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.     

Molecular heterogeneity of the fragile X syndrome.

The fragile X syndrome is an X-linked disorder which has been shown to be associated with the length variation of a DNA fragment containing a CGG trinucleotide repeat element at or close to the fragile site. Phenotypically normal carriers of the disorder generally have a smaller length variation than affected individuals. We have cloned the region in cosmids and defined the area containing the amplified sequence. We have used probes from the region to analyse the mutation in families. We show that the mutation evolves in different ways in different individuals of the same family. In addition we show that not all fragile X positive individuals show this amplification of DNA sequence even though they show expression of the fragile site at levels greater than 25%. One patient has alterations in the region adjacent to the CGG repeat elements. Three patients in fragile X families have the normal fragment with amplification in a small population of their cells. These observations indicate that there is molecular heterogeneity in the fragile X syndrome and that the DNA fragment length variation is not the only sequence responsible for the expression of the fragile site or the disease phenotype.     

Dissociation between mental retardation and fragile site expression in a family with fragile X-linked mental retardation

Summary We report an extended family in which two brothers with a fragile X chromosome are mentally retarded while a third brother with the fragile site is both phenotypically and mentally normal. The study of six probes detecting restriction fragment length polymorphisms on either sides of the fragile site Xq27 confirmed that the fragile X regions inherited by these three brothers were identical from DXS 102 to the telomere. These data highlight the heterogeneity of the fragile X syndrome, which is discussed in the framework of the different hypotheses previously proposed.     

A male with two different familial autosomal fragile sites and a cytogenetically abnormal offspring

During the course of routine prenatal cytogenetics, a male with 2 different autosomal fragile sites (FS) was detected. The FS were at 9p21 and 12q13 and his sister also had both fragile sites, inherited from an obligate carrier father. He was the father of a foetus with an abnormal chromosome complement 46,XY/47,XY,+frag. The origin of the fragment could not be determined.     

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.     

New informative polymorphism at the DXS304 locus,a close distal marker for the fragile X locus

Summary The polymorphic DNA marker DXS304 detected by probe U6.2 has recently been shown to be closer to the fragile X locus than previously available markers. Its usefulness has however been limited by its relatively low heterozygosity. We have isolated, by cosmid cloning, a 67 kilobase region around probe U6.2 and have characterized a new probe (U6.2-20E) that detects BanI and BstEII restriction fragment length polymorphisms (RFLPs). The BanI RFLP has a heterozygosity of 0.49 and is in partial linkage disequilibrium with the previously described polymorphism, with a combined heterozygosity of 0.63. Furthermore, we have found that the U6.2 original probe, which probably detects an insertion-deletion polymorphism, is also informative in BanI digests. Thus, the two informative RFLPs at the DXS304 locus can be conveniently tested in a single hybridization with a single digest. An updated linkage analysis confirms that DXS304 is distal to the fragile X locus. This informative locus can now be used effectively for genetic mapping of the Xq27–q28 region, and for diagnostic applications in fragile X or Hunter syndrome families.     

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.     

Regional localization of chromosome 3-specific DNA fragments by using a hybrid cell deletion mapping panel.

A series of human chromosome 3-specific DNA fragments isolated and characterized from a lamda phage genomic library were regionally localized on human chromosome 3. This was accomplished using filter hybridization blot analysis of a human chromosome 3 hybrid cell deletion mapping panel. Twenty-three new anonymous DNA fragments were assigned to one of four physical regions of chromosome 3. Seventeen DNA fragments were mapped to the long arm of chromosome 3, including one DNA fragment that demonstrated a restriction fragment length polymorphism (RFLP). Five DNA fragments were assigned to 3p14.2----pter, including one highly polymorphic fragment sublocalized at 3p25----pter by in situ hybridization. This DNA fragment is the second reported distal 3p polymorphic probe. One DNA fragment was localized to 3p14----p14.2. In addition, three fragments previously assigned to chromosome 3 were confirmed. Polymorphic DNA probes DNF15S2 (formerly D1S1) and D3S2 were mapped to 3p14.2----pter. The previous 3p25 in situ localization of the c-raf-1 oncogene was supported by deletion panel mapping. The physical localization of these twenty-three new DNA fragments has more than doubled the number of cloned DNA fragments assigned to chromosome 3. These and future regional assignments of DNA fragment probes will facilitate construction of both a physical and genetic linkage map of chromosome 3. They may also be useful in characterizing the chromosomal and molecular aberrations involved in small-cell lung cancer (SCLC), renal cell carcinoma, other malignancies, and the 3p14.2 common fragile site.     

DNA instability at chromosomal fragile sites in cancer

Human chromosomal fragile sites are specific genomic regions which exhibit gaps or breaks on metaphase chromosomes following conditions of partial replication stress. Fragile sites often coincide with genes that are frequently rearranged or deleted in human cancers, with over half of cancer-specific translocations containing breakpoints within fragile sites. But until recently, little direct evidence existed linking fragile site breakage to the formation of cancer-causing chromosomal aberrations. Studies have revealed that DNA breakage at fragile sites can induce formation of RET/PTC rearrangements, and deletions within the FHIT gene, resembling those observed in human tumors. These findings demonstrate the important role of fragile sites in cancer development, suggesting that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. It is hypothesized that under conditions of replication stress, stable secondary structures form at fragile sites and stall replication fork progress, ultimately resulting in DNA breaks. A recent study examining an FRA16B fragment confirmed the formation of secondary structure and DNA polymerase stalling within this sequence in vitro, as well as reduced replication efficiency and increased instability in human cells. Polymerase stalling during synthesis of FRA16D has also been demonstrated. The ATR DNA damage checkpoint pathway plays a critical role in maintaining stability at fragile sites. Recent findings have confirmed binding of the ATR protein to three regions of FRA3B under conditions of mild replication stress. This review will discuss recent advances made in understanding the role and mechanism of fragile sites in cancer development.     

Linkage and recombination between fragile X-linked mental retardation and the factor IX gene

Summary Linkage analysis on a family with fragile X-linked mental retardation was performed using a Taq 1 restriction fragment length polymorphism detected by a cloned human coagulation factor IX cDNA. Two affected brothers in this sibship were found to have different factor IX RFLP alleles, indicating a recombinational event occurred between the two genes. Our data therefore indicate that the gene responsible for fragile X-linked mental retardation is not as tightly linked to the factor IX gene as the previously published data may suggest.     

New distal marker closely linked to the fragile X locus

Summary We have isolated II-10, a new X-chromosomal probe that identifies a highly informative two-allele TaqI restriction fragment length polymorphism at locus DXS466. Using somatic cell hybrids containing distinct portions of the long arm of the X chromosome, we could localize DXS466 between DXS296 and DXS304, both of which are closely linked distal markers for fragile X. This regional localization was supported by the analysis, in fragile X families, of recombination events between these three loci, the fragile X locus and locus DXS52, the latter being located at a more distal position. DXS466 is closely linked to the fragile X locus with a peak lod score of 7.79 at a recombination fraction of 0.02. Heterozygosity of DXS466 is approximately 50%. Its close proximity and relatively high informativity make DXS466 a valuable new diagnostic DNA marker for fragile X.     

Triradial configurations indicate that expression of the fragile site at Xq27 is non-lethal

The staining pattern of BUdR-substituted sister chromatids in triradial configurations associated with the fragile site at Xq27 is reported. The pattern observed in 2nd- and 3rd-generation cells is compatible with the hypothesis that the triradial configurations are caused by mitotic non-disjunction of the distal fragment in the previous mitotic division. This in turn indicates that cells can survive expression of the fragile site in a previous cell cycle.     

Induction of a BrdU-enhanceable fragile site-like lesion and sister chromatid exchanges at 11q23.1 in EBV-transformed lymphoblastoid cell lines.

We examined the expression of a fragile site-like lesion and induction of sister chromatid exchanges (SCEs) at 11q23.1 in EBV-transformed lymphoblastoid cell lines derived from carriers of distamycin A-inducible fragile sites and ataxia telangiectasia patients. The fragile site-like lesion at 11q23.1 was found to be BrdU-enhanceable in all cell lines examined, and the expression frequencies increased linearly with the rates of BrdU substitution in replicated DNA. In addition, an increased frequency of SCEs was observed at 11q23.1 on the expressed chromosome. Thus, the BrdU-enhanceable fragile site-like lesion at 11q23.1 is a "hot spot" for the formation of SCEs, as has been reported for other rare and common fragile sites.     

Genotype mosaicism in fragile X fetal tissues

Summary The 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.     

Spontaneous fragility of an abnormally wide secondary constriction region in a human chromosome No. 9

Summary In an infant with clinically and cytologically typical Down's syndrome a chromosome No. 9 was found exhibiting an exceptionally large paracentric constriction which proved to be spontaneously fragile. In cells in which this chromosome was broken the centric portion was present whereas the acentric fragment was absent. A No. 9 chromosome with identical morphology was found in the karyotype of the father but no fragility was evident. Autoradiography revealed pronounced late replication in the constriction region, suggesting a duplication or insertion of heterochromatic material. Clinically there was no evidence for any phenotypic expression of this cytological anomaly.Supported in part by the Swiss National Foundation     

Founder effect in a Belgian-Dutch fragile X population

For many years, the high prevalence of the fragile X syndrome was thought to be caused by a high mutation frequency. The recent isolation of the FMR1 gene and identification of the most prevalent mutation enable a more precise study of the fragile X mutation. As the vast majority of fragile X patients show amplification of an unstable trinucleotide repeat, DNA studies can now trace back the origin of the fragile X mutation. To date, de novo mutations leading to amplification of the CGG repeat have not yet been detected. Recently, linkage disequilibrium was found in the Australian and US populations between the fragile X mutation and adjacent polymorphic markers, suggesting a founder effect of the fragile X mutation. We present here a molecular study of Belgian and Dutch fragile X families. No de novo mutations could be found in 54 of these families. Moreover, we found significant (P < 0.0001) linkage disequilibrium in 68 unrelated fragile X patients between the fragile X mutation and an adjacent polymorphic microsatellite at DXS548. This suggests that a founder effect of the fragile X mutation also exists in the Belgian and Dutch populations. Both the absence of new mutations and the presence of linkage disequilibrium suggest that a few ancestral mutations are responsible for most of the patients with fragile X syndrome.     

Regional localization for HLA by recombination with a fragile site at 6p23.

A family with a fragile site on chromosome 6 at band p23 was examined for recombination between the fragile site and HLA. Recombination was observed in four of the 20 offspring in whom it could occur. The estimate of the genetic length of chromosome between the fragile site and HLA is 20 centimorgans (cM) with a lower 95% probability limit of 8.5 cM, placing HLA proximal to the midpoint of 6p22. The most likely regional localization is at 6p21.3, which agrees closely with methods that do not involve recombination with the fragile site. This fragile site does not measurably disrupt recombination frequency, and the allele predisposing to expression of the fragile site is situated at the fragile site.     

脆性X综合征的基因诊断与产前诊断

为了探讨简便、快速、准确、价廉的脆性X综合征的诊断方法,对6个智能低下家系进行了细胞遗传学检查,以及PCR直接扩增FMR1 5^'端(CGG)_{n<\sub>重复序列、RT-PCR扩增FMR1基因的cDNA序列的分子遗传学检查。A家系先证者脆性X染色体高表达（35/273）,分子遗传学检查证实为脆性X综合征全突变患者;B家系先证者及其母亲无脆性X染色体表达,分子遗传学检查证实为非脆性X综合征患者;C家系的男性胎儿脆性X染色体表达（5/93）,先证者及其母亲未发现脆性X染色体,分子遗传学检查证实男性胎儿为脆性X综合征全突变患者,其母亲为前突变携带者,哥哥为嵌合体患者;D家系先证者脆性X染色体高表达17%,其姐姐脆性X染色体5%,分子遗传学检查证实先证者为脆性X综合征全突变患者,其姐姐为嵌合体患者;E家系先证者及其母亲,F家系先证者发现可疑脆性X染色体,分子遗传学检查证实为非脆性X综合征家系。结论： PCR直接扩增FMR1基因(CGG)n<\sub>重复序列联合RT-PCR扩增FMR1基因cDNA 序列简便、快速、价廉。可用于脆性X综合征的筛查、诊断及产前诊断,有推广应用价值。}