Mark C. Hirst

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.

PHYSICAL MAPPING ACROSS THE FRAGILE X : HYPERMETHYLATION AND CLINICAL EXPRESSION OF THE FRAGILE X SYNDROME

The most common genetic cause of mental retardation after Downs syndrome, the fragile X syndrome, is associated with the occurrence of a fragile site at Xq27.3. This X-linked disease is intriguing because transmission can occur through phenotypically normal males. Theories to explain this unusual phenomenon include genomic rearrangements and methylation changes associated with a local block of reactivation of the X chromosome. Using microdissected markers close to the fragile site, we have been able to test these hypotheses. We present evidence for the association of methylation with the expression of the disease. However, there is no simple relationship between the degree of methylation and either the level of expression of the fragile site or the severity of the clinical phenotype.

MicroRNA-155 negatively affects blood–brain barrier function during neuroinflammation

Blood–brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses, although their role in central nervous system (CNS) microvascular disorders is largely unknown. We have identified miR‐155 as a critical miRNA in neuroinflammation at the BBB. miR‐155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR‐155 reduced CNS extravasation of systemic tracers, both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR‐155 was strongly and rapidly upregulated by inflammatory cytokines. miR‐155 up‐regulation mimicked cytokine‐induced alterations in junctional organization and permeability, whereas inhibition of endogenous miR‐155 partially prevented a cytokine‐induced increase in permeability. Furthermore, miR‐155 modulated brain endothelial barrier function by targeting not only cell–cell complex molecules such as annexin‐2 and claudin‐1, but also focal adhesion components such as DOCK‐1 and syntenin‐1. We propose that brain endothelial miR‐155 is a negative regulator of BBB function that may constitute a novel therapeutic target for CNS neuroinflammatory disorders.—Lopez‐Ramirez, M. A., Wu, D., Pryce, G., Simpson, J. E., Reijerkerk, A., King‐Robson, J., Kay, O, de Vries, H. E., Hirst, M. C., Sharrack, B., Baker D., Male, D. K., Michael, G. J., Romero, I. A. MicroRNA‐155 negatively affects blood–brain barrier function during neuroinflammation. FASEB J. 28, 2551–2565 (2014). www.fasebj.org

Stability of the Human Fragile X (CGG)n Triplet Repeat Array in Saccharomyces cerevisiae Deficient in Aspects of DNA Metabolism

ABSTRACT 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 withEscherichia 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 andDIN7 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.

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 ragile X ental etardation gene (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.

Genotype mosaicism in fragile X fetal tissues

SummaryThe fragile X syndrome is one of the most common familial causes of mental retardation. It is associated with the expression of a fragile site at Xq27.3, although not all individuals carrying the mutation are fragile-X-positive. Recently, the mutation causing this disease has been identified as the amplification of, or insertion into, a CGG repeat sequence at the fragile site. The mutated chromosome can be recognised by the decrease in mobility of the EcoRI fragment that covers the mutated region. Analysis of lymphocytes of affected males often gives a number of different sized fragments indicating somatic heterogeneity. We have investigated this mosaicism in various tissues of an affected fetus in order to determine the extent of the variation between tissues, and to ascertain how to interpret the results in lymphocytes. Our results suggest that the heterogeneity occurs in all fetal tissues, but that the pattern of fragments observed varies between tissues. Methylation across the region also varies. These differences may be reflected in the cellular phenotypes and may influence the ultimate expression of the clinical phenotype.

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.

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.

Abnormal cortical synaptic plasticity in a mouse model of Huntington's disease.

Huntingtons disease is a fatal neurodegenerative disorder characterised by a progressive motor, psychiatric and cognitive decline and associated with a marked loss of neurons in the cortex and striatum of affected individuals. The disease is inherited in an autosomal dominant fashion and is caused by a trinucleotide (CAG) repeat expansion in the gene encoding the protein huntingtin. Predictive genetic testing has revealed early cognitive deficits in asymptomatic gene carriers such as altered working memory, executive function and recognition memory. The perirhinal cortex is believed to process aspects of recognition memory. Evidence from primate studies suggests that decrements in neuronal firing within this cortical region encode recognition memory and that the underlying mechanism is an activity-dependent long-term depression (LTD) of excitatory neurotransmission, the converse of long-term potentiation (LTP). We have used the R6/1 mouse model of HD to assess synaptic plasticity in the perirhinal cortex. This mouse model provides an ideal tool for investigating early and progressive changes in synaptic function in HD. We report here that LTD at perirhinal synapses is markedly reduced in R6/1 mice. We also provide evidence to suggest that a reduction in dopamine D2 receptor signalling may be implicated.

Genotype prediction in the fragile X syndrome.

Fragile X positive, mentally retarded males have been shown to have an insertion or amplification of DNA sequences at, or close to, the site of expression of the fragile site. We show here the application of the detection of such changes to the diagnosis of affected males and female carriers and the identification of normal transmitting males. One fragile X negative male with the clinical features of the Martin-Bell syndrome also possesses an inserted/amplified DNA sequence. The implications of these results for screening for the fragile X syndrome are discussed.