Newcastle Mitochondrial NSCT lab User Manual 2013
Contents
1. Mitochondrial Research 4 Floor Cookson Building The Medical School Newcastle University Framlington Place Newcastle upon Tyne NE2 4HH Blood or any fluid samples should be in an appropriate container wrapped in absorbent material and placed inside a sturdy plastic container e g biobottle for transport Page 4 of 11 Date of original issue March 2007 Revised version April 2013 NEWCASTLE MITOCHONDRIAL NSCT DIAGNOSTIC LABORATORY Rare Mitochondrial Disorders Service for Adults and Children MUSCLE BIOPSY DISPATCH INSTRUCTIONS NOTE We are not able to accept samples designated or suspected as being High Risk i e from patients with HIV hepatitis CJD polio etc 1 Please inform the Service Administrator or laboratory of your intention to send a sample on dry ice before it leaves your laboratory either by phone or by e mail Please note we cannot accept responsibility for the safety of samples sent without the prior knowledge of our unit 2 When you send the samples please include a covering letter with the names and addresses of any additional people who will require copies of letters or reports More clinical information is always helpful in addition to that provided on the referral form Please send a copy of the muscle histopathology report if available 3 The muscle sample must be sent in dry ice using a recognised courier TNT or FedEx for door to door delivery A next day before 12 noon delivery2. chain complexes I II II and IV individually and compare these activities to the activity of the mitochondrial matrix marker enzyme citrate synthase an indicator of mitochondrial mass within the sample Page 3 of 11 Date of original issue March 2007 Revised version April 2013 NEWCASTLE MITOCHONDRIAL NSCT DIAGNOSTIC LABORATORY Rare Mitochondrial Disorders Service for Adults and Children An unfixed snap frozen muscle sample is required for these investigations preferably unmounted and free from OCT and cork Ideally between 100 150 mg muscle tissue is preferred to be able to perform these assays with 60 mg tissue the absolute minimum amount of sample we will accept Molecular genetic analysis for suspected mitochondrial disorders The laboratory offers a range of tests for both mtDNA mutations ranging from point mutation analysis to whole mitochondrial genome sequencing and the analysis of several nuclear genes involved in mtDNA maintenance For many common mtDNA point mutations genetic screening can be undertaken using blood DNA although the use of alternative sources such as urinary cell sediment DNA samples are recommended for some mutations in particular the m 3243A gt G mutation For the study of possible mtDNA rearrangements and whole mitochondrial genome muscle DNA is essential Samples for genetic analysis Muscle If a muscle biopsy is already being referred for either histochemical or biochemical assessment we are able to
3. D mtDNA rearrangement disorders Kearns Sayre Pearsons syndrome Mitochondrial deafness mutations MTRNRI1 12S rRNA gene including m 1555A gt G mutation and MTTS tRNAS UCN gene MTND gene sequencing patients with isolated complex I deficiency or LHON patients in whom common mutations previously excluded MTCYB gene sequencing patients with isolated complex III deficiency Mitochondrial genome sequencing Screen for rare or novel pathogenic mtDNA mutations Single fibre real time PCR Quantification and screening of mtDNA rearrangements in individual muscle fibres COX deficient and COX positive by real time PCR mtDNA depletion syndromes Assessment of mtDNA copy number to screen for possible mtDNA depletion syndromes POLGI mutation screening POLG mutations cause a phenotypic spectrum ranging from severe encephalopathy and liver failure typical of Alpers syndrome to late onset PEO ataxia myopathy and epilepsy associated with multiple mtDNA deletions and depletion PEO 1 Twinkle Patients with dominant recessive PEO and multiple mtDNA deletions Page 7 of 11 TYPE OF TEST SAMPLE NEEDED Long range PCR and or Southern CPEO blotting of affected tissues to investigate single mtDNA and secondary multiple mtDNA deletions Muscle DNA essential blood DNA for children with Pearsons syndrome Sequencing of both genes Blood DNA samples appropriate for this analysis Sequencing of all mitochondrial e
4. NEWCASTLE MITOCHONDRIAL NSCT DIAGNOSTIC LABORATORY Rare Mitochondrial Disorders Service for Adults and Children i x CPA S Accredited Medical Laboratory Reference No 3118 The Newcastle upon Tyne Hospitals NHS NHS Foundation Trust Newcastle Mitochondrial NSCT Diagnostic Service Wellcome Trust Centre for Mitochondrial Research 4 Floor Cookson Building The Medical School Newcastle University Framlington Place Newcastle upon Tyne NE2 4HH Telephone 0191 2824375 Fax 0191 2824373 www mitochondrialncg nhs uk USER MANUAL for General Laboratory Information Location Service Hours Head of Laboratory Clinical Service Adults Clinical Service Children Clinical Scientists Biomedical Scientists Genetic Technologist Main laboratory Room M4012 Medical School Main Office Room M4028 Medical School Monday Friday 08 00 to 17 00 Professor Rob Taylor Tel 0191 2223685 direct line E mail r w taylor ncl ac uk or robert taylor5 nuth nhs uk or robert taylor10 nhs net Professor Doug Turnbull Tel 0191 2824375 E mail doug turnbull nuth nhs uk or doug turnbull nhs net Dr Andrew Schaefer Tel 0191 2824375 E mail andrew schaefer nuth nhs uk or andrew schaefer nhs net Dr Grainne Gorman Tel 0191 2824375 E mail grainne gorman ncl ac uk or grainne gorman nhs net Dr Robert McFarland Tel 0191 2824375 E mail robert mcfarland ncl ac uk or robert mcfar
5. d Scotland Rare Mitochondrial Disorders Service for Adults and Children This multi disciplinary service will encompass all aspects of diagnosis muscle biopsy histochemistry biochemistry and molecular genetics utilising specialist clinical and laboratory skills available at the three component centres and complementing the routine analysis of common mtDNA mutations that is available throughout the UK at many regional genetics laboratories For many patients with suspected mitochondrial disease it is not possible to make a diagnosis based solely on molecular genetic testing in blood DNA and so for those patients where more detailed investigations are required a comprehensive diagnostic service is now offered which combines clinical investigations histochemical and histological analysis of patient muscle biopsies measurement of respiratory chain complex activities together with screening of both mtDNA and nuclear encoded mitochondrial genes Services offered by the Newcastle Mitochondrial NSCT Laboratory CLINICAL SERVICES Outpatient Consultations are available for patients and families with suspected or proven mitochondrial disease Clinical examination may confirm the suspicion of mitochondrial disease or suggest an alternative diagnosis Clear plans of investigation or patient management will be established during the clinic appointment with close collaboration between the referring consultant and the patients own general practit
6. equencing of the entire coding region Blood DNA samples appropriate and required Sequencing of the entire coding regions Blood DNA samples appropriate and Sequencing of the entire coding region Blood DNA samples appropriate and required Sequencing of the entire coding regions Blood DNA samples appropriate and required Sequencing of the entire AGK coding region Blood DNA samples appropriate and required Sequencing of the entire NFU coding region Blood DNA samples appropriate and required Sequencing of the entire MTO coding region Blood DNA samples appropriate and required Targeted Haloplex Panels for Nuclear complex I structural genes and mtDNA maintenance genes Please contact Professor Taylor to discuss CVB and prenatal testing for nuclear and mtDNA mutations PGD for specific mtDNA mutations Page 9 of 11 Please contact Professor Taylor to discuss individual cases please note that there is a charge applicable for this testing as this is not covered under our NSCT agreement Please contact Professor Taylor and Dr McFarland to discuss individual cases please note that there is a charge applicable for this testing as this is not covered under our NSCT agreement Date of original issue March 2007 Revised version April 2013 NEWCASTLE MITOCHONDRIAL NSCT DIAGNOSTIC LABORATORY Rare Mitochondrial Disorders Service for Adults and Children Sample Reporting Times Enzyme histochemistry With
7. es appropriate and required Sequencing of the entire RARS2 coding region Blood DNA samples appropriate and required Sequencing of the entire AARS2 coding region Blood DNA samples appropriate and required Sequencing of the entire coding region of nine structural genes Blood DNA samples appropriate and required Date of original issue March 2007 Revised version April 2013 NEWCASTLE MITOCHONDRIAL NSCT DIAGNOSTIC LABORATORY Rare Mitochondrial Disorders Service for Adults and Children ACAD9 complex I assembly factor Patients with isolated complex I deficiency often associated with cardiac phenotype Complex II structural and assembly genes Patients with evidence of isolated complex II deficiency Sequencing of the four structural genes SDHA SDHB SDHC SDHD and known assembly factors SDHAF and SDHAF2 Nuclear complex III genes Patients with isolated complex III deficiency are screened for mutations in the BCS L gene a known complex II assembly factor TMEM70 ATPAF2 and ATPS5E Patients with suspected complex V deficiency AGK Patients with multiple respiratory chain complex deficiencies and Sengers syndrome cataracts HCM and elevated lactate NFUI Patients with multiple respiratory chain complex deficiencies complex I II and III FeS cluster scaffold gene defect MTOI Patients with multiple respiratory chain complex Deficiencies hypertrophic cardiomyopathy and and elevated lactate S
8. extract DNA either directly from cryostat cut sections or from a residual nuclear pellet generated during the preparation of an enriched mitochondrial fraction for enzyme studies The details for dispatch of muscle biopsy samples on dry ice are given later in this manual Blood Extracted DNA or 2 3 x 4 5ml EDTA blood purple top tubes should be sent for analysis please use EDTA as anticoagulant rather than heparin which precludes molecular genetic testing Mix the blood well after taking but do not freeze these samples can be stored at 4 C for 1 2 days if required without problems and should be shipped at room temperature Urine Urine samples 40 50ml should be collected into sterile universal or falcon type tubes and forwarded to the lab at room temperature do not freeze To ensure the best possible yield of epithelial cells from these samples an early morning urine sample first pass is preferable Other tissues Other tissue samples can be accepted for DNA analysis including pathology blocks cultured cells buccal mouth scrapes and hair samples The investigation of several non invasive DNA samples can be particularly useful in determining the pathogenic behaviour of specific or novel mtDNA mutations Please contact the laboratory for specific instructions as to what we require and how to send these samples Please forward the samples to Professor Rob Taylor Newcastle Mitochondrial NSCT Laboratory Wellcome Trust Centre for
9. ians wishing to refer patients for a clinical opinion either as outpatients or inpatients please write to Professor Turnbull adult referrals or Dr McFarland paediatric referrals at the following address Newcastle Mitochondrial NSCT Laboratory Wellcome Trust Centre for Mitochondrial Research 4 Floor Cookson Building The Medical School Newcastle University Framlington Place Newcastle upon Tyne NE2 4HH Tel 0191 2824375 Fax 0191 2824373 E mail jane brown nuth nhs uk or jane brown38 nhs net LABORATORY SERVICES Histological and Histochemical analysis of muscle biopsy samples An unfixed frozen muscle sample in transverse orientation measuring approximately 3mm x 3mm x 3mm 25 mg is the minimum required for mitochondrial histochemical analysis which will include cytochrome c oxidase COX staining succinate dehydrogenase SDH staining and sequential COX SDH assays to thoroughly investigate the possibility of low levels of COX deficient fibre As appropriate in specific cases other tissues are accepted for the investigation of mitochondrial histochemical activities including heart e g suspected mitochondrial cardiomyopathies and liver e g Alpers Syndrome Details for the dispatch of frozen tissues are given later in this manual Respiratory chain enzyme measurements in muscle biopsy samples To fully assess mitochondrial respiratory chain activity in frozen tissue samples we assay the activities of respiratory
10. idase COX activity Sequential COX SDH activities Modified Gomori trichrome staining Succinate dehydrogenase SDH activity MITOCHONDRIAL RESPIRATORY CHAIN ANALYSES Each frozen sample is assessed for the following Citrate Synthase MOLECULAR GENETIC ANALYSES MUTATION DISORDER TO BE TESTED m 3243A gt G point mutation MELAS syndrome maternally inherited diabetes and deafness MIDD m 8344A gt G m 8356T gt C and m 8363G gt A mutations MERRF syndrome m 8993T gt G C and m 9176T gt G C mutations NARP and MILS maternally inherited Leigh Syndrome LHON mutations Page 6 of 11 Complex I NADH ubiquinone oxidoreductase activity Complex II Succinate ubiquinone oxidoreductase activity Complex III Ubiquinol cytochrome c oxidoreductase activity Complex IV Cytochrome c oxidase activity TYPE OF TEST SAMPLE NEEDED Pyrosequencing to allow estimation of mtDNA heteroplasmy Blood and or urine DNA muscle if available sequencing of entire MTTK tRNA gene for known pathogenic mutations Blood DNA or muscle if available sequencing of the mitochondrial MTATP6 and MTATPS genes Blood DNA appropriate screening of 3 primary LHON mutations m 3460G gt A m 11778G gt A and m 14484T gt C using blood DNA Date of original issue March 2007 Revised version April 2013 NEWCASTLE MITOCHONDRIAL NSCT DIAGNOSTIC LABORATORY Rare Mitochondrial Disorders Service for Adults and Children MUTATION DISORDER TO BE TESTE
11. in 2 weeks Respiratory chain enzyme analysis Within 4 6 weeks please contact the laboratory if urgent analysis is required Urgent molecular diagnostic tests Within 7 working days PCR based CVB analysis 24 48 hour turnaround Non urgent samples Within 4 8 weeks Mitochondrial genome sequencing 8 10 weeks POLGI sequencing common mutations screened within 4 weeks Whole gene 8 12 weeks Nuclear maintenance gene analysis If relevant these will be assessed sequentially with the first gene reported within an 8 week timeframe Nuclear complex I gene analysis 8 12 weeks for full panel of nine genes Other nuclear genes Within 4 8 weeks External Quality Control We participate in the following external quality assessment schemes UKNEQAS for Molecular Genetics Mitochondrial Disorders European Molecular Genetics Quality Network Molecular Genetic Sequencing Scheme UKNEQAS for Cytopathology Technique There are currently no external schemes available in the UK for mitochondrial respiratory chain enzyme analysis but we have developed an annual sample exchange programme with other NCG service laboratories including Professor Simon Heales Dr Iain Hargreaves lab at UCLH and Dr Garry Brown s lab at Oxford Page 10 of 11 Date of original issue March 2007 Revised version April 2013 NEWCASTLE MITOCHONDRIAL NSCT DIAGNOSTIC LABORATORY Rare Mitochondrial Disorders Service for Adults and Children User Satisfaction Survey for Laboratory Se
12. ioner Whenever possible follow up will be at local hospitals although some patients with particularly rare conditions or where local services are limited may require longer term follow up in Newcastle Consultations are also available for women with proven mtDNA disease or who are carriers of pathogenic mtDNA mutations and who require advice concerning the possible transmission of the mtDNA mutation to their children Patients or families in which mitochondrial respiratory chain Page 2 of 11 Date of original issue March 2007 Revised version April 2013 NEWCASTLE MITOCHONDRIAL NSCT DIAGNOSTIC LABORATORY Rare Mitochondrial Disorders Service for Adults and Children disease is confirmed but in whom the genetic diagnosis has not been established or with mutations in known nuclear genes also have access to this service Inpatient Day case and inpatient services are available for patients who have to travel for specific diagnostic investigations and are available for both children and adults Muscle biopsies are not always available in certain hospitals and these can be arranged to be performed in Newcastle as part of the diagnostic work up Nurse Specialist Catherine Feeney Tel 0191 2821740 E mail catherine feeney nuth nhs uk or catherine feeney nhs net Secretaries Sue Callender sue callender nuth nhs uk or susan callender nhs net Bernadette Caygill bernadette caygill nuth nhs uk or bernadette caygill nhs net For clinic
13. land nuth nhs uk or r mcfarland nhs net Dr Emma Watson Tel 0191 2228877 Dr Langping He Tel 0191 2228877 Mrs Charlotte Alston Tel 0191 2228877 Dr Steven Hardy Tel 0191 2228877 Miss Jessica Gabriel Tel 0191 2228877 Mr Gavin Falkous Tel 0191 2228877 Mrs Angela Baker Tel 0191 2228877 Dr Kate Hickman Tel 0191 2228877 Mrs Karen Baty Tel 0191 2228877 Medical Laboratory Assistant Mr Aaron Potts Tel 0191 2228877 Quality Manager Northern Genetics Service Service Administrator Page 1 of 11 Mr Alan Smith Tel 0191 2418715 E mail alan smith nuth nhs uk Mrs Jane Brown Tel 0191 2824375 E mail jane brown nuth nhs uk or jane brown38 nhs net Date of original issue March 2007 Revised version April 2013 NEWCASTLE MITOCHONDRIAL NSCT DIAGNOSTIC LABORATORY Rare Mitochondrial Disorders Service for Adults and Children Background Information to the Service Mitochondria are ubiquitous organelles that contain their own genetic complement the mitochondrial genome mtDNA Although intimately involved in many cellular processes their principal task is to provide the energy necessary for normal cell functioning and maintenance Disruption of this energy supply can have devastating effects for the cell organ and individual One important consequence of mitochondrial ubiquity is that mitochondrial disease can affect virtually any organ and present with a plethora of symptoms and signs to a varie
14. ncoded complex I genes DNA from affected tissue e g muscle essential in cases of complex I deficiency patients with LHON can be assessed in blood Sequencing of mitochondrial encoded cytochrome b gene DNA from affected tissue e g muscle essential DNA from affected tissue e g muscle essential An unfixed frozen muscle biopsy sample in transverse orientation to allow histochemical analysis and isolation of single cells by laser microdissection Taqman real time PCR assay which coamplifies nuclear 18S rRNA and mtDNA MTND1 genes DNA from affected tissues e g muscle liver essential Sequencing of the entire POLG coding region Blood DNA samples appropriate and required for complete gene analysis Sequencing of the entire PEO coding region Blood DNA samples appropriate and required Date of original issue March 2007 Revised version April 2013 NEWCASTLE MITOCHONDRIAL NSCT DIAGNOSTIC LABORATORY Rare Mitochondrial Disorders Service for Adults and Children MUTATION DISORDER TO BE TESTED SLC25A4 ANTI Patients with dominant PEO and multiple mtDNA deletions POLG2 Patients with dominant PEO and multiple mtDNA deletions TK2 and RRM2B Patients with dominant recessive PEO and multiple mtDNA deletions TK2 and RRM2B Patients with evidence of mtDNA depletion in skeletal muscle DGUOK and MPV17 Patients with evidence of mtDNA depletion in liver SUCLA2 and SUCLGI1 Patients with evidence of mtDNA de
15. pletion and methylmalonic aciduria TRMU Patients with evidence of acute liver disease and respiratory chain deficiency DARS2 Patients with mitochondrial leukoencephalopathy brain stem and spinal cord involvement lactic acidosis LBSL RARS2 Patients with multiple respiratory chain complex deficiencies and pontocerebellar hyoplasia AARS2 Patients with multiple respiratory chain complex deficiencies and hypertrophic cardiomyopathy Nuclear complex I structural genes A panel of 10 genes can be screened in patients with isolated complex I deficiency NDUFV1 NDUFV2 NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS6 NDUFS7 NDUFS8 NDUFAB1 Page 8 of 11 TYPE OF TEST SAMPLE NEEDED Sequencing of the entire SLC25A4 coding region Blood DNA samples appropriate and required Sequencing of the entire POLG2 coding region Blood DNA samples appropriate and required Sequencing of the entire 7K2 RRM2B coding regions Blood DNA samples appropriate and required Sequencing of the entire TK2 and RRM2B coding regions Blood DNA samples appropriate and required Sequencing of the entire DGUOK and MPVI17 coding regions Blood DNA samples appropriate and required Sequencing of the entire SUCLA2 and SUCLGI coding regions Blood DNA samples appropriate and required Sequencing of the entire TRMU coding region Blood DNA samples appropriate and required Sequencing of the entire DARS2 coding region Blood DNA sampl
16. rvices As part of our quality management system and to ensure that we are meeting the needs of our users we are always keen to receive any comments you may have on the quality of the service we provide and would welcome any suggestions on ways in which we might be able to improve the service Please feel free to contact the laboratory Quality Manager alan smith nuth nhs uk with any suggestions an electronic questionnaire is available directly by email should this be required CPA Accreditation Status The Newcastle Mitochondrial Diagnostic Service Laboratory is accredited by Clinical Pathology Accreditation UK Ltd and operates a quality management system in accordance with CPA Standards for the Medical Laboratory CPA reference 3118 United Kingdom Genetic Testing Network The Newcastle Mitochondrial Diagnostic Service Laboratory is registered with UKGTN as a provider of genetic testing for mitochondrial diseases Page 11 of 11 Date of original issue March 2007 Revised version April 2013
17. service should be used Please ensure that sufficient dry ice is used to last 48 hours at least 10kg but dependent on size of polystyrene container Ensure that no absorbent material is insulating the primary vessel from the dry ice Do not use polystyrene chips or freezing blocks to fill the polystyrene container 4 Address the package to Professor Rob Taylor Newcastle Mitochondrial NSCT Laboratory Wellcome Trust Centre for Mitochondrial Research 4 Floor Cookson Building The Medical School Newcastle University Framlington Place Newcastle upon Tyne NE2 4HH It is helpful to place address labels on more than one side of the box because the courier usually puts their own label on top 5 Please try and send samples on a Monday Tuesday or Wednesday Do not send samples to arrive on a Friday since delays may well move the delivery of your package into the weekend Our unit is not staffed at these times and your sample will thaw and be lost Page 5 of 11 Date of original issue March 2007 Revised version April 2013 NEWCASTLE MITOCHONDRIAL NSCT DIAGNOSTIC LABORATORY Rare Mitochondrial Disorders Service for Adults and Children Current list of tests available at the Newcastle Mitochondrial NSCT Diagnostic Laboratory Please contact the lab directly if further details are required HISTOLOGICAL HISTOCHEMICAL ANALYSES Each frozen sample is assessed for the following H amp E staining of cryostat cut sections Cytochrome c ox
18. ty of specialties These are truly multi system diseases with significant morbidity and mortality Over the last two decades mutations in both mtDNA and nuclear DNA nDNA have been identified as causative in a number of the mitochondrial clinical syndromes although for mtDNA mutations in particular this relationship between genotype and phenotype is often far from straightforward A number of epidemiological studies have been undertaken to assess the prevalence of mitochondrial disease and whilst rare these conditions have a major impact on both the community and individual families Finally there is increasing awareness by clinicians experienced in the management of patients with mitochondrial disease that many aspects of mitochondrial disease can be helped or prevented by early diagnosis and subsequent care The Newcastle Mitochondrial Diagnostic Laboratory is an affiliated laboratory of the Northern Regional Genetics Service and is situated within the research laboratory space of the University s Mitochondrial Research Group in the Medical School In partnership with teams at Queen Square Institute of Neurology UCLH NHS Foundation Trust and the Oxford Medical Genetics Laboratories Churchill Hospital Oxford Radcliffe Hospitals NHS Trust the Newcastle lab has been awarded National Specialist Commissioning NSCT funding to provide a comprehensive diagnostic and clinical management service for patients with mitochondrial disease in England an
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