Seeing the whole picture – integrated view of CNVs, AOH, and sequence variants for Improved results

Alessio Venier1, Andrea O’Hara1, Soheil Shams1

1 BioDiscovery, Inc. 715 North Douglas Street, El Segundo, CA, 90245

Given the separate technologies used to detect CNVs, AOH, and Sequence Variants (SNP Microarrays for CNVs and AOH, and NGS for Sequence Variants), this data commonly has been reviewed in isolated silos. This segregation has also been exaggerated by the separate expertise in Cytogenomics and Molecular Genetics. However, as the fields are now coming together, the importance of an integrated view of the data has become even more apparent. Here we present a new system that can integrate data from any array as well as NGS platforms to create a single genomic view of structural changes in a sample. The data is presented in a view familiar to cytogenomicists with aberrations displayed across an ideogram with supporting evidence for each call (e.g. probes from arrays and reads from NGS). Historical data and outside database knowledge is integrated into this view allowing the analyst access to all information needed to make assessments of the results. We will demonstrate the utility of this system using a few samples with compound heterozygous aberrations that are detected using different technologies. This includes a case with a microdeletion detected by custom exon array and SNV detected with a custom targeted NGS panel. Another case includes a variant in a large region of homozygosity.


Dr. Soheil Shams is the founder and President of BioDiscovery, Inc. a privately held bioinformatics company based in El Segundo, California established in 1997. He received his Masters and Ph.D. degrees from University of Southern California in 1986 and 1992 respectively in the field of Computer Engineering. He has been a pioneer in the field of microarray image and data processing having invented many of the basic approaches to array analysis resulting in numerous issued US patents. Under the direction of Dr. Shams, BioDiscovery has played a pioneering role in development of software tools for conducting array based research and more recently in clinical application of arrays in cytogenetic and molecular genetic diagnostics as well as Next Generation

Sequencing (NGS) data analysis. Prior to founding BioDiscovery, he was a Sr. Member of Staff at Hughes Research Laboratories (HRL) in Malibu and taught undergraduate and graduate classes in Artificial Intelligence, Machine Perception, and Data Mining at UCLA. His research interests span a wide range with concentration on pattern recognition technologies and parallel processing architectures. He has worked with many of the pioneering scientist in microarray research and has authored over 50 technical publications and book chapters.

Limitation of molecular karyotyping: Discordance between molecular vs. conventional cytogenetics

Shea Ming Lim, Toni Saville, Pauline Dalzell, Christopher Lucas, Joan Junio, Krystle Standen, Genevieve Temple, Louise Carey

1Molecular and Cytogenetics Unit, SEALS Pathology, Prince of Wales Hospital, Randwick

Introduction: Most laboratories are moving towards molecular based cytogenetics such as QF-PCR, CGH microarray, and MLPA as their first line of prenatal testing, therefore conventional cytogenetics is not routinely performed, and is often supplementary to molecular karyotyping.

Methods: We performed a retrospective audit of 648 fetoplacental referrals (322 prenatal chorionic villus samples and 326 product of conceptions) received in 2016 to identify cases in which cytogenetic discordance between molecular and conventional cytogenetic testing were reported. All prenatal CVS referrals received standard QF-PCR and CGH microarray testing, while the majority of POC referrals received QF-PCR and subtelomere MLPA testing. Conventional karyotype was only performed in a small number of these cases. The main indication for conventional karyotyping is to rule out Robertsonian translocation following a trisomy 13 or 21 result, or as a confirmatory test for any CNVs reported. POC referrals with 3 or more miscarriages also received a conventional karyotype in addition to a molecular karyotype.

Results: We identified 9 cases (9/648; 1.38%) where a discordance between molecular and conventional karyotype was observed.  The findings of these cases are presented in this study, with a discussion of the limitations of molecular testing over conventional cytogenetics.

Conclusion:  Conventional cytogenetics is still a useful tool in the diagnosis of prenatal cytogenetic abnormalities. However, consideration needs to be given to the resources available in each laboratory.



Graduated from Charles Sturt University, Wagga Wagga, Australia with Bachelor of Medical and Applied Biotechnology in 2007 and Bachelor of Medical Science (Pathology) in 2008. Started working for SEALS Pathology, Prince of Wales Hospital in 2009 and has been in the Molecular and Cytogenetics Unit since 2011.

Therapy-related Myeloid Leukaemia in Patients Treated for Breast Cancer

Siobhan Battersby, Praveen Sharma, Dorothy Hung, Luke St Heaps, Dale Wright

Sydney Genome Diagnostics, Children’s Hospital at Westmead, NSW, Australia


The recurrent t(8;21) abnormality found in primary acute myeloid leukaemia (AML) is associated with a favourable prognosis. In therapy-related myeloid leukaemia (t-AML), previous exposure to topoisomerase-II inhibitors tends to be associated with secondary abnormalities involving balanced rearrangements at 11q23 (KMT2A) and 21q22 (RUNX1). Breast cancer is often treated with topoisomerase-II inhibitors and t-AML can be associated with secondary acquired abnormalities.

Aim: We present two cases identified with t(8;21) following cytotoxic therapy for breast cancer.


Case 1: 51yo female with pancytopenia and blasts on peripheral blood. The patient was 2 years post chemotherapy for breast cancer.

Case 2: 61yo female with blasts on peripheral blood and previously diagnosed with breast cancer.

These patients were investigated by karyotyping of 24hr and 48hr synchronised cultures, initiated and harvested according to standard laboratory protocols. Twenty G-band cells were analysed. FISH was performed using the t(8;21) probe for RUNX1/RUNX1T1 (VYSIS).


Case 1 showed a karyotype involving 45,X,-X,t(8;21)(q22;q22,del(9)(q22q32)[15]/46,XX[5]. FISH results failed. Case 2 showed a more complex karyotype involving 46,XX,t(8;17;21)(q22;q23;q22),t(10;20)(p15;p11.2)[14]/46,XX[6]. FISH results showed gene fusion of RUNX1/RUNX1T1 probe.


The finding of the t(8;21) and variant t(8;17;21) with RUNX1/RUNX1T1 gene fusion is consistent with a secondary abnormality associated with t-AML following breast cancer treatment. Although the t(8;21) has a favourable prognosis in primary AML, when found as a secondary abnormality it portends to a more unfavourable outcome.



Hospital Scientist (8th year) currently working in cytogenetics at The Children’s Hospital at Westmead. Completed a Bachelor of Science in 2008 and will be sitting the MHGSA exam in March this year.

Manual vs Automated Plasma Cell (CD138+) Enrichment: A Validation Study.

El-Hajj Racha, St. Heaps Luke, Clark Alissa and Wright Dale.

Sydney Genome Diagnostics, Cytogenetic Department, The Children’s Hospital at Westmead, NSW, Australia.



Multiple myeloma (MM) is a plasma cell (PC) neoplasm in which the surface antigen CD138 (syndecan-1) is highly expressed. We have previously shown that FISH gives higher abnormalities rates on uncultured CD138+ enriched cells compared to routine cultured bone marrow cells (50.8% vs 7.4%, P<0.001). In our laboratory, CD138+ cell enrichment has subsequently become standard practice for FISH and chromosome microarray testing for MM patients.


To validate and implement the AutoMACS Pro Separator for automated enrichment of CD138+ PCs.


To date, eleven bone marrow samples have been enriched using the MACS whole Blood CD138 MicroBeads human kit (Miltenyi Biotec), which was performed in parallel with manual vs. autoMACS Pro Separator (Miltenyi Biotec) protocols, according to the manufacturer’s instructions. FISH was performed using the probes IGH/CCND1, IGH/FGFR3, IGH/MAF, IGH break-apart, TP53/D17Z1 and/or CKS1B/CDKN2C (Carl Zeiss). The proportion of FISH abnormal cells between the two methods was compared using the paired-means t-test with a significance level α=0.05. Hands-in processing time was also evaluated.


The mean proportion of abnormal cells by FISH for each method was 77.7% vs 86.4% for the manual vs. automated method, respectively. The increased yield (8.8%) was marginally significant (t=2.3, df=10, P=0.0442). Hands-on time taken to perform the manual vs. automated method was 140min vs. 55min, respectively.


Although there was a marginally increased yield of abnormal CD138+ cells (8.8%) using the autoMACS Pro, we considered there to be no real practical difference given the small sample size of the study. More importantly, when processing time was considered, the autoMACS Pro reduced processing by ~45 minutes. Furthermore, up to six samples can be batch processed. Although sample recruitment is ongoing, the results to date indicate the benefits of automation for CD138+ cell enrichment in a busy laboratory.

Inverted duplicated deleted 8p: How Microarray presents a prettier picture.

Mahony Fenn1, Ryan Hedley1, Dr. Melody Caramins2, Dr. Nicole Chia3

1 Western Diagnostic Pathology, 74 McCoy St, Myaree, WA, 6154,

2 Genomic Diagnostics, 60 Waterloo Road, Macquarie Park, NSW, 2113

3 Queensland Medical Laboraory, Metroplex on Gateway, 11 Riverview Pl, Murarrie, QLD, 4172


The distal short arm of chromosome 8 is a well described hotspot for chromosomal rearrangement resulting in inversion, duplication and deletion. Until recently these have been detected by conventional chromosome analysis with subjective interpretation of chromosomal bands. The implementation of molecular karyotyping by chromosome microarray has elucidated these rearrangements with respect to the genomic location of the duplicated and deleted segments. Several mechanisms have been described which hypothesise the derivation of these rearrangements. In general these stem from the formation of a dicentric chromosome with a subsequent breakage leading to the classical invdupdel(8p). Parental paracentric inversions within 8p23 and Olfactory receptor (OR) gene clusters (including low copy repeat regions REPP and REPD) have been highlighted as the major triggers involved in the generation of the dicentric chromosome.

The subsequent breakage of the dicentric chromosome will often leave this derivative without a telomere resulting in chromosome instability. Stability is regained by restoration of the telomere. This can be done through telomere healing with the addition of telomeric sequences or by telomere capture whereby telomeres are sourced from another chromosome.

Here we describe 5 case studies of invdupdel(8p) and demonstrate the presence of the dicentric chromosome consistent with the early hypothesis and 2 cases showing telomere capture as the mechanism of telomere repair. This study highlights the value of the information provided by molecular karyotyping for the detection of chromosomal anomalies and additional insight of the mode of derivation.



I am one of the senior scientists at WDP and the 2IC of the Cytogenetics dept at WDP. I have worked in the field of cytogenetics for just over 15 years. Our main areas of interest currently at WDP are fertility, prenatal and paediatrics. We use conventional cytogenetics, FISH, QFPCR and microarray as our main techniques of analysis.

Targeted Molecular Testing for Cancer Therapy

Shravan K Yellenki, Mioara Gavrila, Elizabeth M Algar

Genetics and Molecular Pathology, Monash Health, 246 Clayton Rd, Clayton, VICTORIA


1 in 3 Australian men and 1 in 4 Australian women will be diagnosed with cancer before the age of 75. Cancer treatment has changed significantly over the past 10 years with the introduction of targeted therapies that result in less severe adverse effects compared to traditional chemotherapy. The ascertainment of genetic alterations in cancer is of increasing importance for informing the choice of targeted therapies.

Targeted molecular testing in adult cancer was implemented in the Genetics and Molecular Pathology laboratory at Monash Health at the end of 2014. In two years, 800 molecular tests have been performed on 600 tumour specimens from lung, colorectal, melanoma, thyroid and brain cancer. On average, 45% of mutation screening requests have been for EGFR in lung cancer, 22% for KRAS in colorectal cancer,10% for NRAS in colorectal cancer, melanoma or thyroid cancer, and 24% for BRAF in melanoma, thyroid or brain cancer.

Our method of choice for mutation screening has been the CE IVD Vienna Labs Strip Assay. These are mutant-enriched PCR-based assays that employ reverse hybridisation to detect clinically actionable mutations in EGFR (exons 18-21), KRAS (exon 2,3,4), NRAS (exon 2,3,4) and BRAF (exon 15). The assay utilizes 1-10ng of DNA and reliably detects to a sensitivity of 5% allowing testing of very small biopsy and cytology samples with low tumour content. A minimal equipment outlay is all that is required for set-up. Between 96-99% of clinically relevant mutations are covered by the assays. In our experience, the Strip Assay offers a flexible, rapid and sensitive test platform that although widely used in Europe has not been widely adopted in Australia yet for clinical testing.



Mioara is a senior scientist in the Genetics and Molecular Pathology laboratory at Monash Health. Previously she was in charge of Molecular Genetics Department at Australian Clinical Labs. She has 18 year experience in public and private NATA accredited molecular pathology services.

Undetectable levels of UNCONJUGATED ESTRIOL in maternal serum have led to a diagnosis of X-LINKED ICHTHYOSIS in a male fetus.

Dannielle Ghezzi1, Khoa Lam1, Wendy Waters2, Sarah Smith2, Enzo Ranieri1, Dr Michael Metz1, Dr Janice Fletcher1, Peter Sharp3


1 Antenatal Screening Laboratory , Department of Biochemical Genetics, Directorate of Genetics & Molecular Pathology, SA Pathology, Women’s and Children’s Hospital, Adelaide, South Australia 5006, Australia

2 Department of Cytogenetics, Directorate of Genetics & Molecular Pathology, SA Pathology, Women’s and Children’s Hospital, Adelaide, South Australia 5006, Australia

3 Metabolic Laboratory, Department of Biochemical Genetics, Directorate of Genetics & Molecular Pathology, SA Pathology, Women’s and Children’s Hospital, Adelaide, South Australia 5006, Australia


The South Australian Maternal Serum Antenatal Screening (SAMSAS) laboratory provides antenatal screening to the women of South Australia, Tasmania and Northern Territory. In combination with the Nuchal Translucency (NT) scan provided between 11 weeks + 2 days (11w2d) to 14 weeks, SAMSAS tests maternal serum for biochemical markers and generates a risk of aneuploidy and/or open neural tube defects using a SAMSAS  developed ‘triple test’ algorithm.

Non-invasive Prenatal testing (NIPT) is becoming the preferred screening test for common aneuploidies in place of the biochemical ‘triple test’ screen. We present a case of a microdeletion that was identified through the biochemical testing method, which currently is undetectable by commercial aneuploidy NIPT.

Unconjugated Estriol (uE3) is a marker used in the second trimester biochemical screen. A patient aged 19 years (G1P0), showed undetectable serum levels of uE3 at 14w1d, 15w2d and 17w0d of gestation. The levels of uE3 were persistently undetectable by the Siemens Immulite2000 Immunoassay platform (LLD< 0.24nmol/L). The requested repeat analysis confirmed the low uE3 levels where metabolic studies for the exclusion of Smith-Lemli-Opitz (SLO) Syndrome were performed. SLO was exclude by testing on amniotic fluid revealing low levels of the 7-dehydrocholesterol, the precursor of cholesterol in the steroid biosynthesis pathway.

The patient was also referred for cytogenetic testing by prenatal microarray with particular concern for X-linked Ichthyosis. The microarray that was performed on amniocytes identified a male fetus with a 1.6Mb interstitial microdeletion at Xp22.31. This involved the STS gene which codes for the Steroid Sulfatase enzyme (EC; an important step in the biosynthesis of UE3 and a diagnosis of X-linked Ichthyosis was made. The mother was referred for FISH testing and was found to be a carrier of this deletion which could potentially affect future pregnancies.

This highlights the importance of a biochemical screen of maternal serum and the collaboration between testing laboratories to provide additional clinical information on a pregnancy.



The South Australian Maternal Serum Antenatal Screening (SAMSAS) laboratory provides antenatal screening to the women of South Australia, Tasmania and Northern Territory. In combination with the Nuchal Translucency (NT) scan provided between 11 weeks + 2 days (11w2d) to 14 weeks, SAMSAS tests maternal serum for biochemical markers and generates a risk of aneuploidy and/or open neural tube defects using a SAMSAS  developed ‘triple test’ algorithm.

Phenotype Risk Assessment in a couple with Multiple Alpha and Beta-Globin Variants.

Wendy M Hutchison1, Kerryn M Weekes1, Jeremy N Wells1, Ruoxin Li1, Nicholas Clark1, Anita Feigin2,3, Asif Alam1, Elizabeth Algar4, Zane Kaplan2

1 Thalassaemia & Haemophilia Molecular Reference Laboratory, Level 3 Monash Medical Centre 246 Clayton Road, CLAYTON, VIC, 3168,

2 Medical Therapy Unit, Level 2 Monash Medical Centre 246 Clayton Road, CLAYTON, VIC, 3168

3 Genetics Services, Monash Medical Centre 246 Clayton Road, CLAYTON, VIC, 3168

4 Genetics and Molecular Pathology, Level 3 Monash Medical Centre 246 Clayton Road, CLAYTON, VIC, 3168


Copy number variations in the α-globin genes are the result of unequal crossover between homologous segments in the α-globin gene cluster that misalign during meiosis. The reduction or increase in α-globin gene copy number leads to an imbalance of α and β-globin chains in the haemoglobin tetramer and consequently ameliorates or exacerbates

β thalassaemia clinical symptoms.

We describe a couple with one partner, with transfusion-dependent β thalassaemia major, who is a compound heterozygote for two beta-globin splice site mutations and heterozygous for both the anti3.7 alpha-globin gene triplication and the alpha-globin -3.7 single gene deletion mutation. The second partner is heterozygous for the anti3.7 alpha-globin gene triplication.

Possible genotype combinations and clinical phenotypic risks are discussed.



After many years working in Molecular Genetics Research Wendy moved to the Thalassaemia and Haemophilia Molecular Reference laboratory at Monash Health in 2013.

Two Cases of the Rare but Emerging Syndrome Associated with the Recurrent “3q13.2q13.31 Microdeletion”.

Sharanbeer Kaur, Gregory Peters, Dale Wright

Sydney Genome Diagnostics, The Children’s Hospital Westmead, NSW, 2145


The recurrent 3q13.2q31.31 microdeletion has been associated with a rare but emerging syndrome with clinical features overlapping Primrose syndrome. Herein, two cases are described.


Case 1: 9 year old female with moderate global delay. Case 2: 4 year old female with global development delay (motor and language), macrocephaly and ‘distinctive appearance’. CGH microarray was performed using an 8x60K ISCA design (Agilent Technologies), and data analysed using the ADM-2 algorithm with copy number abnormalities (CNA) calls based on five consecutive probes (Cytogenomics v2.9.2.4). UCSC genome browser [hg19] was used to evaluate CNA pathogenicity and segmental duplications >1000bp. Parental follow-up studies were requested to investigate inheritance.


Case 1 and 2 both showed heterozygous deletions [~3.31Mb] within chromosome band 3q13.2q13.31, extending from coordinates 112.18Mb to 115.49Mb. This included 29 genes, from BTLA to GAP43. Two of the 29 genes are associated with OMIM-listed disease; DRD3 and ZBTB20. No flanking segmental duplications were identified. Parental studies for Case 2 were negative, indicating a de novo mutation. Parental samples for Case 1 have not been received.


The recurrent 3q13.2q13.31 deletion was identified in two cases. Case 2 was de novo and Case 1 of unknown inheritance. Clinical features of the emerging syndrome include development delay, hypotonia, high-arched palate, increased occipitofrontal circumference and distinctive facial features; short philtrum and protruding lips. A smallest region of overlap has been defined as ~0.6Mb, which includes five genes: DRD3, ZNF80, TIGIT, MIR568 and ZBTB20. Primrose syndrome is caused by heterozygous mutations in ZBTB20. DRD3 is associated with schizophrenia. Both are considered candidate genes with respect to development delay, psychiatric features, and associated structural brain malformations. Recurrent deletions typically arise via non-homologous allelic recombination facilitated by flanking segmental duplications, however none were identified >1000bp. This may suggest involvement of smaller duplicated segments, or an alternative mechanism.



I am a Trainee Hospital Scientist at the Children’s Hospital Westmead under the Sydney Genome Diagnostics Program.

Detection of parental mosaicism following non-mosaic findings in their offspring on array

Louise Korte1, Sarah Higgins1, Jillian Nicholl1, Alison Attwood1, Ryan Storer1, Sarah Smith1, Yvonne Hull1, Sue Brown1, Rhonda Hutchinson1, Christopher Barnett2, Jan Liebelt2

1SA Pathology, Cytogenetics Unit, Department of Genetic Medicine, WCH, 72 King William Road, North Adelaide, SA, 5006

2SA Clinical Genetics Services, WCH, 72 King William Road, North Adelaide, SA, 5006


Microarray technology is now widely used to diagnose pathogenic copy number variants (CNVs) in neurological disorders (Intellectual disability, autism, schizophrenia etc.).  We routinely ask for parental follow up to determine if changes are inherited or de novo and to clarify the clinical significance of the majority of CNVs we detect. We will present four cases where SNP array has identified a CNV in the proband and parental follow up has shown mosaicism. This significantly increases the possibility of recurrence of another similarly affected child. Three of these inherited changes were not detectable by routine cytogenetic analysis. This illustrates the value of FISH investigation for parental follow up.

Case 1: An additional supernumerary bisatellited chromosome 22 marker was detected, consistent with a diagnosis of Cat Eye Syndrome. Family studies showed the marker chromosome 22 was maternally inherited, present in 67% of metaphases examined.

Case 2: A 2Mb deletion of chromosome 17p11.2 involving the RAI1 gene was detected, consistent with a diagnosis of Smith-Magenis syndrome.  The deletion was maternally inherited, present in 64% of cells examined.

Case 3: 4.2Mb duplication at chromosome 1q43-q44 involving the AKT3 gene was detected.   A low level of mosaicism was detected in the mother, however FISH results fell outside the reporting guidelines.  A Masked array showed a slight divergence of the B allele frequency, which supports this FISH result.

Case 4: A 560kb deletion of chromosome 2p16.3, involving the NRNX1 gene was detected. The deletion was maternally inherited, present in approximately 30% of cells examined.



Cytogenetics laboratory at the women’s and children’s hospital

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