Sandra Ramos Grade A Project St George’s Hospital, London

1. Analysis of three genes from the RAS-MAPK signalling pathway that are causative of Noonan/LEOPARD syndromes Sandra Ramos Grade A Project St George’s Hospital, London

2. Aims of the Project • Extend existing Noonan/LEOPARD syndrome screen to include new genes • Test the LightScanner™ (HRM) as a pre-screening tool • Investigate genotype-phenotype correlations • Determine optimal NS/LS future testing strategy

3. Noonan syndrome (NS) • Autosomal dominant • Incidence of 1 in 1000 to 1 in 2500 • Clinically heterogeneous disorder characterized by: - distinct facial features - short stature - congenital heart defects - skeletal abnormalities - bleeding problems Taken from London Medical Database

4. LEOPARD syndrome (LS) • Rare autosomal dominant disease • Characterized by: - Lentigines - ECG conduction abnormalities - Ocular hypertelorism - Pulmonary stenosis - Abnormalities of genitalia - Retardation of growth - Deafness Taken from E. J. of Human Genetics (2004) 12, 1069–1072

5. Molecular Genetics of NS • Caused by missense gain-of-function mutations in RAS-MAPK pathway • ~ 60% of Noonan syndrome cases are sporadic, presumed to be the result of de novo mutations Molecular Genetics of LS • Caused by loss of function/dominant negative mutations affecting the catalytic activity of PTPN11

6. HRAS KRAS Noonan syndrome MEK LEOPARD syndrome ERK RAS-MAPK Signalling Pathway RTK SOS1 Shc Grb2 Gab2 40 - 50% SHP-2 RAF1 BRAF ~ 90% Transcription of Target Genes

7. SOS1 gene • SOS1 is located on chromosome 2p22.1 and encodes a major RAS-GEF • Consists of 23 exons of which 9 have reported mutations • Variants disrupt autoinhibition RAS-GEF activity

8. HRAS KRAS Noonan syndrome MEK LEOPARD syndrome ERK RAS-MAPK Signalling Pathway 5 - 10% RTK SOS1 Shc Grb2 Gab2 SHP-2 RAF1 BRAF Transcription of Target Genes

9. KRAS gene • KRAS is located on chromosome 12p12.1 • Encodes a small G protein that is activated by the exchange of bound GDP for GTP • Consists of six exons but RNA splicing reveals two different transcripts • in 98% of transcripts exon 4a is spliced out and exon 4b is translated into protein

10. HRAS KRAS Noonan syndrome MEK LEOPARD syndrome ERK RAS-MAPK Signalling Pathway RTK ~ 1% SOS1 Shc Grb2 Gab2 SHP-2 RAF1 BRAF Transcription of Target Genes

11. RAF1 gene • RAF1 is located on chromosome 3p25 and encodes serine-threonine protein kinase that activates MEK1 and MEK2. • Consists of 17 exons of which 3 have reported mutations • Mutations alter autoinhibition of RAF1

12. HRAS KRAS Noonan syndrome MEK LEOPARD syndrome ERK RAS-MAPK Signalling Pathway RTK SOS1 Shc Grb2 Gab2 3 - 8% SHP-2 RAF1 BRAF Transcription of Target Genes

13. WAVE v LightScanner™ • Primers designed using LightScanner™ primer design software • CADAMA HotShot mastermix • Idaho Technologies designed Touchdown PCR program • Amplified products were successfully analysed using dHPLC (WAVE) and bidirectional sequencing (ABI3730) SOS1 primers

14. WAVE v LightScanner™ results SOS1 exon 13 LS trace SOS1 exon 13 WAVE trace LightScanner™ software missed SOS1 exon 13 variant control (black arrow) Wave traces for SOS1 exon 13 normal samples and 1 variant control (black arrow)

15. WAVE v LightScanner™ results SOS1 exon 10 LS trace SOS1 exon 10 WAVE trace Visual checks difficult by the lack of uniformity/normalisation in traces

16. WAVE v LightScanner™ results SOS1 exon 16 variant control only detected when sensitivity is increased to 2.40

17. WAVE v LightScanner™ conclusions

18. Testing • Cohort of 110 patients from SEEGEN region referred for NS/LS testing • All negative for PTPN11 mutations • Screened exons with reported mutations only • - SOS1 – 9 Exons (3,6,7,8,10,11,13,14 & 16) • - RAF1 – 3 Exons (6,13 & 16) • - KRAS – 5 Exons (1,2,3,4a & 4b) • Samples pre-screened on the Transgenomic WAVE and variants sequenced using ABI3730

19. Results 110 patients screened for SOS1, RAF1 and KRAS SOS1 • 7 missense variants identified of which • 5 previously reported mutations • and • 2 novel missense variants • The prevalence of SOS1 mutations found is 6.4% RAF1 • 4 missense variants identified of which • 3 previously reported mutations • and • 1 novel missense variant • The prevalence of RAF1 mutations found is 3.7% • No mutations found in KRAS gene

20. Genotype-Phenotype Clinical features of NS individuals with SOS1 mutations VSD/ASD (Ventriculal/Atrial septal defect); PVS (Pulmonary valve stenosis); LD (learning difficulties)

21. Genotype-Phenotype Clinical features of NS/LS individuals with RAF1 mutations HOCM (Hypertrophic obstructive cardiomyopathy); PS (Pulmonary stenosis); LD (learning difficulties)

22. SOS1 Mutations RAF1 Mutations Genotype-phenotype

23. Logging-in / Extraction NS/LS Testing Strategy • STAGE 1 • dHPLC analysis of exons 2, 3, 4, 7, 8, 12 and 13 of PTPN11 • Bidirectional Sequencing of variants (ABI 3730) ~ 40-50% NS cases ~ 90% LS cases ~ 10 % NS cases Exceptional LS cases • STAGE 2 • dHPLC analysis of exons 3, 6, 10 of SOS1 and 6, 13, 16 of RAF1 • Bidirectional Sequencing of variants (ABI 3730) STAGE 3 • dHPLC analysis of remaining exons of SOS1 and all exons of KRAS • Bidirectional Sequencing of variants (ABI 3730) ~ 1-3 % NS cases

24. Conclusions • Three genes analysed and 9 mutations (plus 3 novel variants) detected in SOS1/RAF1 from 110 samples • Overall pick up rate for our cohort is ~10% • No mutations identified in KRAS • dHPLC WAVE is a more robust pre-screening method compared to LightScanner™ HRM • Complex genotype-phenotype correlation • Three stage screening strategy designed for NS/LS referrals from April 2008

25. Further Work Taken fromEMBO reports 6, 12, 1169–1175 (2005)

26. Acknowledgements Thank you: • John Short • Navaratnam Elanko • Roy Poh • Sally Cottrell • Rohan Taylor • Professor Michael Patton • Kamini Kalidas (Clinical Developmental Sciences, St George’s University of London) • IDEAS Knowledge Park for funding this project and all staff at Molecular Genetics Lab at St George’s