SW Thames Molecular Genetics Diagnostic Laboratory Analysis of three genes Analysis of three genes from the RAS-MAPK from the RAS-MAPK signalling pathway that signalling pathway that are causative of are causative of Noonan/LEOPARD syndromes Noonan/LEOPARD syndromes Sandra Ramos Sandra Ramos Grade A Project Grade A Project St George’s Hospital, St George’s Hospital, London London
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SW Thames Molecular Genetics Diagnostic Laboratory Analysis of three genes from the RAS-MAPK signalling pathway that are causative of Noonan/LEOPARD syndromes.
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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
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
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
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
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
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
WAVE v LightScannerWAVE v LightScanner™™• Primers designed using LightScannerPrimers designed using LightScanner™™ primer design primer design softwaresoftware
• 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)
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
WAVE v LightScanner™ resultsWAVE v LightScanner™ results
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
Samples pre-screened on the Transgenomic WAVE Samples pre-screened on the Transgenomic WAVE and variants sequenced using ABI3730and variants sequenced using ABI3730
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
• Three stage screening strategy designed for NS/LS referrals from Three stage screening strategy designed for NS/LS referrals from April 2008April 2008
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