Wu Y, Xia Y, Li P, Qu HQ, Liu Y, Yang Y, Lin J, Zheng M, Tian L, Wu Z, Huang S, Qin X, Zhou X, Chen S, Liu Y, Wang Y, Li X, Zeng H, Hakonarson H, Zhuang J.
Orphanet J Rare Dis. 2020 Apr 22;15(1):101. doi: 10.1186/s13023-020-01378-9.
PMID: 32321550 Free PMC Article
Select item 32308111
Background: Rubinstein-Taybi syndrome (RTS) is a rare, congenital, plurimalformative, and neurodevelopmental disorder. Previous studies have reported that large deletions contribute to more severe RTS phenotypes than those caused by CREBBP point mutations, suggesting a concurrent pathogenetic role of flanking genes, typical of contiguous gene syndromes, but the detailed genetics are unclear.
Results: This study presented a rare case of Rubinstein-Taybi (RT) syndrome with serious cardiac abnormalities. Based on the clinical and genetic analysis of the patient, the ADCY9 gene deletion was highlighted as a plausible explanation of cardiac abnormalities. In adcy9 morphant zebrafish, cardiac malformation was observed. Immunofluorescence study disclosed increased macrophage migration and cardiac apoptosis. RNA sequencing in zebrafish model highlighted the changes of a number of genes, including increased expression of the mmp9 gene which encodes a matrix metalloproteinase with the main function to degrade and remodel extracellular matrix.
Conclusions: In this study, we identified a plausible new candidate gene ADCY9 of CHD through the clinical and genetic analysis of a rare case of Rubinstein-Taybi (RT) syndrome with serious cardiac abnormalities. By functional study of zebrafish, we demonstrated that deletion of adcy9 is the causation for the cardiac abnormalities. Cardiac apoptosis and increased expression of the MMP9 gene are involved in the pathogenesis.
Fig. 1 Phenotypes of adcy9 zebrafish morphants. (a–f) Gross morphology at 3-dpf. Compared with control MO, knock down adcy9 present pericardial oedema (b, d, f, red arrow) (Heart beat is visible in the control fish, but is abnormal in adcy9-e3i3-MO injected fish (Supplementary Movie 1, Supplementary Movie 2). The bar graph in Panel G shows the percentage of embryos with development defects. h A time-course plot of percent survival in control vs. adcy9 morphants for 5 days
Fig. 2 Effects of adcy9 knock down on macrophage migration. a–f Representative bright field and fluorescent images of TG (zlyz: EGFP) larvae at 6 days postfertilization (dpf). Control MO injected fish show the normal distribution of labeled cells (b and c). Compared with control fish, embryos injected with adcy9-e3i3-MO present potent macrophage migration in heart (e and f, red circle area). g–k Quantification of the macrophage number at heart shows a 13.3-fold increased in adcy9 morphants. Columns, mean; bars, SEM (n = 10; Student’s t test; ***P < 0.0001;). dpf, days post fertilization. l Endogenous efnb2a, COX2, mmp9, ptp-rb and pik3r2 in control and adcy9 morphants assessed by qRT-PCR (n = 6–10 individual embryos). ns, not significant
Fig. 3 Morpholino knock down of adcy9 induces potent apoptosis in heart. Control-MO injected embryos and embryos injected with adcy9-e3i3-MO were stained with acridine orange (AO) at 3-dpf. Heart apoptotic cells are visible as bright green spots or red spots (f–h), and less bright homogenous green staining is unspecific background staining. a–d Control-MO injected zebrafish exhibited few or no apoptotic cells in heart. In contrast, significantly increased staining was observed throughout the heart in zebrafish injected with 4 ng adcy9-e3i3-MO (F-H). The red boxed regions are shown at higher magnification in the right panels. i Quantification of apoptosis particle number at heart shows a 66.1-fold increased in adcy9 morphants. Error bars, s.e.m.; ***P < 0.0001(n = 10; Student’s t test;); a–h: lateral view, anterior, left. Dpf, days post fertilization. j Endogenous tnfsf10l3, hdr, tnfrsfa, caspase-8, tp53, pmaip1, bbc3, badb, baxa, baxb, caspase-3a and caspase-a in control and adcy9 morphants assessed by qRT-PCR (n = 6–10 individual embryos). ns, not significant
Fig. 4 Scatter plot for KEGG enrichment results. The Gene Ratio is the ratio of differentially expressed gene numbers annotated in this pathway term to all gene numbers annotated in this pathway term. The greater the Gene Ratio, the greater the degree of pathway enrichment. A negLog10_Q value is the corrected p value ranging from 1 to 10, and a higher value indicates greater pathway enrichment