Development of a Diagnostic Service for Pseudohypoparathyroidism type 1b

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Development of a DiagnosticService for
Pseudohypoparathyroidism type 1b

• Jennifer Greatwood

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Pseudohypoparathyroidism (PHP)

• Introduction- Pseudohypoparathyroidism
• Screening strategy
• Results
• Conclusion

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Pseudohypoparathyroidism (PHP)
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 Albrights
hereditary osteodystrophy (AHO).
Brachydactyly- hands/feet
round face, mental retardation
short stature, obesity, short limbs
Characteristic dimpling replacing the knuckles
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PHP variants
PHP (PTH resistance) AHO phenotype
AHO phenotype
PHP (PTH resistance)
PHP type 1a
PPHP Pseudopseudohypoparathyroidism
PHP type 1b
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Parathyroid hormone
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 endorgan resistance to PTH rather
than deficiency of PTH.
The PTH normally mediates its actions via a
Gs?-coupled receptor. In PHP hormone resistance
is due to a deficiency of the Gs? subunit.
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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.

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GNAS locus PHP-1b
Familial PHP-1b LOMM at GNAS exon 1A. This
epigenotype has been associated with maternally
inherited microdeletions in STX16 gene.

• 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.

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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.
If a methylation defect is found
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
STX16
NESP55
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MS-PCR results
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MS-PCR results

• 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.

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Screening strategy for PHP- type 1b
Bisulphite treatment of DNA

Methylation-specific PCR
If a methylation defect is found
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
STX16
NESP55
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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.

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In conclusion..

• 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.

We are now offering a molecular genetic
diagnostic service for PHP-1b at the WRGL Please
contact the lab for further information.
wessex.genetics_at_Salisbury.nhs.uk
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Acknowledgments

• Dr D Mackay
• Jonathan Callaway
• Dr D Robinson
• Dr J Harvey