Pathogenic variants in SMARCA5, a chromatin remodeler, cause a range of syndromic neurodevelopmental features

Abstract

Intellectual disability encompasses a wide spectrum of neurodevelopmental disorders, with many linked genetic loci. However, the underlying molecular mechanism for more than 50% of the patients remains elusive. We describe pathogenic variants in SMARCA5, encoding the ATPase motor of the ISWI chromatin remodeler, as a cause of a previously unidentified neurodevelopmental disorder, identifying 12 individuals with de novo or dominantly segregating rare heterozygous variants. Accompanying phenotypes include mild developmental delay, frequent postnatal short stature and microcephaly, and recurrent dysmorphic features. Loss of function of the SMARCA5 Drosophila ortholog Iswi led to smaller body size, reduced sensory dendrite complexity, and tiling defects in larvae. In adult flies, Iswi neural knockdown caused decreased brain size, aberrant mushroom body morphology, and abnormal locomotor function. Iswi loss of function was rescued by wild-type but not mutant SMARCA5. Our results demonstrate that SMARCA5 pathogenic variants cause a neurodevelopmental syndrome with mild facial dysmorphia.

Fig. 1. Facial photographs of affected individuals with SMARCA5 variants showing dysmorphisms.

Individual identifiers correlate with those in table S1. Note the similarities in facial appearance with blepharophimosis, short palpebral fissures, periorbital fullness, epicanthal folds, wide and/or high nasal bridge, short and/or flat philtrum, thin upper lip, and arched eyebrows. Individual 1: 3.7-year-old male; individual 4: 18-year-old male; individual 5: 39-year-old female; individual 7: 9.5-year-old female; individual 10: 3-year-old male; individual 11: 3.6-year-old male. Photo credits: M. A. Deardorff, Children’s Hospital Los Angeles; A. Kurolap and D. Tiosano, Ruth Rappaport Children’s Hospital; M. J. M. Nowaczyk, McMaster University; Y. Huang, University of California, Los Angeles; N. Boy, University Hospital Heidelberg.

Fig. 2. Molecular genetic findings.

(A) An intolerance landscape plot generated by MetaDome for SMARCA5 variant analysis (top panel) and a schematic outline of SMARCA5 protein showing conserved variants identified in individuals 1 to 12 and ClinVar at the bottom. (B) RT-PCR analysis of individual 1’s fibroblasts and lymphoblastoid cell line showing the SMARCA5 exon 7 skipping in both cell types because of a splice-altering variant.

Fig. 3. Iswi LoF leads to developmental defects in Drosophila larvae.

(A and B) Compared with WT, larval body size is significantly smaller in Iswi² third-instar larvae, which can be rescued by the expression of SMARCA5^WT by Act5C-Gal4. (A) Images showing larvae of different genotypes. Scale bar, 200 μm. (B) Quantification of larval body length. N = 30 to 41 larvae. (C and D) In the brain lobes of Iswi² larvae, the number of pH3⁺ positive mitotic cells is significantly decreased. N = 12 to 17 brain lobes. Expressing SMARCA5^WT by elav>Gal4 is sufficient to rescue the decreased proliferation in Iswi LoF larvae. (C) The brains were dissected from third-instar larvae and stained with pH3. Scale bar, 50 μm. (D) Quantification of pH3⁺ positive mitotic cells; P = 0.0090. (E and F) In Iswi², C4da ddaC and v’ada neurons exhibit dendritic tiling defects. SMARCA5^WT, but not patient variants, partially rescues the tiling defects. Scale bar, 50 μm. (E) The red dashed circles outline the gap area between C4da ddaC and v’ada neurons. (F) Quantification of the gap in C4da neuron dendritic field; N = 17 to 34 dendritic fields from 4 to 5 larvae. All data are means ± SEM. The data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test; **P < 0.01, ***P < 0.001.

Fig. 4. Iswi neural-specific knockdown causes abnormal brain structure and motor deficits in adult files.

(A and B) Iswi neural-specific knockdown leads to smaller brain size compared with control flies. (A) Representative images of fly brains of control and Iswi knockdown flies. Scale bar, 100 μm. (B) Quantification of adult fly brain volume. N = 6, P = 0.0002. (C to E) Iswi neural-specific knockdown affects mushroom body structure in adult flies. While expressing SMARCA5^WT partially rescues the structure, the patient variants are not able to rescue mushroom body morphology. Cyan and magenta dashed lines outline the vertical lobe and horizontal lobe of the mushroom body in the right hemisphere, respectively. N = 11 to 35 brains. (C) Representative images of fly brains from different groups. Mushroom bodies are marked by FasII staining. Scale bar, 100 μm. (D) Quantification of vertical lobe volume. (E) Quantification of horizontal lobe volume. SMARCA5^WT rescue significantly increases the horizontal lobe volume in Iswi knockdown flies. (F) The negative geotaxis test shows that Iswi knockdown leads to locomotion deficit in flies. Expression of SMARCA5^WT in Iswi knockdown flies is able to rescue the climbing deficit to control levels, while SMARCA5^R592Q and SMARCA5^268-319del mutants fail to rescue. N = 3 to 12 groups, with each group containing 10 flies. All data are means ± SEM. The data were analyzed by unpaired t test or one-way ANOVA followed by Tukey’s multiple comparison test; ***P < 0.001.