Molecular genotype-phenotype correlation in ACTB- and ACTG1-related non-muscle actinopathies

Figures

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  Figure 1. Definition of the haploinsufficiency of ACTB and ACTG1 (A) Number and distribution of truncating variants (start loss, stop gain, frameshift) in ACTB. (B) Facial gestalt in individuals with the ACTB whole-gene deletions (1 and 2) and familial presentation of the ACTB stop gain (3–5). Note the resemblance of the facial gestalt of the last individual (6), although he carries a missense variant in ACTB. (C) Segregation and intrafamilial variability of microdeletion of ACTG1 with the pedigree with the affected aunt (II.4) and nephew (III.4) with unilateral polymicrogyria on the left; +/− indicates the carrier status. Right: the UCSC genome hg38 browser view demonstrating a microdeletion encompassing ACTG1 and FSCB2; coordinates of the last normal probes are shown in red with the precise breakpoint mapping in blue (first and last missing bases), including junction sequence shown below.

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  Figure 2. Missense variants in ACTB and ACTG1 result in several distinct disorders (A) Number and distribution of missense variants in ACTB and ACTG1 resulting in the Baraitser-Winter cerebrofrontofacial syndrome (BWCFF); ACTB (blue bar) and ACTG1 (orange bar) with every 50th amino acid numbered in both gene models. The borders of coding exons are marked with dashed lines. (B) Representative facial gestalt in individuals with the BWCFF syndrome. Shown are individuals #1 (180-G), #2 (194-G), #3 (23-B), #4 (ID_09), #5 (47-B), and #6 (45-B), with subjects’ IDs corresponding to those listed in Table S2. Note that individuals #5 and #6 carry the same ACTB hotspot variant at position Arg196, and that individuals in images 5 and 6 carry the same ACTB hotspot variant at position Arg196. (C) Number, distribution, and clinical consequence of the missense variants in ACTB and ACTG1 not resulting in BWCFF; gene models and amino acid numbering are as described above. (D) Representative facial gestalt in individuals with unspecified non-muscle actinopathies (unNMA) due to variants in ACTG1 (1–4) and ACTB (5 and 6). Note that images 3 and 4 show affected individuals from the same family. Shown are…

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  Figure 3. Broad clinical spectrum of non-muscle actinopathies (A) Distribution of 290 pathogenic variants in ACTB and ACTG1 within different disorders of NMA spectrum. BWCFF, Baraitser-Winter cerebrofrontofacial syndrome; del, whole-gene deletion; MV, missense variant; pLoF, putative loss of function. (B) t-SNE visualization of three major NMA phenotypes in GestaltMatcher analysis. NMA-BWCFF indicates three individuals who were not reliably classified as BWCFF or unNMA based on the facial gestalt. Note that GestaltMatcher analysis allocated all three individuals within the BWCFF group. (C) Phenogrid demonstrating frequencies of selected clinical features in individuals with pLoF in ACTB, microdeletions encompassing ACTB (ACTB del) or ACTG1 (ACTG1 del), and non-truncating ACTB variants resulting in unstable protein (ACTB func.LoF), BWCFF, dystonia-deafness syndrome (DD), hearing loss (HL), and unspecified non-muscle actinopathy (unNMA). Individuals with incomplete clinical information were counted as “clinical feature not present” except for gastrointestinal (GI) anomalies. For GI anomalies, individuals with unknown status were excluded from the calculation. The presence of dystonia was…

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  Figure 4. ACTB MVs associated with LoF phenotype result in abnormal folding or impact actin thermal stability (A) Schematic of the purification strategy for recombinant human βCYA and γCYA (adapted from Greve and Manstein40). (B) SDS gels show purified WT and variant actin isoforms with the exact purification yield listed in the right panel; note three recombinant βCYA mutant variants could not be purified. Examples of the immunoblot of Sf9 insect cell lysate are presented in Figure S4. (C) Differential scanning fluorimetry was used to assess the thermal stability of Mg2+-ATP-G-actin. Representative traces of experiments with βCYA WT and variants (left) and γCYA (right) in the presence of 0.1 μM Mg2+-ATP. Data are shown as the first derivative of the obtained protein melting traces. The point of thermal denaturation (TM) is determined from the peak of the function. Final concentration of actin is 0.2 mg mL−1. Four independent experiments were performed. Means and SD are shown in the right panel.

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  Figure 5. NMA variants result in diverse polymerization anomalies (A) Salt-induced actin polymerization assessed by pyrene assay. Scatterplot demonstrates distribution of the measured half-times from the individual experiments with 2 μM βCYA WT, γCYA WT, and mutant isoforms. Means and SD are indicated; representative traces are shown in Figure S14. (B) Same experiment as in (A) repeated with a 1:1 mixture of the WT and mutant proteins. (C) Dilution-induced actin filament depolymerization assessed by pyrene assay. Scatterplot demonstrates distribution of the measured half-times from the individual experiments with 20 μM F-actin that is rapidly diluted to 0.2 μM to induce depolymerization. Representative traces are shown in Figure S14. (D) Summary of the actin mutants characterized in the current and previous studies. Previously published mutants are marked with a silcrow with the following references: βS368Lfs∗13,33 βR183W,34 and γK118M.42 n.a., not assessable.

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  Figure 6. Diagnostic workflow supporting clinical interpretation of ACTB/ACTG1 variants ∗ACTG1 nonsense or frameshift variants are likely to be classified as benign by ACMG criteria. ∗∗Further studies are required to prove the existence of this disorder; deletions usually involve additional flanking genes and likely have variable penetrance. ∗∗∗Specific facial dysmorphism (typically including hypertelorism, high-arched eyebrows, ptosis, long palpebral fissures with everted lower lid, broad nasal tip, long smooth philtrum, large mouth with thin upper lip, grooved chin, and large vertically oriented ears) and/or frontal predominant pachygyria with or without other brain and inner organ malformations or minor anomalies. #Specific facial dysmorphism typical for ACTB LoF (long face, straight eyebrows, deep-set eyes, epicanthus, narrow or flat nasal bridge with broad nasal tip, and large mouth) without pachygyria; ∧Potential revision of the clinical diagnosis during follow-up.