Fragile X Syndrome is caused by the expansion of an unstable CGG-repeat tract in the 5-UTR of the FMR1 gene , which generally results in transcriptional silencing and consequent absence of the FMR1 protein.To date , the smallest premutation allele reported to expand to a full mutation allele in a single generation is 59 CGG repeats. Here , we report a single-generation expansion to a full mutation allele (male with ϳ538 CCG repeats) from a mother who is a carrier of a premutation allele of 56 CGG repeats. Furthermore, the maternal grandfather was a carrier of a gray (or intermediate)-zone allele (45 to 54 repeats) of 52 CGG repeats. Thus , in this family , a gray-zone allele expanded to the full mutation range in two generations. Interestingly , the two AGG interruptions present in the grandfather's allele were absent in the mother's premutation allele. These observations underscore the need to consider carriers of alleles of greater than 55 CGG repeats as being at risk for transmission of a full mutation allele in a single generation , and those with even smaller alleles in the gray zone as being at risk of having grandchildren with full mutation alleles. Fragile X Syndrome (OMIM #300624) is the most common form of inherited mental impairment and the leading known heritable form of autism.1 The disorder is caused by a trinucleotide (CGG) repeat expansion to greater than 200 repeats (full mutation) in the 5Ј-untranslated region of the fragile X mental retardation 1 (FMR1) gene, which generally results in transcriptional silencing and absence of the FMR1 protein.2,3,4 The fragile X syndrome phenotype includes a large spectrum of involvement including mental retardation, developmental and speech delay, physical abnormalities such as large or prominent ears, long and narrow jaw, connective tissue problems, and macro-orchidism. 5 The behavioral phenotype of fragile X syndrome is also characterized by autistic symptoms in approximately 25% to 33%, including social and communication deficits, stereotypic behavior, social anxiety, withdrawal, hyperarousal, unusual responses to sensory stimuli, gaze aversion, inattention, impulsivity, and hyperactivity. 1,6,7,8,9,10 Transmission of a full mutation occurs exclusively from mothers who are carriers of either a full mutation or a premutation (55 to 200 CGG repeats) FMR1 allele. For mothers who are carriers of premutation alleles, the risk of transmitting an allele in the full mutation range is a function of the repeat length. Nolin and colleagues 11 reviewed the propensity for premutation-to-full mutation expansion in more than 1500 transmissions from female carriers of premutation alleles. They showed that the smallest premutation allele leading to a full mutation offspring in a single generation was 59 CGG repeats, which was observed for two female carriers. For slightly smaller alleles (45 to 54 CGG repeats; "gray zone"), the extent of repeat instability is currently unknown. However, due to the observed repeat instability for alleles in this size range, 12,13,14 gray-zo...

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that affects carriers of premutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. The presence of elevated levels of expanded mRNA found in premutation carriers is believed to be the basis for the pathogenesis in FXTAS, but the exact mechanisms by which the mRNA causes toxicity are not known. In particular, it is not clear whether there is a threshold for a CGG-repeat number below which no cellular dysregulation occurs, or whether toxicity depends on mRNA concentration. We have developed a doxycycline-inducible episomal system that allows us to study separately the effects of CGG-repeat number and mRNA concentration (at fixed CGG-repeat length) in neuroblastoma-derived SK cells. Our findings show that there is a CGG-repeat size threshold for toxicity that lies between 62 and 95 CGG repeats. Interestingly, for repeat sizes of 95 CGG and above, there is a clear negative correlation between mRNA concentration and cell viability. Taken together, our results provide evidence for an RNA-toxicity model with primary dependence on CGG-repeat size and secondary dependence on mRNA concentration, thus formally ruling out any simple titration model that operates in the absence of either protein-binding cooperativity or some form of length-dependent RNA structural transition.

5′- and 3′-untranslated regions (UTRs) are important regulators of gene expression and play key roles in disease progression and susceptibility. The 5′-UTR of the fragile X mental retardation 1 (FMR1) gene contains a CGG repeat element that is expanded (>200 CGG repeats; full mutation) and methylated in fragile X syndrome (FXS), the most common form of inherited intellectual disability (ID) and known cause of autism. Significant phenotypic involvement has also emerged in some individuals with the premutation (55–200 CGG repeats), including fragile X-associated premature ovarian insufficiency (FXPOI) in females, and the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS), in older adult carriers. Here, we show that FMR1 mRNA in human and mouse brain is expressed as a combination of multiple isoforms that use alternative transcriptional start sites and different polyadenylation sites. Furthermore, we have identified a novel human transcription start site used in brain but not in lymphoblastoid cells, and have detected FMR1 isoforms generated through the use of both canonical and non-canonical polyadenylation signals. Importantly, in both human and mouse, a specific regulation of the UTRs is observed in brain of FMR1 premutation alleles, suggesting that the transcript variants may play a role in premutation-related pathologies.