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

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, n...

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Published inClinical genetics Vol. 85; no. 5; pp. 423 - 432
Main Authors Ramos, E.I., Bien-Willner, G.A., Li, J., Hughes, A.E.O., Giacalone, J., Chasnoff, S., Kulkarni, S., Parmacek, M., Cole, F.S., Druley, T.E.
Format Journal Article
LanguageEnglish
Published Oxford, UK Blackwell Publishing Ltd 01.05.2014
Subjects
Animals
Binding sites
Binding Sites - genetics
Brain research
CArG-box binding factor
Cell Nucleus - genetics
Cyclin-Dependent Kinases - biosynthesis
Cyclin-Dependent Kinases - genetics
exome
Gene expression
Gene Expression Regulation
Genetic diversity
Humans
Mice
microcephaly
Microcephaly - genetics
Microcephaly - pathology
MKL2
Neurological disorders
Original
Polymorphism, Single Nucleotide
Serum Response Factor - genetics
Serum Response Factor - metabolism
SRF
Transcription factors
Transcription Factors - genetics
Transcriptional Activation - genetics
MKL2
microcephaly
CArG-box binding factor
exome
SRF
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Summary: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.
Bibliography:ark:/67375/WNG-K9MJFZC6-V
ArticleID:CGE12197
istex:A80D9674389D627CE6413537DB7F946383BAD145
Fig. S1. University of California at Santa Cruz Genome Browser snapshot of the upstream paternal deletion in cis with the paternal variant MKL2 allele. Twenty-four CArG boxes upstream of MKL2 are lost in the paternal 185 kb deletion. Many of which overlie regions of high regulatory potential (peaks along the 'ESPERR Regulatory Potential' track).Fig. S2. Relative brain cortical gene expression. Relative expression of 27 genes that included those known to be involved in the SRF:MKL2 pathway, those previously identified in cases of primary microcephaly, and CPPED1 located in the upstream chromosome 16 deletion and the housekeeping genes ACTB and PPIB to normalize expression was measured using the Quantigene Plex 2.0 Assay (Affymetrix). Thirteen individuals were surveyed: three normal brain anatomy fetal controls, three affected probands, and seven fetal specimens with pathology-diagnosed microcephaly. For each specimen, total RNA was extracted from an aggregate of six FFPE cerebral cortex cross sections affixed to glass slides according to the manufacturer's protocol. The average cross-sectional area was 27.6 cm2 (range 9.0-49.5). Lysates of each sample were prepared by incubating each sample at 65°C for 6 h with 1 min of full speed vortexing every 60 min. Target-specific probes were designed and provided by Affymetrix. Target hybridization and signal amplification were performed according to manufacturer's protocol. Signal measurements were made using a Luminex instrument (Austin, TX). Transcripts from each gene in all samples were measured in triplicate, and results expressed as the geometric mean normalized to an aggregate of ACTB and PPIB expression. Controls were six fetal brain tissue specimens without a pathology diagnosis of microcephaly. 'Family' is an average of gene expression from all three affected probands. 'Other' is an average of 33 other fetal cases with a pathology diagnosis of microcephaly. *p < 0.05; **p < 0.005. The red * is for comparison of 'other' microcephaly cases to normal controls.Table S1. Based positions analyzed in the paternal sperm exome.Table S2. Sequencing results summary of MKL2 and SRF from 51 unrelated and geographically distinct primary microcephaly cases. Twelve cases were found to harbor sequence variants. The minor allele frequency, conservation (PhyloP) score and 7-species regulatory potential (ESPERR) score are listed.Table S3. PCR primers for candidate region amplification and Sanger sequencing.
National Institutes of Health - No. R01 HL065174; No. R01 HL082747
The authors declare no competing interest.
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Conflict of interest: The authors declare no competing interest.
ISSN:0009-9163
1399-0004
DOI:10.1111/cge.12197