1. Axenfeld-Rieger Syndrome: A New UKGTN Service CMGS Spring Meeting Tuesday 13 th April 2010 Kenneth Smith Bristol Genetics Laboratory North Bristol NHS Trust - Bristol Genetics Laboratory

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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.pdfFunctional 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.pdfGenotype-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

8. 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

9. 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; Axenfeld Rieger Syndrome TWIST1; Saethre Chotzen Syndrome (Oxford) PITX2; Axenfeld Rieger Syndrome FOXL2; Blepharophimosis GPR143; Ocular Albinism type 1 FOXC2; Distichiasis syndrome (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

10. 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

11. 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) PITX2_Ex2 PITX2_Ex3 PITX2_Ex4.2 FOXC1_Ex1.1 FOXC1_Ex1.2 FOXC1_Ex1.3 PITX2_Ex4.1 FOXC1_IVS1.1 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

12. 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). PITX2 FOXC1 CONTROL Probes C226 reference probe PITX2 North Bristol NHS Trust - Bristol Genetics Laboratory

13. 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. North Bristol NHS Trust - Bristol Genetics Laboratory

14. 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. GeneMutatoin Class Mutation % of PITX2/FOXC1 mutations FOXC1DosageDeletion31% PITX2DosageDeletion8% FOXC1Nonsensec.99_108del, p.Gly34fsThrX8 38% FOXC1Missensec.310A>T, p.Ile104Phe FOXC1Missensec.889C>T, p.Pro297Ser FOXC1Nonsensec.821dupC, p.Ser276fs29X FOXC1Missensec.254C>T, p.Ala85Val PITX2Indelc.652_654 delTAC insAA 23% PITX2Missensec.191C>T, p.Pro64Leu North Bristol NHS Trust - Bristol Genetics Laboratory

15. Case 1: FOXC1 deletion 13 year old girl (II:1) referred from clinical genetics in Belfast. II:1 II:2 I:1 I:2 Posterior embryotoxon Iris hypolplasia Hypodontia 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

16. 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 sequencetttcgttttcag AGAAAGA Mutant sequence tttcgttttcaa AGAAAGA 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”

17. 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

18. 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

19. 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