axenfeld-rieger syndrome: a new ukgtn service cmgs spring meeting tuesday 13 th april 2010 kenneth...
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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
• Axenfeld-Rieger Syndrome is a rare eye disorder (1/250,000).
• Autosomal dominant inheritance.
• 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;
Rieger anomaly
corectopia Iris Hypoplasia
Rieger anomaly
corectopia
Peters’ anomaly
corectopia
• 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).
(Perveen et al, 2000)
corneal opacity polycoria
North Bristol NHS Trust - Bristol Genetics Laboratory
• Individuals with Anterior Segment Dysgenesis (ASD) frequently develop elevated intraocular pressure and sight threatening glaucoma (50-75%) (Strungaru et al, 2007).
• Identifying the genetic basis of the disorder allows;
– appropriate glaucoma surveillance and treatment.
– potential cardiac problems to be assessed and treated appropriately.
The term “Axenfeld-Rieger Syndrome”
Aqueous humour flow.
Increased ocular pressure damages optic nerve
Impaired outflow
ASD
North Bristol NHS Trust - Bristol Genetics Laboratory
Vitreous humour
Optic nerve
Lens
Iris
Anterior chamber (contain aqueous humour) Retina
Anterior segment
Posterior segment
Axenfeld-Rieger Syndrome Loci
• There are 5 reported loci which have been associated with ARS;
PITX2 (4q25)
FOXC1 (6p25)
PAX6 (11p13)
(16q24)
(13q14)
• 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
• 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.
Exon 1
c.1 c.1662
Forkhead domain
• PITX2 Mutations– > 40 mutations reported to date– Missense, nonsense, splice-site mutations, insertion/deletions.– Whole gene deletions.– Majority of mutations reside in the homeodomain.
Exons 1 2 3 4a 4b 5 6
Homeodomain
c.1 c.816 (PITX2a)
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.
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
Polycoria; multiple pupillary openings
North Bristol NHS Trust - Bristol Genetics Laboratory
ARS Testing Strategy
Clinical diagnosis of ARS
Ocular and non-ocular symptoms or
polycoria
PITX2 point mutation analysis (DNA Seq)
-ve
Only ocular symptoms (not
polycoria)
FOXC1 point mutation analysis
(DNA Seq)
-ve
Diagnosis remains on a clinical basis
-ve
Duplication/deletion FOXC1/PITX2(MLPA)
Report+ve
Report+ve
Report+ve
North Bristol NHS Trust - Bristol Genetics Laboratory
ARS testing - Dosage Analysis by MLPA
• 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
FOXC1;
Axenfe
ld R
ieger Syndro
me
TWIST1
; Saeth
re C
hotzen S
yndrom
e
(Oxfo
rd)
PITX2;
Axenfe
ld R
ieger Syndro
me
FOXL2;
Blepharo
phimosis
GPR143
; Ocular A
lbin
ism ty
pe 1
FOXC2;
Distic
hiasis syndrom
e
(St.
Georges)
• 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
• The p300-A1 kits contain reference probes and quality control probes.
• These are spaced allowing the addition of “home made” probes.
• 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.
p300-A1 Reference Kit
Additional probe region
North Bristol NHS Trust - Bristol Genetics Laboratory
MLPA Design, Optimisation and Validation
• 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)
PIT
X2_
Ex2
PIT
X2_
Ex3
PIT
X2_
Ex4
.2
FO
XC
1_E
x1.1
FO
XC
1_E
x1.2
FO
XC
1_E
x1.3
PIT
X2_
Ex4
.1
FO
XC
1_IV
S1.
1
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
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)
– Whole gene deletion of PITX2 (University Hospital Ghent, Belgium – P054 MLPA kit).
FOXC1 deletion
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
PITX2
Ex2
PITX2
Ex3
PITX2
Ex4.1
PITX2
Ex4.2
FOXC1 Ex1
.1
FOXC1 Ex1
.2
FOXC1 Ex1
.3
FOXC1_IV
S1.1
p300
-A1
D88
p300
-A1
D184
p300
-A1
L92
p300
-A1
C109
p300
-A1
C129
p300
-A1
C148
p300
-A1
C172
p300
-A1
C178
p300
-A1
C190
p300
-A1
C196
p300
-A1
C202
p300
-A1
C214
p300
-A1
C220
p300
-A1
C226
(4q2
5)
p300
-A1
C232
p300
-A1
C244
p300
-A1
C250
p300-1 ARS PROBE
NO
RM
AL
ISE
D D
OS
AG
E
PITX2 FOXC1 CONTROL Probes
PITX2 deletion
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
PITX2
Ex2
PITX2
Ex3
PITX2
Ex4.1
PITX2
Ex4.2
FOXC1 Ex1
.1
FOXC1 Ex1
.2
FOXC1 Ex1
.3
FOXC1_IV
S1.1
p300
-A1
D88
p300
-A1
D184
p300
-A1
L92
p300
-A1
C109
p300
-A1
C129
p300
-A1
C148
p300
-A1
C172
p300
-A1
C178
p300
-A1
C190
p300
-A1
C196
p300
-A1
C202
p300
-A1
C214
p300
-A1
C220
p300
-A1
C226
(4q2
5)
p300
-A1
C232
p300
-A1
C244
p300
-A1
C250
p300-1 ARS PROBE
NO
RM
AL
ISE
D D
OS
AG
E
PITX2 FOXC1 CONTROL Probes
C226 reference probe
C226 reference probe PITX2
North Bristol NHS Trust - Bristol Genetics Laboratory
ARS testing - Point Mutation Analysis by DNA Sequencing
• 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.
Exon 1
c.1 c.1662
6 overlapping amplicons
Exons 1 2 3 4a 4b 5 6
c.1 c.816 (PITX2a)
3 amplicons
PITX2
FOXC1
North Bristol NHS Trust - Bristol Genetics Laboratory
ARS Service: Results to Date
• 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 % of PITX2/FOXC1 mutations
FOXC1 Dosage Deletion 31%
PITX2 Dosage Deletion 8%
FOXC1 Nonsense c.99_108del, p.Gly34fsThrX8
38%
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 insAA23%
PITX2 Missense c.191C>T, p.Pro64Leu
North Bristol NHS Trust - Bristol Genetics Laboratory
Case 1: FOXC1 deletion
• 13 year old girl (II:1) referred from clinical genetics in Belfast.
II:1 II:2
I:1 I:2Posterior embryotoxon
Iris hypolplasiaHypodontia
• Dosage analysis identified a deletion of FOXC1.– Enhanced surveillance for glaucoma.
– Referral to cardiology.
– ? Prenatal testing in the future.
• 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
• 44 year old male (I:1) referred from Bristol Eye Hospital.– Bilateral secondary glaucoma.
– Daughter similar ocular features, developmental delay and hydrocephalus.
II:1 II:2
I:1 I: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.
North Bristol NHS Trust - Bristol Genetics Laboratory
Wildtype sequence tttcgttttcagAGAAAGA
Mutant sequence tttcgttttcaaAGAAAGA
WT Splice acceptor site
Mutant Splice acceptor site
c.47-1G>A
Maciolek et al, 2006 c.47-1G>T
“All sequences showed that splicing was shifted 2 nt downstream to the next available "AG" dinucleotide”
Case 3: FOXC1 Unclassified Variant• 23 year old male referred from Cardiff clinical genetics.
• Point mutation analysis of FOXC1 identified a UV; c.254C>T, p.Ala85Val.
• Evidence FOR pathogenicity
– Not recorded in SNPdb
– 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.
Exon 1
c.1 c.1662
c.254C>T, p.Ala85Val
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