FMR1 Transcription Research Articles

Understanding decreased fertility in women carriers of the FMR1 premutation: a possible mechanism for Fragile X-Associated Primary Ovarian Insufficiency (FXPOI).

Fragile X syndrome (FXS) and its associated disorders are caused by the expansion of the CGG repeat in the 5′ untranslated region of the fragile X mental retardation 1 gene (FMR1). The full mutation, defined as >200 cytosine-guanine-guanine (CGG) triplet repeats, causes FXS. Individuals with 55–199 CGG repeats, classified as premutation carriers, are affected by two distinct disorders depending on their premutation status. Disorders associated with premutation carriers include: Fragile X-associated Tremor Ataxia Syndrome (FXTAS) and Fragile X-associated Primary Ovarian Insufficiency (FXPOI). The molecular similarities of FXTAS and FXPOI (e.g. overabundance of FMR1 transcript and intranuclear inclusions) suggest that similar molecular mechanisms underlie both FXTAS and FXPOI. The current hypothesis describes the underlying mechanism for FXTAS as an mRNA gain-of-function mutation, however the underlying mechanism for FXPOI remains unresolved. New data suggests that repeat associated non-AUG (RAN) translation could underlie FXPOI.

The multiple molecular facets of fragile X-associated tremor/ataxia syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset inherited neurodegenerative disorder characterized by intentional tremor, gait ataxia, autonomic dysfunction, and cognitive decline. FXTAS is caused by the presence of a long CGG repeat tract in the 5′ UTR of the FMR1 gene. In contrast to Fragile X syndrome, in which the FMR1 gene harbors over 200 CGG repeats but is transcriptionally silent, the clinical features of FXTAS arise from a toxic gain of function of the elevated levels of FMR1 transcript containing the long CGG tract. However, how this RNA leads to neuronal cell dysfunction is unknown. Here, we discuss the latest advances in the current understanding of the possible molecular basis of FXTAS.

Polycomb group complexes are recruited to reactivated FMR1 alleles in Fragile X syndrome in response to FMR1 transcription.

The FMR1 gene is subject to repeat mediated-gene silencing when the CGG-repeat tract in the 5' UTR exceeds 200 repeat units. This results in Fragile X syndrome, the most common heritable cause of intellectual disability and a major cause of autism spectrum disorders. The mechanism of gene silencing is not fully understood, and efforts to reverse this gene silencing have had limited success. Here, we show that the level of trimethylation of histone H3 on lysine 27, a hallmark of the activity of EZH2, a component of repressive Polycomb Group (PcG) complexes like PRC2, is increased on reactivation of the silenced allele by either the DNA demethylating agent 5-azadeoxycytidine or the SIRT1 inhibitor splitomicin. The level of H3K27me3 increases and decreases in parallel with the FMR1 mRNA level. Furthermore, reducing the levels of the FMR1 mRNA reduces the accumulation of H3K27me3. This suggests a model for FMR1 gene silencing in which the FMR1 mRNA generated from the reactivated allele acts in cis to repress its own transcription via the recruitment of PcG complexes to the FMR1 locus.

Alterations in the Sp1 binding and Fmr-1 gene expression in the cortex of the brain during maturation and aging of mouse.

Fragile X mental retardation protein (FMRP) has been implicated in learning, memory and cognition, therefore, information on alterations in FMRP expression during maturation and aging may provide a clue towards understanding mechanisms of age-dependent cognitive changes in the brain. In the present paper, we have studied Fmr-1 gene expression and its correlation with interaction of a tans-acting factor Sp1with Fmr-1 promoter in the cerebral cortex of female mice at post natal period during maturation and aging. Our data reveal that level of Fmr-1 transcript in the cerebral cortex is significantly up regulated at day 7 after birth compared to day 0 (the day of birth) and is gradually down regulated from day 15 onward to old age. The pattern of Fmr-1 transcript levels corresponds with the level of FMRP, however, its level is significantly up regulated in old age compared to adult mice. Our EMSA data revealed the formation of a single complex as a result of binding of Sp1with Fmr-1 promoter sequence. Its intensity gradually decreased from the day 0 (day of birth) till day 15, remained unaltered in young, significantly decreased in adult and significantly increased in old age. Our data suggests that age-dependent alteration in the Fmr-1 gene expression is associated with Sp1 interaction with Fmr-1 promoter which in turn might be related with cognitive development during brain maturation and aging.

Impaired sensorimotor gating in Fmr1 knock out and Fragile X premutation model mice

Fragile X syndrome (FXS) is a common inherited cause of intellectual disability that results from a CGG repeat expansion in the FMR1 gene. Large repeat expansions trigger both transcriptional and translational suppression of Fragile X protein (FMRP) production. Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is an allelic neurodegenerative disease caused by smaller "pre-mutation" CGG repeat expansions that enhance FMR1 transcription but lead to translational inefficiency and reduced FMRP expression in animal models. Sensorimotor gating as measured by pre-pulse inhibition (PPI) is altered in both FXS patients and Fmr1 knock out (KO) mice. Similarly, FXTAS patients have demonstrated PPI deficits. Recent work suggests there may be overlapping synaptic defects between Fmr1 KO and CGG knock-in premutation mouse models (CGG KI). We therefore sought to interrogate PPI in CGG KI mice. Using a quiet PPI protocol more akin to human testing conditions, we find that Fmr1 KO animals have significantly impaired PPI. Using this same protocol, we find CGG KI mice demonstrate an age-dependent impairment in PPI compared to wild type (WT) controls. This study describes a novel phenotype in CGG KI mice that can be used in future therapeutic development targeting premutation associated symptoms.

Epigenetics, fragile X syndrome and transcriptional therapy

Epigenetics refers to the study of heritable changes in gene expression that occur without a change in DNA sequence. Epigenetic mechanisms therefore include all transcriptional controls that determine how genes are expressed during development and differentiation, but also in individual cells responding to environmental stimuli. The purpose of this review is to examine the basic principles of epigenetic mechanisms and their contribution to human disorders with a particular focus on fragile X syndrome (FXS), the most common monogenic form of developmental cognitive impairment. FXS represents a prototype of the so-called repeat expansion disorders due to "dynamic" mutations, namely the expansion (known as "full mutation") of a CGG repeat in the 5'UTR of the FMR1 gene. This genetic anomaly is accompanied by epigenetic modifications (mainly DNA methylation and histone deacetylation), resulting in the inactivation of the FMR1 gene. The presence of an intact FMR1 coding sequence allowed pharmacological reactivation of gene transcription, particularly through the use of the DNA demethylating agent 5'-aza-2'-deoxycytydine and/or inhibitors of histone deacetylases. These treatments suggested that DNA methylation is dominant over histone acetylation in silencing the FMR1 gene. The importance of DNA methylation in repressing FMR1 transcription is confirmed by the existence of rare unaffected males carrying unmethylated full mutations. Finally, we address the potential use of epigenetic approaches to targeted treatment of other genetic conditions.

Methotrexate treatment of FraX fibroblasts results in FMR1 transcription but not in detectable FMR1 protein levels

BackgroundFragile X syndrome is caused by the loss of FMRP expression due to methylation of the FMR1 promoter. Treatment of fragile X syndrome patients’ lymphoblastoid cells with 5-azadeoxycytidine results in demethylation of the promoter and reactivation of the gene. The aim of the study was to analyze if methotrexate, an agent which also reduces DNA methylation but with less toxicity than 5-azadeoxycytidine, has therapeutic potential in fragile X syndrome.MethodsFibroblasts of fragile X syndrome patients were treated with methotrexate in concentrations ranging from 1 to 4 μg/ml for up to 14 days. FMR1 and FMRP expression were analyzed by quantitative PCR and western blotting.ResultsFMR1 mRNA was detected and levels correlated positively with methotrexate concentrations and time of treatment, but western blotting did not show detectable FMRP levels.ConclusionsWe show that it is possible to reactivate FMR1 transcription in fibroblasts of fragile X syndrome patients by treatment with methotrexate. However, we were not able to show FMRP expression, possibly due to the reduced translation efficacy caused by the triplet repeat extension. Unless FMR1 reactivation is more effective in vivo our results indicate that methotrexate has no role in the treatment of fragile X syndrome.

Role of CTCF protein in regulating FMR1 locus transcription.

Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, is caused by epigenetic silencing of the FMR1 gene, through expansion and methylation of a CGG triplet repeat (methylated full mutation). An antisense transcript (FMR1-AS1), starting from both promoter and intron 2 of the FMR1 gene, was demonstrated in transcriptionally active alleles, but not in silent FXS alleles. Moreover, a DNA methylation boundary, which is lost in FXS, was recently identified upstream of the FMR1 gene. Several nuclear proteins bind to this region, like the insulator protein CTCF. Here we demonstrate for the first time that rare unmethylated full mutation (UFM) alleles present the same boundary described in wild type (WT) alleles and that CTCF binds to this region, as well as to the FMR1 gene promoter, exon 1 and intron 2 binding sites. Contrariwise, DNA methylation prevents CTCF binding to FXS alleles. Drug-induced CpGs demethylation does not restore this binding. CTCF knock-down experiments clearly established that CTCF does not act as insulator at the active FMR1 locus, despite the presence of a CGG expansion. CTCF depletion induces heterochromatinic histone configuration of the FMR1 locus and results in reduction of FMR1 transcription, which however is not accompanied by spreading of DNA methylation towards the FMR1 promoter. CTCF depletion is also associated with FMR1-AS1 mRNA reduction. Antisense RNA, like sense transcript, is upregulated in UFM and absent in FXS cells and its splicing is correlated to that of the FMR1-mRNA. We conclude that CTCF has a complex role in regulating FMR1 expression, probably through the organization of chromatin loops between sense/antisense transcriptional regulatory regions, as suggested by bioinformatics analysis.

Epigenetic mechanisms of gene expression regulation in neurological diseases.

Neurological diseases are a heterogenous group of disorders that are related to alterations in nervous system function. The genetic background of neurological diseases is heterogenous and may include chromosomal aberrations, specific gene mutations and epigenetic defects. This review is aimed at presenting of selected diseases that are associated with different epigenetic alterations. The imprinting defects on chromosome 15 are the cause of Prader-Willi and Angelman syndromes that both are characterized by intellectual disability, developmental delay and specific behavioral phenotype. Besides the imprinting defect, these diseases can also be caused by deletion of chromosome 15 or uniparental disomy. Aberrant epigenetic regulation is also specific for Fragile X syndrome that is caused by expansion of CGG repeats in the FMR1 gene that leads to global methylation of the promoter region and repression of FMR1 transcription. A number of neurological diseases, mainly associated with intellectual impairment, may be caused by mutations in genes encoding proteins involved in epigenetic regulation. The number of such diseases is rapidly growing thanks to the implementation of genomic sequencing for the identification of their molecular causes. One of the best known diseases linked to defects in epigenetic modifiers is Rett syndrome caused by a mutation in the MECP2 gene or its variant - Rett-like syndrome caused by a mutation in CDKL5 or FOXG1 genes. As the epigenetic signature is potentially reversible, much attention is focused on possible therapies with drugs that influence DNA or histone modifications. This is especially important in the case of neurological disorders in which epigenetic changes are observed as the effect of the disease.

Behavioral and Synaptic Circuit Features in a Zebrafish Model of Fragile X Syndrome

Fragile X syndrome (FXS) is the most frequent inherited form of human mental retardation. It is characterized by cognitive impairment and physical and behavioral problems and is caused by the silencing of fmr1 transcription and the absence of the fmr1 protein (FMRP). Recently, animal models of FXS have greatly facilitated the investigation of the molecular and cellular mechanisms of this loss-of-function disorder. The present study was aimed to further characterize the role of FMRP in behavior and synaptic function by using fmr1 knockout zebrafish. In adult zebrafish, we found that fmr1 knockout produces the anxiolytic-like responses of increased exploratory behavior in light/dark and open-field tests and avoidance learning impairment. Furthermore, electrophysiological recordings from telencephalic slice preparations of knockout fish displayed markedly reduced long-term potentiation and enhanced long-term depression compared to wild-type fish; however, basal glutamatergic transmission and presynaptic function at the lateral (Dl) and medial (Dm) division of the dorsal telencephalon synapse remained normal. Taken together, our study not only evaluates the mechanism of FRMP but also suggests that zebrafish have valuable potential as a complementary vertebrate model in studying the molecular pathogenesis of human fragile X syndrome.

Fmr1 Transcript Isoforms: Association with Polyribosomes; Regional and Developmental Expression in Mouse Brain

The primary transcript of the mammalian Fragile X Mental Retardation-1 gene (Fmr1), like many transcripts in the central nervous system, is alternatively spliced to yield mRNAs encoding multiple proteins, which can possess quite different biochemical properties. Despite the fact that the relative levels of the 12 Fmr1 transcript isoforms examined here vary by as much as two orders of magnitude amongst themselves in both adult and embryonic mouse brain, all are associated with polyribosomes, consistent with translation into the corresponding isoforms of the protein product, FMRP (Fragile X Mental Retardation Protein). Employing the RiboTag methodology developed in our laboratory, the relative proportions of the 7 most abundant transcript isoforms were measured specifically in neurons and found to be similar to those identified in whole brain. Measurements of isoform profiles across 11 regions of adult brain yielded similar distributions, with the exceptions of the hippocampus and the olfactory bulb. These two regions differ from most of the brain in relative amounts of transcripts encoding an alternate form of one of the KH RNA binding domains. A possible relationship between patterns of expression in the hippocampus and olfactory bulb and the presence of neuroblasts in these two regions is suggested by the isoform patterns in early embryonic brain and in cultured neural progenitor cells. These results demonstrate that the relative levels of the Fmr1 isoforms are modulated according to developmental stage, highlighting the complex ramifications of losing all the protein isoforms in individuals with Fragile X Syndrome. It should also be noted that, of the eight most prominent FMRP isoforms (1–3, 6–9 and 12) in mouse, only two have the major site of phosphorylation at Ser-499, which is thought to be involved in some of the regulatory interactions of this protein.

Impaired activity-dependent FMRP translation and enhanced mGluR-dependent LTD in Fragile X premutation mice

Fragile X premutation-associated disorders, including Fragile X-associated Tremor Ataxia Syndrome, result from unmethylated CGG repeat expansions in the 5' untranslated region (UTR) of the FMR1 gene. Premutation-sized repeats increase FMR1 transcription but impair rapid translation of the Fragile X mental retardation protein (FMRP), which is absent in Fragile X Syndrome (FXS). Normally, FMRP binds to RNA and regulates metabotropic glutamate receptor (mGluR)-mediated synaptic translation, allowing for dendritic synthesis of several proteins. FMRP itself is also synthesized at synapses in response to mGluR activation. However, the role of activity-dependent translation of FMRP in synaptic plasticity and Fragile X-premutation-associated disorders is unknown. To investigate this question, we utilized a CGG knock-in mouse model of the Fragile X premutation with 120-150 CGG repeats in the mouse Fmr1 5' UTR. These mice exhibit increased Fmr1 mRNA production but impaired FMRP translational efficiency, leading to a modest reduction in basal FMRP expression. Cultured hippocampal neurons and synaptoneurosomes derived from CGG KI mice demonstrate impaired FMRP translation in response to the group I mGluR agonist 3,5-dihydroxyphenylglycine. Electrophysiological analysis reveals enhanced mGluR-mediated long-term depression (mGluR-LTD) at CA3-CA1 synapses in acute hippocampal slices prepared from CGG KI mice relative to wild-type littermates, similar to Fmr1 knockout mice. However, unlike mGluR-LTD in mice completely lacking FMRP, mGluR-LTD in CGG knock-in mice remains dependent on new protein synthesis. These studies demonstrate partially overlapping synaptic plasticity phenotypes in mouse models of FXS and Fragile X premutation disorders and support a role for activity-dependent synthesis of FMRP in enduring forms of synaptic plasticity.

Gender-Specific Effect of Mthfr Genotype and Neonatal Vigabatrin Interaction on Synaptic Proteins in Mouse Cortex

The enzyme methylenetetrahydrofolate reductase (MTHFR) is a part of the homocysteine and folate metabolic pathways, affecting the methylations of DNA, RNA, and proteins. Mthfr deficiency was reported as a risk factor for neurodevelopmental disorders such as autism spectrum disorder and schizophrenia. Neonatal disruption of the GABAergic system is also associated with behavioral outcomes. The interaction between the epigenetic influence of Mthfr deficiency and neonatal exposure to the GABA potentiating drug vigabatrin (GVG) in mice has been shown to have gender-dependent effects on mice anxiety and to have memory impairment effects in a gender-independent manner. Here we show that Mthfr deficiency interacts with neonatal GABA potentiation to alter social behavior in female, but not male, mice. This impairment was associated with a gender-dependent enhancement of proteins implicated in excitatory synapse plasticity in the female cortex. Reelin and fragile X mental retardation 1 protein (FMRP) levels and membrane GluR1/GluR2 ratios were elevated in wild-type mice treated neonatally with GVG and in Mthfr+/- mice treated with saline, but not in Mthfr+/- mice treated with GVG, compared with control groups (wild type treated with saline). A minor influence on the levels of these proteins was observed in male mice cortices, possibly due to high basal protein levels. Interaction between gender, genotype, and treatment was also observed in the GABA pathway. In female mice, GABA Aα2/gephyrin ratios were suppressed in all test groups; in male mice, a genotype-specific enhancement of GABA Aα2/gephyrin was observed. The lack of an effect on either reln or Fmr1 transcription suggests post-transcriptional regulation of these genes. Taken together, these findings suggest that Mthfr deficiency may interact with neonatal GABA potentiation in a gender-dependent manner to interrupt synaptic function. This may illustrate a possible mechanism for the epigenetic involvement of Mthfr deficiency in neurodevelopmental disorders.

NMDA receptor hypofunction in the dentate gyrus and impaired context discrimination in adult Fmr1 knockout mice

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability in humans. This X-linked disorder is caused by the transcriptional repression of a single gene, Fmr1. The loss of Fmr1 transcription prevents the production of Fragile X mental retardation protein (FMRP) which in turn disrupts the expression of a variety of key synaptic proteins that appear to be important for intellectual ability. A clear link between synaptic dysfunction and behavioral impairment has been elusive, despite the fact that several animal models of FXS have been generated. Here we report that Fmr1 knockout mice exhibit impaired bidirectional synaptic plasticity in the dentate gyrus (DG) of the hippocampus. These deficits are associated with a novel decrease in functional NMDARs (N-methyl-D-aspartate receptors). In addition, mice lacking the Fmr1 gene show impaired performance in a context discrimination task that normally requires functional NMDARs in the DG. These data indicate that Fmr1 deletion results in significant NMDAR-dependent electrophysiological and behavioral impairments specific to the DG.

Identification of novel FMR1 variants by massively parallel sequencing in developmentally delayed males.

Fragile X syndrome (FXS), the most common inherited form of developmental delay, is typically caused by CGG-repeat expansion in FMR1. However, little attention has been paid to sequence variants in FMR1. Through the use of pooled-template massively parallel sequencing, we identified 130 novel FMR1 sequence variants in a population of 963 developmentally delayed males without CGG-repeat expansion mutations. Among these, we identified a novel missense change, p.R138Q, which alters a conserved residue in the nuclear localization signal of FMRP. We have also identified three promoter mutations in this population, all of which significantly reduce in vitro levels of FMR1 transcription. Additionally, we identified 10 noncoding variants of possible functional significance in the introns and 3'-untranslated region of FMR1, including two predicted splice site mutations. These findings greatly expand the catalog of known FMR1 sequence variants and suggest that FMR1 sequence variants may represent an important cause of developmental delay.

Preparation and validation of the first WHO international genetic reference panel for Fragile X syndrome

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by expansion of a trinucleotide (CGG)n repeat sequence in the 5' untranslated region of the FMR1 gene, resulting in promoter hypermethylation and suppression of FMR1 transcription. Additionally, pre-mutation alleles in carrier males and females may result in Fragile X tremor ataxia syndrome and primary ovarian insufficiency, respectively. Fragile X is one of the most commonly requested molecular genetic tests worldwide. Quality assessment schemes have identified a wide disparity in allele sizing between laboratories. It is therefore important that clinical laboratories have access to characterized reference materials (RMs) to aid accurate allele sizing and diagnosis. With this in mind, a panel of genotyping RMs for Fragile X syndrome has been developed, which should be stable over many years and available to all diagnostic laboratories. Immortalized cell lines were produced by Epstein-Barr virus transformation of lymphocytes from consenting patients. Genomic DNA was extracted in bulk and RM aliquots were freeze-dried in glass ampoules. Twenty-one laboratories from seventeen countries participated in a collaborative study to assess their suitability. Participants evaluated the samples (blinded, in triplicate) in their routine methods alongside in-house and commercial controls. The panel of five genomic DNA samples was endorsed by the European Society of Human Genetics and approved as an International Standard by the Expert Committee on Biological Standardization at the World Health Organization.

Treatment with valproic acid ameliorates ADHD symptoms in fragile X syndrome boys

Fragile X syndrome (FXS) is the leading cause of inherited mental retardation, due to expansion and methylation of the CGG sequence at the 5' UTR of the FMR1 gene. Around 90% of affected boys present with attention deficit hyperactivity disorder (ADHD), while this percentage is lower in FXS girls (35-47%). Treatment of these behavioral symptoms is critical for many families. In an attempt at identifying drugs capable of restoring the activity of the FMR1 gene, we investigated the use of valproic acid (VPA), a well-known antiepileptic drug, also used as a mood stabilizer and in migraine therapy. It is described as an inhibitor of histone deacetylase (HDAC) and, possibly, as a DNA demethylating agent. In an in vitro study we observed that treatment of lymphoblastoid cells from FXS patients with VPA caused a modest reactivation of FMR1 transcription and increased levels of histone acetylation, confirming the histone hyperacetylating effect, but not its putative DNA demethylating activity. On the basis of these findings, we decided to evaluate the in vivo efficacy of VPA on ADHD symptoms in FXS patients. We observed an improvement in the adaptive behavior, defined as the performance of daily activities required for personal and social competence, due to a significant reduction in hyperactivity after VPA treatment. This treatment could be considered as an alternative to that with stimulants, whose efficacy in patients with FXS needs to be confirmed by further studies.

Comparative analysis of DNA methylation in transgenic mice with unstable CGG repeats from FMR1 gene

Methylation of CpG sequences in and around CGG triplet repeats in FMR1 gene has strong correlation with manifestation of the fragile X syndrome in human patients. In contrast, we have observed a lack of correlation between repeat instability and DNA methylation in three different transgenic mouse models harboring unstable CGG repeats. Further we have demonstrated that the endogenous copy of mouse Fmr1 gene remains unmethylated both in males and females. These results imply that methylation and repeat instability are independent events and raise the possibility that methylation could also result in repression of FMR1 transcription in the absence of repeat expansion.

Examination of FMR1 transcript and protein levels among 74 premutation carriers

Fragile X-associated disorders are caused by a CGG trinucleotide repeat expansion in the 5'-untranslated region of the FMR1 gene. Expansion of the CGG trinucleotide repeats to >200 copies (that is, a full mutation) induces methylation of the FMR1 gene, with transcriptional silencing being the eventual outcome. Previous data have shown that FMR1 premutation carriers (individuals with 55-199 repeats) have increased FMR1 mRNA levels with decreased protein (fragile X mental retardation protein (FMRP)) levels. However, the point at which this translational inefficiency occurs, given the increased transcription mechanism, has not yet been explored and remains to be elucidated. We examined the repeat length group, FMR1 transcript and FMRP levels in 74 males with a wide range of repeat lengths using analysis of covariance to better characterize this association. Results showed that the mean FMRP level among carriers with 80-89 repeats was significantly higher than the mean levels among lower (54-79) and higher (90-120) premutation carriers, in spite of the increasing transcript level with repeat length. Taken together, these results suggest that the 80-89-repeat group may lead to different properties that increase the efficiency of translation compared with other premutation repeat size groups.

Fragile X prenatal analyses show full mutation females at high risk for mosaic Turner syndrome: Fragile X leads to chromosome loss

The fragile X mutation is an expansion of a CGG triplet repeat in the 5' untranslated region of the FMR1 gene. Expansion to >200 repeats (the "full mutation") silences FMR1 transcription and leads to the fragile X mental retardation syndrome in males and in some females. It also affects the structure of the mitotic chromosome as evidenced by a folate sensitive fragile site. Isolated cases of 45,X/46,XX (mosaic Turner syndrome) in full mutation females have been reported but an increased prevalence was not apparent from these reports. PCR and Southern analysis of the CGG repeat in 423 prenatal female samples identified 106 full mutation cases. Surprisingly five of these had 45,X/4,6XX mosaicism while none of the other 317 female fetuses did. In two of the five cases >or=50% of the cells were reported to be 45,X and in the other three, <or=14%. We investigated the association of the full mutation and mosaic Turner syndrome using polymorphisms and single cell cloning to identify the X chromosome that was lost. Our analysis indicates that at least four of the five full mutation mosaicism cases were due to loss of the maternal, full mutation chromosome. These data indicate that 45,X/46,XX mosaicism is much more common than expected in fragile X full mutation females. Moreover, they suggest that the presence of the fragile X full mutation on a chromosome may predispose it to loss during mitosis, possibly due to the altered structure of the metaphase fragile X chromosome.