Evaluation of the fragile X (FRAXA) syndrome with methylation-sensitive PCR

Abstract

The fragile X (FRAXA) syndrome is the most common form of inherited mental retardation in males. Its peculiar pattern of inheritance results from the parent of origin-specific expansion of a CGG-repeat within the FMR1 gene on the X chromosome. In patients, gene function is abolished by hypermethylation of the promoter and the massively expanded repeat. We have developed a methylation-sensitive polymerase chain reaction (MS-PCR) strategy that combines repeat-length and methylation analysis of the CGG-repeat and the promoters of the FMR1 and XIST genes. The allelic methylation of the latter opposes that of the FMR promoter and serves as an internal control and standard for semiquantitative analyses. This system enables the delineation of 11 distinct patterns encountered in nonaffected, carrier, and affected males and females. We have evaluated our system on well-defined samples with different FMR1 mutations and have used it for the diagnostic evaluation of 253 male and 80 female probands. In the male group, we have identified five full mutations, and three gray-zone and premutation alleles with 54, 55, and 62 repeats, respectively. The female group consists of 33 normal homozygote and 41 heterozygote individuals, two of whom harbor a gray-zone allele with 47 repeats, none with a premutation, and six with a full mutation. Our MS-PCR approach allows the currently most comprehensive diagnostic evaluation of the FRAXA syndrome in a cost- and time-efficient fashion. In addition, it is a valuable tool for the analysis of clonality and skewing phenomena in females.

PCR-Based Analysis of Differentially Methylated Regions of GNAS Enables Convenient Diagnostic Testing of Pseudohypoparathyroidism Type Ib

“…Both un- methylated and methylated restriction digests were incubated for 16 h at 37°C. We carried out bisulfite DNA deamination on 500 ng of DNA for 4 h at 55°C with an oil overlay, as has previously been described (18,19 ). After deamination, we purified the bisulfite-treated DNA on QIAquick Spin Columns (Qiagen) and used it for PCRs.…”

Four novel and three recurrent mutations of the BTK gene and pathogenic effects of putative splice mutations

“…To determine the X-inactivation pattern in the mothers who were heterozygous for the protein truncation mutation (1749delT, IVS8 + 1G > A, and c.588G > C) and shown to have null or minimal amount of the mutant transcripts, methylation-specific PCR (MSP) was performed following previously published protocols with some modifications (Kubota et al 1999;Nakayama et al 2000;Weinhausel and Haas 2001). The assays included two steps: bisulfite treatment followed by PCR amplification with primers specific to either methylated or unmethylated DNA.…”

“…The XIST promoter served as an internal control. The XIST gene promoter is methylated on the active X chromosome, therefore the allelic methylation pattern opposes that of the FMR1 promoter (Weinhausel and Haas 2001). Polymorphism of the trinucleotide repeats was intended for use in the determination of percentage methylation for each site.…”

Síndrome de tremor e ataxia associada ao X frágil: rastreamento por PCR em amostra de idosos

“…Os alelos com mais de 200 repetições, conhecidos como mutações completas, determinam a síndrome do X frágil. A presença da mutação leva à inativação do gene FMR1 decorrente da metilação na região promotora e, consequentemente, ausência de FMRP 4,6 . Na faixa entre 55 e 200 repetições CGG estão os alelos pré-mutados.…”