title: Allelic dropout can cause false-positive results for Prader-Willi and Angelman syndrome testing.
abstract: 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.
authors.affiliation: Emory Genetics Laboratory, Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, USA.
authors.forename: Syed
authors.initials: S
authors.lastname: Hussain Askree
authors.forename: Lawrence N
authors.initials: LN
authors.lastname: Hjelm
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ali Pervaiz
authors.forename: Margaret
authors.initials: M
authors.lastname: Adam
authors.forename: Lora J H
authors.initials: LJ
authors.lastname: Bean
authors.forename: Madhuri
authors.initials: M
authors.lastname: Hedge
authors.forename: Bradford
authors.initials: B
authors.lastname: Coffee
mesh_terms: D000328:Adult; D000483:Alleles; D017204:Angelman Syndrome / Q000175:diagnosis* / Q000235:genetics*; D001483:Base Sequence; D002675:Child, Preschool; D002884:Chromosomes, Human, Pair 15 / Q000235:genetics; D019175:DNA Methylation; D003951:Diagnostic Errors; D005260:Female; D018392:Genomic Imprinting; D006801:Humans; D008297:Male; D025202:Molecular Diagnostic Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011218:Prader-Willi Syndrome / Q000175:diagnosis* / Q000235:genetics*; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics; D016415:Sequence Alignment; D044767:Ubiquitin-Protein Ligases / Q000235:genetics; D055517:snRNP Core Proteins / Q000235:genetics
chemical_list: D017411:Ribonucleoproteins, Small Nuclear; D055517:snRNP Core Proteins; C471639:UBE3A protein, human; D044767:Ubiquitin-Protein Ligases