Download Development and validation of a diagnostic service for epimutations

Development of a Diagnostic
Service for
Pseudohypoparathyroidism type 1b
Jennifer Greatwood
Pseudohypoparathyroidism (PHP)
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Introduction- Pseudohypoparathyroidism
Screening strategy
Results
Conclusion
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 Albright’s hereditary osteodystrophy (AHO).
Brachydactylyhands/feet
short stature, obesity,
short limbs
round face,
mental retardation
Characteristic ‘dimpling’
replacing the knuckles
PHP variants
PHP
(PTH resistance)
+
AHO
phenotype
PHP type 1a
AHO
PHP
phenotype
(PTH resistance)
PPHP
PHP type 1b
Pseudopseudohypoparathyroidism
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 end–organ 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.
GNAS locus
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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.
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.
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
NESP55
STX16
Microsatellite analysis to test for
paternal uniparental disomy of 20q
MS-PCR results
MS-PCR results
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6/8 patients referred for PHP showed complete LOM at the
GNAS exon 1A DMR.
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Additional methylation defects at the upstream DMRs
NESP55 and NESPAS were also observed in all 6 patients.
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4/6 cases showed complete hypomethylation at GNAS &
NESPAS and complete hypermethylation at NESP55.
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2/6 cases still had residual methylation at NESPAS.
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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.
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
NESP55
STX16
Microsatellite analysis to test for
paternal uniparental disomy of 20q
Results continued…..
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No evidence of known microdeletions in NESP55 (sporadic).
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No evidence of UPD.
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No known microdeletions in STX16 found (familial).
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All 6 cases are believed to be caused by an epigenetic
abnormality.
In conclusion…..
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PHP type-1b is characterised by hypocalcaemia and
hyperphosphataemia due to end-organ resistance to PTH, which
acts via a Gs- coupled receptor.
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PHP-1b is associated with epimutations at the GNAS locus on
chromosome 20q13.
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6/8 patients had methylation defects at the GNAS locus which
were consistent with a diagnosis of PHP-1b.
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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.
[email protected]
Acknowledgments
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Dr D Mackay
Jonathan Callaway
Dr D Robinson
Dr J Harvey