A Novel Phenotype Associated with ACTB Mutations Sharissa

Thrombocytopenia Microcephaly Syndrome - a novel phenotype associated with ACTB mutations Sharissa L. Latham#,*,1, Nadja Ehmke#,2,3, Patrick Y.A. Reinke1,4, Manuel H. Taft1, Michael J. Lyons5, Michael J Friez5, Jennifer A. Lee5, Ramona Hecker6, Michael C. Fruehwald7, Kerstin Becker8, Teresa M. Neuhann8, Denise Horn2, Evelin Schrock9, Katharina Sarnow9, Konrad Grützmann10, Luzie Gawehn9, Barbara Klink9, Andreas Rump9, Christine Chaponnier11, Ralf J. Knöfler12, Dietmar J. Manstein*,1,4 and Natalia Di Donato*,9 Supplementary notes. Variant ACTB mRNA and β-CYA are present in patient primary fibroblasts All ACTB-TMS patients display heterozygous ACTB mutations. We confirm by RNA-Seq that these ACTB-TMS mutations do not result in nonsense-mediated mRNA decay and show that the variant mRNA accounts for 50% of the ACTB mRNA present in P3 and P5 primary fibroblasts (Supplementary Fig. 7a). With mass spectrometry, we can identify the altered C-terminal frame shift peptide in P3 only (Supplementary Fig. 7b). The 4 amino acid deletion of P5 could not be detected by this method. However, this peptide region was also not detected in the control. Whole-transcriptome analysis (RNA-Seq) To further understand the mechanisms of action, we studied the effect of ACTB-TMS mutations on the global gene expression profile of fibroblasts obtained from P3, P5 and a healthy control. RNA was isolated from three independent fibroblast cell culture flasks for each individual and subsequent whole transcriptome sequencing was performed. Principal component analysis showed clear grouping of replicates for one individual and clear separation between samples from different individuals. Sample distance analysis and unsupervised hierarchical clustering showed a clear separation between patient samples and control in two main clusters, with a further separation between the two patients within the ACTB-mutation cluster (Supplementary Figures 8). Gene expression analysis revealed 2912 significantly differentially expressed genes (DEG, Benjamini-Hochberg adjusted-value ≤ 0.01, FC >1) 1 when comparing both patients with mutation vs control, illustrating the far-reaching impact of ACTB- TMS mutations also on the transcriptome. Interestingly, P5 had many more significantly deregulated genes compared to normal control than P3 (10750 versus 8381 genes, respectively; DEG, Benjamini- Hochberg adjusted-value ≤ 0.01, FC >1), which is in line with the more severe phenotype. Pathway analysis using all genes significantly differentially expressed between both patients’ fibroblasts compared to control showed, as expected, overexpression of protein coding genes associated with the KEGG “regulation of actin cytoskeleton” pathway in both ACTB-TMS cell lines (Supplementary Fig. 9). In addition, we also observed significant upregulation in the “cell cycle” pathway. 86 protein coding genes were massively overexpressed in both ACTB-TMS cell lines with the log2 FC > 4 (Supplementary table 4). 18 of 86 were part of the KEGG cell cycle, DNA replication, RNA transport and DNA repair pathways. 19 further genes encode for kinetochore complex components, centrosomal proteins and proteins involved in spindle formation and checkpoint activity. We also found significant overexpression of 9 different kinesin family members that are microtubule- dependent motors, also required for spindle formation. We hypothesize that this strong overexpression of multiple genes encoding nuclear and cytoplasmic proteins involved in DNA replication and cell division is due to the impaired function of nuclear β-actin1. Specific characterization of nuclear β- actin was beyond the scope of the study and will be assessed in the follow up projects. In Silico analysis of mutation-induced structural changes in β-CYAP3 and β-CYAP5 Structural changes in β-CYAP3 and β-CYAP5occur in the region of actin SD1 that is formed by residues 338-374. This region is highly conserved in all actin isoforms (Supplementary Fig. 1). The P3 mutation changes 26 amino acids and removes 19 residues (Fig. 5a, left). A deletion of four amino acids in P5 results in a shift of residues G342-S358 by ~5Å, while residues K359-F377 are predicted to be structurally unaffected (Fig. 5a, right). In both P3 and P5, the binding interfaces for interaction with the CM loop (T417 – T432) and supporting loop (E542 – L550) of myosin are affected. The CM loop is essential for strong binding of myosin’s upper 50kDa domain to actin via hydrophobic (NM-2C: V422, V427, A430; β-CYA: V30, 2 P333, Y337) and electrostatic interactions (NM-2C: K429; β-CYA: D25, E334). The absence of C- terminal residues P333, E334 and Y337 in β-CYAP3 is predicted to perturb CM loop binding. A recent cryo-EM structure of the human cytoplasmic actomyosin complex shows that this binding interface is stabilized by internal interactions of W3402. This residue interacts with V9, D11 and K18 of the central SD1 β-sheet and forms ring stacking interactions with P27. Our model predicts that absence of W340 in both β-CYAP3 and β-CYAP5 perturbs the CM loop binding surface (Fig. 5b, top). This view receives further support from the results of MD-simulations that compare the folding stability and behavior of β-CYAWT and β-CYAP5. Mutations within the supporting loop alter the actin-activated ATPase activity, actin affinity and in vitro sliding motility of a Dictyostelium discoideum myosin 2 motor domain construct3. Based on biochemical results, the supporting loop was reported to interact with actin N-terminal residues4. However, this interpretation is not compatible with the structure of a human rigor actomyosin complex that was obtained using electron cryomicroscopy2. The structure shows two actin C-terminal glutamines (Q353, Q354) to be within the interaction range of this NM-2 loop. According to our modelling results, these residues are despite the frameshift spatially conserved in β-CYAP3, but out of the interaction range in the case of β-CYAP5 (Fig. 5b, bottom). For the actin-α-actinin complex, a low resolution cryo-EM density is available (PDB: 3LUE, described in 5), which shows that the binding sites for α-actinin and myosin overlap. The actin binding domain of α-actinin is commonly referred to as calponin homology domain, as it shares its actin binding domain with a large superfamily of proteins including calponin, dystonin, dystrophin, filamin, plectin, and spectrin. The interface includes the interaction of β-CYA SD1 I345 with F101 of α- actinin. For both β-CYAP3 and β-CYAP5 the hydrophobic interactions of I345 are conserved in the form of leucine residues (Fig. 5c). However, in β-CYAP3, D349 and D351 are within the interaction range of α-actinin H102 and D95, introducing additional electrostatic interactions (Fig. 5c, left). Regarding α-SMA, despite conservation of C-terminal residues between α-SMA and β-CYA, N- terminal residues are highly divergent (α-SMA: MCEEEDSTAL, β-CYA: MDDDIAAL). These residues are also part of the interaction interfaces of actin with NM-2 (NM2C: R661-R664 within 3 loop2/W-helix) and α-actinin (R93, K96) and may also contribute to the phenotype observed in sub- nuclear bundles. 4 List of Supplementary Figures and Tables Supplementary Figure 1. Schematic representation of ACTB-TMS mutations Supplementary Figure 2. Peripheral blood and bone marrow smears of affected individuals with ACTB-TMS Supplementary Figure 3. Craniofacial appearance of affected individuals with ACTB-TMS Supplementary Figure 4. Characterization of ACTB-TMS patient derived primary fibroblasts Supplementary Figure 5. Localization of actin isoforms in ACTB-TMS patient primary fibroblasts Supplementary Figure 6. Assessment of ABP in ACTB-TMS primary fibroblasts and peripheral thrombocytes Supplementary Figure 7. ACTB-TMS variant mRNA and protein expression Supplementary Figure 8. Whole-transcriptome analysis of control, P3 and P5 primary dermal fibroblasts Supplementary Figure 9. KEGG pathways analysis of RNA-Seq data Supplementary Table 1. Detailed clinical information for ACTB-TMS patients Supplementary Table 2. Protein coding genes significantly deregulated in ACTB-TMS P3 vs. healthy control primary fibroblasts Supplementary Table 3. Protein coding genes significantly deregulated in ACTB-TMS P5 vs. healthy control primary fibroblasts Supplementary Table 4. Protein coding genes upregulated (log2 FC >4) in both ACTB-TMS patient fibroblasts Supplementary Table 5. List of ABP assessed in ACTB-TMS patient fibroblasts Supplementary Table 6. Assessment of 124 ABP encoding genes in P3 and P5 vs. healthy control fibroblasts Supplementary Table 7. Summary of ACTB-TMS P3 and P5 clinical, cellular and structural data Supplementary Table 8. List of antibodies used in this study 5 α-SMAWT 311 377 γ-C YAWT 309 375 β-C YAWT 309 375 319 329 338 348 358 368 β-C YAP1/P2 309 375 β-C YAP3/P4 309 357 β-C YAP5 309 371 β-C YAP6 309 379 β-C YAPN 309 374 Unique to α-SMA Patient mutations Conserved glutamines Supplementary Figure 1. Schematic representation of ACTB-TMS mutations Peptide sequence alignment of wildtype (WT) α-SMA, γ-CYA, β-CYA and ACTB-TMS patient β-CYA isoforms starting at β-CYAWT residue 309. 6 Supplementary Figure 2. Peripheral blood and bone marrow smears of affected individuals with ACTB-TMS. (a-c) May-Gruenwald-Giemsa-stained stained peripheral blood smears of (a) P3, (b) P4 and (c) P5, with anisocytosis demonstrating normal sized thrombocytes (arrowheads) and macrothrombocytes (arrows); (d) May-Gruenwald-Giemsa-stained bone marrow smear of P5 demonstrating numerous megakaryocytes (arrows). 7 Supplementary

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