Parent-of-origin testing for 15q11-q13 gains by quantitative DNA methylation analysis.

The Prader-Willi/Angelman syndrome critical region (PWS/ASCR), located at chromosome 15q11-q13, is associated with several diseases. Absence of paternally expressed genes in this region cause Prader-Willi syndrome (PWS), whereas absence of the maternally expressed UBE3A gene causes Angelman syndrome (AS). In addition, duplications and triplications of this region are also associated with distinct clinical features, indicating that the overexpression of genes within the PWS/ASCR can also lead to distinct phenotypes. Maternally inherited increases in copy number generally lead to a more severe phenotype do than paternally inherited increases. We describe a real-time methylation-sensitive PCR (Q-MSP) assay that quantifies methylation at the promoter of the differentially methylated SNRPN gene located within the PWS/ASCR. Q-MSP can detect both PWS and AS, as well as determine the parent of origin for the allele that carries the PWS/ASCR gains. In addition, Q-MSP requires only a small amount of DNA, is amenable to high-throughput analysis, and can be used in clinical testing as a reflex test to determine the parent of origin after identification of a gain of this region on chromosome 15.

Allelic dropout can cause false-positive results for Prader-Willi and Angelman syndrome testing.

The diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing. Our laboratory uses a standard methylation-sensitive PCR (MSP) to target the differentially methylated SNRPN gene to test for Prader-Willi syndrome (PWS) and Angelman syndrome. One patient, a 27-month-old female, who lacked the classical clinical features of PWS, but had a molecular diagnosis of PWS by MSP by another laboratory, had repeat testing in our laboratory. Testing by MSP in our laboratory also identified an apparent loss of the unmethylated paternal allele, consistent with a diagnosis of PWS. Confirmatory testing using Southern blot analysis with a methylation-sensitive restriction enzyme showed a normal pattern of methylation, detecting both the methylated maternal and unmethylated paternal alleles. To investigate these discrepant results, we amplified and sequenced the SNRPN locus in this patient and identified a single nucleotide change within the binding site for the unmethylated DNA-specific primer. These results indicate this nucleotide change led to allelic dropout in the MSP analysis, yielding the false-positive result. Subsequently, MSP analysis using an alternate primer set that was developed by our laboratory detected both methylated and unmethylated alleles. These findings illustrate that allelic dropout due to the presence of rare polymorphisms can cause false-positive results in commonly used MSP assays and lead to molecular misdiagnosis.

A simple method to confirm and size deletion, duplication, and insertion mutations detected by sequence analysis.

Characterizing heterozygous insertions or deletions in genes by PCR and Sanger sequencing can be a challenge due to overlapping sequencing traces produced by overlapping templates. This is particularly problematic for clinical diagnostic laboratories, because mutations must be precisely characterized. Although the mutation detection software used by clinical diagnostic laboratories reliably identifies small insertions and deletions, overlapping deletions and insertions on opposite chromosomes, complex rearrangements, and insertions or deletions close to the primer sites may be missed. Here we describe a rapid, simple method to confirm and precisely characterize deletions and insertions using a capillary-based gel electrophoresis system. This technique has been applied to a series of patients with deletion, duplication, or insertion mutations identified by sequencing, as well as to patients with repeat tract polymorphisms, to demonstrate the utility of this method.

Mosaic FMR1 deletion causes fragile X syndrome and can lead to molecular misdiagnosis: a case report and review of the literature.

The most common cause of fragile X syndrome is expansion of a CGG trinucleotide repeat in the 5'UTR of FMR1. This expansion leads to transcriptional silencing of the gene. However, other mutational mechanisms, such as deletions of FMR1, also cause fragile X syndrome. The result is the same for both the expansion mediated silencing and deletion, absence of the gene product, FMRP. We report here on an 11-year-old boy with a cognitive and behavioral profile with features compatible with, but not specific to, fragile X syndrome. A mosaic deletion of 1,013,395 bp was found using high-density X chromosome microarray analysis followed by sequencing of the deletion breakpoints. We review the literature of FMR1 deletions and present this case in the context of other FMR1 deletions having mental retardation that may or may not have the classic fragile X phenotype.

Characterization of an unusual deletion of the galactose-1-phosphate uridyl transferase (GALT) gene.

PURPOSE: We previously reported a deletion of the Galactose-1-Phosphate Uridyl Transferase (GALT) gene. This deletion can cause apparent homozygosity for variants located on the opposite allele, potentially resulting in a discrepancy between the biochemical phenotype and the apparent genotype in an individual. The purpose of this study was to determine the deletion breakpoints, allowing the development of a rapid and reliable molecular test for the mutation. METHODS: A Polymerase Chain Reaction walking strategy was used to map the 5' and 3' breakpoints. The junction fragment was amplified and sequenced to precisely characterize the deletion breakpoints. RESULTS: The deletion has a bipartite structure involving two large segments of the GALT gene, while retaining a short internal segment of the gene. Molecular characterization allowed the development of a deletion specific Polymerase Chain Reaction-based assay. In 25 individuals who had a biochemical carrier galactosemia phenotype, but tested negative for 8 common GALT gene variants, 3 carried this deletion. CONCLUSION: This deletion occurs at an appreciable frequency and should be considered when there is a discrepancy between the genotype and biochemical phenotype. Many of the individuals carrying the allele were of Ashkenazi Jewish ancestry suggesting that the deletion may be a common cause of galactosemia in that population.