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Conference Paper: Identifying Genetic Mutations in Patients with Rasopathies Using a Next Generation Sequencing Diagnostic Pipeline in Hong Kong

TitleIdentifying Genetic Mutations in Patients with Rasopathies Using a Next Generation Sequencing Diagnostic Pipeline in Hong Kong
Authors
Issue Date2014
PublisherMedcom Limited. The Journal's web site is located at http://www.hkjpaed.org/index.asp
Citation
The 2013 Annual Scientific Meeting of the Hong Kong College of Paediatricians, Hong Kong, China, 7 December 2013. In Hong Kong Journal of Paediatrics (New series), 2014, v. 19 n. 2, p. 117-118 How to Cite?
AbstractBackground and aims: RASopathies are a group of developmental syndromes, namely Noonan, Costello, Cardio-facio-cutaneous, Legius, and LEOPARD syndromes, collectively affecting 1 in 2000 livebirths. They were caused by mutations in genes involved in the RAS/ MAPK signaling pathway. Since they are characterised by clinical overlap and genetic heterogeneity, diagnoses are often challenging. We aim to develop a NGS-based strategy for molecular diagnosis of RASopathies. Method: Targeted NGS of 13 genes (A2ML1, BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, RAF1, SHOC2, SOS1, and SPRED1) in the RAS/MAPK pathway, where the targeted enrichment panel covered 98% of the gene coding regions, was performed on 57 RASopathies which were previously tested negative for mutation in PTPN11 and HRAS. Positive controls were run in parallel. Results: The average read-depth in the regions of interest was >500X, with 99% of target bases reaching minimal coverage of 30X. Eighteen known pathogenic mutations (SOS1, n=6; RAF1, n=2; KRAS, n=3; BRAF, n=2; SHOC2, n=2; or MAP2K1, n=3) were detected in 18/57 (32%) patients. Three novel mutations (1 nonsense and 2 missense) were found in four patients. All detected mutations were confirmed by Sanger sequencing. The novel missense mutations are in-silico demonstrated to be deleterious and are absent in unaffected control populations. Detailed genotype-phenotype correlation analysis is in progress. Functional analysis using Elk-1 reporter system and zebrafish modeling is underway to examine the pathogenicity of these novel mutations. Conclusion: To our knowledge, this study has the largest sample size of PTPN11 and HRAS negative patients from Hong Kong who received diagnosis of RASopathies from clinical geneticists. Our study has demonstrated that the strategy involving targeted NGS analysis can achieve an addition detection rate of 32%, showing an improvement over the conventional Sanger sequencing analysis merely of PTPN11 and HRAS mutations for RASopathies. Clinical correlations, customised bioinformatics pipelines and follow-up molecular characterisation in cell cultures or animal models are important to delineate the pathogenic role of novel mutations identified by NGS. Acknowledgement: We would like to thank the families for their participation and the SK Medical Foundation and SK Yee Medical Research Fund for financial support.
Persistent Identifierhttp://hdl.handle.net/10722/193284
ISSN
2015 Impact Factor: 0.194
2015 SCImago Journal Rankings: 0.123

 

DC FieldValueLanguage
dc.contributor.authorLo, IFMen_US
dc.contributor.authorLeung, KCen_US
dc.contributor.authorLuk, HMen_US
dc.contributor.authorTang, VHMen_US
dc.contributor.authorGao, Wen_US
dc.contributor.authorWong, WLen_US
dc.contributor.authorTang, LYFen_US
dc.contributor.authorChu, WYen_US
dc.contributor.authorKan, SYAen_US
dc.contributor.authorTang, MHYen_US
dc.contributor.authorLam, STSen_US
dc.contributor.authorYang, Wen_US
dc.contributor.authorMa, CHen_US
dc.contributor.authorJin, Den_US
dc.contributor.authorChan, YKen_US
dc.contributor.authorChung, BHYen_US
dc.date.accessioned2013-12-20T02:43:50Z-
dc.date.available2013-12-20T02:43:50Z-
dc.date.issued2014en_US
dc.identifier.citationThe 2013 Annual Scientific Meeting of the Hong Kong College of Paediatricians, Hong Kong, China, 7 December 2013. In Hong Kong Journal of Paediatrics (New series), 2014, v. 19 n. 2, p. 117-118en_US
dc.identifier.issn1013-9923-
dc.identifier.urihttp://hdl.handle.net/10722/193284-
dc.description.abstractBackground and aims: RASopathies are a group of developmental syndromes, namely Noonan, Costello, Cardio-facio-cutaneous, Legius, and LEOPARD syndromes, collectively affecting 1 in 2000 livebirths. They were caused by mutations in genes involved in the RAS/ MAPK signaling pathway. Since they are characterised by clinical overlap and genetic heterogeneity, diagnoses are often challenging. We aim to develop a NGS-based strategy for molecular diagnosis of RASopathies. Method: Targeted NGS of 13 genes (A2ML1, BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, RAF1, SHOC2, SOS1, and SPRED1) in the RAS/MAPK pathway, where the targeted enrichment panel covered 98% of the gene coding regions, was performed on 57 RASopathies which were previously tested negative for mutation in PTPN11 and HRAS. Positive controls were run in parallel. Results: The average read-depth in the regions of interest was >500X, with 99% of target bases reaching minimal coverage of 30X. Eighteen known pathogenic mutations (SOS1, n=6; RAF1, n=2; KRAS, n=3; BRAF, n=2; SHOC2, n=2; or MAP2K1, n=3) were detected in 18/57 (32%) patients. Three novel mutations (1 nonsense and 2 missense) were found in four patients. All detected mutations were confirmed by Sanger sequencing. The novel missense mutations are in-silico demonstrated to be deleterious and are absent in unaffected control populations. Detailed genotype-phenotype correlation analysis is in progress. Functional analysis using Elk-1 reporter system and zebrafish modeling is underway to examine the pathogenicity of these novel mutations. Conclusion: To our knowledge, this study has the largest sample size of PTPN11 and HRAS negative patients from Hong Kong who received diagnosis of RASopathies from clinical geneticists. Our study has demonstrated that the strategy involving targeted NGS analysis can achieve an addition detection rate of 32%, showing an improvement over the conventional Sanger sequencing analysis merely of PTPN11 and HRAS mutations for RASopathies. Clinical correlations, customised bioinformatics pipelines and follow-up molecular characterisation in cell cultures or animal models are important to delineate the pathogenic role of novel mutations identified by NGS. Acknowledgement: We would like to thank the families for their participation and the SK Medical Foundation and SK Yee Medical Research Fund for financial support.-
dc.languageengen_US
dc.publisherMedcom Limited. The Journal's web site is located at http://www.hkjpaed.org/index.asp-
dc.relation.ispartofHong Kong Journal of Paediatrics (New series)en_US
dc.titleIdentifying Genetic Mutations in Patients with Rasopathies Using a Next Generation Sequencing Diagnostic Pipeline in Hong Kongen_US
dc.typeConference_Paperen_US
dc.identifier.emailLeung, KC: gleungkc@hku.hken_US
dc.identifier.emailTang, VHM: tanghmv@hku.hken_US
dc.identifier.emailGao, W: gaoww@hku.hken_US
dc.identifier.emailWong, WL: wlapwong@hku.hken_US
dc.identifier.emailChu, WY: chuwyy@hku.hken_US
dc.identifier.emailKan, SYA: kansya@hku.hken_US
dc.identifier.emailTang, MHY: mhytang@hkucc.hku.hken_US
dc.identifier.emailYang, W: yangwl@hkucc.hku.hken_US
dc.identifier.emailMa, CH: alvinma@hku.hken_US
dc.identifier.emailJin, D: dyjin@hku.hken_US
dc.identifier.emailChan, YK: ykchanc@hku.hken_US
dc.identifier.emailChung, BHY: bhychung@hku.hken_US
dc.identifier.authorityTang, MHY=rp01701en_US
dc.identifier.authorityYang, W=rp00524en_US
dc.identifier.authorityMa, CH=rp01810en_US
dc.identifier.authorityJin, D=rp00452en_US
dc.identifier.authorityChan, YK=rp00453en_US
dc.identifier.authorityChung, BHY=rp00473en_US
dc.identifier.hkuros227033en_US
dc.identifier.volume19-
dc.identifier.issue2-
dc.identifier.spage117en_US
dc.identifier.epage118en_US
dc.publisher.placeHong Kong-

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