis by long range PCR to examine the STX16 and NESP55 genes for known microdeletions Microdeletion analysis by long range PCR to examine the STX16 and NESP55 genes for known microdeletions Microsatellite analysis to test for paternal uniparental disomy of 20q Microsatellite analysis to test for paternal uniparental disomy of 20q If a methylation defect is found: NESP55 STX16

9. MS-PCR results

10. 6/8 patients referred for PHP showed complete LOM at the GNAS exon 1A DMR. Additional methylation defects at the upstream DMRs NESP55 and NESPAS were also observed in all 6 patients. 4/6 cases showed complete hypomethylation at GNAS & NESPAS and complete hypermethylation at NESP55. 2/6 cases still had residual methylation at NESPAS. This epigenotype is associated with sporadic forms of PHP-1b and can be due to: - maternally inherited microdeletions in NESP55 - paternal UPD of 20q or - an epimutation affecting the GNAS locus.

11. Screening strategy for PHP- type 1b Bisulphite treatment of DNA Methylation-specific PCR Microdeletion analysis by long range PCR to examine the STX16 and NESP55 genes for known microdeletions Microdeletion analysis by long range PCR to examine the STX16 and NESP55 genes for known microdeletions Microsatellite analysis to test for paternal uniparental disomy of 20q Microsatellite analysis to test for paternal uniparental disomy of 20q If a methylation defect is found: NESP55 STX16

12. Results continued….. No evidence of known microdeletions in NESP55 (sporadic). No evidence of UPD. No known microdeletions in STX16 found (familial). All 6 cases are believed to be caused by an epigenetic abnormality.

13. PHP type-1b is characterised by hypocalcaemia and hyperphosphataemia due to end-organ resistance to PTH, which acts via a Gs  - coupled receptor. PHP-1b is associated with epimutations at the GNAS locus on chromosome 20q13. 6/8 patients had methylation defects at the GNAS locus which were consistent with a diagnosis of PHP-1b. This test has been validated and is now available as a diagnostic test. In conclusion….. We are now offering a molecular genetic diagnostic service for PHP-1b at the WRGL Please contact the lab for further information. wessex.genetics@Salisbury.nhs.uk

14. Acknowledgments Dr D Mackay Jonathan Callaway Dr D Robinson Dr J Harvey