Rapid preparation of diagnostic probes for the fragile X syndrome by direct PCR amplification of human chromosomal DNA

Yamauchi, Masatake; Seki, Naohiko; Hori, Tada-aki

doi:10.1007/bf01900713

Cited by 7 publications

(5 citation statements)

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“…The full mutation is associated with methylation in the CpG island and failure of expression of the FMR-1 gene [Pieretti et al, 19911. In general, the p(CCG)n repeat in the fragile X chromosome usually increases in size when transmitted by female carriers, while there is little alteration or reduction in size when transmitted through a male Hori et al, 19931. There is no evidence for any de novo mutation, all probands having parents who themselves have a premutation or full mutation [Yu et al, 1992;Rousseau et al, 1991;Smits et al, 19931. The increasing size of the unstable repeat through an unaffected pedigree is, therefore, accompanied by an increase in the percentage of individuals expressing the Hecht, 1985;Hori et al, 19881 and by DNA analysis using pPCRfxl as a probe [Yamauchi et al, 1992;Suzumori et al, 1993;Yamauchi et al, 19931.…”

Section: Introductionmentioning

confidence: 99%

Haplotype analysis at the FRAXA locus in the Japanese population

et al. 1994

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Fragile X syndrome, one of the most common human genetic diseases, is characterized by a unique genetic mechanism which involves dynamic mutation in a heritable unstable DNA sequence, a p(CCG)n repeat, in the FRAXA locus. It has recently been suggested that a few founder chromosomes are responsible for most fragile X mutations in the Caucasian population. In order to investigate the origin of the fragile X mutations in the Japanese population, we analyzed haplotypes of the FRAXA locus in 40 unrelated fragile X chromosomes and 142 normal X chromosomes in Japanese males, by using two polymorphic AC repeats, FRAXAC1 and FRAXAC2, which flank the fragile site. This analysis provided evidence for founder fragile X chromosomes in the Japanese population, similar to that in Caucasians, although different haplotypes are involved. The distribution of normal allele size of the p(CCG)n repeat among the X chromosomes in the Japanese population is very similar to that reported for Caucasians, except that the most frequent copy number (n = 28) is one copy less than that in Caucasians and that there is an additional peak at 35 copies. There is significant correlation between FRAXAC alleles and the p(CCG)n repeat copy number in non-fragile X chromosomes, however, alleles with more than 31 copies of the p(CCG)n repeat do not segregate with either of the fragile X common FRAXAC haplotypes.

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Section: Introductionmentioning

confidence: 99%

Haplotype analysis at the FRAXA locus in the Japanese population

et al. 1994

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“…The probe was prepared using PCR technique (Yamauchi et al 1992), and partially overlaps with other DNA probes (i.e. pfxa3 by Yu et al 1991 and 0 x 0 3 by Nakahori et al 1991).…”

Section: Resultsmentioning

confidence: 99%

“…We prepared a diagnostic DNA probe using the PCR technique. The details were described in our previous report (Yamauchi et al 1992). In brief, the 451 bp-nucleotide sequence lying within the 1 kb-PstI fragment which contains the (CCG)n repeat was directly amplified from human chromosomal DNA, using nucleotide sequence information reported previously , Fu et al 1991).…”

Section: Diagnostic Dna Probementioning

confidence: 99%

“…Seven micrograms of DNA were digested to completion with appropriate restriction endonuclease(s): PstI for genotype analysis of the dynamic mutation and EcoRI + EagI for analysis of the methylation pattern. Agarose gel electrophoresis (0.8% for EcoRI+EagI, and 1.0% for PstI) and subsequent Southern blot hydridization using pPCRfxl as a probe were performed as described previously (Yamauchi et al 1992).…”

Section: Southern Blot Hydridizationmentioning

confidence: 99%

“…The pPCRfxl carries the 451 bp-nucleotide sequence lying within the 1 kb-Pstl fragment which contains the (CCG)n repeat. This DNA probe can be used to identify the dynamic mutations involved in the fragile X syndrome (Yamauchi et al 1992, Hori et al 1993. We report here the application of pPCRfx1 to the prenatal diagnosis of a fetus which was at risk of having the fragile X mutation.…”

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Prenatal diagnosis of fragile X syndrome by direct detection of the dynamic mutation due to an unstable DNA sequence

Yamauchi¹,

Nagata

Seki

et al. 1993

Clinical Genetics

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Yamauchi M, Nagata S, Seki N, Toyama Y, Harada N, Niikawa N, Masuno I, Kajii T, Hori T. Prenatal diagnosis of fragile X syndrome by direct detection of the dynamic mutation due to an unstable DNA sequence. Clin Genet 1993: 44: 169–172. © Munksgaard, 1993 The fragile X syndrome is the most common familial form of mental retardation. The mutation causing the syndrome is dynamic mutation due to an unstable DNA (CCG)n repeat localized at Xq27.3. We have previously reported a PCR procedure to prepare a diagnostic probe, pPCRfx1, which can be used to determine the genotype of fragile X mutation individuals by Southern blot analysis. In the present study, pPCRfx1 was applied to the prenatal diagnosis, using chorionic villus cells, of a fetus which was at risk of having fragile X syndrome. In the PstI assay, the Southern blot showed the typical pattern of a female carrier with the full mutation. Analysis of the DNA methylation patterns by EcoRI + EagI assay showed that the EagI restriction site was not methylated on the mutated X chromosome of chorionic villi, but the sites were totally methylated in the brain and other tissues of the fetus. Thus the fetus was diagnosed to be a heterozygous female carrier of the dynamic mutation involved in the fragile X syndrome.

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