1. Diagnosing VLCAD (abolishing thermolabile mutant protein) Dr Simon Olpin Sheffield Children’s Hospital UK

2. Assays Fibroblast acylcarnitine profiling Fibroblast acylcarnitine profiling –Fibroblasts incubated - 96 hrs, 400 µmol/L L-carnitine, 200 µmol/L palmitate –Profiles by MS/MS Compare mean results to simultaneous controls Compare mean results to simultaneous controls VLCAD assay using ferrocenium & HPLC detection of product VLCAD assay using ferrocenium & HPLC detection of product –Compare mean results to simultaneous controls Tritium release assays using [9,10-H 3 ]myristate, palmitate & oleate Tritium release assays using [9,10-H 3 ]myristate, palmitate & oleate –Incubate for 2 hours – results as pmol/mg fibroblast protein /min –Express results as % activity of 4-8 simultaneous control cell lines –Assay each cell line 2-5 times Flux assay used to investigate any patient with possible FAOD. Flux assay used to investigate any patient with possible FAOD. No NBS for VLCAD in UK. No NBS for VLCAD in UK. 06/09/12VLCAD

3. 06/09/12VLCAD Fibroblast acylcarnitines - Increased species as compared to controls for a range of fat oxidation disorders (45 patient cell lines in total) See infantile & myopathic VLCAD Fold increases (normal range mean + 2 S.D.)

4. 06/09/12VLCAD n=16 n=4 n=12 n=5

5. 06/09/12VLCAD

6. 06/09/12VLCAD @26 o C fibroblasts from patients with adult- onset myopathic disease show significantly greater oleate flux activity than fibroblasts from severe infantile patients – “mutant protein is functional”

7. 06/09/12VLCAD @41 o C mutant protein from “mild” myopathic patients is largely abolished i.e. similar flux to patients with minimal residual VLCAD activity

8. 06/09/12VLCAD Fat oxidation flux assays Controls v infantile VLCAD’s @37 o C

9. 06/09/12VLCAD Fat oxidation flux assays Controls v infantile plus myopathic VLCAD’s @37 o C

11. 06/09/12VLCAD Fat oxidation flux assays Myopathic VLCAD’s plus Newborn screened false positives at 41 o C

12. 06/09/12VLCAD Patient C14:1 <0.6 Day 1Mutation/change Oleate @ 41degrees % controls HPLC % controls 11.11* c.848T>C only 6687 21.21c.1316G>A only 7032 30.79* c.848T>C only 134145 4N/A 1 mutation only (not stated) 8338 5N/A c.1405C>T (pathogenic) c.686G>A ( novel) 6689 60.913 SNP's93231 False positive VLCAD on NBS - fibroblast cell lines * c.848T>C = p.Val283Ala

13. 06/09/12VLCAD Fat oxidation flux assays Controls v myopathic, screened positive & obligate (?)heterozygous/symptomatic @41C

14. 06/09/12VLCAD Difficult cases Patient 1 - father of infant who died at 18 months (infant previously well) – no mutation in father found (infant - oleate 9% at 41 o C) Patient 1 - father of infant who died at 18 months (infant previously well) – no mutation in father found (infant - oleate 9% at 41 o C) HPLC assay (father) – 87% HPLC assay (father) – 87% Patient 2 - mother of same infant – common mutation c.848T>C (only) in her & infant. Patient 2 - mother of same infant – common mutation c.848T>C (only) in her & infant. HPLC assay (mother) - 30% HPLC assay (mother) - 30% Patient 3 - 21 year old man, cyclical vomiting, bilateral optic atrophy, cystic lesions in brain, mild developmental delay, no hypoglycaemia, no muscle disease. No abnormal metabolites. c.1019G>T (G340V) plus c.753-27C>T substitution. HPLC assay 18% Patient 3 - 21 year old man, cyclical vomiting, bilateral optic atrophy, cystic lesions in brain, mild developmental delay, no hypoglycaemia, no muscle disease. No abnormal metabolites. c.1019G>T (G340V) plus c.753-27C>T substitution. HPLC assay 18% Patient 4 – 30 year old male – previously seen by rheumatology for swelling & pain in arms. Acute hyperthermia & rhabdomyolysis following unacustomed 7 Km run, became encephalopathic – hospitalised & developed multi-organ failure – now epileptic. No clear biochemical abnormalities. Arg613Trp - known pathogenic mutation only. HPLC assay 38% Patient 4 – 30 year old male – previously seen by rheumatology for swelling & pain in arms. Acute hyperthermia & rhabdomyolysis following unacustomed 7 Km run, became encephalopathic – hospitalised & developed multi-organ failure – now epileptic. No clear biochemical abnormalities. Arg613Trp - known pathogenic mutation only. HPLC assay 38% 1 2 3 4

15. 06/09/12VLCAD Myopathic patients 22 year old male – Black Belt Karate 22 year old male – Black Belt Karate Acute admission to hospital with hypoglycaemia as infant. Revived with glucose. Mum always gave sugary drinks when unwell. Self selected low fat diet (12-15% fat calories) Acylcarnitines – reported “normal” even on review Presented with rhabdomyolysis as young adult when doing exhibition Karate - ↓↓ sleep & food intake – 2 mutations (oleate 29% @37C) Two siblings – normal acylcarnitines. (Previously Hewson et al ASHG poster 2006) Two siblings – normal acylcarnitines. (Previously Hewson et al ASHG poster 2006) Patient 1 – hypoketotic hypoglycaemia at 21 months. From adolescence began to have myalgia following prolonged exercise. (32% oleate @37C, 26% oleate at 41C ) HPLC assay - 24%. Diagnosed on oleate assay at 13 years – three mutations identified Patient 2 – similarly diagnosed at 19 years – same mutations low oleate – active sportsman – never any VLCAD ascribable symptoms (40% oleate @37C, 23% oleate @41C) HPLC assay - 29%

16. 06/09/12VLCAD Myopathic patients 64 year old lady – history of recurrent rhabdomyolysis 64 year old lady – history of recurrent rhabdomyolysis Extensively investigated – only recently diagnosed! Oleate 48% @37C, 15% @41C, HPLC – 35% C.753-2A>C plus p.Val283Ala (mild mutation said ~20% residual activity) (Val283Ala plus in cis Val317Ala – severe! Infantile presentation) 30 year old female – rhabdomyolysis following caesarean section. Rhabdomyolysis some months later following Christmas shopping. (Ck~32,000 on both occasions) 30 year old female – rhabdomyolysis following caesarean section. Rhabdomyolysis some months later following Christmas shopping. (Ck~32,000 on both occasions) Previously active with no muscle pains or weakness. However in childhood diagnosed with Reye syndrome & hypoglycaemia. Oleate 6% @ 37C! Acylcarnitines clearly abnormal ↑↑ C14:1. Now very restricted due to poor tolerance of any sustained muscular effort. Now very restricted due to poor tolerance of any sustained muscular effort.

17. 06/09/12VLCAD Diagnosing VLCAD All three methods described detect most VLCAD patients All three methods described detect most VLCAD patients Select the most reliable method for your laboratory Select the most reliable method for your laboratory In our hands our global flux assay @41C detects all VLCAD’s & generally discriminates between carriers & symptomatic VLCAD’s In our hands our global flux assay @41C detects all VLCAD’s & generally discriminates between carriers & symptomatic VLCAD’s

18. Sheffield Children’s Hospital/UK Shirley Clark, Helen Hind Joanne Croft, Nigel Manning Camilla Scott, Jim Bonham Richard Kirk, Jenny Watkinson Jane Dalley, Mark Sharrard Reena Sharma Liz Allen Rodney Pollitt International Brage Andresen – Denmark Rikke Olsen – Denmark Neils Gregersen - Denmark Aneal Khan - Alberta Eileen Treacy - Dublin Ronald Wanders – Amsterdam Jean Bastin - Paris