PMC3741656_01
Heritable pulmonary arterial hypertension (HPAH) kindred for the patient with the W13X mutation. Boxes represent males and circles females. Solid boxes and circles indicated positive bone morphogenetic protein receptor type 2 (BMPR2) mutation status and disease. Solid circle in the box represent unaffected mutation carrier. Diagonal line through a box or circle represents a person who is deceased. The arrow indicates the patient analyzed in this study. Numbers adjoining symbols indicate age of the person.
nihms497337f1.jpg
PMC3741656_01_nihms497337f1.jpg
F1
Specifically, his maternal grandmother died at 38 years of age from heart failure of unknown cause, his mother was diagnosed with and died of PAH at age 35 years, and two female siblings died of PAH in the post-partum period at ages 27 and 28 (Fig. 1).
Presence of a W13X mutation that is not subject to nonsense mediated decay (NMD). (a) Heterozygous G>A (W13X) substitution in the 13th codon (TGG>TGA) detected in cDNA of cultured lymphocyte (CL) cells derived from PPH151 (proband) and incubated with puromycin to inhibit NMD. (b) Mutated transcript can still be detected in the puromycin untreated PPH151cells, suggesting that the presence of this premature termination codon (PTC) did not result in the complete degradation of the mutated transcript. Electropherograms (c) and (d) show a control CL cell line PPH143 selected for a positive NMD response secondary to being heterozygous for a different PTC gene mutation. It behaves in the expected manner and causes degradation of the transcript expressed from the mutated allele in the absence of puromycin.
nihms497337f2.jpg
PMC3741656_01_nihms497337f2.jpg
F2
This PTC would be predicted to activate the NMD pathway and thus give rise to a null allele (Fig. 2a) in the absence of puromycin. Surprisingly, when these CLs were grown in the absence of puromycin (resulting in normal activity of the NMD pathway), we still detected nearly equal levels of transcripts from both the normal and the PTC-containing BMPR2 alleles (Fig. 2b), compared to a control sample that contains a PTC that results in the activation of the NMD pathway (Fig. 2c,d). These data complemented the transcript data presented in Figure 2 and showed the presence of a full-length 113 kD BMPR-II product, as well as a much shorter 96 kD BMPR-II product (Fig. 4a lane 3).
Strong Kozak sequences are present downstream to the premature termination codon (PTC). cDNA sequence downstream of the W13X mutation was analyzed and 27 in-frame AUG codons identified (only six are shown). NetStart 1.0 was used to determine translation initiation scores for the wild-type AUG start site and the downstream AUGs. Protein size was estimated using the ProtParam tool.
nihms497337f3.jpg
PMC3741656_01_nihms497337f3.jpg
F3
Gentamicin treatment results in a full-length bone morphogenetic protein receptor type II (BMPR2-II) product. (a) Western blot analysis in control and patient (proband)-derived cultured lymphocyte (CL) cells. The W13X mutation led to translation reinitiation downstream from the original start site. This resulted in an increased amount of a smaller (96 kD) protein product and a decreased amount of the full-length BMPR-II product (lane 3). Gentamicin treatment allowed for translational read-through of the premature termination codon (PTC), resulting in decreased 96 kD product and a corresponding increase in the full-length 115 kD product (lane 4). (b) BMPR-II signaling is also increased in the gentamicin treated CLs, derived from the proband, as shown by increased phospho-Smad1/5/8 by western blot analysis. Western blot quantification was done using ImageJ software (Collins TJ; 2007 BioTechniques 43 25-30). p values were calculated by Student's t -test. Statistical analysis was done with Prism 5 software suite for Mac.
nihms497337f4.jpg
PMC3741656_01_nihms497337f4.jpg
F4
As shown in Figure 4a, lanes three and four, gentamicin treatment decreased levels of the truncated (96 kD) BMPR-II product but increased full-length (113 kD) BMPR-II product levels. As shown in Figure 4b, gentamicin treatment resulted in increased signaling through the BMPR-II pathway as indicated by increased Smad1 phosphorylation.