Absence of expression of the FMR-1 gene in fragile X syndrome

We previously reported the isolation of a gene (FMR-1) expressed in brain at the fragile X locus. One exon of this gene lies within an EcoRI fragment that exhibits length variation in fragile X patients. This exon also contains the CGG repeat within the CpG island hypermethylated in fragile X patients. To study the involvement of the FMR-1 gene in the fragile X syndrome, its expression was studied in lymphoblastoid cell lines and leukocytes derived from patients and normal controls. FMR-1 mRNA was absent in the majority of male fragile X patients, suggesting a close involvement of this gene in development of the syndrome. Normal individuals and carriers all show expression. The methylation status of the BssHII site at the CpG island was also studied by Southern blot analysis of DNA from patients, carriers, and controls. The minority of fragile X affected males that show expression of FMR-1 demonstrated an associated incomplete methylation of the BssHII site.     

Molecular genetic analysis of the FMR-1 gene in a large collection of autistic patients

A genetic etiology in autism is now strongly supported by family and twin studies. A 3:1 ratio of affected males to females suggests the involvement of at least one X-linked locus in the disease. Several reports have indicated an association of the fragile X chromosomal anomaly at Xq27.3 (FRAXA) with autism, whereas others have not supported this finding. We have so far collected blood from 105 simplex and 18 multiplex families and have assessed 141 patients by using the Autism Diagnostic Interview-Revised (ADI-R), the Autism Diagnostic Observation Scale, and psychometric tests. All four ADI-R algorithm criteria were met by 131 patients (93%), whereas 10 patients (7%) showed a broader phenotype of autism. Southern blot analysis was performed with three different enzymes, and filters were hybridized to an FMR-1-specific probe to detect amplification of the CCG repeat at FRAXA, to the complete FMR-1 cDNA probe, and to additional probes from the neighborhood of the gene. No significant changes were found in 139 patients (99%) from 122 families, other than the normal variations in the population. In the case of one multiplex familiy with three children showing no dysmorphic features of the fragile X syndrome (one male meeting 3 out of 4 ADI-algorithm criteria, one normal male with slight learning disability but negative ADI-R testing, and one fully autistic female), the FRAXA full-mutation-specific CCG-repeat expansion in the genotype was not correlated with the autism phenotype. Further analysis revealed a mosaic pattern of methylation at the FMR-1 gene locus in the two sons of the family, indicating at least a partly functional gene. Therefore, we conclude that the association of autism with fragile X at Xq27.3 is non-existent and exclude this location as a candidate gene region for autism. Received: 25 October 1996 / Accepted: 10 March 1997     

Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome.

Fragile X syndrome is the most frequent form of inherited mental retardation and is associated with a fragile site at Xq27.3. We identified human YAC clones that span fragile X site-induced translocation breakpoints coincident with the fragile X site. A gene (FMR-1) was identified within a four cosmid contig of YAC DNA that expresses a 4.8 kb message in human brain. Within a 7.4 kb EcoRI genomic fragment, containing FMR-1 exonic sequences distal to a CpG island previously shown to be hypermethylated in fragile X patients, is a fragile X site-induced breakpoint cluster region that exhibits length variation in fragile X chromosomes. This fragment contains a lengthy CGG repeat that is 250 bp distal of the CpG island and maps within a FMR-1 exon. Localization of the brain-expressed FMR-1 gene to this EcoRI fragment suggests the involvement of this gene in the phenotypic expression of the fragile X syndrome.     

Fragile X syndrome and the (CGG)n mutation: two families with discordant MZ twins.

The fragile X phenotype has been found, in the majority of cases, to be due to the expansion of a CGG repeat in the 5'-UTR region of the FMR-1 gene, accompanied by methylation of the adjacent CpG island and inactivation of the FMR-1 gene. Although several important aspects of the genetics of fragile X have been resolved, it remains to be elucidated at which stage in development the transition from the premutation to the full mutation occurs. We present two families in which discordance between two sets of MZ twins illustrates two important genetic points. In one family, two affected MZ brothers differed in the number of CGG repeats, demonstrating in vivo mitotic instability of this CGG repeat and suggesting that the transition to the full mutation occurred postzygotically. In the second family, two MZ sisters had the same number of repeats, but only one was mentally retarded. When the methylation status of the FMR-1 CpG island was studied, we found that the majority of normal chromosomes had been inactivated in the affected twin, thus leading to the expression of the fragile X phenotype.     

Molecular diagnosis of the fragile X and FRAXE syndromes in patients with mental retardation of unknown cause in Mexico

The fragile X syndrome (Fra-X) is the most common cause of inherited mental retardation with X-linked semi-dominant inheritance. The prevalence of Fra-X in the Mexican population is unknown. The aim of this population screening study was to determine if Fra-X or FRAXE mutations are the cause of a number of cases of mental retardation in a sample of Mexican children with mental retardation of unknown cause (MRUC) and to stress the importance of performing molecular analysis of the FMR-1 gene in all patients with MRUC. We report here the direct analysis of CGG and GCC repeats within the FMR-1 and FMR-2 genes, respectively, in 62 unrelated patients with MRUC. Two male index cases had the CGG expansion, although they did not express the Xq27.3 fragile site cytogenetically. Fra-X diagnosis was highly suspected on a clinical basis in one of the patients, but not in the other. Both mothers were found to be premutation carriers. The molecular studies of FMR-1 showed that the proportion of MRUC patients with Fra-X is 3.2%. This frequency was not significantly different to that reported in most populations. As reported in other series, no patients with FRAXE were found in our sample. Our findings confirm that the molecular analysis of the FMR-1 gene is necessary in MRUC patients to achieve unequivocal diagnosis of fragile X syndrome, carrier premutation detection and for accurate genetic counseling.     

A microdeletion of less than 250 kb, including the proximal part of the FMR-1 gene and the fragile-X site, in a male with the clinical phenotype of fragile-X syndrome

A gene designated "FMR-1" has been isolated at the fragile-X locus. One exon of this gene is carried on a 5.1-kb EcoRI fragment that exhibits length variation in fragile-X patients because of amplification of or insertion into a CGG-repeat sequence. This repeat probably represents the fragile site. The EcoRI fragment also includes an HTF island that is hypermethylated in fragile-X patients showing absence of FMR-1 mRNA. In this paper, we present further evidence that the FMR-1 gene is involved in the clinical manifestation of the fragile-X syndrome and also in the expression of the cellular phenotype. A deletion including the HTF island and exons of the FMR-1 gene was detected in a fragile X-negative mentally retarded male who presented the clinical phenotype of the fragile-X syndrome. The deletion involves less than 250 kb of genomic DNA, including DXS548 and at least five exons of the FMR-1 gene. These data support the hypothesis that loss of function of the FMR-1 gene leads to the clinical phenotype of the fragile-X syndrome. In the fragile-X syndrome, there are pathogenetic mechanisms other than amplification of the CGG repeat that do have the same phenotypic consequences.     

应用多聚酶链式反应快速分析FMR-1基因突变

为了建立一种在先天性智力低下患儿中快速分析脆性X综合征智力低下基因1(Fragile X mental retardation gene 1.FMR-1)突变的方法,对先天性智力低下儿童进行脆性X综合征的大面积筛查和诊断,应用复式多聚酶链式反应一次性扩增FMR-1基因的(CGG)n的重复区,分析CGG重复序列的大小,判断FMR-1基因状态(正常、突变前、突变后),对脆性X综合征可疑患儿快速筛查,在113倒不明原因的先天性智力低下患儿中,分析有脆性X综合征携带者(FMR-1基因前突变者)7例(2男5女),脆性X综合征患者(FMR-1基因突变者)5例,应用多聚酶链式反应可以对脆性X综合征可疑患儿进行大面积初筛,确定携带者和患者。     

Review: The fragile X syndrome: Isolation of the FMR-1 gene and characterization of the fragile X mutation

Fragile X syndrome, associated with the fragile X chromosome, is the most common cause of familial mental retardation. A breakthrough has been made in molecular biological research into the fragile X site. In this review we describe the molecular investigations that have led to the isolation of the FMR-1 gene. The nature of the fragile X mutation as well as the implications of the DNA test for the mutation are discussed.     

Noninvasive test for fragile X syndrome, using hair root analysis.

Identification of the FMR1 gene and the repeat-amplification mechanism causing fragile X syndrome led to development of reliable DNA-based diagnostic methods, including Southern blot hybridization and PCR. Both methods are performed on DNA isolated from peripheral blood cells and measure the repeat size in FMR1. Using an immunocytochemical technique on blood smears, we recently developed a novel test for identification of patients with fragile X syndrome. This method, also called "antibody test," uses monoclonal antibodies against the FMR1 gene product (FMRP) and is based on absence of FMRP in patients' cells. Here we describe a new diagnostic test to identify male patients with fragile X syndrome, on the basis of lack of FMRP in their hair roots. Expression of FMRP in hair roots was studied by use of an FMRP-specific antibody test, and the percentage of FMRP-expressing hair roots in controls and in male fragile X patients was determined. Control individuals showed clear expression of FMRP in nearly every hair root, whereas male fragile X patients lacked expression of FMRP in almost all their hair roots. Mentally retarded female patients with a full mutation showed FMRP expression in only some of their hair roots (<55%), and no overlap with normal female controls was observed. The advantages of this test are (1) plucking of hair follicles does no appreciable harm to the mentally retarded patient, (2) hairs can be sent in a simple envelope to a diagnostic center, and (3) the result of the test is available within 5 h of plucking. In addition, this test enabled us to identify two fragile X patients who did not show the full mutation by analysis of DNA isolated from blood cells.     

Evidence that methylation of the FMR-I locus is responsible for variable phenotypic expression of the fragile X syndrome.

DNA at the FMR-1 locus was analyzed by Southern blot using probe StB12.3 in an unusual fragile X family with six brothers, three of whom are affected with fragile X to varying degrees, two of whom are nonpenetrant carriers, and one of whom is unaffected. Fragile X chromosome studies, detailed physical examinations, and psychological testing were completed on all six. Two of the affected brothers and the two nonpenetrant brothers were found to be methylation mosaics. The three affected males spanned the phenotypic and cognitive spectrum of the fragile X syndrome. A correlation was seen between the degree of methylation and the phenotypic expression identified in the three affected males. The two males initially classified as nonpenetrant were found to have mild phenotypic expression which consisted of minor cognitive deficits and a partial physical phenotype. These two, who were negative on fragile X chromosome studies, were found on DNA analysis to have large broad smears, with approximately 97% of the DNA unmethylated. The results described here indicate that some "nonpenetrant" carrier males may have varying amounts of methylation of the FMR-1 region, which can result in mild expression of the fragile X syndrome. The apparently mild phenotypic and cognitive expression of the fragile X syndrome in the two males, initially classified as nonpenetrant, who are mosaic for hypermethylation of an expansion of the CGG repeat in the premutation range, indicates that expression of the syndrome is not confined to males with large, hypermethylated expansions (full mutation) but has instead a gradient effect with a threshold for the full expression of the phenotype.     

Polymerase chain reaction analysis of fragile X mutations

Summary The mutation that underlies the fragile X syndrome is presumed to be a large expansion in the number of CGG repeats within the gene FMR-1. The unusually GC-rich composition of the expanded region has impeded attempts to amplify it by the polymerase chain reaction (PCR). We have developed a PCR protocol that successfully amplifies the (CGG)_n region in normal, carrier and affected individuals. The PCR analysis of several large fragile X families is presented. The PCR results agree with those obtained by direct genomic Southern blot analyses. These favorable comparisons suggest that the PCR assay may be suitable for rapid testing for fragile X mutations and premutations and genetic screening of at-risk individuals.     

The fragile X syndrome in Finland: demonstration of a founder effect by analysis of microsatellite haplotypes

Microsatellite markers RS46 (DXS548) and FRAXAC2 flanking the fragile X mutation, an expansion of a (CGG)_n repeat within the FMR-1 gene, were typed in 60 unrelated northern and eastern Finnish fragile X families and in a control population from the same geographical region. A significant difference was found in allelic and haplotypic distributions between the normal X and fragile X chromosomes. Evidence for a strong founder effect was detected, with the haplotype 196-153 being present on 80% of the fragile X chromosomes, but on only 8% of the normal X chromosomes. In addition to this major haplotype, four minor haplotypes were found on the fragile X chromosomes. These results suggest that the majority of present-day fragile X mutations in Finland may have a common initial ancestor, probably from the 16th century.     

Molecular analysis of the (CGG)n expansion in the FMR-1 gene in 59 Spanish fragile X syndrome families

The fragile X mental retardation syndrome is caused by an expansion of a trinucleotide repeat (CGG)_n in the FMR-1 gene. Molecular genetic study of fragile X provides accurate diagnosis and facilitates genetic counseling in families with affected members. We present here the molecular study of 59 Spanish fragile X syndrome families using probe StB 12.3 and the polymerase chain reaction (PCR) of the (CGG)_n repeat sequence of the FMR-1 gene. The results obtained have allowed us to characterize 455 individuals, including eight prenatal diagnoses. The clinical diagnosis of fragile X in 89 affected males was confirmed, 137 female carriers were identified (48 of whom were mentally retarded), 176 individuals at risk were found not to have the expansion, and 12 cases of normal transmitting males (NTM) were detected. In the sample studied, no de novo mutations were detected, nor any mutation different from that described for the (CGG)_n expansion. One nonmentally retarded male was detected as having an unmethylated CpG island for the FMR-1 gene, but with more than 200 CGG repeats (high functioning male). The analysis of the (CGG)_n repeat in 208 normal chromosomes gave an allele distribution similar to that in other Caucasoid population groups, with alleles of 29 and 30 CGG repeats accounting for 46% of the chromosomes. The combination of Southern analysis and PCR of the (CGG)_n repeat is highly efficient for diagnosis, compared with cytogenetic techniques, especially in the detection of female carriers, NTMs, and prenatal diagnosis, enabling accurate genetic counseling to be provided in all cases.     

Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection

Molecular genetic analysis of individuals from 6 Egyptian and 33 German families with fragile X syndrome and 240 further patients with mental retardation was performed applying a completely non-radioactive system. The aim of our study was the development of a non-radioactive detection method and its implementation in molecular diagnosis of the fragile X syndrome. Furthermore, we wanted to assess differences in the mutation sizes between Egyptian and German patients and between Egyptian and German carriers of a premutation. Using non-radioactive polymerase chain reaction (PCR), agarose gel electrophoresis and blotting of the PCR products, followed by hybridisation with a digoxigenin-labelled oligonucleotide probe (CGG)₅ and chemiluminescent detection, we identified the fragile X full mutation (amplification of a CGG repeat in the FMR-1 gene ranging from several hundred to several thousand repeat units) in all patients. We observed no differences in the length of the CGG repeat between the Egyptian and German patients and carriers, respectively. However, in one prenatal diagnosis, we detected only one normal sized allele in a female fetus using the PCR-agarose assay, whereas Southern blot analysis with the digoxigenin labelled probe StB 12.3 revealed presence of a full mutation. Our newly established nonradioactive genomic blotting method is based on the conventional radioactive Southern blot analysis. Labelling of the probe StB 12.3 with digoxigenin via PCR allowed the detection of normal, premutated and fully mutated alleles. For exact sizing of small premutated or large normal alleles, we separated digoxigenin labelled PCR products through denaturing poly-acrylamide gelelectrophoresis (PAGE) and transfered them to a nylon membrane using a gel dryer. The blotted PCR-fragments can easily be detected with alkaline phosphate-labelled anti-digoxigenin antibody. The number of trinucleotide repeat units can be determined by scoring the detected bands against a digoxigenated M13 sequencing ladder. Our newly developed digoxigenin/chemiluminescence approach using PCR and Southern blot analysis provides reliable results for routine detection of full fragile X mutations and premutations.     

Molecular Analysis and Test of Linkage between the FMR-1 Gene and Infantile Autism in Multiplex Families

Approximately 2%–5% of autistic children show cytogenetic evidence of the fragile X syndrome. This report tests whether infantile autism in multiplex autism families arises from an unusual manifestion of the fragile X syndrome. This could arise either by expansion of the (CGG)n trinucleotide repeat in FMR-1 or from a mutation elsewhere in the gene. We studied 35 families that met stringent criteria for multiplex autism. Amplification of the trinucleotide repeat and analysis of methylation status were performed in 79 autistic children and in 31 of their unaffected siblings, by Southern blot analysis. No examples of amplified repeats were seen in the autistic or control children or in their parents or grandparents. We next examined the hypothesis that there was a mutation elsewhere in the FMR-1 gene, by linkage analysis in 32 of these families. We tested four different dominant models and a recessive model. Linkage to FMR-1 could be excluded (lod score between −24 and −62) in all models by using probes DXS548, FRAXAC1, and FRAXAC2 and the CGG repeat itself. Tests for heterogeneity in this sample were negative, and the occurrence of positive lod scores in this data set could be attributed to chance. Analysis of the data by the affected-sib method also did not show evidence for linkage of any marker to autism. These results enable us to reject the hypothesis that multiplex autism arises from expansion of the (CGG)n trinucleotide repeat in FMR-1. Further, because the overall lod scores for all probes in all models tested were highly negative, linkage to FMR-1 can also be ruled out in multiplex autistic families.     

FRAXE expansion is not a common etiological factor among developmentally delayed males.

Expansion of a (CGG)n trinucleotide repeat unit at FRAXE, a newly defined fragile site distal to FRAXA, at Xq28, is reported to be associated with mild mental retardation. Three hundred developmentally delayed male patients referred for fragile X testing but negative for the FMR-1 gene trinucleotide expansion were screened for the FRAXE expansion. This group of patients had a wide range of intellectual or behavioral problems and included 19 patients who had low-level fragile site expression detected cytogenetically at Xq27-q28. None of the patients tested positive for the FRAXE expansion. These results suggest that FRAXE is not a common etiological factor among this group of patients. The data support the hypothesis that FRAXE is either very rare or a benign fragile site that is not associated with any clinical phenotype, similar to the FRAXF and FRA16A sites.     

Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.

Fragile X syndrome results from mutations in a (CGG)n repeat found in the coding sequence of the FMR-1 gene. Analysis of length variation in this region in normal individuals shows a range of allele sizes varying from a low of 6 to a high of 54 repeats. Premutations showing no phenotypic effect in fragile X families range in size from 52 to over 200 repeats. All alleles with greater than 52 repeats, including those identified in a normal family, are meiotically unstable with a mutation frequency of one, while 75 meioses of alleles of 46 repeats and below have shown no mutation. Premutation alleles are also mitotically unstable as mosaicism is observed. The risk of expansion during oogenesis to the full mutation associated with mental retardation increases with the number of repeats, and this variation in risk accounts for the Sherman paradox.     

FMR-1基因中CGG重复序列扩增条件的优化

目的:优化PCR扩增条件,建立一种有效检测脆性X综合征的方法。方法:在常规PCR的基础上,采用耐高温酶替代法、碱基替代法,同时加入有机溶剂DMSO等,对表型正常的人群进行FMR1基因CGG重复序列检测。结果:改良PCR法可以提高G C富集区扩增效率,并取得了较好的效果。结论:建立了一种扩增FMR-1基因中CGG重复序列的可行方法。     

Identification of small molecules rescuing fragile X syndrome phenotypes in Drosophila

Fragile X syndrome is caused by the functional loss of the fragile X mental retardation 1 (FMR1) gene. Deletion of the FMR1 ortholog in Drosophila melanogaster (Fmr1) recapitulates many phenotypes associated with fragile X syndrome. We have discovered that Fmr1 mutant Drosophila die during development when reared on food containing increased levels of glutamate, which is consistent with the theory that FMR1 loss results in excess glutamate signaling. Using this lethal phenotype, we screened a chemical library of 2,000 compounds and identified nine molecules that rescued the lethality, including three that implicate the GABAergic inhibitory pathway. Indeed, GABA treatment rescued several known Fmr1 mutant phenotypes in flies, including mushroom bodies defects, excess Futsch translation and abnormal male courtship behavior. These data are consistent with GABAergic inhibition of the enhanced excitatory pathway in fragile X syndrome. In addition, our screen reveals that the muscarinic cholinergic receptors may have a role in fragile X syndrome in parallel to the GABAergic pathway. These results point to potential therapeutic approaches for treating fragile X syndrome.