Pathology Student

Today’s post, authored by a very smart guest cytogeneticist, nicely describes array-based comparative genomic hybridization, a very cool new DNA test that gives us a way to detect genetic abnormalities that are too small to be seen under the microscope. A student wanted to know more about why array CGH can only detect unbalanced rearrangements (like deletions) but not balanced rearrangements (like inversions).

Q. With regard to array CGH, I do not understand why balanced rearrangements could not be detected. Why can’t they make a probe for an inversion of a few genes or an insertion of a gene? I guess I do not see how these would be any different from making a probe for a deletion or duplication. I am sure I am missing something though.

A. Array-based CGH is a DNA based test that, in a much-simplified nutshell, looks at the quantity of DNA in a patient vs the quantity of DNA in a specimen derived from a pool of normal controls. Thousands of different probes from loci spanning the genome are present on a chip. If there is LESS DNA in the patient than the control for a particular probe, the a-CGH will show a DELETION of material from the patient for that particular locus; if there is MORE DNA in the patient than the control for a particular probe, the a-CGH will show a GAIN of material from the patient for that locus. In a balanced translocation, there is NO gain or loss of material, so the probes will show that the patient and the control have equal amounts of DNA in those translocated regions.

To detect a translocation, then, one would need to do a G-banded chromosomal analysis (i.e, look at the chromosomes under the microscope, the “old-fashioned” way). In that way, the material exchanged between the chromosomes involved in the translocation could be identified because they would LOOK different than their normal homologs — but because the translocation is balanced, there is NO gain or loss of DNA in this exchange, so array-CGH would not detect any genetic imbalance. In cancer cases, in which the genetic abnormalities involved in certain translocations have been well characterized (e.g., the 9;22 translocation in chronic myeloid leukemia involves breakage and rejoining of the ABL gene on chromosome 9q34 and the BCR gene on chromosome 22q11.2), FISH probes can be developed because we know the gene sequences of ABL and BCR. In contrast, however, for a constitutional balanced translocation that is passed on through a family or develops de novo in a patient, we don’t know what those genes are — so we don’t know the base-pair sequences that would enable us to develop a FISH probe. For these cases, then, we are limited to characterizing the abnormality as best we can by means of a G-banded chromosomal analysis.

At the risk of complicating this picture, I will add one further little scenario. Not all translocations that LOOK balanced in a G-banded chromosomal analysis really ARE balanced at the level of the DNA sequences. That is, in the process of formation of the translocation, sometimes very small amounts of DNA can be gained or duplicated at the breakpoints of the translocation. These amounts of DNA are way too small to be detected under the microscope in a G-banded chromosomal analysis (remember, the limit of detection for our eyes at the microscope is about 3 megabases of DNA (i.e. 3 million base pairs)). Gains or losses of DNA at the breakpoint of these apparently balanced translocations that are SMALLER than about 3 MB would NOT be detected in a G-banded chromosomal analysis. For this reason, if a patient is diagnosed with a de novo (not inherited from the parents) translocation that LOOKS balanced under the microscope (via a G-banded chromosomal analysis), we would still recommend that a-CGH be performed — because a-CGH would be able to detect any such small imbalances at the breakpoints of the translocation.

Image credit: DNA spiral by Charles Jencks, Kew Gardens, UK (by mira66, http://www.flickr.com/photos/21804434@N02/3707633630/, under CC license).

We were talking about developmental pathology the other day in class – trisomies, sex chromosome numerical abnormalities, microdeletion syndromes etc. – and the term “uniparental disomy” came up. Someone asked, what is that, and how do you get it? Great question! Before we get to the answer, let’s take a look at some of the syndromes that are caused by this abnormality.

The most well-known syndromes caused by uniparental disomy are Prader-Willi and Angelman syndromes. These are both microdeletion syndromes, meaning that the patient has a little deletion within a chromosome that is so tiny that it is often not visible by regular G-banding techniques (you need to use FISH).  Both Prader-Willi and Angelman syndromes are caused by tiny deletions in chromosome 15. The interesting thing is that if the deletion is in dad’s chromosome 15, the baby develops Prader-Willi syndrome, but if the deletion is in mom’s chromosome 15, the baby develops Angelman syndrome. Patients with these syndromes have uniparental disomy, meaning that they have inherited both copies of the abnormal chromosome 15 from one parent (instead of one from mom and one from dad). We’ll discuss how this can happen in a minute.

The two syndromes are surprisingly different. Patients with Angelman syndrome have mental retardation, a jerky, puppet-like gait, inappropriate outbursts of laughter, and severe speech problems. Patients with Prader-Willi syndrome have poor muscle tone and poor feeding as babies, but later develop an obsession with food, resulting in extreme food-seeking behavior and obesity.

Back to the question. Uniparental disomy means that you inherit two copies of a particular chromosome from one of your parents, and no copy from the other parent. It can happen in three ways, all of which involve two consecutive mistakes in cell division.

1. Trisomic rescue. This happens when you get a trisomy (as happens when the chromosomes don’t split up the way they should during meiosis, and you end up with two copies of some chromosome from mom and one from dad – or vice versa), and then you lose one of those three chromosomes (the “odd one out”, the lone one from one of the parents). You’re left with two chromosomes from one parent, and none from the other.

2. Monosomic rescue. This happens when you have a monosomic zygote (only one copy of a particular chromosome – the other parent’s dropped out), and that chromosome duplicates itself.

3. Gamete complementation. This is when you have a gamete with two copies of a chromosome (should have only one), and it gets fertilized with a gamete that happens to have no copies of that chromosome.

Here’s a good reference with diagrams. The more I read about this stuff, the more amazed I am that things usually go right.

The image above is “Boy with a Puppet” or “A child with a drawing” by Giovanni Francesco Caroto. Dr. Harry Angelman, a pediatrician working in England, first reported three children with what is now known as Angelman syndrome in 1965. While vacationing in Italy, Angelman saw the painting.  The boy’s laughing face reminded him of his three patients, as did the puppet in the drawing, which reminded him of the fact that all three patients exhibited jerky movements. He subsequently described the children with this syndrome as “puppet children,” a title that was obviously not very pleasing. The name of the syndrome was later changed to Angelman syndrome.