FRAGILE X SYNDROME MOLECULAR DIAGNOSTIC TESTING
This molecular test detects the DNA expansion observed in approximately 99% of Fragile X Syndrome carriers or affected individuals. Normal individuals have between about 6 and 50 FMR1 CGG repeats. The FMR1 CGG repeats tend to undergo expansion when repeat numbers exceed about 50. Premutation carrier females and normal transmitting males have between about 50 and 200 repeats. Affected males typically have many more than 200 repeats. Females with an X chromosome having greater than 200 FMR1 CGG repeats may or may not be affected depending on X chromosome inactivation patterns. FMR1 CGG repeats of about 50 to 200 show a dynamic instability directly related to their length, to the sex of the transmitting individual, and to subtle DNA sequence variation within the repeat itself. Premutation alleles tend to be unstable when transmitted by females and stable when transmitted by males (stability in males may be a consequence of selection against expanded FMR1 CGG alleles during spermatogenesis). Repeats greater than about 90-100 repeats have nearly a 100% risk of expansion into the affected range when transmitted by a female. The FMR1 CGG repeat demonstrates significant somatic instability when repeat sizes enter the premutation range. Premutation and larger sized CGG repeats determined from peripheral blood DNA may not reflect FMR1 CGG repeat sizes in other tissues.
Press here for a comprehensive clinical genetics review of Fragile X Syndrome from "Genline".
For a recent collection of papers on clinical/molecular/behavioral investigations of Fragile X Syndrome see Amer. J. Med. Genet. (1996): Volume 64 Number 2.
Testing for the FMR1 CGG repeat expansion is most readily performed by Southern blot analysis of EcoR1 and Eag 1 (or a selection of other methylation sensitive restriction enzymes) double-digested DNA. The probe pStB12.3 detects variable fragments depending on the sex and FMR1 status of the individual.
Examples of diagnostic Southern blot patterns are illustrated below:
The positions of normal alleles are indicated by the yellow arrows while the positions of diagnostic alleles are indicated by the red arrows.
- Lane 1: Normal male 2.8 Kbp unmethylated EcoR1/Eag1 fragment.
- Lane 2: Premutation male Slightly increased size 2.8 Kbp EcoR1/Eag1 fragment due to slight increase in CGG repeat number.
- Lanes 3 and 4: Full mutation affected males Large increases in sizes of 2. 8 EcoR1/Eag1 fragments due to large increase in CGG repeat number and complete methylation of this CGG repeat.
- Lane 5: Normal female 5.2 Kbp methylated (inactive X chromosome) and 2.8 Kbp unmethylated (active X chromosome) EcoR1/Eag1 fragments.
- Lane 6: Premutation carrier female Slightly increased size 2.8 Kbp and 5.2 Kbp EcoR1/Eag1 fragments due to slight increase in the FMR1 CGG repeat number carried by one X chromosome. Some methylated and some unmethylated.
- Lanes 7 and 8: Full mutation carrier females Large increases in sizes of 5.2 EcoR1/Eag1 fragment due to large increase in CGG repeat number and partial methylation of this CGG repeat. Normal 2.8 Kbp EcoR1/Eag1 fragment also present. About 50 -80% of these individuals will show some degree of mental impairment.
Premutation carriers of the smaller FMR1 CGG repeat expansions detected by the Southern blot analysis are further evaluated by sizing on a DNA sequencing gel. This method determines more precisely the size of the normal and expanded alleles and, especially in the case of female carriers, provides additional information about the subsequent risk of expansion of the premutation allele upon transmission. As can be seen in the figure below, the CGG repeat alleles of both normal transmitting males (lanes 3 and 5) and premutation carrier females (lanes 2, 4, and 6) are detected by this method. A normal female with alleles of 23 and 30 repeats is shown in lane 1. Comparing the right and left panels shows that normal alleles and male premutation alleles up to at least 100 CGG repeats are easily detected but that detection of the expanded CGG repeats of female premutation carriers (lanes 2, 4, and 6) require longer autoradiographic exposure due to the under-representation in the PCR reaction of these larger alleles in females. The females shown in lanes 4 and 6 are the daughters of the normal transmitting male shown in lane 5. The size of the premutation allele transmitted from the father to his daughters has remained stable. One of his daughters had previously had a son affected with Fragile X Syndrome due to full mutation expansion of this premutation allele.