Molecular findings
The variants occurred de novo in 12 individuals, were maternally inherited in Individuals K and L, and paternally inherited in individual O. One parent of affected Individual T, Individual U, showed a low level of mosaicism for the variant (with only 2 out of 298 sequencing reads for this variant found in her blood). Nine individuals had unknown modes of inheritance. A majority, 20, are truncating variants (frameshift or nonsense), and five are missense (with three of five belonging to the same family). Twenty-one distinct variants were identified (Table1), with locations shown in Fig.2 [18].
The coding exons for ANKRD11 are depicted to scale. Abbreviations: aa amino acid. The figure was made using: https://www.cbioportal.org/mutation_mapper.
Truncating variants are classified by ACMG criteria [19] as: PVS1 null variant (nonsense, frameshift) in a gene where loss of function is a known mechanism of disease. Some variants are classified as PS2 De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. One missense variants in our cohort (p. (Val586Met) was seen in a heterozygous control individual in the Genome Aggregation Database (GnomAD), thus calling into question its pathogenicity. It is also formally possible that the one individual in GnomAD might be mildly affected. The mother with this variant (individual M) has a very mild phenotype whereas her children (individuals K and L) have phenotypes more consistent with KBG syndrome. However, a recent preprint [20] demonstrated that some missense variants do impair ANKRD11 ability and/or stability, but that these variants mainly localize in the Repression Domain 2. Those authors also tested one variant in the Repression domain 1 (p.Leu509Pro), which turned out to have no effect on ANKRD11 stability or activity. The p.(Val586Met) variant of individuals K, L, and M also falls within the Repression Domain 1, and it has a borderline CADD score (23.9) and is not as highly conserved as the other missense variants. In addition, the affected nucleotides and corresponding amino acid are also not highly conserved when the sequence is aligned with other species. Per DeepGestalt, these individuals (K, L, M) did not have KBG syndrome listed in their top 30 differentials. Segregation analysis with the mother and sister of Individual M is not yet available. While the mother has very mild clinical features of KBG syndrome, the sister (aunt of Individuals K and L) is potentially reporting more severe symptoms. Ultimately, the pathogenicity of the variant (p.(Val586Met)) is still uncertain.
A different missense variant (p. Arg2536Gln) arose de novo and was initially classified as a variant of uncertain significance because it had not been previously reported. However, it has been reclassified because of new information available: two additional patients carrying the variant. One is reported in Clinvar (https://www.ncbi.nlm.nih.gov/clinvar/variation/1012410/?new_evidence=false), a patient in whom the variant was maternally inherited (referred to as Individual Z in Supplementary Information), but who was unavailable for videoconferencing. In the other previously reported patient, the variant has arisen de novo and was classified as pathogenic [21]. Although a more extensive cosegregation of the patient reported in Clinvar is not available, since phenotypes characteristic of KBG syndrome are seen in three individuals possessing this variant, the variant is reclassified to likely pathogenic. Further details about these cases can be found in Supplemental Text and Case Summaries.
As of April 2022, there are 429 putative missense or non-frameshift deletion, substitution or insertion variants in ANKRD11 submitted to ClinVar [22], with many of these listed as variants of uncertain significance (Supplementary Table2), with bioinformatic analyses providing a suggested consensus classification for each variant.
Median age of the 25 individuals was 11 years and average age was 15 years (range=159). One comes from a consanguineous family, roughly half (n=12) had a history of congenital abnormalities in the family, and eight had relatives with intellectual disabilities.
The parents of individuals B, D, T, and Y had histories of miscarriage. The variant was de novo for individual B, whereas the parent of individual T (Individual U) was mosaic for the missense variant (as noted above). The mother of individual Z has a history of several miscarriages early in pregnancy around six weeks of age. The inheritance pattern is unknown for individuals D and Y.
The parents in this study (M, P, U) generally had mild phenotypic features. Individual M, the mother of K and L, possessed some distinct facial traits (e.g., thick eyebrows, anteverted nares, broad nasal base), however, the overall constellation of features was not typical of KBG syndrome. She did not present with common features such as developmental delay, macrodontia, or short stature. Conversely, individual P, the father of O, presented with global developmental delay, macrodontia, and short stature among other common traits of KBG syndrome. Lastly, individual U, the mother of T, had mild facial features (e.g., synophrys, thick eyebrow, wide nasal bridge, prominent nasal tip) with speech delays and seizures in childhood.
The overall frequency of certain phenotypic features is shown in Table2, and these are reviewed in further detail in the following sections.
Height at the time of videoconference clustered into 398th centile (44%), below 3rd centile (24%) and above 98th centile (12%) with a median height of 140.0 29.4cm. Weights at time of videoconference clustered into 3-98th centile (48%), below the 3rd centile (20%), and above 98th centile (4%), with a median weight of 27.8 29.1kg. Of the three individuals who had heights above the 98th centile at time of videoconference, one had been put on growth hormone for approximately 24 years (Individual J) (Table3). Birth length clustered into 398th centile (44%), above 98th centile (8%), and below 3rd centile (8%), with a median length of 49.0 6.3cm. Birth weight clustered between 398th centile (64%), and below the 3rd centile (16%) with a median birth weight of 3 0.7kg.
The photographs with permission for publication are shown in Fig.3. At least one distinctive facial feature common to KBG patients was present in every individual interviewed. Defining facial characteristics include thick eyebrows with synophrys, prominent eyelashes, wide nose, thin upper lip vermillion, and macrodontia. Many have a triangular face or pointed chin and a broad or prominent forehead.
Characteristic features include bushy eyebrows (A, C, D, E, I, K, M, O, P, R, T, U, V, Y), long eyelashes (C, D, I, L, O, P, S, X,), triangular face (A, G, K, R, V) and most had a wide nasal bridge or tip and a thin upper vermillion.
Pairwise ranks of the 25 photos in Fig.4 suggest most patients described in this analysis share similar facial phenotypes. In a gallery of 3533 images with 816 different disorders and 25 KBG patients, 15 out of 25 KBG patients had at least one other KBG patient in their top-10 rank, and 21 out of 25 patients had at least one other patient in their top-30 rank. Other than U being an outlier, there was a cluster containing the set of patients with three sub-clusters (P, J, F, and M), (O, H, R, Y, V, G, and I), and (Q, S, D, and E). Patient U was an outlier, perhaps due to the low-level mosaicism for this variant. No clear clusters were seen when segregated by type of genetic variant (missense, frameshift, nonsense). The similarity between family members is a known confounder in the analysis of syndromic similarity. On average, family members with the same disorder are closer in the clinical face phenotype space than unrelated individuals with the same disorder. That said, in one family, we do not see an increased similarity between M, K, and L.
Sub-cluster P, J, F, M present with synophrys and wide noses. Sub-cluster O, H, R, Y, V, G, I present with thick eyebrows, prominent/broad nasal tips, macrodontia, triangular faces and pointed chins. Sub-cluster Q, S, D, E present with anteverted nares, broad nasal tips, and macrodontia. Link: https://db.gestaltmatcher.org/; individual links to each patient in Supplemental Text. Note: Individual E did not consent to having their photo published, however, a frontal photo was input into the GestaltMatcher and DeepGestalt algorithms.
KBG syndrome was recommended among the top 30 syndromes and ranked as the first (i.e., most likely) diagnosis for 28% (n=7) of individuals, second for 40% (n=10), and third or fourth for 12% (n=3). Overall, 80% (n=20) of patients photos analyzed had KBG syndrome ranked in their top-five potential diagnoses out of the 30 possible suggested syndromes from among the 300+ syndromes currently recognized by the DeepGestalt algorithm. Among the 20 with KBG in the top-five rank, seven had a high gestalt score, 10 had medium gestalt, and three had low gestalt. Fourteen had a medium feature score, five had a low score, and one was unranked for features of KBG (see Supplementary Table3). Individuals B, F, and J initially submitted photos where they were wearing glasses. After analyzing photos without glasses, the ranking of KBG surprisingly dropped from two to six for individual B and from two to three for individual J. Ranking did not change for individual F. While KBG ranking fluctuated, the gestalt and feature levels did not change between the photos with and without glasses for any of the three individuals.
Five individuals (K, L, M, P, U) did not have KBG syndrome appear as a differential diagnosis out of 30. First ranked diagnoses instead included Cornelia de Lange, Williams-Beuren, Rubinstein-Taybi, Angelman, and mucopolysaccharidosis. Notably, Individual P was 5560 years old at the time of the videoconference whereas Individual U was 3035 years old, and both of them initially submitted pictures of themselves around those ages. These ages fall above our median age of 11 years and the age at which most individuals are diagnosed with KBG syndrome. DeepGestalt relies on the photos that it is trained on, so older age photos may not perform as well. Additionally, individual U has very low-level mosaicism for this variant, potentially resulting in lower phenotypic expression of facial features. The other three individuals who were unranked (K, L, and M) are all from the same family and possess the same missense variant (Table1) with questionable pathogenicity.
With PEDIA score, the disease-causing gene ANKRD11 is ranked at the first place in 18 out of 25 (top-1 accuracy: 72%). When looking at the top-10 genes, ANKRD11 is listed in the top-10 genes in 22 out of 25 (top-10 accuracy: 88%). All have ANKRD11 in their top-30 genes.
Eight reported an intelligence quotient (IQ) score, with a mean of 734.84 (range=6480) as measured by the Weschler Intelligence Scale (3rd to 5th edition). A majority, 68% are considered mildly to moderately intellectually disabled based on level of functioning. Global developmental delays prior to 5 years were seen in 68% (n=17), with nine being classified as mild. Median age of crawling onset was 12 months (range=924) (n=8), walking onset 22 months (range=12.536) (n=10), and speech onset 30 months (range=1936) (n=6). Selective mutism and absent speech were observed in three individuals.
Common types of seizures reported included myoclonic, tonic-clonic, and absence with no specific type predominating [23]. Electroencephalogram (EEG) abnormalities were documented in three of 11 individuals with seizures. According to maternal report, Individual E was meeting speech and motor milestones until the onset of myoclonic seizures, complex partial seizures, and verbal tonic seizures with respiratory distress around 0.52 years of age. Similarly, individuals H, K, R, S, T, U, X, and Y reported histories of various types of seizures and concurrent speech and motor delays. Brain abnormalities detected on magnetic resonance imaging (MRI) included pineal cyst, arachnoid cyst, choroid plexus cyst, subdural hemorrhage, and small pituitary gland.
Abnormal mood included abnormal emotion or affect, depression, and/or anxiety, self-injurious behavior including self-biting. Individuals E, O, Q, and R report absent or high pain threshold. O has a history of a fractured foot and a dislocated kneecap with bone scans showing normal density. Impaired tactile sensation was reported in two individuals (M,S).
Six had chronic otitis media, with five of six having concurrent hearing impairment. Those experiencing chronic otitis media likewise had a preauricular pit, abnormal or blocked Eustachian tubes, abnormality of the tympanic membrane, enlarged vestibular aqueduct, choanal atresia, and increased size of nasopharyngeal adenoids. Hearing loss and recurrent infections including sinus, chronic ear, and upper respiratory infections were present in four individuals (O, P, Q, Y). Of the six with palatal anomalies, four had difficulties feeding.
Of note, individual A was diagnosed with osteopenia, and later osteoporosis, at 1520 years with low bone mineral densitometry in the lumbar spine, hip, and femoral neck. An x-ray of his left hand and wrist was performed which revealed physeal closure of the bones, excluding delayed bone maturation. Individual S has visible sacral dimple and was referred to neurology for gait disturbance and urinary incontinency. MRI of her lumbar spine revealed a tethered spinal cord.
Cardiac abnormalities were seen in approximately half the participants and while many resolved without the need for surgical intervention, individual K had Tetralogy of Fallot with pulmonary valve-sparing surgical repair at ~36 months of age. Individual T had mitral valve repair at around one year of age.
Participants F, M, S, T, U had presumed diagnoses of abdominal migraines, characterized by stomach pain, nausea, and vomiting. In F, the abdominal migraines were accompanied by cyclic vomiting syndrome. Reports described her episode as significant pain causing writhing with soft, nontender abdomen normal bowel sounds on examination.
Short stature is a common phenotype in those with KBG syndrome with up to 66% below the 10th centile in height [5]. Individuals H, J, and O were administered growth hormone. J was born with a length below 1st centile and weight at 57th centile. After receiving somatropin injections from 3.5 years to 5.7 years of age, his height is at the 13th centile and weight is at 24th centile. O was given growth hormone from approximately 6 years to 11 with positive improvement in weight (11th percentile at birth and is now at 45th percentile). Efficacy of hormone supplementation is unknown for H. Reports of precocious puberty, immunodeficiency, recurring infections, allergies are also common.
Urogenital disorders were seen in 48% (n=12) of individuals, with seven being female and five being male. Of note, four males were diagnosed with cryptorchidism. Other diagnoses included abnormalities of the urethra and/or bladder, recurrent urinary tract infection, pollakiuria, polyuria, and enuresis.
A majority (56%) reported abnormalities of skin, nails, and hair, which included: hirsutism, low anterior hairline or abnormal hair whorl, cellulitis, keratosis pilaris, acne and dry skin, psoriasiform dermatitis, eczema, fingernail dysplasia, and recurrent fungal infections.
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