Cytogenetics

Welcome to the Cytogenetics Department at The Children's Hospital at Westmead (CHW).  The Cytogenetics and the Molecular Genetics laboratory form the Sydney Genome Diagnostics (SGD) group at CHW.  We provide a NATA accredited service for the diagnosis of a wide range of constitutional (blood), prenatal and acquired [leukaemia, lymphoma and tumour] chromosome disorders.

We perform G-band karyotyping, fluorescence in-situ hybridsation (FISH), CGH and SNP Chromosome Microarray [CMA], quantitative fluorescent-PCR prenatal aneuploidy screening (QF – PCR) and RT-PCR testing (soft-tissue tumour biopsy specimens only).

These services are extended to all States and forms part of both the CHW Division of Laboratory Services and the Western Sydney Genetics Program.

 

Cytogenetics Senior Staff contact list:

Head of Department (Principal Scientist): Mr Dale Wright
B.App.Sc, MSc, FHGSA (Cytogenetics). FFSc RCPA

Ph: 9845-3221
Fax: 9845-3238    Email: Dale.Wright@health.nsw.gov.au

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Deputy Head of Department [2IC] (Senior Scientist): Mr Luke St. Heaps
BSc, MSc(Med)

Ph: 9845 3220
Fax: 9845 3238    Email: Luke.stheaps@health.nsw.gov.au

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Chromosome Microarray/RT-PCR (Senior Scientist): Mr Artur Darmanian
BSc, MSc

Ph: 9845 3228
Fax: 9845 3238    Email: Artur.Darmanian@health.nsw.gov.au

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Oncology Microarray/Oncology Cytogenetics (Senior Scientist): Ms Dorothy Hung
B.App.Sc, MSc, FHGSA (Cytogenetics). FFSc RCPA

Ph: 9845 3223
Fax: 9845 3238     Email: Dorothy.Hung@health.nsw.gov.au

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Oncology and Blood Cytogenetics (Senior Scientist): Mr Praveen Sharma
B.Sc, MScM, MHGSA (Cytogenetics)

Ph: 9845 3224
Fax: 9845 3238     Email: Praveen.Sharma@health.nsw.gov.au

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Prenatal Microarray/QF-PCR (Senior Scientist): Mr Con Ngo
B.Sc. FHGSA (Cytogenetics)

Ph: 9845 3231
Fax: 9845 3238    Email: Con.ngo@health.nsw.gov.au

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Specimen Collection

Test Required

Sample Type

Specimen Requirements

Chromosome microarray

Peripheral Blood

3-10mL collected in purple top (EDTA) tube

DNA

Amniotic Fluid *

Chorionic Villi *

Bone Marrow

Solid Tumour

Minimum of 1.0μg of DNA (concentration between 100 and 500 ng/μL)

15-20mL collected in sterile transport tube

15-40mg collected in sterile transport tube with tissue culture media

2-4mL collected in either Lithium Heparin or EDTA

1mm3 piece of tissue or core [needle] biopsy in sterile saline.

G-band karyotype

Peripheral Blood

2-10mL collected in green top lithium heparin tube

 

Bone Marrow

2-4mL collected in green top lithium heparin tube

Solid Tumour

0.5cm3 in transport media

Chromosome Fragility Testing

Peripheral Blood

5-10mL collected in green top lithium heparin tube

FISH

Peripheral Blood

2-10mL collected in green top lithium heparin tube

Touch Imprints

3-4 slides in 95% ethanol

FACS Sorted Cells

100,000-200,000 cells in PBS

Fixed cell suspension

Formalin Fixed Paraffin Embedded Tissue

3-4 + charged slides with tissue cut at 4µm and an H&E slide indicating the region of interest (please contact the laboratory for details)

 QF-PCR

Amniotic Fluid *

15-20mL collected in sterile transport tube

Chorionic Villi *

15-40mg collected in sterile transport tube with tissue culture media

RT-PCR

Tumour

0.5cm³ snap frozen in liquid nitrogen

Cell Culture & Cryopreservation Facility

Contact Biochemical Genetics: (02) 9845 3654

DNA Storage

Contact Molecular Genetics: (02) 9845 3244

Constitutional G-Band Karyotype

Apparently balanced translocation t(2;7)(q14.2;p22) in a female with a history of infertility and miscarriage.

A karyotype can be performed for a wide variety of indications to look for chromosome abnormalities (constitutional karyotype) by culturing a blood sample and preparing metaphase chromosomes. We more commonly perform blood karyotyping for investigation of infertility looking for balanced structural rearrangements e.g. reciprocal translocations, sex chromosome aneuploidies (e.g. 45,X or 47,XXY), chromosome mosaicism, and aiding in the diagnosis of chromosome breakage syndromes such as Bloom's syndrome, Fanconi's anaemia and ataxia-telangiectasia.

Oncology chromosome abnormalities

 Apparently balanced translocation t(9;22)(q34;q11.2) in a female with Chronic Myeloid Leukaemia (CML).

Cancer represents a malignant form of rapid cell growth, which can be the result of an acquired chromosome or genetic abnormality seen in chromosomes or DNA. In 1960, a small marker chromosome named the Philadelphia chromosome was discovered in patients with Chronic Myeloid Leukaemia (CML) and this was the first consistent chromosomal abnormality found in human cancer. The term Philidelphia Chromosome is no longer used and is now more correctly referred to as the t(9;22) that is typically seen in CML.

Since this discovery, there have been numerous specific chromosome abnormalities associated with certain leukaemia and pre-leukemic conditions. Such abnormalities have important diagnostic and prognostic implications that can impact patient treatment. G-band karyotyping and/or FISH can be used for their detection.

FISH (Fluorescence in-situ Hybridisation)

ABNORMAL FISH Result showing rearrangement of the gene MYC in leukaemia. The normal intact MYC gene is indicated by the red and green signals close together, where the rearranged gene is indicated by the separated signals. We can describe this abnormality as follows: nuc ish(MYCx2)(5'MYC sep 3'MYCx1).

Fluorescent in-situ hybridisation, or FISH, is a technique that can determine the presence, location and/or structural integrity of genes on chromosomes.  In FISH, a DNA probe to a region or gene of interest is fluorescently coloured and is allowed to attach (hybridise) to the region or gene in a sample; usually chromosomes that are fixed to a microscope slide.  The presence of the attached probe in cells is determined by fluorescent microscopy.  FISH can detect a wide range of constitutional and haematological abnormalities that may or may not be detected by standard chromosomal analysis; e.g. rearrangement of the MYC gene in leukaemia. There are a variety of different FISH probes and reasons for FISH testing.

We have a wide range of ‘home grown’ and commercially available FISH probes, which can be used for confirmation of chromosome microarray findings, characterisation of structural chromosome abnormalities, investigation of mosaicism.  However, we currently primarily perform FISH for diagnosis and confirmation of recurrent structural rearrangements/ gene amplifications in haematological malignancies [myeloid and lymphoid leukaemias, MDS, MPD etc] and solid tumours.

HAEMATOLOGICAL

Chromosomal Abnormalities

MDS

EGR1/D5S721

-5/5q-

CSF1R

-5/5q-

7cen/ELN/LIMK1/D7S613

-7/7q-

8cen/MYC

Trisomy 8

11cen

Trisomy 11

D13S319/LAMP1/D12Z1

-13/13q-

D20S108

-20/20q-

AML

RUNX1T1/RUNX1

t(8;21)

KMT2A (MLL)

t(11;v)

PML/RARα

t(15;17)

CBFß

t(16;v)

7cen/ELN/LIMK1/D7S613

-7/7q-

MPD

BCR/ABL1

t(9;22)

EGR1/D5S721

-5/5q-

7cen/ELN/LIMK1/D7S613

-7/7q-

D20S108

-20/20q-

D13S319/LAMP1/D12Z1

-13/13q-

CDKN2A/9cen

Trisomy 9

ALL

BCR/ABL1

t(9;22)

KMT2A (MLL)

t(v;11q23)

ETV6/RUNX1

t(12;21)

10 & 7/17 centromere

Hypodiploidy/Hyperdiploidy

   

CLL & Multiple Myeloma panel

D13S319/LAMP1/CEP12

-13/13q-/+12

TP53/ATM

IgH Break-Apart

17p-/11q-

t(14;v)

FGFR3/IgH

t(4;14)

CCND1/IgH

IgH/MAF

IgH/MAFB

t(11;14)

t(14;16)

t(14;20)

Lymphoma

BCL6

t(3;v)

MYC

t(8;v)

MYC/IgH

t(8;14)

CCND1/IgH

t(11;14)

IgH/BCL2

t(14;18)

 

 

Prenatal Diagnosis

Prenatal Diagnosis is performed to detect chromosome abnormalities in a pregnancy; the most common abnormality being trisomy 21 or Down syndrome.  Prenatal diagnosis can be performed in the first trimester of pregnancy, around 11-12 weeks, after biopsy of chorionic villus (CVS) of the placenta or in the second trimester from 15 weeks onward by amniocentesis - sampling Amniotic fluid (AF).  These tissues contain cells and genetic material from the foetus, DNA from CVS and AF samples can be extracted for chromosome microarray testing. 

 

Prenatal chromosome microarray result showing gain [trisomy] of the whole of chromosome 21. Trisomy 21 causes Down syndrome. The ISCN 2013 description: arr(21)x3 (trisomy 21)

In prenatal diagnosis, routine G-banded karyotype testing has been the gold standard for detection of foetal chromosome abnormalities during pregnancy since the 1970s.  However, many children with birth defects have chromosome microdeletions/microduplications that are not visible by karyotyping due to the detectable size limit for these abnormalities being ~5-10Mb of the genome.  Therefore, chromosome microarray testing in prenatal diagnosis is being increasingly applied and has shown an ~7-10% increased detection rate of clinically significant chromosome abnormalities over karyotyping among high-risk pregnancies with fetal ultrasound anomalies. Prenatal microarray testing on direct CVS or AF DNA extractions usually takes ~7-10 days in our laboratory.

Prenatal aneuploidy screening result using the QF-PCR technique, which shows abnormal results for chromosome 21 and indicates trisomy 21. Three markers show a triallelic results [3 peaks] and three other makers show unbalanced peaks in a 2:1 ratio. The ISCN 2013 description: rsa(21)x3,(13,18,X)x2 (trisomy 21 female)

Alternatively, we can perform a rapid diagnosis for the most common abnormalities (rapid aneuploidy detection) using a technique called Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR). Rapid aneuploidy detection involves using several STR markers from chromosomes 13,18, 21 and X together with Amelogenin and SRY to assess the gender and copy number for the chromosomes tested.  The QF-PCR result for the 5 most common chromosome abnormalities (aneuploidies) can be obtained in 1-2 days.

Chromosome Microarray

The ISCN 2013 Nomenclature: arr 16p11,2(29,592,783-30,190,568)x1

(Example of a 0.60Mb deletion on the chromosome 16 band p11.2 representing the 16p11.2 recurrent microdeletion/duplication syndrome", which has been associated with 1% of unexplained, idiopathic, non-syndrome autism)

Comparative Genomic Hybridisation (CGH ) by Chromosome Microarray can be used to scan the genome for gains and losses of chromosomal material, of size ≥10-50Kb .  Chromosome Microarray Analysis (CMA) is now the recommended test for individuals with unexplained development delay/ intellectual deficit, behavioural and autistic spectrum disorders, and/or congenital abnormalities. CMA offers a much higher diagnostic yield (15-20%) than G-banded karyotyping (3%) for chromosome abnormalities amongst such individuals.  This increased diagnostic yield essentially reflects the ability of this technology to detect chromosome copy number change smaller than 5-10Mb of the genome, which is the detection limit of the conventional G-band karyotype.

In our laboratory, CMA performed by comparative genomic hybridisation (CGH) or Single Nucleotide Polymorphism (SNP) array methodologies is now routine for investigation of prenatal specimens (amniotic fluid and chorionic villus), pregnancy loss tissues from spontaneous abortions and/or still births, and certain malignant disorders (Multiple Myeloma) and paediatric solid tumours.

RT-PCR

 

The RT-PCR result showing the types I, II & III EWSR1/FLI1 fusion transcripts [t(11;22)] seen in Ewing sarcoma.

The diagnosis of soft tissue tumours can be challenging and the molecular diagnosis can be important for treatment. RT-PCR can detect specific chromosomal translocations and gene fusions for an accurate diagnosis and be useful where the tumour histology is inconclusive or equivocal.

Tumour types

Chromosome Translocation
(including variants)

Fusion transcript
(including variants)

Burkitt's Lymphoma

t(8;14)(q24;q32)

MYC-IgH

Clear cell sarcoma

t(12;22)(q13;q12)

EWSR1-ATF1

Ewing's sarcoma

t(21;22)(q22;q12)

EWSR1-ERG

"

t(17;22)(q12;q12)

EWSR1-4

"

t(2;22)(q33;q12)

EWSR1-FEV

"

t(11;22)(q24;q12)

EWSR1-FLI1

Liposarcoma, myxoid

t(12;16)(q13;p11)

FUS-CHOP

"

t(12;22)(q13;q12)

EWSR1-CHOP

Alveolar Rhabdomyosarcoma

t(2;13)(q35;q14)

PAX3-FKHR

"

t(1;13)(p36;q14)

PAX7-FKHR

"

t(2;2)(q35;p23)

PAX3-NCOA1

Synovial sarcoma

t(X;18)(p11;q11)

SYT-SSX1

"

t(X;18)(p11;q11)

SYT-SSX2

"

t(X;18)(p11;q11)

SYT-SSX4

Desmoplastic small round cell tumour

t(11;22)(p13;q12)

EWSR1-WT1

Small round cell tumour

22q12 rearrangement

EWSR1-ZSG