14q12 and severe Rett-like phenotypes: new clinical insights and physical mapping of FOXG1-regulatory elements

Abstract

The Forkhead box G1 (FOXG1) gene has been implicated in severe Rett-like phenotypes. It encodes the Forkhead box protein G1, a winged-helix transcriptional repressor critical for forebrain development. Recently, the core FOXG1 syndrome was defined as postnatal microcephaly, severe mental retardation, absent language, dyskinesia, and dysgenesis of the corpus callosum. We present seven additional patients with a severe Rett-like neurodevelopment disorder associated with de novo FOXG1 point mutations (two cases) or 14q12 deletions (five cases). We expand the mutational spectrum in patients with FOXG1-related encephalopathies and precise the core FOXG1 syndrome phenotype. Dysgenesis of the corpus callosum and dyskinesia are not always present in FOXG1-mutated patients. We believe that the FOXG1 gene should be considered in severely mentally retarded patients (no speech-language) with severe acquired microcephaly (-4 to-6 SD) and few clinical features suggestive of Rett syndrome. Interestingly enough, three 14q12 deletions that do not include the FOXG1 gene are associated with phenotypes very reminiscent to that of FOXG1-mutation-positive patients. We physically mapped a putative long-range FOXG1-regulatory element in a 0.43 Mb DNA segment encompassing the PRKD1 locus. In fibroblast cells, a cis-acting regulatory sequence located more than 0.6 Mb away from FOXG1 acts as a silencer at the transcriptional level. These data are important for clinicians and for molecular biologists involved in the management of patients with severe encephalopathies compatible with a FOXG1-related phenotype.

Figure 1

Physical mapping of FOXG1-regulatory elements in 14q12. (a) CNVs in 14q12 and FOXG1-related encephalopathies. We identified two point mutations in FOXG1 (patients 1 and 2) and five CNVs in 14q12 encompassing or close to FOXG1 (patients 3–7). The detailed view of the 14q12 region is derived from the NCBI genome browser (hg18, March 2006 assembly). Genes in 14q12 are represented by arrows (5′→3′ orientation). Deletions are depicted by horizontal black lines and duplication is depicted by an horizontal black dashed line. Vertical dashed black lines depict the location of 14q12 breakpoint in patients with a t(2;14). The minimal deleted region (28 945 423–29 372 834) is materialised by diagonal lines. (b) Bioinformatic predictions of putative cis-regulatory elements for the FOXG1 gene. In the minimal deleted segment, the FOXG1/PRKD1 intergenic region contains four HCNEs (HCNE1: 28 963 948-28 965 333; HCNE2: 28 981 071-28 982 265; HCNE3: 29 030 242-29 031 756; HCNE4: 29 066 922-29 068 642) identified by using the ESPRR computational method. The conservation throughout rhesus monkey, dog, mouse, opossum,and chicken of fragments >100 bp at 70% identity are indicated in red using the ECR browser (http://ecrbrowser.dcode.org).

Figure 2

Clinical pictures of six out of seven patients presented in this study. Pictures (front view) of patients P1 (a), P2 (b), P4 (c), P5 (d), P6 (e), and P7 (f). No common facial feature is remarkable. Notice that patients are shown at very different ages.

Figure 3

Quantification of the expression of FOXG1 at the mRNA and protein level. (a) Quantification of FOXG1 expression by RT-qPCR on total RNAs extracted from fibroblast cells (patients 3, 6, 7, and 8) or from whole saliva (patient 5). FOXG1 mRNA level is increased in patients 3, 5, and 7 with a 14q12 deletion close to FOXG1. Conversely, the FOXG1 expression is decreased in patient 8 (duplication including FOXG1) and in patient 6 (deletion including FOXG1). qPCRs have been performed in triplicate. (b) Quantification of FOXG1 expression by western blot analysis on total proteins extracted from fibroblast cells (patients 3, 6, 7, and 8). The RT-qPCR results were confirmed by the quantification of the endogenous FOXG1 protein in patients 3, 6, 7, and 8 by immunoblotting with FOXG1 antibody. Densitometric quantification of the relative abundance of FOXG1 in each patient is presented in a bar graph. P: patient. C: control. β-actin was used as a standard in the quantification of the relative abundance of endogenous FOXG1.