Proposal form for the evaluation of a genetic test for NHS Service Gene Dossier Test Disease Population Triad Disease name and description (please provide any alternative names you wish listed) (A)-Testing Criteria Axenfeld-Reiger Syndrome - Reiger Syndrome Type 1 Axenfeld-Reiger malformation (ARM) is a rare AD disorder affecting the anterior eye structures derived from the periocular mesenchyme. OMIM number for disease #180500 Gene name and description (please provide any alternative names you wish listed) The diagnosis refers to a heterogeneous group of dominant presentations including; IGD-Iridogoniodysgenesis IH-Iris Hypoplasia Corectopia ( eccentric pupil) Polycoria ( multiple pupils due to iris holes ) PAS-Peripheral Anterior Synechiae PE-Posterior Embryotoxon Patients with ARM may also present with systemic malformations with incomplete penetrance and variable expressivity e.g. Maxillary Hypoplasia Hypo and microdontia Cardiac defects In rare cases there may be other additional features. The eye related features are highly penetrant and have been associated with approx 50% risk of development of glaucoma which can lead to blindness (75% in cases with PITX2 or FOXC1 mutations). This usually develops in adolescence or rarely in infancy but can present later in life indicating that patients with ARM remain with a lifetime risk of glaucoma. Forkhead Box C1; FOXC1 Forkhead Drosophila,Homolog-Like 7;FKHL7 Forkhead-Related Activator 3;FREAC3 FOXC1 encodes a virtually ubiquitously expressed forkhead transcription factor distinguished by a characteristic 100-amino acid DNA-binding motif. Paired-Like Homeodomain Transcription Factor 2;PITX2 Pituitary Homeobox2;PTX2 Reig Bicoid-Related homeobox transcription factor 1;REIG1 REIG PITX2 is a paired-bicoid HD protein that is expressed during ocular development and is considered part of a signalling cascade modulating cell proliferation, differentiation and morphogenesis. 1
OMIM number for Gene #601090 FOXC1 #601542 PITX2 Mutational spectrum for which you test Technical Method (s) FOX C1 Point mutations/small deletions/insertions and gross rearrangements- whole gene duplications/deletions. PITX2 Point mutations/small deletions/duplications and gross rearrangements- whole gene deletions/microdeletions To our knowledge all reported mutations are in these categories. MLPA is the validated in house method for gene dosage analysis; a kit is available from MRC Holland. Real Time Quantitative PCR (patent negotiations pending) is also used at BGL and can be validated for this gene, or junction fragment analysis. Sequence analysis for all coding exons and intron-exon junctions. Validation Process Note please explain how this test has been validated for use in your laboratory HT automated sequence analysis (Beckman NX robotics/abi3730/ mutation surveyor/ positive and negative control samples) is the validated screening technology in routine use at BGL. Primers (SNP checked) designed to cover all coding regions). BGL participates in an annual EMQN EQA schemes for DNA sequencing. Are you providing this test already? If yes, how many reports have you produced? Please give the number of mutation positive/negative samples you have reported For how long have you been providing this service? Is there specialised local clinical/research expertise for this disease? Are you testing for other genes/diseases closely allied to this one? Please give details No. This service is being developed to support local Clinical and Research expertise in Axenfeld-Reiger malformation and Glaucoma. Dr Amanda Churchill is a Clinical Research specialist at Bristol Eye Hospital and leads a team currently offering dhplc analysis of both genes on a research basis. Yes No Please provide details Dr Amanda Churchill with Dr Ruth Newbury-Ecob hold a Genetics Eye clinic monthly with dedicated genetic counsellor. PAX6, CYP1B1 and MYOC screening using dhplc is also undertaken by Amanda Churchill and offered on a research basis for individuals attending the Genetic Eye Clinic with anterior segment anomalies or early onset glaucoma. 2
Your Activity How many tests do you (intend to) provide annually in your laboratory? Based on experience how many tests will be required nationally (UK)? Please identify the information on which this is based Rare condition no more than 30 tests expected for UK and abroad.. Rare condition <10 new cases per year. With family studies this may extend to 15-20 tests per year Bristol Specialist Eye Clinic receives 2-3 referrals per year. 3
Epidemiology Estimated prevalence of disease in the general UK population Please identify the information on which this is based ARM is associated with mutations in the PITX2 gene (4q25 and FOXC1 gene (6p25) with additional gene loci mapping to 13q14 and16q24. An isolated case has been reported with PAX6 mutations. Lines et al indicate approx 40% ARM cases have mutations in either FOXC1 or PITX2. Strungaru et al Canadian study reports a cohort of 126 ARM patients representing 20 probands.71% of cases 13/20 harboured FOXC1 mutations and 29% of cases 7/20 harboured PITX2 mutations. In combination with the Lines data this would suggest approx 27% AR cases have mutations in FOXC1 and approx 13% AR cases have mutations in PITX2. Weisschuh et al indicate that PITX mutations account for 2/14 (14%) and FOXC1 mutations 7/14 (50%) German AR cases. References: Lines et al 2004 Characterisation and prevalence of PITX2 microdeletions and mutations in Axenfeld-Reiger malformations Invest Opthalmol Vis Sci;45:828-833 Strungaru et al 2007 Genotype-Phenotype Correlations in Axenfeld-Reiger Malformation and Glaucoma Patients with FOXC1 and PITX2 Mutations Invest Opthal Vis Sci 48:1:228-237 Weisschuh et al 2006 Novel mutations of FOXC1 and PITX2 in patients with Axenfeld-Reiger malformations. Invest Opthalmol Vis Sci 27:4:3846-3852. Meyer-Marcotty (2008) Morphology of the sella turcica in Axenfeld-reiger syndrome with PITX2 mutation. J Oral pathol. Med Mar 10 [E pub ahead of print]. Estimated gene frequency (Carrier frequency or allele frequency) Please identify the information on which this is based Incidence for AR in UK is unknown. Meyer-Marcotty et al (2008) give an incidence for in the German population of 1:200,000 It is thought that the UK incidence is similar or slightly higher. Estimated penetrance Please identify the information on which this is based For ARO patients with a defined FOXC1 or PITX2 mutation penetrance of eye related anomalies occurs is very high. The incidence of elevated Intra ocular pressure (IOP) and glaucoma is lower in patients with FOXC1 mutations than PITX2 4
mutations and treatment is therefore more likely to be successful. There is a diversity in clinical presentation with FOXC1 mutations including iris hypoplasia, corectopia, peripheral anterior synechiae and posterior embryotoxon. Patients with a FOXC1 duplication typically had IGD malformations and a more severe prognosis in glaucoma development than FOXC1 mutations, but not as severe as PITX2 mutation carriers. Systemic malformations present with incomplete penetrance and variable expressivity. Patients with non-ocular findings are more likely to have PITX2 mutations than FOXC1 mutations and both are more likely than a FOXC1 duplication. Strungaru et al indicate that 75% 71/95 patients with PITX2 or FOXC1 mutations had glaucoma that developed in adolescence or early childhood. PITX2 mutations have a more severe prognosis for glaucoma development. Target Population The essential clinical or family history features defining the target population must be described. (C)-Testing Criteria Clinical phenotype of Axenberg-Reiger syndrome Individuals should fulfil at least 2 of the following criteria: Posterior embryotoxon Iris hypoplasia Correctopia/Polycoria Childhood but not congenital glaucoma Cardiac abnormalities OR Autosomal Dominant inheritance with one of the above ocular features excluding glaucoma AND Cytogenetic investigations negative Additional supporting evidence for targeting PITX2 Teeth abnormalities and/or umbilical hernia Estimated prevalence of disease in the target population Approximately 13% AR cases have mutations in PITX2 and 27% AR cases have mutations in FOXC1. Extrapolating this data suggests that 18% of FOXC1 negative AR cases will have PITX2 mutations. Will be refined as further data becomes available. 5
Intended Use (Please use the questions in Annex A to inform your answers) Please tick the relevant clinical purpose of testing Diagnosis Treatment Prognosis & Management Presymptomatic testing Risk Assessment YES Confirmation of clinical diagnosis It is suggested that patients with FOXC1 mutations have better response to treatment as incidence of elevated IOP is lower. There is some evidence that glaucoma risk may be higher with PITX2 mutations than FOXC1, and that PITX2 cases may benefit from closer monitoring through more frequent examination, and from more aggressive treatment. Current practice is to monitor all individuals whilst awaiting more refined data. Genotype /phenotype will inform risk of non-ocular findings, for instance, individuals with positive mutations may be referred for cardiac screening Negative tests for familial mutations will negate clinical investigations. Establish genetic cause in this heterogeneous condition facilitating family testing and prenatal diagnosis if appropriate. NO 6
Test Characteristics Analytical sensitivity and specificity This should be based on your own laboratory data for the specific test being applied for or the analytical sensitivity and specificity of the method/technique to be used in the case of a test yet to be set up. If a number of genes will be tested, please include your testing strategy and data on the expected proportions of positive results for each part of the process. It may be helpful to include a diagram to illustrate the testing strategy. FOXC1 Exonic or splice site point mutations, small insertions or deletions have been described, causing either protein truncation or functional defects. Whole gene duplications have also been described. PITX2 Strungaru et al 2007 indicate that over 30 exonic or splice site point mutations or small insertions or deletions have been described, causing either protein truncation or functional defects to the homeodomain. Gross rearrangements deletions and translocations have also been described Strategy: Quantitative analysis for FOXC1 and PITX2 by MLPA or qrt PCR or JF anaysis. Cases negative for the quantitative assay proceed to HT Automated Sequence analysis in this laboratory: Sensitivity 99-100%. To our knowledge all of the above variants would be detected using this combinatorial approach. Specificity 100%. Clinical sensitivity and specificity of test in target population The clinical sensitivity of a test is the probability of a positive test result when disease is known to be present; the clinical specificity is the probability of a negative test result when disease is known to be absent. The denominator in this case is the number with the disease (for sensitivity) or the number without disease (for specificity) Assuming HT sequencing and quantitative analysis has a sensitivity of 99% for known pathogenic mutations for FOXC1 this will be 27% in all AR cases. For PITX2 13% for all AR cases and 18% for FOXC1 negative AR cases. Specificity for a known pathogenic mutation should be close to 100%. 7
Clinical validity (positive and negative predictive value in the target population) The clinical validity of a genetic test is a measure of how well the test predicts the presence or absence of the phenotype, clinical disease or predisposition. It is measured by its positive predictive value (the probability of getting the disease given a positive test) and negative predictive value (the probability of not getting the disease given a negative test). The denominator in this case is the number of people with a positive or a negative test respectively - not the number with or without the disease. The clinical validity may be calculated knowing the sensitivity and the specificity and the prevalence of the disease in the population being studied. Positive and negative predictive values depend critically on the prevalence of the disease in the test population Positive predictive value of test should be close to 100%. Negative predictive value of known familial mutations should be close to 100%. A negative diagnostic test result ( PITX2 and FOXC1 ) has a negative predictive value of 60% as it does not exclude AR due to mutations in other associated genes which have yet to be elucidated. 8
Clinical utility of test in target population (Please refer to Appendix A) Please provide a full description of the clinical care pathway for those individuals undergoing testing. This should include details of which medical specialties will be able to refer for testing. (B)-Testing Criteria AR is a genetically heterogeneous condition. Genetic testing; Enables a molecular confirmation of diagnosis, Enables genetic risk to be determined enabling carrier testing and prenatal diagnosis. How will the test add to the management of the patient or alter clinical outcome? What impact will this test have on the NHS i.e. by removing the need for alternative management and/or investigations for this clinical population? Is there an alternative means of diagnosis or prediction that does not involve molecular diagnosis? If so (and in particular if there is a biochemical test) please state the added advantage of the molecular test Are there specific ethical, legal or social issues with this test? Please complete the referral pathway diagram on the following page and the testing criteria form. 9
Referral Pathway Template TARGET POPULATION Clinical phenotype of Axenberg-Reiger syndrome Individuals should fulfil at least 2 of the following criteria: Posterior embryotoxon, Iris hypoplasia, Correctopia/Polycoria, Childhood but not congenital glaucoma, cardiac abnormalities. Hypodontia/abnormal dentition and/or umbilical hernia suggest reverse order screening. OR Autosomal Dominant inheritance with one of the above ocular features excluding glaucoma AND Cytogenetic investigations negative See testing pathway diagram. WHAT TYPE AND LEVEL OF PROFESSIONAL OR REFERRER DO YOU ACCEPT SAMPLES FROM? - Clinical Geneticist - Genetic Ophthalmologist PLEASE PROVIDE DETAILS OF HOW REFERRALS WILL BE ASSESSED FOR APPROPRIATENESS? Clinical detail as per testing criteria to confirm that referrals are appropriate. HOW MANY TESTS DO YOU EXPECT TO PERFORM ANNUALLY? 15-20 for UK 10
UKGTN Testing criteria for disease: Name of Disease(s): Reiger syndrome Type 1; RIEG (180500) Name of gene(s): Forkhead Box C1;FOXC1 (601090), Paired-Like Homeodomain transcription factor; PITX2 (601542) Patient name: Patient postcode: Date of birth: NHS number: Name of referrer: Title/Position: Lab ID: Referrals will only be accepted from one of the following: Referrer Tick if this refers to you. Genetic Ophthalmologist Clinical Geneticist Minimum criteria required for testing to be appropriate as stated in the Gene Dossier: Criteria Tick if this patient meets criteria Clinical phenotype of Axenberg-Reiger syndrome: Individuals should fulfil at least 2 of the following criteria: Posterior embryotoxon Iris hypoplasia Correctopia/Polycoria Childhood but not congenital glaucoma Cardiac abnormalities OR Autosomal Dominant inheritance with one of the above ocular features excluding glaucoma AND Cytogenetic investigations negative Additional supporting evidence for preferential targeting of PITX2 Abnormal dentition and/or umbilical hernia Polycoria. Severe prognosis for glaucoma development. If the sample does not fulfil the clinical criteria or you are not one of the specified types of referrer and you still feel that testing should be performed please contact the laboratory to discuss testing of the sample. 11