If you wanted to introduce a new service what factors would you consider? Ruth E Sutton

1. If you wanted to introduce a new service what factors would you consider? Ruth E Sutton Laboratory Management 2007

2. Hitting government targets Clinical utility Use of other disciplines Robotics Publications and Research. Setting up a new diagnostic service Quality assurance Screening method Funding and staffing

3. Clinical Perspective: Is there a local need for the service? Talk to clinical groups discuss priorities. Make use of local expertise. More widespread need for the service? Any other centres already up and running. Is there a need for a clinical questionnaire to reduce inappropriate referrals. Important for a disorder which has an overlapping spectrum of symptoms resulting in differential diagnoses. (ie neural tube defects) Clinical sensitivity: does a gene or set of genes account for a high % of the mutation spectrum for the disorder in question? (Marfan syndrome FBN1 ~100% if follow appropriate clinical guidelines). Potential pick up rate. Clinical specificity: probability of a negative result when a disease is not present. Any statistics available regarding prevalence? High/low What would be the clinical outcome of such a genetic diagnosis? improve family planning, more directed treatment or cure, early treatment increases survival ie CF.

4. Research What is the basis for the service? What is known about the disorder and the genes involved. Is there a comprehensive piece of research linking mutation of a gene or genes to the disorder in question, maybe a local research group. Is the inheritance (monogenic/polygenic/heterogeneous nature) of the disorder known. Local research interest often means access to positive samples to validate any new service. The gene or genes involved: Conserved domains, important functions. Alu repeats? Prone to deletion and duplication and therefore need for dosage assay such as MLPA. Does it have homology to others? Need for nested long range PCR ie SMN1/2.

5. Funding and Staffing: Gene dossier written to present to the UKGTN. Offer the service nationally and charge a competitive rate. If service introduced is there enough staff to cover, if not will the service cover the cost of additional staffing. Other Disciplines Can other disciplines be used to direct genetic testing ie biochemistry, histochemistry used in mitochondrial genetic diagnosis and LGMD service. Will a biochemical result suffice, often genetic testing offers further advantages such as carrier testing, prenatal diagnosis, pre-symptomatic testing.

6. Choice of screening method: Efficiency What equipment is available in the laboratory already? Validated for others services. What other services are provided in the laboratory? Can this new service be integrated in order to save money, make more efficient use of equipment and staff time. For example standardisation of PCR conditions across a number of services, use of N13 tagged sequencing. Smaller gene screens could be batched together. Direct sequencing vs pre- screening techniques: How big is the gene, or genes? % GC content, difficult melting profile can hinder pre-screening such as lightscanner techniques. High number of exons not suitable then this tilts balance in favour of direct sequencing. SNP content of the gene, if high this can restrict use of pre-screening methods.

7. High number of referrals may be more amenable to a pre-screening method. Could you be missing something by using a pre-screening technique. Direct sequencing could be more appropriate for smaller genes. Draw backs can be the need to report numerous unclassified variants. Validation process is important: Assessing false negative/false positive rates. Use of a blind study to determine sensitivity of the test. Specificity: the test should give a negative result when a mutation is not present. Reproducibility- failure rate should be low in order to keep costs low and reduce turnaround times. When service is up and running, each run should include appropriate controls.

8. Diagnostic strategy: Common mutation analysis: Use of lighcycler probes, RFLP assay, direct sequencing, Fluorescent ABI assay to detect fragment size reduction or increase. Dosage analysis such as MLPA (if appropriate). If an in-house method is designed is it appropriate to include additional genes for other disorders? Mutational hotspot regions: for example the CFH gene, exons 18-23. Regions of the gene involved in important protein domains, conserved across species. Interim report, if negative offer full screening at an additional cost. Full screen: covering remaining exons and maybe non-coding promoter regions, UTRs. Cost: set up and ongoing Reagents and staffing costs should be considered. High referral number with low pick up – cheap test but with no loss of test integrity. Low referral rate – cost of more expensive test can be absorbed.

9. Government Targets: Need to consider most efficient use of plates by controlling number of patients per panel or adding more than one gene to a plate. Use of reflex testing to increase testing period? Batching of samples for a disorder or with other disorders with similar diagnostic strategies. Use of robotics: When do they become useful? Replication of panels across a plate, minimise plating errors and enable other work to be carried out in the mean time. PCR and sequencing clean ups. Tracking of samples for audit purposes. Draw backs: Reproducibility, handling of small sample quantity, and not useful in small gene screens.

10. Ongoing QA issues SNP checks for primer sequences. Running positive and negative control on each run. Use of plates: sample mix up? Cherry picking can be difficult for large panels. Choose an assay technique involving minimal tube transfers. If confirming research findings, ask for fresh samples. Common analysis standards: Use of DD/DQ fragments in synthetic MLPA kits to determine quality and correct sample amount. Use of standard deviation values in the analysis. Appropriate sequencing quality scores. Default setting for in silico programmes, need for standardisation in the use of these types of programmes. Lack of functional studies is always a problem, prediction software can be difficult to interpret correctly.