Saira Yousoof

Children's Medical Research Institute

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Robyn V. Jamieson

University of Sydney

Anson Cheng

University of Rochester

Alan Ma

Sydney Children’s Hospitals Network

John Grigg

University of Sydney

Benjamin M. Nash

Children's Hospital at Westmead

Meredith Wilson

Children's Hospital at Westmead

Ivan Prokudin

Children's Medical Research Institute

I. Christopher Lloyd

University College London

Elizabeth Farnsworth

Children's Hospital at Westmead

Daniel Speidel

Children's Medical Research Institute

Cooperative Institutions

University of Sydney

Children's Hospital at Westmead

Genome Institute of Singapore

Sydney Children’s Hospitals Network

Children's Medical Research Institute

UNSW Sydney

Agency for Science, Technology and Research

National University of Singapore

Sydney Children's Hospital

Royal Children's Hospital

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A comparison study of droplet digital PCR and qPCR for EGFR T790M detection in plasma of NSCLC patients

DOAJ (DOAJ: Directory of Open Access Journals) (2017)

Chien Wen Tay Che Zairieha Raja Iswara Saira Yousoof Yoke Kqueen Cheah

Background: Specific mutations in epidermal growth factor receptor ( EGFR ) are predictive for response to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) patients. However, most patients acquire resistance to the first generation-TKIs and in about half of these patients the resistance is driven by EGFR T790M mutation. Recently, third generation-TKIs are shown to be effective in patients harbouring this mutation. Although re-biopsy of tumour tissue is regarded as a gold standard in recurrent and NSCLC patients, the procedure can be challenging and hazardous, and tumour heterogeneity between primary and metastatic sites can be a limiting factor for accurate mutational analysis. In recent years, liquid biopsy for the detection of circulating tumour DNA (ctDNA) has been used for EGFR mutation analysis using qualitative PCR assays. Since the detection rate of qPCR for T790M is only 30-50%, droplet digital PCR (ddPCR) has emerged as a more reliable assay for the detection of T790M in ctDNA. In the present study, we compared the performance of ddPCR and qPCR in plasma of patients with NSCLC. Methods: Plasma were collected from NSCLC patients diagnosed in the Subang Jaya Medical Centre. Extracted ctDNA were analysed for three common EGFR mutations (exon 19 deletions, L858R, T790M) using both Droplet Digital PCR (ddPCR TM ; BioRad) and qPCR (PANAMutyper ™ R EGFR). Results : In our study, a preliminary assessment using both platforms was performed with 25 EGFR positive plasma samples. Of these, 11 samples had tumour tissues previously tested for EGFR mutations with qPCR (PNAClamp EGFR test kit). We retrospectively applied ddPCR and qPCR assays to detect exon 19 deletions, L858R and T790M in all the plasma samples. EGFR mutation analysis based on tumour biopsy samples were used as the reference standard. Overall, our results showed about 70% concordance between these two assays. Digital PCR appeared to have a greater sensitivity for T790M mutation (40%) compared to qPCR (28%). Conclusion : Droplet digital PCR may have good application prospects in monitoring patients with EGFR T790M mutation in selecting patients for third generation EGFR -TKI treatment.

T790M

Liquid biopsy

Resistance mutation

Osimertinib

Afatinib

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Mutations inSIPA1L3cause eye defects through disruption of cell polarity and cytoskeleton organization

Human Molecular Genetics (2015)

Rebecca Greenlees Marija Mihelec Saira Yousoof Daniel Speidel Selwin K. Wu

Correct morphogenesis and differentiation are critical in development and maintenance of the lens, which is a classic model system for epithelial development and disease. Through germline genomic analyses in patients with lens and eye abnormalities, we discovered functional mutations in the Signal Induced Proliferation Associated 1 Like 3 (SIPA1L3) gene, which encodes a previously uncharacterized member of the Signal Induced Proliferation Associated 1 (SIPA1 or SPA1) family, with a role in Rap1 signalling. Patient 1, with a de novo balanced translocation, 46,XY,t(2;19)(q37.3;q13.1), had lens and ocular anterior segment abnormalities. Breakpoint mapping revealed transection of SIPA1L3 at 19q13.1 and reduced SIPA1L3 expression in patient lymphoblasts. SIPA1L3 downregulation in 3D cell culture revealed morphogenetic and cell polarity abnormalities. Decreased expression of Sipa1l3 in zebrafish and mouse caused severe lens and eye abnormalities. Sipa1l3−/− mice showed disrupted epithelial cell organization and polarity and, notably, abnormal epithelial to mesenchymal transition in the lens. Patient 2 with cataracts was heterozygous for a missense variant in SIPA1L3, c.442G>T, p.Asp148Tyr. Examination of the p.Asp148Tyr mutation in an epithelial cell line showed abnormal clustering of actin stress fibres and decreased formation of adherens junctions. Our findings show that abnormalities of SIPA1L3 in human, zebrafish and mouse contribute to lens and eye defects, and we identify a critical role for SIPA1L3 in epithelial cell morphogenesis, polarity, adhesion and cytoskeletal organization.

Adherens junction

Cell polarity

10.1093/hmg/ddv298

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Citations (39)

Detection of Chromosomal Breakpoints in Patients with Developmental Delay and Speech Disorders

PLoS ONE (2014)

Kagistia Hana Utami Axel M. Hillmer Irène Aksoy Elaine Guo Yan Chew Audrey S.M. Teo

Delineating candidate genes at the chromosomal breakpoint regions in the apparently balanced chromosome rearrangements (ABCR) has been shown to be more effective with the emergence of next-generation sequencing (NGS) technologies. We employed a large-insert (7–11 kb) paired-end tag sequencing technology (DNA-PET) to systematically analyze genome of four patients harbouring cytogenetically defined ABCR with neurodevelopmental symptoms, including developmental delay (DD) and speech disorders. We characterized structural variants (SVs) specific to each individual, including those matching the chromosomal breakpoints. Refinement of these regions by Sanger sequencing resulted in the identification of five disrupted genes in three individuals: guanine nucleotide binding protein, q polypeptide (GNAQ), RNA-binding protein, fox-1 homolog (RBFOX3), unc-5 homolog D (C.elegans) (UNC5D), transmembrane protein 47 (TMEM47), and X-linked inhibitor of apoptosis (XIAP). Among them, XIAP is the causative gene for the immunodeficiency phenotype seen in the patient. The remaining genes displayed specific expression in the fetal brain and have known biologically relevant functions in brain development, suggesting putative candidate genes for neurodevelopmental phenotypes. This study demonstrates the application of NGS technologies in mapping individual gene disruptions in ABCR as a resource for deciphering candidate genes in human neurodevelopmental disorders (NDDs).

Sanger sequencing

Breakpoint

Candidate gene

10.1371/journal.pone.0090852

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Citations (51)

Revealing hidden genetic diagnoses in the ocular anterior segment disorders

Genetics in Medicine (2020)

Alan Ma Saira Yousoof John Grigg Maree Flaherty André E. Minoche

Ocular anterior segment disorders (ASDs) are clinically and genetically heterogeneous, and genetic diagnosis often remains elusive. In this study, we demonstrate the value of a combined analysis protocol using phenotypic, genomic, and pedigree structure data to achieve a genetic conclusion.We utilized a combination of chromosome microarray, exome sequencing, and genome sequencing with structural variant and trio analysis to investigate a cohort of 41 predominantly sporadic cases.We identified likely causative variants in 54% (22/41) of cases, including 51% (19/37) of sporadic cases and 75% (3/4) of cases initially referred as familial ASD. Two-thirds of sporadic cases were found to have heterozygous variants, which in most cases were de novo. Approximately one-third (7/22) of genetic diagnoses were found in rarely reported or recently identified ASD genes including PXDN, GJA8, COL4A1, ITPR1, CPAMD8, as well as the new phenotypic association of Axenfeld-Rieger anomaly with a homozygous ADAMTS17 variant. The remainder of the variants were in key ASD genes including FOXC1, PITX2, CYP1B1, FOXE3, and PAX6.We demonstrate the benefit of detailed phenotypic, genomic, variant, and segregation analysis to uncover some of the previously "hidden" heritable answers in several rarely reported and newly identified ocular ASD-related disease genes.

PAX6

Genetic Analysis

Candidate gene

Exome

10.1038/s41436-020-0854-x

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Citations (42)