TREACHER COLLINS SYNDROME 2; TCS2 TREACHER COLLINS SYNDROME 3; TCS3 database (alternative names will be listed on the UKGTN website)

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1 Proposal form for the evaluation of a genetic test for NHS Service Gene Dossier/Additional Provider TEST DISEASE/CONDITION POPULATION TRIAD Submitting laboratory: Oxford RGC Approved: September Disease/condition approved name and symbol as published on the OMIM TREACHER COLLINS SYNDROME 2; TCS2 TREACHER COLLINS SYNDROME 3; TCS3 database (alternative names will be listed on the UKGTN website) 2. OMIM number for disease/condition (TCS2) and (TCS3) 3. Disease/condition please provide a brief description of the characteristics of the disease/condition and prognosis for affected individuals. Please provide this information in laymen s terms. Treacher Collins syndrome is a disorder of craniofacial development characterized by a combination of bilateral downward slanting of the palpebral fissures, colobomas of the lower eyelids with a paucity of eyelashes medial to the defect, hypoplasia of the facial bones, cleft palate, malformation of the external ears, atresia of the external auditory canals, and bilateral conductive hearing loss (Dauwerse et al., 2011). There are at least three different types (TCS1-3) caused by mutations in the TCOF1, POLR1D and POLR1C genes respectively. 4. Disease/condition mode of inheritance Autosomal dominant (TCS2) Autosomal recessive (TCS3) 5. Gene approved name(s) and symbol as polymerase (RNA) I polypeptide D, 16kDa; POLR1D published on HUGO database (alternative polymerase (RNA) I polypeptide C, 30kDa; POLR1C names will be listed on the UKGTN website) 6. OMIM number for gene(s) (POLR1D) (POLR1C) 7. Gene description(s) POLR1D is located on 13q12.2 and has two major transcripts. Isoform 1 (NM_ ) has 2 coding exons (exons 2 and 3) and encodes a 133 amino acid protein. Isoform 2 (NM_ ) has 3 coding exons (exons 2, 4 and 5 also known as 1a, 2a and 3a (Dauwerse et al 2011, Nat Genet 43: 20-22)) and encodes a 122 amino acid protein. POLR1D encodes polymerase (RNA) I polypeptide D; one of two proteins that make up the α-subunit of the DNA dependent RNA polymerases I and III. Heterozygous POLR1D mutations were identified in 20 of 252 TCOF1 negative TCS patients, consistent with autosomal dominant inheritance. All variants found to date have been found in isoform 1. (Dauwerse et al 2011). POLR1C is located on 6p21.1, has 9 coding exons and encodes a 346 amino acid protein, polymerase (RNA) I polypeptide C; the second protein forming the α-subunit of the DNA dependent RNA polymerases I and III. Compound heterozygous POLR1C mutations were identified in 3 of 252 TCOF1 negative TCS patients, consistent with autosomal recessive inheritance (Dauwerse et al 2011).

2 7b. Number of amplicons to provide this test 7c. MolU/Cyto band that this test is assigned to 8. Mutational spectrum for which you test including details of known common mutations 13 MolU band D 2012/13 MolU Band E 2013/14 Nonsense mutations, small insertions, deletions and splicing mutations resulting in premature termination of translation. Missense mutations also occur. Mutations found in both POLR1D and POLR1C are consistent with loss of function. We have developed an MLPA test to identify whole exon deletions and duplications. 9. Technical method(s) Bidirectional fluorescent sequencing and MLPA. 10. Validation process Please explain how this test has been validated for use in your laboratory 11a. Are you providing this test already? Yes 11b. If yes, how many reports have you produced? Bidirectional fluorescent sequencing is used by our laboratory for mutation scanning of several genes including the skeletal genes ROR2, GLI3, HOXD13, TCOF1 and FLNA. Prior to use all primers were checked for SNPs and 2 normal controls sequenced to confirm specific amplification. Confirmation of known mutations using controls was also carried out. MLPA was validated by checking all probe binding sites for SNPs and by testing both normal and deletion controls. 11c. Number of reports mutation positive 6 for POLR1D, 0 for POLR1C 11d. Number of reports mutation negative For how long have you been providing this service? 13a. Is there specialised local clinical/research expertise for this disease? 23 1 month Yes 13b. If yes, please provide details Professor Andrew Wilkie s research group at the Weatherall Institute of Molecular Medicine has worked on many of the skeletal and craniofacial genes. The Oxford craniofacial unit provides multi-disciplinary management of associated craniofacial problems. 14. Are you testing for other genes/diseases/conditions closely allied to this one? Please give details Your current activity If applicable - How many tests do you currently provide annually in your laboratory? Yes TCOF1 which is mutated in autosomal dominant TCS type1. We also provide a NSCT-funded service for other craniofacial syndromes with craniosynostosis, however Treacher Collins syndrome is not included in the NSCT funding. We test approximately 30 cases annually for TCOF1. Of these approximately 48% are found to be negative and would be candidates for POLR1D and POLR1C analysis. 15a. Index cases Approx 14 (based on current TCOF1 activity)

3 15b. Family members where mutation is known Your capacity if Gene Dossier approved How many tests will you be able to provide annually in your laboratory if this gene dossier is approved and recommended for NHS funding? Approx 4 (based on current TCOF1 activity and 20% pick-up rate) We would have the capacity to sequence at least all of our current TCOF1 negative cases with a TCS phenotype, as well as any additional cases with a clearly recessive inheritance pattern that have not been previously analysed. 16a. Index cases At least 14 16b. Family members where mutation is known Based on experience how many tests will be required nationally (UK wide) per annum? Please identify the information on which this is based At least 4 17a. Index cases Approx 14 17b. Family members where mutation is known 18. National activity (England, Scotland, Wales & Northern Ireland) If your laboratory is unable to provide the full national need please could you provide information on how the national requirement may be met. For example, are you aware of any other labs (UKGTN members or otherwise) offering this test to NHS patients on a local area basis only? This question has been included in order to gauge if there could be any issues in equity of access for NHS patients. It is appreciated that some laboratories may not be able to answer this question. If this is the case please write unknown. Approx 4 Our laboratory should be able to cover the National need we are not aware of any other UK laboratory that offers this test.

4 EPIDEMIOLOGY 19. Estimated prevalence of condition in the general UK population Please identify the information on which this is based 20. Estimated gene frequency (Carrier frequency or allele frequency) Please identify the information on which this is based 21. Estimated penetrance Please identify the information on which this is based 22. Estimated prevalence of condition in the target population. The target population is the group of people that meet the minimum criteria as listed in the Testing Criteria. INTENDED USE Assuming 15 new TCOF1 cases each year (52% of 30 cases tested), the prevalence of TCS1 is approximately 1 in 50,000. The prevalence of TCS2/TCS3 is approximately 1 in 500,000, with TCS2 accounting for the majority of these cases. Although our overall detection rate is 52% for TCOF1, this becomes 70% if we use data from clear clinically defined TCS cases only (Bowman et al 2012, Eur J Hum Genet; advance online publication 8 Feb 2012). POLR1D and POLR1C mutations account for at least a further 5% of these cases, with POLR1D accounting for the majority (Dauwerse et al 2011, our data). Nonpenetrance may occur with POLR1D giving a carrier frequency greater than 1 in 500,000. POLR1C has a carrier frequency less than 1 in 700. Non-penetrance has been described in 3 out of 5 families affected with TCS2 described thus far (Dauwerse et al 2011).Overall it is recognised that in Treacher Collins there is extremely variable penetrance. for TCOF1 variants which comprise the majority of cases. There is insufficient information to determine if this will also be the case for POLR1D and POLR1C mutations. From our data, of the suspected TCS cases negative for TCOF1, POLR1D mutations are expected in approximately 20%, and POLR1C mutations in less than 1%. Our data suggests that in clinically defined cases the pick-up rate may be as high as 25% in those cases that are TCOF1 negative. 23. Please tick the relevant clinical purpose of testing Diagnosis Yes No Treatment Yes No Prognosis & management Yes No Presymptomatic testing Yes No Carrier testing for family members Yes No Prenatal testing Yes No

5 TEST CHARACTERISTICS 24. 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. Sensitivity of combined sequencing & MLPA approach ~99%. Specificity >99% (no rare ambiguous single nucleotide polymorphisms have been documented that might lead to allele dropout). 25. Clinical sensitivity and specificity of test in target population The clinical sensitivity of a test is the probability of a positive test result when condition 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 condition (for specificity). Due to genetic heterogeneity and variable phenotype the clinical sensitivity is predicted to be about 20% in TCOF1 negative patients. Despite non-penetrance the clinical specificity is expected to approach 100% for POLR1D. 26. 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 condition or predisposition. It is measured by its positive predictive value (the probability of getting the condition given a positive test) and negative predictive value (the probability of not getting the condition given a negative test). Negative predictive values approach 100% if both sequencing and MLPA are undertaken. Positive predictive value will be less than 100% due to non-penetrance. Phenotypically normal individuals with a positive family history are tested as, if they are found to have the mutation, they may be at risk of having a more severely affected child (there is non-penetrance as well as phenotypic variability). As the TCS phenotype manifests during embryonic development, older individuals who are phenotypically normal are likely to remain so - therefore the positive predictive value will be less than 100%. 27. Testing pathway for tests where more than one gene is to be tested Please include your testing strategy if more than one gene will be tested and data on the expected proportions of positive results for each part of the process. Please illustrate this with a flow diagram. All TCS referrals will initially be tested for TCOF1 (positive in ~52% of all cases, Bowman et al 2012). If negative, then cases will be tested for both POLR1D and POLR1C. If there is a clear autosomal dominant inheritance pattern then only POLR1D will be tested. CLINICAL UTILITY 28. How will the test add to the management of the patient or alter clinical outcome? Main benefits of positive test result are confirmation of diagnosis, delineation of inheritance pattern in the extended family and detection of asymptomatic carriers. A confirmed diagnosis indicates a need for hearing assessment if not previously undertaken. Genetic counselling: enables recurrence risk to be provided to families. Prenatal testing would be available. Negative test result in the proband would indicate that further loci (currently unknown) could be tested. One of the main values of the test is for those people who carry the gene but do not show clinical signs but would benefit from testing to determine recurrence risks. 29. How will the availability of this test impact on patient and family life? No further molecular investigation required if mutation identified. Once diagnosis is confirmed accurate recurrence risks can be given and accurate and earlier prenatal diagnosis offered.

6 30. Benefits of the test Please provide a summary of the overall benefits of this test. Benefits of positive test result include confirmation of diagnosis, accurate assessment of recurrence risks, cascade screening of family members, earlier and more accurate prenatal diagnosis. Negative test result indicates that the diagnosis should be reconsidered and/or further loci tested. 31. 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. No other biochemical testing is available. Variation in expressivity can make clinical diagnosis challenging and molecular test can provide objective confirmation. 32. Please describe any specific ethical, legal or social issues with this particular test. Variability in clinical phenotype may cause a problem in interpreting prenatal results, but generally mutations within a family result in a similar disease severity. Carrier testing in minors may be an issue for POLR1C mutations. 33. The Testing Criteria must be completed where Testing Criteria are not already available. If Testing Criteria are available, do you agree with them Yes/No If No: Please propose alternative Testing Criteria AND please explain here the reasons for the changes. N/A 34. Savings or investment per annum in the diagnostic pathway based on national expected activity, cost of diagnostics avoided and cost of genetic test. Please show calculations. Cost of POLR1C and POLR1D analysis 510 Cost savings are for those that test positive as the following tests would no longer be required following a positive test result: acgh ( 500), acrofacial dysostoses such as Nager and Miller syndrome ( 1000), oculoauriculovertebral spectrum eg hemifacial microsomia, Goldenhar syndrome ( 400), karyotype ( 4180) Therefore each positive case could result in cost savings of = 1570 Estimated 3 positive cases per year indicating potential annual cost savings of List the diagnostic tests/procedures that would no longer be required with costs. These would no longer be required after a positive result: Costs and type of imaging procedures Costs and types of laboratory pathology tests (other than molecular/cyto genetic proposed in this gene dossier) Karyotype Array CGH Miller syndrome DHODH gene Goldenhar syndrome SALL1 gene Costs and types of physiological tests (e.g. ECG) Cost and types of other investigations/procedures (e.g. biopsy) Total cost tests/procedures no longer required after a positive result: 2080

7 36. REAL LIFE CASE STUDY In collaboration with the clinical lead, describe a real case example to illustrate how the test would improve patient experience. This female proband has a clinical diagnosis of Treacher Collins syndrome, presenting with the classical features of malar hypoplasia, lower eyelid coloboma, mandibular hypoplasia, macrostomia, malformation of the auricle and ear tags. The proband presented to our laboratory for testing at the age of 23 years. She had had 3 miscarriages and was pregnant for a fourth time. The hope was that a mutation would be identified in the TCOF1 gene allowing prenatal diagnosis to be carried out. Chromosome analysis had been carried out previously and was normal. Unfortunately no mutation was identified in the TCOF1 gene, and so we were unable to offer prenatal testing by molecular analysis. Subsequent analysis of the POLR1D and POLR1C genes identified a mutation in the POLR1D gene, confirming a diagnosis of TCS2. There are no cost savings in this example, however, prenatal diagnosis is now available for future pregnancies of this lady. 37. For the case example, if there are cost savings, please provide these below: PRE GENETIC TEST Costs and type of imaging procedures Costs and type of laboratory pathology tests (other than molecular/cyto genetic proposed in this gene dossier) Costs and type of physiological tests (e.g. ECG) Cost and type of other investigations/procedures (e.g. biopsy) Cost outpatient consultations (genetics and non genetics) Total cost pre genetic test POST GENETIC TEST Costs and type of imaging procedures Costs and types laboratory pathology tests (other than molecular/cyto genetic proposed in this gene dossier) Cost of genetic test proposing in this gene dossier Costs and type of physiological tests (e.g. ECG) Cost and type of other investigations/procedures (e.g. biopsy) Cost outpatient consultations (genetics and non genetics) Total cost post genetic test 38. Estimated savings for case example described

8 UKGTN Testing Criteria Approved name and symbol of disease/condition(s): Treacher Collins Syndrome 2; TCS2 Treacher Collins Syndrome 3; TCS3 Approved name and symbol of gene(s): polymerase (RNA) I polypeptide D, 16kDa; POLR1D polymerase (RNA) I polypeptide C, 30kDa; POLR1C Patient name: Patient postcode: OMIM number(s): (TCS2) (TCS3) OMIM number(s): (POLR1D) (POLR1C) Date of birth: NHS number: Name of referrer: Title/Position: Referrals will only be accepted from one of the following: Referrer Consultant Geneticist Lab ID: Tick if this refers to you. Minimum criteria required for testing to be appropriate as stated in the Gene Dossier: Criteria Tick if this patient meets criteria Individuals suspected of having Treacher-Collins syndrome, preferably after assessment by an experienced dysmorphologist. Individuals should fulfil the following criteria: 1. Have TWO of the following major clinical features: - Hypoplasia of the zygomatic bones and mandible - Absent, small or malformed ears, or rotated ears - lower eyelid abnormality - family history consistent with autosomal dominant inheritance 2. OR have THREE of the following minor clinical features - atresia or stenosis of the external auditory canals - conductive hearing loss - ophthalmologic defect - cleft palate with or without cleft lip - preauricular hair displacement - airway abnormality - delayed motor or speech development - family history consistent with recessive inheritance OR At risk family members where familial mutation is known 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