A second hypermutable DNA sequence is located within the fragile X gene

The molecular basis for the Fragile X syndrome is a large expansion of an unstable triplet repeat (CGG)n at the 5{prime} untranslated end of the FMR-1 gene leading to a lack of gene expression. We have discovered that another DNA sequence located within the gene is hypermutable. The FRAXAC2 locus is a microsatellite marker located 10 kb downstream to the (CGG)n within FMR-1. PCR analysis of FRAXAC2 from 265 chromosomes showed 261 had lengths that differed by single bps from 146 to 153 bps and four had 155, 157, 158, and 159 bps. Sequence analysis showed three variable length subregions: a GT repeat followed by a constant C, a TA repeat and a poly(T), which could be represented as a (GT)x-C-(TA)y-(T)z complex, with x values of 13-17 and 19; y values of 6-8; and a z values 10-20 and 25, with a total of 40 different alleles and a heterozygosity of 90.1%. Inheritance studies of FRAXAC2 in 32 fragile X families showed 4 mutations among 2 fragile X families including 3 of 67 from female-carrier fragile X chromosomes and 1 of 54 from their normal chromosomes. Thus, 3.3% (4/121) of the fragile X derived meioses were unstable. No mutation wasmore » detected from 25 normal male spouses and 112 CEPH family meioses. The hypermutable FRAXAC2 appears to be the first identified microsatellite with three variable parts. The finding of two highly mutable sequences near each other and within the same gene suggests a related mutational mechanism exists with two targets in the FMR-1 gene.« less