1. Thomas Linton-Willoughby
Birkbeck College, University of London; Student Number: 13003444
Acrodysostosis diagnosis: Can a definitive diagnosis be achieved
using molecular genetic tools?
• 17 Variants Identified
• 3 PRKAR1A
• 14 PDE4D
• 15 Benign,
• 2 Likely Pathogenic
L312F, L319P
Not Previously Discovered
Methods and Analysis
Key Findings
Figure 2: The PTH signal transduction pathway :
GNAS (Gs-α) and PRKAR1A mutations  PTH resistance
PDE4D mutations  cAMP resistance. (Lee et al., 2012).
Acknowledgments
•Mrs Lucy Jenkins, Interim Director
(GOSH) genetics (Funding)
•Ms Ann-Marie Differ, Principal Scientist
(GOSH), (Lab Supervisor)
•Dr Richard Rayne, (Birkbeck) IRP tutor
•Prof Nicholas Keep, (Birkbeck ISMB)
(Variant Interpretation Support)
.
Figure 1: ACRDYS skeletal
abnormalities; Brachycephaly (L)
Brachydactyly (R)
c.956T>C
p.(Leu319Pro) Het
• Disease Causing
(Mutation Taster)
• Tolerated (SIFT)
• Probably Damaging
(Poly Phen)
c.934C>T
p.(Leu312Phe) Het
• Disease Causing
(SIFT & Mutation Taster)
• Probably Damaging
(Poly Phen)
Literature cited
Lee, H. et al., (2012). Exome sequencing identifies
PDE4D mutations in acrodysostosis. The American
Journal of Human Genetics, 90(4):746–751.
Linglart, A. et al., (2012). PRKAR1A and PDE4D
mutations cause acrodysostosis but two distinct
syndromes with or without GPCR-signalling hormone
resistance. Journal of Clinical Endocrinology
& Metabolism, 97(12):E2328–E2338.
Michot, C. et al., (2012). Exome sequencing identifies
PDE4D mutations as another cause of
acrodysostosis. American Journal of Human Genetics,
90:740–5.
Mika, D. and Conti, M. (2015). PDE4D
phosphorylation: A coincidence detector
integrating multiple signalling pathways. Cellular
Signalling. [online] Available from:
http://www.sciencedirect.com/science/article/pii/S
0898656815003137 [accessed 16/02/2016].
Wang, H. et al., (2007). Structural insight into
substrate specificity of phosphodiesterase 10.
Proceedings of the National Academy of Science
USA, 104(14):5782–5787.
Introduction Interpretation of Findings
• L312F pushes a neighbouring helix away (Fig. 3),
the mutant F ring occupies a larger space (Fig. 4)
altering cAMP binding by closing the domain,
reducing cAMP entry, or opening it and increasing
cAMP off rate.
• L312F pushes an auto-inhibitory region (Tyr621)
into the active site, further reducing cAMP entry.
• L319P is likely to unwind the last turn of the helix in
which it is located (Fig. 3) altering the level of auto-
inhibition, with uncertain effect.
• L319F was not predicted to cause structural
problems (Fig. 5). However, the amino acid
changes from non-polar to polar, the charge
surrounding the cAMP binding domain may alter
binding affinity.
• The PDE4D cAMP binding domain is published
within amino acids p159-372 (Wang et al., 2007)
and p239-579 (Mika & Conti, 2015), supporting the
association of L312F and L319P with cAMP
degradation and the ACRDYS phenotype.
• Published ACRDYS PDE4D mutations are found
between p190-228 and p590-673 (Linglart et al.,
2012, Lee et al., 2012 and Michot et al., 2012).
The identified mutations fall outside these regions,
suggesting they may not be causative.
• Acrodysostosis, ACRDYS
(MIM 101800) is an autosomal
dominant genetic condition
characterised by
- Skeletal (Fig. 1)
- Endocrine
- Neurological abnormalities
• ACRDYS is caused by signalling defects within the
parathyroid hormone (PTH) signalling pathway (Fig.2)
which regulates cellular cyclic adenosine mono-
phosphate (cAMP) levels.
• Sufferers can demonstrate PTH or cAMP elevation,
due to varying pathway resistance.
• Cases are sporadic due to de novo gene mutations in
PRKAR1A (MIM 188830)  PTH resistance and
PDE4D (MIM 600123)  cAMP resistance.
• Albright’s hereditary osteodystrophy (AHO) (MIM
103580), caused by GNAS (MIM 13920) gene
mutations  PTH resistance, overlaps phenotypically.
• Misdiagnoses delay patient treatment
In the absence of offered ACRDYS gene testing, a
phenotypic cohort at Great Ormond Street Hospital
(GOSH), identified as negative for GNAS mutations was
studied. PRKAR1A and PDE4D genes were tested by
Sanger sequencing and variants identified.
Outcome
• A national diagnostic genetic testing strategy was
established to help differentiate AHO and ACRDYS.
• 2 likely causative mutations were found , indicating
clinical misdiagnoses.
• The Mutations could not be conclusively linked to
the phenotype, but are highly likely to be causative.
• Further studies could investigate variant effect
on cAMP degradation, within GM bacteria.
Figure 4: L312F, L electron
cloud (blue), F (brown)
Figure 5: L319P
electron cloud
6. Variant Interpretation
Alamut, PolyPhen2, ExAC
1. DNA Extraction
From blood in EDTA
(Chemagic Star)
2. Spectrophotometry
Purified DNA quality checked (Nanodrop)
3. PCR Exon amplification
(Biorad Tetrad)
4. PCR purification
Robotic bead + ethanol
(Beckman NXp, AMPure,
Clean SEQ XP)
5. Sanger Sequencing
ABi 3730 and Sequence
Scanning; Mutation
Surveyor
7. Insilico Interpretation
(Wincoot)
Figure 3: Ribbons Diagram;
L312 (254LEU), L319 (261
LEU), cAMP (green and red)