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Department of Human Genetics, Radboudumc, Nijmegen, The NetherlandsDonders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands
Correspondence and requests for materials should be addressed to Charlotte W. Ockeloen, Department of Genetics, Radboudumc, P.O. Box 9101, 6500 HB, Nijmegen The Netherlands
Department of Human Genetics, Radboudumc, Nijmegen, The NetherlandsRadboud Institute for Molecular Life Sciences, Radboudumc, Nijmegen, The Netherlands
Department of Human Genetics, Radboudumc, Nijmegen, The NetherlandsDonders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands
Department of Human Genetics, Radboudumc, Nijmegen, The NetherlandsDonders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands
Department of Clinical Genetics, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United KingdomClinical Genetics, Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom
Genetic Laboratory of Rare and Autoinflammatory Diseases, Department of Medical Genetics, Rare Diseases and Personalized Medicine, Centre Hospitalier Universitaire de Montpellier, Montpellier, France
Service de Génétique, Hospices Civils de Lyon, Bron, FranceInstitut NeuroMyoGene, CNRS UMR5310, INSERM U1217, Université Claude Bernard Lyon 1, Lyon, France
Génétique des Anomalies du Développement, Université de Bourgogne Franche-Comté, UMR1231-Inserm, Dijon, FranceLaboratoire de Génétique Chromosomique et Moléculaire, UF6254 Innovation en Diagnostic Génomique des Maladies Rares, Centre Hospitalier Universitaire de Dijon, Dijon, France
Génétique des Anomalies du Développement, Université de Bourgogne Franche-Comté, UMR1231-Inserm, Dijon, FranceCentre de Génétique et Centre de Référence Anomalies du Développement et Syndromes Malformatifs de l'Interrégion Est, Centre Hospitalier Universitaire Dijon, Dijon, FranceFédération Hospitalo-Universitaire Médecine Translationnelle et Anomalies du Développement (TRANSLAD), Centre Hospitalier Universitaire Dijon, Dijon, France
Department of Plastic and Reconstructive Surgery and Hand Surgery, Dutch Craniofacial Center, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The NetherlandsDepartment of Bioinformatics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indiana University, Indianapolis, INDepartment of Epidemiology, Richard M. Fairbanks School of Public Health, Indiana University, Indianapolis, IN
Department of Clinical and Molecular Genetics, Vall d'Hebron University Hospital and Medicine Genetics Group, Vall d'Hebron Research Institute, Barcelona, Spain
Service de Génétique, Hospices Civils de Lyon, Bron, FranceInstitut NeuroMyoGene, CNRS UMR5310, INSERM U1217, Université Claude Bernard Lyon 1, Lyon, France
Department of Plastic and Reconstructive Surgery and Hand Surgery, Dutch Craniofacial Center, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
Service de Génétique - Centre de Référence Anomalies du Développement, Hospices Civils de Lyon, Bron, FranceÉquipe GENDEV, Centre de Recherche en Neurosciences de Lyon, INSERM U1028 CNRS UMR5292, Université Claude Bernard Lyon 1, Lyon, France
Department of Human Genetics, Radboudumc, Nijmegen, The NetherlandsDonders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands
Génétique des Anomalies du Développement, Université de Bourgogne Franche-Comté, UMR1231-Inserm, Dijon, FranceCentre de Génétique et Centre de Référence Anomalies du Développement et Syndromes Malformatifs de l'Interrégion Est, Centre Hospitalier Universitaire Dijon, Dijon, France
Department of Human Genetics, Radboudumc, Nijmegen, The NetherlandsDepartment of Clinical Genetics, Maastricht University Medical Center+, Maastricht University, Maastricht, The Netherlands
Department of Clinical and Molecular Genetics, Vall d'Hebron University Hospital and Medicine Genetics Group, Vall d'Hebron Research Institute, Barcelona, Spain
Centre de Génétique et Centre de Référence Anomalies du Développement et Syndromes Malformatifs de l'Interrégion Est, Centre Hospitalier Universitaire Dijon, Dijon, France
Génétique des Anomalies du Développement, Université de Bourgogne Franche-Comté, UMR1231-Inserm, Dijon, FranceLaboratoire de Génétique Chromosomique et Moléculaire, UF6254 Innovation en Diagnostic Génomique des Maladies Rares, Centre Hospitalier Universitaire de Dijon, Dijon, France
Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indiana University, Indianapolis, INDepartment of Pediatrics, Indiana University School of Medicine, Indianapolis, IN
Centre de Génétique et Centre de Référence Anomalies du Développement et Syndromes Malformatifs de l'Interrégion Est, Centre Hospitalier Universitaire Dijon, Dijon, France
Department of Human Genetics, Radboudumc, Nijmegen, The NetherlandsRadboud Institute for Molecular Life Sciences, Radboudumc, Nijmegen, The Netherlands
Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The NetherlandsLanguage and Genetics Department, Max Planck Institute for Psycholinguistics, Nijmegen, The Netherlands
Department of Human Genetics, Radboudumc, Nijmegen, The NetherlandsDonders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands
Department of Human Genetics, Radboudumc, Nijmegen, The NetherlandsDonders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The NetherlandsCenter of Excellence for Neuropsychiatry, Vincent van Gogh Institute for Psychiatry, Venray, The Netherlands
Although haploinsufficiency of ANKRD11 is among the most common genetic causes of neurodevelopmental disorders, the role of rare ANKRD11 missense variation remains unclear. We characterized clinical, molecular, and functional spectra of ANKRD11 missense variants.
Methods
We collected clinical information of individuals with ANKRD11 missense variants and evaluated phenotypic fit to KBG syndrome. We assessed pathogenicity of variants through in silico analyses and cell-based experiments.
Results
We identified 20 unique, mostly de novo, ANKRD11 missense variants in 29 individuals, presenting with syndromic neurodevelopmental disorders similar to KBG syndrome caused by ANKRD11 protein truncating variants or 16q24.3 microdeletions. Missense variants significantly clustered in repression domain 2 at the ANKRD11 C-terminus. Of the 10 functionally studied missense variants, 6 reduced ANKRD11 stability. One variant caused decreased proteasome degradation and loss of ANKRD11 transcriptional activity.
Conclusion
Our study indicates that pathogenic heterozygous ANKRD11 missense variants cause the clinically recognizable KBG syndrome. Disrupted transrepression capacity and reduced protein stability each independently lead to ANKRD11 loss-of-function, consistent with haploinsufficiency. This highlights the diagnostic relevance of ANKRD11 missense variants, but also poses diagnostic challenges because the KBG-associated phenotype may be mild and inherited pathogenic ANKRD11 (missense) variants are increasingly observed, warranting stringent variant classification and careful phenotyping.
KBG syndrome (OMIM 148050) is an autosomal dominant neurodevelopmental disorder (NDD) typically characterized by mild intellectual disability (ID) or developmental delay, macrodontia of upper central permanent incisors, mild skeletal anomalies, behavioral disturbances, and distinctive craniofacial features.
there is considerable clinical variability and none of the KBG features is pathognomonic. Hence, despite being described as a clinical entity since 1975,
The exact prevalence of KBG syndrome is not established but it is thought to be a relatively common cause of genetic NDDs, with the associated gene (ANKRD11) in the top 3 of mutated genes in NDD cohorts accounting for 0.5% to 1% of diagnoses.
KBG syndrome is caused by heterozygous protein truncating variants (PTVs) in ANKRD11 (encoding ANKRD11) or by 16q24.3 microdeletions encompassing (part of) ANKRD11. PTVs and microdeletions explain all cases in 4 previously described KBG cohorts,
whereas in the general population, ANKRD11 shows strong constraint against loss-of-function variation (probability of being loss-of-function intolerant [pLI] = 1; observed/expected [o/e] = 0.05 [0.02-0.11]; gnomAD v2.1.1).
Also consistent with haploinsufficiency is the finding that ANKRD11 mutated with p.(Lys1347del) or p.(Leu2143Val) shows reduced transcriptional activity on the p21 promotor in cell-based systems, that can be rescued by wild type but not mutated ANKRD11.
showing (co)regulatory effects on various sets of genes. These include genes encoding signaling molecules, chromatin remodelers, and transcriptional regulators,
controlling histone acetylation and gene expression during neural development. ANKRD11 contains 3 transcriptional regulatory domains: 1 activation domain and 2 repression domains (RDs). The RDs, located at the N-terminus (RD1) and C-terminus of ANKRD11 (RD2), functionally outweigh the activation domain, because full-length ANKRD11 functions as a repressor of ligand-dependent transcription.
Whereas PTVs in ANKRD11 are a well-recognized cause of KBG syndrome, the role of rare missense variants remains ambiguous. Contrary to what is seen for PTVs, constraint metrics based on the general population indicate that missense variants tend to be well-tolerated (z-score –0.55; o/e = 1.04 [1-1.08]).
There are numerous entries of ANKRD11 missense variants in ClinVar (access date August 20, 2021) but only approximately 6% are classified as (likely) pathogenic and almost half as variants of uncertain significance.
listed in Supplemental Table 1. Missense variants are reported with varying levels of evidence on pathogenicity, and functional studies have only been performed for p.(Leu2143Val), showing a loss-of-function effect.
The Yoda mutant mouse (C3H.Cg-Ankrd11Yod/H, p.[Glu2502Lys]) carries an Ankrd11 missense variant and shows phenotypic overlap with core features of KBG syndrome, including reduced body size and craniofacial abnormalities such as shortened snouts with deformed nasal bones, wider skulls, and failure of cranial sutures to close.
On the cellular level, the heterozygous Yoda variant causes similar cellular perturbations of abnormal neuronal precursor proliferation and localization of neurons as seen for Ankrd11 knockdown,
Because the N-terminal ankyrin repeats are unaffected by the variant, dimerization of wild-type and mutant Ankrd11 was hypothesized to result in decreased degradation of both proteins, potentially implicating such dominant-negative mechanism.
So far, a dominant-negative mechanism has not been confirmed in additional studies. In general, consequences of ANKRD11 missense variants on clinical phenotypes and protein function are largely unknown.
We characterized genotypes, phenotypes, and functional consequences associated with ANKRD11 missense variants by describing a cohort of 29 individuals. Most individuals exhibit both characteristic facial appearance and other KBG-associated features, fitting well within the clinical spectrum described for KBG syndrome. We showed that missense variants in ANKRD11 significantly cluster in the C-terminal RD2, with an overrepresentation of mutated arginine residues. Missense variants result in a loss of normal ANKRD11 function, either caused by reduced protein stability with normal or increased proteasome degradation or caused by a loss of transrepression capacity with decreased proteasome degradation. Our findings are consistent with ANKRD11 haploinsufficiency, mechanistically underlying KBG syndrome caused by PTVs or 16q24.3 microdeletions.
Materials and Methods
Clinical and InSilico characterization
Identification and clinical characterization of individuals and in silico analyses of (likely) pathogenic ANKRD11 missense variants are described in the Supplementary Methods. Variants were annotated in the context of genome build GRCh37/Hg19, using transcript reference sequence NM_013275.6 and protein reference sequence NP_037407.4. Human Phenotype Ontology (HPO)-based
using clinical data of 29 individuals with ANKRD11 missense variants and 35 individuals with ANKRD11 PTVs or microdeletions (Supplemental Table 2A-C, Supplemental JSON) after grouping HPO-data on the basis of semantic similarity (Supplemental Table 3A).
excluding 4 familial variants. The Supplementary Methods contain details of both clustering analyses. P values < .05 were considered significant.
Missense permutation analysis
To test whether the observed number of variants affecting arginine residues was significantly greater than expected by chance, ANKRD11 (ENST00000301030.10/NM_013275.6) was mutated in silico and output was annotated using Ensembl Variant Effect Predictor v104.
To generate an expected missense distribution, sets of 17 missense variants in RD2 and 8 missense variants outside RD2 (based on 25 independently observed missense variants, excluding 4 familial variants) were randomly sampled 100,000 times using per-nucleotide mutation rates as weights.
Number of missense variants affecting arginine residues inside and outside RD2 were counted per iteration. P values were computed using a permutation test by ranking the observed number of variants affecting arginine residues within the set of 100,000 expected values. P values < .05 were considered significant.
Cell culture and transfection
HEK293T/17 cells (CRL-11268, ATCC) were cultured in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 100 U/mL penicillin-streptomycin (Thermo Fisher Scientific) at 37°C and 5% carbon dioxide. For immunofluorescence analysis, cells were seeded onto coverslips coated with 100 μg/mL poly-D-lysine (Merck, Millipore). Transfections were performed using GeneJuice (Merck, Millipore) following the manufacturer’s instructions or polyethyenimine in 3:1 ratio with total mass of DNA transfected.
DNA constructs and site-directed mutagenesis
Full-length wild-type ANKRD11 construct fused to a C-terminal Myc-DDK tag under a human cytomegalovirus (CMV) promoter (pCMV-Entry-ANKRD11) was purchased from Origene (RC211717). To generate an N-terminal EGFP-tag, sequence encoding EGFP was cut from a pEGFP-C2 vector (Clontech) and subcloned into the pCMV-Entry-ANKRD11 plasmid using KpnI/NdeI restriction sites. Constructs (pCMV-Entry-EGFP-ANKRD11) carrying (in frame) ANKRD11 missense variants were generated using a QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent) following the manufacturer’s protocol. All constructs were verified using Sanger sequencing. Supplemental Table 4 lists primer sequences.
Fluorescence imaging of subcellular localization
HEK 293T/17 cells grown on poly-D-lysine-coated coverslips were transiently transfected with 500 ng of pCMV-EGFP-ANKRD11 constructs 24 hours after seeding. Cells were fixated 48 hours after transfection using 4% paraformaldehyde solution (Electron Microscopy Supplies Ltd) for 20 minutes at room temperature. Hoechst 33342 (Invitrogen) was used for nuclear staining, before mounting with VECTASHIELD Antifade Mounting Medium (Vectorlab). Fluorescence images were obtained using an LSM880 AxioObserved confocal microscope (Zeiss). For images of single nuclei, the Airyscan unit (Zeiss) was used with a 4.0 zoom factor. Images were analyzed using the ImageJ “Analyze particle” plugin.
Fluorescence-based quantification of protein stability and degradation
HEK293/T17 cells were transfected in triplicate in clear-bottomed black 96-well plates with EGFP-tagged ANKRD11 variants. After 48 hours, cycloheximide (Sigma Aldrich) at 50 μg/mL or MG132 (R&D Systems) at 5 μg/mL was added. Cells were incubated at 37°C with 5% carbon dioxide in the Infinite M200PRO microplate reader (Tecan), and fluorescence intensity of EGFP (excitation: 503 nm, emission: 540 nm) was measured over 24 hours at 3-hour intervals. Statistical analysis was performed using 2-way analysis of variance followed by Dunnett’s correction for multiple testing. P values < .05 were considered significant.
Luciferase reporter assays
We used firefly luciferase reporters: pGL2-p21 promoter-Luc and WWP-Luc carrying the promoter region of CDKN1A/P21. Reporters were gifts from Martin Walsh (Addgene plasmid #33021; http://n2t.net/addgene:33021; RRID:Addgene_33021)
respectively. HEK293/T17 cells were seeded in clear-bottomed white 96-well plates (Greiner Bio-One). Cells were cotransfected with 320 ng of firefly luciferase reporter construct, 6.5 ng of pGL4.74 Renilla luciferase normalization control, and 1000 ng of an EGFP-ANKRD11 expression construct or empty EGFP expression vector. After 48 hours, firefly luciferase and Renilla luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions on an Infinite F Plex microplate reader (Tecan). Statistical analysis was performed using a 1-way analysis of variance followed by Dunnett’s post hoc test. P values < .05 were considered significant.
Results
ANKRD11 missense variants cause syndromic neurodevelopmental phenotypes
we identified 29 individuals with rare missense variants in ANKRD11 (Figure 1; Supplemental Table 2A). The cohort consisted of 24 unrelated individuals with ANKRD11 missense variants—of which 20 variants occurred de novo—and 1 family with 5 affected individuals from 3 generations (Supplemental Figure 1). For 5 individuals, inheritance status of the variant could not be established. The cohort comprises 18 males and 11 females, with an age range of 7 months to 73 years. We observed syndromic neurodevelopmental phenotypes, summarized in Table 1 with detailed data compiled in Supplemental Table 2A and B and Supplementary Results. Most frequent phenotypic features were facial dysmorphisms (28/29 [96.6%]; fitting characteristic dysmorphisms of KBG, 23/29 [79.3%]; Figure 2A), behavioral disturbances (25/28 [89.3%]), neurodevelopmental delay (26/28 [92.9%]; speech delay, 23/26 [88.5%]; motor delay, 20/27 [74.1%]), mild to moderate ID (22/27 [81.5%]; borderline, 2/27 [7.4%]; mild, 15/27 [55.6%]; moderate, 4/27 [14.8%]; unknown severity, 3/27 [11.1%]), abnormal dentition (21/26, [80.8%]; macrodontia of upper central incisors, 13/24 [54.2%]; other dental abnormalities, 14/21 [66.7%]; Figure 2B), and hand abnormalities (20/25 [80%]; Figure 2C). Of note, individual 19 also carried a likely pathogenic variant in ARID2, implicated in Coffin-Siris syndrome 6 (OMIM 617808), and in the family of 5 individuals an additional ANKRD11 variant of uncertain significance, p.(Pro61Ser), was observed in cis with the pathogenic p.(Arg2579His) variant.
Figure 1Missense variants cluster in the intrinsic repressor domain 2 in the C-terminus of the ANKRD11 protein. A. Schematic representation of ANKRD11 (UniProt: Q6UB99, having 100% sequence identity with NP_037407.4) indicating the location of variants included in this study. De novo variants are indicated by black disks, inherited variants by gray disks, variants with unknown inheritance by white disks, and variants marked with # are included in functional assays. The ANKRD11 protein sequence (2663 amino acids) contains an ankyrin-repeat domain (ANK; purple; amino acids 133-296), 4 PEST sequences (gray; amino acids 286-324, 1796-1806, 2146-2212, and 2222-2297), 4 bipartite nuclear localization signals (NLS; black; amino acids 1184-1200, 1208-1236, 1358-1374, 1640-1656), 2 intrinsic repressor domains (RD) (RD1; blue; amino acids 318-611 and RD2; green; amino acids 2369-2663), and an activator domain (AD1; pink; amino acids 2076-2145). An overview with variant details per subject is provided in Supplemental Table 2A, with details on variant interpretation in Supplemental Table 5. B. MetaDome analysis of the ANKRD11 missense variants. Overview of the ANKRD11 protein (NP_037407.4) tolerance landscape visualized via the MetaDome web server version 1.0.1. The green and blue peaks correspond to regions more tolerant to missense variation and the red valleys indicate intolerant regions. C. Sequence alignment of the region containing part of ANKRD11 amino acid sequence in human (UniProt: Q6UB99), chimpanzee (A0A2I3TR65), mouse (E9Q4F7), chicken (A0A3Q2UE98), African clawed frog (A0A1L8GEN1), and zebrafish (E7F5R3). Residues in which missense variants were found are highlighted in gray. D-boxes, destruction box motifs.
Figure 2Clinical evaluation of individuals with ANKRD11 missense variants. A. Facial photographs of affected individuals. Features of most individuals fit the characteristic facial gestalt of KBG syndrome (individuals 1, 2, 8, 10, 15, 17, 23, 24, 28). Individuals display a triangular face (individuals 4, 10, 15, 23, 24, 28), with full eyebrows and synophrys (individuals 1, 23, 28). Both downslant (individuals 3, 10) and upslant (individuals 1, 2, 8, 15, 21, 23, 28) of palpebral fissures can be observed. Individuals 2, 4, 8, 10, 15, 21, and 23 exhibit a low nasal bridge, with an upturned nasal tip and triangular-shaped nasal tip in individuals 4, 8, 10, 15, 17, 23, and 24. In individuals 1, 2, 4, 10, 15, and 23 a smooth philtrum is observed, with a thin upper lip in individuals 1, 2, 4, 15, 23, and 24. From the side, low-set, simple ears (individuals 2, 4, 23, 24, 28) and a flat facial profile (individuals 1, 2, 23, 24) can be observed. B. Photographs of teeth, showing macrodontia of central permanent incisors in individuals 8, 10, and 21, with teeth discolorization in individuals 21 and 22, and widely spaced teeth in individuals 4, 8, and 23. C. Photographs of hands, showing short palms (individuals 1, 2, 3, 17, 21, 22, 23, 28) and short fingers (individuals 21, 22, 24, 28) with tapering (individual 2, 21, 22, 23, 24) and clinodactyly of the fifth digit (individual 1, 2, 3, 22, 24, 28). D. Visualization of Human Phenotype Ontology-based clustering analysis, indicating that the clustering algorithm recognizes a difference between individuals with missense variants and individuals with PTVs or 16q24.3 microdeletions. PC, principal component; PTV, protein truncating variant.
ANKRD11 missense variants are predicted deleterious and cluster at C-terminal RD2
We found 20 unique missense variants (Figure 1; Supplemental Table 5) significantly clustering at the highly intolerant C-terminal RD2 (P = 9.99e-9), with recurrence of p.(Arg2512Gln), p.(Glu2522Lys), p.(Arg2579His), p.(Arg2585Cys), and p.(Leu2605Arg). In addition, the arginine residues at p.2512, p.2536, and p.2579 were affected by 2 different missense variants, and p.(Glu2522Lys) is equivalent to the orthologous Ankrd11 p.(Glu2502Lys) in the Yoda mouse (Supplemental Figure 2). Most of observed variants are predicted deleterious and affect conserved and intolerant residues (Figure 1B and C), with no predicted effects on premessenger RNA splicing (Supplemental Table 5). Two variants, p.(Glu2522Lys) and p.(Arg2523Trp), affect a ProViz
predicted (low consensus similarity) D-box (Supplemental Table 6; Supplemental Figure 3) and 2 variants, p.(Arg2512Leu) and p.(Arg2512Gln), are located at an additional RxxL-motif, marked as D-box in literature.
We observed a significantly greater number of missense variants affecting arginine residues within RD2 (12/17 variants [70.6%]) than would be expected by chance (P = 1.00e-4), whereas such enrichment was not observed outside RD2 (0/8 variants; 0%; P = .37), visualized in Supplemental Figure 4. We therefore hypothesized that generally, ANKRD11 missense variants affecting arginine residues in RD2 are likely to be pathogenic.
Phenotypes associated with ANKRD11 missense variants fit the KBG-associated clinical spectrum
On the basis of 4 large published cohorts together describing 135 individuals with KBG syndrome caused by PTVs or 16q24.3 microdeletions affecting ANKRD11,
we assessed phenotypic fit of each of the individuals in the missense cohort to the clinical spectrum associated with KBG syndrome. The majority of individuals (23/29 [79.3%]) exhibited dysmorphisms fitting the characteristic facial gestalt by which KBG syndrome can be recognized (Figure 2A and B; Supplemental Table 2A and B, Supplemental Table 5). Indeed, 2 individuals in the cohort (individual 6 and 14) were diagnostically evaluated through targeted ANKRD11 Sanger sequencing because of a high clinical suspicion. Of the 29 individuals, 22 (75.9%) met the diagnostic criteria described for KBG syndrome
(Supplemental Table 2A). After review of the observed phenotypes by expert clinicians, it was concluded that almost all individuals fit the KBG-associated phenotypic spectrum. These included many individuals not fully meeting the diagnostic criteria, either because some features of KBG syndrome not captured in the diagnostic criteria (eg, delayed bone age and congenital heart defects) were seen or because the characteristic facial appearance of KBG syndrome was observed. Only for individuals 3 and 29, carrying p.(Leu509Pro) and p.(Leu2605Arg), phenotypic fit to the clinical spectrum of KBG syndrome was considered poor.
We next compared the group of individuals with missense variants with the collective KBG cohorts
and found that frequencies of most KBG-associated features observed in the missense cohort lie within the range of frequencies of these features seen in the group of individuals with PTVs or microdeletions (Table 2). We therefore hypothesized that KBG syndrome resulting from missense variants is indistinguishable from KBG syndrome caused by ANKRD11 PTVs or microdeletions.
Table 2Comparison of clinical features between individuals with ANKRD11 missense variants and previously reported KBG cohorts
) of 29 individuals with missense variants and 35 individuals with KBG syndrome caused by ANKRD11 PTVs or microdeletions affecting ANKRD11 only. Applying a Partitioning Around Medoids clustering algorithm
on 68 features derived from HPO-data resulted in correct classification of 40 of 64 individuals as either belonging to the PTV or missense variant group (P = .04396; Figure 2D; Supplemental Table 3A-C), indicating that the algorithm recognizes a difference between the group with PTVs or microdeletions and the group with missense variants. This challenges the clinical observation that pathogenic ANKRD11 missense variants and PTVs or microdeletions cause the same clinical entity.
ANKRD11 missense variants act via 2 distinct loss-of-function mechanisms
We continued by studying the functional consequences on protein localization, protein stability, proteasome degradation, and transcriptional activity of a subset of the observed ANKRD11 missense variants using HEK293T/17 cells transiently transfected with mutant ANKRD11. To obtain a comprehensive insight of the spectrum of variants, we examined variants spread across ANKRD11, including p.(Leu509Pro) located in RD1; p.(Thr1721Met), p.(Ala2017Thr), and p.(Asp2178Tyr) outside known protein domains; and several variants (p.(Arg2512Gln), p.(Arg2523Trp), p.(Arg2536Trp), p.(Arg2536Pro), p.(Arg2579His), and p.(Arg2585Cys)) in RD2.
We first assessed the effect of missense variants on subcellular localization of ANKRD11. When transiently expressed as EGFP-fusion proteins in HEK239T/17 cells, wild-type ANKRD11 localized to the nucleus in a homogeneous speckle-like pattern, consistent with previous findings in non-neuronal cell lines
None of the tested missense variants affected nuclear localization of ANKRD11 (Figure 3A, Supplemental Figure 5), contrasting with the nucleolar mislocalization of mutant Ankrd11 reported in Yoda mice.
Figure 3ANKRD11 variants in RD2 result in reduced protein stability and impaired proteasome degradation or loss of CDKN1A/P21 transcriptional repression. A. Direct fluorescence imaging of cells expressing EGFP-tagged variants of the ANKRD11 protein using confocal microscopy. Wild type and all variants showed a speckle-like pattern in the nucleus. Nuclei are stained with Hoechst 33342 (blue). Protein domains in which variants are located are indicated. Results are representative of 3 independent experiments. Scale bar = 5 μm. B. Relative fluorescence intensity of EGFP-tagged ANKRD11 variants overexpressed in HEK293T/17 cells treated with translation inhibitor cycloheximide (CHX; 50 μg/mL) shown in upper panels and with proteasome inhibitor MG132 (5 μg/mL) in the lower panels. Equal volume of dimethyl sulfoxide was used as a vehicle control. Fluorescence intensity was measured for 24 hours with 3-hour intervals. Values are expressed relative to t = 0 hour and represent the mean ± SD of 3 independent experiments, each preformed in triplicate (∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001; 2-way analysis of variance and a post hoc Dunnett’s test). C-D. Results of luciferase assay with constructs containing WT and ANKRD11 variants and 2 firefly luciferase reporter constructs with a CDKN1A/P21 promoter. Values are expressed relative to the control condition that used a EGFP-C2 construct without ANKRD11 and represent the mean ± SD of 4 (C) or 5 (D) independent experiments, each performed in triplicate (∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001 vs WT; 1-way analysis of variance and a post hoc Dunnett’s test). RD, repressor domain; WT, wild type.
we hypothesized that missense variants might alter ANKRD11 stability, possibly via altered proteasome degradation. Moreover, 4 variants are located at putative destruction motifs (Supplemental Table 6, Supplemental Figure 3). To assess ANKRD11 protein stability, we treated HEK239T/17 cells expressing EGFP-tagged ANKRD11 with cycloheximide to inhibit translation and measured relative fluorescence intensity over 24 hours. We found that all variants in RD2 except p.(Arg2585Cys) showed reduced protein stability compared with wild type, whereas among variants outside the RD2, only p.(Ala2017Thr) was less stable (Figure 3B, Supplemental Figure 6 and 7). All other variants outside RD2 showed stability similar to wild type. We next examined the effect of missense variants on proteasome-mediated degradation after treating EGFP-ANKRD11 expressing cells with proteasome inhibitor MG132. Only 2 variants, both located in RD2, affected proteasome degradation, showing opposite directions of effect; whereas p.(Arg2523Trp) displayed increased proteasome degradation, p.(Arg2585Cys) showed decreased proteasome degradation (Figure 3B).
To study the effects of missense variation on transcriptional activity of ANKRD11, we performed luciferase reporter assays with the CDKN1A/P21 promoter, a known downstream target. Of all tested variants, only p.(Arg2585Cys) affected transcriptional activity, leading to a loss of transcriptional repression on CDKN1A/P21 (Figure 3C and D).
Taken together, our cell-based assays indicate that most missense variants yield a loss-of-function effect, either resulting from a reduced dosage of ANKRD11 due to decreased protein stability with or without increased proteasome degradation or through a loss of transrepressive activity. Three variants, p.(Leu509Pro), p.(Thr1721Met), and p.(Asp2178Tyr), did not result in aberrations in any of the tested protein functions and are therefore classified as of uncertain significance, although the individuals carrying p.(Thr1721Met) and p.(Asp2178Tyr) exhibit typical KBG features (individual 6 and 8, respectively). All evidence per observed missense variant including classification on the basis of American College of Medical Genetics and Genomics/Association for Molecular Pathology criteria are provided in Supplemental Table 5.
Discussion
Although KBG syndrome has been clinically recognized for almost 50 years
the role of rare missense variation in ANKRD11 remained unclear. We characterized clinical, molecular, and functional spectra of ANKRD11 missense variants by collecting information for 29 individuals and assessing effects of missense variation on ANKRD11 functions. We show that almost all individuals carrying rare heterozygous predicted damaging ANKRD11 missense variants fit well within the clinical spectrum described for KBG syndrome. Missense variants mainly affect the C-terminal RD2 with an overrepresentation of mutated arginine residues. Based on cellular assays, missense variants result in loss-of-function of ANKRD11, either by impaired protein stability or reduced transcriptional activity, consistent with ANKRD11 haploinsufficiency causing KBG syndrome through PTVs and microdeletions.
Most individuals presented with characteristics fitting the KBG-associated phenotypic spectrum and, from a clinical perspective, individuals with KBG syndrome caused by ANKRD11 missense variants or by PTVs or microdeletions are indistinguishable. However, unexpectedly, HPO-based clustering analysis showed a difference between the groups. Possibly, ascertainment bias influenced this analysis, because recognizing pathogenicity for missense variants is more challenging than that for PTVs. In addition, of the 7 individuals with missense variants who did not meet the KBG diagnostic criteria, 6 were correctly assigned to the missense cluster (Supplemental Table 3B), potentially (in part) driving the observed difference. Finally, the cohort of cases with PTVs was obtained from 1 expert health care center, whereas the missense cohort represents an international collaboration. Larger cohorts are needed to assess whether there are indeed phenotypic differences or whether these results can be explained by cohort effects.
The clinical variability of KBG syndrome is noteworthy, showing considerable phenotypic differences between affected individuals within the same family or between unrelated individuals with the same variant. This variability is best shown by comparing individuals 13 and 14, carrying de novo p.(Glu2522Lys). Although both presented with macrodontia and the characteristic facial appearance, individual 13 exhibited moderate ID, behavioral disturbances, hypotonia, a duplex kidney, strabismus, and normal growth, whereas individual 14 had normal intelligence, no neurobehavioral abnormalities, a submucous cleft palate, moderate hearing loss, mild growth hormone deficiency, and microcephaly. The family with 5 affected individuals also is a key example, in whom 2 presented with macrodontia (individual 21 and 24) and 3 exhibited the characteristic facial gestalt (individuals 20, 23, and 24). We therefore argue not to rule out pathogenicity for individual ANKRD11 missense variants on inheritance or clinical grounds only. Also for the 2 individuals (individual 3 and 29) not clearly exhibiting symptoms of KBG syndrome, the variants are classified as of uncertain significance and pathogenic when applying American College of Medical Genetics and Genomics/Association for Molecular Pathology criteria (Supplemental Table 5; p.(Leu509Pro) and p.(Leu2605Arg), respectively).
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
ANKRD11 shows significant regional differences in missense depletion in the general population, with 3 distinct regions of missense tolerance: p.1-p.415 with modest regional missense depletion (o/e = 0.51), p.416-p.2276 tolerating missense variation (o/e = 1.1), and p.2277-p.2664 showing high missense depletion (o/e = 0.11) (unpublished data, Samocha KE, Kosmicki JA, Karczewski KJ, et al. 2017. https://doi.org/10.1101/148353). Consistently, in our cohort, we observed that variants significantly clustered in the highly depleted C-terminal region, which was previously suggested to be implicated in the mechanism underlying KBG syndrome,
although we also observed missense variants in the tolerant middle and N-terminal depleted regions. The proportion of independently mutated arginine residues is remarkable (total cohort 12/25 [48%]; RD2, 12/17 [70.6]) and more pronounced than the overrepresentation of mutated arginine residues seen for pathogenic variants underlying genetic disorders in general (15%-20%
Therefore, we hypothesize that the molecular underpinnings of the observed overrepresentation of mutated arginine residues lies in the 3-dimensional structure of ANKRD11, which could not be taken into account, because the crystal structure of ANKRD11 is largely uncharacterized and ab initio models are unreliable, despite recent advances in the field.
However, on the basis of our in silico studies, we argue that if missense variants in ANKRD11 affect an arginine residue in the C-terminal RD2, it is suggestive for pathogenicity.
Regarding functional impact, most tested missense variants resulted in reduced protein stability, but only for p.(Arg2523Trp), it could be explained by increased proteasome degradation. We hypothesize that variants reducing protein stability without impairment of proteasome degradation affect other mechanisms implicated in protein homeostasis that could be activated by ubiquitination (eg, autophagy). Of note, p.(Arg2523Trp) is located at a putative D-box possibly affecting ANKRD11 ubiquitination and subsequent proteasome degradation. However, the other tested variant at a D-box, p.(Arg2512Gln), showed no impairment of proteasome degradation, which challenges the previous suggestion that disruption of the C-terminal D-boxes is the pathophysiological mechanism underlying KBG syndrome.
In contrast, the only other variant with altered proteasome degradation, p.(Arg2585Cys), showed reduced proteasome degradation and might slightly increase protein stability, although the latter was not statistically significant. The p.(Arg2585Cys) variant is also the only tested variant resulting in reduced transcriptional repression on CDKN1A/P21, consistent with previous observations for p.(Leu2143Val).
These findings suggest that p.(Arg2585Cys) results in loss-of-function, despite a potential accumulation of mutant ANKRD11 that contrasts with the dosage reduction seen for the other tested variants. This is further supported by phenotypes of the 2 individuals carrying p.(Arg2585Cys), which are characteristic for KBG syndrome, without apparent differences from phenotypes of individuals with missense variants impairing ANKRD11 stability. Finally, we did not observe changes in ANKRD11 subcellular localization for the assessed missense variants, contrary to what has been reported for Yoda mice with p.(Glu2502Lys).
The 3 variants not showing aberrations in any of our assays are all located outside RD2 and classified as variants of uncertain significance (Supplemental Table 5). Of the 4 tested variants located outside RD2, only p.(Ala2017Thr) affected the assessed protein functions, whereas all tested variants inside RD2 did affect protein function. It is therefore possible that variants outside RD2 exert effects on ANKRD11 functions not captured by our studies. Alternatively, they might alter premessenger RNA splicing (Supplemental Figure 8)
On the basis of the role of ANKRD11 in chromatin remodeling, evaluating transcriptomic and epigenetic profiles of individuals or cell-models could increase understanding of the effects of missense variants in the various domains.
In conclusion, our study showed that (de novo) pathogenic missense variants in ANKRD11 cause the clinically recognizable KBG syndrome with a similar phenotypic spectrum as previously observed for PTVs and microdeletions affecting ANKRD11. We showed that loss of transrepression capacity and reduced protein stability are independent molecular mechanisms by which missense variants cause a functional loss of ANKRD11. These findings add to the mechanistic complexity underlying ANKRD11 haploinsufficiency, already comprising deletion of the locus,
although effects of the latter on protein stability and function have not been elucidated. Because inheritance of pathogenic variants in ANKRD11 is regularly observed owing to the variability of the associated phenotype, missense variants pose diagnostic challenges, warranting stringent variant classification and careful phenotyping. However, because KBG syndrome is a relatively common cause of genetic NDDs, the involvement of ANKRD11 missense variants in cohorts of undiagnosed individuals with NDD should be considerable.
H.Z.E. and K.G.M. are employees of GeneDx, Inc. All other authors declare no conflicts of interest.
Acknowledgments
We are very grateful to all individuals and their families for their participation in this study. This work was financially supported by Aspasia grants of the Dutch Research Council (015.014.036 to T.K. and 015.014.066 to L.E.L.M.V.), Netherlands Organization for Health Research and Development (91718310 to T.K.), and the Max Planck Society (M.M.K.W., S.E.F.). Individual 4 was sequenced at the Scottish Genomes Partnership. The Scottish Genomes Partnership was funded by the Chief Scientist Office of the Scottish Government Health Directorates (SGP/1) and the Medical Research Council Whole Genome Sequencing for Health and Wealth Initiative (MC/PC/15080). The Deciphering Developmental Disorders study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003). This study makes use of Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (https://www.deciphergenomics.org/), which is funded by Wellcome. See Deciphering Developmental Disorders study8 or https://www.ddduk.org/access.html for full acknowledgment.
In addition, the collaborations in this study were facilitated by ERN ITHACA, one of the 24 European Reference Networks (ERNs) approved by the ERN Board of Member States, cofunded by European Commission. The aims of this study contribute to the Solve-RD project (E.d.B., A.S.D.P., L.F., C.G., T.K., A.V., L.E.L.M.V.), which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement number 779257.
We obtained informed consent to publish unidentifiable data for all individuals reported in this study. Specific consent was obtained for publication of clinical photographs. Consent procedures were in accordance with the Declaration of Helsinki and local ethical guidelines of the participating centers. The institutional review board 'Commissie Mensgebonden Onderzoek Regio Arnhem-Nijmegen' approved this study under number 2011/188 and 2022-13611. Number 2011/188 refers to performing diagnostic exome sequencing. Discovery of novel syndromes and description of clinical cohorts from this series can be taken as such. Number 2022-13611 refers to publishing Human Phenotype Ontology data for individuals in Biobank Genetics and Rare Diseases and Biobank Intellectual Disability of the Radboudumc. All the appropriate institutional forms have been archived locally.
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
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