Increase of FMRP expression, raised levels of FMR1 mRNA, and clonal selection in proliferating cells with unmethylated fragile X repeat expansions: aclue to the sex bias in the transmission of full mutations?

Fragile X syndrome is a triplet repeat disorder caused by expansions of a C GG repeat in the fragile X mental retardation gene (FMR1) to more than 220 triplets (full mutation) that usually coincide with hypermethylation and tr anscriptional silencing. The disease phenotype results from deficiency or l oss of FMR1 protein (FMRP) and occurs in both sexes. The underlying full mu tations arise exclusively on transmission from a mother who carries a premu tation allele (60-200 CGGs). While the absolute requirement of female trans mission could result from different mechanisms, current evidence favours se lection or contraction processes acting at gametogenesis of pre- and full m utation males. To address these questions experimentally, we used a model s ystem of cultured fibroblasts from a male who presented heterogeneous unmet hylated expansions in the pre- and full mutation size range. On continual c ell proliferation to 30 doublings we reexamined the behaviour of the expand ed repeats on Southern blots and also determined the expression of the FMR1 gene by FMRP immunocytochemistry, western analysis, and RT-PCR. With incre asing population doublings, expansion patterns changed and showed accumulat ion of shorter alleles. The FMRP levels were below normal but increased con tinuously while the cells that were immunoreactive for FMRP accumulated. Th e level of FMR1 mRNA was raised with even higher levels of mRNA measured at higher passages. Current results support the theory of a selection advanta ge of FMRP positive over FMRP deficient cells. During extensive proliferati on of spermatogonia in fragile X males, this selection mechanism would even tually replace all full mutations by shorter alleles allowing more efficien t FMRP translation. At the proliferation of oogonia of carrier females, the same mechanism would, in theory, favour transmission of any expanded FMR1 allele on inactive X chromosomes.