F. Tassone

Elevated Levels of FMR1 mRNA in Carrier Males: A New Mechanism of Involvement in the Fragile-X Syndrome

Fragile-X syndrome is a trinucleotide-repeat-expansion disorder in which the clinical phenotype is believed to result from transcriptional silencing of the fragile-X mental retardation 1 (FMR1) gene as the number of CGG repeats exceeds approximately 200. For premutation alleles ( approximately 55-200 repeats), no abnormalities in FMR1-gene expression have been described, despite growing evidence of clinical involvement in premutation carriers. To address this (apparent) paradox, we have determined, for 16 carrier males (55-192 repeats), the relative levels of leukocyte FMR1 mRNA, by use of automated fluorescence-detection reverse transcriptase-PCR, and the percent of lymphocytes that are immunoreactive for FMR1 protein (FMRP). For some alleles with>100 repeats, there was a reduction in the number of FMRP-positive cells. Unexpectedly, FMR1 mRNA levels were elevated at least fivefold within this same range. No significant increase in FMR1 mRNA stability was observed in a lymphoblastoid cell line (160 repeats) derived from one of the carrier males, suggesting that the increased message levels are due to an increased rate of transcription. Current results support a mechanism of involvement in premutation carriers, in which reduced translational efficiency is at least partially compensated through increased transcriptional activity. Thus, diminished translational efficiency may be important throughout much of the premutation range, with a mechanistic switch occurring in the full-mutation range as the FMR1 gene is silenced.

Transcription of the FMR1 gene in individuals with fragile X syndrome

Fragile X syndrome generally arises as a consequence of a large expansion of a CGG trinucleotide repeat element that is located in the GC-rich promoter region of the fragile X mental retardation gene (FMR1). In the conventional model for fragile X, clinical involvement arises as a consequence of silencing of the FMR1 gene, with the attendant loss of FMR1 protein (FMRP). However, it has recently been demonstrated that most males with large premutation alleles (100-200 repeats), or with unmethylated full mutation alleles, have FMR1 mRNA levels that are higher than normal, despite reduced levels of FMRP. In the current work, we extend and confirm these observations using quantitative (fluorescent) reverse transcription polymerase chain reaction on larger sample populations, establishing that even for smaller premutation alleles (55-100 repeats) the mRNA levels are significantly elevated (mean 2.1-fold elevation; P = 3.9 x 10(-3)), relative to normal controls. Thus, an abnormal molecular phenotype is established close to the upper end of the normal range. We also demonstrate that the levels of FMR1 mRNA are elevated in females with premutation alleles; however, the mRNA levels are more varied than in the males, and are attenuated in a manner that is consistent with the fraction of normal alleles that are active in any given individual. Finally, we demonstrate that in lymphoblastoid cells derived from a patient with a severe form of fragile X caused by a point mutation in the second KH domain of the gene, but with a normal CGG element (25 repeats), the FMR1 mRNA level is normal. Thus, although models in which FMRP level (or level of function) modulates transcriptional activity remain viable, other explanations for the elevated message levels, including direct (cis) effects of the CGG element on transcription, must also be considered.

Aging in Individuals With the FMR1 Mutation

Individuals with fragile X mental retardation 1 (FMR1) premutation (55 to 200 CGG repeats) are typically unaffected by fragile X syndrome. However, a subgroup of older males with the premutation have developed a neurological syndrome, which usually begins between 50 and 70 years and is associated with a progressive intention tremor and/or ataxia manifested by balance problems, frequent falling, and Parkinsonian symptoms, such as masked facies, intermittent resting tremor, and mild rigidity. This finding has been termed the fragile X-associated tremor/ataxia syndrome (FXTAS) and has brought focus to the aging process in individuals with the FMR1 mutation. The premutation is associated with elevated messenger RNA levels leading to the formation of intranuclear inclusions in neurons and astrocytes associated with FXTAS. This review is a summary of our experience with FXTAS in male carriers of the premutation.

Intranuclear inclusions in neural cells with premutation alleles in fragile X associated tremor/ataxia syndrome

Fragile X syndrome is generally considered to be a non-progressive neurodevelopmental disorder in which carriers of premutation alleles (~55 to 200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene are largely unaffected. However, we have recently identified a new syndrome among male carriers, characterised by tremor and/or ataxia, cognitive deficits, parkinsonism, and autonomic dysfunction.1,5,6,8,10 Neuroradiological findings include moderate to severe cortical atrophy (cerebral and cerebellar cortical volume loss) and characteristic hyperintensities on T2-weighted MR imaging of the deep cerebellar white matter and middle cerebellar peduncles.2,8 The disorder, termed fragile X associated tremor/ataxia syndrome,8 has been observed thus far almost exclusively in older adult male premutation carriers of more than 50 years of age. Eosinophilic intranuclear inclusions are broadly distributed in both neurones and astrocytes throughout the cerebrum and brain stem of all persons with fragile X associated tremor/ataxia syndrome (FXTAS) examined to date (8/8),5,7 with the greatest numbers of neuronal inclusions found in the hippocampus. No inclusions were detected in the Purkinje cells, although axonal degeneration and Purkinje cell loss were prominent findings in the cerebellum.5 Premutation carriers generally have elevated FMR1 mRNA levels, suggesting that FXTAS could result from a toxic gain of function of the FMR1 mRNA.5,6,8 To determine whether inclusion formation and clinical involvement are associated with allelic mosaicism, we defined the size of the CGG expansion and the relative expression levels of the FMR1 gene in various regions of the brain of a male premutation carrier who died with neurological symptoms consistent with FXTAS, and whose neural cells possessed numerous intranuclear inclusions. Analysis of multiple brain regions was undertaken to demonstrate that premutation alleles were directly associated with inclusion formation, and that full mutation alleles, even if present at …

FMR1 CGG repeat length predicts motor dysfunction in premutation carriers

Background: Fragile X–associated tremor/ataxia syndrome (FXTAS) is a recently described, underrecognized neurodegenerative disorder of aging fragile X mental retardation 1 (FMR1) premutation carriers, particularly men. Core motor features are action tremor, gait ataxia, and parkinsonism. Carriers have expanded CGG repeats (55 to 200); larger expansions cause fragile X syndrome, the most common heritable cause of mental retardation and autism. This study determines whether CGG repeat length correlates with severity and type of motor dysfunction in premutation carriers. Methods: Persons aged ≥50 years with a family history of fragile X syndrome underwent structured videotaping. Movement disorder neurologists, blinded to carrier status, scored the tapes using modified standardized rating scales. CGG repeat length analyses for women incorporated the activation ratio, which measures the percentage of normal active chromosome X alleles. Results: Male carriers (n = 54) had significantly worse total motor scores, especially in tremor and ataxia, than age-matched male noncarriers (n = 51). There was a trend toward a difference between women carriers (n = 82) and noncarriers (n = 39). In men, increasing CGG repeat correlated with greater impairment in all motor signs. In women, when activation ratio was considered, increasing CGG correlated with greater ataxia. Conclusions: CGG repeat size is significantly associated with overall motor impairment in premutation carriers. Whereas this association is most pronounced for men and covers overall motor impairment—tremor, ataxia, and parkinsonism—the association exists for ataxia among women carriers. This is the first report of a significant correlation between the premutation status and a motor feature of fragile X–associated tremor/ataxia syndrome in women. GLOSSARY: AR = activation ratio; FXTAS = fragile X-associated tremor/ataxia syndrome; MCP = middle cerebellar peduncle; mRNA = messenger RNA.

A majority of fragile X males with methylated, full mutation alleles have significant levels of FMR1 messenger RNA

FMR1 mRNA levels were determined in peripheral blood leucocytes for 48 fragile X males with methylated, full mutation alleles that are resistant to cleavage by methylation sensitive enzymes. Using quantitative (fluorescence) RT-PCR, we observed that more than half of these males produceFMR1 mRNA, with some mRNA levels approaching those found in normal subjects. In none of the samples analysed was there any evidence of premutation alleles. These results suggest that the assumed relationship between enzyme resistance andFMR1 gene silencing may not be generally valid. Despite the presence of FMR1 mRNA in some subjects, no FMRP production was detected by either immunocytochemistry or western blotting. The low/absent FMRP levels are probably a reflection of a post-trancriptional effect such as a defect in translation.

Spastic paraparesis, cerebellar ataxia, and intention tremor: a severe variant of FXTAS?

Fragile X associated tremor/ataxia syndrome (FXTAS) is a recently identified neurodegenerative disorder affecting older adult males with pre-mutation alleles of the fragile X mental retardation 1 ( FMR1 ) gene.1–3 These male carriers, in their fifties and older, develop progressive intention tremor, cerebellar ataxia, progressive cognitive difficulties, and variable features including peripheral neuropathy, lower limb proximal muscle weakness, and autonomic dysfunction. Radiological signs include global brain atrophy and white matter disease characterised by hyperintensities in the middle cerebellar peduncles (MCP) on T2 sequence with scattered periventricular disease, corresponding to spongiform changes and demyelination on neuropathological studies.2 Here we report three pre-mutation carriers (two sisters and one sporadic case) presenting with spastic paraparesis, cerebellar ataxia, and intention tremor, and having neuroimaging features suggestive of a severe variant of FXTAS. A random X inactivation pattern was found in the two affected sisters. The third female sibling was unaffected and her inactivation pattern was significantly skewed. This suggests that the X inactivation ratio may be responsible for the incomplete penetrance of the neurological symptoms in this family. ### Case 1 (II-2) The first sister developed spasticity and balance difficulty in the third decade of her life. During this time, she underwent neurological evaluation. She was well oriented, with intact speech and cognitive function. Examination of the lower extremity showed paraparesis, mild spasticity, and positive Babinski. The upper extremities showed diminished co-ordination and intention tremor. EEG, ECG fundoscopy, electromyography (EMG), and nerve conduction studies were normal. Cerebrospinal fluid (CSF) was normal, with absence of oligoclonal bands. Muscle biopsy showed a chronic neurogenic process. She had anxiety and mild depression. ### Case 2 (II-3) The other sister gradually developed spasticity with dragging of the toes while walking, lower limb weakness, and difficulty with balance. Neurological evaluation at 35 years of age showed normal cognitive functions. Examination of the lower extremities, showed paraparesis, …

FMR1 premutation in females diagnosed with multiple sclerosis.

Fragile X associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that affects individuals with premutation alleles of the fragile X mental retardation ( FMR1 ) gene. The clinical features of FXTAS include intention tremor, ataxia, parkinsonism, peripheral neuropathy, autonomic dysfunction and cognitive impairment.1 Such symptoms are accompanied by characteristic MRI focal areas of increased T2 signal in the middle cerebellar peduncles (MCP).1 In this report, we describe two female patients with FXTAS who were initially diagnosed with multiple sclerosis (MS) and treated accordingly but who also met diagnostic criteria for FXTAS. Their clinical features and MRI appearance are compared with other female FXTAS cases previously reported without a diagnosis of MS. A recent report of a female who died of MS and who also had FXTAS inclusions suggests that these two disorders can occur together.2 ### Patient No 1 Case 1 is a 48-year-old female premutation carrier with a history of MS which began at the age of 33 years. Initially, her handwriting quality declined, and she subsequently became ataxic. A combination of weakness, clumsiness and ataxia forced her to use a wheelchair beginning in her late thirties. During that same period, she was diagnosed with MS following a CSF study which showed elevated IgG (9.8 mg/dl; reference range 0.5–6.1 mg/dl) without oligoclonal bands or myelin basic protein. Her course was consistent with relapsing–remitting MS with early secondary progression, and she was treated …

Tissue-specific methylation differences in a fragile X premutation carrier.

Methylation of a premutation was found in a small percentage of blood cells in a male premutation carrier for the FMR1 mutation. To investigate the inter‐tissue heterogeneity and possible clinical implications of this finding, fibroblast cells from the subject were also studied. Although the premutation size was found to be the same in leukocytes and fibroblasts, the methylation pattern was different. In cultured fibroblasts, the premutation was completely unmethylated, as is typical of premutations, whereas methylation of the premutation was detected in a small percentage of lymphocytes. However, the change in methylation did not affect the FMR1 protein (FMRP) expression, as immunocytochemical analysis of FMRP performed on cultured skin fibroblasts and a blood smear revealed normal levels of expression in both tissues.