Transcript Axenfeld-Rieger Syndrome

Axenfeld-Rieger Syndrome:
A New UKGTN Service
CMGS Spring Meeting
Tuesday 13th April 2010
Kenneth Smith
Bristol Genetics Laboratory
North Bristol NHS Trust - Bristol Genetics Laboratory
UKGTN and Genetic Ophthalmology
• In April 2008, the UKGTN published a review of service provision within
genetic ophthalmology.
• The report highlighted that some genetic testing in ophthalmology existed
in a research setting with no provision for transferring to mainstream
genetic testing.
• Bristol Eye Hospital (BEH) was in this position having established a
service Axenfeld-Rieger syndrome during a period of research.
– WAVE (scanning)  point mutation analysis (PITX2 and FOXC1)
• Bristol Genetics Laboratory submitted a UKGTN gene dossier to develop
this as an NHS service.
• This would hopefully secure the long term future of the service and
mediate nationwide access to testing via the UKGTN.
North Bristol NHS Trust - Bristol Genetics Laboratory
Axenfeld-Rieger Syndrome
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•
Axenfeld-Rieger Syndrome is a rare eye disorder (1/250,000).
Autosomal dominant inheritance.
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•
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The disorder is genetically and phenotypically heterogeneous.
Axenfeld-Rieger syndrome is a form of anterior segment dysgenesis (ASD).
Affected individuals display a characteristic spectrum of ocular anomalies;
Peters’ anomaly
corneal opacity
Rieger anomaly
corectopia
corectopia
Rieger anomaly
Iris Hypoplasia
corectopia
polycoria
(Perveen et al, 2000)
•
Systemic features can include cardiac defects, dental anomalies, craniofacial
anomalies and umbilical defects.
•
Additional features of ARS can include sensorineural hearing loss and Dandy
Walker Malformation (FOXC1 mutations).
North Bristol NHS Trust - Bristol Genetics Laboratory
The term “Axenfeld-Rieger Syndrome”
•
Individuals with Anterior Segment Dysgenesis (ASD) frequently develop elevated
intraocular pressure and sight threatening glaucoma (50-75%) (Strungaru et al, 2007).
Anterior chamber
(contain aqueous
humour)
Vitreous humour
ASD
Retina
Lens
Iris
Aqueous
humour flow.
Impaired
outflow
Optic nerve
Anterior
segment
•
Posterior
segment
Identifying the genetic basis of the disorder allows;
– appropriate glaucoma surveillance and treatment.
– potential cardiac problems to be assessed and treated appropriately.
North Bristol NHS Trust - Bristol Genetics Laboratory
Increased ocular
pressure damages
optic nerve
Axenfeld-Rieger Syndrome Loci
•
There are 5 reported loci which have been associated with ARS;
FOXC1 (6p25)
PAX6 (11p13)
PITX2 (4q25)
(13q14)
(16q24)
•
•
•
RIEG was identified and cloned in 1996, and later renamed PITX2.
In 1998, FOXC1 was identified as the second loci of ARS.
Point mutations and copy number variations of both PITX2 and FOXC1 have been
reported to cause ARS (40% of cases).
•
•
Conflicting reports over PAX6 association with ARS phenotype.
Two loci have been identified but the genes remain unknown.
North Bristol NHS Trust - Bristol Genetics Laboratory
ARS Genetics – FOXC1 and PITX2 Mutations
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Exon
1
c.1
c.1662
FOXC1 Mutations
– > 44 mutations reported to date.
– Missense, nonsense, insertion/deletions
– Also whole gene duplications and deletions.
– Majority of mutations reside in the fork head domain.
Forkhead
domain
Exons
1
2
c.1
3
4a
4b
5
6
c.816
(PITX2a)
Homeodomain
•
PITX2 Mutations
– > 40 mutations reported to date
– Missense, nonsense, splice-site mutations, insertion/deletions.
– Whole gene deletions.
– Majority of mutations reside in the homeodomain.
North Bristol NHS Trust - Bristol Genetics Laboratory
ARS Genetics – FOXC1 and PITX2 Proteins
•
FOXC1 and PITX2 proteins are both transcription factors.
– FOXC1 and PITX2 proteins could physically interact and that this interaction was a
requirement of normal eye development.
– PITX2 could negatively regulate the action of FOXC1 to transcribe target genes.
– FOXC1 and PITX2 proteins were co-expressed in specific populations of periocular
mesenchyme cells which give rise to the anterior segment of the eye.
– Degree of co-expression varied depending on the structure the cells give rise to.
– This point may explain one of the few genotype/phenotype correlations in ARS.
Iris; Expression PITX2 +++++, FOXC1 +.
Polycoria is a feature of PITX2 mutations but not FOXC1.
Polycoria; multiple pupillary openings
Berry et al, 2006 Functional Interaction Between FOXC1 and PITX2
Underlie the Sensitivity to FOXC1 Gene Dose in ARS and Anterior
Segment Dysgenesis.pdf
Walter et al, 2007 Genotype-Phenotype Correlations in Axenfeld-Rieger
Malformation and Glaucoma Patients with FOXC1 and PITX2
Mutations.pdf
North Bristol NHS Trust - Bristol Genetics Laboratory
ARS Testing Strategy
Clinical diagnosis of ARS
Duplication/deletion FOXC1/PITX2
(MLPA)
-ve
Report
+ve
+ve
Report
-ve
Ocular and non-ocular
symptoms or polycoria
Only ocular symptoms
(not polycoria)
PITX2 point mutation
analysis (DNA Seq)
FOXC1 point mutation
analysis (DNA Seq)
-ve
Diagnosis remains on
a clinical basis
North Bristol NHS Trust - Bristol Genetics Laboratory
+ve
Report
ARS testing - Dosage Analysis by MLPA
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•
Being a rare condition MRC-Holland do not offer a kit exclusively for ARS.
P054 kit for Ophthalmogenetic Abnormalities exists but consider inappropriate.
p054 MLPA Kit for Ophthalmogenetic Abnormalities
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Approached MRC-Holland about a reference probe only kit.
Recently market the p200 and p300 reference kits.
North Bristol NHS Trust - Bristol Genetics Laboratory
MLPA using the p300-A1 Reference Kit
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The p300-A1 kits contain reference probes and quality control probes.
These are spaced allowing the addition of “home made” probes.
p300-A1 Reference Kit
Additional probe region
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This approach allows further probes to be added if other dosage sensitive genes
are identified causing ARS.
Cost of kit can be spread over several rare disease tests.
Currently being trialled by BGL;
‒ Thrombocytopenia and Absent Radius (TAR) syndrome.
‒ aCGH follow-up.
North Bristol NHS Trust - Bristol Genetics Laboratory
MLPA Design, Optimisation and Validation
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Probes were designed according to the MRC-Holland protocol and ordered from
Sigma-Aldrich.
The final probe mix contained:
– 4 probes for FOXC1 (3 exonic and 1 intronic)
– 4 probes for PITX2 (4 exonic)
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PITX2_Ex4.2
FOXC1_Ex1.3
FOXC1_IVS1.1
PITX2_Ex4.1
FOXC1_Ex1.2
PITX2_Ex3
FOXC1_Ex1.1
PITX2_Ex2
p300-A1 Reference Kit with custom FOXC1 and PITX2 Probes
Normal controls were selected by demonstration of heterozygosity in both genes.
Positive controls were obtained from several sources.
Assay was validated based on observed and expected results being concordant.
– 3 FOXC1 deletions
– 1 PITX2 deletion
North Bristol NHS Trust - Bristol Genetics Laboratory
PITX2 FOXC1
2
4.
1
3
2
Ex
Ex
Ex
2
2
Ex
FO
4.
2
XC
1
Ex
FO
1.
1
XC
1
Ex
FO
1.
2
XC
1
FO
Ex
1.
XC
3
1_
IV
S1
p3
.1
00
-A
1
p3
D8
00
8
-A
1
D1
p3
84
00
-A
1
p3
L9
00
2
-A
1
C1
p3
09
00
-A
1
C1
p3
29
00
-A
1
C1
p3
48
00
-A
1
C1
p3
72
00
-A
1
C1
p3
78
00
-A
1
C1
p3
90
00
-A
1
C1
p3
96
00
-A
1
C2
p3
02
00
-A
1
C2
p3
14
0
p3
000
A1
-A
C2
1
C2
20
26
(4
p3
q2
00
5)
-A
1
C2
p3
32
00
-A
1
C2
p3
44
00
-A
1
C2
50
PI
TX
PI
TX
2
NORMALISED DOSAGE
(University Hospital Ghent, Belgium – P054 MLPA kit).
PITX2 FOXC1
2
Ex
3
Ex
2
Ex
2
2
4.
PI
1
TX
2
Ex
FO
4.
XC
2
1
Ex
FO
1.
XC
1
1
Ex
FO
1.
2
XC
1
FO
Ex
1.
XC
3
1_
IV
S1
p3
.1
00
-A
1
p3
D8
00
8
-A
1
D1
p3
84
00
-A
1
p3
L9
00
2
-A
1
C1
p3
09
00
-A
1
C1
p3
00
29
-A
1
C1
p3
48
00
-A
1
C1
p3
72
00
-A
1
C1
p3
00
78
-A
1
C1
p3
90
00
-A
1
C1
p3
96
00
-A
1
C2
p3
02
00
-A
1
C2
p3
00
14
p3
-A
00
1
-A
C2
1
C2
20
26
(4
p3
q2
00
5)
-A
1
C2
p3
00
32
-A
1
C2
p3
44
00
-A
1
C2
50
PI
TX
PI
TX
PI
TX
NORMALISED DOSAGE
•
PI
TX
PI
TX
MLPA Design, Optimisation and Validation
Example data from MLPA validation:
– Isolated whole gene deletion of FOXC1 (Lehmann et al, 2008, HMG, 2008, Vol. 17, No. 22)
FOXC1 deletion
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
1.80
PITX2 deletion
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
p300-1 ARS PROBE
CONTROL Probes
C226 reference probe
North Bristol NHS Trust - Bristol Genetics Laboratory
p300-1 ARS PROBE
CONTROL Probes
– Whole gene deletion of PITX2
C226 reference probe
PITX2
ARS testing - Point Mutation Analysis by DNA Sequencing
FOXC1
Exon
c.1
1
c.1662
6 overlapping amplicons
PITX2
Exons
1
2
c.1
3
4a
4b
5
6
c.816
(PITX2a)
3 amplicons
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BGL offers complete sequencing of the coding region (+/-20nt) for each gene.
‒
FOXC1 is sequenced in 6 overlapping fragments
‒
PITX2 is sequenced in 3 fragments.
North Bristol NHS Trust - Bristol Genetics Laboratory
ARS Service: Results to Date
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To date we have completed testing in 36 patients with a clinical diagnosis of ARS.
– Patients from BEH cohort and UKGTN referrals.
– Identified 13 mutations in either FOXC1 or PITX2 (36% pick-up rate).
– Frequency of different mutation class consistent with literature.
Gene
Mutatoin
Class
Mutation
FOXC1
Dosage
Deletion
31%
PITX2
Dosage
Deletion
8%
FOXC1
Nonsense
c.99_108del, p.Gly34fsThrX8
FOXC1
Missense
c.310A>T, p.Ile104Phe
FOXC1
Missense
c.889C>T, p.Pro297Ser
FOXC1
Nonsense
c.821dupC, p.Ser276fs29X
FOXC1
Missense
c.254C>T, p.Ala85Val
PITX2
Indel
c.652_654 delTAC insAA
PITX2
Missense
c.191C>T, p.Pro64Leu
% of PITX2/FOXC1
mutations
North Bristol NHS Trust - Bristol Genetics Laboratory
38%
23%
Case 1: FOXC1 deletion
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13 year old girl (II:1) referred from clinical genetics in Belfast.
I:1
I:2
Posterior embryotoxon
Iris hypolplasia
II:1
•
Hypodontia
II:2
Dosage analysis identified a deletion of FOXC1.
– Enhanced surveillance for glaucoma.
– Referral to cardiology.
– ? Prenatal testing in the future.
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I:2 and II:2 came forward for testing.
– Although not thought to be affected ocular defects can be very subtle.
– Cases of ARS have been reported where ocular features have been identified secondary
to cardiac defects.
– Both had normal dosage of FOXC1.
North Bristol NHS Trust - Bristol Genetics Laboratory
Case 2: PITX2 Splice-site Mutation
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44 year old male (I:1) referred from Bristol Eye Hospital.
– Bilateral secondary glaucoma.
– Daughter similar ocular features, developmental delay and hydrocephalus.
I:1
I:2
II:1
II:2
– PITX2 point mutation analysis identified a splice-site mutation in PITX2 c.47-1G>A.
– Previously reported mutations affecting this position c.47-1G>T and c.47-1G>C.
– Functional studies in these cases report that protein is poor expressed and truncated.
WT Splice acceptor site
Wildtype sequence
tttcgttttcagAGAAAGA
c.47-1G>A
Mutant sequence
Maciolek et al, 2006
c.47-1G>T
“All sequences showed that splicing was shifted 2 nt
downstream to the next available "AG" dinucleotide”
tttcgttttcaaAGAAAGA
Mutant Splice acceptor site
North Bristol NHS Trust - Bristol Genetics Laboratory
Case 3: FOXC1 Unclassified Variant
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23 year old male referred from Cardiff clinical genetics.
•
Point mutation analysis of FOXC1 identified a UV; c.254C>T, p.Ala85Val.
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Exon
1
c.1
c.1662
Evidence FOR pathogenicity
– Not recorded in SNPdb
c.254C>T, p.Ala85Val
– Variant resides in the functional forkhead domain of the protein.
– Aminoacid is highly conserved among species available for comparison.
– reports in the literature of a mutation affecting the same codon but a different nucleotide (c.253G>C,
p.Ala85Pro) associated with eye and heart defects in two family members.
•
Evidence AGAINST pathogenicity.
– There is only a small physiochemical difference between alanine and valine.
•
This was reported as possibly pathogenic and the clinician was advised to forward parental samples to the
laboratory to assist with interpretation.
Peters’ anomaly
North Bristol NHS Trust - Bristol Genetics Laboratory
Conclusion
• BGL now offers mutation analysis of PITX2 and FOXC1
causing Axenfeld Rieger syndrome.
– Fulfilled the objectives set out in the gene dossier.
– Developed a research service in to mainstream genetic testing and
mediating nationwide access via the UKGTN.
– Results to date are consistent with those reported in the literature.
– Assay allows for expansion with minimum increase in resources.
• Genetic testing allows:
– Appropriate glaucoma surveillance and treatment.
– Presymptomatic referral to cardiology.
North Bristol NHS Trust - Bristol Genetics Laboratory
Acknowledgments
Bristol Genetics Laboratory
– Maggie Williams and Thalia Antoniadi
– Technical staff
Bristol Eye Hospital
– Amanda Churchill and Jim Carter
University of Alberta, Department of Ophthalmology and Medical Genetics
– Professor Ordan Lehmann
University Hospital Ghent, Centre for Medical Genetics
– Dr Elfride De Baere
North Bristol NHS Trust - Bristol Genetics Laboratory