July is Fragile X Syndrome Awareness Month! In addition, yesterday we celebrated the Fragile X Awareness Day, and for this reason, we would like to share with you a Q&A section that we have prepared about our Fragile X Syndrome Kits of the Adellgene product line.
Here are some of the questions we have compiled, with answers from our Field Application Specialist, María Martínez Monge.
- What is the role of the AGG intersections in the development of the Fragile X disease? Is it possible to detect these intersections with the Adellgene® Fragile X screening and Adellgene® Fragile X kits?
- The AGG triplets inserted within the CGG repeats region seem to confer stability to the DNA, decreasing the risk of expansion in the offspring. Thus, those women who do not have AGG intersections, present a higher risk of transmitting CGG expansions to their descendants.
- The AGG intersections are not detectable with the Adellgene® Fragile X screening but the Adellgene® Fragile X kit does allow to detect them.
- Can the Adellgene® Fragile X screening and Adellgene® Fragile X be used to analyse chorionic samples for a prenatal test?
- The Adellgene® Fragile X screening and Adellgene® Fragile X kits have initially been designed to be used with DNA samples, extracted from whole blood. Nevertheless, chorionic samples can also be tested with either of these two kits. In any case, it is worth bearing in mind that these types of samples usually render lower quality, compared to those of DNA samples extracted from whole blood. Given that these kits are based on a quantitative technique, results are highly dependent on the quantity and purity of DNA used. Therefore, whatever the type of sample tested, it is always recommended to measure these parameters before proceeding with the protocol.
- What is the difference between the Adellgene® Fragile X screening and Adellgene® Fragile X kits?
- The Adellgene® Fragile X screening kit allows for the quantification and visualization of up to 200 CGG triplet repeats. Thus, with this kit, samples corresponding to healthy or premutated males and heterozygous healthy or premutated females can be resolved. With this kit, samples corresponding to homozygous healthy females or full-mutated males cannot be resolved and need to be confirmed with the Adellgene® Fragile X kit or with an alternative method.
- With the Adellgene® Fragile X kit full mutations can be visualized, thus allowing the resolution of all types of samples.
- What techniques are the Adellgene Fragile X and Fragile X Screening kits based on?
- Both kits are based on an amplification of the fragments of interest by a PCR, followed by a fragment analysis by a capillary electrophoresis. In the case of the Adellgene® Fragile X Screening kit, the amplification is performed by means of a conventional PCR, in which a forward and a reverse primer are used. On the other hand, the Adellgene® Fragile X kit is based on a modification of the conventional PCR, known as a TP-PCR (Triplet Repeat Primed PCR). In this type of PCR, the reverse primer is complementary to the CGG repeat region, thus having multiple hybridization sites. This way, DNA fragments of different sizes are obtained (depending on the place where the TP-primer has hybridized on). These fragments will later be order by size in the capillary electrophoresis. This design allows to better cover the CGG repeat region, enabling the detection of full expansions.
- Do the Adellgene® Fragile X and Fragile X screening kits allow to know the exact number of repeats that a patient has?
- Both the Adellgene® Fragile X Screening and the Adellgene® Fragile X kits allow to quantify up to 200 CGG triplet repeats. Furthermore, the Adellgene® Fragile X kit allows to visualize expansions of over 200 CGG repeats, but it does not allow to quantify them. Nonetheless, when the expansions fall within the full mutated range (>200 CGG repeats), determining the exact number of repeats has no clinical relevance, since all patients that fall in this range will be phenotypically affected.
- In any case, as it happens with any quantitative technique, an error range is accepted in the determination of the number of triplet repeats. This way, for healthy alleles, an error of ±1 triplet repeats is accepted, whereas for premutated and full mutated alleles an error of ±3 repeats is accepted.