1. Expanded PLA2G6 Copy Number Variant Analysis in Patients with Infantile Neuroaxonal Dystrophy (INAD) Danielle Crompton, P. K. Rehal, L. MacPherson, K. Foster, P. Lunt, A. Hughes, A. F. Brady, M. G. Pike, S. De Gressi, N. V. Morgan, C. Hardy, M. Smith, F. Macdonald, E. R. Maher, M. A. Kurian

2. Phospholipase associated neurodegeneration (PLAN) Heterogeneous group of autosomal recessive disorders associated with mutations in the PLA2G6 gene 3 clinically distinct PLAN phenotypes: Classical INAD (infantile onset PLAN) Classical INAD (infantile onset PLAN) Atypical INAD/NBIA Adult onset dystonia-parkinsonism

3. Classical INAD Progressive psychomotor regression Infantile onset truncal hypotonia Cerebellar signs Pyramidal tract features Extrapyramidal tract features Bulbar dysfunction Mean age of death 9.1 years Kurian et al 2008, Gregory et al 2008

4. Classical INAD

5. Patient, Age 2.9 years MRI Brain Scan

6. PLA2G6 Mutations Mutation pick up rate in classical INAD ~85% Multiple explanations Project hypothesis: Intragenic deletions and duplications of PLA2G6 may account for a proportion of mutations that are undetectable by direct sequencing Project aims: Detect such mutations, detail the extent of any CNV and develop a test that can be used on prenatal samples

7. Patients in Study West Midlands Regional Genetics Service Identified 4 classical INAD cases Both disease-causing mutations not identified on direct sequencing

8. Molecular genetic investigation – patient 1 Direct sequencing of PLA2G6 in patient 1 Heterozygous c.1674delG; p.Leu560TrpfsX5 Control Patient 1 Mother of patient 1

9. Molecular genetic investigation – patient 1 SNP analysis Gap PCR Dosage PCR – no evidence of exon 13, 14 and 15 deletion RNA studies – Primers in exons 11 and 17 - No deletion of exons 13, 14 and 15

10. Molecular genetic investigation – patient 2 PLA2G6– no pathogenic mutations by sequencing PCR failed to amplify exons 5 and 6 - homozygously deleted? SNP analysis of parents - heterozygous deletion of PLA2G6 exons 5 and 6? Patient 2 hypothesised to be homozygous for a deletion of exons 5 and 6 Patient 2: Consanguineous Irish traveller family

11. MLPA using specifically designed probes Normal controls Patient 2

12. Molecular genetic investigation – patient 2,3 and 4 Patient 3,4: Dizygotic twins from separate Irish traveller family PCR non-amplification of exons 5 and 6 PCR product for exon 6 amplification PCR product for exon 5 amplification C C C P2 P3 P4 N

13. MLPA analysis in patient 1 Heterozygous duplication of exons 4, 5, 6 and 7 of the PLA2G6 gene Control Father of Patient 1 Patient 1

14. RNA analysis for patient 1

15. MLPA analysis for patient 2,3,4 Homozygous deletion of exons 5 and 6 of the PLA2G6 gene Control Patients 2,3,4 Parents of Patient 2

16. Determination of the genomic deletion breakpoint in patient 2 PCR amplification of introns 4 and 6 of the PLA2G6 gene

17. Breakpoint Characterisation Schematic representation of the deletion breakpoint in patient 2,3,4

18. The Alu recombination event between Alu repeats in intron 4 and intron 6

19. Summary of MLPA findings Patient 1HeterozygousDuplication of exons 4,5,6,7 p.Lys360LeufsX22 Patient 2HomozygousDeletion of exons 5,6 p.Leu204_Glu298del95 Patient 3HomozygousDeletion of exons 5,6 p.Leu204_Glu298del95 Patient 4HomozygousDeletion of exons 5,6 p.Leu204_Glu298del95

20. Conclusions First reported use of MLPA in PLA2G6 analysis CNVs have a role in the pathogenesis of PLAN CNVs account for ~ 12.5% PLA2G6 mutations identified in the UK diagnostic screening service Such definitive diagnosis avoids unnecessary neurological investigation, improves the quality of genetic counselling and provide more accurate prenatal diagnoses Provide greater understanding of disease mechanism as well as genotype-phenotype correlations We propose that MLPA is a second-line diagnostic tool and adjunct to exon-by-exon sequencing in identifying novel PLA2G6 mutations

22. Acknowledgements Manju A Kurian Pauline Rehal Sequencing team at the West Midlands Regional Genetics Birmingham Children’s Hospital Research Foundation