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. 2017 Dec 7;101(6):965–976. doi: 10.1016/j.ajhg.2017.11.007

Figure 2.

Figure 2

AEI of PEX6 in Affected Individuals and Control Individuals with the Heterozygous 3′ UTR Variant PEX6 c.∗442_445delTAAA

(A and B) Allelic expression of PEX6 mRNA in cells of affected individuals and the mother of affected individual 2. PEX6 cDNA and genomic DNA were Sanger sequenced and the peak height of both PEX6 alleles were used to determine the relative abundance of each allele (see representative sequence reads in A). Mean c.2578T allele peak ratios in the different cell lines (B), expressed as the ratio of the mutant allele peak height (c.2578T) over the total peak height (c.2578T+c.2578C) as described in Moncini et al.,14 depicted as mean with standard deviation of 6–19 analyzed electropherograms per cell line (or gDNA group) using 2–3 different primer sets. While the PEX6 allele ratio is in balance in genomic DNA of the affected individuals and genomic DNA and cDNA of the mother of individual P2 (i.e., equal peak heights), the mutant allele ratio in cDNA of all affected individuals is significantly increased, demonstrating the AEI of PEX6.

(C and D) PEX6 mRNA expression in fibroblasts of affected individuals and fibroblasts of 22 unrelated control individuals with different combinations of the PEX6 c.∗442_445delTAAA allele (homozygous for the allele without the deletion [“TAAA / TAAA”], homozygous for the PEX6 c.∗442_445delTAAA allele [“delTAAA / delTAAA”] or compound heterozygous [“TAAA / delTAAA”]).

(C) Mean allele peak ratios for the PEX6 c.∗442_445delTAAA allele in cDNA of cells of the affected individuals and control cells heterozygous and homozygous for this allele, confirming that the AEI of PEX6 occurs only in the heterozygous cells. Shown are the peak ratios of the c.2816A variant, which is a common polymorphism (MAF 0.39 [ExAc] / 0.33 [1000 Genomes]) that is in cis with PEX6 c.∗442_445delTAAA (see also Table 3). The peak ratios were determined as described in (B) and Moncini et al.14 and are presented as mean with standard deviation based on analysis of 5–10 electropherograms from 4–7 different cell lines per allele combination.

(D) Results of quantitative RT-PCR experiments, demonstrating an increased level of total PEX6 mRNA but no expression of longer PEX6 c.∗1_462 mRNA in cells homozygous for the PEX6 c.∗442_445delTAAA. Depicted are the PEX6 mRNA levels normalized to reference gene expression or as ratio PEX6 c.∗1_462 over total PEX6, with “TAAA / TAAA” values set as 1, as mean with standard deviation determined in 4–13 cell lines per allele combination.

Statistical analyses for (B) and (C) were performed using one-sample t tests with a theoretical mean of 0.5, and for (D) using Mann-Whitney U tests (∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, ns not significant).