ABSTRACT
In this study, we report a familial inversion of chromosome 18, inv(18)(p11.31q21.33), in both members of a consanguineous couple. Their first child had inherited one balanced pericentric inversion along with a recombinant chromosome 18 resulting in dup(18q)/del(18p), and had mild dysmorphic features in the absence of mental and developmental retardation. The second child had received two recombinant chromosomes 18, from the mother a derivative chromosome 18 with dup(18p)/del(18q) and from the father a derivative chromosome 18 with dup(18q)/del(18p). The aberration was prenatally detected; however, as the two opposite aneuploidies were thought to compensate each other, the family decided to carry on with the pregnancy, knowing that uniparental disomy for the segments outside the inversion could have an adverse influence on the development of the child. Uniparental disomy was confirmed by SNP arrays. The child, who has been followed up until the age of 20 months, is healthy and normal. It seems to be the first reported case with two opposite recombinant chromosomes that compensate each other and lead to segmental uniparental disomy for two segments on the chromosome, one maternal and the other paternal.
Subject(s)
Chromosome Inversion , Chromosomes, Human, Pair 18 , Uniparental Disomy , Child , Child, Preschool , Consanguinity , Female , Humans , In Situ Hybridization, Fluorescence , Male , PedigreeABSTRACT
BACKGROUND: DNA methylation analysis currently requires complex multistep procedures based on bisulfite conversion of unmethylated cytosines or on methylation-sensitive endonucleases. To facilitate DNA methylation analysis, we have developed a quantitative 1-step assay for DNA methylation analysis. METHODS: The assay is based on combining methylation-sensitive FastDigest(R) endonuclease digestion and quantitative real-time PCR (qPCR) in a single reaction. The first step consists of DNA digestion, followed by endonuclease inactivation and qPCR. The degree of DNA methylation is evaluated by comparing the quantification cycles of a reaction containing a methylation-sensitive endonuclease with the reaction of a sham mixture containing no endonuclease. Control reactions interrogating an unmethylated locus allow the detection and correction of artifacts caused by endonuclease inhibitors, while simultaneously permitting copy number assessment of the locus of interest. RESULTS: With our novel approach, we correctly diagnosed the imprinting disorders Prader-Willi syndrome and Angelman syndrome in 35 individuals by measuring methylation levels and copy numbers for the SNRPN (small nuclear ribonucleoprotein polypeptide N) promoter. We also demonstrated that the proposed correction model significantly (P < 0.05) increases the assay's accuracy with low-quality DNA, allowing analysis of DNA samples with decreased digestibility, as is often the case in retrospective studies. CONCLUSIONS: Our novel DNA methylation assay reduces both the hands-on time and errors caused by handling and pipetting and allows methylation analyses to be completed within 90 min after DNA extraction. Combined with its precision and reliability, these features make the assay well suited for diagnostic procedures as well as high-throughput analyses.
Subject(s)
Angelman Syndrome/diagnosis , DNA Methylation , DNA/analysis , Prader-Willi Syndrome/diagnosis , snRNP Core Proteins/genetics , Angelman Syndrome/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , DNA Restriction Enzymes/chemistry , Feasibility Studies , Gene Dosage , Genetic Loci , Genome, Human , Genomic Imprinting , Humans , Polymerase Chain Reaction/methods , Prader-Willi Syndrome/genetics , Promoter Regions, GeneticABSTRACT
We describe a young woman with Prader-Willi syndrome (PWS) due to a mosaic imprinting defect. Three independent assays revealed a reduced proportion of nonmethylated SNURF-SNRPN alleles in peripheral blood DNA: methylation-specific PCR followed by denaturing high-performance liquid chromatography (MSP/DHPLC), methylation-sensitive restriction enzyme analysis and methylation-specific real-time PCR analysis. Microsatellite analysis and fluorescence in situ hybridisation revealed apparently normal chromosomes 15 of biparental origin. Based on the MSP/DHPLC and real-time PCR results, we estimate that approximately 50% of the patient's blood cells have an imprinting defect and 50% of the cells are normal. Apart from a rather normal facial appearance, the proband has typical features of PWS.
