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. 2017 Sep 7;101(3):466-477.
doi: 10.1016/j.ajhg.2017.08.007.

RAC1 Missense Mutations in Developmental Disorders with Diverse Phenotypes

Margot R F Reijnders  1 , Nurhuda M Ansor  2 , Maria Kousi  3 , Wyatt W Yue  4 , Perciliz L Tan  3 , Katie Clarkson  5 , Jill Clayton-Smith  6 , Ken Corning  5 , Julie R Jones  5 , Wayne W K Lam  7 , Grazia M S Mancini  8 , Carlo Marcelis  1 , Shehla Mohammed  9 , Rolph Pfundt  1 , Maian Roifman  10 , Ronald Cohn  11 , David Chitayat  10 ; Deciphering Developmental Disorders Study; Tom H Millard  12 , Nicholas Katsanis  3 , Han G Brunner  13 , Siddharth Banka  14
Affiliations

RAC1 Missense Mutations in Developmental Disorders with Diverse Phenotypes

Margot R F Reijnders et al. Am J Hum Genet. .

Abstract

RAC1 is a widely studied Rho GTPase, a class of molecules that modulate numerous cellular functions essential for normal development. RAC1 is highly conserved across species and is under strict mutational constraint. We report seven individuals with distinct de novo missense RAC1 mutations and varying degrees of developmental delay, brain malformations, and additional phenotypes. Four individuals, each harboring one of c.53G>A (p.Cys18Tyr), c.116A>G (p.Asn39Ser), c.218C>T (p.Pro73Leu), and c.470G>A (p.Cys157Tyr) variants, were microcephalic, with head circumferences between -2.5 to -5 SD. In contrast, two individuals with c.151G>A (p.Val51Met) and c.151G>C (p.Val51Leu) alleles were macrocephalic with head circumferences of +4.16 and +4.5 SD. One individual harboring a c.190T>G (p.Tyr64Asp) allele had head circumference in the normal range. Collectively, we observed an extraordinary spread of ∼10 SD of head circumferences orchestrated by distinct mutations in the same gene. In silico modeling, mouse fibroblasts spreading assays, and in vivo overexpression assays using zebrafish as a surrogate model demonstrated that the p.Cys18Tyr and p.Asn39Ser RAC1 variants function as dominant-negative alleles and result in microcephaly, reduced neuronal proliferation, and cerebellar abnormalities in vivo. Conversely, the p.Tyr64Asp substitution is constitutively active. The remaining mutations are probably weakly dominant negative or their effects are context dependent. These findings highlight the importance of RAC1 in neuronal development. Along with TRIO and HACE1, a sub-category of rare developmental disorders is emerging with RAC1 as the central player. We show that ultra-rare disorders caused by private, non-recurrent missense mutations that result in varying phenotypes are challenging to dissect, but can be delineated through focused international collaboration.

Keywords: HACE1; RAC1; Rho GTPase; TRIO; cerebellar abnormalities; developmental disorders; intellectual disability; macrocephaly; microcephaly; neuronal proliferation.

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Figures

Figure 1
Figure 1
Properties of Germline RAC1 Variants (A) RAC1 is located on human chromosome 7p22.1. Exon-intron structure of the two protein-coding RAC1 transcripts is shown. The ubiquitously expressed RAC1 transcript is composed of six exons (blue vertical lines). RAC1B (mainly expressed in gastrointestinal and epithelial cells) contains an additional exon (exon 4; gray vertical line). (B) Schematic of RAC1, with the coding exons shown as blue rectangles and the introns as blue lines. Exon 4 that is expressed only in the longer RAC1B transcript is shown as a gray rectangle. The red triangles represent the positions of the mutations identified in this study. The red oval within exon 4 shows the two splice donor variants detected in the ExAC database. The purple ovals represent the missense variants in the ExAC database. (C) Amino acid sequence of the RAC1 across positions p.1-p.192. Red amino acids are highly conserved in >90% of the RAS family members and blue amino acids represent RAS protein G box consensus residues. Positions of the identified missense mutations (in red) are marked with dotted lines and the high conservation among different species of these amino acids is shown in the rectangle below. (D) Crystal structure of human RAC1 showing the position of the identified missense variants presented in this study. The sites of substitution are indicated by sticks in spheres and are color coded according to their putative impact on guanine nucleotide binding (cyan), protein-protein interactions (yellow), and structural stability (red). For reference, the non-hydrolyzable GTP analog GMPPNP bound to the structure is shown in sticks. The structure was analyzed and this figure was generated using the program ICM-Pro v3.8 (Molsoft, LLC).
Figure 2
Figure 2
Facial Dysmorphism and Brain Abnormalities Observed in Individuals with RAC1 Mutations (A) Frontal and lateral photographs of the faces of individuals 1–3 and 5–7. Individuals 1, 2, and 3 were microcephalic, individual 5 had a normal occipital-frontal circumference, and individuals 6 and 7 had macrocephaly. Variable degrees of arched eyebrows, dysplastic ears, prominent nasal bridge, and overhanging columella were noted. Individuals with macrocephaly have prominent broad foreheads, slightly up-slanted palpebral fissures, open mouths, and “scooped out” appearance on lateral view. (B) Panel 1 shows brain MRI images of individual 1: axial T2 images showing dysgenetic vermis and right cerebellar hemisphere (left), cystis lesions in the right frontal deep white matter (middle, arrow), and sagittal T1 images (right) showing enlarged cisterna magna (arrow). Panel 2 shows brain MRI images of individual 2: axial T2 images showing hypoplastic cerebellar vermis (left), slightly enlarged lateral ventricles (middle), and sagittal T1 images (right) showing hypoplastic corpus callosum, pons (arrow head), hypoplastic lower vermis, enlarged 4th ventricle, and cisterna magna (arrow). Panel 6 shows brain MRI image of individual 6: axial T2 image showing non-specific white matter changes in the frontal and parietal lobes. No hydrocephalus or extra-axial fluid was observed. Panel 7 shows brain MRI of individual 7: T2 weighted MRI images. Left: axial image showing very discrete bilateral patchy high intensity signal changes in the deep white matter of the frontal and parietal lobes; right: mid-sagittal section showing, except for megalencephaly, normal brain structures.
Figure 3
Figure 3
Distinct RAC1 Mutations Genocopy Dominant-Negative or Constitutively Active Variants (A) NIH 3T3 fibroblasts transfected with 500 ng of DNA per coverslip expressing the indicated Rac1 mutants and were fixed 30 min after plating onto fibronectin and stained with Alexa-568 phalloidin to reveal the actin cytoskeleton and anti-GFP to reveal the expressed construct. Cells were imaged by confocal microscopy and divided into the three indicated categories. At least 50 cells were analyzed for each dataset except p.Asn39Ser, p.Tyr64Asp, p.Cys157Tyr (40 cells), and p.Gln61Leu (25 cells). Data were pooled from three independent experiments. (B) Graph showing mean circularity index of cells expressing indicated Rac1 mutants. Error bars indicate SEM. Datasets statistically analyzed using ANOVA test with Dunnett’s correction for multiple comparisons. Asterisks indicate datasets significantly different to WT (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (C) Images of cells expressing the indicated RAC1 mutation. In each case the cell is a representative example of the most common morphological category for that mutation (see A). Top panels show Alexa-568 phalloidin staining to reveal F-actin distribution, while the panels immediately below show a magnified view of the boxed region from the top panels to highlight the morphology of actin protrusions at the cell periphery. The third row of panels show the GFP channel of the same cells to reveal the distribution of the expressed RAC1 mutation, and the bottom panels show a merge of the F-actin (red) and GFP (green) channels. All scale bars indicate 10 μm.
Figure 4
Figure 4
Overexpression of Human RAC1 p.Cys18Tyr and p.Asn39Ser Cause Microcephaly, while p.Gln61Leu Causes the Mirror Phenotype in Zebrafish Embryos Functional assessment of de novo variants in RAC1 by in vivo complementation in zebrafish larvae. (A) Dorsal view of 5 days post fertilization (dpf) control and overexpressant larvae. For each experiment, embryos were injected with either WT or mutant RAC1 human mRNA message. The embryos were allowed to grow to 5 dpf and imaged live for head size. The midbrain area between the eyes highlighted with the dashed red line was measured for every imaged embryo, to produce a quantitative score. (B) Bar graph of normalized values showing the quantification of the head size phenotype in control embryos and embryos injected with either WT or mutant human RAC1 message, from two plotted experiments. Statistical analyses were performed by Student’s t test. (C) Dorsal view of 2 dpf control and overexpressant larvae, fixed in Dent’s fixative and whole-mount immuno-stained with an anti-phospho histone 3 (PH3) antibody that marks proliferating cells. For each experiment, embryos were injected with either WT or mutant RAC1 human mRNA message. The embryos were allowed to grow to 2 dpf and subsequently were fixed, stained, and imaged. Embryos were imaged dorsally and z stacks were acquired every 100 nm. The z stacks were collapsed to produce an extended depth of focus (EDF) image that was then processed for scoring. The PH3-positive cells were counted for every embryo imaged using the ITCN plugin from ImageJ, to produce a quantitative score. (D) Bar graph of normalized values showing the quantification of the proliferating cell count phenotype, across three biological replicas. Statistical analyses for the neuronal proliferation experiments were performed by Student’s t test. (E) Dorsal view of 3 dpf control and overexpressant larvae for RAC1 WT and mutant conditions. The area of the cerebellum consisting of the neuronal axons that cross the midline is highlighted with a green dashed box. At least 50 embryos per condition were imaged live and evaluated at 3 dpf for the depletion of axons within the area highlighted. (F) Bar graph of cumulative plotted experiments across three biological replicas showing percentages of embryos with cerebellar defects. Statistical analyses for the cerebellar integrity assay using a χ2 test. Error bars define 95% confidence interval.
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