Mutations in ANKRD11 cause KBG syndrome, characterized by intellectual disability, skeletal malformations, and macrodontia

Abstract

KBG syndrome is characterized by intellectual disability associated with macrodontia of the upper central incisors as well as distinct craniofacial findings, short stature, and skeletal anomalies. Although believed to be genetic in origin, the specific underlying defect is unknown. Through whole-exome sequencing, we identified deleterious heterozygous mutations in ANKRD11 encoding ankyrin repeat domain 11, also known as ankyrin repeat-containing cofactor 1. A splice-site mutation, c.7570-1G>C (p.Glu2524_Lys2525del), cosegregated with the disease in a family with three affected members, whereas in a simplex case a de novo truncating mutation, c.2305delT (p.Ser769GlnfsX8), was detected. Sanger sequencing revealed additional de novo truncating ANKRD11 mutations in three other simplex cases. ANKRD11 is known to interact with nuclear receptor complexes to modify transcriptional activation. We demonstrated that ANKRD11 localizes mainly to the nuclei of neurons and accumulates in discrete inclusions when neurons are depolarized, suggesting that it plays a role in neural plasticity. Our results demonstrate that mutations in ANKRD11 cause KBG syndrome and outline a fundamental role of ANKRD11 in craniofacial, dental, skeletal, and central nervous system development and function.

Figure 1

Phenotypic Features of KBG Syndrome Written consents were obtained from all participants for publication of clinical images. Distinct facial features of KBG syndrome include a triangular face, low anterior and posterior hairlines, synophrys, hypertelorism, eyelid ptosis, a broad and high nasal bridge, an upturned nose, a long philtrum, and wide permanent central incisors (≥10 mm in males and ≥9.7 mm in females). Hand anomalies include brachydactyly, clinodactyly, and cutaneous syndactyly. Photographs were taken when individuals in family 1 were 25 years old (II-1), 22 years old (II-2), and 46 years old (I-1) and individual 1 in family 2 was 9 years old. Facial and dental pictures were taken when individual 1 in family 3 was 6 and 11 years old, respectively. Individual 1 in family 4 was 21 years old and individual 1 in family 5 was 9 years old when photographs were taken.

Figure 2

ANKRD1 Mutations (A) Sanger results of identified mutations. Affected residues are underlined or indicated with arrows. (B) Quantitative PCR results of ANKRD11 in individual 1 of family 2 with a heterozygous c.2305delT mutation (left) and his unaffected father with homozygous wild-type alleles (right). (C) Sanger sequencing of ANKRD11 cDNA showing a heterozygous c.7570-1G>C mutation in individual II-1 in family 1. The deleted six base pairs correspond to two amino acids (red). (D) The alignment of a region from the C-terminal repression domain of ANKRD11 showing the localization of deleted amino acids (E and K) in family 1 as well as the mutation site in Yoda mice.

Figure 3

ANKRD11 Domains and Identified Mutations The positions of the five identified mutations in ANKRD11 domains. The two mutations identified with exome sequencing are shown with blue characters.

Figure 4

Activity-Dependent Nuclear Aggregation of ANKRD11 (A) RT-PCR expression analysis of ANKRD11 in adult human brain cDNA. (1) DNA control sample, (2) cDNA sample with reverse transcriptase, and (3) cDNA sample without reverse transcriptase in RT reaction. (B) Representative image of neonatal cortical neurons transfected with GFP-ANKRD11 along with DsRed-MECP2. Postnatal day 1 neurons (cultured in vitro for 5 days) were cotransfected with GFP-ANKRD11 and Ds-Red-MECP2. Twenty-four hours after transfection, 50 mM KCl (or 5mM to controls plates) was added to the culture media for 90 min, cells were fixed, stained with antibodies to tyrosinated tubulin (blue), and imaged on a confocal microscope. (C) Higher magnification image of cotransfected neurons to observe GFP-ANKRD11 nuclear inclusions (arrows). GFP-ANKRD11 (green) and DsRed-MeCP2 (red) nuclear aggregates do not colocalize. (D) Representative glial cells (identified by their lack of tyrosinated-tubulin staining) from the same cultures as in (B). Note that in glial cells, ANKRD11 nuclear inclusions form regardless of KCl addition.