7 research outputs found
Holistic Health Status Questionnaire: developing a measure from a Hong Kong Chinese population
Massive parallel sequencing for the genetic diagnosis of rasopathies: a study of 58 Chinese patients in Hong Kong
Poster Presentatio
Identifying genetic mutations in patients with Rasopathies using a Next Generation Sequencing Diagnostic Pipeline in Hong Kong
Background 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