Whole-exome sequencing identifies mutated c12orf57 in recessive corpus callosum hypoplasia

Abstract

The corpus callosum is the principal cerebral commissure connecting the right and left hemispheres. The development of the corpus callosum is under tight genetic control, as demonstrated by abnormalities in its development in more than 1,000 genetic syndromes. We recruited more than 25 families in which members affected with corpus callosum hypoplasia (CCH) lacked syndromic features and had consanguineous parents, suggesting recessive causes. Exome sequence analysis identified C12orf57 mutations at the initiator methionine codon in four different families. C12orf57 is ubiquitously expressed and encodes a poorly annotated 126 amino acid protein of unknown function. This protein is without significant paralogs but has been tightly conserved across evolution. Our data suggest that this conserved gene is required for development of the human corpus callosum.

Figure 1

Families 542, 1414, 1183, and EZ1 with a Homozygous C12orf57 Mutation Encoding a p.Met1? Substitution Leading to CCH (A–D) Pedigrees demonstrating affected and unaffected individuals, all from first-cousin marriages. An asterisk indicates that DNA was ascertained. (E) Multipoint linkage plot resulting from the analysis of a 5K SNP array performed in all members of 542. Highest pLOD scores were found in chromosomes 3, 10, and 12. A red arrow indicates the location of C12orf57. (F) Invariant conservation of the Met1 amino acid position of the predicted protein (red arrow). Nonconserved amino acids are in gray.

Figure 2

Brain Imaging Demonstrates a Specific CC Abnormality in C12orf57-Mutated Individuals Upper image: midline sagittal MRI (where available). Lower image: axial MRI Images show CCH (arrows) in 542-III-3, 542-III-5, 1414-III-1, 1183-III-5, and EZ1-III-5 and complete agenesis in 542-III-9, 1414-III-2, and 1183-III-6. The bottom panel shows a prominent fornix (arrow) and unique appearance of the third ventricle (large arrowhead). Individual 1183-III-6 showed a colloid cyst, in addition to complete agenesis of the CC. No sagittal images were available for individual 1414-III-1, only a CT was available for individual 1183-III-5, and no imaging was available for individuals EZ1-III-5 and EZ1-III-6.

Figure 3

C12orf57 Transcript Expression in Human Tissues and Evidence for a Single Transcript Bearing the p.Met1? Substitution in CCH (A) qPCR of C12orf57 transcript normalized with GAPDH in adult human testis, liver, kidney, skeletal muscle, lung, heart, NPCs, cerebellum, forebrain, and fetal brain total cDNA. Error bars represent the standard deviation between two independent PCR products for C12orf57 cDNA. (B) ENCODE project track of UCSC Genome Browser shows transcriptional levels (from RNaseq), RNApol positioning, and H3K4me3 enrichment (from ChIPseq) for three C12orf57 cDNAs (represented by Genebank IDs AK310266, BI601148, and BC0092) with the most relevant alternative start codon, suggesting that the transcript in which Met1 is substituted (red arrow) in CCH is the major active transcript. (C) Schematic representation of C12orf57 transcripts Primer positioning is indicated by green arrows, and the substituted Met1 is indicated by a red arrow. (D) PCR of C12orf57 exons in human adult brain, fetal brain, and NPCs from total poly-A reverse-transcribed cDNA compared with total genomic DNA (bottom). Numbers refer to the primer pairs in (C). (E) qPCR of C12orf57 exon 2, representative of AK310266 and BI601148, and exon 4, representative of BC00925 transcripts, from human fetal brain cDNA. (F) RT-PCR from human fetal brain cDNA was performed with the indicated combination of primers and is consistent with the idea that the Met1 substituted in CCH derives the major active transcript.

Figure 4

C12ORF57-EGFP Localization Is Cytoplasmic, and c.1A>G Mutation Results in Reduced Translational Efficiency (A) Cytoplasmic localization of C12ORF57-EGFP fusion protein compared with the diffuse localization of EGFP in transiently transfected COS7 cells. (B) Cytoplasmic localization of C12ORF57-EGFP in human NPCs. C12orf57-EGFP was subcloned in pINDUCER20 and transduced to NPCs, expression was induced with 30 ng/ml doxycycline for 24 hr, and NPCs were immunostained with Nestin antibody (red). (C) Comparison between wild-type and c.1A>G C12ORF57-EGFP translated protein levels on transduced NPCs 1 week after positive selection with 200 μg/ml G418 and 24 hr after induction with 30 ng/ml doxycycline. Cells transduced with the mutant construct show notably reduced protein levels. (D) Immunoblot with EGFP antibody in wild-type and c.1A>G C12ORF57 transduced NPCs treated as in (C). Severely reduced protein levels occur in the presence of the mutation. GAPDH was used as a loading control. The scale bar represents 20 μm.