CYTOGENETICS DEPARTMENT

MOLECULAR CYTOGENETICS (FISH)

Molecular cytogenetics (also known as Fluorescence In Situ Hybridisation) represents a means of increasing the speed, sensitivity and specificity of conventional cytogenetics.
These tests can usually be carried out on the sample supplied for routine cytogenetics. When in doubt, consult the laboratory, as there are many options.

Some applications are described below, but please contact the department for specific information, as FISH is a rapidly evolving area.

The technique takes advantage of an intrinsic property of DNA. When similar sequences are rendered single-stranded, they will anneal, or "hybridise" together. By labelling probe sequences of interest with suitable fluorochromes, we can visualise specific sequences on a slide of patient material, using image analysis software.

Figure 1. Cytogenetics microscope setup.

Figure 2. FISH detects the presence of both chromosome22 copies (green) each with an orange signal, hybrisised to the loci within the DiGeorge critical region, indicating a normal result.

Figure 3. FISH detects a deletion of the elastin gene on chromosome 7 in a patient with Williams Syndrome.

Used in special cases where the referring clinician suspects a specific syndrome. These are often not detectable by conventional cytogenetic methods. Syndromes for which a FISH test is available include: Williams, Prader-Willi, Angelman, Miller-Dieker, Smith-Magenis, Di George

Figure 4. FISH in aneuploidy screening

FISH techniques for aneuploidy screening of prenatal samples have now largely been superceded by QF-PCR, although there may be exceptions. Consult the section on amniocentesis samples above. A similar approach can be used to detect changes in chromosome number in other clinical settings: e.g.: the acquisition of an extra copy of chromosome 12 in chronic lymphocytic leukaemia or the detection of ploidy changes in fixed paraffin embedded tissue sections.

Figure 5. Detection of Gene Rearrangements in Cancer

Oncogenic "fusion genes" may be created by rearrangement of the genetic material. This is a recognised cause of many cancers and can be highly specific. By designing two-colour FISH probes to both gene partners in a fusion (usually a cancer gene and a promoter gene) the novel sequences may be identified by the close juxtaposition of signals. The haematological malignancies have been the most extensively studied to date.

Gene fusions currently detectable by FISH include:

• BCR/ABL in chronic myeloid leukaemia
• PML/RARA in acute promyelocytic leukaemia
• TEL/AML in acute lymphoblastic leukaemia
• t(14;18) in follicular lymphoma

Figure 6. FISH detection of increased HER2 copy number in breast cancer.

Array Comparative Genomic Hybridisation: (enquiries x36765)

During 2007 the department introduced the new technique of array comparative genomic hybridisation (aCGH), also known as microarray or CHIP analysis. For constitutional cases tests would require referral via Consultant Clinical Geneticists as gatekeepers of the service.  Diagnostic criteria are generally for children with moderate to severe retardation and associated significant malformation.

Basis of aCGH test:
Array comparative genomic hybridisation is performed using a BlueGnome CytoChip array.  Test DNA is referenced against same sex control DNA in dye-swap hybridisations and data analysed with BlueFuse software.  Copy number changes are sized according to DECIPHER convention.

This is a new diagnostic test for constitutional studies, which is still under development, and there are currently no nationally accepted best practice guidelines.  However, using the analysis protocol described above, we are confident of the quality of this result.  The array has a backbone clone set with higher coverage across known disease related regions, and subtelomeric regions. The resolution of the arrays is increased periodically as new versions are brought out, and this is documented in all clinical reports.  Please note aCGH will not detect balanced rearrangements and is limited in detection of mosaicism, and for these reasons there may be cases where a different strategy is more appropriate.  Please contact the department if you wish to discuss these tests.

Other types of array (eg: cancer, microdeletions) are under development, and we will keep you informed of progress in these areas.

Figure 7. Array CGH uses the same principle as conventional CGH (left) but uses arrays of target clones spotted onto a slide chip (right).