Impaired long-term potentiation in the prefrontal cortex of Huntington's disease mouse models: rescue by D1 dopamine receptor activation.

BACKGROUND: The introduction of gene testing for Huntington's disease (HD) has enabled the neuropsychiatric and cognitive profiling of human gene carriers prior to the onset of overt motor and cognitive symptoms. Such studies reveal an early decline in working memory and executive function, altered EEG and a loss of striatal dopamine receptors. Working memory is processed in the prefrontal cortex and modulated by extrinsic dopaminergic inputs. OBJECTIVE: We sought to study excitatory synaptic function and plasticity in the medial prefrontal cortex of mouse models of HD. METHODS: We have used 2 mouse models of HD, carrying 89 and 116 CAG repeats (corresponding to a preclinical and symptomatic state, respectively) and performed electrophysiological field recording in coronal slices of the medial prefrontal cortex. RESULTS: We report that short-term synaptic plasticity and long-term potentiation (LTP) are impaired and that the severity of impairment is correlated with the size of the CAG repeat. Remarkably, the deficits in LTP and short-term plasticity are reversed in the presence of a D(1) dopamine receptor agonist (SKF38393). CONCLUSION: In a previous study, we demonstrated that a deficit in long-term depression (LTD) in the perirhinal cortex could also be reversed by a dopamine agonist. These and our current data indicate that inadequate dopaminergic modulation of cortical synaptic function is an early event in HD and may provide a route for the alleviation of cognitive dysfunction.

Instability of a (CGG)98 repeat in the Fmr1 promoter.

Fragile X syndrome is one of 14 trinucleotide repeat diseases. It arises due to expansion of a CGG repeat which is present in the 5'-untranslated region of the FMR1 gene, disruption of which leads to mental retardation. The mechanisms involved in trinucleotide repeat expansion are poorly understood and to date, transgenic mouse models containing transgenic expanded CGG repeats have failed to reproduce the instability seen in humans. As both cis-acting factors and the genomic context of the CGG repeat are thought to play a role in expansion, we have now generated a knock-in mouse Fmr1 gene in which the murine (CGG)8 repeat has been exchanged with a human (CGG)98 repeat. Unlike other CGG transgenic models, this model shows moderate CGG repeat instability upon both in maternal and paternal transmission. This model will now enable us to study the timing and the mechanism of repeat expansion in mice.

Stability of the human fragile X (CGG)(n) triplet repeat array in Saccharomyces cerevisiae deficient in aspects of DNA metabolism.

Expanded trinucleotide repeats underlie a growing number of human diseases. The human FMR1 (CGG)(n) array can exhibit genetic instability characterized by progressive expansion over several generations leading to gene silencing and the development of the fragile X syndrome. While expansion is dependent upon the length of uninterrupted (CGG)(n), instability occurs in a limited germ line and early developmental window, suggesting that lineage-specific expression of other factors determines the cellular environment permissive for expansion. To identify these factors, we have established normal- and premutation-length human FMR1 (CGG)(n) arrays in the yeast Saccharomyces cerevisiae and assessed the frequency of length changes greater than 5 triplets in cells deficient in various DNA repair and replication functions. In contrast to previous studies with Escherichia coli, we observed a low frequency of orientation-dependent large expansions in arrays carrying long uninterrupted (CGG)(n) arrays in a wild-type background. This frequency was unaffected by deletion of several DNA mismatch repair genes or deletion of the EXO1 and DIN7 genes and was not enhanced through meiosis in a wild-type background. Array contraction occurred in an orientation-dependent manner in most mutant backgrounds, but loss of the Sgs1p resulted in a generalized increase in array stability in both orientations. In contrast, FMR1 arrays had a 10-fold-elevated frequency of expansion in a rad27 background, providing evidence for a role in lagging-strand Okazaki fragment processing in (CGG)(n) triplet repeat expansion.

Cloned human FMR1 trinucleotide repeats exhibit a length- and orientation-dependent instability suggestive of in vivo lagging strand secondary structure.

The normal human FMR1 gene contains a genetically stable (CGG) n trinucleotide repeat which usually carries interspersed AGG triplets. An increase in repeat number and the loss of interspersions results in array instability, predominantly expansion, leading to FMR1 gene silencing. Instability is directly related to the length of the uninterrupted (CGG) n repeat and is widely assumed to be related to an increased propensity to form G-rich secondary structures which lead to expansion through replication slippage. In order to investigate this we have cloned human FMR1 arrays with internal structures representing the normal, intermediate and unstable states. In one replicative orientation, arrays show a length-dependent instability, deletions occurring in a polar manner. With longer arrays these extend into the FMR1 5'-flanking DNA, terminating at either of two short CGG triplet arrays. The orientation-dependent instability suggests that secondary structure forms in the G-rich lagging strand template, resolution of which results in intra-array deletion. These data provide direct in vivo evidence for a G-rich lagging strand secondary structure which is believed to be involved in the process of triplet expansion in humans.

Sequence analysis of long FMR1 arrays in the Japanese population: insights into the generation of long (CGG)n tracts.

The human fragile-X syndrome is associated with expansions of a (CGG)n triplet repeat within the FMR1 gene. Whilst normal FMR1 arrays consist of variable numbers of (CGG)7-13 blocks punctuated with single AGG triplets, unstable arrays contain longer blocks of uninterrupted (CGG)n. The degree of instability, and subsequent risk of expansion to the fragile-X mutation, is dependent upon the length of this uninterrupted repeat. Detailed analyses of normal FMR1 array structures suggest that longer uninterrupted blocks of repeat could arise either through a process of gradual slippage or a more dramatic loss of an intervening AGG triplet. Up to 15% of Japanese and Chinese individuals have FMR1 triplet arrays centred on 36 repeats in length, a modal group not found in Caucasians. As longer FMR1 arrays have been associated with high-risk fragile-X haplotypes in some populations, we investigated the nature of these larger arrays. Sequence analysis revealed that the unusual length is due to the presence of a novel (CGG)6 block within the array. Several haplotypically related arrays contain blocks of (CGG)16 or (CGG)15, consistent with the fusion of adjacent (CGG)9 and (CGG)6 blocks after loss of the intervening AGG triplet. This is compatible with inferences from the Caucasian population that AGG loss is a mechanism by which long blocks of identical repeats are generated.

Nucleosome assembly on methylated CGG triplet repeats in the fragile X mental retardation gene 1 promoter.

Expansion and methylation of CGG repeat sequences is associated with Fragile X syndrome in humans. We have examined the consequences of CGG repeat expansion and methylation for nucleosome assembly and positioning on the Fragile X Mental Retardation gene 1 (FMR1) gene. Short unmethylated CGG repeats are not particularly favored in terms of affinity for the histone octamer or for positioning of the reconstituted nucleosome. However, upon methylation their affinity for the histone octamer increases and a highly positioned nucleosome assembles with the repeat sequences found adjacent to the nucleosomal dyad. Expansion of these CGG repeats abolishes the preferential nucleosome assembly due to methylation. Thus, the expansion and methylation of these triplet repeats can alter the functional organization of chromatin, which may contribute to alterations in the expression of the FMR1 gene and the disease phenotype.

Analysis of germline variation at the FMR1 CGG repeat shows variation in the normal-premutated borderline range.

In order to characterize the dynamics of CGG repeat instability at the fragile X syndrome locus (FMR1 gene), we have used small pool PCR to estimate the mutation rate within germline (sperm) and somatic tissue (leukocytes) of two normal males, one carrying the most common 29 CGG repeats allele, the other carrying a borderline normal-premutated allele of 55 repeats. Large contractions and moderate expansions of the repeat were found in sperm and blood for the 55 repeat allele while almost no variation was found in sperm or blood with the 29 repeat allele. Somatic blood DNA exhibited fewer expansions and contractions than sperm. Contractions were more frequent than expansions, and all the expansions were found in the +4 to +10 repeats range, while most of the contractions were found in the -10 to -30 range, suggesting that a subset of contractions results from a distinct mechanism. These results also suggest that the dynamics of the CGG repeat could be partly due to germline instability within the high normal or premutated ranges.

Unusual (CGG)n expansion and recombination in a family with fragile X and DiGeorge syndrome.

In a fragile X family referred for prenatal diagnosis, the female fetus did not inherit the full fragile X mutation from her mother, but an unexpected expansion within the normal range of CGG repeats from 29 to 39 was observed in the paternal X chromosome. Also, a rare recombination between DXS548 and FRAXAC1 was recorded in the maternal meiosis. Follow up of the neonate confirmed the same DNA genotype as in the CVS, but the child died of DiGeorge syndrome after four days and was subsequently found to carry a microdeletion of chromosome 22 using probe cEO. It is suggested that in this family the deletion of chromosome 22 is likely to be a chance event but the rare recombinant and the fragile X mutation might be causally related.

DNA testing for fragile X syndrome in schools for learning difficulties.

Fragile X syndrome is the most common inherited cause of mental retardation. Early diagnosis is important not only for appropriate management of individuals but also to identify carriers who are unaware of their high risk of having an affected child. The disorder is associated with a cytogenetically visible fragile site (FRAXA) at Xq27.3, caused by amplification of a (CGG)n repeat sequence within the gene at this locus designated FMR1. Clinical and molecular studies have been undertaken to screen for fragile X syndrome in 154 children with moderate and severe learning difficulties of previously unknown origin. Southern blot analysis of peripheral blood showed the characteristic abnormally large (CGG)n repeat sequence associated with fragile X syndrome in four of the 154 children. The findings were confirmed by cytogenetic observation of the fragile site and by further molecular studies. The families of the affected children were offered genetic counselling and DNA tests to determine their carrier status. These findings show that there are still unrecognised cases of fragile X syndrome. Given the difficulty of making a clinical diagnosis and the implications for families when the diagnosis is missed, screening in high risk populations may be justified. The issues involved in screening all children in special schools for fragile X syndrome are discussed.

The cloning of FRAXF: trinucleotide repeat expansion and methylation at a third fragile site in distal Xqter.

Three fragile sites, FRAXA, FRAXE and FRAXF lie in the Xq27-28 region of the human X chromosome. The expression of FRAXA is associated with the fragile X syndrome, the most prevalent form of inherited mental retardation whilst the expression of FRAXE is associated with a rarer and comparatively milder form of mental handicap. Both the FRAXA and FRAXE sites have been cloned and the fragile site expression found to be due to the expansion of analogous CGG/GCC trinucleotide repeat arrays. We describe here the cloning of the third fragile site, FRAXF, and demonstrate that it involves the expansion of a (GCCGTC)n(GCC)n compound array. PCR analyses across the repeat of normal individuals show that the number of triplets in the array ranges from 12-26 and the most common allele consists of 14 triplet units. Sequencing analyses show that 95% of normal individuals have three copies of the GCCGTC motif and in these individuals, the size variation observed by PCR is due to copy number alterations in the GCC array. In a cytogenetically positive male with developmental delay, the array is expanded by > 900 triplets and the adjacent CpG-rich region is methylated. The array is also expanded in cytogenetically positive carrier females from the family originally used to define the FRAXF site. We conclude that the expanded array corresponds to the FRAXF fragile site.

Precursor arrays for triplet repeat expansion at the fragile X locus.

To determine factors governing triplet repeat expansion at FMR1, we need to understand the basis of normal variation. We have sequenced the FMR1 repeat from 102 normal X chromosomes and show that most are interrupted with a regularly spaced AGG trinucleotide giving an ordered structure to the array. Five types of arrays were identified consisting of varying numbers of a core unit with consensus [AGG(CGG)9]. Additional variation in the length of the (CGG)n portion within each unit generates the continuum of lengths seen on normal chromosomes. Ten per cent contain long, uninterrupted tracts of (CGG)n, and their lengths suggest they have arisen by the loss of AGG triplets from longer interrupted arrays. Haplotype analysis of arrays carrying long, uninterrupted (CGG)n tracts suggests that they occur more frequently on genetic backgrounds which are more highly represented on fragile X chromosomes. These arrays may well be precursors from which the larger fragile X associated arrays have arisen by further expansion.

Triplet repeat expansion at the FRAXE locus and X-linked mild mental handicap.

We have recently shown that the expression of the FRAXE fragile site in Xq28 is associated with the expansion of a GCC trinucleotide repeat. In the families studied, FRAXE expression is also associated with mild mental handicap. Here we present data on families that previously had been diagnosed as having the fragile X syndrome but that later were found to be negative for trinucleotide repeat expansion at the FRAXA locus. In these families we demonstrate the presence of a GCC trinucleotide repeat expansion at the FRAXE locus. Studies of the FRAXE locus of normal individuals show that they have 6-25 copies of the repeat, whereas affected individuals have > 200 copies. As in the fragile X syndrome, the amplified CpG residues are methylated in affected males.

Origins of the fragile X syndrome mutation.

The fragile X syndrome is a common cause of mental impairment. In view of the low reproductive fitness of affected males, the high incidence of the syndrome has been suggested to be the result of a high rate of new mutations occurring exclusively in the male germline. Extensive family studies, however, have failed to identify any cases of a new mutation. Alternatively, it has been suggested that a selective advantage of unaffected heterozygotes may, in part, explain the high incidence of the syndrome. Molecular investigations have shown that the syndrome is caused by the amplification of a CGG trinucleotide repeat in the FMR-1 gene which leads to the loss of gene expression. Further to this, genetic studies have suggested that there is evidence of linkage disequilibrium between the fragile X disease locus and flanking polymorphic markers. More recently, this analysis has been extended and has led to the observation that a large number of fragile X chromosomes appear to be lineage descendants of founder mutation events. Here, we present a study of the FRAXAC1 polymorphic marker in our patient cohort. We find that its allele distribution is strikingly different on fragile X chromosomes, confirming the earlier observations and giving further support to the suggestions of a fragile X founder effect.

Trinucleotide repeat amplification and hypermethylation of a CpG island in FRAXE mental retardation.

We have cloned the fragile site FRAXE and demonstrate that individuals with this fragile site possess amplifications of a GCC repeat adjacent to a CpG island in Xq28 of the human X chromosome. Normal individuals have 6-25 copies of the GCC repeat, whereas mentally retarded, FRAXE-positive individuals have > 200 copies and also have methylation at the CpG island. This situation is similar to that seen at the FRAXA locus and is another example in which a trinucleotide repeat expansion is associated with a human genetic disorder. In contrast with the fragile X syndrome, the GCC repeat can expand or contract and is equally unstable when passed through the male or female line. These results also have implications for the understanding of chromosome fragility.

Identification of the FRAXE fragile site in two families ascertained for X linked mental retardation.

Chromosome fragility in two families not exhibiting amplification of the CGG trinucleotide associated with the fragile X site has been examined. Fluorescence in situ hybridisation with cosmid DNA from loci immediately flanking FRAXA and other distal loci have confirmed that cytogenetic fragility in these subjects is the result of expression of a new folate sensitive fragile X site, FRAXE.

The identification of a third fragile site, FRAXF, in Xq27--q28 distal to both FRAXA and FRAXE.

FRAXA is unique amongst fragile sites in that it is intimately involved with a specific clinical phenotype, the fragile X syndrome. Whilst the majority of fragile X individuals have been found to have a characteristic mutation in the FMR1 gene, a small proportion of individuals exhibiting fragility have no such mutation. Investigation of the site of chromosome fragility in these FMR1 mutation negative, fragile X site positive individuals, has identified a second site of fragility, FRAXE. However, the presence of FRAXE has not explained all such cases. Here we describe a fragile X site positive, FMR1 mutation negative family, in which chromosome fragility is not due to the FRAXA or FRAXE but is due to a third site designated FRAXF. Using fluorescent in situ hybridisation (FISH) this site is shown to lie over 1Mb distal to FRAXA. The identification of a third fragile site in this small region of the X chromosome provides an opportunity to extend our studies of the molecular nature of chromosome fragility.

The fragile X syndrome.

An amplification of a highly unstable DNA element has been identified at the fragile X locus in Xq27.3. This sequence appears to be both the source of the primary mutation causing the fragile X syndrome, apparently having its causative effect through the methylation of the FMR-1 HTF island and the region of cytogenetic fragility. The direct analysis of the genotype of carrier and affected individuals can be used as a direct diagnosis tool which will improve both the accuracy and speed of diagnosis. The identification of hereditary unstable DNA in a disease with such a wide level of non-penetrance and variable phenotype may give clues as to the basis of non-penetrance in other human genetic disorders.