Heterozygous variant at nucleotide position 875+11A>T in exon 6A cystic fibrosis transmembrane conductance regulator gene induces 852del22 mutation false-positivity by line probe assay

Abstract

Objective: To explain the lack of genotype-phenotype correlation observed in a patient double heterozygous for the 852del22 and F508del mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Design: Case report.

Setting: Medical laboratory department.

Patient(s): A 42-year-old asymptomatic patient underwent genetic screening for in vitro fertilization (IVF).

Intervention(s): CFTR genetic screening (commercial kit aimed at detecting 57 mutations), segregation analysis, evaluation of the polymerase chain reaction (PCR) products using a denaturing high performance liquid chromatography (DHPLC), and sequence analysis.

Main outcome measure(s): To avoid diagnostic errors and improve genetic counseling.

Result(s): Segregation analysis allowed us to establish that the mutations were in trans. Analysis of the PCR products using a DHPLC apparatus showed a heteroduplex formation indicative of a heterozygous variant in exon 6A. Direct sequencing characterized the heterozygous variant as an A to T transversion at nucleotide position 875+11. Therefore, the change of one single nucleotide in a portion surrounding the 852del22 mutation facilitated the aspecific interaction between the commercial oligonucleotide probe and the amplified genomic DNA, which explains the 852del22 mutation false molecular positivity that was detected by the line probe assay.

Conclusion(s): The individualization of 852del22 mutation by a standard genetic panel should be confirmed by more extensive analyses.