Deletion of KDM6A, a histone demethylase interacting with MLL2, in three patients with Kabuki syndrome

Abstract

Kabuki syndrome (KS) is a rare genetic disease that causes developmental delay and congenital anomalies. Since the identification of MLL2 mutations as the primary cause of KS, such mutations have been identified in 56%-76% of affected individuals, suggesting that there may be additional genes associated with KS. Here, we describe three KS individuals with de novo partial or complete deletions of an X chromosome gene, KDM6A, that encodes a histone demethylase that interacts with MLL2. Although KDM6A escapes X inactivation, we found a skewed X inactivation pattern, in which the deleted X chromosome was inactivated in the majority of the cells. This study identifies KDM6A mutations as another cause of KS and highlights the growing role of histone methylases and histone demethylases in multiple-congenital-anomaly and intellectual-disability syndromes.

Figure 1

Region Xp11.3 Showing the Patients' Deletions Region Xp11.3 shows the deletions drawn from the UCSC Genome Browser (GRCh37/hg19) for patients 1, 2, and 3. The black full tracks represent each patient's deletion, and each patient's number is above his or her respective track. The deletion in patient 1 spans 283.5 kb from base 44,941,324 to base 45,224,829, patient 2's deletion spans 815.7 kb from base 44,377,858 to base 45,193,629, and patient 3's deletion spans 45.4 kb from base 44,866,302 to base 44,912,718. The genes in the area are noted below the deletion tracks. CNVs in the Database of Genomic Variants are shown on the bottom lines. There are no previous reports of KDM6A copy-number changes.

Figure 2

Facial Appearance in Affected Individuals (A) Patient 1. (B) Patient 2. (C) Patient 3. Note the long palpebral fissures in patients 1 and 2 and the arched eyebrows in patients 1 (mild) and 3.

Figure 3

Appearance of Feet in Affected Individuals (A) Patient 1. (B) Patient 2. Note the long halluces in both patients.

Figure 4

Image of FISH Study on X Chromosomes Differentially Labeled with 5-BrdU An image of a FISH study shows X chromosomes differentially labeled by the incorporation of 5-BrdU. The inactive X chromosome (in the white circle) appears brighter than the active X chromosome. Xqter subtelomeric probes hybridize to both X chromosomes (blue arrows). The KDM6A signal (white arrow) is absent from the inactive X chromosome and is present on the active X chromosome. For this experiment, peripheral blood lymphocytes from patients 1 and 2 were stimulated with phytohemagglutinin and were cultured for 72 hr. In order to identify the late-replicating inactive X chromosome, we treated the cells with 5-BrdU (30 μg/ml) 5 hr prior to harvesting. Colcemid was then added to a concentration of 0.2 μg/ml, and 1 hr later, metaphase preparations were produced via standard procedures involving swelling in 75 mM KCl and fixing in 3:1 methanol/acetic acid. We performed FISH analysis by simultaneously using a subtelomeric Xqter probe labeled with SpectrumOrange (Vysis) to indicate the X chromosomes (blue arrow) and RP11-435K1 labeled with SpectrumOrange (AmpliTech) to distinguish between the deleted and nondeleted X chromosomes (white arrow). To facilitate the detection of incorporated 5-BrdU, we denatured cellular DNA in 2N HCl for 30 min at 37°C. 5-BrdU was then labeled with a 5-BrdU-specific monoclonal antibody conjugated to fluorescein (Roche) (1 μg/ml). Finally, we counterstained the DNA by applying an antifade solution containing 0.1 μg/ml DAPI.