Homozygous STIL mutation causes holoprosencephaly and microcephaly in two siblings

Abstract

Holoprosencephaly (HPE) is a frequent congenital malformation of the brain characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been identified in HPE patients that account for only 30% of HPE cases, suggesting the existence of other HPE genes. Data from homozygosity mapping and whole-exome sequencing in a consanguineous Turkish family were combined to identify a homozygous missense mutation (c.2150G>A; p.Gly717Glu) in STIL, common to the two affected children. STIL has a role in centriole formation and has previously been described in rare cases of microcephaly. Rescue experiments in U2OS cells showed that the STIL p.Gly717Glu mutation was not able to fully restore the centriole duplication failure following depletion of endogenous STIL protein indicating the deleterious role of the mutation. In situ hybridization experiments using chick embryos demonstrated that expression of Stil was in accordance with a function during early patterning of the forebrain. It is only the second time that a STIL homozygous mutation causing a recessive form of HPE was reported. This result also supports the genetic heterogeneity of HPE and increases the panel of genes to be tested for HPE diagnosis.

Fig 1. Pedigree of the consanguineous family, brain MRI of the affected siblings, Sanger validation of the c.2150G>A (p.Gly717Glu) STIL mutation, and schematic report of all STIL mutations reported so far.

(A) Pedigree of the inbred family. Closed symbols indicate individuals affected with holoprosencephaly. Family members marked with an asterisk were analyzed by whole exome sequencing. (B) Coronal (on the left) and axial (on the right) brain MRI in individuals II3 and II5 at 12 and 5 years old respectively. II3: lobar HPE, the arrow in the coronal section shows the corpus callosum, and the arrows in the axial section show the absence of visualization of frontal horns, and a partial agenesis of the corpus callosum; II5: semi-lobar HPE, the arrow on axial MRI shows the absence of occipital lobe and a large unilateral temporal and occipital fluid cavity communicating. (C) Sanger validation was performed for the 3 available individuals I2, II3 and II5. The c.2150G>A mutation in STIL revealed a segregation with HPE in the two affected children. (D) Distribution of mutations previously reported in the literature on STIL protein. All mutations were present in a homozygous state [21,23,29] except those represented under the protein, which were two compound heterozygous mutations [28]. The p.Leu1218* mutation was found twice in two different families. The mutation reported in this study is p.Gly717Glu (in red) and is located in the central domain of the protein. Three important domains were represented here, the CPAP binding domain from amino acid 429 to 448, the coiled-coil domain (CC) from amino acid 720 to 750 and the KEN box, located between amino acids 1243–1245.

Fig 2. p.Gly717Glu cannot fully restore STIL depletion in synchronized U2OS cells.

(A) Protocol used to assay the centriole duplication potential of WT and mutant STIL proteins. U2OS were treated with STIL RNAi, and transfected 24h later with GFP-STIL WT or GFP-STIL p.Gly717Glu constructs in aphidicolin containing medium (4 μg/ml). Cells were fixed and counted 36h later to allow centriole duplication. (B) Percentages of S phase cells containing <4 or ≥4 centrioles following control RNAi (scrambled) and STIL RNAi, followed or not by transfection with GFP-STIL WT or GFP-STIL p.Gly717Glu (p<0,001***). (C) Examples of S phase-arrested cells following different treatments. A control cell with 4 centrioles (top left panel), a STIL RNAi treated cell with 2 centrioles (top right panel), a STIL-depleted cell expressing GFP-STIL WT with 4 centrioles (bottom left), and a STIL-depleted cell expressing GFP-STIL p.Gly717Glu with 2 centrioles are displayed (bottom right). Centrin is shown in red (and in monochrome in the insets), DNA is blue (top panels) and GFP is green (bottom panels). The white arrowheads indicate the centriole region. The bar represents 10 μm.

Fig 3. Stil was specifically expressed in the developing chick forebrain.

In situ hybridization analysis of Stil (A, C-E) was performed on whole-mount chick embryos at developmental stages HH8, HH10 and HH16. Expression of Shh (B) was analyzed at HH8. A, B and D are dorsal views, E is a lateral view. (C) Flat-mounted preparation of the forebrain of the above embryo (A). Brackets indicate the compartments of the brain. d, dorsal border of the forebrain; fp, floor plate; n, neurectoderm; ot, otic vesicle; op optic vesicle, sp; spinal cord.