Poster
27. Assessment of the Fragile X PCR assay in routine diagnostic practice
Z. Mušová1, S. Bendová1, K. Pavlíková1, P. Hedvičáková1, E. Hlavová2, A. Křepelová1
1
Ústav biologie a lékařské genetiky, FN Motol Praha;
2
Klinika sv. Klimenta Praha – GENNET
zuzana.musova@lfmotol.cuni.cz
Here we present our initial assessment of the Fragile X PCR kit (Abbott) in diagnostic
practice. A sample genotype was determined using an in-house PCR protocol (ABI 2720) and
Southern blot analysis, while the assay was utilised in subsequent prenatal and postnatal
diagnoses. In 14 male samples, 7 premutations and 7 full mutations, and in 18 female
samples, 10 premutations and 8 full mutations were detected. All expansions were
independently confirmed and detection of PCR products within the premutation range was
robust. However, 6 out of 15 full mutation samples were not visible on the agarose gel, and
two of them were not even conclusive on ABI 3100. In order to assess reliability of the TR/X
ratio we have analyzed the homozygous or heterozygous status in a set of 23 females with
known genotypes. A discrepancy was found in 6 out of 23 cases (26 %), which is in general
agreement with previous reports. Subsequently, these results were taken into account in 3
different prenatal diagnostic cases. In two instances the results were negative, while in one
case the expansion was exactly at the premutation / full mutation threshold (199 cgg+/-3cgg).
There is a necessity to examine its methylation status in order to distinguish the affected from
the healthy carrier. In summary, this assay is a promising product which allows rapid, albeit
incomplete FraX diagnostics. However, an in-house diagnostic validation is necessary for its
optimalisation, and manufacturer instructions should be strictly adhered to in order to assure
consistent results.
Podporováno z výzkumného záměru VZ MZO 00064203.