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. 2015 Aug 1;24(15):4443-53.
doi: 10.1093/hmg/ddv180. Epub 2015 May 13.

Kabuki syndrome genes KMT2D and KDM6A: functional analyses demonstrate critical roles in craniofacial, heart and brain development

Peter M Van Laarhoven  1 , Leif R Neitzel  1 , Anita M Quintana  1 , Elizabeth A Geiger  1 , Elaine H Zackai  2 , David E Clouthier  3 , Kristin B Artinger  4 , Jeffrey E Ming  2 , Tamim H Shaikh  5
Affiliations

Kabuki syndrome genes KMT2D and KDM6A: functional analyses demonstrate critical roles in craniofacial, heart and brain development

Peter M Van Laarhoven et al. Hum Mol Genet. .

Abstract

Kabuki syndrome (KS) is a rare multiple congenital anomaly syndrome characterized by distinctive facial features, global developmental delay, intellectual disability and cardiovascular and musculoskeletal abnormalities. While mutations in KMT2D have been identified in a majority of KS patients, a few patients have mutations in KDM6A. We analyzed 40 individuals clinically diagnosed with KS for mutations in KMT2D and KDM6A. Mutations were detected in KMT2D in 12 and KDM6A in 4 cases, respectively. Observed mutations included single-nucleotide variations and indels leading to frame shifts, nonsense, missense or splice-site alterations. In two cases, we discovered overlapping chromosome X microdeletions containing KDM6A. To further elucidate the functional roles of KMT2D and KDM6A, we knocked down the expression of their orthologs in zebrafish. Following knockdown of kmt2d and the two zebrafish paralogs kdm6a and kdm6al, we analyzed morphants for developmental abnormalities in tissues that are affected in individuals with KS, including craniofacial structures, heart and brain. The kmt2d morphants exhibited severe abnormalities in all tissues examined. Although the kdm6a and kdm6al morphants had similar brain abnormalities, kdm6a morphants exhibited craniofacial phenotypes, whereas kdm6al morphants had prominent defects in heart development. Our results provide further support for the similar roles of KMT2D and KDM6A in the etiology of KS by using a vertebrate model organism to provide direct evidence of their roles in the development of organs and tissues affected in KS patients.

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Figures

Figure 1.
Figure 1.
Spectrum of mutations in subjects diagnosed with KS. (A) An ideogram of chromosome X is shown, highlighting a region of Xp11.3–p11.23 with a red box. An expanded view of this region is shown including the genes and chromosomal coordinates. The microdeletions detected in Subjects 1 and 2 are shown as black bars. (B) The KMT2D and KDM6A proteins are shown as gray bars. The functional domains in each protein are indicated by colored bars. The location of alterations, in both proteins, detected in Subjects 3–16 are indicated by vertical black lines.
Figure 2.
Figure 2.
Defects in craniofacial development observed in morphant zebrafish embryos. Five day post-fertilization embryos were treated with alcian and alizarin dyes, which stain cartilage blue and bone red. Both ventral and left sagittal views focused on the visceral cranial cartilages at 10× magnification are shown. (A) Un-injected, wild-type control; (B) A typical kmt2d morphant; (C) A typical kdm6a morphant; (D) A kdm6a morphant coinjected with human KDM6A; (E) A typical kdm6al morphant. Bb, basibranchial; Cb, ceratobranchial arches 3–7; Ch, ceratohyal; O, opercle; Eth, ethmoid plate; M, Meckel's cartilage.
Figure 3.
Figure 3.
Defects in heart looping observed in morphant zebrafish embryos. Images shown of hearts from 48 h post-fertilization zebrafish embryos in transgenic line Tg(szhand2:mCherry; cmlc2:gfp)co10 which expresses green fluorescent protein (GFP) under the cmlc2 promoter were analyzed. The looping angle of the heart in the embryos was measured as shown (inset). The measured looping angles are shown as a bar graph for the wild-type and morphant embryos along with a kdm6al morphant coinjected with human KDM6A mRNA. The number of embryos (n) tested in each category are shown. Error bars represent the standard error of the mean. The bottom panel shows representative GFP-expressing hearts from each of the respective categories in the graph.
Figure 4.
Figure 4.
Defects in brain morphology observed in morphant zebrafish embryos. Hematoxylin and eosin (H&E) stained transverse sections of the zebrafish containing elements of both fore- and mid-brain at 48 h post-fertilization. (A) Wild-type control, (B) a typical kmt2d morphant, (C) a typical kdm6a morphant, (D) a typical kdm6a morphant coinjected with human KDM6A mRNA, (E) a typical kdm6al morphant and (F) a typical kdm6al morphant coinjected with human KDM6A mRNA. H, hypothalamus; MT, midbrain tegmentum; OT, optic tectum.
Figure 5.
Figure 5.
Expression of sox2 and huc in morphant zebrafish embryo brains. Transverse sections through the brain of 48 h post-fertilization zebrafish embryos immunofluorescently labeled with anti-sox2 (green) and anti-huc (red) antibodies. (A) Wild-type control, (B) a typical kmt2d morphant, (C) a typical kdm6a morphant, (D) a typical kdm6a morphant coinjected with human KDM6A mRNA, (E) a typical kdm6al morphant and (F) a typical kdm6al morphant coinjected with human KDM6A mRNA.
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