Introduction: Cystic fibrosis is an autosomal recessive chloride ion channelopathy causing disruption of exocrine function of the pancreas, intestinal glands, biliary tree, bronchial glands and sweat glands. Pre-conception carrier screening for cystic fibrosis is common.
The molecular findings from an ARMS assay that required detailed analysis to arrive at the underlying molecular mechanism are presented.
Methods: The Elucigene CFEU2v1 kit detects 50 of the most common variants found in European populations, as well as the intron 9 (previously intron 8) polyT tract and adjacent TG repeat. It is a multiplex-fluorescent ARMS PCR assay which has two panels of primers – one panel targeting the specific variants and a second panel targeting the equivalent wild types, allowing the differentiation of variants in the heterozygous and homozygous states.
The assay also has the added benefit of being able to detect the presence though not the genomic location of small insertions and deletions within the amplicons as well as some variants other than the 50 that are targeted in the assay. Insertions and deletions alter the size of the PCR product produced while variants that fall under the assay primers can disrupt primer binding and thus reduce the amount of product produced. Both of these can be visualised on the assay electropherograms.
Results: A double peak was identified at one locus, suggesting an insertion or deletion, and a half-height peak at another locus, suggesting a variant disrupting the binding of one of the primers.
Following Sanger sequencing of all exons thought to potentially contain these two variants, only one variant was identified – CFTR:c.3874-64_3874-51del – a 14bp benign intronic deletion. This deletion was the cause of both anomalies in this individual’s electropherograms. The single deletion appeared to cause two mutational signals by altering the size of one amplicon.
Monica is a Hospital Scientist in Molecular Genetics at SEALS Pathology, Prince of Wales Hospital. She is the team leader for the Community Genetics section of the laboratory and focuses on cystic fibrosis and other community-based screening.