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Mar 1, 2009 (Vol. 29, No. 5)

Shining a Different Light on Genetic Diversity

Copy Number Variations Are Poised to Rival Single Nucleotide Polymorphisms in Importance for Clinical Applications

  • Exploring Copy Number Variations

    Click Image To Enlarge +
    The integrated fluidic circuit system provides a platform to examine copy number variation.(Fluidigm)

    Several approaches are currently available to explore copy number variations, and understanding their limitations is essential. Real-time PCR, for example, is user-friendly, has a very dynamic range, requires small sample sizes, and provides extremely good resolution when discriminating 50% or 25% differences in copy numbers but, beyond that point, technical considerations and biological errors often affect resolution.

    Fluidigm’s integrated fluidic circuit system is a platform to examine gene- and sequence-specific copy number variations. “With our digital array platform, because we are looking at end-point images, there is literally no limit to the difference in copy numbers that we can detect,” reveals Ramesh Ramakrishnan, Ph.D., director of molecular biology.

    This nanofluidic platform enables the isolation and amplification of single DNA molecules, a technique known as digital PCR, and accurate DNA quantitation is based on the random partitioning of DNA molecules into an array that can have more than 9,000 chambers or wells.

    Individual samples are subsequently PCR amplified, and the initial concentration of a specific sequence is calculated based on the number of positive chambers that contain at least one copy of the desired specific sequence, using an algorithm relying on probability theory and statistics.

    The output, in the form of “relative copy number,” represents the ratio between the copy number of the gene of interest and that of a reference gene, which is always 1. The relative copy number is always 1 for single copy genes, higher than 1 for duplications, and values lower than 1 are indicative of deletions.

    The nanofluidic platform provides a stronger discrimination power as compared to quantitative PCR, explains Dr. Ramakrishnan as exemplified by its ability to differentiate between six and seven copies of a target gene, which corresponds to as little as a 15% difference in gene copy number and, importantly, can be tailored to any gene or sequence.

    An increasing number of conditions affecting various organs and systems are impacted by copy number variations. Chack-Yung Yu, D.Phil., professor of pediatrics, molecular virology, immunology and medical genetics at the Ohio State University, is using Southern blot analyses and TaqMan-based real-time PCR assays to examine copy number variations for complement C4, a key immune effector protein of the classical complement activation pathway.

    “Microarrays tell us that there is a copy number variation,” says Dr. Yu, “but they don’t give us the actual or detailed description, especially when it is continuous variation. We know that there are many common CNVs, and microarrays give us an idea of where they are, but a lot of work needs to be done to figure out what genes are involved, how they change, and which genes are functional and which are non-functional.”

    Different human populations harbor different numbers of C4 gene copies on chromosome 6. A quantitative correlation exists between the gene copy number and plasma C4 protein expression levels, with lower protein levels in individuals harboring fewer gene copies and more abundant protein levels in those with more copies.

    Additionally, qualitative diversity provides an additional level of complexity, such that, when multiple copies of C4 genes are present, there is a chance that they will undergo mutations or polymorphisms, and despite a 99.9% identity, subtle variations caused by sometimes as few as two to three amino acid changes in various places can provide new functions for the protein.

    “The gene products also tend to vary subtly; they will be picked up by the same polyclonal antibodies, but when you look at the sequences of the genes, you will see differences,” says Dr. Yu.

    For the C4 protein, two classes have been described, the acidic C4A and the basic C4B, with only four differences at the amino acid level defining each class, but functionally they generate different proteins. The Yu group recently demonstrated that C4A gene copy number variations are associated with susceptibility to systemic lupus erythematosus among European Americans.

    The situation gets even more complex. In a phenomenon known as segmental variation, often genes do not vary alone in their copy numbers, but neighboring genes are affected as well. Duplications of the C4 genes are always associated with duplications of the RP (or STK19) gene at the 5´ end, encoding a serine/threonine nuclear protein kinase, and of the CYP21 and TNX genes, at the 3´ end, encoding cytochrome P450 21-hydroxylase and the extracellular matrix protein tenascin, respectively.

    Whenever such segmental duplications occur, some of the duplicated genes are always functional, but other genes acquire mutations, turn into pseudogenes, and may be recombined into the progeny during the meiotic crossover.

    “It is one way to generate diversity,” explains Dr. Yu, “but it is also a way to get into trouble, because functional genes acquire mutations.” One of the genes in the immediate vicinity of C4, CYP21, encodes an essential enzyme involved in the biosynthesis of cortisone and mineralocorticoids, and its deficiency results in excessive male sexual hormone and congenital adrenal hyperplasia.


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