sandra ramos grade a project st george’s hospital, london

SW Thames Molecular Genetics Diagnostic Laboratory

Analysis of three genes from Analysis of three genes from the RAS-MAPK signalling the RAS-MAPK signalling

pathway that are causative of pathway that are causative of Noonan/LEOPARD syndromesNoonan/LEOPARD syndromes

Sandra RamosSandra RamosGrade A ProjectGrade A Project

St George’s Hospital, LondonSt George’s Hospital, London


Aims of the ProjectAims of the Project• Extend existing Noonan/LEOPARD syndrome Extend existing Noonan/LEOPARD syndrome

screen to include new genesscreen to include new genes • Test the LightScanner™ (HRM) as aTest the LightScanner™ (HRM) as a pre-screening toolpre-screening tool • Investigate genotype-phenotype correlationsInvestigate genotype-phenotype correlations

• Determine optimal NS/LS future testing Determine optimal NS/LS future testing strategystrategy


Noonan syndrome (NS)Noonan syndrome (NS)• Autosomal dominant Autosomal dominant • Incidence of 1 in 1000 to 1 in 2500Incidence of 1 in 1000 to 1 in 2500

• Clinically heterogeneous disorderClinically heterogeneous disorder characterized characterized by:by:

- distinct facial features- distinct facial features - short statureshort stature - congenital heart defects- congenital heart defects - skeletal abnormalities- skeletal abnormalities - bleeding problems- bleeding problems

Taken from London Medical Database


LEOPARD syndrome (LS)LEOPARD syndrome (LS)• Rare autosomal dominant diseaseRare autosomal dominant disease

• CCharacterized by:haracterized by:

- - LLentiginesentigines

- - EECG conduction abnormalities CG conduction abnormalities

- - OOcular hypertelorism cular hypertelorism

- - PPulmonary stenosis ulmonary stenosis

- - AAbnormalities of genitalia bnormalities of genitalia

- - RRetardation of growth etardation of growth

- - DDeafness eafness Taken from E. J. of Human Genetics (2004) 12, 1069–1072


Molecular Genetics of NSMolecular Genetics of NS• Caused by missense gain-of-function mutations Caused by missense gain-of-function mutations in RAS-MAPK pathwayin RAS-MAPK pathway

• ~ 60% of Noonan syndrome cases are sporadic, ~ 60% of Noonan syndrome cases are sporadic, presumed to be the result of presumed to be the result of de novode novo mutations mutations

Molecular Genetics of LSMolecular Genetics of LS

• Caused by loss of function/dominant negative Caused by loss of function/dominant negative mutations affecting the catalytic activity of mutations affecting the catalytic activity of PTPN11PTPN11


RAS-MAPK Signalling PathwayRAS-MAPK Signalling Pathway

HRASHRAS KRASKRAS

RAF1RAF1

MEKMEK

ERKERK

Transcription of Target GenesTranscription of Target Genes

SHP-2SHP-2

GabGab22

ShcShcGrbGrb22

SOS1SOS1

Noonan syndromeNoonan syndrome

LEOPARD syndromeLEOPARD syndrome

RTKRTK

BRAFBRAF

40 - 50%40 - 50%

~ 90%~ 90%


SOS1SOS1 gene gene• SOS1SOS1 is located on chromosome 2p22.1 and is located on chromosome 2p22.1 and encodes a major RAS-GEFencodes a major RAS-GEF • Consists of 23 exons of which 9 have reported Consists of 23 exons of which 9 have reported mutationsmutations

• Variants disrupt autoinhibition RAS-GEF activityVariants disrupt autoinhibition RAS-GEF activity



HRASHRAS KRASKRAS

RARAFF11

MEKMEK

ERKERK


SHPSHP-2-2

GabGab22

ShcShcGrbGrb22

SOS1SOS1



RTKRTK

BRAFBRAF

5 - 10%5 - 10%


KRAS KRAS genegene• KRASKRAS is located on chromosome 12p12.1 is located on chromosome 12p12.1

• Encodes a small G protein that is activated by the Encodes a small G protein that is activated by the exchange of bound GDP for GTPexchange of bound GDP for GTP

• Consists of six exons but RNA splicing reveals two Consists of six exons but RNA splicing reveals two different transcriptsdifferent transcripts– in 98% of transcripts exon 4a is spliced out and exon 4b is translated in 98% of transcripts exon 4a is spliced out and exon 4b is translated

into proteininto protein



HRASHRAS KRASKRAS

RARAFF11

MEKMEK

ERKERK


SHPSHP-2-2

GabGab22

ShcShcGrbGrb22

SOS1SOS1



RTKRTK

BRAFBRAF

~ 1%~ 1%


RAF1RAF1 gene gene• RAF1RAF1 is located on chromosome 3p25 and is located on chromosome 3p25 and encodes serine-threonine protein kinase that encodes serine-threonine protein kinase that activates activates MEK1MEK1 and and MEK2MEK2..

• Consists of 17 exons of which 3 have Consists of 17 exons of which 3 have reported mutationsreported mutations

• Mutations alter autoinhibition of Mutations alter autoinhibition of RAF1RAF1



HRASHRAS KRASKRAS

RARAFF11

MEKMEK

ERKERK


SHPSHP-2-2

GabGab22

ShcShcGrbGrb22

SOS1SOS1



RTKRTK

BRAFBRAF

3 - 8%3 - 8%


WAVE v LightScannerWAVE v LightScanner™™• Primers designed using LightScannerPrimers designed using LightScanner™™ primer design primer design softwaresoftware

• CADAMA HotShot mastermixCADAMA HotShot mastermix

• Idaho Technologies designed Touchdown PCR programIdaho Technologies designed Touchdown PCR program

• Amplified products were successfully analysed using dHPLC Amplified products were successfully analysed using dHPLC (WAVE) and bidirectional sequencing (ABI3730)(WAVE) and bidirectional sequencing (ABI3730)

SOS1 primers


WAVE v LightScanner™ resultsWAVE v LightScanner™ results

Wave traces for Wave traces for SOS1SOS1 exon 13 exon 13 normal samples and 1 variant normal samples and 1 variant control (black arrow)control (black arrow)

LightScannerLightScanner™™ software software missed missed SOS1SOS1 exon 13 exon 13 variant control (black arrow)variant control (black arrow)

SOS1SOS1 exon 13 LS trace exon 13 LS trace SOS1SOS1 exon 13 WAVE trace exon 13 WAVE trace



Visual checks difficult by the lack of uniformity/normalisation in Visual checks difficult by the lack of uniformity/normalisation in tracestraces

SOS1SOS1 exon 10 LS trace exon 10 LS trace SSOS1 exon 10 WAVE trace



SOS1SOS1 exon 16 variant control only detected when sensitivity exon 16 variant control only detected when sensitivity is increased to 2.40is increased to 2.40


WAVE v LightScanner™ conclusionsWAVE v LightScanner™ conclusions

LightScannerLightScanner™™ dHPLC WAVEdHPLC WAVE

False Variants False Variants 10/77 (12.9%)10/77 (12.9%) 1/77 (1.2%)1/77 (1.2%)

Positive Controls Positive Controls detecteddetected

5/9 (55%)5/9 (55%) 9/9 (100%)9/9 (100%)

FailsFails 1/88 (1.1%)1/88 (1.1%) 3/88 (3.4%)3/88 (3.4%)


• Cohort of 110 patients from SEEGEN region referred Cohort of 110 patients from SEEGEN region referred for NS/LS testingfor NS/LS testing

• All negative for All negative for PTPN11PTPN11 mutations mutations

• Screened exons with reported mutations onlyScreened exons with reported mutations only- SOS1 - SOS1 – 9 Exons (3,6,7,8,10,11,13,14 & 16)– 9 Exons (3,6,7,8,10,11,13,14 & 16)- RAF1 - RAF1 – 3 Exons (6,13 & 16)– 3 Exons (6,13 & 16)- KRAS- KRAS – 5 Exons (1,2,3,4a & 4b) – 5 Exons (1,2,3,4a & 4b)

Samples pre-screened on the Transgenomic WAVE Samples pre-screened on the Transgenomic WAVE and variants sequenced using ABI3730and variants sequenced using ABI3730

TestingTesting


ResultsResults

SOS1SOS1

7 missense variants identified7 missense variants identified

of whichof which 5 previously reported mutations5 previously reported mutations

andand 2 novel missense variants 2 novel missense variants

The prevalence of The prevalence of SOS1SOS1

mutations found is mutations found is 6.4%6.4%

RAF1RAF1

4 missense variants identified4 missense variants identified

of whichof which 3 previously reported mutations3 previously reported mutations

andand 1 novel missense variant1 novel missense variant

The prevalence of The prevalence of RAF1RAF1

mutations found is mutations found is 3.7%3.7%

110 patients screened for 110 patients screened for SOS1SOS1, , RAF1RAF1 and and KRASKRAS

No mutations found in No mutations found in KRASKRAS gene gene


Genotype-PhenotypeGenotype-Phenotype

Noonan Noonan SyndromeSyndrome

Patient 1Patient 1 Patient 2Patient 2 Patient 3Patient 3 Patient 4Patient 4 Patient 5Patient 5 Patient 6Patient 6

GenotypeGenotype 1655G>C1655G>C

R552TR552T

1654A>G1654A>G

R552TR552T

1655G>T1655G>T

R552TR552T

1300G>A1300G>A

G434RG434R

305C>G305C>G

P102RP102R

1867T>A1867T>A

F623IF623I

Sex/AgeSex/Age M/2YM/2Y M/5YM/5Y F/14YF/14Y M/2YM/2Y F/13YF/13Y M/22YM/22Y

Cardiac Cardiac DefectDefect

-- VSD, VSD, Mild Mild PVSPVS

PVSPVS PVSPVS n.d.n.d. ASDASD

HCMHCM

Short Short StatureStature

-- ++ ++ n.dn.d ++ ++

Facial Facial FeaturesFeatures

++ -- n.d.n.d. ++ n.d.n.d. ++

Mental Mental RetardationRetardation

-- -- n.d.n.d. n.dn.d LDLD --

OthersOthers Ptosis,Ptosis,

lymphoedemalymphoedema

Hydrops, Hydrops, speech speech delaydelay

Dev. Dev. delay, low delay, low factor VIIIfactor VIII

Pectus Pectus excavatum,excavatum,

keratosiskeratosis

Clinical features of NS individuals with Clinical features of NS individuals with SOS1SOS1 mutations mutations

VSD/ASD (Ventriculal/Atrial septal defect); PVS (Pulmonary valve stenosis); LD (learning difficulties)VSD/ASD (Ventriculal/Atrial septal defect); PVS (Pulmonary valve stenosis); LD (learning difficulties)


Genotype-PhenotypeGenotype-Phenotype

Patient 1Patient 1

LEOPARDLEOPARD

Patient 2Patient 2

Noonan/ Noonan/ LEOPARDLEOPARD

Patient 3 Patient 3 NoonanNoonan

Patient4 Patient4 NoonanNoonan

GenotypeGenotype 781C>G781C>G

P261AP261A

770C>T770C>T

S257LS257L

770C>T770C>T

S257LS257L

1835C>G1835C>G

S612C (NV)S612C (NV)

Sex/AgeSex/Age M/14YM/14Y F/ 5monthsF/ 5months

deceaseddeceased

F/17YF/17Y M/52YM/52Y

Cardiac DefectCardiac Defect HOCMHOCM HOCM, HOCM, PSPS HOCMHOCM --

Short StatureShort Stature ++ N/AN/A ++ ++

Facial FeaturesFacial Features ++ -- ++ ++

Mental RetardationMental Retardation -- N/AN/A LDLD LDLD

OthersOthers Neck webbing, Neck webbing, lentigines, lentigines, subaortic subaortic stenosisstenosis

Chronic lung Chronic lung disease, disease, failure to failure to

thrivethrive

HypertelorismHypertelorism Bilateral Bilateral ptosis, pectus ptosis, pectus excavatum, excavatum,

cryptorchidismcryptorchidism

Clinical features of NS/LS individuals with Clinical features of NS/LS individuals with RAF1RAF1 mutations mutations

HOCM (Hypertrophic obstructive cardiomyopathy); PS (Pulmonary stenosis); LD (learning difficultiesHOCM (Hypertrophic obstructive cardiomyopathy); PS (Pulmonary stenosis); LD (learning difficulties)


Genotype-phenotypeGenotype-phenotype

Present Present StudyStudy

Tartaglia Tartaglia et. et. al.al.

Pulmonary Pulmonary StenosisStenosis

2/6 (33%)2/6 (33%) 10/16 (63%)10/16 (63%)

HCM/HOCMHCM/HOCM 1/6 (16%)1/6 (16%) 2/16 (12.5%)2/16 (12.5%)

Atrial/ventricular Atrial/ventricular Septal DefectSeptal Defect

1/6 (16%)1/6 (16%) 4/16 (25%)4/16 (25%)

Short StatureShort Stature 4/6 (66%)4/6 (66%) 2/15 (13%)2/15 (13%)

Mental retardationMental retardation 1/6 (16%)1/6 (16%) 1/16 (6%)1/16 (6%)

Thorax deformityThorax deformity 2/6 (33%)2/6 (33%) 16/16 (100%)16/16 (100%)

CryptorchidismCryptorchidism 2/4 (50%)2/4 (50%) n.d.n.d.

SOS1SOS1 Mutations Mutations RAF1RAF1 Mutations Mutations

Present Present StudyStudy

Razzaque Razzaque

et. al.et. al.

1/4 (25%)1/4 (25%) n.d.n.d.

3/4 (75%)3/4 (75%) 8/10 (80%)8/10 (80%)

3/4 (75%)3/4 (75%) 6/10 (60%)6/10 (60%)

3/4*(75%)3/4*(75%) 9/10 (90%)9/10 (90%)

2/4*(50%)2/4*(50%) 8/10 (80%)8/10 (80%)

(3 LD)(3 LD)

1/4 (25%)1/4 (25%) 5/10 (50%)5/10 (50%)

1/2 (50%)1/2 (50%) 2/8 (25%)2/8 (25%)


NS/LS NS/LS Testing Testing StrategyStrategy

STAGE 1 dHPLC analysis of exons 2, 3, 4, 7, 8,

12 and 13 of PTPN11 Bidirectional Sequencing of variants

(ABI 3730)

Logging-in / Extraction

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)

~ 40-50% 40-50% NS casesNS cases

~ 90% LS ~ 90% LS casescases

~ 10 % NS ~ 10 % NS casescases

Exceptional Exceptional LS casesLS cases

~ 1-3 % NS ~ 1-3 % NS casescases


ConclusionsConclusions• Three genes analysed and 9 mutations (plus 3 novel variants) Three genes analysed and 9 mutations (plus 3 novel variants)

detected in detected in SOS1SOS1//RAF1RAF1 from 110 samples from 110 samples

• Overall pick up rate for our cohort is ~10%Overall pick up rate for our cohort is ~10%

• No mutations identified in No mutations identified in KRASKRAS

• dHPLC WAVE is a more robust pre-screening method compared dHPLC WAVE is a more robust pre-screening method compared to LightScannerto LightScanner™ HRM™ HRM

• Complex genotype-phenotype correlation Complex genotype-phenotype correlation

• Three stage screening strategy designed for NS/LS referrals from Three stage screening strategy designed for NS/LS referrals from April 2008April 2008


Further WorkFurther Work

Taken from EMBO reports 6, 12, 1169–1175 (2005)


AcknowledgementsAcknowledgementsThank you: Thank you: • John ShortJohn Short• Navaratnam ElankoNavaratnam Elanko• Roy PohRoy Poh• Sally CottrellSally Cottrell• Rohan Taylor Rohan Taylor • Professor Michael PattonProfessor Michael Patton

• Kamini Kalidas (Clinical Developmental Sciences, St Kamini Kalidas (Clinical Developmental Sciences, St George’s University of London)George’s University of London)

• IDEAS Knowledge Park for funding this projectIDEAS Knowledge Park for funding this project

and all staff at Molecular Genetics Lab at St George’sand all staff at Molecular Genetics Lab at St George’s

sandra ramos grade a project st george’s hospital, london

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