Identification of Two Novel ANKRD11 Mutations: Highlighting Incomplete Penetrance in KBG Syndrome

Dear Editor,

KBG syndrome is a rare autosomal dominant genetic condition caused by 16q24.3 microdeletion involving ANKRD11 or its pathogenic variants [1, 2]. The protein encoded by ANKRD11 is an ankyrin repeat-containing cofactor that controls neurogenesis in the embryonic brain, explaining the intellectual disability associated with this disorder [3]. We report two novel ANKRD11 mutations found in two patients with typical KBG syndrome features, including developmental delay, intellectual disability, and macrodontia of the upper central incisors (Table 1). The first patient harbored a de novo null mutation, whereas the second patient harbored a missense mutation inherited from her healthy mother. In view of our findings and given that inherited pathogenic ANKRD11 variants are increasingly observed, we compared our findings (case Nos. 1 & 2) with those of other studies (case Nos. 3-21) (Table 1). This study was reviewed and approved by the Rabat Ethics Committee for Biomedical Research (approval number: CERB-38-23).

Table 1.

Clinical features and molecular data of reported patients with inherited ANKRD11 mutations in this study and the literature

	This study		Zhang, et al. (2021) [3]		Parenti, et al. (2021) [7]
No. case	1	2	3	4	5	6	7
No. case in publication	1	2	1	2	4	12	13
Age at evaluation	8 yrs	11 yrs	NA	NA	9 yrs	11 yrs	1 yr 9 months
Sex	M	F	F	F	F	F	M
Craniofacial abnormalities	Brachycephaly Low anterior hairline Triangular face Thick eyebrows Hypertelorism Prominent eyes Anteverted nostrils Broad nasal tip Large anteverted ears	Low anterior hairline Thick eyebrows Long eyelashes Low nasal bridge Flat nose Prominent ears Thin upper lip Short neck	Triangular face Large cheek bone Low nasal bridge	Hypertelorism Low nasal bridge	Thick eyebrows Synophrys Anteverted nostrils Long philtrum Large mouth	Triangular face Low anterior hairline Thick and arched eyebrows Long eyelashes Anteverted nostrils Broad nasal tip Long philtrum	Flat face Thick and arched eyebrows Synophrys Depressed nasal bridge Anteverted nostrils
Neurodevelopmental abnormalities	Developmental delay Hyperactivity Aggressivity	Developmental delay Mild intellectual disability	NA	NA	Intellectual disability Behavioral problems	Intellectual disability Hyperactivity Seizures	Intellectual disability Behavioral problems
Dental/oral abnormalities	Macrodontia of central incisors Dental malposition	Macrodontia of central incisors	-	-	Macrodontia of central incisors	Macrodontia of central incisors	NA
Endocrinological abnormalities/growth	-	Short stature	Short stature	Short stature	-	-	Short stature
Skeletal/extremity abnormalities	-	Fifth finger clinodactyly	Delayed bone age	Advanced bone age	Small hands and feet Brachydactyly Proximally set thumbs Fifth finger clinodactyly	Delayed bone age Brachydactyly Proximally set thumbs	Small hands and feet Fifth finger clinodactyly
Ocular abnormalities	-	-	-	Strabismus	NA	Retinopathy Strabismus	Iris coloboma
Otorhinolaryngological abnormalities	-	-	-	-	NA	-	-
Other features	Hypospadias Interventricular communication	-	-	-	-	-	Cryptorchidism Epicanthus
	This study		Zhang, et al. (2021) [3]		Parenti, et al. (2021) [7]
Clinical features of transmitting parent	Mother -	Not inherited -	Mother Short stature	Mother Short stature	Mother Short stature Deep-set eyes Depressed nasal bridge Large mouth Proximally set thumbs Fifth finger clinodactyly Incomplete prono-supination of the elbow	Mother NA	Mother Low anterior hairline Arched and thick eyebrows Synophrys Long eyelashes Anteverted nostrils Large mouth Macrodontia of central incisors Mild intellectual disability Myopia
Reference sequence	NM_001256183.2	NM_001256183.2	NM_013275.6	NM_013275.6	NM_013275.6	NM_013275.6	NM_013275.6
Nucleotide change	c.1669C>G	c.3907del	c.4039_4041del	c.6427C>G	c.2398_2401del	c.7470 + 2T>C	c.1381_1384del
Nature of mutation	Missense	Frameshift	In frame	Missense	Frameshift	Splicing	Frameshift
Predicted protein change	p.Pro557Ala	p.Val1303Serfs*15	p.Lys1347del	p.Leu2143Val	p.Glu800Asnfs*62	-	p.Glu461Glnfs*48
Protein domain	RD1	Between RD1 and AD	Between RD1 and AD	AD	Between RD1 and AD	-	RD1
	Li, et al. (2021) [5]	Scarano, et al. (2019) [8]	Goldenberg, et al. (2016) [1]		Low, et al. (2016) [9]		Murray, et al. (2017) [10]
No. case	8	9	10	11	12	13	14
No. case in publication	2	9	16	5	6	7	AU12.1
Age at evaluation	2 yrs	NA	2 yrs	21 yrs	19 yrs	12 yrs	NA
Sex	M	F	F	F	F	F	F
Craniofacial abnormalities	Round face Large forehead Long eyelashes Prominent anteverted ears	Coarse face Microcephaly Nose abnormalities Long philtrum Large ears	Round face Synophrys Hypertelorism Prominent nose High nasal bridge	Unspecified	Unspecified	Unspecified	Low hairline Ptosis Broad eyebrows Anteverted nares Broad nasal tip Low-set ears Webbed neck
Neurodevelopmental abnormalities	-	Developmental delay Intellectual disability Behavioral problems Brain imaging anomalies	Developmental delay Behavioral problems Epilepsy EEG anomalies	Learning difficulties Seizures Behavioral problems	Learning difficulties Behavioral problems	Learning difficulties Seizures Behavioral problems	Developmental delay Hypotonia
	Li, et al. (2021) [5]	Scarano, et al. (2019) [8]	Goldenberg, et al. (2016) [1]		Low, et al. (2016) [9]		Murray, et al. (2017) [10]
Dental/oral abnormalities	Marked enamel hypoplasia	Macrodontia Oral abnormalities	-	Macrodontia	Macrodontia Overcrowding	Macrodontia	Dental abnormalities
Endocrinological abnormalities /growth	Short stature	Short stature	Short stature	Short stature	Short stature	-	-
Skeletal/extremity abnormalities	Fifth finger clinodactyly	Spinal abnormalities Costal abnormalities Joint stiffness Brachydactyly Clinodactyly	NA	Hand abnormalities Toe syndactyly (2nd and 3rd toes) Dysplastic 3, 4, 5 toenails	Hand abnormalities	Hand abnormalities	Delayed bone age Brachydactyly
Ocular abnormalities	-	NA	NA	-	-	-	Bilateral strabismus
Otorhinolaryngological abnormalities	-	Hearing loss	-	-	-	-	Left-sided conductive hearing loss
Other features	-	-	-	-	Delayed closure of fontanelle (age >3 yrs)	Severe constipation	Patent foramen ovale Malrotated bowel
Clinical features of transmitting parent	Mother Short stature Round face Prominent anteverted ears Macrodontia of central incisors Learning difficulties	Mother Milder phenotype	Mother Short stature Hand abnormalities Developmental delay Learning difficulties Sleep disorder	Father Small feet Hand abnormalities Learning difficulties			Father Large forehead Learning difficulties Dental abnormalities
Reference sequence	NM_001256183.2	NM_013275.5	NM_013275.5	NM_013275.5			NM001256182.1
Nucleotide change	c.7407dupC	c.3339G>A	c.1381_1384del	c.1903_1907del			c.3442G>A
Nature of mutation	Frameshift	Nonsense	Frameshift	Frameshift			Missense
Predicted protein change	p. P2530Rfs*61	p.Trp1113*	p.Glu461Glnfs*48	p.Lys635fs*26			p.Gly1148Ser
Protein domain	RD2	Between RD1 and AD	RD1	Between RD1 and AD			Between RD1 and AD
	Murray, et al. (2017) [10]			Kim, et al. (2015) [11]		Sirmaci, et al. (2011) [2]
No. case	15	16	17	18	19	20	21
No. case in publication	AU1.1	AU1.2	AU3.1	1	2	II-1	II-2
Age at evaluation	NA	NA	NA	11 months	23 months	NA	NA
Sex	M	M	F	M	F	M	M
Craniofacial abnormalities	Large forehead Triangular face Low anterior hairline Ptosis Downslanting palpebral fissures Long philtrum Short neck	Brachy turricephaly Large forehead Downslanting palpebral fissures Anteverted nares Long philtrum	Large forehead Hypertelorism Synophrys Anteverted nares High nasal bridge Dysplastic left ear Thick vermillion Short philtrum	Brachycephaly Triangular face Wide eyebrows Synophrys/ Ptosis Anteverted nostrils Long philtrum Prominent ears		Brachycephaly Triangular face Low anterior and posterior hairlines Synophrys/ptosis/hypertelorism Prominent nasal bridge/anteverted nostrils Long philtrum Large and prominent ears
Neurodevelopmental abnormalities	Developmental delay Hyperactivity	Developmental delay Hypotonia Sleep disorder Small optic nerves	Developmental delay Hyperactivity Aggressivity	Developmental delay EEG abnormalities	Developmental delay EEG abnormalities	Seizures Intellectual disability	Seizures Intellectual disability Hyperactivity
Dental/oral abnormalities	Macrodontia Crowding Premature loss of permanent teeth High arched and narrow palate	Primary dentition Bifid uvula	Macrodontia	High arched palate	Macrodontia High arched palate	Macrodontia	Macrodontia
Endocrinological abnormalities/growth	-	NA	Short stature	Short stature	Short stature	Short stature	Short stature
Skeletal/extremity abnormalities	Delayed bone maturation Bilateral cutaneous syndactyly 2 – 3 fingers Bilateral cutaneous syndactyly 2 – 3 toes	Delayed bone age Mild osteopenia Fifth finger brachydactyly	Fifth finger clinodactyly	Delayed bone age	Delayed bone age	Delayed bone age Short hands Fifth finger clinodactyly Accessory cervical ribs	Delayed bone age short hands Fifth finger clinodactyly Closed spina bifida
Ocular abnormalities	-	-	Left-sided strabismus	NA	NA	NA	NA
Otorhinolaryngological abnormalities	Bilateral hearing loss	-	-	NA	NA	NA	NA
Other features	Bilateral cryptorchidism	Ventricular septal defect and patent foramen ovale	-	Inguinal hernia	-	Cryptorchidism	Cryptorchidism
		Murray, et al. (2017) [10]		Kim, et al. (2015) [11]		Sirmaci, et al. (2011) [2]
Clinical features of transmitting parent		Mother Triangular face Short neck Hypertelorism/downslanting palpebral fissures Low anterior hairline Anteverted low-set ears Synophrys/myopia/astigmatism/night blindness Macrodontia of central incisors/high arched palate Learning difficulty/behavioral problems	Mother Brachycephaly Hypertelorism Synophrys High nasal bridge Macrodontia Learning difficulties	Mother Short stature Triangular face/brachycephaly Broad eyebrows/Synophrys/ptosis Prominent ears Long philtrum Macrodontia Intellectual disability		Father Short stature Triangular face/brachycephaly/low anterior and posterior hairlines Synophrys/long palpebral fissures/hypertelorism/ptosis Prominent nasal bridge/anteverted nostrils Large and prominent ears Long philtrum Macrodontia Short hands/Fifth finger clinodactyly Thoracic kyphosis/closed spina bifida/cryptorchidism Intellectual disability
Reference sequence		NM001256182.1	NM001256182.1	NM_013275.5		NM_013275.4
Nucleotide change		c.1903_1907 del	c.4406G>A	c.2395-2398del		c.7570-1G>C
Nature of mutation		Frameshift	Nonsense	Frameshift		Splicing
Predicted protein change		p.Lys635GInfs*26	p.Trp1469	p.Glu800Asnfs*62		p.Glu2524_Lys2525del
Protein domain		Between RD1 and AD	Between RD1 and AD	RD2		C-Ter

Abbreviations: F, female; M, male; NA, not available; RD2, repression domain 2; AD, activation domain; EEG, electroencephalogram.

After obtaining informed consent from their parents, we performed clinical exome sequencing on samples obtained from both patients in the BioAnalytica-Geno type laboratory using the Clinical Exome Solution v2 kit (SOPHiA GENETICS, Rolle, Switzerland) on the Illumina NextSeq platform (Illumina, Rolle, Switzerland). Bioinformatics and segregation analysis were performed in the department of medical genetics of the National Institute of Health of Rabat-Morocco. The study lasted 22 months (From June 2021 to April 2023).

Bioinformatics analysis revealed the presence of the monoallelic variants ANKRD11(NM_001256183.2):c.3907del(p.Val1303Serfs*15) in patient 1 and ANKRD11(NM_001256183.2):c.1669C>G(p.Pro557Ala) in patient 2, which were classified as likely pathogenic and variant of uncertain significance, respectively, according to the American College of Medical Genetics and Genomics guidelines [4]. These are rare polymorphisms in the Moroccan population as they were not found in 384 chromosomes (in an in-house database). Moreover, they were not found in the public Leiden Open Variation Database. Segregation was analyzed using Sanger sequencing (Fig. 1A–D).

Fig. 1.

Genetic analysis of the detected variants involved in our study. (A) The pedigree of patient 1’s family. (B) Electropherograms showing the monoallelic variant ANKRD11:c.3907del in patient 1 (II-3) and its absence in his parents (I-1 & I-2). (C) The pedigree of patient 2’s family. (D) Electropherograms showing the monoallelic mutation ANKRD11:c.1669C>G in patient 2 (II-2) and her mother (I-1) and its absence in her father (I-2). (E) Amino acid alignment of the ANKRD11 protein showing the high conservation of the residue Pro557 (highlighted by a red box) among different mammals. (F) The schematic diagram of the ANKRD11 protein structure showing the location of the two variants on protein level. ANKRD11 protein contains 2,663 residues and 4 functional domains: ANK: ankyrin domains, RD1: repression domain-1, AD: activation domain, RD2: repression domain-2. Patient 1 harbors p.Pro557Ala located on the RD1 domain. Patient 2 harbors p.Val1303Serfs*15 located between RD1 and AD domains.

The p.Pro557Ala variant in patient 2 was assessed as deleterious based on the results of several in silico pathogenicity prediction analyses (Polyphen-2: probably damaging; PANTHER-PSEP: probably damaging; MuPro: decreases the protein stability; IMutant3.0: decreases the protein stability). The proline residue at position 557 is highly conserved among mammals (Fig. 1E).

KBG syndrome is caused by the aberrant accumulation of abnormal proteins that lack C-terminal regions in cells. Intact repression function of the ANKRD11 protein is critical for normal intelligence development. Patients with null variants disrupting either all functional domains or repression domain (RD) 2 alone have more severe intellectual disability than patients with null variants disrupting both the activation domain (AD) and RD2 [5]. This can be explained by the repression effect of RD1 being weaker than the activation effect of the AD. A recent study showed that missense mutations result in phenotypes that fit the clinical spectrum of KBG syndrome, exerting a loss-of-function effect by either disrupting the transrepression capacity or reducing the protein stability [6]. p.Val1303Serfs*15 is located between RD1 and the AD, whereas p.Pro557Ala is located on RD1, which explains the mild intellectual disability in patient 2 (Fig. 1F). The other two reported inherited missense mutations (case Nos. 4 and 14) were located on the AD and between RD1 and the AD, respectively (Table 1). The remaining patients harbored protein-truncating mutations.

The most common craniofacial features in patients with inherited ANKRD11 mutations were broad eyebrows, prominent/anteverted ears, and nose abnormalities, such as a depressed nasal bridge and anteverted nares. Sixty-five percent of patients with inherited ANKRD11 mutations had macrodontia. Regarding neurodevelopmental abnormalities, 45% of patients had developmental delay, 45% had intellectual disability and/or learning difficulties, and 40% had seizures or other electroencephalogram anomalies. Behavioral problems, hyperactivity, or aggressivity were often observed in patients. Remarkably, 75% of patients had hand abnormalities, such as clinodactyly, brachydactyly, or syndactyly. Delayed bone age and short stature were found in 45% and 75% of patients, respectively. Intrafamilial variability was commonly reported (case Nos. 11–16) [1-3, 5, 7-11].

Clinical features appeared to be less severe in all patients’ parents. All parents carrying null ANKRD11 mutations had several craniofacial features and intellectual disability or learning difficulties. Except for our patient’s mother, parents with missense or in-frame mutations (case Nos. 3, 4, and 14) presented with milder phenotypes, mainly including short stature (Table 1). The heights of the parents of case Nos. 3 and 4 (–1.5 SD and –0.8 SD, respectively) did not fit the diagnosis of short stature. Together, these results demonstrate intrafamilial variability in KBG syndrome, highlighting the incomplete penetrance of missense mutations, particularly, those in RD1, in KBG syndrome as suggested by Zhang, et al. [3].

In conclusion, we reported two novel ANKRD11 mutations in two patients with typical KBG syndrome phenotypes, expanding the molecular and clinical spectrum of this rare genetic disorder. Importantly, our results support low and incomplete penetrance and variable expressivity in this rare neurodevelopmental disorder and pave the way to novel experimental approaches.

Funding Statement

RESEARCH FUNDING None declared.