Genetic variation in MKL2 and decreased downstream PCTAIRE1 expression in extreme, fatal primary human microcephaly

Abstract

The genetic mechanisms driving normal brain development remain largely unknown. We performed genomic and immunohistochemical characterization of a novel, fatal human phenotype including extreme microcephaly with cerebral growth arrest at 14-18 weeks gestation in three full sisters born to healthy, non-consanguineous parents. Analysis of index cases and parents included familial exome sequencing, karyotyping, and genome-wide single nucleotide polymorphism (SNP) array. From proband, control and unrelated microcephalic fetal cortical tissue, we compared gene expression of RNA and targeted immunohistochemistry. Each daughter was homozygous for a rare, non-synonymous, deleterious variant in the MKL2 gene and heterozygous for a private 185 kb deletion on the paternal allele, upstream and in cis with his MKL2 variant allele, eliminating 24 CArG transcription factor binding sites and MIR4718. MKL1 was underexpressed in probands. Dysfunction of MKL2 and its transcriptional coactivation partner, serum response factor (SRF), was supported by a decrease in gene and protein expression of PCTAIRE1, a downstream target of MKL2:SRF heterodimer transcriptional activation, previously shown to result in severe microcephaly in murine models. While disruption of the MKL2:SRF axis has been associated with severe microcephaly and disordered brain development in multiple model systems, the role of this transcription factor complex has not been previously demonstrated in human brain development.

Keywords: CArG-box binding factor; MKL2; SRF; exome; microcephaly.

© 2013 The Authors. Clinical Genetics published by John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Figures

Figure 1

Representative non-contrast T2 brain MRI of one proband. These images are from Proband 2, born at 37 4/7 EGA and taken on day of life two, one day prior to expiration and two days prior to neuroautopsy. These images show sagittal (a), axial (b) and coronal (c) views. In all three probands, MRI demonstrated significant disruption of normal brain architecture including extreme microcephaly, marked cerebellar hypoplasia, and complete lissencephaly with failure of normal opercularization. Although the hypothalamus and optic nerves were present, the pituitary fossa was filled by fat signal, and the brainstem and cerebellum were hypoplastic.

Figure 2

Relative brain cortical gene expression. Controls were three fetal brain tissue specimens without a pathology diagnosis of microcephaly. ‘Affected Family’ is an average of gene expression from all three affected probands.

Figure 3

Comparative immunostaining between a non-microcephalic control and proband. Proband specimen is from neuroautopsy after expiration on day of life 4. (a, b) H&E staining of cerebrum (×100). In control, note the junction between white and gray matter, which is less evident on the proband image (arrow). (c, d) DAPI staining (×100) showing equivalent cell numbers between proband and control. (e, f) Nuclear MKL2 immunostaining (×100) is observed in a subpopulation of neurons of control and mutant brains (not all cells in either sample). (g, h) Anti-PCTAIRE1 (CDK16) staining of control and proband cortex (×40).

Figure 4

Proposed partial genetic model for variant haploinsufficiency.