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
SW Thames Molecular Genetics Diagnostic Laboratory
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
SW Thames Molecular Genetics Diagnostic Laboratory
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
SW Thames Molecular Genetics Diagnostic Laboratory
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
SW Thames Molecular Genetics Diagnostic Laboratory
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
SW Thames Molecular Genetics Diagnostic Laboratory
RAS-MAPK Signalling Pathway
Noonan syndrome
RTK
SOS1
Shc
Grb2
HRAS
KRAS
Gab2
40 - 50%
SHP-2
RAF1 BRAF
~ 90%
MEK
LEOPARD syndrome
ERK
Transcription of Target Genes
SW Thames Molecular Genetics Diagnostic Laboratory
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
SW Thames Molecular Genetics Diagnostic Laboratory
RAS-MAPK Signalling Pathway
Noonan syndrome
5 - 10%
RTK
SOS1
Shc
Grb2
HRAS
KRAS
Gab2
SHP-2
RAF1 BRAF
MEK
LEOPARD syndrome
ERK
Transcription of Target Genes
SW Thames Molecular Genetics Diagnostic Laboratory
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
SW Thames Molecular Genetics Diagnostic Laboratory
RAS-MAPK Signalling Pathway
Noonan syndrome
RTK
~ 1%
SOS1
Shc
Grb2
HRAS
KRAS
Gab2
SHP-2
RAF1 BRAF
MEK
LEOPARD syndrome
ERK
Transcription of Target Genes
SW Thames Molecular Genetics Diagnostic Laboratory
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
SW Thames Molecular Genetics Diagnostic Laboratory
RAS-MAPK Signalling Pathway
Noonan syndrome
RTK
SOS1
Shc
Grb2
HRAS
KRAS
Gab2
3 - 8%
SHP-2
RAF1 BRAF
MEK
LEOPARD syndrome
ERK
Transcription of Target Genes
SW Thames Molecular Genetics Diagnostic Laboratory
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
SW Thames Molecular Genetics Diagnostic Laboratory
WAVE v LightScanner™ results
SOS1 exon 13 LS trace
LightScanner™ software
missed SOS1 exon 13
variant control (black arrow)
SW Thames Molecular Genetics Diagnostic Laboratory
SOS1 exon 13 WAVE trace
Wave traces for SOS1 exon 13
normal samples and 1 variant
control (black arrow)
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
SW Thames Molecular Genetics Diagnostic Laboratory
WAVE v LightScanner™ results
SOS1 exon 16 variant control only detected when sensitivity
is increased to 2.40
SW Thames Molecular Genetics Diagnostic Laboratory
WAVE v LightScanner™ conclusions
LightScanner™
dHPLC WAVE
False Variants
10/77 (12.9%)
1/77 (1.2%)
Positive Controls
detected
5/9 (55%)
9/9 (100%)
Fails
1/88 (1.1%)
3/88 (3.4%)
SW Thames Molecular Genetics Diagnostic Laboratory
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
SW Thames Molecular Genetics Diagnostic Laboratory
Results
110 patients screened for SOS1, RAF1 and KRAS
SOS1
RAF1
7 missense variants identified
of which
 5 previously reported mutations
and
 2 novel missense variants
4 missense variants identified
of which
 3 previously reported mutations
and
 1 novel missense variant


The prevalence of SOS1
mutations found is 6.4%

The prevalence of RAF1
mutations found is 3.7%

 No mutations found in KRAS gene
SW Thames Molecular Genetics Diagnostic Laboratory
Genotype-Phenotype
Clinical features of NS individuals with SOS1 mutations
Noonan
Syndrome
Patient 1
Patient 2 Patient 3 Patient 4 Patient 5
Genotype
1655G>C
R552T
1654A>G
R552T
1655G>T
R552T
1300G>A
G434R
305C>G
P102R
1867T>A
F623I
Sex/Age
M/2Y
M/5Y
F/14Y
M/2Y
F/13Y
M/22Y
Cardiac
Defect
-
VSD, Mild
PVS
PVS
PVS
n.d.
ASD
HCM
Short
Stature
-
+
+
n.d
+
+
Facial
Features
+
-
n.d.
+
n.d.
+
Mental
Retardation
-
-
n.d.
n.d
LD
-
Ptosis,
lymphoedema
Hydrops,
speech
delay
Others
Dev.
delay, low
factor VIII
Patient 6
Pectus
excavatum,
keratosis
VSD/ASD (Ventriculal/Atrial septal defect); PVS (Pulmonary valve stenosis); LD (learning difficulties)
SW Thames Molecular Genetics Diagnostic Laboratory
Genotype-Phenotype
Clinical features of NS/LS individuals with RAF1 mutations
Patient 1
LEOPARD
Patient 2
Noonan/
LEOPARD
Patient 3
Noonan
Patient4
Noonan
781C>G
P261A
770C>T
S257L
770C>T
S257L
1835C>G
S612C (NV)
Sex/Age
M/14Y
F/ 5months
deceased
F/17Y
M/52Y
Cardiac Defect
HOCM
HOCM, PS
HOCM
-
Short Stature
+
N/A
+
+
Facial Features
+
-
+
+
Mental Retardation
-
N/A
LD
LD
Neck webbing,
lentigines,
subaortic
stenosis
Chronic lung
disease,
failure to
thrive
Hypertelorism
Bilateral
ptosis, pectus
excavatum,
cryptorchidism
Genotype
Others
HOCM (Hypertrophic obstructive cardiomyopathy); PS (Pulmonary stenosis); LD (learning difficulties)
SW Thames Molecular Genetics Diagnostic Laboratory
Genotype-phenotype
SOS1 Mutations
RAF1 Mutations
Present
Study
Tartaglia et.
al.
Present
Study
Razzaque
et. al.
Pulmonary
Stenosis
2/6 (33%)
10/16 (63%)
1/4 (25%)
n.d.
HCM/HOCM
1/6 (16%)
2/16 (12.5%)
3/4 (75%)
8/10 (80%)
Atrial/ventricular
Septal Defect
1/6 (16%)
4/16 (25%)
3/4 (75%)
6/10 (60%)
Short Stature
4/6 (66%)
2/15 (13%)
3/4*(75%)
9/10 (90%)
Mental retardation
1/6 (16%)
1/16 (6%)
2/4*(50%)
8/10 (80%)
(3 LD)
Thorax deformity
2/6 (33%)
16/16 (100%)
1/4 (25%)
5/10 (50%)
Cryptorchidism
2/4 (50%)
n.d.
1/2 (50%)
2/8 (25%)
SW Thames Molecular Genetics Diagnostic Laboratory
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)
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)
SW Thames Molecular Genetics Diagnostic Laboratory
~ 40-50%
NS cases
~ 90% LS
cases
~ 10 % NS
cases
Exceptional
LS cases
~ 1-3 % NS
cases
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
SW Thames Molecular Genetics Diagnostic Laboratory
Further Work
Taken from EMBO reports 6, 12, 1169–1175 (2005)
SW Thames Molecular Genetics Diagnostic Laboratory
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
SW Thames Molecular Genetics Diagnostic Laboratory