1/9. Rare variants in the promoter of the fragile x syndrome gene (FMR1).fragile x syndrome, the most common form of familial mental retardation, is mainly caused by the expansion of an unstable region of CGG repeats in the 5' untranslated region of the FMR1 (Fragile X Mental Retardation-1) gene. Molecular tools to detect an abnormal CGG expansion in FMR1 include Southern blot hybridization and PCR amplification. Southern blotting with the StB12.3 probe and Eco RI/Eag I double digestion is widely used as a routine test for fragile x syndrome diagnosis in laboratories around the world. A patient with mental retardation of unknown origin showed absence of digestion for Eag I due to a -149C-->G substitution in the CpG island of the FMR1 gene, which destroys that restriction enzyme site. Screening for other changes around that region also detected a -154insGGC in a patient with a phenotype highly suggestive of fragile X syndrome but without CGG expansion. Expression studies did not show any abnormal changes in FMR1 function. In summary, we have identified two different changes (a C to G substitution at -149 and a GGC insertion at -154) in the promoter of the FMR1 gene. These are the first variants described in the promoter of the FMR1 gene.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
2/9. Identification of small FRAXA premutations.BACKGROUND: Diagnosis of fragile x syndrome in mentally retarded individuals is satisfactorily achieved using a Southern blot test that detects the typical triplet repeat expansion (>200 repeats) within the FMR1 gene. All such individuals inherit the mutation from a carrier, who usually shows a lower triplet repeat number and may be asymptomatic. Having identified a fragile X proband, it is necessary to identify related carriers of this familial X-linked dominant mutation to provide family counseling and testing. methods AND RESULTS: For one family in which a fragile XA repeat expansion occurs, Southern blot hybridization did not give accurate sizing data because of the very small premutation associated with the unstable allele. PCR sizing methods and linkage analysis were adapted to identify family members with the premutation. CONCLUSION: Although most carriers can be detected using Southern blot and/or direct PCR sizing tests, very small expansions (55-70 repeats) are difficult to distinguish from larger, normal alleles. We have used linkage analysis in combination with direct allele analysis to identify carriers of very small expansions of a fragile x chromosome in a four-generation family.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
3/9. Characterisation of a new rare fragile site easily confused with the fragile X.A new fragile site (FRAXE) in Xq28 is described. It appears to be a typical folate sensitive fragile site. The fragile site is not associated with mental retardation, it does not give abnormal results when subjected to Southern analysis with probe pfxa3 which detects the unstable dna sequence characteristic of fragile x syndrome. in situ hybridization mapping locates the fragile site between 150 kb and 600 kb distal to FRAXA. The distinction between the two fragile sites is important clinically since cytogenetic detection of FRAXE, without molecular analysis, could result in misdiagnosis of fragile x syndrome.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
4/9. Fragile X genotype characterized by an unstable region of dna.dna sequences have been located at the fragile X site by in situ hybridization and by the mapping of breakpoints in two somatic cell hybrids that were constructed to break at the fragile site. These hybrids were found to have breakpoints in a common 5-kilobase Eco RI restriction fragment. When this fragment was used as a probe on the chromosomal dna of normal and fragile X genotype individuals, alterations in the mobility of the sequences detected by the probe were found only in fragile X genotype dna. These sequences were of an increased size in all fragile X individuals and varied within families, indicating that the region was unstable. This probe provides a means with which to analyze fragile X pedigrees and is a diagnostic reagent for the fragile X genotype.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
5/9. No mental retardation in a man with 40% abnormal methylation at the FMR-1 locus and transmission of sperm cell mutations as premutations.We report the case of a mentally normal male carrier of a fragile X full mutation with a 'methylation mosaic' hybridization pattern, who carried premutation-size mutations in his sperm cells and transmitted one of them to a daughter. Clinical evaluation of the father revealed a phenotype resembling that of the fragile x syndrome but without mental impairment and 4% fragile sites on cytogenetic analysis. Direct FRAXA genotyping revealed 40% abnormal methylation at the classical EagI FMR-1 restriction site, a delta of 400 bp to 1400 bp associated, in the non-methylated region, to a widely spread smear. This is thus a rare occurrence of a male carrier of a fragile X full mutation with significant methylation of the EagI site and no mental impairment. A premutation of 400 bp was detected in his daughter's leukocytes and analysis of sperm cells from the father revealed a normal spermogram and only 400 bp premutations. This is the first documented case of transmission (with analysis of sperm dna) of a fragile X mutation from a male carrier of a full fragile X mutation to a girl. This may be due to the early setting apart of progenitor germ cells in males having inherited a premutation from their mother. It is more likely that there could be a strong selection process favouring those cells with a reverted full mutation which produced a small and unmethylated FMR-1 CpG island allowing for re-expression of the FMR-1 gene, especially in male germ cells.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
6/9. sex determination of human embryos using the polymerase chain reaction and confirmation by fluorescence in situ hybridization.OBJECTIVE: To use fluorescence in situ hybridization to corroborate the polymerase chain reaction (PCR) preimplantation diagnosis of human embryos in three couples carrying a chromosome X-linked disease. SETTING: Clinical and research IVF laboratories. patients: Individuals undergoing preimplantation diagnosis. RESULTS: Four ETs were performed in couples undergoing preimplantation diagnosis by multiplex PCR or fluorescence in situ hybridization, resulting in the birth of two normal female twins. The result of another is pending. A total of 22 embryos were analyzed by PCR. Embryos that were diagnosed as being at risk of carrying the genetic abnormality (n = 8), embryos that failed diagnosis (n = 4), and genetically normal embryos that arrested development (n = 4) were further analyzed by fluorescence in situ hybridization. The sex of all 16 embryos was determined and confirmed the previous 12 preimplantation diagnoses by multiplex PCR. In addition, fluorescence in situ hybridization analysis allowed the detection of two aneuploid embryos, one XO and one XXY, previously diagnosed by PCR as a normal female and male. Two mosaics were also detected. CONCLUSION: polymerase chain reaction and fluorescence in situ hybridization are possible for preimplantation sex determination in cases of genetic sex-linked disease. fluorescence in situ hybridization, however, supplies additional information about sex chromosome aneuploidy and is not susceptible to contamination or misdiagnosis of monosomy X.- - - - - - - - - - ranking = 10keywords = hybridization (Clic here for more details about this article) |
7/9. Segregation of the fragile X mutation from a male with a full mutation: unusual somatic instability in the FMR-1 locus.fragile x syndrome is associated with an unstable CGG-repeat in the FMR-1 gene. There are few reports of affected males transmitting the FMR-1 gene to offspring. We report on a family in which the propositus and his twin sister each had a full mutation with abnormal methylation. Their mother had an FMR-1 allele in the normal range and a large premutation, with normal methylation. The maternal grandmother had two normal FMR-1 alleles. The maternal grandfather had an unusual somatic FMR-1 pattern, with allele size ranging from premutation to full mutation. No allele was detectable by PCR analysis. Multiple Southern blot analyses identified a hybridization pattern that originated at a distinct premutation band and extended into the full mutation range. methylation studies revealed a mosaic pattern with both unmethylated premutations and methylated full mutations. This individual declined formal evaluation but did not finish high school and has difficulty in reading and writing. The size of the premutation FMR-1 allele passed to his daughter is larger than his most prominent premutation allele. This is most likely due to gonadal mosaicism similar to that in his peripheral lymphocytes. Alternatively, this expansion event may have occurred during his daughter's early embryonic development and this large premutation allele is mitotically unstable. This pattern of FMR-1 alleles in a presumably mildly affected male is highly unusual. These findings are consistent with the absence of transmission of a full fragile X mutation through an expressing male. Studies of tissue specific FMR-1 allele expansion and FMR-1 protein expression on this individual should help to determine the correlation of the molecular findings with the phenotypic effects.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
8/9. Disappearing trisomy 8 mosaicism.A case is presented of a patient with disappearing trisomy 8 mosaicism initially thought to have stigmata of the fragile x syndrome. This case is interesting for two reasons. First, it demonstrates the occurrence of "disappearing mosaicism," a phenomenon first described by LaMarche et al, in 1967. Our patient, initially studied in 1991 by two laboratories and found to be mosaic for chromosome 8 trisomy, was apparently normal by both GTG-banding and fluorescent in situ hybridization (FISH) when studied in 1996. Second, this case further underscores the fact that except under special circumstances, it is important that GTG-banding analysis be performed so that the entire human genome be examined in addition to scoring for the fragile X mutation on Xq27.3. In a recent review of the existing database at rhode island Hospital on chromosomal abnormalities found in patients referred because of a question of the fragile x syndrome during the period from January 1, 1990 to June 30, 1995, it was found that the frequency of other chromosomal abnormalities in patients referred because of a question of fragile x syndrome equaled or exceeded that of patients found to be positive for fragile X. Our figures, consistent with those reported in the literature, underscore the value of routine karyotyping in this population of patients.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
9/9. Deletions in Xq26.3-q27.3 including FMR1 result in a severe phenotype in a male and variable phenotypes in females depending upon the X inactivation pattern.High resolution cytogenetics, microsatellite marker analyses, and fluorescence in situ hybridization were used to define Xq deletions encompassing the fragile X gene, FMR1, detected in individuals from two unrelated families. In family 1, a 19-year-old male had facial features consistent with fragile x syndrome; however, his profound mental and growth retardation, small testes, and lover limb skeletal defects and contractures demonstrated a more severe phenotype, suggestive of a contiguous gene syndrome. A cytogenetic deletion including Xq26.3-q27.3 was observed in the proband, his phenotypically normal mother, and his learning-disabled non-dysmorphic sister. methylation analyses at the FMR1 and androgen receptor loci indicated that the deleted X was inactive in > 95% of his mother's white blood cells and 80-85% of the sister's leukocytes. The proximal breakpoint for the deletion was approximately 10 Mb centromeric to FMR1, and the distal breakpoint mapped 1 Mb distal to FMR1. This deletion, encompassing approximately 13 Mb of dna, is the largest deletion including FMR1 reported to date. In the second family, a slightly smaller deletion was detected. A female with moderate to severe mental retardation, seizures, and hypothyroidism, had a de novo cytogenetic deletion extending from Xq26.3 to q27.3, which removed approximately 12 Mb of dna around the FMR1 gene. Cytogenetic, and molecular data revealed that approximately 50% of her white blood cells contained an active deleted X. These findings indicate that males with deletions including Xq26.3-q27.3 may exhibit a more severe phenotype than typical fragile X males, and females with similar deletions may have an abnormal phenotype if the deleted X remains active in a significant proportion of the cells. Thus, important genes for intellectual and neurological development, in addition to FMR1, may reside in Xq26.3-q27.3. One candidate gene in this region, SOX3, is thought to be involved in neuronal development and its loss may partly explain the more severe phenotypes of our patients.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |