Proposal form for the evaluation of a genetic test for NHS Service Gene Dossier Test Disease Population Triad Disease name Genetic Causes of Hypothyroidism 1. Loss of function mutations in TSHR cause thyroid stimulating hormone (TSH, thyrotropin) receptor resistance. When heterozygous this most commonly causes subclinical hypothyroidism. 2. Individuals with two total loss-of-function mutations present with congenital hypothyroidism. 3. Congenital hyperthyrotropinaemia has also been reported, and is due to the presence of TSHR with decreased functional activity. OMIM number for disease #275200 Disease alternative names please provide any alternative names you wish listed Disease please provide a brief description of the disease characteristics Sub-clinical hypothyroidism Congenital hypothyroidism Congenital hyperthyrotropinaemia Subclinical hypothyroidism is a biochemical diagnosis, defined as an increased serum TSH when freet4 is within reference limits. The commonest causes are usually inadequately treated hypothyroidism and autoimmune hypothyroidism or Haashimotos disease. Subclinical hypothyroidism should be confirmed by repeat biochemical tests after 3-6 months. TSHR loss-of-function mutations have also been associated with non-autoimmune subclinical hypothyroidism. TSHR gene sequencing to identify loss of function mutations may be of use in the investigation of selected patients: 1. Patients with persistently increased serum TSH concentrations in whom autoimmune thyroid disease has been excluded who have a family history of increased serum TSH in first degree relatives. 2. Children or infants with congenital hyperthyrotropinaemia, with normally sited thyroid glands (small/normal size) who are clinically euthyroid with increased serum TSH, but normal free thyroxine concentrations. These patients may not respond well to thyroxine therapy. Becoming clinically hyperthyroid when sufficient thyroxine to bring TSH down into the reference range is given. Disease - mode of inheritance Autosomal dominant subclinical hypothyroidism / congenital hyperthyrotropinaemia Autosomal recessive congenital hypothyroidism Gene name(s) TSHR OMIM number for gene(s) 603372 Gene alternative names Thyrotropin receptor gene please provide any alternative names you wish listed 1
Gene description(s) (including number of amplicons). Mutational spectrum for which you test including details of known common mutations. Technical Method (s) Validation Process Note: please explain how this test has been validated for use in your laboratory 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 The TSHR gene has a chromosomal location of 14q31 encompassing a 190-kb gene arranged in 10 exons, This encodes a 764 amino acid G protein coupled receptor with a predicted molecular weight of 86.83 kda. The gene is amplified in 10 amplicons. Those patients with subclinical hypothyroidism are initially screened for the p.trp546x variant. Complete gene sequencing can be undertaken if required. Complete sequencing will identify missense, nonsense, splice and small deletions/insertions Denaturing HPLC and/or sequencing SNP analysis of primers Optimisation of PCRs Sequencing of normal and affected individuals with comparison to a reference sequence Sequencing is used for mutation screening in the laboratory for a number of genes Participation in EQA (EMQN) for sequencing Yes If Yes: Number of reports issued:36 Number of reports mutation positive:10 Number of reports mutation negative:26 3 years Yes Please provide details There are strong links with Dr Marian Ludgate, Reader, at Cardiff University School of Medicine who has an extensive research track record in this field. Local expertise is available from Dr Carol Evans, Consultant Clinical Scientist and Head of the Regional Endocrine Laboratory and Director of the SAS Endocrine Service in the Department of Medical Biochemistry & Immunology at the University Hospital of Wales. The Regional Endocrine Laboratory and SAS Endocrine Service provides a comprehensive endocrine service and includes an advisory function performed and co-ordinated by the Director, supported by Clinical Associates who are expert endocrinologists. Consultant paediatricians Prof John Gregory and Dr Justin Warner are Clinical Associates with experience of management of thyroid dysfunction in childhood. We offer a service for the molecular characterisation of hyperthyroidism which includes investigation of the TSHR for gain of function mutations associated with gestational hyperthyroidism and non-autoimmune hyperthyroidism. The Regional Endocrine Laboratory and SAS Endocrine Service provides comprehensive laboratory support for the investigation of thyroid disease including provision of thyroid function tests 2
(freet4, TSH, FreeT3 and thyroid peroxidise antibodies) and specialist thyroid tests (thyroglobulin, thyroglobulin antibodies and TSHR antibodies (TRAb)) Your Current Activity If applicable - How many tests do you currently provide annually in your laboratory? 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? Based on experience how many tests will be required nationally (UK wide)? Please identify the information on which this is based The Department of Medical Biochemistry & Immunology houses the All Wales Hypothyroid Screening Laboratory. Exon 10+9: 1 Exon 10: 1 p.trp546x variant: 10 Index cases:10 (If full gene sequencing were required) Index cases: 10 (for exons 9 and 10) Index cases for the common p.trp546x variant: 30 Family members where mutation is known:30 It is not possible to estimate the number of tests required nationally as the prevalence of the disorder is not known. 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. 3
Epidemiology Estimated prevalence of disease in the general UK population Please identify the information on which this is based Estimated gene frequency (Carrier frequency or allele frequency) The prevalence is not known. Studies of subclinical hypothyroidism in the US report a prevalence of 4 to 10% of the adult population but patients with anti TPO antibodies were not excluded. Surks MI (2004) Subclinical thyroid disease JAMA 291:228-238 From our own results a single mutation was identified in 10 of 32 patients with subclinical hypothyroidism. Please identify the information on which this is based Estimated penetrance Please identify the information on which this is based Target Population Description of the population to which this test will apply (i.e. description of the population as defined by the minimum criteria listed in the testing criteria) Estimated prevalence of disease in the target population Loss of function mutations in the TSHR gene have shown variable expression among family members making penetrance difficult to estimate. Camilot M (2005) Thyrotropin receptor gene mutations and TSH resistance: variable expressivity in the heterozygotes Clin Endocrinol 63:146-151 Population 1: Non autoimmune subclinical hypothyroidism Patients with persistently increased serum TSH concentrations in whom autoimmune thyroid disease has been excluded who have a family history of increased serum TSH in first degree relatives. Population 2: Congenital hyperthyrotropinaemia Children or infants with congenital hyperthyrotropinaemia, with normally sited thyroid glands (small/normal size) who are clinically euthyroid with increased serum TSH, but normal free thyroxine concentrations. These are infants identified by new born screening that may not respond well to thyroxine treatment. The prevalence is not known. From our own results a single mutation was identified in 10 of 32 patients referred according to the above criteria for population 1. Intended Use (Please use the questions in Annex A to inform your answers) Please tick the relevant clinical purpose of testing YES NO Diagnosis Treatment Prognosis & Management Presymptomatic testing Risk Assessment for family members Risk Assessment prenatal testing 4
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. 32 patients with subclinical hypothyroidism were analysed for the common p.trp546x mutation. This mutation was identified in 10 patients. From these figures the sensitivity was 26% of those patients with a clinical diagnosis of subclinical hypothyroidism. Of a group of 4 patients who requested one or more exons of the TSHR gene sequenced no mutations were identified. 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) 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). Testing pathway 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. This can be added to the document as a separate sheet if necessary. The specificity of fluorescent sequencing analysis is greater than 99% The clinical sensitivity ie patients with subclinical hypothyroidism and have a mutation in the TSHR gene is estimated to be 11%. Camilot M (2005) Thyrotropin receptor gene mutations and TSH resistance: variable expressivity in the heterozygotes Clin Endocrinol 63:146-151 The clinical specificity is approximately 100% The disease phenotype is variable. The risk of family members in whom a familial mutation has been excluded developing subclinical hypothyroidism is significantly reduced although the possibility of sporadic mutations cannot be excluded. Only one gene is to be tested however the testing strategy is given below. A step-wise approach to screening the TSHR gene will be taken. In the first instance samples will be screened for the W546X mutation which is relatively common in the Welsh population. If this is not present, the clinician can request analysis for the remaining TSHR exons. Sequencing of the entire gene can be arranged if required. 5
Clinical utility of test in target population (Please refer to Appendix A) Please provide a description of the clinical care pathway. TSHR gene sequencing to identify loss of function mutations may be of use in the investigation of selected patients: 1. Patients with persistent subclinical hypothyroidism (slightly increased serum TSH concentrations with FreeT4 within the normal range). In whom autoimmune thyroid disease has been excluded and who have a family history of increased serum TSH in first degree relatives. Locantore P, Evans C, Zhang L, Warner J, Gregory J W, John R, Lazarus J H, Ludgate M. (2007) W546X mutation of the thyrotropin receptor, causes subclinical hypothyroidism in various clinical settings. Clinical Endocrinology. 67: 317. Jordan N, Williams N, Gregory JW, Evans C, Owen M and Ludgate M. (2003). The W546X Mutation of the Thyrotropin Receptor Gene: Potential Major Contributor to Thyroid Dysfunction in a Caucasian Population. Journal of Clinical Endocrinology and Metabolism. J Clin Endocrinol Metab. 88 1002-1005. 2. Children or infants with congenital hyperthyrotropinaemia, with normally sited thyroid glands (small/normal size) who are clinically euthyroid with increased serum TSH, but normal free thyroxine concentrations. Careful management of these patients is required. They may not respond well to thyroxine therapy. Although some thyroxine treatment to achieve some degree of TSH suppression may be desirable to prevent pituitary hyperplasia. 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? Please provide evidence from your own service. Clifton-Bligh RJ, Gregory JW, Ludgate M, John R, Persani C, Asteria P, Beck-Peccoz P & Chatterjee VKK. Two Novel Mutations in the Thyrotropin (TSH) receptor Gene in a Child with Resistance to TSH. JCEM. 1997 82, 1094-1100. Confirmation of the cause of subclinical hypothyroidism will aid management decisions. Follow up with repeated thyroid function tests will not be required in patient with a heterozygous loss of function mutation in the TSHR. Genetic analysis can confirm congenital hyperthyrotropinaemia, a defect due to the presence of TSHR with decreased functional activity. These patients require careful management. Some long term suppression of TSH secretion is desirable without rendering the patient clinically thyrotoxic. Patients with mild subclinical hypothyroidism due to TSHR loss-offunction mutations do not require thyroxine and do not require long term monitoring of their thyroid function. Children or infants with congenital hyperthyrotropinaemia may be to be identified as hypothyroid by newborn screening. They may become clinically hyperthyroid when given enough thyroxine to bring their TSH concentration into the normal range. The thyroid function profile in these infants or children may be consistent with poor compliance with thyroxine treatment (Increased TSH with FreeT4 within the reference range). The genetic test confirms the cause. 6
What are the consequences of not doing this genetic test. Commissioners have asked for specific information to support introduction of tests. Utility of test in the NHS In a couple of sentences explain the utility of this test for the disease(s) 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 Please describe any specific ethical, legal or social issues with this particular test? Patients with congenital hyperthyrotropinaemia require careful management. The patients are likely to be suspected of poor compliance with thyroxine therapy unless an alternative cause for their thyroid function test results is found. To confirm the cause of hyperthyrotropinaemia to ensure appropriate management of patients. No None known 7
UKGTN Testing criteria Name of Disease(s): HYPOTHYROIDISM, CONGENITAL, NONGOITROUS, 1; CHNG1 (275200) Name of gene(s): thyroid stimulating hormone receptor; TSHR (603372) 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 Consultant Paediatrician Consultant Clinical Geneticist Consultant Endocrinologist Tick if this refers to you. Minimum criteria required for testing to be appropriate as stated in the Gene Dossier: Criteria Clinically euthyroid children with increased serum TSH, but normal free thyroxine concentrations OR Infants identified with congenital hypothyroidism by newborn screening who respond poorly to thyroxine treatment. Patients with persistently increased serum TSH concentrations in whom autoimmune thyroid disease has been excluded AND who have a family history of increased serum TSH in first degree relatives Tick if this patient meets criteria 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. 8