The authors declare no competing interest.
Genetic variation in MKL2 and decreased downstream PCTAIRE1 expression in extreme, fatal primary human microcephaly
Article first published online: 18 JUN 2013
© 2013 The Authors. Clinical Genetics published by John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Volume 85, Issue 5, pages 423–432, May 2014
How to Cite
Ramos, E.I., Bien-Willner, G.A., Li, J., Hughes, A.E.O., Giacalone, J., Chasnoff, S., Kulkarni, S., Parmacek, M., Cole, F.S. and Druley, T.E. (2014), Genetic variation in MKL2 and decreased downstream PCTAIRE1 expression in extreme, fatal primary human microcephaly. Clinical Genetics, 85: 423–432. doi: 10.1111/cge.12197
- Issue published online: 1 APR 2014
- Article first published online: 18 JUN 2013
- Accepted manuscript online: 21 MAY 2013 12:14PM EST
- Manuscript Revised: 16 MAY 2013
- Manuscript Accepted: 16 MAY 2013
- Manuscript Received: 14 MAR 2013
- National Institutes of Health. Grant Numbers: R01 HL065174, R01 HL082747
|cge12197-sup-0001-FigureS1.doc||Word document||80K||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).|
|cge12197-sup-0002-FigureS2.doc||Word document||285K||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.|
|cge12197-sup-0003-TableS1.doc||Word document||331K||Table S1. Based positions analyzed in the paternal sperm exome.|
|cge12197-sup-0004-TableS2.doc||Word document||57K||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.|
|cge12197-sup-0005-TableS3.doc||Word document||601K||Table S3. PCR primers for candidate region amplification and Sanger sequencing.|
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