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Missense variants in ANKRD11 cause KBG syndrome by impairment of stability or transcriptional activity of the encoded protein

Open AccessPublished:July 14, 2022DOI:https://doi.org/10.1016/j.gim.2022.06.007

      Abstract

      Purpose

      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.

      Keywords

      Introduction

      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.
      • Herrmann J.
      • Pallister P.D.
      • Tiddy W.
      • Opitz J.M.
      The KBG syndrome-a syndrome of short stature, characteristic facies, mental retardation, macrodontia and skeletal anomalies.
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      • Goldenberg A.
      • Riccardi F.
      • Tessier A.
      • et al.
      Clinical and molecular findings in 39 patients with KBG syndrome caused by deletion or mutation of ANKRD11.
      • Low K.
      • Ashraf T.
      • Canham N.
      • et al.
      Clinical and genetic aspects of KBG syndrome.
      • Gnazzo M.
      • Lepri F.R.
      • Dentici M.L.
      • et al.
      KBG syndrome: common and uncommon clinical features based on 31 new patients.
      • Skjei K.L.
      • Martin M.M.
      • Slavotinek A.M.
      KBG syndrome: report of twins, neurological characteristics, and delineation of diagnostic criteria.
      Although KBG syndrome is considered a clinically recognizable syndrome with macrodontia as its most defining trait,
      • Skjei K.L.
      • Martin M.M.
      • Slavotinek A.M.
      KBG syndrome: report of twins, neurological characteristics, and delineation of diagnostic criteria.
      there is considerable clinical variability and none of the KBG features is pathognomonic. Hence, despite being described as a clinical entity since 1975,
      • Herrmann J.
      • Pallister P.D.
      • Tiddy W.
      • Opitz J.M.
      The KBG syndrome-a syndrome of short stature, characteristic facies, mental retardation, macrodontia and skeletal anomalies.
      KBG syndrome was underdiagnosed before causative ANKRD11 variants were discovered.
      • Sirmaci A.
      • Spiliopoulos M.
      • Brancati F.
      • et al.
      Mutations in ANKRD11 cause KBG syndrome, characterized by intellectual disability, skeletal malformations, and macrodontia.
      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.
      Deciphering Developmental Disorders Study
      Large-scale discovery of novel genetic causes of developmental disorders.
      ,
      • Kaplanis J.
      • Samocha K.E.
      • Wiel L.
      • et al.
      Evidence for 28 genetic disorders discovered by combining healthcare and research data.
      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,
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      • Goldenberg A.
      • Riccardi F.
      • Tessier A.
      • et al.
      Clinical and molecular findings in 39 patients with KBG syndrome caused by deletion or mutation of ANKRD11.
      • Low K.
      • Ashraf T.
      • Canham N.
      • et al.
      Clinical and genetic aspects of KBG syndrome.
      • Gnazzo M.
      • Lepri F.R.
      • Dentici M.L.
      • et al.
      KBG syndrome: common and uncommon clinical features based on 31 new patients.
      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).
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans.
      Therefore, haploinsufficiency of ANKRD11 is commonly accepted as mechanism of pathogenicity for KBG syndrome.
      • Morel Swols D.
      • Foster 2nd, J.
      • Tekin M.
      KBG syndrome.
      This is supported by observations of reduced amounts of ANKRD11 messenger RNA and protein when the gene contains a PTV,
      • Cucco F.
      • Sarogni P.
      • Rossato S.
      • et al.
      Pathogenic variants in EP300 and ANKRD11 in patients with phenotypes overlapping Cornelia de Lange syndrome.
      suggesting that variants trigger the nonsense-mediated decay (NMD) pathway,
      • Cucco F.
      • Sarogni P.
      • Rossato S.
      • et al.
      Pathogenic variants in EP300 and ANKRD11 in patients with phenotypes overlapping Cornelia de Lange syndrome.
      although PTVs leading to (partial) escape from NMD have also been described.
      • Sirmaci A.
      • Spiliopoulos M.
      • Brancati F.
      • et al.
      Mutations in ANKRD11 cause KBG syndrome, characterized by intellectual disability, skeletal malformations, and macrodontia.
      ,
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.
      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.
      • Zhang T.
      • Yang Y.
      • Yin X.
      • et al.
      Two loss-of-function ANKRD11 variants in Chinese patients with short stature and a possible molecular pathway.
      ANKRD11 is ubiquitously expressed and localizes mainly to the nucleus in a homogenous pattern. ANKRD11 is a crucial regulator of neuronal development
      • Sirmaci A.
      • Spiliopoulos M.
      • Brancati F.
      • et al.
      Mutations in ANKRD11 cause KBG syndrome, characterized by intellectual disability, skeletal malformations, and macrodontia.
      ,
      • Gallagher D.
      • Voronova A.
      • Zander M.A.
      • et al.
      Ankrd11 is a chromatin regulator involved in autism that is essential for neural development.
      that interacts with coactivators and corepressors of transcription,
      • Zhang A.
      • Yeung P.L.
      • Li C.W.
      • et al.
      Identification of a novel family of ankyrin repeats containing cofactors for p160 nuclear receptor coactivators.
      showing (co)regulatory effects on various sets of genes. These include genes encoding signaling molecules, chromatin remodelers, and transcriptional regulators,
      • Gallagher D.
      • Voronova A.
      • Zander M.A.
      • et al.
      Ankrd11 is a chromatin regulator involved in autism that is essential for neural development.
      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.
      • Zhang A.
      • Li C.W.
      • Chen J.D.
      Characterization of transcriptional regulatory domains of ankyrin repeat cofactor-1.
      Interaction of ANKRD11 with other proteins and homodimerization are mediated through ankyrin repeats, located at the N-terminus.
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.
      The C-terminal part of ANKRD11, containing (predicted) destruction box motifs (D-boxes), was suggested to be critical for its degradation.
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.
      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]).
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans.
      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.
      • Landrum M.J.
      • Lee J.M.
      • Benson M.
      • et al.
      ClinVar: improving access to variant interpretations and supporting evidence.
      In the literature, approximately 2.6% of (de novo) variants in ANKRD11 are missense variants,
      • Hanly C.
      • Shah H.
      • Au P.Y.B.
      • Murias K.
      Description of neurodevelopmental phenotypes associated with 10 genetic neurodevelopmental disorders: A scoping review.
      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.
      • Zhang T.
      • Yang Y.
      • Yin X.
      • et al.
      Two loss-of-function ANKRD11 variants in Chinese patients with short stature and a possible molecular pathway.
      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.
      • Barbaric I.
      • Perry M.J.
      • Dear T.N.
      • et al.
      An ENU-induced mutation in the Ankrd11 gene results in an osteopenia-like phenotype in the mouse mutant Yoda.
      In addition, Yoda mice show behavioral abnormalities reflective of cognitive dysfunction.
      • Gallagher D.
      • Voronova A.
      • Zander M.A.
      • et al.
      Ankrd11 is a chromatin regulator involved in autism that is essential for neural development.
      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,
      • Gallagher D.
      • Voronova A.
      • Zander M.A.
      • et al.
      Ankrd11 is a chromatin regulator involved in autism that is essential for neural development.
      suggesting a loss-of-function mechanism. However, a dominant-negative mechanism has also been hypothesized to contribute to the Yoda mouse phenotype.
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.
      Ankrd11 was shown to mislocalize to the nucleolus, possibly resulting from diminished degradation.
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.
      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.
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.
      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 In Silico 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
      • Köhler S.
      • Carmody L.
      • Vasilevsky N.
      • et al.
      Expansion of the Human Phenotype Ontology (HPO) knowledge base and resources.
      clustering was performed as previously described
      • den Hoed J.
      • de Boer E.
      • Voisin N.
      • et al.
      Mutation-specific pathophysiological mechanisms define different neurodevelopmental disorders associated with SATB1 dysfunction.
      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).
      • Deng Y.
      • Gao L.
      • Wang B.
      • Guo X.
      HPOSim: an R package for phenotypic similarity measure and enrichment analysis based on the human phenotype ontology.
      ,
      • Wang J.Z.
      • Du Z.
      • Payattakool R.
      • Yu P.S.
      • Chen C.F.
      A new method to measure the semantic similarity of GO terms.
      Spatial clustering of independently observed missense variants (25/29) was performed as previously described,
      • Lelieveld S.H.
      • Wiel L.
      • Venselaar H.
      • et al.
      Spatial clustering of de novo missense mutations identifies candidate neurodevelopmental disorder-associated genes.
      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.
      • McLaren W.
      • Gil L.
      • Hunt S.E.
      • et al.
      The Ensembl Variant Effect Predictor.
      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.
      • Nachman M.W.
      • Crowell S.L.
      Estimate of the mutation rate per nucleotide in humans.
      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)
      • Nishio H.
      • Walsh M.J.
      CCAAT displacement protein/cut homolog recruits G9a histone lysine methyltransferase to repress transcription.
      and Bert Vogelstein (Addgene plasmid 16451; http://n2t.net/addgene:16451; RRID:Addgene_16451),
      • el-Deiry W.S.
      • Tokino T.
      • Velculescu V.E.
      • et al.
      WAF1, a potential mediator of p53 tumor suppression.
      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

      Through international collaborations
      • Sobreira N.
      • Schiettecatte F.
      • Valle D.
      • Hamosh A.
      GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.
      ,
      • Firth H.V.
      • Richards S.M.
      • Bevan A.P.
      • et al.
      DECIPHER: database of chromosomal imbalance and phenotype in humans using Ensembl resources.
      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 thumbnail gr1
      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 , with details on variant interpretation in . 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.
      Table 1Summary of observed clinical features
      Features Associated With KBG Syndrome%Present/Total Assessed
      Macrodontia upper central incisors
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      54.213/24
      Additional dental abnormalities
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      66.714/21
      Fitting the KBG characteristic facial appearance
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      79.323/29
      Hand abnormalities
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      8020/25
      Postnatal short stature
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      (<–2 SD)
      53.615/28
      Delayed bone age
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      57.18/14
      Costovertebral anomalies
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      7.11/14
      ID
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      81.522/27
       Normal IQ11.13/27
       ID of unknown severity11.13/27
       Borderline7.42/27
       Mild55.615/27
       Moderate14.84/27
      Developmental delay
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      92.926/28
      Seizures
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      22.26/27
      Behavioral abnormalities
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      89.325/28
       ADHD/hyperactive behavior69.218/26
       Autism spectrum disorder369/25
       Anxiety37.59/24
      Cryptorchidism
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      203/15
      Congenital heart defect
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      328/25
      Palate defect
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      7.12/28
      Hearing loss
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      39.311/28
      First degree relative with KBG syndrome
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      19.25/26
      Large fontanelle at birth
      • Low K.
      • Ashraf T.
      • Canham N.
      • et al.
      Clinical and genetic aspects of KBG syndrome.
      18.22/11
      Feeding difficulties
      • Low K.
      • Ashraf T.
      • Canham N.
      • et al.
      Clinical and genetic aspects of KBG syndrome.
      33.39/27
      Precocious puberty
      • Goldenberg A.
      • Riccardi F.
      • Tessier A.
      • et al.
      Clinical and molecular findings in 39 patients with KBG syndrome caused by deletion or mutation of ANKRD11.
      5.91/17
      Meeting diagnostic criteria of KBG syndrome
      • Low K.
      • Ashraf T.
      • Canham N.
      • et al.
      Clinical and genetic aspects of KBG syndrome.
      75.922/29
      Other features
       Motor delay74.120/27
       Speech delay88.523/26
       Hypotonia41.710/24
       Hypertonia/spasticity123/25
       Sleep disturbances30.88/26
       Abnormal brain MRI203/15
       Abnormalities during pregnancy45.811/24
       Abnormalities during delivery57.715/26
       Macrocephaly (>2 SD)3.71/27
       Microcephaly (<–2 SD)22.26/27
       Vision abnormalities5013/26
       Gastrointestinal abnormalities328/25
       Endocrine/metabolic abnormalities25.97/27
       Immunologic abnormalities18.55/27
       Skin/hair/nail abnormalities57.715/26
      ADHD, attention deficit hyperactivity disorder; ID, intellectual disability; IQ, intelligence quoteint; MRI, magnetic resonance imaging.
      Figure thumbnail gr2
      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
      • Jehl P.
      • Manguy J.
      • Shields D.C.
      • Higgins D.G.
      • Davey N.E.
      ProViz-a web-based visualization tool to investigate the functional and evolutionary features of protein sequences.
      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.
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.
      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,
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      • Goldenberg A.
      • Riccardi F.
      • Tessier A.
      • et al.
      Clinical and molecular findings in 39 patients with KBG syndrome caused by deletion or mutation of ANKRD11.
      • Low K.
      • Ashraf T.
      • Canham N.
      • et al.
      Clinical and genetic aspects of KBG syndrome.
      • Gnazzo M.
      • Lepri F.R.
      • Dentici M.L.
      • et al.
      KBG syndrome: common and uncommon clinical features based on 31 new patients.
      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
      • Low K.
      • Ashraf T.
      • Canham N.
      • et al.
      Clinical and genetic aspects of 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
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      • Goldenberg A.
      • Riccardi F.
      • Tessier A.
      • et al.
      Clinical and molecular findings in 39 patients with KBG syndrome caused by deletion or mutation of ANKRD11.
      • Low K.
      • Ashraf T.
      • Canham N.
      • et al.
      Clinical and genetic aspects of KBG syndrome.
      • Gnazzo M.
      • Lepri F.R.
      • Dentici M.L.
      • et al.
      KBG syndrome: common and uncommon clinical features based on 31 new patients.
      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
      PublicationOckeloen et al
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      Low et al
      • Low K.
      • Ashraf T.
      • Canham N.
      • et al.
      Clinical and genetic aspects of KBG syndrome.
      Goldenberg et al
      • Goldenberg A.
      • Riccardi F.
      • Tessier A.
      • et al.
      Clinical and molecular findings in 39 patients with KBG syndrome caused by deletion or mutation of ANKRD11.
      Gnazzo et al
      • Gnazzo M.
      • Lepri F.R.
      • Dentici M.L.
      • et al.
      KBG syndrome: common and uncommon clinical features based on 31 new patients.
      Cumulative Frequencies in Published Cohorts
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      • Goldenberg A.
      • Riccardi F.
      • Tessier A.
      • et al.
      Clinical and molecular findings in 39 patients with KBG syndrome caused by deletion or mutation of ANKRD11.
      • Low K.
      • Ashraf T.
      • Canham N.
      • et al.
      Clinical and genetic aspects of KBG syndrome.
      • Gnazzo M.
      • Lepri F.R.
      • Dentici M.L.
      • et al.
      KBG syndrome: common and uncommon clinical features based on 31 new patients.
      This Publication
      Variant TypePTVs + MicrodeletionsPTVsPTVs + MicrodeletionsPTVs + MicrodeletionsPTVs + MicrodeletionsMissense Variants
      Features Associated With KBG Syndrome%Present/Total Assessed%Present/Total Assessed%Present/Total Assessed%Present/Total Assessed%Present/Total AssessedFrequency Range (%)%Present/Total Assessed
      Macrodontia upper central incisors
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      75.825/3385.223/2769.218/2676.723/3076.789/11669.2-85.254.213/24
      Additional dental abnormalities
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      48.516/33NANANANANANA48.516/3348.566.714/21
      Fitting characteristic facial appearance of KBG
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      93.931/3340.613/3210039/3910031/3184.4114/13540.6-10079.323/29
      Hand abnormalities
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      81.827/3346.915/3269.723/3367.721/3166.786/12946.9-81.18020/25
      Postnatal short stature
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      54.518/3328.19/3240.515/3758.118/3145.160/13328.1-58.153.615/28
      Delayed bone age
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      15.25/33603/566.78/12NANA3216/5015.2-66.757.18/14
      Costovertebral anomalies
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      30.310/33NANANANANANA30.310/3330.37.11/14
      ID
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      60.620/33NANA609/1559.113/226042/7059.1-60.681.522/27
      Learning disability or developmental delay
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      93.931/3310032/3294.333/3596.830/3196.2126/13193.9-10092.926/28
      Seizures
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      27.39/3343.814/3231.612/3816.15/3129.940/13416.1-43.822.26/27
      Behavioral abnormalities
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      78.826/3310030/3051.419/37NANA7575/10051.4-10089.325/28
      Cryptorchidism
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      408/2031.35/1618.83/1613.32/1526.918/6713.3-40203/15
      Congenital heart defect
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      18.26/3312.54/3225.610/3935.511/312331/13512.5-35.5328/25
      Palate defect
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      21.27/3312.54/325.12/3900/319.613/1350-21.27.12/28
      Hearing loss
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      24.28/33258/3230.611/3619.46/312533/13219.4-30.639.311/28
      First degree relative with KBG syndrome
      • Ockeloen C.W.
      • Willemsen M.H.
      • de Munnik S.
      • et al.
      Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
      52.610/1921.97/3220.58/396.52/3122.327/1216.5-52.619.25/26
      Large fontanelle at birth
      • Low K.
      • Ashraf T.
      • Canham N.
      • et al.
      Clinical and genetic aspects of KBG syndrome.
      NANA21.97/32NANANANA21.97/3221.918.22/11
      Feeding difficulties
      • Low K.
      • Ashraf T.
      • Canham N.
      • et al.
      Clinical and genetic aspects of KBG syndrome.
      NANA31.310/32NANA35.511/3133.321/6331.3-35.333.39/27
      Precocious puberty
      • Goldenberg A.
      • Riccardi F.
      • Tessier A.
      • et al.
      Clinical and molecular findings in 39 patients with KBG syndrome caused by deletion or mutation of ANKRD11.
      NANANANA15.65/325.61/18126/505.6-15.65.91/17
      ID, intellectual disability; NA, not available; PTV, protein truncating variant.
      To quantitatively investigate this hypothesis, we compared standardized clinical data (Supplemental JSON
      • Köhler S.
      • Carmody L.
      • Vasilevsky N.
      • et al.
      Expansion of the Human Phenotype Ontology (HPO) knowledge base and resources.
      ) 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

      Kaufman L, Rousseeuw PJ. Clustering by means of medoids. In: Proceedings of the Statistical Data Analysis Based on the L1 Norm Conference August 31, 1987 (Vol. 31). https://wis.kuleuven.be/stat/robust/papers/publications-1987/kaufmanrousseeuw-clusteringbymedoids-l1norm-1987.pdf

      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
      • Zhang A.
      • Yeung P.L.
      • Li C.W.
      • et al.
      Identification of a novel family of ankyrin repeats containing cofactors for p160 nuclear receptor coactivators.
      and mouse neocortical neurons overexpressing wild-type Ankrd11.
      • Sirmaci A.
      • Spiliopoulos M.
      • Brancati F.
      • et al.
      Mutations in ANKRD11 cause KBG syndrome, characterized by intellectual disability, skeletal malformations, and macrodontia.
      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.
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.
      Figure thumbnail gr3
      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.
      On the basis of spatial clustering at the C-terminus, which is critical for ANKRD11 degradation,
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.
      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
      • Herrmann J.
      • Pallister P.D.
      • Tiddy W.
      • Opitz J.M.
      The KBG syndrome-a syndrome of short stature, characteristic facies, mental retardation, macrodontia and skeletal anomalies.
      and PTVs and microdeletions affecting ANKRD11 have been robustly implicated in its etiology since 2011,
      • Sirmaci A.
      • Spiliopoulos M.
      • Brancati F.
      • et al.
      Mutations in ANKRD11 cause KBG syndrome, characterized by intellectual disability, skeletal malformations, and macrodontia.
      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).
      • Richards S.
      • Aziz N.
      • Bale S.
      • et al.
      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,
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to 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%
      • Schulze K.V.
      • Hanchard N.A.
      • Wangler M.F.
      Biases in arginine codon usage correlate with genetic disease risk.
      ,
      • Khan S.
      • Vihinen M.
      Spectrum of disease-causing mutations in protein secondary structures.
      ). Arginine is also the most frequently mutated residue in all secondary structures when considering pathogenic variants.
      • Khan S.
      • Vihinen M.
      Spectrum of disease-causing mutations in protein secondary structures.
      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.
      • Jumper J.
      • Evans R.
      • Pritzel A.
      • et al.
      Highly accurate protein structure prediction with AlphaFold.
      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.
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to 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).
      • Zhang T.
      • Yang Y.
      • Yin X.
      • et al.
      Two loss-of-function ANKRD11 variants in Chinese patients with short stature and a possible molecular pathway.
      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).
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.
      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)
      • Fadaie Z.
      • Khan M.
      • Del Pozo-Valero M.
      • et al.
      Identification of splice defects due to noncanonical splice site or deep-intronic variants in ABCA4.
      despite low SpliceAI scores.
      • Jaganathan K.
      • Kyriazopoulou Panagiotopoulou S.
      • McRae J.F.
      • et al.
      Predicting splicing from primary sequence with deep learning.
      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,
      • Goldenberg A.
      • Riccardi F.
      • Tessier A.
      • et al.
      Clinical and molecular findings in 39 patients with KBG syndrome caused by deletion or mutation of ANKRD11.
      putative null alleles,
      • Cucco F.
      • Sarogni P.
      • Rossato S.
      • et al.
      Pathogenic variants in EP300 and ANKRD11 in patients with phenotypes overlapping Cornelia de Lange syndrome.
      and PTVs escaping the NMD pathway,
      • Sirmaci A.
      • Spiliopoulos M.
      • Brancati F.
      • et al.
      Mutations in ANKRD11 cause KBG syndrome, characterized by intellectual disability, skeletal malformations, and macrodontia.
      ,
      • Walz K.
      • Cohen D.
      • Neilsen P.M.
      • et al.
      Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.
      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.

      Data Availability

      Code used for spatial clustering is shared at https://github.com/laurensvdwiel/SpatialClustering. Code used for permutation testing is available at https://github.com/jhampstead/ANKRD11-simulations. All available phenotypic information in Human Phenotype Ontology terminology is shared as a supplement (Supplemental JSON). Model and code for Human Phenotype Ontology-based clustering analysis are available at https://github.com/ldingemans/HPO_clustering_Wang.

      Conflict of Interest

      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.

      Author Information

      Conceptualization: C.W.O., M.M.K.W., T.K.; Data Curation: E.d.B., C.W.O., D.R., T.A., R.B., M.B.-H., B.A., N.C., A.-S.D.-P., O.D., C.D., F.E., H.Z.E., L.F., S.F.B., D.G., J.A.C.G., B.M.H., U.K., A.L.-A., G.L., S.A.L., I.M.J.M., R.M.G., K.G.M., S.O., R.P., A.P., J.v.R., G.W.E.S., E.S., A.S., P.J.v.d.S., A.P.A.S., S.M.A.S., I.V., E.V.-D., A.V., S.M.W., M.W., K.J.L., T.K.; Formal Analysis: E.d.B., M.M.K.W., C.W.O., L.E.L.M.V., S.E.F., T.K.; Investigation: R.A.K., E.d.B, M.M.K.W., C.W.O., T.K.; Software: L.L., A.J.M.D., J.E.H., C.G.; Writing-original draft: E.d.B., M.M.K.W., C.W.O., T.K.; Supervision: C.W.O., M.M.K.W., T.K.; All authors contributed to the final version of the manuscript.

      Ethics Declaration

      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.

      Additional Information

      References

        • Herrmann J.
        • Pallister P.D.
        • Tiddy W.
        • Opitz J.M.
        The KBG syndrome-a syndrome of short stature, characteristic facies, mental retardation, macrodontia and skeletal anomalies.
        Birth Defects Orig Artic Ser. 1975; 11: 7-18
        • Ockeloen C.W.
        • Willemsen M.H.
        • de Munnik S.
        • et al.
        Further delineation of the KBG syndrome caused by ANKRD11 aberrations.
        Eur J Hum Genet. 2015; 23 (Published correction appears in Eur J Hum Genet. 2015;23(9):1270.): 1176-1185
        • Goldenberg A.
        • Riccardi F.
        • Tessier A.
        • et al.
        Clinical and molecular findings in 39 patients with KBG syndrome caused by deletion or mutation of ANKRD11.
        Am J Med Genet A. 2016; 170: 2847-2859https://doi.org/10.1002/ajmg.a.37878
        • Low K.
        • Ashraf T.
        • Canham N.
        • et al.
        Clinical and genetic aspects of KBG syndrome.
        Am J Med Genet A. 2016; 170: 2835-2846https://doi.org/10.1002/ajmg.a.37842
        • Gnazzo M.
        • Lepri F.R.
        • Dentici M.L.
        • et al.
        KBG syndrome: common and uncommon clinical features based on 31 new patients.
        Am J Med Genet A. 2020; 182: 1073-1083https://doi.org/10.1002/ajmg.a.61524
        • Skjei K.L.
        • Martin M.M.
        • Slavotinek A.M.
        KBG syndrome: report of twins, neurological characteristics, and delineation of diagnostic criteria.
        Am J Med Genet A. 2007; 143A: 292-300https://doi.org/10.1002/ajmg.a.31597
        • Sirmaci A.
        • Spiliopoulos M.
        • Brancati F.
        • et al.
        Mutations in ANKRD11 cause KBG syndrome, characterized by intellectual disability, skeletal malformations, and macrodontia.
        Am J Hum Genet. 2011; 89: 289-294https://doi.org/10.1016/j.ajhg.2011.06.007
        • Deciphering Developmental Disorders Study
        Large-scale discovery of novel genetic causes of developmental disorders.
        Nature. 2015; 519: 223-228https://doi.org/10.1038/nature14135
        • Kaplanis J.
        • Samocha K.E.
        • Wiel L.
        • et al.
        Evidence for 28 genetic disorders discovered by combining healthcare and research data.
        Nature. 2020; 586: 757-762https://doi.org/10.1038/s41586-020-2832-5
        • Karczewski K.J.
        • Francioli L.C.
        • Tiao G.
        • et al.
        The mutational constraint spectrum quantified from variation in 141,456 humans.
        Nature. 2020; 581 (Published correction appears in Nature. 2021;590(7846):E53. Published correction appears in Nature. 2021;597(7874):E3-E4.): 434-443
        • Morel Swols D.
        • Foster 2nd, J.
        • Tekin M.
        KBG syndrome.
        Orphanet J Rare Dis. 2017; 12: 183https://doi.org/10.1186/s13023-017-0736-8
        • Cucco F.
        • Sarogni P.
        • Rossato S.
        • et al.
        Pathogenic variants in EP300 and ANKRD11 in patients with phenotypes overlapping Cornelia de Lange syndrome.
        Am J Med Genet A. 2020; 182: 1690-1696https://doi.org/10.1002/ajmg.a.61611
        • Walz K.
        • Cohen D.
        • Neilsen P.M.
        • et al.
        Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.
        Hum Genet. 2015; 134: 181-190https://doi.org/10.1007/s00439-014-1509-2
        • Zhang T.
        • Yang Y.
        • Yin X.
        • et al.
        Two loss-of-function ANKRD11 variants in Chinese patients with short stature and a possible molecular pathway.
        Am J Med Genet A. 2021; 185: 710-718https://doi.org/10.1002/ajmg.a.62024
        • Gallagher D.
        • Voronova A.
        • Zander M.A.
        • et al.
        Ankrd11 is a chromatin regulator involved in autism that is essential for neural development.
        Dev Cell. 2015; 32: 31-42https://doi.org/10.1016/j.devcel.2014.11.031
        • Zhang A.
        • Yeung P.L.
        • Li C.W.
        • et al.
        Identification of a novel family of ankyrin repeats containing cofactors for p160 nuclear receptor coactivators.
        J Biol Chem. 2004; 279: 33799-33805https://doi.org/10.1074/jbc.M403997200
        • Zhang A.
        • Li C.W.
        • Chen J.D.
        Characterization of transcriptional regulatory domains of ankyrin repeat cofactor-1.
        Biochem Biophys Res Commun. 2007; 358: 1034-1040https://doi.org/10.1016/j.bbrc.2007.05.017
        • Landrum M.J.
        • Lee J.M.
        • Benson M.
        • et al.
        ClinVar: improving access to variant interpretations and supporting evidence.
        Nucleic Acids Res. 2018; 46: D1062-D1067https://doi.org/10.1093/nar/gkx1153
        • Hanly C.
        • Shah H.
        • Au P.Y.B.
        • Murias K.
        Description of neurodevelopmental phenotypes associated with 10 genetic neurodevelopmental disorders: A scoping review.
        Clin Genet. 2021; 99: 335-346https://doi.org/10.1111/cge.13882
        • Barbaric I.
        • Perry M.J.
        • Dear T.N.
        • et al.
        An ENU-induced mutation in the Ankrd11 gene results in an osteopenia-like phenotype in the mouse mutant Yoda.
        Physiol Genomics. 2008; 32: 311-321https://doi.org/10.1152/physiolgenomics.00116.2007
        • Köhler S.
        • Carmody L.
        • Vasilevsky N.
        • et al.
        Expansion of the Human Phenotype Ontology (HPO) knowledge base and resources.
        Nucleic Acids Res. 2019; 47: D1018-D1027https://doi.org/10.1093/nar/gky1105
        • den Hoed J.
        • de Boer E.
        • Voisin N.
        • et al.
        Mutation-specific pathophysiological mechanisms define different neurodevelopmental disorders associated with SATB1 dysfunction.
        Am J Hum Genet. 2021; 108: 346-356https://doi.org/10.1016/j.ajhg.2021.01.007
        • Deng Y.
        • Gao L.
        • Wang B.
        • Guo X.
        HPOSim: an R package for phenotypic similarity measure and enrichment analysis based on the human phenotype ontology.
        PLoS One. 2015; 10e0115692https://doi.org/10.1371/journal.pone.0115692
        • Wang J.Z.
        • Du Z.
        • Payattakool R.
        • Yu P.S.
        • Chen C.F.
        A new method to measure the semantic similarity of GO terms.
        Bioinformatics. 2007; 23: 1274-1281https://doi.org/10.1093/bioinformatics/btm087
        • Lelieveld S.H.
        • Wiel L.
        • Venselaar H.
        • et al.
        Spatial clustering of de novo missense mutations identifies candidate neurodevelopmental disorder-associated genes.
        Am J Hum Genet. 2017; 101: 478-484https://doi.org/10.1016/j.ajhg.2017.08.004
        • McLaren W.
        • Gil L.
        • Hunt S.E.
        • et al.
        The Ensembl Variant Effect Predictor.
        Genome Biol. 2016; 17: 122https://doi.org/10.1186/s13059-016-0974-4
        • Nachman M.W.
        • Crowell S.L.
        Estimate of the mutation rate per nucleotide in humans.
        Genetics. 2000; 156: 297-304https://doi.org/10.1093/genetics/156.1.297
        • Nishio H.
        • Walsh M.J.
        CCAAT displacement protein/cut homolog recruits G9a histone lysine methyltransferase to repress transcription.
        Proc Natl Acad Sci U S A. 2004; 101: 11257-11262https://doi.org/10.1073/pnas.0401343101
        • el-Deiry W.S.
        • Tokino T.
        • Velculescu V.E.
        • et al.
        WAF1, a potential mediator of p53 tumor suppression.
        Cell. 1993; 75: 817-825https://doi.org/10.1016/0092-8674(93)90500-p
        • Sobreira N.
        • Schiettecatte F.
        • Valle D.
        • Hamosh A.
        GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.
        Hum Mutat. 2015; 36: 928-930https://doi.org/10.1002/humu.22844
        • Firth H.V.
        • Richards S.M.
        • Bevan A.P.
        • et al.
        DECIPHER: database of chromosomal imbalance and phenotype in humans using Ensembl resources.
        Am J Hum Genet. 2009; 84: 524-533https://doi.org/10.1016/j.ajhg.2009.03.010
        • Jehl P.
        • Manguy J.
        • Shields D.C.
        • Higgins D.G.
        • Davey N.E.
        ProViz-a web-based visualization tool to investigate the functional and evolutionary features of protein sequences.
        Nucleic Acids Res. 2016; 44: W11-W15https://doi.org/10.1093/nar/gkw265
      1. Kaufman L, Rousseeuw PJ. Clustering by means of medoids. In: Proceedings of the Statistical Data Analysis Based on the L1 Norm Conference August 31, 1987 (Vol. 31). https://wis.kuleuven.be/stat/robust/papers/publications-1987/kaufmanrousseeuw-clusteringbymedoids-l1norm-1987.pdf

        • Richards S.
        • Aziz N.
        • Bale S.
        • et al.
        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.
        Genet Med. 2015; 17: 405-424https://doi.org/10.1038/gim.2015.30
        • Schulze K.V.
        • Hanchard N.A.
        • Wangler M.F.
        Biases in arginine codon usage correlate with genetic disease risk.
        Genet Med. 2020; 22: 1407-1412https://doi.org/10.1038/s41436-020-0813-6
        • Khan S.
        • Vihinen M.
        Spectrum of disease-causing mutations in protein secondary structures.
        BMC Struct Biol. 2007; 7: 56https://doi.org/10.1186/1472-6807-7-56
        • Jumper J.
        • Evans R.
        • Pritzel A.
        • et al.
        Highly accurate protein structure prediction with AlphaFold.
        Nature. 2021; 596: 583-589https://doi.org/10.1038/s41586-021-03819-2
        • Fadaie Z.
        • Khan M.
        • Del Pozo-Valero M.
        • et al.
        Identification of splice defects due to noncanonical splice site or deep-intronic variants in ABCA4.
        Hum Mutat. 2019; 40: 2365-2376https://doi.org/10.1002/humu.23890
        • Jaganathan K.
        • Kyriazopoulou Panagiotopoulou S.
        • McRae J.F.
        • et al.
        Predicting splicing from primary sequence with deep learning.
        Cell. 2019; 176: 535-548.e24https://doi.org/10.1016/j.cell.2018.12.015