Oncology and Constitutional FISH Probe Catalogue 2012 / 2013

Transcription

1 Oncology and Constitutional FISH Probe Catalogue 2012 / 2013

2 cytocell.com Our website provides a detailed account of our comprehensive range of FISH probes, devices and accessories. It also provides details of your local contact from our network of international distributors who will be happy to take your order. For a complete list of worldwide distributors please go to:

3 Introduction An Introduction to Cytocell Cytocell s FISH business is now the oldest and most experienced in the world. From humble beginnings, the first products were developed 20 years ago in Birmingham Heartlands Hospital, England, where, in 1991, the patented process, Chromoprobe, was developed. Initially, thanks to Chromoprobe, all the products were available reversibly dried onto the surface of special glass coverslips or as arrays of probes on 24 square glass devices. From 1994 these were developed, manufactured and supplied from our new premises in Banbury, near Oxford. In 2006, we moved to our current premises in Cambridge, England and this year we will continue to invest in our manufacturing and R&D facilities by expanding into a new purpose built building. Over the last 20 years, we have actively listened to and acted upon your needs and requirements leading to the development of our current portfolio of FISH probes, these are available in a convenient liquid format and packaged in kit sizes appropriate to your laboratory workload. At Cytocell we have a long history of collaborating with world-renowned experts. As well as providing clean and quick FISH tests, the development of our product portfolio has led to a number of industry Firsts, each of which has helped to make FISH a more robust and customer friendly technique: 1st to develop directly labelled probes 1st to provide co-denaturation protocols 1st to develop subtelomere probes 1st to develop a single slide assay for subtelomeric aberrations 1st to provide a screening solution for all human chromosomes on a single slide This latest catalogue is our largest to date and contains the single largest collection of FISH probes available from one supplier. Cytocell is committed to the future development and expansion of our probe catalogue through the use of our proprietary sequence data and exclusive BAC collection. Under the banner of myprobes, Cytocell is proud to offer our FISH expertise and services to our customers for the development of custom built probes. Using our myprobes service and its new website ( we can supply FISH probes tailored to your exact needs and specification, from establishing new diagnostic tests to research into genetic aberrations in cancer. So celebrate our 20 th anniversary with us and enjoy looking through our catalogue as we look forward together at the next 20 years and consider what we may achieve together! 1st to provide two-probes-in-one-test solutions We are justifiably proud of our achievements over the last 20 years and equally as proud to have served you, our customers. Dr. Martin Lawrie Managing Director, Cytocell Ltd. With the changing face of FISH testing over the last 5 years, Cytocell has continued to innovate and move with the times so that now we have a comprehensive Haematology and Pathology portfolio to complement our Classical Cytogenetics catalogue. Cytocell FISH probes are developed and produced in the UK 3

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5 Our products Our products Cytocell s DNA diagnostic Fluorescence In Situ Hybridisation (FISH) kits are aimed at two disciplines; Constitutional cytogenetics and Oncology/Pathology cytogenetics. We offer three key product ranges: Chromoprobe Multiprobe Macroarray Range Using our unique Chromoprobe process, multiple probes are reversibly bound to a glass slide providing a convenient screening tool for genetic abnormalities across multiple chromosomes. Key applications include detection of chromosomal rearrangements and as a diagnostic / prognostic tool in the detection of various Leukaemias. Aquarius Range Aquarius probes are directly labelled liquid probes provided in hybridisation solution. The probes are accompanied by DAPI counterstain to provide a complete Fluorescence In Situ Hybridisation kit. myprobes myprobes is a custom FISH probe design and manufacture service, which utilises Cytocell s proprietary BAC clone collection. If a product you require does not appear in the catalogue, do not hesitate to contact Cytocell with your request. Patents and Trademarks Aquarius, Chromoprobe, Cytocell, Chromoprobe Multiprobe and myprobes are registered trademarks of Cytocell Ltd. The Chromoprobe principle is covered by international patents WO , EP The design of the Multiprobe is a registered design and is also covered by a Design Patent No. 420,745. Cytocell is registered with the MHRA (Medicines and Healthcare products Regulatory Agency) and all Cytocell products are CE marked in accordance with the In-Vitro Diagnostic Medical Devices Directive 98/79/EC, and the 2002 Regulations. Cytocell operates a Quality Management System that have been assessed and certified to both EN ISO 9001:2008 and EN ISO 13485:2003. Please check our website for the latest probe ranges available from Cytocell. Disclaimer Every attempt has been made to ensure that the information in this catalogue is correct at time of going to print. Although every care has been taken in preparing this publication, no responsibility or liability will be accepted by Cytocell Ltd or its employees for its accuracy or completeness. vers001/

6 Contents - Oncology and Constitutional FISH Contents Aquarius Haematology 11 AML1 Breakapart 12 AML1/ETO Translocation, Dual Fusion 13 ATM Deletion 14 BCR/ABL and BCR/ABL Plus Translocation, Dual Fusion 16 BCL6 Breakapart 17 CBFβ /MYH11 Translocation, Dual Fusion 18 CKS1B/CDKN2C (P18) Amplification/Deletion 19 cmyc Breakapart 20 Deletion 13q14.3, D13S319 and D13S25 22 Del (5q) Deletion 23 Del (7q) Deletion 24 Del (20q) Deletion 25 E2A Breakapart 26 EVI1 Breakapart 27 FIP1L1/CHIC2/PDGFRA Deletion/Fusion 28 IGH Breakapart 29 IGH/BCL2 Translocation, Dual Fusion 30 IGH/CCND1 Translocation, Dual Fusion 31 IGH/CCND3 Translocation, Dual Fusion 32 IGH/cMYC Translocation, Dual Fusion 33 IGH/FGFR3 Translocation, Dual Fusion 34 IGH/MAF Translocation, Dual Fusion 35 IGH/MAFB Translocation, Dual Fusion 36 IGH/MYEOV Translocation, Dual Fusion 37 IGL and IGK Breakapart 38 MLL Breakapart 39 MYB Deletion 40 P16 Deletion 41 P53 Deletion 42 P53/ATM Probe Combination 43 PDGFRB Breakapart 44 PML/RARα Translocation, Dual Fusion 45 TCL1 Breakapart 46 TCRAD Breakapart 47 TCRB Breakapart 48 TEL/AML1 Translocation, Dual Fusion 49 TLX1 Breakapart 50 TLX3 Breakapart 51 CLL Screening panel Multiprobe Haematology 56 Chromoprobe Multiprobe ALL 58 Chromoprobe Multiprobe CLL 60 Chromoprobe Multiprobe AML/MDS Aquarius Pathology 65 CHOP Breakapart 66 C-MET Amplification 67 EGFR Amplification 68 EWSR1 Products 70 FGFR1 Breakapart/Amplification 71 HER2 Amplification 72 MALT1 Breakapart 73 MDM2 Amplification 74 N-MYC Amplification 75 PAX3 & PAX7 Breakapart 76 RB1 Deletion 77 SRD (CHD5) Deletion 78 SYT Breakapart 79 TOP2A Amplification/Deletion 80 ZNF217 Amplification 6

7 Contents - Oncology and Constitutional FISH Aquarius Prenatal Paints 83 Aquarius Prenatal Enumeration Kits Aquarius Microdeletion 112 Painting Probes 113 Chromoprobe Multiprobe OctoChrome 114 Summary of Painting Probes 87 Alagille (JAG1) 88 Angelman (UBE3A/D15S10) 89 CHARGE 90 Cri-Du-Chat and SOTOS Probe Combination 91 DiGeorge II (10p14) 92 DiGeorge and 22q13.3 Deletion Syndrome Probe Combinations 94 Kallmann (KAL1) and Steroid Sulphatase Deficiency (STS) Probe Combination 95 Langer-Giedion 96 Monosomy 1p36 97 Neurofibromatosis Type 1 98 Prader-Willi/Angelman (SNRPN) 99 Rubinstein-Taybi 100 Saethre-Chotzen/Williams-Beuren Probe Combination 101 SHOX 102 Smith-Magenis (RAI1 and FLII)/Miller-Dieker Probe Combinations 103 SRY 104 Williams-Beuren 105 Wolf-Hirschhorn 106 XIST Satellites 116 Satellite Enumeration Probes 117 Dual Labelled Satellite Probe Sets 117 Acro-P-Arm Probe Accessories 120 Accessories Ordering Information 122 Ordering Information 128 Index by Chromosome Number 130 Index by Probe Name 132 Haematology products by disease state Subtelomere Specific Probes 108 Subtelomere Specific Probes 109 Chromoprobe Multiprobe -T Applications 110 Chromoprobe Multiprobe -T Design 110 Aquarius Subtelomere Specific Probes 7

8 Aquarius Overview The Aquarius range consists of directly labelled FISH probes in a liquid format. All Aquarius probes are supplied in hybridisation solution; either in a ready-to-use format or require a simple dilution with hybridisation solution which is conveniently supplied in the kit. The probes are additionally packaged with DAPI counterstain and comprehensive 'Instructions for Use'. Aside from the standard 5 and 10 test kits, Prenatal probes are also available in 30 and 50 test formats. The procedure is simple: 1 Spot slide with cell suspension and dehydrate. 2 Apply Aquarius probe onto dehydrated cell sample. 3 Place coverslip on slide and seal. Denature on a hotplate and hybridise overnight. 4 Wash with rapid formamide-free stringency washes. Counterstain and view by fluorescence microscopy. 8

9 Haematology Aquarius Haematology

10 Aquarius Haematology Contents 11 AML1 Breakapart 12 AML1/ETO Translocation, Dual Fusion 13 ATM Deletion 14 BCR/ABL and BCR/ABL Plus Translocation, Dual Fusion 16 BCL6 Breakapart 17 CBFβ/MYH11 Translocation, Dual Fusion 18 CKS1B/CDKN2C (P18) Amplification/Deletion 19 cmyc Breakapart 20 Deletion 13q14.3, D13S319 and D13S25 22 Del (5q) Deletion 23 Del (7q) Deletion 24 Del (20q) Deletion 25 E2A Breakapart 26 EVI1 Breakapart 27 FIP1L1/CHIC2/PDGFRA Deletion/Fusion 28 IGH Breakapart 29 IGH/BCL2 Translocation, Dual Fusion 30 IGH/CCND1 Translocation, Dual Fusion 31 IGH/CCND3 Translocation, Dual Fusion 32 IGH/cMYC Translocation, Dual Fusion 33 IGH/FGFR3 Translocation, Dual Fusion 34 IGH/MAF Translocation, Dual Fusion 35 IGH/MAFB Translocation, Dual Fusion 36 IGH/MYEOV Translocation, Dual Fusion 37 IGL and IGK Breakapart 38 MLL Breakapart 39 MYB Deletion 40 P16 Deletion 41 P53 Deletion 42 P53/ATM Probe Combination 43 PDGFRB Breakapart 44 PML/RARα Translocation, Dual Fusion 45 TCL1 Breakapart 46 TCRAD Breakapart 47 TCRB Breakapart 48 TEL/AML1 Translocation, Dual Fusion 49 TLX1 Breakapart 50 TLX3 Breakapart 51 CLL Screening panel Haematology As long ago as the 19th Century, nuclear changes were recognised as being significant in cancer biology. Advances in cytogenetics and molecular cytogenetics in the last century showed that although a number of numerical and structural chromosome changes appeared to be random and non-specific, rearrangements involving individual chromosomes were shown to define specific abnormalities in individual tumour types. Fluorescence In Situ Hybridisation (FISH) using locus-specific probes which are capable of defining these stereotypic structural rearrangements has now become a routine diagnostic test in the clinical laboratory. The technique has thus been shown to be useful in the management of cancer patients. Cytocell offers a range of FISH probes specific for a number of haematological malignancies which are available in the Aquarius liquid format. These probes are directly labelled, ready to use in hybridisation buffer and available in economical 5, and larger 10 test kits. The protocol is rapid and simple and has been developed to allow co-denaturation of the FISH probe and target DNA simultaneously. The probe mixtures are designed for Fluorescence In Situ Hybridisation of interphase cells and metaphase chromosomes from cultured peripheral blood cells or cultured bone marrow samples. Which condition is associated with which FISH probe? Use our handy haematology key to find out. Look for the key on each haematology product. ALL Haematology key: Acute Lymphoblastic Leukaemia CLL AML CML MM L Chronic Lymphocytic Leukaemia Acute Myeloid Leukaemia Chronic Myeloid Leukaemia Multiple Myeloma Lymphoma 10

11 Cat. No. LPH 027-S Cat. No. LPH 027 Aquarius Haematology AML1 Breakapart The AML1 (CBFA2 or RUNX1) gene is the most frequent target of chromosomal rearrangements observed in human acute leukaemia. The most common rearrangements are the TEL/AML and AML/ETO fusions. The TEL(ETV6)/AML1 fusion is brought about by the t(12;21)(p13;q22) translocation which is observed in 21% of childhood B-ALL cases 1, whilst the AML/ETO fusion is the result of the t(8;21)(q22;q22) translocation observed in ~40% of AML M2 and 7% of AML cases overall. Both of these rearrangements provide for a favourable outcome although the TEL/AML1 fusion is associated with late relapse. The gene is also, however, rearranged in many other rare translocations including the partner chromosomes 1, 2, 3, 4, 6, 9, 16, 20 and X. AML ALL 1. Jamil A et al., Cancer Genet Cytogenet 2000;122(2): Niini T, Haematologica 2000;85(4): García-Casado Z et al., Cancer Genet Cytogenet 2006;170(2): Robinson HM et al., Leukemia 2003;17(11): Mikhail FM et al., Leukemia 2002;16(4): Rearrangements of the RUNX1 gene are not restricted to translocations, however. Using FISH, gene amplifications have also been found in childhood ALL 2,3. Amplification of the gene has further been shown to be associated with poorer outcome in ALL 4. One mechanism for this is thought to be because of the overexpression of RUNX1 (AML1). 5 The Cytocell AML breakapart product has been designed to show gross changes in the AML1 gene. Prior knowledge of the partner chromosome(s) involved is therefore not necessary as a rearrangement will be clearly visible in non-dividing cells as well as those at metaphase. AML1 Chromosome 21 DSCR1 (RCAN1) CLIC6 AML1 (RUNX1) D21S326 D21S1706 D21S1969 D21S393 D21S1895 D21S Kb 167Kb 11

12 Aquarius Haematology Cat. No. LPH 026-S Cat. No. LPH 026 AML1/ETO Translocation, Dual Fusion AML1 (or RUNX1 Runt related Transcription Factor 1) is fused with ETO (or MTG8) in the t(8;21)(q22;q22) translocation. The rearrangement is observed in ~40% of AML M2 patients and less frequently in subgroups M1 and M4. Overall 7% of AML cases demonstrate the abnormality, the majority of which are de novo. Additional abnormalities occur in 75% of cases. These may be loss of a sex chromosome, del(9q), trisomy 8 or monosomy 7. Three-way variants of this rearrangement and the t(3;21) (AML1/EVI1) show that juxtaposition of AML1 to the derivative chromosome is consistent and therefore the critical rearrangement 1,2. AML1 is the most common target for translocations in acute myeloid leukaemia. The breakpoint mainly occurs in the intron between exons 5 and 6 just before the transactivation domain. The fusion protein created contains the DNA-binding domain of AML1 fused to the transcription factors ETO or EVI1 (on chromosomes 8 and 3 respectively). The abnormality can give rise to tumourigenic growth through a number of mechanisms. AML1 activates transcription of reporter genes from Cbf, GM-CSF, CSF1R, or TCRβ sites. AML 1. Nucifora G et al., Blood 1995;86(1): Heim and Mitelman, Willey-Liss, Inc AML1 Chromosome 21 DSCR1 CLIC6 AML1 (RUNX1) D21S326 D21S1706 D21S1969 D21S393 D21S1895 D21S Kb 167Kb ETO Chromosome 8 ETO (CBFA2T1) D8S1950 D8S1648 D8S1952 D8S2020 D8S1603 D8S Kb 148Kb 12

13 Cat. No. LPH 011-S Cat. No. LPH 011 Aquarius Haematology ATM Deletion The protein kinase ATM (Ataxia-Telangiectasia Mutated) gene located in 11q22.3 is frequently deleted in cases of B-CLL. The ATM gene is an important checkpoint gene involved in cell damage management. Its function is to assess the level of DNA damage that the cell has received and to attempt repair by phosphorylating key substrates involved in DNA repair. Recently, the ATM/P53 interaction in B-CLL has been shown to have an important impact on the proliferation or otherwise of the cancer. 1 It has been shown that ATM concurrently enhances the phosphorylation of P53 2 should the damage to the cell be so great that it should be destroyed by apoptosis (which is mediated by P53). Deletion of ATM therefore removes this checkpoint activity and hence activation of the P53. Thus, there is no attempt at repairing damaged cells and no apoptosis of these cells despite the P53 protein being present. In the absence of ATM, damaged cells are allowed to proliferate. Deletions of ATM and P53 are the most serious rearrangements involved in CLL and detection of deletions of these genes provides very important information as to the therapy choices for such patients especially since deletions of 11q22.3, and therefore ATM, provide a poor prognosis. CLL 1. Stankovic et al., Blood 2004;103(1): Khanna et al., Nature Genetics 1998;20(4): D11Z1 ATM Chromosome 11 NPAT ATM D11S3347 D11S Kb 13

14 Aquarius Haematology Cat. No. LPH 007-S Cat. No. LPH 007 Cat. No. LPH 038-S Cat. No. LPH 038 BCR/ABL and BCR/ABL Plus Translocation, Dual Fusion The presence of the Philadelphia chromosome (Ph') has important diagnostic and prognostic implications in a number of Haematological disorders. The abnormality is characteristic of Chronic Myeloid Leukaemia (CML), found in around 90% of cases but represents a significant abnormality in 30% of adult, and 2 to 10% of childhood 1, Acute Lymphoblastic Leukaemia (ALL) cases 2,3,4. This rearrangement is also seen in rare cases of Acute Myelogenous Leukaemia (AML) 5. As a result of the Philadelphia translocation, t(9;22)(q34;q11), the ABL1 (Abelson) proto-oncogene and the BCR (Breakpoint Cluster Region) gene fuse, giving rise to the BCR/ABL1 hybrid or fusion gene. The breakpoints in the CML translocation have been located in sub-bands 22q11.21 and 9q The translocation between chromosomes 9 and 22 can be accompanied by loss of proximal sequence (ASS1/ABL1) and distal 22q found in ALL and rare cases of AML. The deletion of 9q encompassing the ASS1 gene is associated with poor prognosis and the time to disease progression on imatinib treatment is shorter. Therefore the establishment of the atypical patterns in the BCR/ABL1 translocation may have clinical diagnostic and prognostic implications. In BCR there are two breakpoint regions, m-bcr and M-BCR. In CML, most translocations involve the Major Breakpoint Cluster Region (M-BCR) located approximately between exons 12 to 16. In ALL, breaks mostly fall in the Minor Breakpoint Cluster Region (m-bcr), located between exons 1 and 2, though the resulting translocation is cytogenetically identical to that of CML. These alternative breakpoints join different exon sets of BCR to a common subset of the exons of the ABL1 resulting in 2 alternative chimeric oncogene products, p210(bcr-abl1) and p185(bcr- ABL1). Both of these fusion proteins possess enhanced tyrosine kinase activity. The resultant oncoproteins are (constitutively active) hyperactive kinases which promote uncontrolled growth of certain blood cells. 7 In ALL, the rearrangement is associated with an extremely poor outcome with an event-free survival (EFS) of 15% or less at 5 years in adult and childhood patients treated with chemotherapy alone 8,9. There are no reports of long-term survivors 10. Allogeneic bone marrow transplantation is the only curative therapy for these patients. Children with this rearrangement are treated on a high-risk protocol, and adults are recommended for immediate bone marrow transplantation. Philadelphia (Ph) chromosomepositive Acute Myeloid Leukaemia (AML) is characterised by its resistance to conventional standard chemotherapy and poor prognosis, so accurate and rapid identification of this chromosomal abnormality is vital. In a small number of cases of ALL, the translocation does not result in a cytogenetically visible Philadelphia chromosome. In these cases, FISH is essential for highlighting the fusion gene Macdonald et al., Molecular Biology of Cancer 2nd Ed BCR/ABL Translocation, Dual Fusion (t(9;22) patient) ALL AML CML 2. Groffen et al., Cell 1984;36: Shtivelman et al., Nature 1985;315: Hermans et al., Cell 1987;51: Nishida et al., Leuk Res 2007;31(3): Prakash O, Yunis JJ, Cancer Genet Cytogenet 1984;11: Goodsell D, The Oncologist 2005;10(9): Fletcher et al., Leuk Lymphoma 1992;8: Secker-Walker et al., Br Med J 1978;2(6151): Wetzler et al., Blood 1999;3(11): Van Rhee et al., Br J Haematol 1995;90:

15 Aquarius Haematology BCR/ABL BCR/ABL Plus Cat. No. LPH 007 Cat. No. LPH 038 BCR Chromosome 22 GNAZ RAB36 BCR IGLL1 D22S1002E D22S257 D22S Kb 148Kb BCR/ABL Cat. No. LPH 007 ABL1 Chromosome 9 ASS1 FUBP3 PRDM12 ABL1 LAMC3 D9S2057 D9S1863 D9S Kb 346Kb 100kb BCR/ABL Plus Cat. No. LPH 038 ABL1 Chromosome 9 ASS1 FUBP3 PRDM12 ABL1 LAMC3 D9S2057 D9S1863 D9S Kb 346Kb 100kb 15

16 Aquarius Haematology Cat. No. LPH 035-S Cat. No. LPH 035 BCL6 Breakapart Rearrangements of the BCL6 gene have been observed in 20-40% of diffuse large B cell lymphoma (DLCL) and in 5-15% of follicular lymphoma (FL) 1,2. This translocation results in the deregulation of BCL6 and may involve the IGH, IGK and IGL genes, although other non-ig partner genes have also been identified 2, 3. BCL6 encodes a 96 kd nuclear phosphoprotein belonging to the pox virus zinc finger (POZ)/zinc finger (ZF) family of transcription factors 4. BCL6 functions as a transcriptional repressor and plays an important regulatory role in lymphoid cell development and function. In the B-cell lineage the BCL6 protein is expressed only in germinal centre B cells 5. Translocations involving band 3q27.3 affect a 4 kb major breakpoint region (MBR) of BCL6 in the first non-coding exon and 5 region of the first intron, which are common in diffuse large B Cell Lymphomas. Recently an alternative breakpoint cluster region (ABR) located between 245 and 285kb 5 of BCL6 was identified in Non- Hodgkins Lymphomas (NHL) which may be preferentially associated with Follicular Lymphoma 6. L 1. Ohno H et al., Leuk Lymphoma 1997;27: Ueda C et al., Leuk Lymphoma 2002;43: Akasaka H et al., Cancer Res 2000;60: Chang CC et al., PNAS 1996;93: Cattoretti G et al., Blood 1995;86: Bosga-Bouwer AG et al., Genes Chrom Cancer 2005;44:301-4 BCL6 Chromosome 3 SST RTP2 BCL6 G67157 D3S Kb 170Kb 16

17 Cat. No. LPH 022-S Cat. No. LPH 022 Aquarius Haematology CBFβ/MYH11 Translocation, Dual Fusion The fusion gene MYH11/CBFβ is created by the inversion inv(16)(p13q22) found in 20% of AML M4 cases. In particular M4 with marked eosinophilia (M4eo) and rarely, in M2, M5 and M4, without eosinophilia. Overall, abnormalities involving 16q22 are seen in 5-10% of AML 1,2,3. Frequently Central Nervous System (CNS) involvement develops, particularly in relapse, however, the complete remission rate is high and the prognosis is better than most of the AML associated abnormalities 3,. The inversion may be missed in poor cytogenetic preparations. FISH probes for 16p13 often show a deletion within 16p13 in addition to the 16p13/16q22 rearrangement (~20% cases). In these patients, the split signal may be lost. Variant rearrangements are t(16;16)(p13;q22) and del(16)(q22). The latter is associated with previous MDS, older age, a complex karyotype and a worse prognosis. Additional abnormalities include +8, +22, del(7q) and +2 which confer no change to the prognosis. AML 1. Heim and Mitelman, Wiley-Liss Inc Monreno-Miralles et al., J Biol Chem 2005;280(48): Huret, Atlas Genet Cytogenet Oncol Haematol 1999 The breakpoints occur in intron 5 of CBFβ and intron 5 of MYH11. The N-terminal of CBFβ fuses to the C-terminal of MYH11 with its multimerisation domain. The resultant chimeric protein reduces the amount of active CBF. An accumulation of CBFβ-MYH11/CBFα multimers in the nucleus also occurs. CBFβ regulates expression of certain ADP-Ribosylation Factors (ARFs) and other TSGs and therefore the fusion protein is thought to repress TSG expression 2,3. CBFB Chromosome 16 D16S301 D16S324 D16S3339 D16S3323 CBFB 617Kb D16S3308 D16S2584E D16S2846 MYH11 Chromosome 16 MYH11 D16S3302 D16S3060 D16S2853 D16S3286 D16S2803 D16S405 D16S3127 D16S Kb 17

18 Aquarius Haematology Cat. No. LPH 039-S Cat. No. LPH 039 CKS1B/CDKN2C (P18) Amplification/Deletion Structural abnormalities of chromosome 1 are frequently detected in multiple myeloma, and have been correlated with more advanced disease 1. Amplification of 1q21 (CKS1B) is one of the most recurrent chromosomal aberrations in multiple myeloma. Over-expression of the CKS1B gene up-regulates cell cycle progression resulting in a more proliferative disease 2. This is related to the advanced phenotype of multiple myeloma and therefore may be associated with poor prognosis and disease progression 3,4. Gain of 1q21 has been linked to inferior survival and further amplification is observed in disease relapse. In has been shown that gain of 1q21 copy number is not an independent prognostic factor in multiple myeloma and is often associated with other chromosomal aberrations most commonly t(4:14) and chromosome 13 deletion 2. The association of 1q21 gain and loss of chromosome 13 has been linked to the risk of conversion to overt disease 5. In a case study of multiple myeloma patients it was discovered that 30% of chromosomal abnormalities mapped to chromosome 1, most up-regulated genes mapped to chromosome 1q and down-regulated genes to chromosome 1p 6. Gains of the long arm 1q are one of the most common genetic abnormalities in multiple myeloma 7 and duplications of the chromosome 1q band are frequently associated with disease progression 8,9. MM 1. Tasaka et al., Br J Haematology 1997;96(1): Fonseca et al., Leukemia 2006;20(11): Cremer FD, Cancer Genet Cytogenet 2005;161(2): Hanamura I, Blood 2006;108(5): Rosinol L, Br J Haematology 2005;130(5): Shaughnessy JD, Blood 2007;109(6): Avet-Loiseau H, Genes Chromosomes Cancer 1997;19(2): Sawyer JR, Blood 1998;91(5): Le Baccon P et al., Genes Chromosomes Cancer 2001;32(3): Kulkari et al., Leukemia 2006;16: In band 1p32.3 CDKN2C (p18) is a tumour suppressor gene responsible for inducing apoptotic cell death and DNA fragmentation 10. It is up-regulated by the expression of the cytokine IL-6 in multiple myeloma and deletion of the gene is associated with a more proliferative disease. Although p18 deletions have been reported to be rare in human malignancy, cytogenetic analysis has shown abnormalities of 1p32-36 in 16% of human multiple myeloma 10. CDKN2C (P18) CKS1B Chromosome 1 FAF1 CDKN2C CKS1B PYOG2 ZBTB7B D1S Kb 180Kb 18

19 Cat. No. LPH 010-S Cat. No. LPH 010 Aquarius Haematology cmyc Breakapart Translocations involving the cmyc oncogene (Avian Myelocytomatosis Viral Oncogene Homologue) are similar in B-ALL as they are in Burkitt s Lymphoma and result in the close juxtaposition of cmyc to IGH, IGL and IGK in t(8;14) 1, t(8;22) and t(2;8) respectively. In each case, this results in deregulation of the cmyc (as a result of being close to the constitutively active Immunoglobulin locus), increased transcription and neoplastic growth 2. The breakpoints involved are widely scattered throughout the gene but the t(8;14) translocation always spares the protein coding exons which become attached to the derived 14. In the t(2;8) and t(8;22) the cmyc remains on chromosome 8 and the Immunoglobulin locus joins it, resulting in the close positioning of the cmyc and Immunoglobulin loci. ALL with these associated translocations (less than 5% of all ALLs) are invariably of the L3 subtype. Most are caused by the t(8;14) (85%), t(8;22) is the next most common at 10% and finally t(2;8) makes up the remaining 5%. Patients with cmyc rearrangements were originally thought to have poor prognoses but they do respond well to intensive chemotherapy affording them an increased survival rate. This shows that cytogenetic confirmation of the rearrangement is necessary to manage the patient effectively 3. ALL L 1. Moore S et al., Cancer Genet Cytogenet 2003;141: Robertson et al., Nature 1983;302(5908): Hoelzer et al., Blood 1996;87(2): MYC Chromosome 8 POU5F1P1 MYC D8S1153 D8S1128 D8S1980 D8S1720 D8S Kb 186Kb 19

20 Aquarius Haematology Cat. No. LPH 006-S Cat. No. LPH 006 Deletion 13q14.3, D13S319 and D13S25 Chromosomal abnormalities on chromosome 13q occur in 16-40% of multiple myeloma cases 1 and are associated with poor prognosis. A case study showed that in 90% of patients, the 13q14 region was affected and 68% also involved the 13q21 region. The critical region in all but 8 patients was located to 13q14 2. Deletions affecting the 13q14 band are also the most frequent genetic abnormalities of B-cell Chronic Lymphocytic Leukaemia (B-CLL) 3. This region is found deleted heterozygously in 30-60% and homozygously in 10-20% of CLL patients 4. Recently though, the survival rate has been shown to be similar 5. Two non-coding RNA genes, DLEU1 and DLEU2, and the genetic marker D13S319, span the pathogenic critical region 13q DLEU1 is considered to be the most likely CLL-associated candidate tumour suppressor gene within the 13q14 region 7. Subsequently the locus D13S319, located between the RB1 gene and D13S25 and within the DLEU1 locus, was found to be deleted in 45% of CLL cases 8. It has also been postulated that a gene telomeric to the D13S319 region encompassing D13S25 and 206XF12 may be important in cases where there are hemizygous deletions, and that this gene is a putative tumour suppressor gene 6. The Cytocell D13S319 deletion probe covers the marker and the centromeric end of the DLEU1 locus. The Cytocell D13S25 deletion probe covers the marker, the telomeric end of DLEU1 and the centromeric end of DLEU7. CLL MM 13q14.3 Deletion 1. Bullrich F et al., Cancer Res 2001;61: Shaughnessy J et al., Blood 2000;96: Juliusson G et al., N Eng J Med 1990;323: Hammarsund M et al., FEBS Letters 2004;556: Van Dyke DL et al., Br J Haematology 2009;148: Liu Y et al., Oncogene 1997;15: Wolf S et al., Hum Mol Genet 2001;10: Liu Y et al., Blood 1995;86: Bullrich F et al., Blood 1996;88(8):

21 Cat. No. LPH 042-S Cat. No. LPH 042 Cat. No. LPH 043-S Cat. No. LPH 043 Aquarius Haematology 13q14.3 Cat. No. LPH q qter Chromosome 13 DLEU1 D13S319 D13S272 D13S25 D13S Kb D13S319 Cat. No. LPH 042 D13S319 13qter Chromosome 13 DLEU5 DLEU2 DLEU1 D13S1220 D13S272 D13S Kb D13S25 Cat. No. LPH 043 D13S25 13qter Chromosome 13 DLEU1 DLEU7 RNASEH2B GUCY1B D13S25 D13S Kb 21

22 Aquarius Haematology Cat. No. LPH 024-S Cat. No. LPH 024 Del (5q) Deletion Deletion of a region of 5q which includes EGR1 is the most common rearrangement in AML and MDS 1,2. Abnormalities of chromosome 5 are found in 42% of therapy related MDS. The deletions are large with breakpoints occurring in bands between 5q11 and 5q35 and are usually interstitial. Two regions, 5q12-14 and 5q31-33, are hot spots for breakpoints but the common deleted region (CDR) in AML and aggressive MDS cases is within 5q31.1 2,3. Early Growth Response 1 (EGR1) maps to this band 3 and loss of the gene may cause tumourigenesis in at least two ways. Firstly, EGR1 directly controls the expression of fibronectin (FN1) through pathways that involve TGFB1 and plasminogen activator-1 (SERPINE1 or PAI1). Together FN1 and PAI1 inhibit cancer cell growth 4. In addition, EGR1 is required for p53-dependent apoptosis through the mediation of Retinoblastoma (RB1) protein. AML 1. Boultwood J et al., Blood 2002;99(12): Charrin C, Atlas Genet Cytogenet Oncol Haematol 1998;2(3): McKusick V, Rasooly R, OMIM last edited Liu C et al., Proc Natl Acad Sci USA 1996;93: p EGR1 Chromosome 5 TAS2R1 CDC25C EGR1 D5S630 D5S2064 D5S1701 D5S500 D5S2415 D5S Kb 184Kb 22

23 Cat. No. LPH 025-S Cat. No. LPH 025 Aquarius Haematology Del (7q) Deletion Abnormalities of chromosome 7 are very common in myeloid malignancies occurring in 5-10% of de novo AML (M4 & M6), ~15% of adult MDS (30% of RAEB/RAEBT), 40% of paediatric MDS and 50% of treatment related AML/MDS. In children, it is often associated with juvenile Chronic Myeloid Leukaemia (jcml) 1,2,3,4. There is also a predominance in leukaemias associated with a constitutional predisposition caused by disorders including neurofibromatosis 1 (NF1), Fanconi Anaemia and possibly Down syndrome which produces a distinct clinical picture known as Monosomy 7 syndrome. Another rearrangement, -5/del(5q), is found as an additional abnormality in 40-60% of secondary MDS cases, +8 is less frequently seen 4. Studies of myeloid disorders involving -7/del(7q) have found that signalling pathways using RAS proteins are affected. There are two commonly deleted regions (CDR), one at 7q22 determined through LOH studies and the use of YAC libraries, the second at 7q ,5,6,7,8. RELN (7q22) encodes a large secreted protein related to extracellular matrix proteins which contains multiple epidermal growth factor (EGF)-like proteins. AML 1. Heim and Mittelman, Willey-Liss, Inc Emerling BM et al., Oncogene 2002;21: Kratz CP et al., Genomics 2001;77(3): Desangles F, Atlas Genet Cytogenet Oncol Haematol Le Beau MM et al., Blood 1996;88(6): Fischer K et al., Blood 1997;89(6): Koike M et al., Leukemia Res 1999;23: Kratz CP et al., Genomics 2001;77(3): q22 7q31 Chromosome 7 RELN ORC5L TES D7S Kb D7S658 D7S2543 D7S2886 D7S Kb 203Kb 23

24 Aquarius Haematology Cat. No. LPH 020-S Cat. No. LPH 020 Del (20q) Deletion Deletions within the long arm of chromosome 20 are found in 4.1% and 1.5% of MDS and AML cases respectively. Del(20q) also occurs in 10% of polycythemia vera patients and other subgroups of MPD 1,2,3. The deletions are predominantly interstitial and the breakpoints usually occur in the region q11.2-q13.1. Often other cytogenetic abnormalities are present such as del(5q), - 7/del(7q), +8, del(18q), +21 and rearrangements of 13q. Due to the relatively small size of the deletion and the lack of banding features on chromosome 20, FISH is particularly useful in detecting this abnormality. The prognosis for MDS cases where del(20q) is the sole abnormality is good. However if secondary abnormalities are present, a poor outcome is indicated. AML patients respond poorly to treatment and have reduced survival rates. The clinical outcome for MPD patients remains unchanged in the presence of the abnormality. AML 1. Patsouris et al., Cancer Genet Cytogenet 2002;138: Bilhou-Nabera, Atlas Genet Cytogenet Oncol Haematol Bench et al., Oncogene 2000;19(34): Li J et al., PNAS 2004;101: Wang et al., Genomics 1999;59: The mechanism of leukaemogenesis associated with del(20q) is unknown, however deletion of a tumour supressor gene (TSG) is thought to cause the increased proliferation of the cancer cells 2. Using RT-PCR analysis, Bench et al identified potential target genes in the region of overlap between the AML/MDS and MPD CDR at band 20q12. Five genes were expressed in both bone marrow and CD34 positive cells. Of the three previously identified genes, h-lmbt regulates chromatin structure during mitosis, SFRS6 encodes a serine rich protein important to regulation of alternative splicing of mrna and MYBL2, a member of the myb transcription factor family, is involved in cell cycle control 2,4,5. 20q12 20q13.12 Chromosome 20 PTPRT MYBL2 D20S591E D20S108 D20S858 D20S43 D20S Kb 139Kb 174Kb 24

25 Cat. No. LPH 019-S Cat. No. LPH 019 Aquarius Haematology E2A Breakapart Translocations involving the E2A (Transcription Factor 3) gene have been generally accepted as non-random chromosome translocations in childhood B-ALL. At present, there are two partner genes, PBX1 and HLF (on chromosomes 1 and 17 respectively) which become fused to E2A as a result of the t(1;19) and t(17;19) translocations forming the E2A/PBX1 and E2A/HLF fusion proteins. The former is more common, being present in about 5% of paediatric ALLs 1, whilst the other is present in some 1%. Both are associated with poor outcome, with the t(1;19) patients being at high risk of relapse. Both fusion proteins have transcriptionally active domains but their true pathway to the leukaemia is yet to be completely understood. ALL 1. Crist et al., Blood 1990;76(1): Izraeli et al., Leukemia 1993;7(5):671-8 Detection of the t(1;19) is best carried out using molecular methods such as FISH as the fusion has been shown to be missed in 20 to 25% of patients by standard cytogenetics 2. E2A Chromosome 19 RKHD1 E2A (TCF3) D19S883 MBD3 RH Kb 189Kb 25

26 Aquarius Haematology Cat. No. LPH 036-S Cat. No. LPH 036 EVI1 Breakapart Chromosome rearrangements involving band 3q26.2 are associated with myeloid malignancies, aberrant expression of the human ecotropic virus integration site 1 (EVI1) gene, an unfavourable prognosis and an aggressive clinical course 1. The EVI1 gene encodes a 1051 amino acid finger protein inappropriately expressed in the leukaemic cells of between 2-5% of AML and MDS patients 2. The EVI1 activation often follows a chromosomal rearrangement involving 3q26, and the two most common abberations are t(3;3)(q21;q26) and inv(3)(q21q26). EVI1 also exists as a longer protein which includes 188 additional amino acids at the N-terminus named MDS1/EVI1. The breakpoints for the translocations and inversions vary considerably. Inv(3) breakpoints are found centromeric to, and including, the EVI1 gene and cover about 600kb. The breakpoints in 3q26.2 translocations are telomeric to the EVI1 gene and cover a region including the telomeric end of the MDS1 gene and the MYNN gene 1. AML 1. Bobadilla D et al., Br J Haematol 2007;136: Soderholm J et al., Leukemia 1997;11:352-8 EVI1 Chromosome 3 EVI1 MYNN LRRC34 D3S3364 D3S1614 D3S3809 D3S1282 D3S3523 D3S Kb 179Kb 156Kb 26

27 Cat. No. LPH 032-S Cat. No. LPH 032 Aquarius Haematology FIP1L1/CHIC2/PDGFRA Deletion/Fusion Deletion of CHIC2 in patients with idiopathic hypereosinophilic syndrome (HES) and chronic eosinophilic leukaemia (CEL) results in the fusion of FIP1L1 (FIP1-like 1) with PDGFRA (platelet derived growth factor receptor alpha) producing a tyrosine kinase which transforms haematopoietic cells. In such patients, this activity is inhibited by imatinib mesylate (Gleevec), which is a tyrosine kinase inhibitor. The diagnosis of the fusion gene can therefore lead to therapeutic choices for the patient 1,2. CML 1. Cools J et al., N Eng J Med 2003;348: Griffin JH et al., PNAS 2003;100: FIP1L1/CHIC2/PDGFRA Chromosome 4 SCFD2 FIP1L1 LNX CHIC2 PDGFRA KIT D4S1514 D4S1036 D4S461 D4S1394 D4S956 D4S1594 D4S1630 D4S Kb 174Kb 174Kb 27

28 Aquarius Haematology Cat. No. LPH 014-S Cat. No. LPH 014 IGH Breakapart In ALL, IGH is most notably involved in rearrangements involving the cmyc oncogene as a result of the t(8;14) translocation 1. However, less common rearrangements of the IGH gene are most often seen in T-ALL but can also be found in B-ALL. There are a number of stereotypical translocations involved in each of the two diseases and more are being described regularly. In T-ALL for example, IGH is observed in the t(14;14)(q11;q32) translocation (or inv(14)(q11q32) rearrangement) 2 and is found in T-cell leukaemia associated with ataxia-telangiectasia (AT). However, rare reports have indicated that this abnormality also occurs in B-ALL. The recurrent t(14;19)(q32;q13) translocation associated with chronic B-cell lymphoproliferative disorders, such as atypical CLL, has also been shown to occur in B-ALL and results in the juxtaposition of the IGH and BCL2 genes and subsequent over expression of BCL3 3. More recently, a report suggested the involvement of IGH in a novel cryptic translocation in paediatric T-cell Acute Lymphoblastic Leukaemia (T-ALL), which also involved TLX3 (HOX11L2) or NKX2-5 (CSX) on 5q35 brought about by a t(5;14)(q35;q32) translocation 4. IGH Breakapart (IGH Translocation patient) ALL CLL MM L 1. Moore et al., Cancer Genet Cytogenet 2003;141(1): Liu et al., Cancer Genet Cytogenet 2004;152: Robinson et al., Genes Chromosomes Cancer 2004;39(1): van Zutven et al., Haematologica 2004;89(6):671-8 In CLL, around 20% of cytogenetically abnormal CLL patients have a detectable 14q+ marker chromosome. The markers are derived from reciprocal translocations involving a number of fusion partners from different chromosomes that fuse with the IGH gene at 14q32. Involvement of chromosomes 1, 2, 5, 6, 7, 8, 9, 11, 12, 13, 14, 17, 18, 19 and 22 have been described and the two most common translocations are IGH/BCL2 involving the t(14;18) translocation and IGH/CCND1 involving the t(11;14) translocation. With all these rearrangements having breakpoints within the IGH gene, we have designed a split probe set for IGH, which can detect any rearrangement. This involves the splitting of the IGH gene in the region between the Constant and Variable segments thereby identifying the IGH translocation partner chromosome in the less common rearrangements of this gene. IGH Chromosome 14 Constant Segment JD IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP Variable Segment D14S1419 D14S1420 D14S Kb 617Kb 28

29 Cat. No. LPH 018-S Cat. No. LPH 018 Aquarius Haematology IGH/BCL2 Translocation, Dual Fusion This is the second most common rearrangement of IGH in CLL and is cytogenetically indistinguishable from the t(14;18) translocation observed in follicular lymphoma. The translocation is thought to be brought about by an error in the joining function of the IGH gene, mediated by the recent observation that both IGH and BCL2 are arranged next to each other in the normal B lymphocyte 1. The translocation breakpoint at the end of the Joining (J) segment, and the subsequent fusion of the BCL2 gene to this region, results in the BCL2 gene coming under the regulatory control of those processes normally involved in maintenance of IGH gene activity. The BCL2 gene itself has been shown to be involved in the regulation of apoptosis. CLL L 1. Roix et al., Nature Genetics 2003;34(3): IGH Chromosome 14 Constant Segment JD IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP Variable Segment D14S1419 D14S1420 D14S Kb 168Kb 617Kb BCL2 Chromosome 18 BCL2 KDSR D18S91 D18S1264 D18S51 D18S87 D18S Kb 100kb 29

30 Aquarius Haematology Cat. No. LPH 021-S Cat. No. LPH 021 IGH/CCND1 Translocation, Dual Fusion Reciprocal translocations involving the IGH and CCND1 (BCL1) loci were frequently reported in B-CLL patients 1. The involvement of the CCND1 (Cyclin D1) gene was initially reported from a cloning study looking at the breakpoints of the translocation. However, it is likely that the initial diagnosis on the samples used for the study should have been Mantle Cell Lymphoma (MCL). The probe set for the t(11;14) translocation has been provided on the CLL panel to enable atypical B-CLL patients to be distinguished from possible MCL patients following the guidelines of the UK ACC Professional Standards Committee. MM L CLL 1. Brizard et al., Leuk Lymphoma 1997;25(5-6): IGH Chromosome 14 Constant Segment JD IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP Variable Segment D14S1419 D14S1420 D14S Kb 168Kb 617Kb CCND1 (BCL1) Chromosome 11 MYEOV CCND1 FGF19 FGF4 FGF3 D11S2663 D11S1100 D11S4095 D11S1076 D11S4381 D11S Kb 159Kb 30

31 Cat. No. LPH 040-S Cat. No. LPH 040 Aquarius Haematology IGH/CCND3 Translocation, Dual Fusion Approximately 40-60% of Multiple Myeloma cases are associated with translocations involving the IgH locus at chromosome 14q32 and one of several partners including CCND1, WHSC1 (MMSET), CCND3, cmaf and MAFB. Cases lacking an IgH translocation are associated with hyperdiploid phenotype 1. CLL MM The CCND3/IGH translocation t(6;14)(p12-p21;q32) has been reported in 3-4% of multiple myeloma tumours 2. However, it is also characteristic of a variety of other B-cell malignancies including plasma cell leukaemia, diffuse large B-cell non-hodgkin lymphoma (DLBCL) and splenic marginal zone lymphomas (SMZLs) 3. Cyclin D3 has been identified as a putative oncogene that is dysregulated as the consequence of the translocation 2. The translocation appears to be mediated by an error in IgH switch recombination because it has been shown that in KMM-1 cell lines, the translocation disrupts a switch sequence in this region and results in juxtaposition of CCND3 with the IGH promoter, thus elevating the levels of CCND3 expression 2. It is thought that this mechanism is similar in all cases of IgH translocation. Most breakpoints appear to be clustered in a region that is fewer than 200kb centromeric to CCND3 2 and it is this region that is flanked by the Cytocell probe. IGH Chromosome Fonseca et al., Cancer Res 2004;64: Shaughnessy et al., Blood 2001;98(1): Soniki et al., Blood 2001;98(9): Constant Segment JD IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP Variable Segment D14S1419 D14S1420 D14S Kb 168Kb 617Kb CCND3 Chromosome 6 CCND3 TAF8 C6orf132 D6S1017 D6S400 D6S Kb 244Kb 31

32 Aquarius Haematology Cat. No. LPH 041-S Cat. No. LPH 041 IGH/cMYC Translocation, Dual Fusion The cmyc/igh translocation t(8;14) and the variant forms t(2;8)(p13;q24) and t(8;22)(q24;q11) are found in Burkitt s lymphoma and mature B-cell or Burkitt s type Acute Lymphoblastic Leukaemia (ALL) 1. The t(8;14) is found in approximately 75% of patients with Burkitt s lymphoma. While the translocation breakpoints on chromosome 14 are clustered to a narrow region 5 of the intron enhancer of the immunoglobulin heavy chain, the breakpoints on chromosome 8 can occur more than 340kb upstream of cmyc with no preferential site 2. The translocations bring cmyc into close proximity of the IgH enhancer and result in the up-regulation of cmyc 3. Over expression of the transcription factor stimulates gene amplification resulting in uncontrolled cell proliferation, which usually occurs in the late event of tumour progression 4. L ALL 1. Berger R, Bernheim A, Cancer Genet Cytogenet 1982;7(3): Joos et al., Human Molecular Genetics 1992;1(8): Erikson J et al., Proc Natl Acad Sci USA 1983;80(3): Shou et al., PNAS 2000;97(1): IGH Chromosome 14 Constant Segment JD IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP Variable Segment D14S1419 D14S1420 D14S Kb 617Kb MYC Chromosome 8 POU5F1P1 MYC D8S1153 D8S1128 D8S490 D8S1087 D8S1980 D8S1720 D8S Kb 186Kb 32

33 Cat. No. LPH 030-S Cat. No. LPH 030 Aquarius Haematology IGH/FGFR3 Translocation, Dual Fusion The t(4;14) (p16.3;q32.3) translocation is found in approximately 10% of myeloma patients and results in the deregulation of at least two genes. These are MMSET (Multiple Myeloma SET domain containing protein also known as WHSC1) and FGFR3 (fibroblast growth factor receptor 3) with the formation of a fusion product between MMSET and the immunoglobulin heavy chain (IGH) locus and overexpression of FGFR3. The majority of the breakpoints on chromosome 4 occur within the MMSET locus, resulting in a fusion gene where the 5 exons of MMSET are replaced with the VDJ region of the heavy chain locus. MM 1. Sibley K et al., Br J Haematol 2002;118: Keats JJ et al., Blood 2003;101(4): FGFR3 lies at least 50kb from the breakpoints after translocation to chromosome 14, and it has been suggested that FGFR3 expression is switched on by close proximity to the strong enhancer elements in the immunoglobulin heavy chain 1. Other studies state that the breakpoints on chromosome 4 lie in a 113kb region between FGFR3 and MMSET exon 5 2. The breakpoint on chromosome 14 is almost exclusively in or near the switch region of the IGH locus. IGH Chromosome 14 Constant Segment JD IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP Variable Segment D14S1419 D14S1420 D14S Kb 168Kb 617Kb FGFR3 Chromosome 4 FGFR3 WHSC2 D4S168 LETM1 MMSET D4S573 D4S2561E (WHSC1) D4S166 D4S1182 D4S43 118Kb 126Kb 33

34 Aquarius Haematology Cat. No. LPH 029-S Cat. No. LPH 029 IGH/MAF Translocation, Dual Fusion The translocation t(14;16) (q32.3;q22) involving c-maf and IGH is present in 25% of myeloma cell lines 1, in 2-6% of primary Multiple Myeloma samples 2,3 and results in high levels of c-maf expression. Patients harbouring t(14;16) appear to have a worse outcome 3,4. The majority of the breakpoints are dispersed over an approximate 500kb region centromeric to the c-maf proto-oncogene at 16q23. Other breakpoints however have been seen telomeric to c-maf 1. Translocation with the centromeric breakpoints places c-maf under the control of the strong 3 IGH enhancer. Recent gene expression profiling of myeloma cell lines revealed that c-maf transactivated cyclin D2 (a promoter of cell cycle progression), thus enhancing proliferation of myeloma cells 4. The putative tumour suppressor WWOX gene spans the common chromosomal fragile site 16D (FRA16D) at chromosome 16q , and this region is a frequent target for loss of heterozygosity and chromosomal rearrangements in ovarian, breast, hepatocellular, prostate carcinomas and other neoplasias 5. WWOX is contained within the breakpoint region of the c-maf-igh translocation. MM 1. Chesi M et al., Blood 1998;91(12): Avet-Loiseau H et al., Blood 2002;99(6): Fonseca R et al., Blood 2003;101(11): Chang H et al., Leukemia 2007;21: Nunez MI et al., BMC Cancer 2005;5:64 IGH Chromosome 14 Constant Segment JD IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP Variable Segment D14S1419 D14S1420 D14S Kb 168Kb 617Kb MAF Chromosome 16 WWOX MAF D16S2793 D16S518 D16S3213 D16S3029 RH69965 D16S3073 D16S Kb 91Kb 119Kb 125Kb 34

35 Cat. No. LPH 044-S Cat. No. LPH 044 Aquarius Haematology IGH/MAFB Translocation, Dual Fusion The t(14:20) translocation is one of seven recurrent IGH translocations that are referred to as primary oncogenic events in around 73% of Multiple Myeloma cases 1. These translocations result in a transcription product that dysregulates further controlling genes such as the cyclin family of cell cycle control genes which then disrupts normal cell division. In the case of the t(14;20) translocation, the reciprocal rearrangement brings a truncated form of the IGH µ-enhancer (Eµ, located between the Joining (J) segments and the constant region of the IGH gene) in close contact with the MAFB gene 2. The resultant fusion and the up-regulated transcription product has been shown to cause overexpression of CyclinD3 in around 7% of tumours 3. Of the seven translocation partners of IGH in Multiple Myeloma (including CCND1, CCND2, CCND3, c-maf, MAFA and FGFR3/MMSET), MAFB has a prevalence of around 2% 4. The three genes in the MAF family provide equally poor prognosis for patients and a similar phenotype, but the MAFB translocation shows a different prevalence in Multiple Myeloma patients (2%) compared to MGUS/SMM cases as around 7% of these tumours have the rearrangement 5. MM 1. Avet-Loiseau H et al., Blood 2002;99(6): Boersma-Vreugdenhil GR et al., Br J Haematol 2004;126: Bergesgagel PL et al., Blood 2005;106(1): Zhan F et al., Blood 2007;109(4): Chng W et al., Best Pract Res Clin Haematol 2007;20: IGH Chromosome 14 Constant Segment JD IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP Variable Segment D14S1419 D14S1420 D14S Kb 617Kb MAFB Chromosome 20 TIMAP MAFB TOP1 PLCG1 195Kb 161Kb 289Kb 35

36 Aquarius Haematology Cat. No. LPH 045-S Cat. No. LPH 045 IGH/MYEOV Translocation, Dual Fusion The most common translocation in Multiple Myeloma is the t(11;14)(q13;q32) which accounts for 15-20% of cases that were identified by FISH 1. These translocations lead to overexpression of Cyclin D1 in about 25% of cases 2 and lead to alterations in the switch regions of the IGH gene 3. Unlike Mantle Cell Lymphoma (MCL), where the breakpoints are clustered in a 1kb region 120kb centromeric of the CCND1 gene (BCL1), the breakpoints in Multiple Myeloma cases are dispersed within a 360kb region between CCND1 and MYEOV (MYEloma OVerexpressed) in band 11q13 4. MYEOV is a putative oncogene, located 360kb away from CCND1 which is thought to be activated in the translocation by it becoming closely associated with IGH enhancers. In contrast to IGH rearrangements in other neoplasms, those found in Multiple Myeloma have IGH breakpoints predominantly in the C/J region which, in the case of MYEOV, brings the MYEOV gene under the control of the 3 Eα1 enhancer 4. In CCND1 translocations, by contrast, the Eµ enhancer controls CCND1 expression. Both of these recombination events provide a poor prognosis for the patient and are thought to be independent. MM 1. Ohno H et al., Leuk Lymphoma 1997;27: Ueda C et al., Leuk Lymphoma 2002;43: Akasaka H et al., Cancer Res 2000;60: Chang CC et al., PNAS 1996;93: IGH Chromosome 14 Constant Segment JD IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP Variable Segment D14S1419 D14S1420 D14S Kb 617Kb MYEOV Chromosome 11 TPCN2 MYEOV CCND1 ORAOV1 FGF4 FGF3 D11S4113 D11S2663 D11S1100 D11S4095 D11S1076 D11S Kb 162Kb 36

37 Cat. No. LPH 033-S Cat. No. LPH 033 Cat. No. LPH 034-S Cat. No. LPH 034 Aquarius Haematology IGL and IGK Breakapart Translocations involving the immunoglobulin loci are recurring events in various subtypes of B cell lymphomas. In addition to translocations involving the IGH locus, variant translocations have been described in 5-10% of B cell neoplasms involving either the immunoglobulin kappa (IGK) light chain locus at 2p11.2 or the lambda light chain (IGL) at 22q11 1,2. The best known translocations involving IG light chain loci are the variant Burkitt s translocations t(2;8)(p12;q24) and t(8;22)(q24;q11) present in up to 25% of all Burkitt s lymphomas 3. Other translocations involve the BCL6 oncogene: the t(2;3) (p12;q27) and t(3;22)(q27;q11) and BCL2 locus: t(2;18)(p12;q21) and t(18;22)(q21;q11) 4,5. L IGK Breakapart Translocations involving the IG light chain loci usually lead to breakage within the joining region of the respective locus 2. IGL consists of 38 potentially active variable (IGLV) gene segments, 35 pseudogenes and seven IGL constant gene segments, each with a joining (J)-segment IGL (J-C). IGK has a largely duplicated structure with duplicate gene regions being % identical. The IGK proximal copy is present in a 542kb contig, with 22 potentially functional variable (IGKV) gene segments and 18 pseudogenes, plus five joining (J)-segments and one IGK constant (IGKC) gene segment. The distal copy is a 433kb contig. with 21 potentially functional IGKV gene segments and 15 pseudogenes. The two contigs. are separated by a DNA sequence of 800kb without any IGKV segments. 1. Poulseu TS et al., Leukemia 2002;16: Martin-Subero JI et al., Int J Cancer 2002;98: Kornblau SM et al., Hematol Oncol 1991;9: Chaganti SR et al., Genes Chromosomes Cancer 1998;23: Tashiro S et al., Oncogene 1992;7:573-7 IGL Cat. No. LPH 033 IGL Chromosome 22 MAPK1 IGLV IGLC BCR D22S446 D22S1003 D22S Kb 307Kb IGK Cat. No. LPH 034 IGK Chromosome 2 CJ IGK Variable Regions Proximal Distal D2S2216 D2S2191 D2S1994 D2S2610 D2S2510 D21S Kb 183Kb 37

38 Aquarius Haematology Cat. No. LPH 013-S Cat. No. LPH 013 MLL Breakapart Rearrangement of the MLL gene at chromosome band 11q23 can be detected in the leukaemia cells of approximately 85% of infants with B-ALL 1,2,3. Translocations involving the MLL (11q23) gene are generally associated with increased risk for treatment failure 4. The most frequently observed of these translocations is the t(4;11) translocation involving the MLL gene and the AFF1 (AF4) gene on chromosome 4. 5,6 A poor outcome for infants with ALL is strongly associated with the presence of this rearrangement in particular. The discovery that a single YAC spanned breakpoints in four of the more common translocations led to the naming of the candidate gene MLL (Myeloid/Lymphoid or Mixed Lineage Leukaemia). The gene has homology with a drosophila gene ( trithorax ) which is highly conserved in humans and gives rise to a protein that can be folded to give six zinc finger domains and is a developmental regulator. The zinc finger domains are translocated to the AFF1, MLLT3 and FEN1P1 genes respectively on the partner chromosomes in the t(4;11), t(9;11) and t(11;19) translocations. Each of the genes involved in these translocations have been shown to have high sequence homology. The MLL gene is necessary to maintain HOX gene expression which is an important gene involved in development. ALL AML 1. Rubnitz et al., Blood 1994;84(2): Secker-Walker et al., Leukaemia 1998;12(5): Rowley, Annu Rev Genet 1998;32: Pui and Evans, New Engl J Med 1998;339(9): Felix and Lange, Oncologist 1999;4(3): Heerema et al., Leukemia 1999;13(5): MLL Chromosome 11 CD3G UBE4A MLL PHLDB1 D11S1374 D11S1933 D11S Kb D11S Kb 38

39 Cat. No. LPH 016-S Cat. No. LPH 016 Aquarius Haematology MYB Deletion Deletions of 6q are the fourth most common chromosome aberrations in B-CLL. In around 10% of cytogenetically abnormal patients, there is a deletion of 6q 1 the breakpoint for which has been variously reported to be in either 6q13, q15 or q21. The MYB gene (Avian Myeloblastosis Viral Oncogene Homolog) is a homologue of the avian v-myb oncogene and has been shown to be expressed in all immature lymphoid and myeloid T cells but not in mature T or B-cells. It is provided as a marker for 6q as it is situated distal to the most proximal breakpoint of 6q (6q21) in 6q23.3. ALL CLL D6Z1 MYB Chromosome 6 MYB D6S1837 D6S1431 AFMA074ZG9 183Kb 39

40 Aquarius Haematology Cat. No. LPH 009-S Cat. No. LPH 009 P16 Deletion Deletions of chromosome 9p21 are implicated in a wide variety of tumours including approximately 10% of paediatric ALL patients, 1 though the incidence is higher in T-ALL 2. The region has been the subject of much study, but deletion of the potential tumour suppressor gene P16 or Cyclin-Dependent Kinase Inhibitor 2a (CDKN2A) was found to take place in 90% of newly diagnosed cases of paediatric ALL showing cytogenetic deletions of 9p21by FISH 3. This study showed that deletion of P16 only, (rather than both P16 (CDKN2A) and P15 (CDKN2B)) was the critical step as one case was found to be deleted for P16 but P15 was present. The deletion is usually homozygous (81% compared to 9% hemizygous) in cases of T-ALL whilst homozygous and hemizygous deletions are roughly equal in B-ALL (23% vs. 20% for homozygous and hemizygous respectively). ALL 1. Heerema et al., Leukemia 1999;13(5): Secker-Walker et al., Br J Haematol 1997;96(3): Okuda et al., Blood 1995;85(9): The gene product inhibits the Cyclin Dependent Kinases CDK4 and CDK6 which are important in controlling the cell cycle from G1 to S phase, so disruptions of this process are likely to result in the proliferation of mutated cells. P16 D9Z3 Chromosome 9 P16 (CDKN2A) CDKN2B D9S2060 D9S974 D9S1605 D9S1604 D9S Kb 100kb 40

41 Cat. No. LPH 017-S Cat. No. LPH 017 Aquarius Haematology P53 Deletion Although previously difficult to detect, the advent of FISH analysis of interphase cells from patients with B-CLL showed that around 17% of patients with the disease have deletions of the P53 gene 1. As with ATM, deletions of P53 have important therapeutic implications to patients with B-CLL. Knowledge of the deletion status of P53 in the patient should mediate the choice of therapy. The P53 gene is a tumour suppressor gene and its product, the P53 protein, is responsible for the death of DNA damaged cells thought to be brought about by its phosphorylation and subsequent removal of its inhibition by MDM2 (Mouse Double Minute 2 Homolog). This phosphorylation is mediated by ATM. In the absence of P53 activity, cells that cannot be repaired by ATM will continue to proliferate in their damaged state. Patients deleted for P53 may be rendered resistant to alkylating chemotherapeutic agents as these are designed to damage the DNA in the cells that P53 would have destroyed. In the absence of P53 therefore, patients treated with these agents will harbour a proliferating population of damaged cells. CLL AML L MM 1. Dohner et al., J Mol Med 1999;77: P53 D17Z1 Chromosome 17 P53 EFNB3 D17S1678 D17S1353 D17S Kb 41

42 Aquarius Haematology Cat. No. LPH 052-S Cat. No. LPH 052 P53/ATM Probe Combination Although previously difficult to detect, the advent of FISH analysis of interphase cells of patients with B-CLL showed that around 17% of patients with the disease have deletions of the P53 gene 1. As with ATM, deletions of P53 have important therapeutic implications to patients with B-CLL. Knowledge of the deletion status of P53 in the patient should mediate the choice of therapy. The P53 gene is a tumour suppressor gene and its product, the P53 protein, is responsible for the death of DNA damaged cells thought to be brought about by its phosphorylation and subsequent removal of its inhibition by MDM2 (Mouse Double Minute 2 Homolog). This phosphorylation is mediated by ATM. In the absence of P53 activity cells that cannot be repaired by ATM will continue to proliferate in their damaged state. Patients deleted for P53 may be rendered resistant to alkylating chemotherapeutic agents as these are designed to damage the DNA in the cells that P53 would have destroyed. In the absence of P53 therefore, patients treated with these agents will harbour a proliferating population of damaged cells. CLL 1. Dohner et al., J Mol Med 1999;77: Stankovic et al., Blood 2004;103(1): Khanna et al., Nature Genetics 1998;20(4): The protein kinase ATM (Ataxia-Telangiectasia Mutated) gene located in 11q22.3 is frequently deleted in cases of B-CLL. ATM Chromosome 11 The ATM gene is an important checkpoint gene involved in cell damage management and its function is to assess the level of DNA damage that the cell has received and to attempt repair by phosphorylating key substrates involved in DNA repair. Recently, the ATM/P53 interaction in B-CLL has been shown to have an important impact on the proliferation or otherwise of the cancer. 2 It has been shown that ATM concurrently enhances the phosphorylation of P53 3 should the damage to the cell be so great that it should be destroyed by apoptosis (which is mediated by P53). Deletion of ATM therefore removes this checkpoint activity and hence activation of P53 gene. Thus, there is no attempt at repairing damaged cells and no apoptosis of these cells despite the P53 protein being present. In the absence of ATM, damaged cells are allowed to proliferate. Deletions of ATM and P53 are the most serious rearrangements involved in CLL and detection of deletions of these genes provides very important information as to the therapy choices for such patients especially since deletions of 11q22.3 and therefore ATM provide a poor prognosis. NPAT P53 ATM D11S3347 D11S Kb Chromosome 17 P53 EFNB3 D17S1678 D17S1353 D17S Kb 42

43 Cat. No. LPH 031-S Cat. No. LPH 031 Aquarius Haematology PDGFRB Breakapart Some cases of Chronic Myelomonocytic Leukaemia (CML) have constitutive activation of the gene for PDGFRB (platelet-derived growth factor receptor beta) which encodes a receptor for tyrosine kinase 1. The activation is usually caused by a t(5;12)(q32;p13) translocation which results in the ETV6 (TEL)-PDGFRB fusion gene. The drug imatinib mesylate (Gleevec) specifically inhibits the kinase activity of PDGFRB and has shown efficiency in the treatment of Chronic Myeloid Leukaemia and gastrointestinal stromal tumours. The breakpoint in PDGFRB is characteristically located in intron It is located 10.7kb from the end of the gene. CML 1. Apperley JF et al., N Engl J Med 2002;347: Tokita K et al., Leukemia 2007;21:190-2 PDGFRB Chromosome 5 CSF1R PDGFRB D5S2618 D5S2619 D5S Kb 154Kb 43

44 Aquarius Haematology Cat. No. LPH 023-S Cat. No. LPH 023 PML/RARα Translocation, Dual Fusion The fusion gene PML/RARα is created by the t(15;17)(q24;q21) translocation found in 98% of AML M3 Acute hypergranular Promyelocytic Leukaemia and 9% of AML overall 1,2,3. The breakpoint in PML is variable between intron 3 and exon 7a which is in contrast to the RARα breakpoint which remains constant in intron 2. Variant translocations include breakpoints 11q23, 5q32 and 11q13. Three-way translocations indicate that the critical event occurs on der(15) which always receives the distal end of chromosome 17. The cells are blocked at the promyelocytic stage of differentiation and then proliferate. The PML protein is a transcription factor and RARα encodes a nuclear receptor. The fusion protein generated, PML-RARα, is a chimeric transcription factor that operates as a dominant negative form of RARα. Gain of function mutation allows repression of multiple genes and recruitment of DNA methyl transferases to promoters allowing prolonged suppression. PML-RARα also activates components of the Wnt signalling pathway promoting stem cell renewal. Immediate treatment is critical as intravascular coagulation causes early death in 10-40% of cases. Terminal differentiation is induced by the use of all-trans-retinoic-acid (ATRA) which reactivates the RARα gene and degrades the PML-RARα fusion, and 80-90% of cases achieve complete remission 1,2,4. Despite the efficacy of ATRA, the death rate is 15-20% but 70% will achieve 3 year survival 2. Additional abnormalities found include trisomy 8, seen in one third of cases, del(7q) and del(9q). AML 1. Licht, Sternberg, The Molecular Pathway of AML ASH Education Book Huret, Chomienne, Atlas Genet Cytogenet Oncol Haematol 1998;2(3): Heim and Mitelman, Willey-Liss, Inc Greaves, BMJ 2002;324(7332):283-7 PML Chromosome 15 D15S188 D15S818 D15S169 PML D15S624E D15S1281 D15S160 D15S965 D15S1326 RARα Chromosome Kb 174Kb CASC3 CDC6 RARα TOP2A IGFBP4 D17S1423E D17S Kb 164Kb 44

45 Cat. No. LPH 046-S Cat. No. LPH 046 Aquarius Haematology TCL1 Breakapart Probe Deregulation of normal transcription is a feature of all acute leukaemias. In T-cell neoplasms, this is brought about by altered expression of normal transcription factor proteins. This is the result of chromosomal translocations resulting in the promoter and enhancer elements of the T-cell receptor 1 genes TRA@ (TCRA), TRB@ (TCRB), TRG@ (TCRG) and TRD@ (TCRD) being brought into close proximity with one of these T-cell receptor genes 1. In T-PLL (a form of mature T-cell proliferation in patients with Ataxia Telangiectasia (AT) 2 ), the TCL1 (T-cell Leukaemia 1A/1B) gene cluster on chromosome 14q32 has been shown to be involved in a number of different chromosome rearrangements including the t(14;14)(q11;q32) and inv(14)(q11;q32), which bring elements of the cluster in to close juxtaposition, to and under the control of the TCR gene promoters and enhancers. There were found to be two breakpoint clusters 3 in the gene cluster, each of which are observed in different neoplasms, though they are both involved in either the inv(14) or t(14;14). The breakpoints are concentrated centromeric and telomeric to the TCL1, TCL6 and TML1 genes. ALL 1. Korsmeyer SJ, Annual Rev Immunol 1992;10: Brito-Babpulle V, Catovsky D, Cancer Genet Cytogenet 1991;55: Saitou et al., Oncogene 2000;19:

46 Aquarius Haematology Cat. No. LPH 047-S Cat. No. LPH 047 TCRAD Breakapart Probe Deregulation of normal transcription is a feature of all acute leukaemias. In T-ALL, this is brought about by altered expression of normal transcription factor proteins, which is in contrast with AML and B-ALL where chimaeric proteins are expressed and provide the drivers for malignancy. T-ALL is a feature of between 12 and 15% of childhood ALL 1. In about 30% of these cases, chromosomal translocations involving the T-cell receptor genes TRA@ (TCRA), TRB@ (TCRB), TRG@ (TCRG) and TRD@ (TCRD) have been described 2. It has been suggested that a number of developmentally important transcription factor genes are dysregulated as a result of being brought into close proximity with the promoter and enhancer elements of one of these T-cell receptor genes due to chromosomal rearrangements 3. This can be more clearly shown to have occurred using FISH compared to conventional cytogenetics 4. The TCRAD complex, on chromosome 14q11 has been shown to be involved in a number of different translocations in T-ALL, including t(10:14)(q24;q11), involving TLX1 (HOX11); t(1:14)(p32;q11), involving TAL1; t(11:14)(p15;q11), involving LMO1 and t(11:14)(p13q11), involving LMO2. The relative frequencies for these rearrangements in children were recently shown to be 7, 3, 2 and 3% respectively 5. ALL 1. Schneider NR et al., Blood 2000;96: Secker-Walker LM, Chromosomes and Genes in Acute Lymphoblastic Leukaemia. New York: Chapman and Hall Korsmeyer SJ, Annual Rev Immunol 1992;10: Gesk S et al., Leukemia 2003;17: Graux C et al., Leukemia 2006;20:

47 Cat. No. LPH 048-S Cat. No. LPH 048 Aquarius Haematology TCRB Breakapart Probe Deregulation of normal transcription is a feature of all acute leukaemias. In T-ALL, this is brought about by altered expression of normal transcription factor proteins, which is in contrast with AML and B-ALL where chimaeric proteins are expressed and provide the drivers for malignancy. T-ALL is a feature of between 12 and 15% of childhood ALL 1. In about 30% of these cases, chromosomal translocations involving the T-cell receptor genes TRA@ (TCRA), TRB@ (TCRB), TRG@ (TCRG) and TRD@ (TCRD) have been described 2. It has been suggested that a number of developmentally important transcription factor genes are dysregulated as a result of being brought into close proximity with the promoter and enhancer elements of one of these T-cell receptor genes due to a chromosomal rearrangement 3. This can be more clearly shown to have occurred using FISH compared to conventional cytogenetics 4. The TCRB T-cell Receptor gene on chromosome 7q34 is rearranged with the genes TLX1, HOX@, LYL1, TAL2, LCK and NOTCH1 following the t(7:10)(q34;q24); t(7;7)(p15;q34); t(7;19)(q34;p13); t(7;9)(q34;q32); t(1;7)(p34;q34) and t(7;9)(q34;q34) translocations respectively. The frequency of rearrangements varies between 7% (for the TLX1 rearrangement) to less than 1% (for TAL2, LYL1, LCK and NOTCH1) 5. ALL 1. Schneider NR et al., Blood 2000;96: Secker-Walker LM, Chromosomes and Genes in Acute Lymphoblastic Leukaemia. New York: Chapman and Hall Korsmeyer SJ, Annual Rev Immunol 1992;10: Gesk S et al., Leukemia 2003;17: Graux C et al., Leukemia 2006;20:

48 Aquarius Haematology Cat. No. LPH 012-S Cat. No. LPH 012 TEL/AML1 Translocation, Dual Fusion The TEL (or ETV6 - Erythroblastosis Variant Gene 6 translocation, ETS) / AML1 (or RUNX1 - Runt-Related Transcription Factor 1) fusion is brought about by the cytogenetically invisible t(12;21) translocation. The rearrangement is the most common in childhood B-ALL and has been detected using FISH in around 21% of cases, 1 compared to a pick-up rate of 0.05% by conventional cytogenetics. The translocation is associated with a favourable outcome though it has also been implicated with late relapse. TEL1 has also been shown to be deleted in some children with ALL where the deletion is cytogenetically invisible but where there is loss of heterozygosity (LOH) of chromosome 12p This deletion is often associated with a TEL1/AML1 translocation 2. Both the TEL1 and AML1 genes encode transcription factors, but TEL1 has been shown to be required specifically for proper transcription during haematopoiesis with the bone marrow. The translocation results in an almost intact AML1 protein fused with part of the TEL1 protein resulting from breakpoints beyond exon 4 in TEL1 and 3 of the AML1 gene. ALL 1. Jamil et al., Cancer Genet Cytogenet 2000;122(2): Raynaud et al., Blood 1996;87(7): TEL1 Chromosome 12 TEL1 (ETV6) D12S827 D12S845 D12S1095 D12S89 D12S1697 D12S98 D12S1898 D12S Kb 168Kb AML1 Chromosome 21 DSCR1 CLIC6 AML1 (RUNX1) D21S326 D21S1706 D21S1969 D21S393 D21S1895 D21S Kb 167Kb 48

49 Cat. No. LPH 049-S Cat. No. LPH 049 Aquarius Haematology TLX1 Breakapart Probe Deregulation of normal transcription is a feature of all acute leukaemias. In T-cell neoplasms, this is brought about by altered expression of normal transcription factor proteins. This is the result of chromosomal translocations resulting in the promoter and enhancer elements of the T-cell receptor genes TRA@ (TCRA), TRB@ (TCRB), TRG@ (TCRG) and TRD@ (TCRD) being brought into close proximity with one of these T-cell receptor genes 1. Murine studies show that expression of mouse homologues of TLX1 (T-Cell Leukaemia Homeobox 1 or HOX11) can immortalise haematopoetic cells in-vitro as the first of a potential two-hit mechanism leading to full malignancy 2. This work suggests that TLX1, which is not expressed in adults but is expressed in 7% of paediatric T-ALL cases 3, is an oncogene which, in separate reports has been shown, through the translocations t(10:14) and t(7;10), to be capable of being dysregulated by close juxtaposition to TCRA/D and TCRB elements respectively 4. Breakpoints are clustered within 10kb centromeric and immediately adjacent to TLX1. ALL 1. Korsmeyer SJ, Annual Rev Immunol 1992;10: Hawley RG et al., Oncogene 1994;9: Cavé H et al., Blood 2004;103(2): Dubé ID et al., Blood 1991;78(11):

50 Aquarius Haematology Cat. No. LPH 050-S Cat. No. LPH 050 TLX3 Breakapart Probe In common with other factors involved in T-ALL, the normal expression of TLX3 (T-Cell Leukaemia Homeobox 3) is disrupted by a chromosomal translocation 1. Unlike TLX1 (HOX11), however, the deregulation of TLX3 is not brought about by close juxtaposition with T-cell receptor genes, instead, it is brought into contact with another gene which is highly expressed in normal T-cell differentiation, BCL11B (B-Cell Lymphoma 11B, or CTIP2) on chromosome 14q The translocation t(5;14)(q35;q32) is generally cryptic and does not actually disrupt TLX3, but RANBP17 (Ran-Binding Protein 17) over a 110kb breakpoint region 2. However, this gene is very close to TLX3 and though RANBP17 expression is not affected by the translocation, TLX3 expression is. Deregulated expression of TLX3 is observed in around 20% of paediatric cases and around 13% of adult T-ALL patients 3,4. ALL 1. Bernard OA et al., Leukaemia 2001;15: Van Zutven et al., Haematologica 2004;89: Cavé H et al., Blood 2004;103(2): Mauviuex et al., Leukemia 2002;16:

51 Cat. No. LPH CLL-S Cat. No. LPH CLL (5x5 tests) (5x10 tests) Aquarius Haematology CLL Screening panel A selection of haematology probes and a classical satellite probe for Chronic Lymphocytic Leukaemia Chromosome 12 Enumeration probe The product is a repeat sequence probe labelled in red which recognises the centromeric repeat sequence D12Z3. This product is also available in 5 (LPH028-S) and 10 (LPH028) test kit sizes and has been optimised for overnight hybridisation. 13q14.3 Deletions in 13q14 are observed in over 20% of B-cell CLL patients, and 64% of these deletions are believed to be the primary event in CLL. Patients with a 13q14.3 deletion have a better prognosis than patients with trisomy 12. P53 (17p13.1) P53 is a tumour suppressor gene that mediates the apoptosis of damaged cells. It has been found that approximately 17% of B-CLL patients have deletions of the P53 gene. Patients exhibiting this genotype are associated with a poor prognosis as they harbour a proliferating population of damaged cells. ATM (11q22.3) The ATM gene is an important regulatory candidate in cell damage management. When it is deleted damaged cells are neither repaired nor apoptosed and are allowed to proliferate. The detection of the ATM deletion in CLL is important as it indicates poor prognosis and can define a patient s therapy. MYB (6q23.3) The MYB gene is essential in haematopoietic cell proliferation and differentiation. It is located on chromosome 6q22 and is a 6q marker. Deletions of 6q occur in around 10% of cytogenetically abnormal patients and have been associated with the pathogenesis of CLL. 51

52 Aquarius Haematology 52

53 Multiprobe Haematology Multiprobe Haematology

54 Multiprobe Haematology Contents 56 Chromoprobe Multiprobe ALL 58 Chromoprobe Multiprobe CLL 60 Chromoprobe Multiprobe AML/MDS Multiprobe Haematology As long ago as the 19th Century, nuclear changes were recognised as being significant in cancer biology. Advances in cytogenetics and molecular cytogenetics in the last century showed that although a number of numerical and structural chromosome changes appeared to be random and non-specific, rearrangements involving individual chromosomes were shown to define specific abnormalities in individual tumour types. Fluorescence In Situ Hybridisation (FISH), using locus-specific probes which are capable of defining these stereotypic structural rearrangements, has now become a routine diagnostic test in the clinical laboratory and the technique has thus been shown to be useful in the management of cancer patients. The Chromoprobe Multiprobe haematology devices are designed for Fluorescence In Situ Hybridisation of interphase cells and metaphase chromosomes from cultured peripheral blood cells or cultured bone marrow samples. 54

55 Multiprobe Overview The Chromoprobe Multiprobe System is an extension of Cytocell s proprietary Chromoprobe technology whereby DNA FISH probes are reversibly bound to the surface of a glass device. The probes attached to the glass device dissolve back into solution once in contact with hybridisation buffer, whilst denaturation of the probes and target DNA occurs simultaneously under the device once heated. This approach both simplifies the whole FISH procedure and renders it safer and quicker to use. This system allows multiple FISH probes to be hybridised on the same slide in a spatially separated manner allowing rapid screening of a patient sample for a number of DNA sequences in a single FISH analysis. Multiprobe Haematology The assay is supplied in a kit format of 2, 5 or 10 tests and includes hybridisation solution, counterstain, template slides, a hybridisation chamber and full instructions for use. The kit even contains a unique liquid crystal display slide surface thermometer for accurate measurement of the temperature of the denaturation surface. The procedure is simple: Soak slides in 100% Methanol, then polish dry with a lint free cloth. Spot 2 µl (or 4 µl for 8 square device) cell sample on alternate squares. Fill in the gaps, then check using phase contrast. Spot 1µl (or 2µl for 8 square device) Hybridisation solution onto each square of the device Carefully lower spotted slide onto the device. Check the temperature of the Hotplate using the Slide Surface Thermometer supplied. Denature slide at 75 C for 2 min. Place slide/device in Hybridisation chamber supplied and float on the surface of a clean water bath at 37 C overnight. Wash in 0.4xSSC at 72 C for 2 min, then 2xSSC/Tween for 30 sec at room temperature. Apply Counterstain supplied and view under fluorescence microscope. 55

56 Cat. No. PMP 030 (2 devices) Cat. No. PMP 032 (10 devices) Cat. No. PMP 031 (5 devices) Cat. No. PMP 033 (20 devices) Multiprobe Haematology Chromoprobe Multiprobe ALL cmyc Breakapart P16 Deletion Around 5% of B-ALL patients with c-myc rearrangements are invariably of the L3 subtype. Of these patients, 85% have a t(8;14), 10% have t(8;22) and 5% have t(2;8). Patients with these rearrangements were thought to have a poor prognosis but they have now been found to respond well to chemotherapy. TEL/AML1 Translocation, Dual Fusion This rearrangement is the most common in childhood B-ALL and has been detected, using FISH, in around 21% of cases, compared to a pick-up rate of 0.05% by conventional cytogenetics. Deletions of chromosome 9p21 are implicated in a wide variety of tumours including approximately 10% of paediatric ALL patients and result in the loss of the P16 (CDKN2A) gene. 80% of patients with a deletion in 9p21 are deleted for P16. MLL Breakapart Rearrangements of the MLL gene at chromosome band 11q23 can be detected in approximately 85% of infants with B-ALL. Translocations involving MLL are generally associated with treatment failure. 1 2 MYC Chromosome 8 P16 D9Z3 Chromosome 9 P16 (CDKN2A) CDKN2B POU5F1P1 MYC D8S1153 D8S1128 D8S1980 D8S1720 D8S1644 D9S2060 D9S974 D9S1605 D9S1604 D9S Kb 173Kb 186Kb 100kb 5 6 TEL1 Chromosome 12 TEL1 (ETV6) MLL D12S827 D12S845 D12S1095 D12S89 D12S1697 D12S98 D12S1898 D12S850 Chromosome Kb 168Kb AML1 CD3G UBE4A MLL PHLDB1 Chromosome 21 D11S1374 D11S1933 D11S3222 D11S Kb 87Kb DSCR1 CLIC6 AML1 (RUNX1) D21S326 D21S1706 D21S1895 D21S1969 D21S393 D21S Kb 167Kb 56

57 E2A Breakapart Hyperdiploidy Multiprobe Haematology Rearrangements of E2A (TCF3) are relatively common in ALL cases with the two types (t(1:19) and t(17;19)) making up about 6% of patients. Detection of the t(1;19) translocation involving E2A is best carried out via FISH as the fusion has been shown to be missed in 20 to 25% of patients by standard cytogenetic techniques. BCR/ABL Translocation, Dual Fusion The stereotypic translocation t(9;22) is most common in CML but it is also observed in around 25% of adult ALL and 2-10% of childhood ALL. There are two breakpoint clusters in the BCR gene, the minor and major breakpoint clusters, mbcr and MBCR respectively. ALL most often results from breakpoints involving mbcr. In a small number of cases of ALL the translocation does not result in a cytogenetically visible Philadelphia chromosome. In these cases, FISH is essential for highlighting the fusion gene. Approximately 30% of cases have cells that exhibit a chromosome number between 51 and 67. Within this region are prognostically significant subgroups, with individuals possessing 51 to 55 chromosomes having a poorer prognosis than those with 56 to 67. The chromosomes that are more readily hyperdiploid are chromosomes 4, 6, 10, 14, 17, 18, 21 and X, with gains of chromosomes 4, 10 and 17 leading to a relatively better prognosis. IGH Breakapart This is most notably involved in rearrangements involving the cmyc oncogene as a result of the t(8;14) translocation. However, less common rearrangements of the IGH gene are most often seen in T-ALL but can also be found in B-ALL. 3 4 D10Z1 Chromosome 10 E2A Chromosome 19 D17Z1 Chromosome 17 D19S883 RKHD1 MBD3 E2A (TCF3) RH98588 CHIC2 Chromosome 4 163Kb 189Kb CHIC2 D4S Kb 7 8 ABL1 Chromosome 9 IGH FUBP3 PRDM12 ABL1 LAMC3 Chromosome 14 D9S1863 D9S Kb 100kb Constant Segment JD Variable Segment BCR Chromosome 22 IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP D14S1419 D14S1420 D14S Kb 617Kb GNAZ RAB36 BCR IGLL1 D22S1002E D22S257 D22S Kb 148Kb 57

58 Cat. No. PMP 018 (2 devices) Cat. No. PMP 016 (10 devices) Cat. No. PMP 017 (5 devices) Cat. No. PMP 020 (20 devices) Multiprobe Haematology Chromoprobe Multiprobe CLL MYB Deletion Chromosome 12 Enumeration Deletions of 6q are the fourth most common chromosome abnormality in B-CLL, being observed in 10% of cytogenetically abnormal patients. The breakpoints for this deletion have been reported to be 6q13, 6q15 or 6q21, and MYB is located distal to 6q21 so is a strong candidate. IGH Breakapart Around 20% of cytogenetically abnormal patients with CLL have a rearrangement of IGH leading to formation of a 14q+ marker chromosome. The most common rearrangements are between CCND1 and BCL2, though there are many other chromosomes involved which a breakapart strategy probe should demonstrate. The chromosome 12 probe on the CLL Panel is a repeat sequence probe, labelled in red, which recognises the centromeric repeat sequence D12Z3. P53 Deletion Along with ATM, P53 deletions are the most serious aberrations in CLL. Since the interaction between ATM and P53 during damage-control and apoptosis of damaged cells is disrupted by deletion of one or other of these genes, damaged cells can proliferate in the patient unchecked, leading to a poor prognosis. 1 2 D6Z1 MYB Chromosome 6 D12Z3 Chromosome 12 MYB D6S1837 D6S1431 AFMA074ZG9 183Kb 5 6 P53 D17Z1 IGH Chromosome 14 Chromosome 17 Constant Segment JD IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP Variable Segment D14S1419 D14S1420 D14S308 P53 EFNB3 D17S1678 D17S1353 D17S Kb 617Kb 159Kb 58

59 ATM Deletion IGH/BCL2 Translocation, Dual Fusion Multiprobe Haematology Deletions of ATM (and P53) are the most serious aberrations involved in B-CLL. The two genes have an important interaction in checkpoint control of patients with the disease. In the absence of ATM, damaged cells are allowed to escape P53-mediated apoptosis and proliferate, leading to poor prognosis for the patient. Although more usually found in follicular lymphoma, where there are rearrangements of IGH in CLL patients, IGH/BCL2 is the second most common. BCL2 gene fragments, as a result of the translocation, become under the regulatory control of IGH elements and as the BCL2 gene is involved in apoptosis, can lead to dysregulation of this process in CLL patients. IGH/CCND1 Translocation, Dual Fusion Although a common translocation in Mantle Cell Lymphoma (MLL), this has been included on the panel as it is an important distinguishing factor between patients with MLL and those with atypical CLL. 13q14.3 Deletion Deletions of this region of chromosome 13 are thought to be the primary cause of CLL and are found in 20% of cases. The deletions in CLL patients are large and cover a number of putative tumour suppressor genes and can be homozygous or heterozygous. Though common and variable, these deletions confer a good prognosis for the patient when seen as the sole deletion in such cases. 3 4 IGH Chromosome 14 D11Z1 ATM Chromosome 11 Constant Segment JD IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP Variable Segment D14S1419 D14S1420 D14S Kb 168Kb 617Kb NPAT ATM BCL2 Chromosome 18 D11S3347 D11S Kb BCL2 KDSR D18S91 D18S1264 D18S51 D18S87 D18S Kb 100kb 7 IGH Chromosome 14 8 Constant Segment JD Variable Segment IGHA2 IGHM D14S1007 IGHE IGHG3 IGHG4 IGHG1 IGHG2 IGHA1 IGHGP D14S1419 D14S1420 D14S308 13q qter 124Kb 168Kb 617Kb Chromosome 13 CCND1 (BCL1) Chromosome 11 DLEU1 FAM10A4 D13S319 D13S272 D13S25 D13S Kb MYEOV CCND1 FGF19 FGF4 FGF3 D11S2663 D11S4095 D11S1100 D11S1076 D11S4381 D11S Kb 159Kb 59

60 Cat. No. PMP 025 (2 devices) Cat. No. PMP 027 (10 devices) Cat. No. PMP 026 (5 devices) Cat. No. PMP 028 (20 devices) Multiprobe Haematology Chromoprobe Multiprobe AML/MDS Del(5q) Deletion This is the most common rearrangement in AML and MDS and involves the deletion of a region of 5q including EGR1. This is thought to be a good marker gene for these patients because, with interaction between it and the fibronectin, TGFB1 and plasminogen activator 1 (SERPINE) genes, it can inhibit cancer growth. PML/RARα Translocation, Dual Fusion A t(15;17) rearrangement results in the fusion of the PML and RARα genes in around 9% of all AML cases (and 98% of AML M3 patients.). Treatment is possible but must be carried out early as intravascular coagulation can cause early death in 10-40% of cases. Following treatment, around 80-90% of cases achieve complete remission. MLL Breakapart Translocations involving the MLL (11q23) gene are generally associated with increased risk for treatment failure, and are found in 33% of AML M4 cases. The MLL gene is necessary to maintain HOX gene expression, which is an important gene involved in development. Del(7q) Deletion Abnormalities of chromosome 7 are common in myeloid malignancies and occur in 5-10% of de novo AML, 15% of adult MDS, 40% of paediatric MDS and 50% of therapy related AML/MDS. The region most involved is 7q22.1 which contains RELN which encodes for secretory proteins similar to epidermal growth factors. 1 2 PML Chromosome 15 5p EGR1 Chromosome 5 PML D15S818 D15S160 D15S188 D15S965 D15S169 D15S1326 D15S624E D15S Kb 174Kb D5S630 TAS2R1 D5S2064 CDC25C EGR1 D5S1701 D5S500 D5S2415 D5S2542 RARα Chromosome Kb 184Kb CASC3 CDC6 RARα TOP2A IGFBP4 D17S1423E D17S Kb 164Kb 5 6 MLL 7q22.17q31 Chromosome 7 Chromosome 11 RELN ORC5 TES CD3G UBE4A D11S1374 D11S1933 D11S3222 MLL D11S3207 PHLDB1 D7S796 D7S Kb D7S2543 D7S2886 D7S Kb 203Kb 90Kb 87Kb 60

61 P53 Deletion AML1/ETO Translocation, Dual Fusion Multiprobe Haematology Deletion of P53 is one of the most serious aberrations. Since the interaction between ATM and P53 during cell damage-control and apoptosis of damaged cells is disrupted by deletion of one or the other of these genes, damaged cells can proliferate in the patient, unchecked, leading to a poor prognosis. CBFβ/MYH11 Translocation, Dual Fusion An inversion found in 20% of AML M4 cases, involving chromosome 16, brings about the fusion of CBFβ with MYH11. Although reasonably common in AML patients, the rearrangement does provide a better prognosis than most other AML associated abnormalities. Around 40% of AML M2 patients show the stereotypic t(8;21) leading to formation of the fusion gene AML/ETO. The fusion gene results in tumourogenic growth through a number of mechanisms and is present in about 7% of all AML cases. Del (20q) Deletion Although less common than Del (5q) and Del (7q), occurring in around 4% of MDS cases and 5% of AML cases, different outcomes are observed in each disease which make it an indicator of poor prognosis in AML but a much better one for MDS patients where it is the sole abnormality. Where MDS patients acquire additional rearrangements, however, the prognosis for such patients is worse. The disorder is thought to be mediated by an as yet undescribed tumour suppressor gene. 3 4 AML1 Chromosome 21 P53 D17Z1 Chromosome 17 DSCR1 CLIC6 AML1 (RUNX1) D21S326 D21S1706 D21S1895 D21S1969 D21S393 D21S Kb 167Kb P53 EFNB3 D17S1678 D17S1353 D17S655 ETO Chromosome 8 159Kb ETO (CBFA2T1) D8S1950 D8S1648 D8S1952 D8S2020 D8S1603 D8S Kb 148Kb 7 CBFB 8 Chromosome 16 CBFB 20q12 20q13.12 D16S301 D16S324 D16S3339 D16S3323 D16S3308 D16S2584E D16S2846 Chromosome Kb MYH11 Chromosome 16 PTPRT MYBL2 D20S591E D20S108 D20S858 D20S43 D20S Kb 139Kb 174Kb MYH11 D16S3302 D16S2853 D16S3292 D16S3060 D16S3286 D16S2803 D16S405 D16S Kb 61

62 Multiprobe Haematology 62

63 Pathology Aquarius Pathology

64 Pathology Aquarius Pathology Contents 65 CHOP Breakapart 66 C-MET Amplification 67 EGFR Amplification 68 EWSR1 Products 70 FGFR1 Breakapart/Amplification 71 HER2 Amplification 72 MALT1 Breakapart 73 MDM2 Amplification 74 N-MYC Amplification 75 PAX3 & PAX7 Breakapart 76 RB1 Deletion 77 SRD (CHD5) Deletion 78 SYT Breakapart 79 TOP2A Amplification/Deletion 80 ZNF217 Amplification The assessment of genetic changes in tissue biopsies can provide important information for prediction of tumour progression. The majority of such changes are either chromosome amplifications, deletions or other complex rearrangements that can be detected using FISH. Current methodologies, namely immunohistochemistry or Southern blotting can provide information at the gene expression level but, when carried out on tissue sections (either cryostat or paraffin embedded), FISH can provide information at a gene level, in situ, at the precise site within the tumour. This can reveal cell-to-cell heterogeneity and enable the detection of small clones of genetically distinct cells. This analysis can be made even more efficient through the use of automatic image analysis systems and software. 64

65 Cat. No. LPS 015-S Cat. No. LPS 015 CHOP Breakapart Myxoid liposarcoma (MLS) is the most common subtype of liposarcoma 1. The TLS-CHOP (FUS-DDIT3) t(12;16)(q13;p11) fusion gene, firstly described by Turc-Carel et al. in , is now well established and present in at least 95% of MLS 3. In rare cases an EWS-CHOP (EWSR1-DDIT3) t(12;22)(q13;q12) translocation has been described 4. The transcription factor gene, CHOP (DDIT3) (CREBP-homologous protein/dna damage inducible transcript 3) is a negative regulator of adipocyte differentiation 5. Aquarius Pathology The TLS (Translocated in Liposarcoma) 6 or FUS 7 is a nuclear RNA-binding protein with extensive sequence similarity to EWS. The TLS-CHOP protein interferes with adipocyte differentiation and favours proliferation over terminal differentiation. 1. Enzinger and Weiss, Soft Tissue Tumors 3rd Ed. St Louis; MO:Mosby, Turc-Carel et al., Can Genet Cytogenet 1986;23: Mitelman, Catalog of chromosome aberrations in cancer. 5th Ed New York: WileyLiss, Inc Antonescu et al., Clin Canc Res 2000;7: Ron, Habener, Genes Dev 1992;6: Crozat et al., Nature 1993;363: Rabbitts et al., Nat Genet 1993;4: CHOP Chromosome 12 R3HDM2 GLI1 CHOP (DDIT3) MARS MBD6 KIF5A D12S1266 D12S1934 D12S1943 D12S2159 D12S1889 D12S Kb 145Kb 100kb 65

66 Cat. No. LPS 004-S Cat. No. LPS 004 C-MET Amplification The c-met (Mesenchymal-Epithelial Transition factor) or Hepatocyte Growth Factor Receptor (HGFR) gene encodes for a transmembrane tyrosine kinase 1 which is a proto-oncogene. Aquarius Pathology The c-met gene regulates both cell motility and cell growth 2 so normal c-met expression allows stem cells and progenitor cells to grow invasively. This invasive growth is required to generate new tissues in an embryo, or in an adult in regenerating tissue such as in the liver or during wound repair 3. c-met has been shown to be overexpressed in many tumours: ovarian 4, breast 5, lung 6, thyroid 7, stomach 8, pancreatic 9,10 and colon cancer 11,12 and this overexpression correlates with a poor prognosis. In breast cancers, c-met was shown to be only co-expressed with HER2 (ERBB2) in 50% of patients, indicating that it has a significant impact on tumour aggressiveness independent of HER Dean M et al., Nature 1985;318: Gherardi E, Stoker M, Cancer Cells 1991;3(6): Boccaccio C, Comoglio PM, Nat Rev Cancer 2006;6(8): Sawada K et al., Cancer Res 2007;67(4): Carracedo A et al., Breast Cancer Res 2009;11: Zucali PA et al., Ann Oncol 2008;19(9): Di Renzo MF et al., Oncogene 1992;7: Kuniyasu H et al., Int J Cancer 1993;55: Ebert M et al., Cancer Res 1994;54: Di Renzo MF et al., Cancer Res 1995;55: Liu C et al., Oncogene 1992;7: Di Renzo MF et al., Clin Cancer Res 1995;1: Lengyel E et al., Int J Canc 2005;113: D7Z1 cmet Chromosome 7 MET CAPZA2 D7S1402 D7S2877 D7S2460 D7S1764 D7S2863 D7S Kb 100kb 66

67 Cat. No. LPS 003-S Cat. No. LPS 003 EGFR Amplification EGFR (also known as HER1) Epidermal Growth Factor Receptor on chromosome band 7p11.2 is a type 1 tyrosine kinase receptor for members of the epidermal growth factor family. Binding of the EGFR to epidermal growth factor protein leads to cell proliferation. Abnormally elevated EGFR kinase activity can lead to proliferative diseases such as non-small-cell lung carcinoma (NSCLC), which accounts for 80-85% of all lung cancers 1, and less frequently breast cancer 2, amongst others. There are three small molecule EGFR kinase inhibitor drugs in clinical use (gefitinib, erlotinib and lapatinib) and several others are currently undergoing clinical development 3. Approximately 10% of lung cancer patients have rapid and dramatic response to these Tyrosine Kinase Inhibitors (TKIs) 4,5. FISH has proven to be useful for determining the status, and therefore selection, of NSCLC patients for treatment with EGFR TKIs Jemal A et al., CA Cancer J Clin 2006;56: Bhargava R et al., Modern Path 2005;18: Hegymegi-Barakonyi B, Curr Opin Mol Ther 2009;11(3): Lynch TJ et al., N E J Med 2004;350: Pao W et al., Proc Natl Acad Sci USA 2004;101: Hirsch FR et al., JCO 2008;26(20): Aquarius Pathology EGFR D7Z1 Chromosome 7 EGFR D7S793 D7S2357 D7S1988 D7S Kb 100kb 67

68 Cat. No. LPS 006-S Cat. No. LPS 006 EWSR1 Products Aquarius Pathology Ewing s sarcoma is the second most frequent primary bone cancer in patients below 20 years of age 1. It is characterised mostly by translocation between the two genes EWSR1 (Ewing s Sarcoma Region 1) and FLI1 (Friend Leukaemia virus Integration 1) in the t(11;22)(q24;q12) translocation found in 85% of such tumours 2. Ten percent of Ewing s sarcoma cases have a translocation involving EWSR1 and ERG (t(21;22) (q22;q12)), and less than 1% of Ewing s sarcoma patients harbour each of the 7p22 (ETV1), 17q12 (E1AF) and 2q36 (FEV) translocations with EWSR1. EWSR1 is also involved in translocations in desmoplasmic small round cell tumour, a subset of angiomatoid fibrous histocytomas, and possibly in myxoid liposarcoma 3,4. The EWSR1 breakapart probe can also be used to distinguish rare soft tissue sarcoma, a clear cell sarcoma from malignant myeloma, which is hard to distinguish histologically and immunohistologically. EWS-ATF (Activating Transcription Factor) translocation t(12;22) (q13;q12) which never happens in the malignant myeloma, has been identified in 70% to over 90% of clear cell sarcoma 5. FISH has been shown to be a more sensitive and reliable method than RT-PCR for the diagnosis of EWS in solid tissues 6. EWSR1/ERG Translocation, Dual Fusion 1. Bernstein M et al., Oncologist 2006;11(5): Turc-Carel C et al., Cancer Genet Cytogenet 1988;32: Borden EC et al., Clin Canc Res 2003;9: Sandberg AA, Bridge JA, Cancer Genet Cytogenet 2000;123: Patel RM et al., Modern Path 2005;18: Bridge RS et al., Modern Path 2006;19:1-8 68

69 Cat. No. LPS 007-S Cat. No. LPS 008-S Cat. No. LPS 007 Cat. No. LPS 008 EWSR1 Cat. No. LPS 006 EWSR1 Chromosome 22 EWSR1 RASL10A Aquarius Pathology D22S991E D22S448 D22S Kb 100kb 151Kb FLI1/EWSR1 Cat. No. LPS 007 FLI1 Chromosome 11 EWSR1 Chromosome 22 ETS1 FLI1 KCNJ1 EWSR1 RASL10A D11S4369 D11S1017 D11S912 D11S3491 D11S4123 D11S4150 D11S3463 D22S991E D22S448 D22S590 94Kb 79Kb 139Kb 151Kb 100kb 100kb EWSR1/ERG Cat. No. LPS 008 EWSR1 Chromosome 22 ERG Chromosome 21 EWSR1 RASL10A KCNJ15 ERG ETS2 D22S991E D22S448 D22S590 D21S339 D21S338 D21S1809 D21S1836 D21S1959 D21S Kb 151Kb 215Kb 154Kb 100kb 100kb 69

70 Cat. No. LPS 018-S Cat. No. LPS 018 FGFR1 Breakapart/Amplification Aquarius Pathology Fibroblast Growth Factor Receptor 1 (FGFR1), located on chromosome 8, was the first gene to be shown to become amplified in some breast cancers 1 and has also been found to be translocated in patients with 8p11 myeloproliferative syndrome 2 Amplification of the gene is thought to be the causative factor in around 10% of breast tumours 3 and has been shown to provide a poor prognosis to patients because over-expression of the gene product can lead to early relapse 4. In common with other genetic defects in lymphoproliferative disorders like the BCR/ABL translocation in CML, the FGFR1 gene contains sequences that could code for a putative tyrosine kinase protein 5,6 which has been implicated myeloproliferative syndrome (also known as EMS or Stem cell leukaemia/lymphoma syndrome, SCLL) marking out the importance of this gene in both solid tumours and cancers of the blood. There are at least 8 partner genes involved with FGFR1 translocations including ZNF198 (the most common 7 ), FOP, CEP110, BCR, HERV-K, FGFR10P2, TIF1 and MYO18A and the breakpoints in FGFR1 have been shown to be withing exon 8 in at least one of these rearrangements. 1. Theillet C, Adelaide J et al., Genes Chromosomes Cancer 1993;7: Macdonald D, Aguiar RC et al., Leukemia 1995;9: Courjal F, Cuny M et al., Cancer Res 1997;57(19): Turner N, Pearson A et al., Cancer Res 2010;70(5): Ruta M, Howk R et al., Oncogene 1988;3: Smedley D, Hamoudi R et al., Hum Mol Genet 1998;7: Xiao S, Nalabolu SR et al., Nature Genet 1998;18:84-7 Cytocell has developed a three-colour combined breakapart and amplification FISH probe for FGFR1 which can be used in either bone marrow samples from myeloproliferative syndrome (EMS) patients or tissue sections in patients where the gene may be amplified. In EMS, the breakapart strategy will show the split of one of the two fusion signals along with a blue 8 centromere to enumerate chromosome 8. In patients that are being tested for amplifications of FGFR1, the un-translocated FGFR1 FISH signal will appear as a fusion probe and this fusion signal will appear amplified. The 8 centromere probe will also act as a control probe in these cases. FGFR1 D8Z2 Chromosome 8 LETM2-202 FGFR1 D8S135 D8S Kb 108Kb 70

71 Cat. No. LPS 001-S Cat. No. LPS 001 HER2 Amplification The HER2 human epidermal growth factor receptor gene (ERBB2 or NEU), located at 17q11-12 is a member of the EGF (epidermal growth factor) receptor family 1. It is activated and amplified in 20-30% of breast cancers 2 and it has been correlated with a poor prognosis for the patient. Monoclonal antibody treatment of amplification patients using Herceptin (Trastuzumab) is effective, increasing the survival time by specifically targeting cells that overexpress the HER2 protein 3 and cleaning them from the system. Similar results have been obtained for a variety of other malignant neoplasms overexpressing HER2 in cancers e.g. ovary 4, stomach 5, salivary gland 6 and in small cell lung cancer Coussens L, Yang-Feng TL et al., Science 1985;230(4730): Slamon DJ et al., Science 1987;235(4785): Kauraniemi P et al., Oncogene 2004;23: Slamon DJ et al., Science 1989;244(4905): Gravalos C, Jimeno A, Ann Oncol 2008;19(9): Vidal L et al., Head Neck 2009;31(8): Sozzi G et al., Cancer Res 1991;52: Aquarius Pathology D17Z1 ERBB2 (Her2) Chromosome 17 PERLD1 ERBB2 GRB7 D17S2147 D17S754 D17S Kb 71

72 Cat. No. LPS 017-S Cat. No. LPS 017 MALT1 Breakapart Aquarius Pathology Extranodal marginal zone B-cell lymphoma (MZBCL) of the MALT (Mucosa-Associated Lymphoid Tissue) type is characterized by two main translocations involving MALT1; t(11;18)(q21;q21) and t(14;18)(q32;q21). Other MALT1 associated translocations are rare. However, there are no MALT1 translocations in nodal or splenic lymphomas 1. The t(11;18) API2/BIRC3 (Apoptosis Inhibitor 2 / Baculovirus IAP repeat containing protein 3 )/MALT1 translocation is present in 30% of cases of MALT lymphoma 2, and the API2-MALT1 fusion protein induces NF-κB (nuclear factor kappa-light-chainenhancer of activated B cells) resulting in transcription activation 3. Eighteen percent of MALT lymphomas harbour the t(14;18) MALT1/IGH translocation 4. The genetic abnormalities in MALT lymphomas reflect their site of origin. The t(14;18) translocation was most frequently found in MALT1 lymphomas of the parotid gland, liver, skin, ocular adnexa; sites rare for the t(11;18). However, the pulmonary and gastrointestinal MALT lymphomas show high frequencies of t(11;18) and absence of t(14;18) 1,4. 1. Murga Penas EM et al., Leukemia 2003;17(11): Auer IA et al., Ann Oncol 1997;8(10): Lucas PC et al., J Biol Chem 2001;276(22): Streubel B et al., Blood 2003;101(6): MALT Chromosome 18 MALT1 D18S1333 D18S881 D18S1117 D18S1129 D18S Kb 132Kb 100kb 72

73 Cat. No. LPS 016-S Cat. No. LPS 016 MDM2 Amplification The MDM2 gene, located at 12q14.3-q15, when overexpressed, enhances the tumourigenic potential of cells. The oncogene product forms a tight complex with the p53 tumour suppressor protein and can inhibit p53 mediated transactivation leading to escape from p53 regulated growth control 1. The Murine Double Minute (MDM2) gene has been shown to be amplified in around 7% of human tumours. Many tumours overexpress MDM2, the most common being soft tissue tumours 20%, osteosarcomas 16% and oesophageal carcinomas 13% 2. The use of FISH in welldifferentiated liposarcoma has been a valuable diagnostic tool 3,4, whilst small-molecule MDM2 inhibitors which could act as an innovative therapeutic tool have also been identified Momanad J et al., Cell 1992;69(7): Momand J, Nuc Acid Res 1998;20(15): Weaver J et al., Modern Path 2008;21(8): Sirvent N et al., Am J Surg Pathol 2007;31(10): Vassilev LT, Trends Mol Med 2007;13(1):23-31 Aquarius Pathology D12Z1 MDM2 Chromosome 12 NUP107 MDM2 CPM D12S kb 146Kb 73

74 Cat. No. LPS 009-S Cat. No. LPS 009 N-MYC Amplification The proto-oncogene N-MYC (MYCN), located on chromosome 2 at 2p24, is a transcription factor which plays a role in regulation of cell growth and proliferation. Aquarius Pathology It is mainly expressed in the developing nervous system and it is critical during neural crest embryogenesis. Myelocytomatosis viral homologue (N-MYC) becomes rapidly down-regulated as tissues become terminally differentiated and growth-arrested 1. Overexpression of N-MYC seems to block differentiation and increase cell proliferation. N-MYC amplification is the most important unfavourable prognostic factor for neuroblastoma and it is amplified in 25% of cases 2. Neuroblastoma is a dominating group of neuron tumours and is one of the most common solid tumours occurring in children Thomas WD et al., Int J of Bioch & Cell Biol 2004;36(5): Seeger RC et al., N Eng J Med 1985;313: Heim and Mitelman, Cancer cytogenetics 2nd Ed MYCN LAF Chromosome 2 MYCN LAF4 (AFF3) D2S2719 RH17899 RH D2S Kb 100kb 214Kb 74

75 Cat. No. LPS 012-S Cat. No. LPS 013-S Cat. No. LPS 012 Cat. No. LPS 013 PAX3 & PAX7 Breakapart Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children. One of the primary histological subtypes is the alveolar RMS (ARMS) 1. The majority (80%) of ARMS are associated with the t(2;13)(q35;q14) translocation and less commonly with t(1;13)(p36;q14), leading to the fusion of transcription factor FKHR (Forkhead in Rhabdomyosarcoma) or FOXO1 (Forkhead Box protein 1) to the transcription factors PAX3 (Paired Box protein 3) and PAX7 respectively. The distinction between t(1;13) and t(2;13) ARMS is important because patients with the t(2;13) have a more adverse outcome then patients with t(1;13) 2. In cases of ARMS patients who are negative for FKHR translocation, two variant PAX3 translocation associated fusion transcripts (PAX3-AFX (FOXO4) and PAX3-NCOA1) could be present 3,4. PAX7 Breakapart 1. Kodet R et al., Am J Surg Pathol 1993;17(5): Sorensen et al., J Clin Oncol 2002;20: Nishio J et al., Lab Investigation 2006;86: Barr et al., Cancer Res 2002;62: Aquarius Pathology PAX3 Cat. No. LPS 012 PAX3 Chromosome 2 PAX3 D2S102 D2S313 D2S2599 D2S Kb 168Kb 100kb PAX7 Cat. No. LPS 013 PAX7 Chromosome 1 IGSF21 KLHDC7A PAX7 TASIR2 D1S1345 D1S2598 D1S2644 D1S1324 D1S3604 D1S Kb 100kb 139Kb 75

76 Cat. No. LPS 011-S Cat. No. LPS 011 RB1 Deletion Aquarius Pathology Retinoblastoma is a cancer of immature retina cells which occurs in infants and small children 1. A 180kb RB1 tumour suppressor gene located on 13q14 may form complexes with oncoproteins and block their tumourigenic activity 2. The RB1 gene also plays a role in secondary tumours arising in retinoblastoma patients, such as osteosarcoma and some soft tissue sarcomas 3. RB1 may also be missing as a result of a progressional tumourigenic event in some leukaemias, as well as in some breast, lung, bladder, oesophagus and prostate cancers Heim and Mitelman, Cancer Cytogenetics 2nd Ed Whyte et al., Nature 1988;334(6178): Draper et al., Br J Cancer 1986;53(5): Cowell, Hogg, Cancer Genet Cytogenet 1992;64(1):1-11 RB1 13qter Chromosome 13 RB1 D13S645 D13S153 D13S1211E D13S kb 262Kb 76

77 Cat. No. LPS 010-S Cat. No. LPS 010 SRD (CHD5) Deletion The 1p36 region is frequently deleted in a broad range of human cancers 1. The Chromodomain Helicase DNA binding domain 5 (CHD5) gene acts as a tumour suppressor at 1p36 and is frequently deleted in human gliomas 2, leukaemia/lymphoma 3 and neuroblastoma 4. Deletion of the short arm of chromosome 1 is one of the most characteristic genetic changes in neuroblastomas, a tumour of the sympathetic nervous system. This is the most common childhood extracranial solid tumour accounting for around 8% 10% of childhood cancers and 15% of childhood cancer deaths 5. The CHD5 gene has been characterised as the lead tumour suppressor candidate from the 1p36 SRD (smallest region of consistent deletion) region in neuroblastoma Bagchi, Mills Canc Res 2008;68(8): Bagchi et al., Cell 2007;128(3): Maser et al., Nature 2007;447: Okawa et al., Oncogene 2008;27: Broudeur and Maris, Neuroblastoma in Principles and Practice of Pediatric Oncology. 5th Ed. Philadelphia, PA: Lippincott: , Fujita et al., J Natl Cancer Inst 2008;100:940-9 Aquarius Pathology SRD 1qter Chromosome 1 KCNAB2 SRD (CHD5) D1S1323 D1S83 D1S286E D1S2870 D1S2531 D1S Kb 100kb 77

78 Cat. No. LPS 014-S Cat. No. LPS 014 SYT Breakapart Synovial sarcomas account for up to 10 percent of soft-tissue sarcomas, typically arising in the para-articular regions in adolescents and young adults 1. Aquarius Pathology A characteristic SYT (SS18) SSX fusion gene resulting from the chromosomal translocation t(x;18)(p11;q11) is detectable in 90% of synovial sarcomas suggesting this is the primary causal event in the sarcoma 2. The translocation fuses the SYT (Synaptotagmin) gene from chromosome 18 to either of two highly homologous genes at Xp11, SSX1 (Synovial Sarcoma X Breakpoint 1) or SSX2, or in less than 1% of cases SSX4 3. SYT SSX1 and SYT SSX2 are thought to disrupt transcription and the expression of specific target genes 4,5. 1. Fletcher et al., World Health Organization Classification of Tumours IARC Press: Lyon, Sreekantaiah et al., Am J Pathol 1994;144: Ladanyi et al., Canc Res 2002;62: Ladanyi et al., Diagn Mol Pathol 1995;4: Sorensen, Triche, Semin Cancer Biol 1996;7:3-14 SYT Chromosome 18 SYT (SS18) TAF4B D18S1359 SGC35446 PMC303366P3 148Kb 100kb 151Kb 78

79 Cat. No. LPS 002-S Cat. No. LPS 002 TOP2A Amplification/ Deletion The Topoisomerase 2A encoding cell-cycle regulated protein gene (TOP2A) is located on 17q12-q21 close to the HER2 (ERBB2) oncogene, and it has a function in DNA replication and transcription of mrna 1,2. Aquarius Pathology Amplification of TOP2A is frequent in breast cancer and is often accompanied by HER2 amplification 3. TOP2A gene alterations are associated with an increased benefit from therapy using the anti-cancer drug anthracycline 4, while patients with tumours normal for TOP2A and HER2 do not appear to have additional benefit from usage of this drug Chen AY, Liu LF, Ann Rev Pharmacol Toxicol 1994;34: Tsai-Pflugfelder M et al., Proc Nat Acad Sci 1988;85: Bofin AM et al., Cytopath 2003;14(6): Larsen A et al., Pharmacol Ther 2003;99(2): O Malley et al., J Natl Cancer Inst 2009;101: D17Z1 TOP2A Chromosome 17 TOP2A D17S1335 D17S kb 164Kb 79

80 Cat. No. LPS 005-S Cat. No. LPS 005 ZNF217 Amplification Aquarius Pathology The Zinc Finger protein 217 gene (ZNF217), which functions as a transcription repressor for a variety of genes 1, is a strong oncogene candidate mapped within 260 kb of the minimum common amplicon at 20q13.2. It drives the 20q13.2 amplification in breast cancer 2, ovarian cancer and other tumours 3,4,5 and is associated with aggressive tumour behaviour 6. It has been shown that the silencing of ZNF217 inhibits ovarian cancer cell growth and the invasive ability in cancer cell lines Quinlan KG et al., Mol Cell Biol 2006;26: Collins C et al., Proc Natl Acad Sci 1998;95: Yang SH et al., Clin Cancer Res 2005;11: Iwabuchi H et al., Cancer Res 1995;55: Bar-Shira A et al., Cancer Res 2002;62: Tanner MM et al., Clin Canc Res 1995;1: Sun G et al., Int J Oncol 2008;32: pter ZNF217 Chromosome 20 ZNF217 D20S183 D20S840 D20S Kb 100kb 80

81 Prenatal Aquarius Prenatal

82 Prenatal Contents 83 Aquarius Prenatal Enumeration Kits Cytocell s prenatal Fluorescence In Situ Hybridisation (FISH) assays are designed for the rapid and accurate detection of the most common foetal chromosome disorders. Aquarius Prenatal Trisomy of chromosome 21 resulting in Down syndrome affects approximately 1 in 1000 newborns. 1 Trisomy of chromosome 18 results in Edwards syndrome and occurs in 1 in live births. 2 The rarest trisomy, trisomy 13, responsible for Patau syndrome occurs in approximately 1 in 16,000 newborns. 2 Aberrant copy numbers of the X and Y chromosomes can lead to various sex chromosome disorders (such as Klinefelter and Turner syndromes). The kits contain fluorescent probes for easy identification of trisomy 21, 18 and 13 present in Down, Edwards and Patau syndrome as well as sex chromosome aneuploidies. Cytocell are able to offer a range of probe combinations. The complete prenatal screen (Cat. No. LPA 001), used to detect all five most common foetal aneuploidies, is available in a range of kit sizes (5, 10, 30 and 50 tests). Prenatal enumeration X, Y and 18 Prenatal enumeration 13 and 21 (trisomy 21) 1. Ohira M et al., Genome Res. 1997;7(1): Abdul Vahab Saad et al., Int J Hum Genet 2010;10(1-3):

83 Cat. No. LPA 001-S Cat. No. LPA 002 * Cat. No. LPA 001 Cat. No. LPA 003 * Cat. No. LPA (30 tests) Cat. No. LPA 004 * Cat. No. LPA (50 tests) Cat. No. LPA 005 * Aquarius Prenatal Enumeration Kits * also available as 5 tests Cat. No. LPA 001 Probe set 1: X centromere Xp11.1-q11.1 (DXZ1) Green Y centromere Yp11.1-q11.1 (DYZ3) Orange 18 centromere 18p11.1-q11.1 (D18Z1) Blue 13q14.2 Chromosome 13 Probe set 2: 13 unique sequence (13q14.2) Green D13S1195 D13S917 ITM2B D13S1155 D13S153 RB1 CHC1L (RCBTB2) D13S915 D13S1218 Aquarius Prenatal 21 unique sequence (21q22.13) Orange 124Kb 128Kb 261Kb Cat. No. LPA 002 X centromere Xp11.1-q11.1 (DXZ1) Green Y centromere Yp11.1-q11.1 (DYZ3) Orange 18 centromere 18p11.1-q11.1 (D18Z1) Blue 21q22.13 Chromosome 21 DYRK1A KCNJ6 DSCR4 DSCR8 D21S270 D21S1867 D21S1972 D21S337 D21S1439 D21S1440 D21S1425 D21S1917 D21S1444 D21S1238 D21S341 D21S1883 Cat. No. LPA Kb 288Kb 106Kb 13 unique sequence (13q14.2) Green 21 unique sequence (21q22.13) Orange Cat. No. LPA 004 D18Z1 Chromosome centromere 18p11.1-q11.1 (D18Z1) Blue Cat. No. LPA 005 DYZ3 Chromosome Y 13 unique sequence (13q14.2) Green 21 unique sequence (21q22.13) Orange 18 centromere 18p11.1-q11.1 (D18Z1) Blue DXZ1 Chromosome X 83

84 Aquarius Prenatal 84

85 Microdeletion Aquarius Microdeletion

86 Microdeletion Aquarius Microdeletion Contents 87 Alagille (JAG1) 88 Angelman (UBE3A/D15S10) 89 CHARGE 90 Cri-Du-Chat and SOTOS Probe Combination 91 DiGeorge II (10p14) 92 DiGeorge and 22q13.3 Deletion Syndrome Probe Combinations 94 Kallmann (KAL1) and Steroid Sulphatase Deficiency (STS) Probe Combination 95 Langer-Giedion 96 Monosomy 1p36 97 Neurofibromatosis Type 1 98 Prader-Willi/Angelman (SNRPN) 99 Rubinstein-Taybi 100 Saethre-Chotzen/Williams-Beuren Combination 101 SHOX 102 Smith-Magenis (RAI1 and FLII)/ Miller-Dieker Probe Combinations 103 SRY 104 Williams-Beuren 105 Wolf-Hirschhorn 106 XIST Microdeletion syndromes are a heterogeneous group of disorders brought about by the deletion of specific regions of chromosomal DNA causing haploinsufficiencies of important genes. These deletions are difficult to visualise using standard cytogenetic techniques, but Fluorescence In Situ Hybridisation (FISH) can resolve these submicroscopic deletions and has therefore become the method of choice for the diagnosis of these disorders. Cytocell s comprehensive range of Microdeletion probes features products for some of the rarest human genetic syndromes. With this in mind, we offer all Microdeletion probes in economical five, or standard ten, test kits. 86

87 Cat. No. LPU 012-S Cat. No. LPU 012 Alagille (JAG1) Alagille syndrome (AGS) is an autosomal dominant disorder characterised by abnormalities of liver, heart, skeleton, eye and face 1. Mutations in the 36kb long human gene Jagged1 (JAG1) localised on 20p12 have been identified as causal for abnormalities found in patients with AGS 3,4. The gene encodes a ligand for the Notch 1 transmembrane receptor which plays a key role in cell fate determination and differentiation 5. The syndrome has also been noted in a patient with a deletion larger than 3Mb including JAG1 and a translocation t(3;20)(q13.3;p12.2) Alagille D et al., J Pediatr 1987;110: Li L et al., Nature Genet 1997;16: Oda T et al., Nature Genet 1997;16: Artavanis-Tsakonas S et al., Science 1999;284: Oda T et al., Human Mutat 2000;16:92 Alagille 20qter Aquarius Microdeletion Chromosome 20 JAG1 D20S507 D20S160 D20S1091 D20S Kb 87

88 Cat. No. LPU 006-S Cat. No. LPU 006 Angelman UBE3A/D15S10 In 70% of patients with Prader-Willi (PWS) or Angelman Syndrome (AS) a large interstitial deletion of 3 4Mb at 15q11 13 is observed 1,2. Aquarius Microdeletion Mutations of the imprinting centre and uniparental disomy account for the remaining cases of PWS. However, 20% of AS show biparental inheritance and normal methylation suggesting the involvement of a single AS gene. The UBE3A gene lies within the minimum AS critical region 3 approximately 400kb telomeric of SNRPN; it shows preferential expression of the maternal allele in the brain 4, and is mutated in 20-30% of AS patients with normal methylation and biparental contribution of 15q It is considered to be one of the AS genes 4. The Angelman Region probe is approximately 108kb of genomic DNA and targets most of the UBE3A gene and includes the locus D15S10. This type of probe may be used to identify deletions of the AS region. It will not detect intragenic deletions or small mutations of UBE3A. The probe may also be used to help determine the nature of a Prader-Willi syndrome deletion detected with the SNRPN/Imprinting Centre probe. Large deletions of 3 4Mb at 15q11 13 will cause the deletion of both probe regions SNRPN/IC and UBE3A/D15S10. Smaller deletions (of approximately 200kb incorporating the IC and SNRPN) will not cause deletion of the UBE3A/D15S10 probe. These deletions may indicate a much higher risk of recurrence and patients could be referred for further investigation Butler MG, Am J Med Genet 1990;35: Clayton-Smith J, Pembrey M, E J Med Genet 1992;29: Sutcliffe JS et al., Genome Res 1997;7: Rougeulle C, Lalande M, Neurogenetics 1998;1: Horsthemke B. University of Essen, Germany, pers comm. UBE3A 15qter Chromosome 15 SNRPN Angelman Syndrome Region UBE3A GABRB3 D15S63 PAR5 IPW PAR1 D15S10 108Kb 88

89 Cat. No. LPU 021-S Cat. No. LPU 021 CHARGE CHARGE syndrome is an autosomal dominant disorder that occurs in 1 in 10,000 births. The acronym CHARGE summarises six main clinical features: ocular Coloboma, Heart defects of any type, Atresia of the choaneae, Retardation, Genital and Ear anomalies 1. The chromodomain 7 gene (CHD7) was identified as causative for the syndrome in 2/3 of patients with a clinical diagnosis of CHARGE. CHD7 is located in 8q12.1 and 8q12.2 and is 188 kb in size. Most mutations are truncating mutations, leading to haploinsufficiency and are mostly de novo. They are found in 53-65% of patients with the syndrome. Microdeletions are found in up to 10% of CHARGE patients 2,3. The CHD7 protein plays a role in chromatin organisation and is a member of the chromodomain helicase DNA-binding (CHD) proteins. The CHD7 has an important function in early embryonic development. It is ubiquitously expressed in several fetal and adult tissues, including those affected in CHARGE syndrome Pagon RA et al., J Pediat 1981;99: Vissers LE et al., Nat Genet 2004;36: Jongmans MC et al., J Med Genet 2005;43: Aquarius Microdeletion D8Z2 CHARGE Chromosome 8 RAB2 CHD7 D8S1929 D8S2048 D8S1986 D8S Kb 89

90 Cat. No. LPU 013-S Cat. No. LPU 013 Cri-Du-Chat and SOTOS Probe Combination Aquarius Microdeletion Cri-Du-Chat syndrome consists of multiple congenital anomalies, mental retardation, microcephaly, abnormal face, and a mewing cry in infants. Cri-Du-Chat Syndrome is associated with deletions, which vary in size, of part of the short arm of chromosome 5 1. The estimated prevalence varies between 1 in 20,000 to 1 in 50,000 births 2. A critical chromosomal region involved in the high-pitched cry is mapped to proximal 5p15 3. The region involved in the remaining features of the syndrome has been mapped to 5p ,5. SOTOS syndrome is a neurological disorder characterised by a distinctive facial appearance; overgrowth in childhood and learning disabilities or delayed development 6. Malignant tumour formation was also reported to be associated with SOTOS syndrome 7. NSD1, which encodes a histone methyltransferase implicated in chromatin regulation 8, was identified as the gene disrupted by the 5q35 breakpoint and its haploinsufficiency is the major cause of SOTOS Lejeune J et al., C R Hebd Seances Acad Sci 1963;257: Niebuhr E et al., Hum Genet 1978;44: Cerruti Mainardi P et al., J Med Genet 2001;38: Overhauser J et al., Hum Mol Genet 1994;3: Wu Q et al., Eur J Hum Genet 2005;13: Cole TR, Hughes HE, J Med Genet 1994;31: Maldonado V et al., Am J Dis Child 1984;138: Tatton-Brown K, Rahman N, Eur J Hum Genet 2007;15: Kurotaki N et al., Nat Genet 2002;30:365-6 Cri-Du-Chat SOTOS Chromosome 5 FLJ25076 NSUN2 FGFR4 NSD1 RGS14 D5S1637E D5S2678 D5S2703 D5S2630 D5S2281E D5S2260E D5S2221E 193Kb CTNND2 167Kb D5S Kb 90

91 Cat. No. LPU 015-S Cat. No. LPU 015 DiGeorge II (10p14) DiGeorge syndrome 1, and a variety of congenital malformation syndromes including Velocardiofacial (VCFS) 2, share the deletion of chromosome 22 at 22q11 2,3,4,5. However, deletions on 10p13.14 have been shown in some patients with DGS 6,7,8. The deletion of the DGS2 locus on 10p may be 50 times less frequent than the deletion of the DGS1 locus on 22q, and may be estimated to occur in 1 in 200,000 live births 9. A gene called BRUNOL3 has been identified within the 300kb minimal region of DGS2 and postulated to be involved in DGS2 deletion 10. It is a candidate gene for the heart defect and thymus hypoplasia/aplasia associated with partial monosomy 10p 10. This gene may be involved in atrial septal defects (ASD) which is a common cardiac anomaly associated with DGS DiGeorge AM, J Pediatr 1965;67: Shprintzen RJ et al., Cleft Palate J 1978;15: Wilson DI et al., J Med Genet 1993;30: Driscoll DA et al., J Med Genet 1992;50: Burn J et al., J Med Genet 1993;30: Schuffenhouer S et al., Ann Genet 1995;38(3): Daw SC et al., Nat Genet 1996;13: Dasouki M et al., Am J Med Genet 1997;73(1): Berend SA et al., Am J Med Genet 2000;91(4): Lichtner P et al., J Mol Med 2002;80: Yatsenko SA et al., Clin Genet 2004;66: Aquarius Microdeletion DiGeorge II D10Z1 Chromosome 10 CUGBP2 (BRUNOL3) D10S2393 D10S2235 D10S2196 D10S1632 D10S1364E D10S Kb 91

92 Cat. No. LPU 004-S Cat. No. LPU 004 DiGeorge and 22q13.3 Deletion Syndrome Probe Combinations DiGeorge Syndrome Aquarius Microdeletion DiGeorge syndrome 1, and a variety of congenital malformation syndromes including Velocardiofacial (VCFS) 2 and Conotruncal Anomaly Face syndromes 3, share the phenotypic features covered by the acronym CATCH22 (Cardiac defects; Abnormal facies; Thymic hypoplasia; Cleft palate; Hypocalcaemia) and deletion of chromosome 22 at 22q11.2 3,4,5. In addition, around 17% of nonsyndromic patients with isolated conotruncal defects have been shown to have a 22q11.2 microdeletion 6. The incidence of these anomalies is estimated to be 1:4000 live births 7, and therefore, the deletion 22q11.2 represents one of the most common genetic defects. A region of 2Mb referred to as the DiGeorge Critical Region (DGCR) is most commonly deleted in up to 90% of patients 5,8,9. Within the DGCR, a minimal critical region of kb has been described 10,11 containing several genes including TUPLE1 (HIRA), TBX1, the Citrate Transport Protein (CTP) and the Clathrin heavy chain genes (CLTD). 22q13.3 Deletion Syndrome 1. Pinsky L, DiGeorge AM, J Pediatr 1965;66: Shprintzen RJ et al., Cleft Palate J 1978;15: Burn J et al., J Med Genet 1993;30: Wilson DI et al., J Med Genet 1993;30: Driscoll DA et al., J Am Hum Genet 1992;50: Goldmuntz E et al., J Med Genet 1993;30: Tezenas Du Montcel S et al., J Med Genet 1996;33: Driscoll DA et al., Am J Med Genet 1992;44(2): Scambler PJ et al., Genomics 1991;10: Halford S et al., Hum Mol Genet 1993;2(12): Carlson C et al., Am J Hum Genet 1997;61: Phelan MC et al., Am J Med Genet 2001;101(2): Prasad C et al., Clin Genet 2000;57(2): Beeckers TM et al., J Neurochem 2002;81(5): Bonaglia MC et al., Am J Hum Genet 2001;69(2): Anderlid BM et al., Hum Genet 2002;110(5): Wilson HL et al., J Med Genet 2003;40(8): Dupont C et al., French Speaking Cytogeneticists Association Congress Luciani J et al., J Med Genet 2003;40(9): Chen CP et al., Prenat Diagn 2003;23(6):504-8 The 22q13.3 deletion syndrome presents a recognisable phenotype characterised by hypotonia, delay or absence of expressive speech, moderate to profound mental retardation, normal to accelerated growth and mild dysmorphic features 12. Some deletions of the terminal region of chromosome 22q are cytogenetically visible. However, a few cases of cryptic deletions have been reported 12,13, suggesting that the actual incidence of 22q telomere deletion may be higher than previously thought. Several observations of patients with 22q13.3 deletion showed that the ProSAP2/SHANK3 19 gene, coding for a structural protein of the postsynaptic density of excitation synapses, and expressed in the cortex and cerebellum of the brain 14, was disrupted 14,15,16 or deleted 17 making it a good candidate gene for this syndrome. The deletion varies widely in size from 130kb to 9Mb 17,18,19. Recent findings therefore recommend the use of 22q subtelomeric probes distal to the ARSA gene for examining all 22q13.3 deletions 19,20. Cytocell now offers three probes for 22q11.2 covering more of this important region. In addition to TUPLE1 (NIRA) and N25, we can now offer a probe for TBX1 which means that there is now a wider range available for characterising the extent of the deletion in DiGeorge patients. 92

93 Cat. No. LPU 010-S Cat. No. LPU 014-S Cat. No. LPU 010 Cat. No. LPU 014 VCFS TUPLE 1 Cat. No. LPU 004 TUPLE1 N85A3 Chromosome 22 DGCR6 TUPLE1 (HIRA) DGCR14 UFD1L DGCR2 CLTCL1 CDC45L D22S75 D22S1095E D22S451 D22S944 SHANK3 RABL2B Telomere ARSA ACR 113Kb 44Kb VCFS N25 Cat. No. LPU 010 N25 N85A3 Chromosome 22 Aquarius Microdeletion DGCR6 N25 (D22S75) DGCR14 HIRA UFD1L DGCR2 CLTCL1 CDC45L D22S75 D22S1095E D22S451 D22S944 SHANK3 RABL2B Telomere ARSA ACR 63Kb 44Kb TBX1 Cat. No. LPU 014 TBX1 N85A3 Chromosome 22 HIRA (TUPLE1) UFD1L TBX1 SEPT5 GNB1L CDC45L D22S1637 D22S1662 D22S1627 DGCR8 SHANK3 RABL2B Telomere ARSA ACR 211Kb 44Kb 93

94 Cat. No. LPU 016-S Cat. No. LPU 016 Kallmann (KAL1) and Steroid Sulphatase Deficiency (STS) Probe Combination Aquarius Microdeletion Kallmann syndrome (KS) is a developmental disease characterised by olfactory deficiency and hypogonadotrophic hypogonadism (HH), which is responsible for the absence of spontaneous puberty 1. Kallmann syndrome is a heterogeneous developmental genetic disorder affecting about 1 in 8,000 males and 1 in 40,000 females 2. Reports indicate three modes of inheritance: X-linked, autosomal dominant and autosomal recessive 134. It has been shown that mutations in KAL1 on Xp22.3 result in the X- linked form 5. KAL1 consists of 14 exons and extends over 200kb 6. KAL1 gene abnormalities reported in patients with KS include missense and nonsense mutations, splice site mutations, intragenic deletions and submicroscopic chromosomal deletions involving the entire KAL1 gene Kallmann FJ et al., Am J Ment Defic 1944;48: Hu Y et al., Int J Biochem Cell Biol 2003;35: Hockaday TD, Postgrad Med J 1966;42: White BJ, Am J Med Genet 1983;15: Hardelin JP et al., Human Mol Genet 1993;2: Del Castillo I et al., Nat Genet 1992;2: Izumi Y et al., Endocr J 2001;48: Wells RS et al., Arch Dermatol 1965;92(1): Koppe G et al., Arch Dis Child 1978;53(10): Hernandez-Martin A et al., Br J Dermatol 1999;141(4): Hazan C et al., Dermatology Online 2005;11(4): Paige DG et al., Br J Dermatol 1994;131(5):622-9 Steroid Sulphatase Deficiency (STS) (also known as X-linked Ichthyosis) 8 is the second most common type of ichthyosis, and one of the most frequent human enzyme deficiency disorders. Deficiency of the STS enzyme is known to be responsible for dark, adhesive and regular scaling of the skin 9. The gene is mapped at the distal part of the short arm of the X chromosome, which escapes X- chromosome inactivation, and has the highest ratio of chromosomal deletions among all genetic disorders 10. Complete deletions have been found in more than 90% of patients 11. The deletions can extend to involve neighbouring genes causing contiguous gene defect. Therefore, STS may be associated with KS 12. Kallmann Steroid Sulphatase DXZ1 X Chromosome HDHD1A STS KAL1 FAM9A DXS7731 DXS7500 DXS7965 DXS6767 DXS7470 DXS278 DXS7053 DXS Kb 334Kb 94

95 Cat. No. LPU 022-S Cat. No. LPU 022 Langer-Giedion Langer-Giedion syndrome (LGS; tricho-rhino-phalangeal syndrome type II) is a contiguous gene deletion syndrome involving bands 8q23.3 and 8q LGS is characterised by cranio-facial and skeletal abnormalities including multiple cartilaginous exostoses and cone-shaped epiphyses. Mental retardation is also a common phenotype. LGS combines the clinical features of two autosomal dominant diseases: tricho-rhino-phalangeal syndrome type 1 (TRPS1) and multiple cartilaginous exostoses (EXT1). TRPS1 gene maps more than 1,000kb proximal to the EXT1 2,3. 1. Buhler, Malik, Am J Med Genet 1984;19: Ludecke HJ et al., Hum Mol Genet 1995;4: Hou et al., Genomics 1995;29(1):87-97 D8Z2 Langer-Giedion Chromosome 8 Aquarius Microdeletion TRPS1 EXT1 D8S565 D8S98 D8S547 D8S1009 D8S1020 D8S527 D8S43 115Kb 131Kb 165Kb 206Kb 95

96 Cat. No. LPU 020-S Cat. No. LPU 020 Monosomy 1p36 Monosomy 1p36 is the most common terminal deletion syndrome, involving 1 in 10,000 births 1. It is characterised by developmental delay, growth abnormalities and craniofacial dysmorphism. Minor cardiac malformation and sensorineural hearing loss and variable opthalomological anomalies have also been observed 2. Deletions range from 1.5 to 10.5Mb 3. SKI, located at distal 1p36, was deleted in all of the individuals tested who had the syndrome 4. The gene is involved in neural tube development and muscle differentiation 5, and deletions in mice have shown phenotypes with some of the features observed in individuals with 1p36 deletion syndrome Shaffer, Lupski et al., Annu Rev Genet 2000;34: Slavotinek A et al., J Med Genet 1999;36: Heilstedt HA et al., Am J Hum Genet 2003;72: Colmenares C, Nat Genet 2002;30: Kaufman CD et al., Mech Dev 2000;95: Berk M et al., Genes Dev 1997;11: Aquarius Microdeletion 1p36 1qter Chromosome 1 GABRD PRKCZ SKI MORN1 D1S1328 D1S243 D1S Kb 96

97 Cat. No. LPU 017-S Cat. No. LPU 017 Neurofibromatosis Type 1 Neurofibromatosis type 1 (NF1) occurs in 1 in 3,000-4,000 people. It is a common autosomal dominant disorder characterised by neurofibromas, café-au-lait spots, freckles, Lisch nodules, bone deformities, learning disabilities, macrocephaly, short stature and predisposition to developing tumours such as myeloid malignancies, gliomas and pheochromocytomas 1,2,3. In most cases the second copy of the NF1 gene becomes altered during a patient s lifetime. This event is most likely necessary to trigger tumour formation 6. NF1 is caused by mutations of the tumour suppressor gene NF1 which spans approximately 280kb and is located at 17q Mutations encompass both single nucleotide substitutions and large genomic deletions. Patients with deletions of the entire gene typically have a more severe presentation than those with intragenic mutations. Approximately 5-20% of patients with NF1 carry a heterozygous deletion and thus lack the NF1 gene, and 11 or more contiguous genes. Most interstitial fusions appear to directly link two regions of high sequence similarity that occur at distances of approximately 400kb and 700kb distal to the NF1 gene Bader JL et al., Ann N J Acad Sci 1986;486: Huston SM and Hughes RA, The neurofibromatosis: a pathogenic and clinical overview. New York: Chapman and Hall Medical Trovo-Marqui AB, Tajara EH et al., Clin Genet 2006;70: Baker D et al., Science 1987;236(4805): Cichowski K, Jacks T et al., Cell 2001;104(4): Side, Shannon et al., The NF1 gene as a tumor suppressor. BiosScientific, Oxford Jenne DE et al., Am J Hum Genet 2001;69: Aquarius Microdeletion 17pter NF1 Chromosome 17 RNF135 NF1 RAB11FIP4 D17S1307 D17S2162 D17S1972 D17S Kb 97

98 Cat. No. LPU 005-S Cat. No. LPU 005 Prader-Willi/Angelman (SNRPN) Aquarius Microdeletion Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS) are distinct neurogenetic disorders caused by the loss of function of genes on chromosome 15 (bands 15q11 13), on either the paternally or maternally inherited chromosome respectively 1. In 70% of patients, a large interstitial deletion of 3-4Mb is observed 1,2. In 2 4% of patients, an imprinting defect is observed of which 25% involve an approximately 200kb deletion of the Imprinting Centre (IC) 3. Uniparental disomy, in which the chromosomes 15 are inherited from the same parent, accounts for most of the remaining patients with PWS and 80% of AS patients. The SNRPN gene is one of four imprinted loci that is expressed from the paternal chromosome 15 region 15q11-13 and maps to the smallest deletion region involved in PWS. Its chromosomal location and imprinting status suggest a possible role in the aetiology of PWS 4. The imprinting centre (IC) maps to a 100kb region proximal to SNRPN. Parental deletions or mutations in the IC impair the imprinting process in 15q11 13 and cause the two distinct diseases in their offspring 5,6. Most of the PWS imprinting deletions involve SNRPN and are approximately 200kb. The AS imprinting deletions are small (approximately 40kb), involve the BD3 region, and do not include SNRPN. 1. Butler MG, Am J Med Genet 1990;35: Clayton-Smith J, Pembrey M, J Med Genet 1992;29: Buiting K et al., Am J Hum Genet 1998;63(1): Glenn C et al., Am J Hum Genet 1996;58: Buiting K et al., Nat Genet 1995;9: Dittrich B et al., Nat Genet 1996;14: SNRPN 15qter Chromosome 15 Imprinting Centre SNRPN UBE3A D15S63 PAR5 PAR1 D15S10 170Kb 98

99 Cat. No. LPU 023-S Cat. No. LPU 023 Rubinstein-Taybi Rubinstein-Taybi is a well-defined condition characterised by short stature, mental retardation, facial abnormalities, and broad thumbs and first toes 1. This condition was syndrome described in 1963 and is an autosomal-dominant disease with a frequency of one individual in 100,000 newborns. Chromosome band 16p13.3 contains the Rubinstein-Taybi syndrome locus, which includes a gene encoding a binding protein for cyclic adenosine monophosphate response element binding protein (CBP, CREBBP). This gene spans approximately 150kb with 31 exons 2, and dosage abnormalities are the common cause of the disease 3. Disruptions of CREBBP, either by gross chromosomal rearrangement or point mutation, are responsible for the phenotype of Rubinstein-Taybi syndrome. Microdeletions including the CREBBP gene account for about 10-15% of RTS cases Rubinstein JH, Taybi H, Am J Dis Child 1963;105: Petrij F et al., J Med Genet 2000;37(3): Stef M et al., Eur J Hum Genet 2007;15(8): Bartsch O et al., Eur J Hum Genet 1999;7(7): Aquarius Microdeletion Rubinstein-Taybi D16Z2 Chromosome 16 DNASE1 TRAP1 CREBBP D16S3382 D16S2906 D16S Kb 153Kb 99

100 Cat. No. LPU 024-S Cat. No. LPU 024 Saethre-Chotzen/Williams -Beuren Combination Aquarius Microdeletion Saethre-Chotzen is one of the most frequent syndromes in congenital malformation. It is characterised by craniofacial and limb abnormalities and the incidence of this rare syndrome is estimated at between 1 in 25-50,000 live births. This autosomal dominant disorder is one of the most difficult to diagnose, and the identification of TWIST1 (a basic helix-loop-helix transcription factor on chromosome band 7p21.1) as a causative gene has proved invaluable for the diagnosis 1,2. Williams-Beuren Syndrome (WBS) is a developmental disorder caused by a microdeletion within band 7q Patients display connective tissue problems; typically supravalvular aortic stenosis (SVAS), growth retardation, renal anomalies, transient hypercalcaemia, hyperacusis and mental retardation 4. Haploinsufficiency of the elastin (ELN) gene has been identified as being responsible for SVAS 5,6. However, of the remaining 15 genes identified within the WBS deletion, none of the remaining clinical features have been conclusively attributed to any one of these genes. The most recent work maps the WBS deletion region to be 1.6Mb 7, and flanked by highly homologous duplicons of kb within which the common breakpoints cluster. The common WBS deletion results from non-homologous recombination between the GTF2I/NCF1 locus and the GTF2IP1/NCF1P1 locus, or rare intrachromosomal exchange between the centromeric GTF2IP2/NCF1 and the telomeric GT2IP2/NCF1P2 duplicons. 1. Howard TD et al., Nat Genet 1997;15: El Ghouzzi V et al., Nat Genet 1997;15: Francke U et al., Hum Mol Genet 1999;8: Prober BR, Dykens EM, Child Adolesc Psychiatr Clin North Am 1996;5: Li DY et al., Hum Mol Genet 1997;6: Tassabehji M et al., Hum Mol Genet 1997;6: Peoples R et al., Am J Hum Genet 2000;66:47-68 HDAC9 TWIST1 FERD3L D7S2495 D7S1459 D7S1683 D7S Kb Saethre-Chotzen Williams-Beuren Chromosome 7 FZD9 WBSCR28 TBL2 WBSCR22 CLDN4 ELN WBSCR1 CYLN2 GTF2IRD1 GTF2I NCF1 WBSCR9 D7S2476 WBSCR27 LIMK1 RFC2 D7S Kb 144Kb 204Kb 100

101 Cat. No. LPU 025-S Cat. No. LPU 025 SHOX The SHOX (Short stature Homeobox-containing gene) is located at Xp22 and Yp11.3 in the pseudoautosomal region (PAR1) of both chromosomes 1 It encodes a transcription factor of 292 and 225 amino acids (SHOXa and SHOXb respectively) whose translated proteins differ in the C-terminal region. SHOX is a cell-specific homeodomain protein involved in cell cycle and growth regulation and activates transcription in osteogenic cells 2. SHOX haploinsufficiency is involved in the aetiology of idiopathic short stature, and the short stature in Turner syndrome. Homozygous loss of the SHOX gene has been correlated with the Langer type mesomelic dysplasia. Subsequently, heterozygous SHOX mutations were also shown to cause Leri-Weill dyschondrosteosis. The incidence of SHOX deficiency is between 1 in 2000 to 1 in 5000 in the general population, and 1 in 40 to 1 in 150 in short people 3,4. 1. Rao E et al., Nat Genet 1997;16: Rao E et al., Hum Mol Genet 2001;10: Leka SK et al., Hormones 2006;5: Jorge AL et al., Clin Endocri 2007;66:130-5 Aquarius Microdeletion SHOX DYZ1 SHOX DXZ1 Chromosome Y Chromosome X SHOX DXYS153 DYS290 DXYS28 DXYS86 DXYS15 164Kb 101

102 Cat. No. LPU 007-S Cat. No. LPU 019-S Cat. No. LPU 007 Cat. No. LPU 019 Smith-Magenis (RAI1 and FLII)/Miller-Dieker Probe Combinations Aquarius Microdeletion Smith-Magenis syndrome (SMS) is a multiple congenital anomaly syndrome characterised by mental retardation, neuro-behavioral abnormalities, sleep disturbances, short stature, minor craniofacial and skeletal anomalies, congenital heart defects and renal anomalies 1,2. It is one of the most frequently observed human microdeletion syndromes and is associated with an interstitial deletion of the chromosome band 17p Molecular studies in SMS patients suggest a common deletion region spanning approximately 700kb 3. The proximal boundary is within a region of overlap between the FLII and LLGL1 genes, and the distal boundary within the PEMT gene 3. Deletions or mutations in RAI1 (Retinoic Acid Induced 1) gene, which lies within the 17p11.2 locus, were associated with the syndrome 3,4,5,6. RAI1 was shown to be the primary gene responsible for most features of SMS 7,8. Miller-Dieker syndrome (MDS) is a multiple malformation characterised by classical lissencephaly, a characteristic facial appearance and sometimes other birth defects 9. It is associated with visible or submicroscopic rearrangements within chromosome band 17p13.3 in almost all cases 10. Isolated lissencephaly sequence (ILS) consists of classical lissencephaly with no other major anomalies 11. Submicroscopic deletions of chromosome 17p13.3 have been detected in almost 40% of these patients 10. MDS is considered a contiguous gene deletion syndrome where deletion of physically contiguous genes leads to the complex phenotypic abnormalities seen in MDS. LIS1 is located at 17p13.3 and recognised as the causative gene responsible for the lissencephaly phenotype in both MDS and ILS 12,13. A deletion in MDS patients always involves LIS1 together with telomeric loci in excess of 250kb Smith ACM et al., Am J Hum Genet 1986;24: Stratton RF et al., Am J Med Genet 1986;24: Vlangos CN et al., Am J Med Genet 2005;132A(3): Girirajan S et al., J Med Genet 2005;42: Bi W et al., Am J Med Genet 2006;140(22): Slager RE et al., Nat Genet 2003;33: Schoumans J et al., Eur J Med Genet 2005;48(3): Girirajan S et al., Genet Med 2006;8(7): Dobyns WB et al., Am J Hum Genet 1991;48: Dobyns WB et al., J Am Med Assoc 1993;270: Dobyns WB et al., Neurology 1992;42: Chong SS et al., Hum Mol Genet 1997;6(2): Lo Nigro C et al., Hum Mol Genet 1997;6(2): FLII Cat. No. LPU 007 RAI1 Cat. No. LPU 019 Miller-Dieker Smith-Magenis Chromosome 17 Miller-Dieker Smith-Magenis Chromosome 17 LIS1 CLU1 RAI1 Common Deletion Region FLII SMCR7 LIS1 CLU1 RAI1 DRG2 FLII SMCR7 D17S379 D17S2027 D17S1566 D17S2111 D17S2021 TOP3A D17S447 D17S1715 D17S379 D17S2027 D17S1566 D17S2111 D17S620 AF021115/ D17S2021 AF D17S Kb 37Kb 38Kb 117Kb 158Kb 102

103 Cat. No. LPU 026-S Cat. No. LPU 026 SRY SRY (sex-determining region Y) located in band Yp11.31 is the genetic master switch of gonadal differentiation. It encodes a transcription factor that is a member of the high mobility group (HMG) -box family of DNA binding proteins 1. In mammals it triggers the development of undifferentiated gonads into testes 2. Human zygotes with mutations in SRY develop into XY females, while XX zygotes with presence of SRY result in a male phenotype with occasional ambiguous genitalia 3,4. Translocations of X and Y are rarely reported but often lead to sex reversal Sinclair AH et al., Nature 1990;346: Koopman P et al., Nature 1991;351: Iida T et al., Hum Mol Genet 1994;3: Kusz K et al., J Med Gene 1999;36: Ellaithi M et al., BMC Ped 2006;6:11 Aquarius Microdeletion SRY DYZ1 Chromosome Y DXZ1 Chromosome X CD99 SRY RPS4Y1 ZFY PAR RH38681 DYS376 DYS242 DYS234 30Kb 50Kb 103

104 Cat. No. LPU 011-S Cat. No. LPU 011 Williams-Beuren Aquarius Microdeletion Williams-Beuren Syndrome (WBS) is a developmental disorder caused by a microdeletion of 7q Patients display connective tissue problems, typically supravalvular aortic stenosis (SVAS), growth retardation, renal anomalies, transient hypercalcaemia, hyperacusis and mental retardation 2. Haploinsufficiency of the elastin (ELN) gene has been identified as responsible for SVAS 3,4. However, of the remaining 15 genes identified within the WBS deletion, none of the remaining clinical features have been conclusively attributed to any one of these genes. The most recent work maps the WBS deletion region to be 1.6Mb 5, and flanked by highly homologous duplicons of kb within which the common breakpoints cluster. The common WBS deletion results from non-homologous recombination between the GTF2I/NCF1 locus and the GTF2IP1/NCF1P1 locus, or rare intrachromosomal exchange between the centromeric GTF2IP2/NCF1 and the telomeric GT2IP2/NCF1P2 duplicons. 1. Francke U et al., Hum Mol Genet 1999;8: Prober BR, Dykens EM, Child Adolesc Psychiatr Clin North Am 1996;5: Li DY et al., Hum Mol Genet 1997;6: Tassabehji M et al., Hum Mol Genet 1997;6: Peoples R et al., Am J Hum Genet 2000;66:47-68 D7Z1 Williams-Beuren Chromosome 7 FZD9 WBSCR28 TBL2 WBSCR22 CLDN4 ELN WBSCR1 CYLN2 GTF2IRD1 GTF2I NCF1 WBSCR9 D7S2476 WBSCR27 LIMK1 RFC2 D7S Kb 144Kb 204Kb 104

105 Cat. No. LPU 009-S Cat. No. LPU 009 Wolf-Hirschhorn Wolf-Hirschhorn syndrome is a multiple malformation syndrome characterised by severe growth deficiency, severe to profound mental retardation with the onset of convulsions in early infancy, microcephaly, sacral dimples and a characteristic face ( Greek helmet appearance ) 1. The phenotype results from the partial deletion of the short arm of chromosome 4 (4p16.3), originally observed as a large cytogenetically visible terminal deletion of 2Mb. Molecular analyses of patients with small terminal and interstitial deletions have allowed for the definition of the Wolf-Hirschhorn Critical Region, which is 165kb and lies between D4S166 and D4S Wilson MG et al., Hum Genet 1981;59: Wright TJ et al., Hum Mol Genet 1997;6(2): Aquarius Microdeletion Wolf-Hirschhorn 4qter Chromosome 4 FGFR3 WHSC2 D4S168 LETM1 WHSC1 D4S166 D4S43 223Kb 105

106 Cat. No. LPU 018-S Cat. No. LPU 018 XIST One X chromosome is inactivated (Xi) in each cell of a female in order to achieve transcriptional balance. An X linked inactivation centre (Xic) is responsible for the initiation of X inactivation. The exact size of the Xic is unclear but it includes the XIST gene (X-inactivation locus) at Xq13.2. Up-regulation of XIST to high levels occurs on the chosen inactive (Xi) chromosome, and repression of XIST on the active (Xa) one 1. Very small ring r(x) chromosomes that do not include XIST have been described with a more severe phenotype in syndromes such as Turner Syndrome 2,3. 1. Boumil RM, Lee JT et al., Hum Mol Genet 2001;10(20): Le Caignec C et al., Prenat Diagn 2003;23(2): Bouayed Abdelmoula N, Ann Genet 2004;47(3): Aquarius Microdeletion DXZ1 XIST X Chromosome CHIC1 XIST FXYD8 DXS8235 DXS9758 DXS7717 DXS Kb 106

107 Subtelomere Subtelomere

108 Contents 108 Subtelomere Specific probes 109 Chromoprobe Multiprobe -T Applications 110 Chromoprobe Multiprobe -T Design 110 Aquarius Subtelomere Specific Probes Subtelomere Specific Probes Chromosomal rearrangements involving the ends of chromosomes have emerged as an important cause of genetic disease given the gene-rich nature of the regions adjacent to the telomeres 1. The importance of such subtelomeric chromosome rearrangements has been clearly shown by their observed association with unexplained mental retardation and congenital abnormalities 2. Individual subtelomere specific probes have been used to focus on particular subtelomeric regions, and have resulted in the establishment of syndromes such as the chromosome 1p36 deletion syndrome 3 and the 22q13.3 deletion syndrome 4. The probes are also finding applications in the investigation of autistic disorders 5, recurrent miscarriages 6 and haematological malignancies 7. Subtelomere Cytocell s subtelomere specific probes are located in the most distal region of chromosome specific DNA on each chromosome. Beyond this unique sequence material is the 100 to 300kb region of telomere associated repeat followed by the cap of between 3 to 20kb of tandemly repeated (TTAGGG)n sequence 8. The probes have been chosen from the most distal unique sequence to provide the best possible specificity, whilst also being applicable for routine use for the examination of subtelomeric enumeration and integrity. The original second-generation set of probes is derived from PAC clones 9 and was established in conjunction with the Institute of Molecular Medicine, part of Oxford University, in the UK 11. Continuing product improvements have led to some substitutions with alternative cosmid (35 40kb) or BAC (150kb) clones to give improved signal strength or chromosome specificity. The average probe size is therefore 100kb, and all have been mapped to within a maximum of 600kb of the true telomere. 108

109 Chromoprobe Multiprobe -T Applications Subtelomeric rearrangements in idiopathic mental retardation and/or in malformation syndromes In the last ten years, 22 studies have reported results from approximately 2500 patients 8,9 which show the importance of subtelomeric chromosome rearrangements as a major cause of mental retardation and/or malformation syndromes. The frequency of subtelomeric rearrangements in the entire group is about 5% but the figure is higher (6.8%) in patients with moderate to severe mental retardation. About half of the positive cases were familial rearrangements which increased significantly the number of informative tests carried out. A total of 15 of these 22 studies were facilitated using the Chromoprobe Multiprobe -T assay, making the Chromoprobe Multiprobe -T the FISH method of choice for the identification of subtle chromosome rearrangements in patients suspected of carrying a chromosome anomaly, but who have an apparently normal karyotype. Characterisation of known abnormalities Subtelomeric chromosome rearrangement screening has been clearly associated with the establishment of new syndromes such as the chromosome 1p36 deletion syndrome 10 and the 22q13.3 deletion syndrome 11. Autistic disorders Several reports have associated subtelomeric chromosomal rearrangements with autistic disorders. Recurrent miscarriages Subtelomeric rearrangements are also being investigated in recurrent miscarriages. Haematological cancers Other clinical applications include the characterisation of known constitutional, and acquired haematological, abnormalities initially detected by standard cytogenetic studies but where further submicroscopic rearrangements cannot be ruled out by the limited resolution of standard karyotyping. Therefore telomere screening provides confirmation and further characterisation in these cases. 1. Saccone S et al., Proc Natl Acad Sci USA 1992;89: Flint J et al., Nat Genet 1997;15(3): Heilstedt HA et al., Clin Genet 2003;64(4): Luciani JJ et al., J Med Genet 2003;40(9): Wolff DJ et al., Genet in Med 2002;4(1): Yakut S et al., Clin Genet 2002;61(1): Tosi S et al., Genes Chrom Cancer 1999;25(4): Moyzis RK et al., Proc Natl Acad Sci USA 1988;85: Knight SJL et al., Am J Hum Genet 2000;67: Institute of Molecular Medicine and National Institute of Health Collaboration, Nat Genet 1997;14: Knight SJL et al., Eur J Hum Genet 1997;5:1-6 Subtelomere 109

110 Cat. No. PMP 009 Cat. No. PMP 008 Cat. No. PMP 007 (2 devices) (5 devices) (10 devices) Chromoprobe Multiprobe -T Design The Chromoprobe Multiprobe -T device is divided into 24 squares. Each square carries subtelomere specific probes for both the p-arm and the q-arm of one of the 23 chromosomes (except for the acrocentric chromosomes). The p-arm and the q-arm probes for each chromosome are labelled in different colours with two spectrally independent fluorophores (green and red respectively). These are located together in the corresponding Multiprobe square. This format provides each other with an internal hybridisation control and effective chromosome identification Subtelomere XY 2QNP Aquarius Subtelomere Specific Probes Cytocell also offers a complete set of subtelomere specific probes available in the Aquarius liquid format. The set identifies 41 of the 46 human subtelomeres with the exclusion of the p-arm telomeres of the acrocentric chromosomes. The probes are available independently and directly labelled in either a red or a green fluorophore (Texas Red or FITC spectra respectively). The probes are supplied in an economical 5 test format and are concentrated to allow the mixing, if required, of up to 3 Aquarius subtelomere specific probes in the same hybridisation. The kits come complete with hybridisation solution, DAPI counterstain and full instructions for use. Please refer to page 125 for details of the Aquarius Subtelomere Specific Probe Range. 110

111 Paints Paints

112 Painting Probes Contents 112 Painting Probes 113 Chromoprobe Multiprobe OctoChrome 114 Summary of Painting Probes Cytocell s Whole Chromosome Painting Probes consist of libraries of DNA sequences derived from flow-sorted chromosomes. These libraries contain sequences stretching over the entire length of the chromosome and provide superior coverage of each human chromosome. Applications include analysis of chromosome partners involved in translocations; identification of the chromosome of origin of marker chromosomes; confirmation of results obtained from M-FISH and SKY testing and may be of particular interest to those studying mutagenesis of human chromosomes as a result of exposure to genotoxic agents. Cytocell offers chromosome painting probes in the Aquarius liquid format (summarised on page 114) and on a Chromoprobe Multiprobe device where FISH probes covering 24 chromosomes are reversibly bound to one 8 square device, illustrated opposite. Paints 112

113 Cat. No. PMP 802 Cat. No. PMP 804 Cat. No. PMP 803 (2 devices) (5 devices) (10 devices) Chromoprobe Multiprobe OctoChrome Cytocell s Chromoprobe Multiprobe OctoChrome combines the utility of an 8 square Multiprobe device and the whole chromosome painting probe (labelled in 3 different colours), to allow all 24 chromosomes to be identified on a single slide. The OctoChrome device allows the simultaneous analysis of the whole genome on one slide in one hybridisation. Each square of the Multiprobe device carries whole chromosome painting probes for three different chromosomes in three different colour fluorophores, red, green and blue (Texas Red, FITC and Coumarin spectra respectively) which are visible simultaneously with a DAPI/FITC/Texas Red triple filter or specific single filters. The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the nonrandom chromosome rearrangements found in the most common leukaemias Paints 113

114 Summary of Painting Probes Cytocell offers a comprehensive range of Whole Chromosome Painting probes available in the Aquarius liquid format. The probes are available in either a red or a green fluorophore (Texas Red or FITC spectra respectively). They are ready to use in hybridisation solution. The kits are supplied in an economical 5 test format and are supplied with DAPI counterstain. Green Whole Chromosome Paints Red Whole Chromosome Paints Paints Cat. No. LPP 01G Cat. No. LPP 02G Cat. No. LPP 03G Cat. No. LPP 04G Cat. No. LPP 05G Cat. No. LPP 06G Cat. No. LPP 07G Cat. No. LPP 08G Cat. No. LPP 09G Cat. No. LPP 10G Cat. No. LPP 11G Cat. No. LPP 12G Cat. No. LPP 13G Cat. No. LPP 14G Cat. No. LPP 15G Cat. No. LPP 16G Cat. No. LPP 17G Cat. No. LPP 18G Cat. No. LPP 19G Cat. No. LPP 20G Cat. No. LPP 21G Cat. No. LPP 22G Cat. No. LPP 0XG Cat. No. LPP 0YG Cat. No. LPP 01R Cat. No. LPP 02R Cat. No. LPP 03R Cat. No. LPP 04R Cat. No. LPP 05R Cat. No. LPP 06R Cat. No. LPP 07R Cat. No. LPP 08R Cat. No. LPP 09R Cat. No. LPP 10R Cat. No. LPP 11R Cat. No. LPP 12R Cat. No. LPP 13R Cat. No. LPP 14R Cat. No. LPP 15R Cat. No. LPP 16R Cat. No. LPP 17R Cat. No. LPP 18R Cat. No. LPP 19R Cat. No. LPP 20R Cat. No. LPP 21R Cat. No. LPP 22R Cat. No. LPP 0XR Cat. No. LPP 0YR 114

115 Satellites Satellites

116 Satellite Enumeration Probes Contents 116 Satellite Enumeration Probes 117 Dual Labelled Satellite Probe Sets 117 Acro-P-Arm Probe Cytocell s Enumeration Satellite probes are chromosome specific sequences generated from highly repeated human satellite DNA located in the centromeric, pericentromeric or heterochromatic regions of each chromosome. These probes allow rapid identification and enumeration of human chromosomes in interphase and metaphase cells of postnatal samples. Cytocell offers a complete range of satellite probes available in the Aquarius liquid format. The probes are available independently and directly labelled in either red or green. They are produced in a concentrated form to allow the mixing, if required, of up to 3 satellite probes in the same hybridisation. The kits are supplied in an economical 5 test format and come complete with hybridisation solution and DAPI counterstain. Green Enumeration Satellite Probes Cat. No. LPE 001G Cat. No. LPE 002G Cat. No. LPE 003G Cat. No. LPE 004G Cat. No. LPE 005G (Chromosome 1,5,19) Cat. No. LPE 006G Cat. No. LPE 007G Cat. No. LPE 008G Cat. No. LPE 009G Cat. No. LPE 010G Cat. No. LPE 011G Cat. No. LPE 012G Red Enumeration Satellite Probes Cat. No. LPE 001R Cat. No. LPE 002R Cat. No. LPE 003R Cat. No. LPE 004R Cat. No. LPE 005R (Chromosome 1,5,19) Cat. No. LPE 006R Cat. No. LPE 007R Cat. No. LPE 008R Cat. No. LPE 009R Cat. No. LPE 010R Cat. No. LPE 011R Cat. No. LPE 012R Satellites Cat. No. LPE 013G (Chromosome 13,21) Cat. No. LPE 014G (Chromosome 14,22) Cat. No. LPE 015G Cat. No. LPE 016G Cat. No. LPE 017G Cat. No. LPE 018G Cat. No. LPE 020G Cat. No. LPE 0XG Cat. No. LPE 0YcG Cat. No. LPE 0YqG Cat. No. LPE 013R (Chromosome 13,21) Cat. No. LPE 014R (Chromosome 14,22) Cat. No. LPE 015R Cat. No. LPE 016R Cat. No. LPE 017R Cat. No. LPE 018R Cat. No. LPE 020R Cat. No. LPE 0XR Cat. No. LPE 0YcR Cat. No. LPE 0YqR 116

117 Cat. No. LPE 0XYc Cat. No. LPE 0XYq Dual Labelled Satellite Probe Sets We also offer two dual labelled X&Y probe sets available in the Aquarius liquid range in a 10 test format. They may be used to identify X & Y human chromosomes in interphase and metaphase cells. XYc: Xp11.1-q11.1 directly labelled with a green fluorophore and Yp11.1-q11.1 with a red fluorophore. XYq: Xp11.1-q11.1 directly labelled with a green fluorophore and Yq12 with a red fluorophore. The probes are designed for use on cultured peripheral blood and bone marrow cells XYq probe (patient after sex-mismatched bone marrow transplant) Acro-P-Arm Probe Cat. No. LPE NOR The Nucleolar Organiser Regions (NOR) contain clusters of genes which code for the three largest structural rrna molecules (5.8S, 18S and 28S). These rrna genes are critically important for the viability of the cell and represent around 0.5% of the human diploid genome. They are found in the short arm of the acrocentric chromosomes and are the region around which the nucleoli develop at the end of mitosis 1. A NOR can translocate to a terminal region of another chromosome. In rare instances this can be pathogenic, particularly when the translocation has led to a deletion at the tip of the recipient chromosome 2. It has been suggested that the restructuring of the rrna genes is the most common chromosomal alteration in adult solid tumours 3. In routine cytogenetic analysis, NOR can be used to delineate marker chromosomes through a process of silver staining the NOR (known as AgNOR staining 4 ). However, the technique relies on translation of protein and if this is not present, conventional silver staining will not stain the NOR. Cytocell s FISH probe has been developed to overcome this problem so that presence of the acrocentric chromosomes in the marker can be accurately confirmed. 1. McClintock B, Z Zellforsch Mikrosk Anat 1934;21: Gardner RJM, Grant R Sutherland, Chromosome abnormalities and genetic counseling, Oxford University Press p Stults DM et al., Cancer Res 2009;69(23): Goodpasture C, Bloom SE, Chromosoma 1975;53:37-50 Satellites 117

118 Satellites 118

119 Accessories Accessories

120 Accessories Cat. No. Description PCA005 PCN003 PCN004 PCN007 PCN008 DES500L DES1000L DFS500L DSS500L HA500L HB500L HA1000L HB1000L PCA003 Rubber Solution Glue 15g Vectashield 10ml Hybridisation Chamber 24 Square Template Slides (x100) 8 Square Template Slides (x100) 0.125µg/ml DAPI 500µl/vial 0.125µg/ml DAPI 1000µ/ vial 1.0 µg/ml DAPI 500µl/vial µg/ml DAPI 500µl/vial Hybridisation Solution A 500µl/vial Hybridisation Solution B 500µl/vial Hybridisation Solution A 1000µl/vial Hybridisation Solution B 1000µl/vial 20x SSC (100ml) ADR123 Detection reagent for indirectly labelled probes (6 month expiry, 2-3 week lead time, min order quantity 10 units) PCN002 Cytocell Slide Surface Thermometer (pack 4) F31001 F31002 F31004 F31036 F51006 F61002 FITC Filter DAPI Filter Texas Red Filter DEAC Filter FITC/Texas Red Dual Filter FITC/DAPI/Texas Red Triple Filter Accessories 120

121 Ordering information Ordering Information

122 Ordering information Aquarius Haematology Probe Range Summary Probe Name Chromosome Region Probe Type Control Probe No. Tests Cat. No.* 13q q14.3 Deletion 13qter 5 or 10 LPH 006 Alpha satellite 12 red for CLL 12p11.1-q11.1 Enumeration N/A 5 or 10 LPH 028 AML1 21q22.12 Breakapart N/A 5 or 10 LPH 027 AML1/ETO Dual Fusion 21q22.12/8q21.3 Translocation N/A 5 or 10 LPH 026 ATM 11q22.3 Deletion D11Z1 5 or 10 LPH 011 BCL6 3q27.3 Breakapart N/A 5 or 10 LPH 035 BCR/ABL Dual Fusion 9q34/22q11 Translocation N/A 5 or 10 LPH 007 BCR/ABL Plus Dual Fusion 9q34/22q11 Translocation/Deletion N/A 5 or 10 LPH 038 CBFβ/MYH11 Dual Fusion 16q22/16p13 Translocation N/A 5 or 10 LPH 022 CKS1B/CDKN2C (P18) 1q21/1p32.3 Amplification/Deletion N/A 5 or 10 LPH 039 cmyc 8q24.21 Breakapart N/A 5 or 10 LPH 010 D13S319 13q14.3 Deletion 13qter 5 or 10 LPH 042 D13S25 13q14.3 Deletion 13qter 5 or 10 LPH 043 Del (5q) 5q31.2 Deletion 5p or 10 LPH 024 Del (7q) 7q22/7q31 Deletion N/A 5 or 10 LPH 025 Del (20q) 20q12/20q13.12 Deletion N/A 5 or 10 LPH 020 E2A 19p13 Breakapart N/A 5 or 10 LPH 019 EVI1 3q26.2 Breakapart N/A 5 or 10 LPH 036 FIP1L1/CHIC2/PDGFRA 4q12 Deletion/Fusion N/A 5 or 10 LPH 032 IGH 14q32.3 Breakapart N/A 5 or 10 LPH 014 IGH/BCL2 Dual Fusion 14q32.3/18q21 Translocation N/A 5 or 10 LPH 018 IGH/CCND1 (BCL1) Dual Fusion 14q32.3/11q13 Translocation N/A 5 or 10 LPH 021 IGH/CCND3 Dual Fusion 14q32.3/6p21 Translocation N/A 5 or 10 LPH 040 IGH/cMYC Dual Fusion 14q32.3/8q24.21 Translocation N/A 5 or 10 LPH 041 IGH/FGFR3 Dual Fusion 14q32.3/4p16.3 Translocation N/A 5 or 10 LPH 030 IGH/MAF Dual Fusion 14q32.3/16q23 Translocation N/A 5 or 10 LPH 029 IGH/MAFB Dual Fusion 14q32.3/20q12 Translocation N/A 5 or 10 LPH 044 IGH/MYEOV Dual Fusion 14q32.3/11q13 Translocation N/A 5 or 10 LPH 045 IGK 2p11.2 Breakapart N/A 5 or 10 LPH 034 IGL 22q11.2 Breakapart N/A 5 or 10 LPH 033 MLL 11q23 Breakapart N/A 5 or 10 LPH 013 MYB 6q23 Deletion D6Z1 5 or 10 LPH 016 P16 9p21 Deletion D9Z3 5 or 10 LPH 009 P53 17p13 Deletion D17Z1 5 or 10 LPH 017 P53/ATM Combination 17p13/11q22.3 Deletion N/A 5 or 10 LPH 052 PDGFRB 5q32 Breakapart N/A 5 or 10 LPH 031 PML/RARα Dual Fusion 15q24.1/17q21 Translocation N/A 5 or 10 LPH 023 TCL1 14q32.13 Breakapart N/A 5 or 10 LPH 046 TCRAD 14q11.2 Breakapart N/A 5 or 10 LPH 047 TCRB 7q34 Breakapart N/A 5 or 10 LPH 048 TEL/AML1 Dual Fusion 12p13.2/21q22.12 Translocation N/A 5 or 10 LPH 012 TLX1 10q24 Breakapart N/A 5 or 10 LPH 049 TLX3 5q35 Breakapart N/A 5 or 10 LPH 050 * for 5 test kit add -S to catalogue number, e.g: LPH ###-S Ordering Information 122

123 Ordering information CLL Screening Panel LPH CLL or LPH CLL-S Product Description Chromosome Region Probe Type No. Tests Control Probe P53 17p13 Deletion D17Z1 5 or 10 ATM 11q22.3 Deletion D11Z1 5 or 10 MYB 6q23 Deletion D6Z1 5 or 10 13q q14.3 Deletion 13qter 5 or 10 Alpha satellite 12 red for CLL 12p11.1-q11.1 Enumeration N/A 5 or 10 Chromoprobe Multiprobe - ALL v2 System Range Summary Probe Name No. Tests Cat. No. Chromoprobe Multiprobe - ALL System 2 PMP 030 Chromoprobe Multiprobe - ALL System 5 PMP 031 Chromoprobe Multiprobe - ALL System 10 PMP 032 Chromoprobe Multiprobe - ALL System 20 PMP 033* Chromoprobe Multiprobe - CLL System Range Summary Probe Name No. Tests Cat. No. Chromoprobe Multiprobe - CLL System 2 PMP 018 Chromoprobe Multiprobe - CLL System 5 PMP 017 Chromoprobe Multiprobe - CLL System 10 PMP 016 Chromoprobe Multiprobe - CLL System 20 PMP 020* Chromoprobe Multiprobe - AML/MDS System Range Summary Probe Name No. Tests Cat. No. Chromoprobe Multiprobe - AML System 2 PMP 025 Chromoprobe Multiprobe - AML System 5 PMP 026 Chromoprobe Multiprobe - AML System 10 PMP 027 Chromoprobe Multiprobe - AML System 20 PMP 028* * supplied as 4 x 5 Multiprobe devices Aquarius Pathology Probe Range Summary Probe Name Chromosome Region Probe Type Control Probe No. Tests Cat. No.* CHOP 12q13 Breakapart N/A 5 or 10 LPS 015 C-MET 7q31 Amplification D7Z1 5 or 10 LPS 004 EGFR 7p11.2 Amplification D7Z1 5 or 10 LPS 003 EWSR1 22q12 Breakapart N/A 5 or 10 LPS 006 EWSR1/ERG Dual Fusion 21q22/22q12 Translocation N/A 5 or 10 LPS 008 FGFR1 8p12 Breakapart/Amplification D8Z2 5 or 10 LPS 018 FLI1/EWSR1 Dual Fusion 11q24/22q12 Translocation N/A 5 or 10 LPS 007 HER2 17q12 Amplification D17Z1 5 or 10 LPS 001 MALT1 18q21 Breakapart N/A 5 or 10 LPS 017 MDM2 12q15 Amplification D12Z1 5 or 10 LPS 016 N-MYC 2p24.3 Amplification LAF (2q11) 5 or 10 LPS 009 PAX3 2q35 Breakapart N/A 5 or 10 LPS 012 PAX7 1p36 Breakapart N/A 5 or 10 LPS 013 RB1 13q14.2 Deletion 13qter 5 or 10 LPS 011 SRD (CHD5) 1p36 Deletion 1qter 5 or 10 LPS 010 SYT 18q11.2 Breakapart N/A 5 or 10 LPS 014 TOP2A 17q21 Amplification/Deletion D17Z1 5 or 10 LPS 002 ZNF217 20q13 Amplification 20pter 5 or 10 LPS 005 * for 5 test kit add -S to catalogue number, e. g: LPS ###-S 123 Ordering Information

124 Ordering information Aquarius Prenatal Probe Range Summary Product Description Locus Chromosome Region No. Tests Cat. No.* Probe Set 1 and 2 5, 10, 30 or 50 LPA 001 X centromere DXZ1 Xp11.1-q11.1 Y centromere DYZ3 Yp11.1-q centromere D18Z1 18p11.1-q unique sequence N/A 13q unique sequence N/A 21q22.13 Probe Set 1 5 or 10 LPA 002 X centromere DXZ1 Xp11.1-q11.1 Y centromere DYZ3 Yp11.1-q centromere D18Z1 18p11.1-q11.1 Probe Set 2 5 or 10 LPA unique sequence N/A 13q unique sequence N/A 21q22.13 Probe Set 3 5 or 10 LPA unique sequence N/A 13q centromere D18Z1 18p11.1-q unique sequence N/A 21q centromere blue D18Z1 18p11.1-q or 10 LPA 004 Ordering Information 124 * for 5, 30 or 50 test kit add -S, -30 or -50 to the catalogue number respectively, e.g: LPA### -S, LPA### -30 or LPA### -50 Aquarius Microdeletion Probe Range Summary Probe Name Chromosome Region Probe Locus Identification Control Locus No. Tests Cat. No.* Alagille (JAG1) 20p12 JAG1 20qter 5 or 10 LPU 012 Angelman (UBE3A/D15S10) 15q11-13 UBE3A/D15S10 15qter 5 or 10 LPU 006 CHARGE 8q12.1-8q12.2 CHD7 D8Z2 5 or 10 LPU 021 Cri-Du-Chat and 5p15.2/5q35 FLJ25076, CTNND2, NSD1 N/A 5 or 10 LPU 013 SOTOS Combination DiGeorge II (10p14) 10p14 CUGBP2 D10Z1 5 or 10 LPU 015 DiGeorge TBX1 and 22q11.21/22q13.3 TBX1, N85A3 N/A 5 or 10 LPU q13.3 Combination DiGeorge/VCFS N25 and 22q11.21/22q13.3 N25/D22S75, N85A3 N/A 5 or 10 LPU q13.3 Combination DiGeorge/VCFS TUPLE1 22q11.21/22q13.3 TUPLE1, N85A3 N/A 5 or 10 LPU 004 and 22q13.3 Combination Kallmann (KAL1) and Steroid Xp22.31 KAL1, STS DXZ1 5 or 10 LPU 016 Sulphatase (STS) Combination Langer-Giedion 8q23.3/8q24.11-q24.12 TRPS1, EXT1 D8Z2 5 or 10 LPU 022 Monosomy 1p36 1p p36 1qter 5 or 10 LPU 020 Neurofibromatosis Type 1 17q11.2 NF1 17pter 5 or 10 LPU 017 Prader-Willi/Angelman (SNRPN) 15q11-13 SNRPN 15qter 5 or 10 LPU 005 Rubinstein-Taybi 16p13.3 CREBBP D16Z2 5 or 10 LPU 023 Saethre-Chotzen /Williams-Beuren 7p21.1/7q11.23 TWIST1, WBSCR N/A 5 or 10 LPU 024 Combination SHOX Xp22.33/Yp11.2 SHOX DXZ1, DYZ1 5 or 10 LPU 025 Smith-Magenis (RAI1)/Miller-Dieker 17p11.2/17p13.3 RAI1, LIS1 N/A 5 or 10 LPU 019 (LIS1) Combination Smith-Magenis (FLII)/Miller-Dieker 17p11.2/17p13.3 FLII, LIS1 N/A 5 or 10 LPU 007 (LIS1) Combination SRY Yp11.2 SRY DXZ1, DYZ1 5 or 10 LPU 026 Williams-Beuren 7q11.23 WBSCR D7Z1 5 or 10 LPU 011 Wolf-Hirschhorn 4p16.3 WHSC1/2 4qter 5 or 10 LPU 009 XIST Xq13.2 XIST DXZ1 5 or 10 LPU 018 * for 5 test kit add -S to catalogue number, e.g: LPU ###-S

125 Ordering information Aquarius Subtelomere Specific Probe Range Summary Probe Clone Name Marker (STS) Accession Max. physical Cat. No.* Specificity Number distance from (if available) Telomere (kb) 1p CEB108 CEB108/T7 - <300 LPT 01PR/G 1q 160H23 1qtel19 D1S LPT 01QR/G 2p dj892g20 2ptel27 D2S LPT 02PR/G 2q dj1011o17 2qtel47 D2S LPT 02QR/G 2q NP 172I13 VIJ-YRM2112 D2S LPT 02QNPR/G 3p dj1186b18 3ptel25 D3S LPT 03PR/G 3q 196F4 3qtel06 D3S LPT 03QR/G 4p 36P21 4ptel04 D4S LPT 04PR/G 4q CTC-963K6 4qtel LPT 04QR/G 5p 189N21 5ptel LPT 05PR/G 5q 240G13 5qtel70 D5S LPT 05QR/G 6p 62I11 6ptel LPT 06PR/G 6q 57H24 6qtel54 D6S LPT 06QR/G 7p 109a6 - G31341 <255 LPT 07PR/G 7q 2000a5 - G31340 <8 LPT 07QR/G 8p dj580l5 8ptel91 D8S LPT 08PR/G 8q 489D14 8qtel11 D8S LPT 08QR/G 9p 43N6 9ptel LPT 09PR/G 9q 112N13 9qtel33 D9S LPT 09QR/G 10p 306F7 10ptel45 D10S LPT 10PR/G 10q 137E24 10qtel24 D10S LPT 10QR/G 11p dj908h22 11ptel03 D11S LPT 11PR/G 11q dj770g7 11qtel38 D11S LPT 11QR/G 12p 496A11 12ptel LPT 12PR/G 12q 221K18 12qtel87 D12S LPT 12QR/G 13q 163C9 13qtel56 D13S LPT 13QR/G 14q dj820m16 14qtel01 D14S LPT 14QR/G 15q 154P1 WI-5214 D15S LPT 15QR/G 16p 121I4 16ptel05 D16S LPT 16PR/G 16q 240G10 16qtel LPT 16QR/G 17p 202L17 17ptel80 D17S LPT 17PR/G 17q 362K14 17qtel13 D17S LPT 17QR/G 18p 74G18 VIJ-YRM2102 D18S LPT 18PR/G 18q dj964m9 18qtel11 D18S LPT 18QR/G 19p dj546c11 19ptel LPT 19PR/G 19q F qtel LPT 19QR/G 20p dj106l1 20pthy33 D20S LPT 20PR/G 20q 81F12 20qtel14-50 LPT 20QR/G 21q 63H24 21qtel07 D21S LPT 21QR/G 22q 99K24 22qtel31 D22S LPT 22QR/G XpYp** 839D20 - DXYS LPT XYPR/G XqYq*** C8.2/1 - DXYS61 <90 LPT XYQR/G * R specifies a red label, G specifies a green label ** This probe is specific for the p-arms of both X and Y *** This probe is specific for the q-arms of both X and Y NP Non Polymorphic 125 Ordering Information

126 Ordering information Chromoprobe Multiprobe -T Subtelomere System Range Summary Probe Name No. Tests Cat. No. Chromoprobe Multiprobe - T System 2 PMP 009 Chromoprobe Multiprobe - T System 5 PMP 008 Chromoprobe Multiprobe - T System 10 PMP 007 Aquarius Whole Chromosome Painting Probe Range Summary Chromosome No. Tests Cat. No.* 1 5 LPP 01R/G 2 5 LPP 02R/G 3 5 LPP 03R/G 4 5 LPP 04R/G 5 5 LPP 05R/G 6 5 LPP 06R/G 7 5 LPP 07R/G 8 5 LPP 08R/G 9 5 LPP 09R/G 10 5 LPP 10R/G 11 5 LPP 11R/G 12 5 LPP 12R/G 13 5 LPP 13R/G 14 5 LPP 14R/G 15 5 LPP 15R/G 16 5 LPP 16R/G 17 5 LPP 17R/G 18 5 LPP 18R/G 19 5 LPP 19R/G 20 5 LPP 20R/G 21 5 LPP 21R/G 22 5 LPP 22R/G X 5 LPP 0XR/G Y 5 LPP 0YR/G * R specifies a red label, G specifies a green label Whole Chromosome Paint Combinations Chromosome Description No. Tests Cat. No.* 1/2/4 Whole chromosome paint combination 1,2,4 10 LPP 124 (3 colour, 3 probe combination) directly labelled 1/2/4 and 3/5/6 Whole chromosome paint combination 1,2,4 and 10 LPP 356 3,5,6 (2 colour, 6 probe combination) indirectly labelled * Minimum order of ten units (100 tests). Orders are subject to 4-6 weeks lead time Ordering Information 126

127 Ordering information Chromoprobe Multiprobe -OctoChrome Paint System Range Summary Probe Name No. Tests Cat. No.* Chromoprobe Multiprobe - OctoChrome System 2 PMP 802 Chromoprobe Multiprobe - OctoChrome System 5 PMP 804 Chromoprobe Multiprobe - OctoChrome System 10 PMP 803 Aquarius Satellite Enumeration Probe Range Summary Chromosome Locus Chromosome Region DNA Class No. Tests Cat. No.* 1 D1Z1 1q12 satellite III 5 LPE 001R/G 2 D2Z2 2p11.1-q11.1 α-satellite 5 LPE 002R/G 3 D3Z1 3p11.1-q11.1 α-satellite 5 LPE 003R/G 4 D4Z1 4p11.1-q11.1 α-satellite 5 LPE 004R/G 1/5/19 D1Z7 1p11.1-q11.1 α-satellite 5 LPE 005R/G D5Z2 5p11.1-q11.1 D19Z3 19p11.1-q D6Z1 6p11.1-q11.1 α-satellite 5 LPE 006R/G 7 D7Z1 7p11.1-q11.1 α-satellite 5 LPE 007R/G 8 D8Z2 8p11.1-q11.1 α-satellite 5 LPE 008R/G 9 D9Z3 9q12 satellite III 5 LPE 009R/G 10 D10Z1 10p11.1-q11.1 α-satellite 5 LPE 010R/G 11 D11Z1 11p11.1-q11.1 α-satellite 5 LPE 011R/G 12 D12Z3 12p11.1-q11.1 α-satellite 5 LPE 012R/G 13/21 D13Z1 13p11.1-q11.1 α-satellite 5 LPE 013R/G D21Z1 21p11.1-q /22 D14Z1 14p11.1-q11.1 α-satellite 5 LPE 014R/G D22Z1 22p11.1-q D15Z4 15p11.1-q11.1 α-satellite 5 LPE 015R/G 16 D16Z2 16p11.1-q11.1 α-satellite 5 LPE 016R/G 17 D17Z1 17p11.1-q11.1 α-satellite 5 LPE 017R/G 18 D18Z1 18p11.1-q11.1 α-satellite 5 LPE 018R/G 20 D20Z1 20p11.1-q11.1 α-satellite 5 LPE 020R/G X DXZ1 Xp11.1-q11.1 α-satellite 5 LPE 0XR/G Yc DYZ3 Yp11.1-q11.1 α-satellite 5 LPE 0YcR/G Yq DYZ1 Yq12 satellite III 5 LPE 0YqR/G XYc Dual Labelled DXZ1 Xp11.1-q11.1 α-satellite 10 LPE 0XYc DYZ3 Yp11.1-q11.1 XYq Dual Labelled DXZ1 Xp11.1-q11.1 α-satellite 10 LPE 0XYq DYZ1 Yq12 satellite III * R specifies a red label and G specifies a green label. Acro-P-Arm Probe Chromosome Colour No. Tests Cat. No. 13, 14, 15, 21, 22 Red 10 LPE NOR 127 Ordering Information

128 Index by Chromosome Number Ordering Information 128 Chromosome Probe Name Page 1-22, X, Y Painting Probes-Aquarius , X, Y Painting Probes-Chromoprobe Multiprobe , X, Y Satellite Probes-Aquarius , X, Y Subtelomere Specific Probes-Aquarius , X, Y Subtelomere Specific Probes-Chromoprobe Multiprobe Monosomy 1p SRD (CHD5) Deletion 77 1 PAX7 Breakapart 75 1 CKS1B/CDKN2C (P18) Amplification/Deletion 18 2 IGK Breakapart 37 2 PAX3 Breakapart 75 2 N-MYC Amplificaton 74 3 BCL6 Breakapart 16 3 EVI1 Breakapart 26 4 Wolf-Hirschhorn FIP1L1/CHIC2/PDGFRA Deletion/Fusion 27 4, 14 IGH/FGFR3 Translocation, Dual Fusion 33 5 Del (5q) Deletion 22 5 Cri-Du-Chat and SOTOS Combination 90 5 PDGFRB Breakapart 43 5 TLX3 Breakapart 50 6 MYB Deletion 39 6, 14 IGH/CCND3 Translocation, Dual Fusion 31 7 Williams-Beuren Del (7q) Deletion 23 7 Saethre-Chotzen/Williams-Beuren Combination EGFR Amplification 67 7 C-MET Amplification 66 7 TCRB Breakapart 47 8, 21 AML1/ETO Translocation, Dual Fusion 12 8 cmyc Breakapart 19 8 CHARGE 89 8 Langer-Giedion 95 8 FGFR1 Breakapart/Amplification 70 8, 14 IGH/cMYC Translocation, Dual Fusion 32 9 P16 Deletion 40 9, 22 BCR/ABL Translocation, Dual Fusion 14 9, 22 BCR/ABL Plus Translocation, Dual Fusion DiGeorge II TLX1 Breakapart ATM Deletion 13 11, 17 P53/ATM Combination MLL Breakapart 38 11, 14 IGH/CCND1 (BCL1) Translocation, Dual Fusion 30 11, 22 FLI1/EWSR1 Translocation, Dual Fusion 68 11, 14 IGH/MYEOV Translocation, Dual Fusion 36 12, 21 TEL/AML1 Translocation, Dual Fusion CHOP Breakapart 65

129 Chromosome Probe Name Page 12 MDM2 Amplification q14.3 Deletion RB1 Deletion D13S319 Deletion D13S25 Deletion IGH Breakapart 28 14, 18 IGH/BCL2 Translocation, Dual Fusion 29 14, 16 IGH/MAF Translocation, Dual Fusion 34 14, 20 IGH/MAFB Translocation, Dual Fusion TCL1 Breakapart TCRAD Breakapart Angelman (UBE3A/D15S10) Prader-Willi/Angelman (SNRPN) 98 15, 17 PML/RARα Translocation, Dual Fusion CBFβ/MYH11 Translocation, Dual Fusion Rubinstein-Taybi Smith-Magenis(RAI1)/Miller-Dieker Combination Smith-Magenis(FLII)/Miller-Dieker Combination P53 Deletion Neurofibromatosis Type HER2 Amplification TOP2A Amplification/Deletion MALT1 Breakapart SYT Breakapart E2A Breakapart Del (20q) Deletion Alagille (JAG1) ZNF217 Amplification AML1 Breakapart 11 21, 22 EWSR1/ERG Translocation, Dual Fusion DiGeorge/VCFS TUPLE1 and 22q13.3 Deletion Syndrome Probe Combination DiGeorge/VCFS N25 and 22q13.3 Deletion Syndrome Probe Combination DiGeorge TBX1 and 22q13.3 Deletion Syndrome Probe Combination IGL Breakapart EWSR1 Breakapart 68 X Kallmann (KAL1) and Steroid Sulphatase (STS) Combination 94 X XIST 106 X, Y SHOX 101 Y SRY 103 Various ALL-Chromoprobe Multiprobe 56 Various AML/MDS-Chromoprobe Multiprobe 60 Various CLL-Chromoprobe Multiprobe 58 Various CLL Screening Panel 51 Various Acro-P-Arm Probe 117 Various Prenatal Enumeration Probes Ordering Information

130 Index by Probe Name Ordering Information 130 Probe Name Chromosome Page 13q14.3 Deletion 13q Acro-P-Arm Probe 13,14,15,21, Alagille (JAG1) 20p12 87 ALL-Chromoprobe Multiprobe Various 56 AML1/ETO Translocation, Dual Fusion 21q22.12/8q AML/MDS-Chromoprobe Multiprobe Various 60 AML1 Breakapart 21q Angelman (UBE3A/D15S10) 15q ATM Deletion 11q BCL6 Breakapart 3q BCR/ABL Plus Translocation, Dual Fusion 9q34/22q11 14 BCR/ABL Translocation, Dual Fusion 9q34/22q11 14 CBFβ/MYH11 Translocation, Dual Fusion 16q22/16p13 17 CHARGE 8q12.1-8q CHOP Breakapart 12q13 65 CKS1B/CDKN2C (P18) Amplification/Deletion 1q21/1p CLL-Chromoprobe Multiprobe Various 58 CLL Screening Panel Various 51 C-MET Amplification 7q31 66 cmyc Breakapart 8q Cri-Du-Chat and SOTOS Combination 5p15.2/5q35 90 D13S25 Deletion 13q D13S319 Deletion 13q Del (5q) Deletion 5q Del (7q) Deletion 7q22/7q31 23 Del (20q) Deletion 20q12/20q DiGeorge II 10p14 91 DiGeorge TBX1 and 22q13.3 Deletion Syndrome Probe Combination 22q11.21/22q DiGeorge/VCFS N25 and 22q13.3 Deletion Syndrome Probe Combination 22q11.21/22q DiGeorge/VCFS TUPLE1 and 22q13.3 Deletion Syndrome Probe Combination 22q11.21/22q EGFR Amplification 7p E2A Breakapart 19p13 25 EVI1 Breakapart 3q EWSR1 Breakapart 22q12 68 EWSR1/ERG Translocation, Dual Fusion 21q22/22q12 68 FGFR1 Breakapart/Amplification 8p12 70 FIP1L1/CHIC2/PDGFRA Deletion/Fusion 4q12 27 FLI1/EWSR1 Translocation, Dual Fusion 11q24/22q12 68 HER2 Amplification 17q12 71 IGH Breakapart 14q IGH/BCL2 Translocation, Dual Fusion 14q32.3/18q21 29 IGH/CCND1 (BCL1) Translocation, Dual Fusion 14q32.3/11q13 30 IGH/CCND3 Translocation, Dual Fusion 14q32.3/6p21 31 IGH/cMYC Translocation, Dual Fusion 14q32.3/8q IGH/FGFR3 Translocation, Dual Fusion 14q32.3.3/4p IGH/MAF Translocation, Dual Fusion 14q32.3/16q23 34

131 Probe Name Chromosome Page IGH/MAFB Translocation, Dual Fusion 14q32.3/16q23 35 IGH/MYEOV Translocation, Dual Fusion 14q32.3/11q13 36 IGK Breakapart 2p IGL Breakapart 22q Kallmann (KAL1) and Steroid Sulphatase (STS) Combination Xp Langer-Giedion 8q23.3/8q24.11-q MALT1 Breakapart 18q21 72 MDM2 Amplification 12q15 73 MLL Breakapart 11q23 38 Monosomy 1p36 1p MYB Deletion 6q23 39 Neurofibromatosis Type 1 17q N-MYC Amplificaton 2p P16 Deletion 9p21 40 P53 Deletion 17p13 41 P53/ATM Combination 11q22.3/17p13 42 Painting Probes-Aquarius 1-22, X, Y 114 Painting Probes-Chromoprobe Multiprobe 1-22, X, Y 113 PAX3 Breakapart 2q35 75 PAX7 Breakapart 1p36 75 PDGFRB Breakapart 5q32 43 PML/RARα Translocation, Dual Fusion 15q24.1/17q21 44 Prader-Willi/Angelman (SNRPN) 15q Prenatal Enumeration Probes 13,18,21, X, Y 82 RB1 Deletion 13q Rubinstein-Taybi 16p Satellite Probes-Aquarius 1-22, X, Y 116 Saethre-Chotzen/Williams-Beuren Combination 7p21.1/7q SHOX Xp22.33/Yp Smith-Magenis(FLII)/Miller-Dieker Combination 17p11.2/17p Smith-Magenis(RAI1)/Miller-Dieker Combination 17p11.2/17p SRD (CHD5) Deletion 1p36 77 SRY Yp Subtelomere Specific Probes-Aquarius 1-22, X, Y 110 Subtelomere Specific Probes-Chromoprobe Multiprobe 1-22, X, Y 110 SYT Breakapart 18q TCL1 Breakapart 14q TCRAD Breakapart 14q TCRB Breakapart 7q34 47 TEL/AML1 Translocation, Dual Fusion 12p13.2/21q TLX1 Breakapart 10q24 49 TLX3 Breakapart 5q35 50 TOP2A Amplification/Deletion 17q21 79 Williams-Beuren 7q Wolf-Hirschhorn 4p XIST Xq ZNF217 Amplification 20q Ordering Information

132 Haematology products by disease state Haematological Malignancy Chromosome Region Cytocell Products Cat. No. Page No. ALL Acute Lymphoblastic Leukaemia (ALL) 21q22.12 AML1 Breakapart LPH q34/22q11 BCR/ABL Translocation LPH q34/22q11 BCR/ABL Plus Translocation/Deletion LPH q24.21 cmyc Breakapart LPH p13 E2A Breakapart LPH q32.3 IGH Breakapart LPH q32.3/8q24.21 IGH/cMYC Translocation LPH q23 MLL Breakapart LPH q23 MYB Deletion LPH p21 P16 Deletion LPH q32.13 TCL1 Breakapart LPH q11.2 TCRAD Breakapart LPH q34 TCRB Breakapart LPH p13.2/21q22.12 TEL/AML1 Translocation LPH q24 TLX1 Breakapart LPH q35 TLX3 Breakapart LPH CLL Chronic Lymphocytic Leukaemia (CLL) 13q q14.3 Deletion LPH p11.1-q11.1 Alpha-satellite 12 for CLL LPH q22.3 ATM Deletion LPH q14.3 D13S25 Deletion LPH q14.3 D13S319 Deletion LPH q32.3 IGH Breakapart LPH q32.3/18q21 IGH/BCL2 Translocation LPH q32.3/11q13 IGH/CCND1 Translocation LPH q32.3/6p21 IGH/CCND3 Translocation LPH q23 MYB Deletion LPH p13 P53 Deletion LPH p13/11q22.3 P53/ATM Combination Probe LPH AML Acute Myeloid Leukaemia (AML) 21q22.12 AML1 Breakapart LPH q22.12/8q21.3 AML1/ETO Translocation LPH q34/22q11 BCR/ABL Translocation LPH q34/22q11 BCR/ABL Plus Translocation/Deletion LPH q22/16p13 CBFβ/MYH11 Translocation LPH q31.2 Del (5q) Deletion LPH q22/7q31 Del (7q) Deletion LPH q12/20q13.12 Del (20q) Deletion LPH q26.2 EVI1 Breakapart LPH q23 MLL Breakapart LPH p13 P53 Deletion LPH q24.1/17q21 PML/RARα Translocation LPH Ordering Information CML MM L 132 Chronic Myeloid Leukaemia (CML) 9q34/22q11 BCR/ABL Translocation LPH q34/22q11 BCR/ABL Plus Translocation/Deletion LPH q12 FIP1L1/CHIC2/PDGFRA Deletion/Fusion LPH q32 PDGFRB Breakapart LPH Multiple Myeloma (MM) 13q q14.3 Deletion LPH q21/1p32.3 CKS1B/CDKN2C Amplification/Deletion LPH q14.3 D13S25 Deletion LPH q14.3 D13S319 Deletion LPH q32.3 IGH Breakapart LPH q32.3/11q13 IGH/CCND1 Translocation LPH q32.3/6p21 IGH/CCND3 Translocation LPH q32.3/4p16.3 IGH/FGFR3 Translocation LPH q32.3/16q23 IGH/MAF Translocation LPH q32.3/20q12 IGH/MAFB Translocation LPH q32.3/11q13 IGH/MYEOV Translocation LPH p13 P53 Deletion LPH Lymphomas (L) 3q27.3 BCL6 Breakapart LPH q24.21 cmyc Breakapart LPH q32.3 IGH Breakapart LPH q32.3/18q21 IGH/BCL2 Translocation LPH q32.3/11q13 IGH/CCND1 Translocation LPH q32.3/8q24.21 IGH/cMYC Translocation LPH p11.2 IGK Breakapart LPH q11.2 IGL Breakapart LPH p13 P53 Deletion LPH

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