Molecular characterization of a 1p36 chromosomal duplication and in utero interference define ENO1 as a candidate gene for polymicrogyria

Abstract

While chromosome 1p36 deletion syndrome is one of the most common terminal subtelomeric microdeletion syndrome, 1p36 microduplications are rare events. Polymicrogyria (PMG) is a brain malformation phenotype frequently present in patients with 1p36 monosomy. The gene whose haploinsufficiency could cause this phenotype remains to be identified. We used high-resolution arrayCGH in patients with various forms of PMG in order to identify chromosomal variants associated to the malformation and characterized the genes included in these regions in vitro and in vivo. We identified the smallest case of 1p36 duplication reported to date in a patient presenting intellectual disability, microcephaly, epilepsy, and perisylvian polymicrogyria. The duplicated segment is intrachromosomal, duplicated in mirror and contains two genes: enolase 1 (ENO1) and RERE, both disrupted by the rearrangement. Gene expression analysis performed using the patient cells revealed a reduced expression, mimicking haploinsufficiency. We performed in situ hybridization to describe the developmental expression profile of the two genes in mouse development. In addition, we used in utero electroporation of shRNAs to show that Eno1 inactivation in the rat causes a brain development defect. These experiments allowed us to define the ENO1 gene as the most likely candidate to contribute to the brain malformation phenotype of the studied patient and consequently a candidate to contribute to the malformations of the cerebral cortex observed in patients with 1p36 monosomy.

Fig. 1. Magnetic resonance imaging of the patient brain showing bilateral perisylvian polymicrogyria (arrows).

Representative sagittal fast spoiled gradient echo (FSPGR) image (a), axial FSPGR image (b) and coronal fast spin echo (FSE) T2 image (c) sections are shown. Notice asymmetry of location and extent of abnormal cortex on both hemispheres and asymmetric ventricles possibly explaining the patients’ hemiparesis in addition to her opercular symptoms and signs.

Fig. 2. Genomic location and size of the duplications.

a Schematic representation of the genomic duplications identified in the patient on chromosomes 1 and 12, following human genome build 38 (GRCh38). Gray bars show the duplicated regions, based on the first and the last duplicated oligonucleotides on the CGH array. Exons are represented as black or gray boxes and an arrow indicates the position of the translation initiation codon and the orientation of transcription. b Fluorescent in situ hybridization performed on metaphases of the patient with probes RP11-651L10 (BAC clone on chromosome 1) and G248P81726 (fosmid clone on chromosome 12), both spanning the duplicated regions. These probes appear as red signals and white arrows show the duplicated chromosomes.

Fig. 3. Structure of the duplicated segments and comparison with published cases.

a Determination of the orientation of the duplicated segments. The three possible configurations are shown: in tandem (1), in mirror at the distal side (2) or in mirror at the proximal side (3). Arrows indicate orientation of PCR primers. Positive PCR amplifications reveal that the 1p36 duplication occurred in mirror while it occurred in tandem in 12p13.1. The positive control corresponds to the amplification of a portion of the TBC1D24 gene on chromosome 16, to check for proper PCR conditions. b Location of the 1p36 duplicated region in the reported patient with respect to previously reported and precisely characterized 1p36 deletion cases with PMG. Deleted regions are represented as black lines, gray lines identify uncertainly delimited deletions. The gray box denotes the previously published minimal critical regions for PGM, located between 1 and 4.8 Mb on chromosome 1.

Fig. 4. In situ hybridization and in utero electroporation.

I. Expression of Eno1, Rere, and Ddx47 in mouse development using in situ hybridization. a–c Sagittal sections of mouse embryo at E12.5 (a), E14.5 (b), and E17.5 (c). d Sagittal and transverse sections of adult brains. Eno1 was strongly expressed in the inner ventricular side of the neopallial cortex at E14.5 (b–I), in the CA2 and CA3 fields of hippocampus and in the cerebral cortex in the adult brain (d-II and d-III, respectively). Rere expression was detected in central nervous system and strongly in the neopallial cortex (outer side) (b-II) and the midbrain (b-III) at E14.5. At E17.5, Rere expression was also detected in the neopallial cortex but at a lower level (c-I). A faint expression of Ddx47 transcript was detected in the urogenital region at E12.5 (a-I). II. Coronal sections of rat brain at E20, five days after electroporation. The presence of RFP alone (a), of a scramble shRNA (b), or shRere-2 (c) does not disturb neuronal migration. The presence of shEno1-1 severely impairs neuronal migration (d). Cells were counted in the different regions of the developing brain: intermediate (IZ) or ventricular (VZ) zones, or the cortical plate (CP) and plotted for comparison (E). Results are reported as mean ± standard error of the mean (SEM), and Student t test was used to test statistical significance, p value = 0.0008 for the significative difference in IZ and VZ and p value = 0.0009 for the significative difference in CP. A p value < 0.001 was considered to be statistically significant. **p < 0.001.