Aminoglycoside-stimulated readthrough of premature termination codons in selected genes involved in primary ciliary dyskinesia

<p>Translational readthrough of premature termination codons (PTCs) induced by pharmacological compounds has proven to be an effective way of restoring functional protein expression and reducing symptoms in several genetic disorders. We tested the potential of different concentrations of several aminoglycosides (AAGs) for promoting PTC-readthrough in 5 genes involved in the pathogenesis of primary ciliary dyskinesia, an inherited disorder caused by the dysfunction of motile cilia and flagella. The efficiency of readthrough stimulation of PTCs cloned in dual reporter vectors was examined in 2 experimental settings: <i>in vitro</i> (transcription/translation system) and <i>ex vivo</i> (transiently transfected epithelial cell line). PTC-readthrough was observed in 5 of the 16 mutations analyzed. UGA codons were more susceptible to AAG-stimulated readthrough than UAG; no suppression of UAA was observed. The efficiency of PTC-readthrough <i>in vitro</i> (from less than 1% to ∼28% of the translation from the corresponding wild-type constructs) differed with the AAG type and concentration, and depended on the combination of AAG and PTC, indicating that each PTC has to be individually tested with a range of stimulating compounds. The maximal values of PTC suppression observed in the <i>ex vivo</i> experiments were, depending on AAG used, 3–5 times lower than the corresponding values <i>in vitro</i>, despite using AAG concentrations that were 2 orders of magnitude higher. This indicates that, while the <i>in vitro</i> system is sufficient to examine the readthrough-susceptibility of PTCs, it is not sufficient to test the compounds potential to stimulate PTC-readthrough in the living cells. Most of the tested compounds (except for G418) at their highest concentrations did not disturb ciliogenesis in the cultures of primary respiratory epithelial cells from healthy donors.</p

Comparison of the efficiency of mutation detection efficiency using INNOLiPA tests and teh follow-up screening (SSCP/HD and sequencing).

<p>Legend: ^a including six alleles with T₅_TG_12–13 in intron 9; ^b including four alleles with T_5or8_TG_12–13 in intron 9; ^c also analyzed by MLPA</p

Distribution of the most frequent CFTR mutations detected in CF patients from Poland, compared with Central and Southeastern European populations.

<p>Legend: Data are given in %. ^a non-INNOLiPA mutations are in bold, and a novel mutation is underlined; ^b very close to the earlier estimate of 54% based on a much smaller study group of PL patients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089094#pone.0089094-Aznarez1" target="_blank">[7]</a>; ^c only selected segments of the gene have been screened; ^d frequency significantly higher or ^e lower than in Polish cohort (p<0.005, Pearsons’s chi square); ^f<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089094#pone.0089094-Bobadilla1" target="_blank">[5]</a>; NA– not analyzed/not available; Poland (mostly Southern and Western Poland; this study); Czech Republic <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089094#pone.0089094-Krenkova1" target="_blank">[21]</a>; Slovakia (based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089094#pone.0089094-Krenkova1" target="_blank">[21]</a>); Germany <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089094#pone.0089094-Dork1" target="_blank">[22]</a>; Lithuania <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089094#pone.0089094-Giannattasio1" target="_blank">[23]</a>; Western Ukraine <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089094#pone.0089094-Makukh1" target="_blank">[24]</a>; East Hungary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089094#pone.0089094-Ivady1" target="_blank">[25]</a>; Romania <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089094#pone.0089094-Frentescu1" target="_blank">[26]</a>; Bulgaria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089094#pone.0089094-Angelicheva1" target="_blank">[27]</a>; Serbia <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089094#pone.0089094-Radivojevic1" target="_blank">[28]</a>; Greece <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089094#pone.0089094-Kanavakis1" target="_blank">[29]</a>.</p

<i>CFTR</i> mutation detection efficiency in PL CF patients from different health centers.

<p>A. Both mutations identified. B. One mutation identified. C. No mutation identified. Diagonal stripes – INNOLiPA mutations; solid black – non-INNOLiPA mutations. Rabka – Institute of Tuberculosis and Lung Diseases in Rabka; Other – other health care centers in Poland.</p

Mutations found in the analyzed cohort of 738 Polish CF patients, sorted according to the position in the gene.

<p>Legend: ^a IL19 i 17 – mutations included in the INNOLiPA tests (see below); CFMDB – non-INNOLiPA mutations present in the CTFR mutation database; novel – mutations first reported in this study; ^b in three chromosomes R668C with G576A in trans; ^c F508del, c.1585-1G>A, G542X, N1303K or c.579+3A>G; ^d F508del, G542X, R553X or N1303K; ^e not pathogenic if not in cis with c.3067-72del6 (l.n.3199del6); ^f not pathogenic – see explanation the text; ^g not pathogenic if not in cis with G1244V.</p>a<p>Mutations detected by two INNOLiPA_CFTR tests (legacy names): IL19 (INNOLiPA_<i>CFTR</i>19): F508del; G542X; N1303K; W1282X; G551D; 1717-1G>A; R553X; <i>CFTR</i>dele2,3(21kb); I507del; 711+1G>T; 3272-26A>G; 3905insT; R560T; 1898+1G>A; S1251N; I148T; 3199del6; 3120+1G>A; Q552X.</p><p>IL17 (INNOLiPA_<i>CFTR</i>17_TnUpdate): 621+1G>T; 3849+10kbC>T; 2183AA>G; 394delTT; 2789+5G>A; R1162X; 3659delC; R117H; R334W; R347P; G85E; 1078delT; A455E; 2143delT; E60X; 2184delA; 711+5G>A; polymorphism 5T/7T/9T.</p

Mutation detection efficiency using INNOLiPA tests<i>: CFTR19</i> and <i>CFTR17TnUpdate</i>.

<p>Legend: ^a I148T included in CFTR-17TnUpdate was not counted as a mutation if not in cis with c.3067-72del6 (l.n. 3199del6); c.1210(-12)T_n site in intron 9 (l.n.IVS8-T_n) without data on the associated TG repeat was not counted as a mutation.</p

Localization of the c.367delC mutation and primers used to analyze <i>ZMYND10</i> exon 4.

<p>Upper panel—exon structure of the <i>ZMYND10</i> gene; lower panel—exon 4; an asterisk indicates position of the homozygous mutation found in patients #683 and #810; the short arrows depict approximate localization of the primers (forward and reverse) used for HRM and SSCP analysis</p

Transmission electron microscope analysis.

<p>Right panel, outer and inner dynein arms (ODA and IDA, black arrows) in the cross-section of cilia from the respiratory epithelium of a healthy individual. Left panel, lack of ODA and IDA (white arrows) in patient #683. Magnification 30,000; lower panel—enlarged view of a single cilium. Black scale bars represent 0,1 μm.</p

Potential miRNA target sites in <i>ZMYND10</i> mRNA.

<p>Potential miRNA target sites predicted using miRDB (database tab: ‘<i>Custom Prediction; Search for unconventional target sites in the coding region or 5'-UTR</i>’) are shown in the mRNA sequence downstream from the c.367delC mutation (spliced exons 4–12 in transcript 001 are shown in alternating blue and black colors; the site of the mutation is double underlined; position of the STOP codons—normal and premature—are underlined in bold). Seed sequences complementary to the highest scoring miRNAs (sequences shown below) are highlighted.</p

<i>ZMYND10</i> mutations identified in different studies.

<p><i>ZMYND10</i> mutations identified in different studies.</p