The c.1364C>A (p.A455E) Mutation in the CFTR Pseudogene Results in an Incorrectly Assigned Carrier Status by a Commonly Used Screening Platform  Kristin K. Deeb, James D. Metcalf, Kaitlin M. Sesock, Junqing Shen, Christine A. Wensel, Larisa I. Rippel, Michelle Smith, Mark S. Chapman, Shulin Zhang  The Journal of Molecular Diagnostics  Volume 17, Issue 4, Pages 360-365 (July 2015) DOI: 10.1016/j.jmoldx.2015.02.005 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Sequence alignment and primer locations of CFTR exon 10 and the CFTR pseudogenes. A: Schematic illustration of the primers spanning exon 10 and adjacent intronic regions of the CFTR gene. Black arrowheads designate primers identical between the CFTR gene and CFTR pseudogene sequences in the human genome; white arrowheads indicate location of primers for specific PCR amplifications of the different regions. The CFTR, CFTRP1, and CFTRP3 primer sequences are listed in Table 1. B: Multiple sequence alignments of CFTR exon 10 (NM_000492.3) and the CFTR pseudogenes CFTRP3 and CFTRP1. The homology between exon 10 of the CFTR gene and CFTRP1 and CFTRP3 is 99% and 97%, respectively. Approximately 95% homology is shared between the two pseudogenes. The exon-intron boundaries of CFTR exon 10 are indicated by brackets. The nucleotide of interest at CFTR coding position 1364 is highlighted by a white arrow. Primers (Table 1) specific for CFTR exon 10 and the CFTR pseudogenes are underlined and noted along the sequences. The CFTR-ex10-3F and CFTR-ex10-3R primers, shown in italics, could anneal to the CFTR gene and the two pseudogenes. The most 3′-nucleotides of the reverse primers, highlighted in gray, do not share homology to the other aligned sequences. The Journal of Molecular Diagnostics 2015 17, 360-365DOI: (10.1016/j.jmoldx.2015.02.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Sequence analysis of CFTR exon 10 and CFTR pseudogenes. Primers specific to the CFTR gene and the CFTRP3 and CFTRP1 pseudogenes are used for PCR amplification and sequence analysis (Table 1). A: PCR amplification and sequence analysis using primers CFTR-ex10-3F and CFTR-ex10-3R (amplicon C). Note that these primers will co-amplify CFTR exon 10, CFTRP3, and CFTRP1 (Figure 1). Two different overlapping sequences are predominantly observed, but three different alleles (C/A/T) are apparent at position c.1364 (red arrow). B: Sequence analysis using primers specific to CFTR exon 10 reveals that the c.1364C>A (p.A455E) mutation is not in the CFTR gene as indicated by asterisks. The c.1364C>A (p.A455E) mutation is present in another patient. C: The C>A nucleotide change is not on the CFTRP3 pseudogene. D: A g.25919527C>A variant on chromosome 20 is observed in the CFTRP1 pseudogene as noted by asterisks. The variant is present in both the proband and his mother. The Journal of Molecular Diagnostics 2015 17, 360-365DOI: (10.1016/j.jmoldx.2015.02.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions