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An alternative to FISH: detecting deletion and duplication carriers within 24 hours
  1. S J White,
  2. E Sterrenburg,
  3. G-J B van Ommen,
  4. J T den Dunnen,
  5. M H Breuning
  1. Human and Clinical Genetics, Leiden University Medical Center, Wassenarrseweg 72, Leiden, the Netherlands
  1. Correspondence to:
 Dr Johan T den Dunnen 
 Human and Clinical Genetics Leiden University Medical Center Wassenarrseweg 72 Leiden, the Netherlands; ddunnenlumc.nl

https://doi.org/10.1136/jmg.40.10.e113

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    A range of genetic disorders has been revealed to be caused by deletions and duplications within the genome.1–3 In addition, computational analysis of the recently completed human genome sequence4 suggests that many more rearrangements might exist. Such rearrangements are either directly involved in genetic disease or may play an important, but yet to be determined, role in human variation and multifactorial diseases. Efficient methods are thus required to screen for and detect such rearrangements.

    While changes of several megabases are usually cytogenetically visible, smaller changes require other methods of analysis. Many techniques have been applied, including dinucleotide repeat polymorphism analysis,5 array comparative genomic hybridisation,6 fluorescent in situ hybridisation (FISH),7,8 quantitative multiplex PCR,9,10 and Southern blotting.11,12 The last three mentioned are the most commonly applied techniques,13 with FISH analysis preferred as the method of choice in many clinical centres. FISH has the advantage that the analysis is visual, with the number of fluorescent signals determining the copy number of the region examined. However, the method is rather laborious, with cell culturing and preparation of metaphase spreads being necessary, but difficult and time consuming steps. FISH is thus expensive and not suitable for high throughput analysis. In addition, as FISH probes are usually artificial chromosomes or cosmids, it precludes the analysis of small rearrangements, and duplications can be difficult to detect.

    Quantitative multiplex PCR seems an attractive alternative. It can co-amplify up to 15 products per sample, with the amount of each product corresponding to the copy number of the locus. However, achieving consistent results has proven to be technically challenging, and the method requires fluorescent labels and sophisticated equipment.

    Southern blotting is more flexible and does not require sophisticated equipment. Its disadvantages are that it is laborious, requiring several …

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