Loss of mutation at the FMR1 locus through multiple exchanges between maternal X chromosomes

The mutation observed in the fragile X syndrome, an X-linked inherited disorder causing mental retardation, is almost exclusively an expanded CGG repeat in the first exon of the FMR1 gene. Here we describe a daughter of a female carrier, who inherited the fragile X premutation chromosome based on haplotype analysis using flanking markers. However, the CGG repeat sequence and the intragenic polymorphic marker FMRb showed the normal maternal alleles, while two other intragenic markers, FMRa and FRAXAC2 and other, more distant markers, showed the risk haplotype. Since FMRa and FRAXAC2 are located in between the markers CGG and FMRb, this results in patches of normal and fragile X sequences in the FMR1 gene of the daughter. This observation is very likely due to gene conversion. As this daughter received a normal CGG repeat region, we expect that her risk to have affected offspring is the same as the population risk. The observed phenomenon would therefore represent a back mutation at the FMR1 locus.     

A deletion of 1.6 kb proximal to the CGG repeat of the FMR1 gene causes the clinical phenotype of the fragile X syndrome

The vast majority of individuals with the fragile X syndrome show expanded stretches of CGG repeats in the 5' non-coding region of FMR1. This expansion coincides with abnormal methylation patterns in that area resulting in the silencing of the FMR1 gene. Evidence is accumulating that this directly causes the fragile X phenotype. Very few other mutations in FMR1, causing the fragile X phenotype have been reported thus far and all concerned isolated cases. We, however, report a family, in which 11 individuals have a deletion of 1.6 kb proximal to the CGG repeat of the FMR1 gene. Although fragile X chromosomes were not detected, all 4 affected males and 2 of the carrier females show characteristics of the fragile X phenotype. Using RT-PCR we could demonstrate that FMR1 is not expressed in the affected males, strongly suggesting that the FMR1 promoter sequences 5' to the CGG repeat are missing. The deletion patients have approximately 45 CGG repeats in their FMR1 gene, though not interspersed by AGG triplets that are usually present in both normal and expanded repeats. It is hypothesized that prior to the occurrence of the deletion, an expansion of the repeat occurred, and that the deletion removed the 5' part of the CGG repeat containing the AGG triplets. Transmission of the deletion through the family could be traced back to the deceased grandfather of the affected males, which supports the hypothesis that the FMR1 gene product is not required for spermatogenesis. Finally, the data provide additional evidence that the fragile X syndrome is a single gene disorder.     

Association of fragile X syndrome with delayed replication of the FMR1 gene

The fragile X syndrome is commonly associated with mutant alleles of the FMR1 gene that are hypermethylated and have large expansions of CGG repeats. We present data here on the replication timing of FMR1 that confirm predictions of delayed replication of alleles from affected males. The normal FMR1 allele replicates late in S phase, while alleles from affected males replicate later, the major peak of replication occurring in the flow cytometry fraction usually referred to as G2/M. The delayed timing of replication is not the direct result of a single replication fork stalling at the expanded CGG repeat, because delayed replication was observed for regions on both sides of the repeat. The domain of altered replication timing includes sites at least 150 kb 5' and 34 kb 3' of the repeat, indicating that genes in addition to FMR1 may be affected.     

CGG repeat interruptions in the FMR1 gene in patients with infantile autism

We determined the CGG repeat length and AGG interruptions in the FMR1 gene in normal Chinese subjects and patients with infantile autism and mild mental retardation. Genomic DNA was investigated by PCR and Southern hybridisation for CGG repeat number and PCR with Mnl I restriction analysis for AGG interruption. Both the normal subjects and the patients with autism have 53 CGG repeats in FMR1, and the majority have two interspersed AGG. Our normal Chinese subjects have a similar number of interspersed AGG as other populations. When compared with the normal subjects, the autism patients have less AGG interruptions and a different pattern of AGG distribution. There was a significant difference in the CGG configurations between normal subjects and patients with autism. The latter had less interspersed AGG, as in fragile X patients, but they did not have fragile X. A study on mentally retarded patients with no infantile autism should also be carried out to ascertain whether mental retardation alone may have contributed to such AGG pattern.     

Characteristics of traditional birth attendants and their beliefs and practices in the Offot Clan, Nigeria

The fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by an expansion of a polymorphic CGG repeat upstream of the coding region in the FMR1 gene. The expansion blocks expression of the FMR1 gene due to methylation of the FMR1 promoter. Functional studies on the FMR1 protein have shown that the protein can bind RNA and might be involved in transport of RNAs from the nucleus to the cytoplasm. A role of FMR1 protein on translation of certain mRNAs has been suggested. An animal model for fragile X syndrome exists and these mice show some behavioural difficulties mimicking the human fragile X syndrome phenotype. This review presents what is known about the protein and what is learned from the animal model for fragile X syndrome.     

Phylogeny of leeches (Hirudinea) based on mitochondrial cytochrome c oxidase subunit I

The fragile X syndrome is the most frequent hereditary form of mental retardation. This X-linked disorder is, in most cases, caused by an unstable and expanding trinucleotide CGG repeat located in the 5'-untranslated region of the gene involved, the fragile X mental retardation 1 (FMR1) gene. Expansion of the CGG repeat to a length of more than 200 trinucleotides results in silencing of the FMR1 gene promoter and, thus, in an inactive gene. The clinical features of male fragile X patients include mental retardation, autistiform behavior, and characteristic facial features. In addition, macroorchidism is observed. To study the role of Sertoli cell proliferation and FSH signal transduction in the occurrence of macroorchidism in fragile X males, we made use of an animal model for the fragile X syndrome, an Fmr1 knockout mouse. The results indicate that in male Fmr1 knockout mice, the rate of Sertoli cell proliferation is increased from embryonic day 12 to 15 days postnatally. The onset and length of the period of Sertoli cell proliferation were not changed compared with those in the control males. Serum levels of FSH, FSH receptor messenger RNA expression, and short term effects of FSH on Sertoli cell function, as measured by down-regulation of FSH receptor messenger RNA, were not changed. We conclude that macroorchidism in Fmr1 knockout male mice is caused by an increased rate of Sertoli cell proliferation. This increase does not appear to be the result of a major change in FSH signal transduction in Fmr1 knockout mice.     

Stability of the FMR1 CGG repeat in a Basque sample

The fragile X syndrome is an X-chromosome-linked dominant disorder with reduced penetrance. It is the most common inherited form of mental retardation. The molecular basis is usually the unstable expansion of a CGG trinucleotide repeat in the 5' untranslated region of the first exon of the FMR1 gene, which resides at chromosome position Xq27.3 and is coincident with the cytogenetic fragile site FRAXA, which characterizes the syndrome. In the Biscay province of the Basque Country the prevalence of FRAXA in a mentally retarded sample of non-Basque origin is in the range of other analyzed Spanish populations. In the sample of Basque origin we have not found FRAXA site expression and the repeat size is in the normal range. Based on this, we have examined FMR1 gene stability in normal individuals of Basque origin from the Biscay province. This study is based on a sample of 242 X chromosomes. The results from the CGG repeat region of FMR1 indicate that a prevalence of predisposing normal alleles toward repeat instability in the Basque population is 0.00% or near to it. This could be 1 of the explanations of the apparently low fragile X syndrome incidence found in the Basque mentally retarded sample analyzed by us. This low incidence does not seem to be associated with the flanking microsatellite markers.     

Reversal of sensorimotor gating abnormalities in Fmr1 knockout mice carrying a human Fmr1 transgene.

Fragile X syndrome is caused by a CGG trinucleotide repeat expansion of the FMR1 gene. Individuals with fragile X display several behavioral abnormalities including hyperactivity, social anxiety, autistic-like features, impaired cognitive processing, and impaired sensorimotor gating. The Fmr1KO mouse model of fragile X exhibits several related behavioral phenotypes such as increased activity and altered social interactions. Individuals with fragile X also have impaired sensorimotor gating as measured using the prepulse inhibition of startle response. The authors have recently shown that Fmr1KO mice with a yeast artificial chromosome containing the human FMR1 gene have corrected or overcorrected abnormal behaviors including hyperactivity and altered social interactions. Here the authors present results from a study examining abnormal sensorimotor gating in Fmr1KO mice. Consistent with previous findings, Fmr1KO mice have increased prepulse inhibition. The KO mice with the yeast artificial chromosome containing the human FMR1 gene had levels of prepulse inhibition comparable to WT mice, indicating not only a correction of this phenotype, but also clearly demonstrating that in mice levels of the fragile X mental retardation protein regulate sensorimotor gating. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Rapid protein sequencing by tandem mass spectrometry and cDNA cloning of p20-CGGBP. A novel protein that binds to the unstable triplet repeat 5'-d(CGG)n-3' in the human FMR1 gene

The autonomous expansion of the unstable 5'-d(CGG)n-3' repeat in the 5'-untranslated region of the human FMR1 gene leads to the fragile X syndrome, one of the most frequent causes of mental retardation in human males. We have recently described the isolation of a protein p20-CGGBP that binds sequence-specifically to the double-stranded trinucleotide repeat 5'-d(CGG)-3' (Deissler, H., Behn-Krappa, A., and Doerfler, W. (1996) J. Biol. Chem. 271, 4327-4334). We demonstrate now that the p20-CGGBP can also bind to an interrupted repeat sequence. Peptide sequence tags of p20-CGGBP obtained by nanoelectrospray mass spectrometry were screened against an expressed sequence tag data base, retrieving a clone that contained the full-length coding sequence for p20-CGGBP. A bacterially expressed fusion protein p20-CGGBP-6xHis exhibits a binding pattern to the double-stranded 5'-d(CGG)n-3' repeat similar to that of the authentic p20-CGGBP. This novel protein lacks any overall homology to other known proteins but carries a putative nuclear localization signal. The p20-CGGBP gene is conserved among mammals but shows no homology to non-vertebrate species. The gene encoding the sequence for the new protein has been mapped to human chromosome 3.     

Transition from normal to premutated alleles in fragile X syndrome results from a multistep process

In fragile X syndrome, the most common cause of inherited mental retardation, phenotypic expression has been linked to a region containing a repetitive sequence, (CGG)n, that appears to lengthen dramatically in fragile X patients and to show length variation in normal individuals. In order to investigate possible mechanisms responsible for further expansion of CGG in the normal population, we selected 31 normal unrelated X chromosomes carrying either the high-risk DX204-AC155 or DX196-AC151 haplotypes, as defined by the flanking microsatellites, DXS548 and FRAXAC2. Nearly 100% of CGGs with more than 35 repeats were found on DX204-AC155 haplotypes, with a mean length significantly higher and much more variable than in normal individuals carrying other haplotypes including the high-risk haplotype DX196-AC151. These findings suggest that the transition from the normal to the abnormal range occurs by a multistep process, a primary event, such as unequal crossing-over, leading to increased size and moderate instability of the repeat, and from which DNA polymerase slippage could lead to recurrent premutations. Our results also suggest that the upper limit of the normal range is roughly 35 repeats in the fragile X gene. The 36-54 repeats range would define an intermediate allele only observed, up to now, in DX204-AC155 fragile X chromosomes.     

Molecular biologic screening test (PCR) for fragile X syndrome

Fragile X syndrome is the most common inherited from of familial mental retardation. It is caused by an expanded CGG repeat in the first exon of the fragile X mental retardation gene. A polymerase chain reaction based technique was used for the identification of full mutations among men. According to our conditions full mutations failed to amplify. An internal control was used at a CG rich region 147 bp upstream of the polymorphic region. The bands were visualised on silver stained polyacrylamide gels. From the 57 individuals studied molecular analysis was performed on 38 males and 16 females. From the 26 males with suspected fragile X syndrome 9 males resulted in no amplification of the 500 kb product, all having a positive cytogenetic result for fragile X syndrome. One cytogeneticly positive male had normal results by molecular studies suggesting a different mutation. All control males had normal results. The results on the 16 females studied were inconclusive. We suggest that our method is highly sensitive and specific for screening males for fragile X syndrome.     

Extended gene diversity at the FMR1 locus and neighbouring CA repeats in a sub-Saharan population

We report on the allele distributions in a normal black African population at two microsatellite loci neighbouring the FRAXA locus and at the CGG repeat in the 5' end of the FMR-1 gene, which causes the fragile X syndrome. The CGG repeat distribution was found to be similar to that of other ethnic groups, as well as to that of other nonhuman primates, possibly predicting a comparable prevalence of fragile X in Africa. Significant linkage disequilibrium has been observed between fragile X mutations and alleles of the DXS548 and FRAXAC1 loci in European and Asian populations, and some founder chromosomes may be extremely old. Those associated with FRAXAC1-A and DXS548-2 alleles are not present in the Asian fragile X samples. We searched for these alleles and their frequency in the well defined Bamileke population of Cameroon. All previously described alleles and some new ones were found in this sample, supporting the hypothesis of their pre-existence and subsequent loss in Asian populations. Finally, the heterozygosity of the Bamileke sample was significantly higher at both marker loci and comparable to that of Europeans at the CGG repeat, confirming the notion that genetic diversity is greater in Africans than in other groups and supporting the view that evolution of modern man started in Africa.     

Progressive spatial processing deficits in a mouse model of the fragile X premutation.

Fragile X associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that is the result of a CGG trinucleotide repeat expansion in the range of 55–200 in the 5′ UTR of the FMR1 gene. To better understand the progression of this disorder, a knock-in (CGG KI) mouse was developed by substituting the mouse CGG8 trinucleotide repeat with an expanded CGG98 repeat from human origin. It has been shown that this mouse shows deficits on the water maze at 52 weeks of age. In the present study, this CGG KI mouse model of FXTAS was tested on behavioral tasks that emphasize spatial information processing. The results demonstrate that at 12 and 24 weeks of age, CGG KI mice were unable to detect a change in the distance between two objects (metric task), but showed intact detection of a transposition of the objects (topological task). At 48 weeks of age, CGG KI mice were unable to detect either change in object location. These data indicate that hippocampal-dependent impairments in spatial processing may occur prior to parietal cortex-dependent impairments in FXTAS. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Mental status and fragile X expression in relation to FMR-1 gene mutation

The fragile X mental retardation syndrome is caused by unstable expansion of a CGG repeat in the FMR-1 gene. Clinical expression is associated with a large expansion of the CGG repeat. The mutation in the FMR-1 gene and the cytogenetic expression of the fragile site at Xq27.3 have been studied in 52 fragile X male patients. The percentage of the cytogenetic expression of the fragile site at Xq27.3 positively correlates with the mean size of the full mutation in the FMR-1 gene (p < 0.0001) irrespective of the presence of additional premutation alleles. We noted a less frequent occurrence of additional premutation alleles in adult patients compared with juveniles, suggesting a continued mitotic instability in life. Additionally, the level of mental retardation has been ascertained in 35 patients using the Stanford-Binet or Terman-Merrill test of general intelligence. The presence of a full mutation in the FMR-1 gene seemed decisive for the occurrence of mental impairment in the patient. No correlation is observed between the degree of mental retardation and the size of the full mutation. The degree of mental retardation seemed not to be influenced by the presence of premutation alleles in part of the cells in addition to a full mutation. One patient is described with the 'Prader-Willi-like' subphenotype of the fragile X syndrome, showing a deletion in the FMR-1 gene in a part of his cells in addition to a full mutation.     

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.