Trisomy 2 mosaicism with high trisomic fraction identified by cell-free DNA screening is associated with an increased risk for adverse feto-placental outcomes

Mark D Pertile1,2, Nicola Flowers1, Grace Shi1, Olivia Giouzeppos1, Shelley Baeffel1, Ian Burns1, Tom Harrington1, Rebecca Manser1, Absera Tsegay1, Ralph Oertel1, Fiona Norris1.

1 Victorian Clinical Genetics Services, Murdoch Childrens Research Institute, Royal Children’s Hospital, Flemington Road, Parkville, VIC, 3052
2 Department of Paediatrics, University of Melbourne, Parkville, VIC, 3010.

Whole genome sequencing (WGS) of maternal plasma cell-free DNA (cfDNA) can potentially evaluate all 24 chromosomes to identify abnormalities of the placenta or fetus. We have systematically analysed WGS data from all chromosomes to identify rare autosomal trisomies (RATs) to improve our understanding of discordant findings and feto-placental biology. Here, we describe our experience with prenatally ascertained trisomy 2 mosaicism.

Trisomy 2 usually presents as a benign mosaic finding at CVS, commonly confined to the chorionic mesenchyme and rarely involving the cytotrophoblast. Involvement of the cytotrophoblast increases the likelihood that the conception is trisomic, with subsequent increased risk for true fetal mosaicism (TFM) and uniparental disomy (UPD) following trisomy rescue.

We have identified six cases of trisomy 2 mosaicism ascertained by cfDNA screening. In five samples, the mean ratio of the trisomic fraction (TF) to fetal fraction (FF) approached 1.0 (TF:FF = 0.83 ± SD 0.09), consistent with very high levels of trisomy 2 cells in placental cytotrophoblast. These pregnancies were associated with TFM, UPD, intrauterine growth restriction (IUGR) and/or intrauterine fetal demise (IUFD), in keeping with a meiotic origin of the trisomy. In the sixth , the mean ratio of the trisomic fraction to fetal fraction was low (TF:FF = 0.25). This pregnancy had normal prenatal investigations and continued to an uncomplicated term delivery. Examination of chorionic villi from the term placenta indicated the trisomy 2 mosaicism was most likely of mitotic (post zygotic) origin.

This data, together with our experience involving other RATs, suggests cfDNA screening can potentially identify those pregnancies at highest risk for relevant fetal-placental complications.



Dr. Mark Pertile is a senior medical scientist who specialises in reproductive cytogenetics and genomics. He is currently the Deputy Director of Laboratories at VCGS, Heads the Division of Reproductive Genetics and is also Head of the NIPT Laboratory. Mark has a long standing interest in early human embryology and development. He works with a team that applies molecular cytogenetics and genomics technologies to help identify the causes and origins of genomic conditions early in pregnancy.

PGD for HLA matching and mutation detection by Karyomapping

Paisu Tang1, Andrea P. Twomey, Anke E. Kohfahl, Sharyn E. Stock-Myer

1 Virtus Diagnostics, Melbourne IVF, 344 Victoria Parade, Victoria, 3002

The most effective treatment option for many acquired and inherited paediatric haematological disorders is Haematopoietic stem cell transplantation (HSCT).  Preimplantation genetic diagnosis (PGD) for HLA typing is an established procedure for assisting a couple to have a healthy sibling who can donate cord blood or haematopoietic stem cells to an ill child. PGD-HLA in Australia is performed under guidelines provided by the NHMRC.

Traditional PGD for HLA typing involves the search for informative STR markers and the establishment of a family-specific multiplex PCR test. While effective, this method typically requires a significant investment of time which is often precious in the case of acute haematological failure such as leukaemia and aplastic anaemia.

The recent availability of Karyomapping, a high resolution SNP array system, has dramatically reduced the test development time in PGD for HLA typing and single gene disorders. Karyomapping eliminates the need for patient-specific tests and allows for the simultaneous analysis of multiple loci and chromosome copy number for all 24 chromosomes in the human genome. Thus it is possible to diagnose the HLA compatibility, mutation status and aneuploidy status of an embryo within the one test.

We report on the application of Karyomapping for beta thalassaemia and HLA typing in 3 families. Each couple had one child affected with beta thalassaemia and their eligibility for PGD-HLA was assessed by our Reproductive Services Clinical Review Committee according to NHMRC guidelines. Overall, a total of 17 embryos were tested with a diagnostic efficiency of 100%.  One family delivered a healthy HLA-matched child, one family has an ongoing pregnancy and the third family is yet to produce a genetically suitable embryo. We discuss the benefits and limitations of Karyomapping for PGD-HLA.


PhD in Genetics, have been working as a PGD scientist at Virtus Diagnostics-Melbourne IVF for 8 years.

PGD for reciprocal translocation and CNVs using Karyomapping

Sharyn Stock-Myer1, Andrea Twomey, Paisu Tang, Anke Kohfahl, Mirjana Martic

1 Virtus Diagnostics, Melbourne IVF, 344 Victoria Parade, East Melbourne 3002,

Pre-implantation Genetic Diagnosis (PGD) allows the diagnosis of known gene disorders from pre-implantation embryos significantly reducing the risk of ongoing affected pregnancy in a couple at known risk for a particular disorder.  PGD for reciprocal translocations typically utilises either array CGH or Next Generation Sequencing to detect unbalanced segregants and allows embryos to be transferred that have normal copy number DNA.  However, using these technologies it is impossible to distinguish embryos that are balanced and have the derivative chromosomes from those that have inherited normal chromosomes.  Additionally, these technologies are unable to reliably detect pathogenic CNVs (below about 5 Mb in size) that may be inherited by embryos from known carrier parents.

Karyomapping, a recently developed PGD technology that utilises a SNP array, was developed as a universal tool for diagnosing single gene disorders by linkage analysis. Our aim was to attempt to use this method to distinguish embryos that are balanced from those that have inherited normal chromosomes, and also to diagnose very small CNVs by linkage (and in the case of deletions, also by direct detection).

To date, we have successfully used Karyomapping to perform PGD for duplications and deletions ranging from 0.2 – 2.5 Mb in size in 13 carrier couples and also for one translocation case.  In this translocation case we were clearly able to see unbalanced segregants, as well distinguish embryos that had inherited balanced segregants with derivative chromosomes from those that had inherited normal chromosomes only.

Karyomapping is a reliable method for performing PGD for CNVs below the level of detection of other technologies.  This method has also been used successfully to perform PGD for a translocation case and can enable the preferential transfer of embryos with normal chromosomes if available and clinically indicated.



Sharyn has been working for Melbourne IVF since 2002 when she was employed to establish PGD services for monogenic patients.  She has continued running this program since that time, and in 2015 took over role of Scientific Director for Pre-implantation Genetics.  She is honoured to be able to help couples in their dream to have a healthy child.

PGD for reciprocal translocations using next-generation sequencing

Mirjana Martic1, Sophie Falle1, Katerina Mitsiou1, Maria Kemeridis1, Sharyn Stock-Myer1

1 Virtus Diagnostics, Melbourne IVF, 344 Victoria Parade, East Melbourne 3002

The 24sure+ BAC array Comparative Genomic Hybridisation (CGH) system has been the first commonly available technology for many years used to detect unbalanced segregants in the embryos of reciprocal translocation carriers in Preimplantation Genetic Diagnosis (PGD) programs.  Next generation sequencing (NGS) based testing has recently been introduced and widely accepted for aneuploidy testing. Here we present the application and limitation of a NGS based chromosome testing system, VeriSeq (Illumina, USA) on single cells and trophectoderm (TE) samples for reciprocal translocation carriers.

Aliquots of whole genome amplified DNA (Sureplex, Illumina, USA) from either single cells or TE samples of embryos diagnosed as unbalanced using arrayCGH, were subjected to NGS. Amplified samples were from carriers of 15 different reciprocal translocations with varied sizes of the unbalanced segments. The chromosome status of these samples was analysed and concordance determined.

Thirteen single blastomeres and 27 TE samples were amplified with 100% efficiency and results were obtained on 100% of amplified samples. NGS confirmed clinical diagnosis predicted by arrayCGH in all samples. Although individual segmental imbalances were detected in all samples, two unbalanced segments smaller than 6 Mb were not as apparent in the NGS profile as they were in the arrayCGH.  Additionally, the NGS system was able to detect full aneuploidy and mosaicism of other chromosomes not involved in the translocation.

The NGS system is a reliable alternative to array CGH to detect large unbalanced chromosome segments as well as full aneuploidy and mosaicism of any other chromosome in single blastomeres and TE samples. In reciprocal translocation carriers in which unbalanced segments are smaller than 6 Mb, trophectoderm biopsy has to be requested and the confirmation of results by array CGH is strongly recommended.


Mirjana is a Preimplantation Genetics Laboratory manager at Virtus Diagnostics. She completed her Medical degree at the University of Zagreb, Croatia and her PhD at the University of Melbourne. Initially, she worked as an embryologist at Melbourne IVF and for the last 13 years she has been working in Preimplantation Genetics.

Assessing unknown sequence variations in thalassaemia diagnosis

Kerryn M Weekes1, Wendy M Hutchison 1, Jeremy N Wells 1, Nicholas Clarke 1, Anastasia Adrahtas1, Lily Li 1, Jesse Pinguinha 1, Asif Alam1, Elizabeth Algar 2, Zane Kaplan.3

1 Thalassaemia and Haemophilia Molecular Reference Laboratory, L3 Monash Medical Centre 246 Clayton Rd Clayton Vic 3168, Email:
2 Genetics and Molecular Pathology, L3 Monash Medical Centre 246 Clayton Rd Clayton Vic 3168

3 Medical Therapy Unit, L2 Monash Medical Centre 246 Clayton Rd Clayton Vic 3168

Thalassaemia and haemoglobinopathies are among the most common monogenic recessive disorders and are caused by mutations in both the alpha and beta globin gene complexes resulting in either decreased globin production or structural anomalies associated with distinct phenotypes. They are among only a few recessive disorders presenting with a mild phenotype in the carrier state that enable carriers to be detected during routine blood tests.

Recent advances in genetic testing for thalassemia have revealed increasing complexity with carriers described with mutations affecting more than one globin gene. Due to the increasing demand for thorough investigation of couples with mild phenotypic changes, many sequence variations in multiple genes are being discovered by our laboratory that are not reported in any of the databases or in scientific literature. The challenge is to evaluate the pathogenicity of these variations so that an accurate risk assessment for a couple can be reported.

We present a cohort of gene sequence variations and the difficulty in categorising them as pathogenic or benign using the current guidelines and standards.


Kerryn is the senior scientist in the Thalassaemia and Haemophilia Molecular Reference Laboratory. This laboratory is the Victorian reference laboratory and provides carrier, proband and prenatal testing for thalassaemia and haemophilia.  Kerryn has been senior scientist for nearly 10 years and during this time the laboratory has grown 10-fold.  She obtained her fellowship of the HGSA in 2016, one of the last candidates to receive a HGSA fellowship.

Fragile X syndrome FMR1 CGG retractions; Not so rare?

David I Francis1, Matthew Hunter2, Yael Prawer2, Zornitza Stark1, Robin Forbes1, David Amor 1,Mike Field3, Carolyn Rogers3, Ralph Oertel1, Sara Cronin1, Amber Boys1, Olivia Giouzeppos1, Essra Bartlett1, David Godler4

1 Victorian Clinical Genetics Service, Murdoch Childrens Research Institute, Flemington Road, Parkville, VIC, 3052.
2 Genetics Dept., Monash Health, Monash Medical Centre, 246 Clayton Rd, Clayton VIC 3168.
3 GOLD Service, PO Box 84, Waratah NSW 2298
4 Murdoch Childrens Research Institute, Flemington Road, Parkville, VIC, 3052.

FMR1 premutation (PM: 55-199 CCGs) alleles are highly unstable upon maternal transmission expanding at high frequency into the full mutation (FM: = or >200 CGGs) range. However there is increasing evidence that FM’s are highly unstable postzygotically, retracting into smaller size alleles, including the intermediate (45-54 CGGs) and normal (<45 CGGs) range. This has important implications for counselling, as mosaic individuals are less severely affected due to expression of FMR1; thought to be silent in non-mosaic fully methylated FM alleles. I present conclusive evidence in monozygous Fragile X twins of postzygotic retraction as well as three other FXS cases demonstrating complete retraction into the normal range. Further evidence of instability is confirmed by analysis of all VCGS male Southern Blot detected positive cases showing a high incidence of PM/FM mosaics of 25%, comparable to the literature which is variable (average of 26%).  This is likely to be an under representation of mosaicism as Southern blot cannot detect mosaic alleles if present in less than 20% of cells (Aliaga et al 2016 Clin Chem). Further studies of 100 males with typical FXS (manuscript in preparation) showed a significantly higher proportion of mosaic FMR1 alleles was detected in FM males using a more sensitive test, termed MS-QMA, than by Southern blot. Together, this suggests that mosaicism is currently under diagnosed by standard testing and retraction from FM to smaller size alleles is not as uncommon as previously thought.


Over 20 years of experience in the Cytogenetics/Molecular diagnostic field. I have published widely in the cyto/molecular area and currently involved in Fragile X syndrome and Prader Willi syndrome research projects. Recently appointed as the Head of the Postnatal Cytogenetics Laboratory at VCGS and was the past/last President of ASoC.

Molecular subtyping of medulloblastoma

R. Fraser1, C.C. Young1, M. Nicola1, J. Suttle1, R. Kenyon 2, N. Manton3, S. Moore1

1 SA Cancer Cytogenetics Unit, Genetics and Molecular Pathology Directorate, SA Pathology, Adelaide, South Australia
2 ACRF Cancer Genome Facility, Adelaide, South Australia
3 Anatomical Pathology Directorate, Women’s and Children’s Hospital, Adelaide, South Australia

Medulloblastoma is a highly malignant embryonal tumour of the cerebellum, and is the most common form of malignant paediatric brain tumour. Treatment involves surgical resection, and in some cases radiation therapy and chemotherapy. Current recurrence free survival is approximately 50-70%. Traditional clinical prognostication and stratification incorporates clinical factors (age, presence of metastases, extent of resection), histological/immunohistochemical subgrouping, and tumour cytogenetics. More recently, molecular profiling of medulloblastoma cases has suggested the existence of distinct subgroups that differ in their demographics, transcriptomes, somatic genetic events, and clinical outcomes. Consensus was reached recently for four main molecular subgroups of medulloblastomoa (ie Wnt, Shh, Group 3, and Group 4) and there is evidence to suggest that some arise from different cellular origins. The demanding nature of tumour culturing and karyotyping has led us to investigate the use of SNP-array analysis to provide critical somatic genetic information to help stratify patients into one of these subgroups. We now report the results of a small pilot study of local medulloblastoma patients for whom we have performed SNP-array and karyotyping and compared our data with the Children’s Oncology Group findings. SNP-array results were easier to interpret than karyotyping, could be performed in a shorter time frame, since there is no need for culture of FISH analysis, and resulted in correct molecular classification of medulloblastoma.


Rachel works as a medical scientist in the cancer cytogenetics laboratory in Adelaide.

Improved detection of cytogenomic prognostic markers by chromosome microarray in a group of clinically heterogeneous neuroblastoma patients

Dale C Wright1, Luke St Heaps1, Geoff McCowage2, Nicole Graf3.

1The Children’s Hospital at Westmead, Sydney Genome Diagnostics, Cytogenetics, Westmead
2The Children’s Hospital at Westmead, Oncology, Westmead
3The Children’s Hospital at Westmead, Anatomical Pathology, Westmead

BACKGOUND: Neuroblastomas show genetic and clinical heterogeneity. They can occur in infants with subsequent spontaneous regression, be localised with favourable outcome, or become refractory to treatment. Prognostic markers include age, histology and tumour stage, and genetic/genomic alterations. The latter includes DNA-ploidy, MYCN amplification (MYCNA), loss of heterozygosity (LOH) of 1p/11q and other segmental chromosome abnormalities (SCA); e.g. loss 17p, gain 1q/17q, among others. LOH/ SCA detection can be performed by FISH and/or microsatellite STR markers but compared to chromosome microarray (CMA) can be laborious. This study aimed to improve prognostic cytogenomic marker detection in neuroblastoma.

METHOD: Thirteen neuroblastoma patients [five retrospective, eight prospective] were investigated with median age at diagnosis 6 months [range: 2 – 72] and varying tumour stage: poorly differentiated, III, IV and IV-S. Fresh/frozen tissue was analysed using a customised 8x60K CGH+SNP cancer microarray [5Mb resolution] (Agilent Technologies). FISH was performed on touch-imprints using MYCN and chromosome 1p [TP73] probes on 12/13 and 2/13 patients, respectively. Karyotyping was attempted on three tumours.

RESULTS: CMA identified ploidy and MCYN status, and other SCAs in 11/13 tumours. One showed no abnormality and one failed. A single MYCNA tumour was diploid with LOH 1p and 12 SCAs. The remaining showed hyperdiploidy only (n=4), hyperdiploidy with SCA (n=5) [four with LOH 11q], hypodiploidy LOH 1p with three SCA (n=1), and diploidy with LOH 1p (n=1). These findings confirmed MYCN FISH, where one showed MYCNA and 9/11 tumours showed increased MYCN copy number (3-6) but NOT amplification. Two showed three 1p copies. Tumour karyotypes showed no abnormality (n=2) or failed (n=1).

CONCLUSION: CMA overcomes the challenges of karyotyping and targeted FISH, providing improved detection of ploidy and cytogenomic markers. However, rapid MYCN FISH remains important. Along with age, tumour stage and MYCN status, CMA can identify ploidy and LOH/SCA that refines patient risk-stratification.


Dale is a Principal Hospital Scientist and Head of Cytogenetics for Sydney Genome Diagnostics at The Children’s Hospital at Westmead, Sydney. He has wide experience in Clinical Cytogenetics, mostly involving infertility, prenatal and preimplantation genetic diagnosis, but more recently has been working with microarrays in multiple myeloma and paediatric solid tumours.

Interpretation of a complex chromosome 18 SNP array pattern in AML

MacKinnon R1,2, Wall M1,2,3

1Victorian Cancer Cytogenetics Service, St Vincent’s Hospital, Melbourne, 2Department of Medicine (St Vincent’s), University of Melbourne, 3St Vincent’s Institute of Medical Research


Abstract to come.


Dr MacKinnon has investigated dicentric chromosome abnormalities in AML and MDS during her time at the Victorian Cancer Cytogenetics Service. Previously she has worked on fragile sites at the Adelaide Children’s Hospital, the Fragile X at the Institute of Molecular Medicine, Oxford, and cattle genomics at the CSIRO.

Inversions within the MSH2 gene

Anna C Ritchie1, Shannon Cowie1, Desirée du Sart1.

1Molecular Genetics Laboratory, VCGS Pathology, Murdoch Childrens Research Institute, Flemington Rd, Parkville, Victoria 3052.

Lynch syndrome is caused by a germline mutation in a mismatch repair gene (MLH1, MSH2, MSH6, PMS2 and EPCAM) resulting in a predisposition to colorectal and other cancers.  Testing of these MMR genes by Next Generation sequencing and MLPA analysis will identify point mutations, small deletions and insertions, splice site mutations as well as intragenic and whole gene deletions and duplications. However, gene rearrangements, such as inversions and chromosome translocations, will not be detected.

A 10Mb inversion within the MSH2 gene was initially identified by Wagner et al. 2002 and a further study by Rhees et al. 2014 found that six out ten previously unexplained MSH2-type Lynch syndrome families had this inversion.  To assist in identifying these mutations, recently two new probes have been introduced into the MCR-Holland P003-D1 MLPA kit to detect this recurrent pathogenic inversion.  The assay focuses on the breakpoint within intron 7 of the MSH2 gene which will bind and produce a peak at 265 and 317nt when the inversion is present.  We have confirmed the same 10Mb inversion by PCR amplification and gel electrophoresis in a family where this inversion was previously identified.  We have also identified another inversion of exons 2 to 6 within the MSH2 gene in a different family with a history of Lynch syndrome, which will not be detected by the MLPA assay.  It is currently unclear how common inversions within the MSH2 gene are and further testing of intronic regions within this gene would be required to gain a better understanding.  We will present our data of screening for the 10Mb inversion and the exon 2 to 6 inversion in mutation-negative Lynch syndrome patients to determine whether these are common inversions and whether a more universal screening method should be developed to identify other inversions within the MSH2 gene.


Anna Ritchie is a medical scientist in the Molecular Genetics laboratory within VCGS pathology in Melbourne.  She is a Grade 2 scientist, obtaining her HGSA membership in 2011, and has been part of the Molecular Genetics team for over 12 years.  Currently, her main role involves the management of the Next Generation Sequencing (NGS) service for Cancer patients, which includes the testing, analysis, curation and reporting of cancer diagnostic patients.  Previously she has been involved with the testing and reporting for Cystic Fibrosis, Mitochondrial disorders, Duchenne/Becker Muscular Dystrophy and predictive testing for various cardiac conditions and cancers


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