Polymerase chain reaction analysis of fragile X mutations

Abstract

The mutation that underlies the fragile X syndrome is presumed to be a large expansion in the number of CGG repeats within the gene FMR-1. The unusually GC-rich composition of the expanded region has impeded attempts to amplify it by the polymerase chain reaction (PCR). We have developed a PCR protocol that successfully amplifies the (CGG)n region in normal, carrier and affected individuals. The PCR analysis of several large fragile X families is presented. The PCR results agree with those obtained by direct genomic Southern blot analyses. These favorable comparisons suggest that the PCR assay may be suitable for rapid testing for fragile X mutations and premutations and genetic screening of at-risk individuals.

Technical Standards and Guidelines for Fragile X: The First of a Series of Disease-Specific Supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics

“…[5][6][7][8] Other primers and methods can be used if equivalence is demonstrated. A particular region to be aware of in primer design is the deletional hotspot.…”

The fragile X syndrome: implications of molecular genetics for the clinical syndrome

“…Since the identification of fragile X mutations several genotyping protocols have been published [20,21,29,52,53]. Although fragile X mutations all affect the same target fragment, they are of many different types.…”

“…What physicians need to know is whether an individual carries a premutation or a full mutation (see genotype-phenotype correlations) because this greatly affects genetic counselling as well as follow up of the case. Some recent papers have proposed using a PCR test for genotypic analysis at the fragile-)< locus [52,53]. Such a method has already been used to measure the exact number of CGG repeats of fragile X mutations with great precision [27,52].…”

“…Furthermore, small target DNA fragments are much more easily amplified, so the performance of the PCR assay for detection of full mutations has not been fully assessed. In particular PCR results may be questioned in fully mutated females (who also have a normal X) and in 'mosaics' (male or female) who could show only a 'premutated' band if the larger mutations are not amplified in the same proportion [53]. Finally, the PCR test does not allow for evaluation of the methylation status of the FMR-I CpG island which is necessary to distinguish between large premutations ( > 400 bp) and small full mutations ( < 600 bp).…”

Genotype-Phenotype Correlates in Fragile X Syndrome

“…Molecular diagnosis of the syndrome can be done using polymerase chain reaction (PCR) to detect the expanded CGG repeats in the FMR1 gene [28,29]. Todorov et al [30] reported fragile X mosaicism in a male by using PCR and methylation-specific multiplex ligationdependent probe amplification (MS-MLPA).…”