De Novo Variants in the F-Box Protein FBXO11 in 20 Individuals with a Variable Neurodevelopmental Disorder
2018; Elsevier BV; Volume: 103; Issue: 2 Linguagem: Inglês
10.1016/j.ajhg.2018.07.003
ISSN1537-6605
AutoresAnne Gregor, Lynette G. Sadleir, Reza Asadollahi, Silvia Azzarello‐Burri, Agatino Battaglia, Lilian Bomme Ousager, Paranchai Boonsawat, Ange‐Line Bruel, Rebecca Buchert, Eduardo Calpena, Benjamin Cogné, Bruno Dallapiccola, Felix Distelmaier, Frances Elmslie, Laurence Faivre, Tobias B. Haack, Victoria Harrison, Alex Henderson, David Hunt, Bertrand Isidor, Pascal Joset, Satoko Kumada, Augusta M.A. Lachmeijer, Melissa Lees, Sally Ann Lynch, Francisco Martı́nez, Naomichi Matsumoto, Carey McDougall, Heather C Mefford, Noriko Miyake, Candace T. Myers, Sébastien Moutton, Addie Nesbitt, Antonio Novelli, Carmen Orellana, Anita Rauch, Mónica Rosello, Ken Saida, Avni Santani, Ajoy Sarkar, Ingrid E. Scheffer, Marwan Shinawi, Katharina Steindl, Joseph D. Symonds, Elaine H. Zackai, André Reis, Heinrich Sticht, Christiane Zweier,
Tópico(s)Ubiquitin and proteasome pathways
ResumoNext-generation sequencing combined with international data sharing has enormously facilitated identification of new disease-associated genes and mutations. This is particularly true for genetically extremely heterogeneous entities such as neurodevelopmental disorders (NDDs). Through exome sequencing and world-wide collaborations, we identified and assembled 20 individuals with de novo variants in FBXO11. They present with mild to severe developmental delay associated with a range of features including short (4/20) or tall (2/20) stature, obesity (5/20), microcephaly (4/19) or macrocephaly (2/19), behavioral problems (17/20), seizures (5/20), cleft lip or palate or bifid uvula (3/20), and minor skeletal anomalies. FBXO11 encodes a member of the F-Box protein family, constituting a subunit of an E3-ubiquitin ligase complex. This complex is involved in ubiquitination and proteasomal degradation and thus in controlling critical biological processes by regulating protein turnover. The identified de novo aberrations comprise two large deletions, ten likely gene disrupting variants, and eight missense variants distributed throughout FBXO11. Structural modeling for missense variants located in the CASH or the Zinc-finger UBR domains suggests destabilization of the protein. This, in combination with the observed spectrum and localization of identified variants and the lack of apparent genotype-phenotype correlations, is compatible with loss of function or haploinsufficiency as an underlying mechanism. We implicate de novo missense and likely gene disrupting variants in FBXO11 in a neurodevelopmental disorder with variable intellectual disability and various other features. Next-generation sequencing combined with international data sharing has enormously facilitated identification of new disease-associated genes and mutations. This is particularly true for genetically extremely heterogeneous entities such as neurodevelopmental disorders (NDDs). Through exome sequencing and world-wide collaborations, we identified and assembled 20 individuals with de novo variants in FBXO11. They present with mild to severe developmental delay associated with a range of features including short (4/20) or tall (2/20) stature, obesity (5/20), microcephaly (4/19) or macrocephaly (2/19), behavioral problems (17/20), seizures (5/20), cleft lip or palate or bifid uvula (3/20), and minor skeletal anomalies. FBXO11 encodes a member of the F-Box protein family, constituting a subunit of an E3-ubiquitin ligase complex. This complex is involved in ubiquitination and proteasomal degradation and thus in controlling critical biological processes by regulating protein turnover. The identified de novo aberrations comprise two large deletions, ten likely gene disrupting variants, and eight missense variants distributed throughout FBXO11. Structural modeling for missense variants located in the CASH or the Zinc-finger UBR domains suggests destabilization of the protein. This, in combination with the observed spectrum and localization of identified variants and the lack of apparent genotype-phenotype correlations, is compatible with loss of function or haploinsufficiency as an underlying mechanism. We implicate de novo missense and likely gene disrupting variants in FBXO11 in a neurodevelopmental disorder with variable intellectual disability and various other features. Neurodevelopmental disorders (NDDs), including intellectual disability (ID), autism spectrum disorders, and developmental and epileptic encephalopathies, are clinically and genetically extremely heterogeneous. Currently, pathogenic variants in more than 1,000 genes have been linked to NDDs (SysID database).1Kochinke K. Zweier C. Nijhof B. Fenckova M. Cizek P. Honti F. Keerthikumar S. Oortveld M.A. Kleefstra T. Kramer J.M. et al.Systematic phenomics analysis deconvolutes genes mutated in intellectual disability into biologically coherent modules.Am. J. Hum. Genet. 2016; 98: 149-164Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar In non-consanguineous populations, de novo mutations have been highlighted as an important contributor.2de Ligt J. Willemsen M.H. van Bon B.W. Kleefstra T. Yntema H.G. Kroes T. Vulto-van Silfhout A.T. Koolen D.A. de Vries P. Gilissen C. et al.Diagnostic exome sequencing in persons with severe intellectual disability.N. Engl. J. Med. 2012; 367: 1921-1929Crossref PubMed Scopus (1129) Google Scholar, 3Deciphering Developmental Disorders S. Deciphering Developmental Disorders StudyPrevalence and architecture of de novo mutations in developmental disorders.Nature. 2017; 542: 433-438Crossref PubMed Scopus (765) Google Scholar, 4Rauch A. Wieczorek D. Graf E. Wieland T. Endele S. Schwarzmayr T. Albrecht B. Bartholdi D. Beygo J. Di Donato N. et al.Range of genetic mutations associated with severe non-syndromic sporadic intellectual disability: an exome sequencing study.Lancet. 2012; 380: 1674-1682Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar World-wide data sharing approaches facilitated by various platforms have further increased the rate of novel disease-gene discovery and the delineation of associated phenotypes. By trio exome sequencing on an Illumina HiSeq 2500 platform and data analysis with an in-house analysis pipeline as described previously,5Hauer N.N. Popp B. Schoeller E. Schuhmann S. Heath K.E. Hisado-Oliva A. Klinger P. Kraus C. Trautmann U. Zenker M. et al.Clinical relevance of systematic phenotyping and exome sequencing in patients with short stature.Genet. Med. 2018; 20: 630-638Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar we identified a de novo missense variant in FBXO11 (MIM: 607871) in a non-verbal individual with severe ID and short stature. So far, only two variants in FBXO11 have been reported as associated with NDDs, a likely gene-disrupting (LGD) de novo variant (c.2738_2739delAT [p.Tyr913∗]) in an individual with developmental delay, microcephaly, and other features6Martínez F. Caro-Llopis A. Roselló M. Oltra S. Mayo S. Monfort S. Orellana C. High diagnostic yield of syndromic intellectual disability by targeted next-generation sequencing.J. Med. Genet. 2017; 54: 87-92Crossref PubMed Scopus (83) Google Scholar and a homozygous missense variant (c.2596G>A [p.Val866Met]) in another individual with severe ID and muscular hypotonia.7Reuter M.S. Tawamie H. Buchert R. Hosny Gebril O. Froukh T. Thiel C. Uebe S. Ekici A.B. Krumbiegel M. Zweier C. et al.Diagnostic yield and novel candidate genes by exome sequencing in 152 consanguineous families with neurodevelopmental disorders.JAMA Psychiatry. 2017; 74: 293-299Crossref PubMed Scopus (129) Google Scholar Searching the DECIPHER Database,3Deciphering Developmental Disorders S. Deciphering Developmental Disorders StudyPrevalence and architecture of de novo mutations in developmental disorders.Nature. 2017; 542: 433-438Crossref PubMed Scopus (765) Google Scholar, 8Firth H.V. Richards S.M. Bevan A.P. Clayton S. Corpas M. Rajan D. Van Vooren S. Moreau Y. Pettett R.M. Carter N.P. DECIPHER: database of chromosomal imbalance and phenotype in humans using Ensembl resources.Am. J. Hum. Genet. 2009; 84: 524-533Abstract Full Text Full Text PDF PubMed Scopus (1167) Google Scholar using GeneMatcher9Sobreira 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: 928-930Crossref PubMed Scopus (819) Google Scholar and personal communication with colleagues enabled us to assemble clinical and mutational details on a total of 20 individuals with de novo variants in FBXO11, including the previously published variant in individual 18.6Martínez F. Caro-Llopis A. Roselló M. Oltra S. Mayo S. Monfort S. Orellana C. High diagnostic yield of syndromic intellectual disability by targeted next-generation sequencing.J. Med. Genet. 2017; 54: 87-92Crossref PubMed Scopus (83) Google Scholar Testing in collaborating centers was performed either in the setting of routine diagnostic testing without the requirement for institutional ethics approval or within research settings approved by the ethical review boards of the respective institutions (e.g., University Erlangen-Nuremberg, Yokohama City University School of Medicine). Informed consent was obtained from the parents or legal guardians of the affected individuals. In two individuals, chromosomal microarray analysis was performed. In 13 individuals, trio exome sequencing with filtering for de novo variants was performed, and in four individuals, affected-only exome sequencing with filtering against lists including confirmed and candidate genes such as SysID1Kochinke K. Zweier C. Nijhof B. Fenckova M. Cizek P. Honti F. Keerthikumar S. Oortveld M.A. Kleefstra T. Kramer J.M. et al.Systematic phenomics analysis deconvolutes genes mutated in intellectual disability into biologically coherent modules.Am. J. Hum. Genet. 2016; 98: 149-164Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar was carried out. Panel sequencing in individual 18 has been described previously6Martínez F. Caro-Llopis A. Roselló M. Oltra S. Mayo S. Monfort S. Orellana C. High diagnostic yield of syndromic intellectual disability by targeted next-generation sequencing.J. Med. Genet. 2017; 54: 87-92Crossref PubMed Scopus (83) Google Scholar (for details see Table S1). De novo occurrence of the FBXO11 variant was shown for all case subjects by trio exome and/or Sanger sequencing (see also Table S1). Clinical information is summarized in Tables 1 and 2. All identified individuals had developmental delay manifested by delayed motor and/or speech milestones and a variable degree of ID ranging from normal IQ with difficulties in specific areas to severe or profound ID. Individuals of school age were attending either special or mainstream schools with additional support. Behavioral abnormalities including autistic features and attention deficits as well as sleeping difficulties were reported in most of the individuals. Additionally, variable aspects such as growth and vision anomalies, hypotonia, and skeletal anomalies occurred. Epilepsy in at least five individuals ranged from developmental and epileptic encephalopathy in individual 7 to a single seizure in individual 15. Facial dysmorphisms were frequently noted. Though certain individuals resembled each other (e.g., individuals I3, I9, and I13) and aspects such as long or downslanting palpebral fissures with laterally everted lids or long eyelashes were frequently observed, overall no distinctive facial gestalt could be delineated (Figure 1A). De novo FBXO11 variants are therefore associated with a variable neurodevelopmental disorder and a range of additional anomalies but without a distinct, recognizable presentation.Table 1Summary of Clinical DetailsMissense (n = 8)Del/LGD (n = 12)Total (n = 20)Developmental delay/IDmild DD, profound IDmild DD, severe ID100%Walking age12 mo–5 y13 mo–6 y12 mo–6 yAge first wordsdelay–no speechdelay–no speechdelay–no speechShort stature3120%Tall stature1110%Obesity1425%Microcephaly1425%Macrocephaly2010%Seizures4 (+1)125%Behavioral anomalies71085%Sleeping difficulties5445%Cleft lip/palate/ bifid uvula2115%Skeletal/hand/feet anomalies5760%Vision anomalies5655%Recurrent infections/otitis media2430%Abbreviations: mo, months; y, years Open table in a new tab Table 2Clinical Details of Individuals with De Novo FBXO11 VariantsIndividual123456789101112131415161718 6Martínez F. Caro-Llopis A. Roselló M. Oltra S. Mayo S. Monfort S. Orellana C. High diagnostic yield of syndromic intellectual disability by targeted next-generation sequencing.J. Med. Genet. 2017; 54: 87-92Crossref PubMed Scopus (83) Google Scholar1920GendermalefemalemalemalefemalefemalemalemalemalefemalemalefemalemalemalemalefemalemalemalemalefemaleAge at last investigation9 y 3 mo8 y7 y 5 mo8 y29 y10 y 10 mo15 y7 y 6 mo3 y 10 mo5 y14 y1 y 6 mo8 y 4 mo9 y 10 mo11 y 5 mo19 y2 y 4 mo9 y 2 mo13 y 6 mo15 y 4 moDe novo variantc.1612A>G (p.Ile538Val)c.414A>T (p.Arg138Ser)c.467A>G (p.Gln156Arg); mosaic (60%)c.1868C>G (p.Thr623Arg)c.2518T>C (p.Ser840Pro)c.2675C>A (p.Ala892Asp)c.2714C>G (p.Pro905Arg)c.2729A>G (p.Asp910Gly)c.319_320del (p.Leu107Alafs∗45)c.506_507del (p.Ser169Leufs∗9)c.1042−1G>C (p.?)c.1260+1G>C (p.?)c.1825_1829del (p.Glu609∗)c.2084−1G>A (p.?)c.2084−6_2085dup (p.Gly697∗)c.2700_2703dup (p.Ala902Ilefs∗4)c.2709dup (p.Glu904∗)c.2738_2739del (p.Tyr913∗)deletionahg19:chr2:g.48045596-48418922; 373,327 bp exons 1–18deletionbhg19:chr2:g.48032122-48698515; 666,394 bp plus 3 genesB. weight (SD)3,230 g (−0.9)4,900 g (3.4)4,000 g (0.9)2,850 g (−1.8)3,175 g (−0.71)3,495 g (0)normalNA2,580 g (−2.4)3,624 g (0.4)3,900 g (0.6)1,795 g (−4)2,670 g (−1.7)3,100 g (−1.2)3232 g (−0.89)2325 g (−2.3)2178 g (−2.6)2500 g (−2.6)2610 g (−2.3)3175 g (−0.7)B. length (SD)51 cm (−0.7)NAnormal52 cm (−0.2)NA51 cm (−0.3)normalNA45 cm (−3.3)51 cm (−0.3)52 cm (−0.2)40 cm (−5.3)47 cm (−2)52 cm (−0.2)NANA45.0 cm (−2.6)48 cm (−2)47 cm (−2.4)NAB. OFC (SD)36 cm (0.3)smallnormal35 cm (−0.5)NA35 cm (0.1)normalNA33 cm (−2)34 cm (−0.7)36 cm (0.3)28.5 cm (−4.9)32 cm (−2.3)33.5 cm (−1.6)NANA32.0 cm (−1.1)32 cm (−2.8)32.4 cm (−2.5)(−0.66)Height (SD)122 cm (−2.7)136.8 cm (1.3)(−0.3)107 cm (−4.6)174 cm (1.29)133 cm (−2)(>2) (tall parents)119.4 cm (−1.6)105.3 cm (0.5)115.5 cm (1.1)194 cm (3.3)79 cm (- 0.9)134 cm (1.3)146 cm (0.8)152.5 cm (0.82)157.7 cm (−1.3)84.5 cm (−1.0)102 cm (−2.1) at 5 y172 cm (1.1)164.9 cm (0)Weight (SD)20 kg (−3.5)31.4 kg (0.8)(1.65)17.1 kg (−4.1)59.3 kg (NA)25 kg (−2.3)(>2)23.6 kg (−0.7)21.6 kg (1.9)29.5 kg (2.7)93 kg (2.1)12.4 kg (1.3)44.1 kg (5.4)88 kg (3)73 kg (2.3)69.9 kg (0.9)11.9 kg (−0.4)14.4 kg (−2.6) at 5 y80 kg (1.7)71.85 kg (1.2)OFC (SD)53 cm (−0.3)51 cm (−0.7)(1.6)47 cm (−4)59 cm (2.44)50.5 cm (−1.9)(>2)NA48 cm (−2.1)50 cm (−0.4)55 cm (−0.3)44.2 cm (−2.2)50 cm (−2.2)56 cm (1.6)54.5 cm (0.32)57.5 cm (1.7)47.5 cm (−0.8)47.5 cm (−2.9) at 5 y54 cm (−0.7)55 cm (0.1)Walking at age17 mo22 mo18 mo5 y16 mo2 y 2 mo12 mo2 y 6 mo13 mo15 mo24 monot yet18 mo4 y14 mo2–3 y2 y 4 mo6 y18 mo2 y 2 moFirst words at age/speech abilitiesno speech, limit. compreh.2 y/speech therapymild speech delay/speech therapy8 y/2 words, compreh. better20 mo2 y/oral motor dyspraxia18 mo/short sentences3 y: no words/signs, good non-verbal commun.23 mo/speech therapy, rhinolalia apertaNAspeech delay13 mo/stagnation, 2 words2–3 y/limit. vocabulary, saccadic speechno speechlimit. vocabulary, short sentences, single wordssentencesno words, several signs28 mo/only disyllabic words, limit. compreh.12 mo/long stagnation, mild oral motor dyspraxiaspeech delayDD/ID (tested)severe (22 mo: 12 mo delay)moderateglobal DD,WPPSI-III-NL:TIQ 98, VIQ 106,PIQ 96profoundmild-moderatemoderatemild-moderatemoderateuneven profile (IQ 75-88)mild-moderatemild-moderatemoderateDD, no ID in WISC-V, reduced comprehension and speed, learning difficultiesseveremoderatemoderatemoderateseveremild-moderatemild-moderateSeizures (onset) (type)noyes (7 y) (focal, nightly)noyes (3 y) (GTCS, absence)yes (4 y) (atonic, GTCS)possibly 1− (14 mo)yes (2 y 10 mo) (myoclonic atonic, myoclonic, GTCS, absence)nononononononoyes (single seizure 5 y)nononononoRegressionnononononoNAyes (3 y)NAnononoNAnonoyes (1 y)noNAnonoNAMRI anomaliesnoreduced white matter bulknonocraniosynostosisNAparenchymal cysts right perioccipital white matterNANANAnonoNA, CT normalNANAprominent posterior cerebral sulci, low brainstem volumenonoright opercular focal dysplasiaNAHypotoniayesyesyessevereyesmildyesseverein infancyNAyesmildyesNAyesyesyesfacialyesSleeping difficultiesneeds little sleepsleep initiation difficultyawakes each nightnosleep initiation difficultynosleep initiation difficultynofrequent awakeningssleep initiation difficultyNAsleep initiation difficulty, frequent awakeningsnonosleep initiation difficulty, awakes each nightNAnonononoBehavioral anomaliesautistic feat., little social interaction, no eye contact, (auto-) aggressivityautistim spectrum disorder, anxiety, panic attacksshort concentration span, attention deficit, limited expression, lacks social intuition4 y: little social interaction, intermittent hand flappingautism, anxietynoautistic featurespoor concentration, sociable, some behavioral issuesemotional immaturity, low attention spanshort attention span, “social and emotional delays”, hand wringinghyperactivity, mood disorderhyperactivitynoanxiety, agressive behaviorautism, ADHD, anxiety, aggressive outbursts, difficulty with social interaction, food seekingdisruptive behavior disorder, picking behaviorsnoautistic feat., stereotypiesrepetitive and ritual sentences, difficulties in social interactionautism, anxietyFacial dysmorphismtriangular face, broad forehead, mild exophthalmos, long eyelashes, mild hypertelorism, large incisorsdeep set eyesperiorbital fullness, mild hypertelorismbrachy- and plagiocephalus, deep set eyes, broad eyebrows, long eyelashes, narrow palpebral fissures, small nose, bulbous nasal tip, anteverted nares, smooth, long philtrum, narrow mouth, thin upper and everted lower vermillion, pointed chin, large pinnaelong facies, micrognathiadownslanting palpebral fissures, long philtrum, low set ears, large eyebrowsnoplagiocephallus, narrow palpebral fissures, blepharochalasis, full cheeks, pointed chinlarge eyes, arched eyebrows, long eyelashes, downslanting palpebral fissures, hypertelorism, prominent nasal bridge, broad nasal tip, smooth, short philtrum, thin upper lip, pointed chin, prominent lower vermillion, large pinnae, rotated ears, prominent tragusdeep-set, hooded eyes, broad nasal bridge, ear pit on left, high arched palatelong face, epicanthic folds, retrognathia, mandibular hypoplasialow-set ears, large eyebrows, downslanting palpebral fissuressharp, tapered eye corners, long eyelashes, supernumerary left superior incisorlong palpebral fissures, simple prominent earsangular eyebrows, broad mouth and philtrum with full lower lip, full cheeks, slightly uplifted ear lobeslarge nose, large lips, hooded eyelids, coarse faciesfrontal bossing, curled hair, highly arched and sparse eyebrows, long eyelashes, downslanting palpebral fissures, depressed nasal ridge,tented upper lipepicanthus, sparse eyebrows, large everted ears, thick lipsdownslanting palpebral fissures, everted lower lip, mild prognathismflat philtrum, cowlickAbnormalities of hands and feetfetal fingerpadsintoeingmild clinodactyly V, mild fetal finger padssmall hands, broad feet, short fingers+toes, narrow end phalanges, clinodactyly V, pits on the back of hands, hyperconvex nailsarachnodactyly, pes planusNAnobilateral camptodactyly finger V, sandal gap, broad thumbs+halluces, unilat. single palmar creasesmall hands, broad distal phalanges, fetal finger pads, broad feet, sandal gapnotapering fingers, flat feet, sandal gapnofetal fingerpads, short hands and fingersNADclinodactyly Vbilateral morton’s toe, small left 3rd toe, broad fingers with hyperconvex nailsdeep plantar creasessmall and cold hands+feet, polysyndactyly digit V left footnotoe syndactyly II/III, sandal gaps, ingrown toe nailsCleft lip or palatenononouvula bifidanoshort uvulanocleft palatenonoNAnocleft lip and alveolar ridgenononononononoSkeletal anomaliesjoint laxitynohypermobilityslender long bones, 2 fractures, osteopenia, delayed bone agesagittal craniosynostosisnononoNAnometopic craniosynostosis, joint hypermobilitynononononononononoRecurrent infections/otitis medianonorespiratory (early childhood adeno tonsillectomy)respiratory tract (early childhood)nonononoNAnoyesnononorecurrent otitis mediarecurrent otitis media, low-frequency hearing loss2 mo–1 y: several otitis medianonoNAVision anomaliesstrabismusright divergent squintnoconvergent strabismus alternansNAstrabismusnoconvergent squint, hypermetropiaconvergent strabismusnohypermetropia, strabismusnormalhypermetropianormalbilateral Duane anomalybilateral degenerative progressive high myopia, strabismusnoconvergent starbismusnormalnormalOther anomalieshairy elbows, constipationsevere hyperemesis gravidarum, low APGARs, brief resuscitation requiredNAfeeding tube until age 8 ysyringomyelia, increased sweating, pneumothorax, marfanoid featuresNAnoatrial septal defect, dry feet with fine creasespectus excavatum of inferior sternumtongue tie (repaired), 1 hypopigmented spot on right shouldercubitus valgus, striae, mild aortic dilatation, luxation of patella, trichonocephalyrecurrent vomitingNAconstipationGORD in infancyasthma, primary amenorrheafeeding difficulitiescryptorchidism, renal ectasianoconstipation, hirsutismVariant nomenclature based on GenBank: NM_001190274.1. Abbreviations: y, years; mo, months; NA, not available or not applicable; SD, standard deviation; limit. compreh., limited comprehension; commun., communication; DD, developmental delay; feat., features; ADHD, attention deficit hyperactivity disorder; GORD, gastresophageal reflux disease; GTCS, generalized tonic-clonic seizures.a hg19:chr2:g.48045596-48418922; 373,327 bpb hg19:chr2:g.48032122-48698515; 666,394 bp Open table in a new tab Abbreviations: mo, months; y, years Variant nomenclature based on GenBank: NM_001190274.1. Abbreviations: y, years; mo, months; NA, not available or not applicable; SD, standard deviation; limit. compreh., limited comprehension; commun., communication; DD, developmental delay; feat., features; ADHD, attention deficit hyperactivity disorder; GORD, gastresophageal reflux disease; GTCS, generalized tonic-clonic seizures. FBXO11 encodes a 927 amino acid F-Box protein which is highly conserved in evolution, reflected in a 100% amino acid identity with Pan troglodytes, 99.5% with mice, and 60% with C. elegans. According to GTEX, it is broadly expressed across various tissues with particularly high levels in the brain. FBXO11 contains an F-Box domain, three carbohydrate binding and hydrolase (CASH) domains, and a zinc finger-UBR domain (Figure 1B). The F-Box motif is shared by more than 60 human F-Box proteins, with FBXL denoting proteins additionally containing leucine-rich repeats, FBXW denoting proteins with additional WD repeats, and FBXO denoting F-Box proteins with either another or no other motif.11Kipreos E.T. Pagano M. The F-box protein family.Genome Biol. 2000; 1 (reviews3002.1–reviews3002.7)Crossref PubMed Google Scholar Several other genes encoding F-Box proteins have been implicated in NDDs so far (Figure S1). Recessive mutations in FBXL4 (MIM: 605654) cause an encephalomyopathic, mitochondrial DNA depletion syndrome (MIM: 615471), a homozygous LGD variant in FBXO31 (MIM: 609102) was identified in one family with non-syndromic mild to moderate ID (MIM: 615979),12Mir A. Sritharan K. Mittal K. Vasli N. Araujo C. Jamil T. Rafiq M.A. Anwar Z. Mikhailov A. Rauf S. et al.Truncation of the E3 ubiquitin ligase component FBXO31 causes non-syndromic autosomal recessive intellectual disability in a Pakistani family.Hum. Genet. 2014; 133: 975-984Crossref PubMed Scopus (21) Google Scholar and a homozygous missense variant in FBXO47 (MIM: 609498) has been found in four affected members of one family with non-syndromic ID.13Harripaul R. Vasli N. Mikhailov A. Rafiq M.A. Mittal K. Windpassinger C. Sheikh T.I. Noor A. Mahmood H. Downey S. et al.Mapping autosomal recessive intellectual disability: combined microarray and exome sequencing identifies 26 novel candidate genes in 192 consanguineous families.Mol. Psychiatry. 2018; 23: 973-984Crossref PubMed Scopus (99) Google Scholar The only dominant variant in another F-box gene reported so far is a de novo missense variant in FBXO10 (MIM: 609092) identified in a single individual with autism-spectrum disorder and an IQ of 67.14O’Roak B.J. Vives L. Girirajan S. Karakoc E. Krumm N. Coe B.P. Levy R. Ko A. Lee C. Smith J.D. et al.Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations.Nature. 2012; 485: 246-250Crossref PubMed Scopus (1567) Google Scholar Interestingly, FBXO10 is the protein most closely related to FBXO11, and the reported de novo FBXO10 variant is in close proximity to the F-Box (Figure S1), similarly positioned to the missense FBXO11 variants in individuals I2 and I3. We assembled two whole or partial gene deletions, eight missense variants, and ten LGD variants. One of the missense variants in I3 was detected in a possible 60% mosaic. All identified LGD variants are distributed throughout the gene/protein, with only one of them located within a functional domain (Figure 1B). They comprise nonsense, splice site, and frameshift variants and most are likely to lead to nonsense-mediated mRNA decay, except for the variants in individuals 16, 17, and 18,6Martínez F. Caro-Llopis A. Roselló M. Oltra S. Mayo S. Monfort S. Orellana C. High diagnostic yield of syndromic intellectual disability by targeted next-generation sequencing.J. Med. Genet. 2017; 54: 87-92Crossref PubMed Scopus (83) Google Scholar which are located in the last exon of FBXO11 and thus potentially escape nonsense-mediated mRNA decay. In combination with observations of larger de novo deletions of FBXO11 in individuals with a phenotype of developmental delay or intellectual disability and variable additional features, our findings are compatible with a loss-of-function mechanism causing haploinsufficiency, though other mechanism such as gain-of-function or dominant-negative effects are possible, particularly for missense variants. This is in accordance with constraint scores from ExAC,15Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. O’Donnell-Luria A.H. Ware J.S. Hill A.J. Cummings B.B. et al.Exome Aggregation ConsortiumAnalysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (6551) Google Scholar which indicate that FBXO11 is a gene highly intolerant to loss-of-function variants with a pLI score of 1. With a z-score of 4.02 it is also intolerant to missense variation. The identified missense variants do not cluster within a particular domain or region of FBXO11 but are also distributed across the protein (Figure 1B). Interpretation regarding pathogenicity is more challenging than for LGD variants. All missense variants affect highly conserved amino acids (Figure 2A), are not contained in GnomAD, and are predicted to be deleterious by at least three in silico prediction programs (Table S1). To further assess possible consequences of the missense variants on protein stability and function, we performed structural modeling. Variants were modeled with Swiss-Model,23Guex N. Peitsch M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9560) Google Scholar and RasMol24Sayle R.A. Milner-White E.J. RASMOL: biomolecular graphics for all.Trends Biochem. Sci. 1995; 20: 374Abstract Full Text PDF PubMed Scopus (2320) Google Scholar was used for structure analysis and visualization. Missense variants for which structural information was available are likely to lead to a destabilization of the protein, which supports loss of function as the most likely disease mechanism. The missense variants p.Ile538Val and p.Thr623Arg are located in the first and second CASH domain of FBXO11, respectively. CASH domains are frequently found in carbohydrate binding proteins and hydrolases and are important for substrate recognition in F-Box proteins.25Duan S. Cermak L. Pagan J.K. Rossi M. Martinengo C. di Celle P.F. Chapuy B. Shipp M. Chiarle R. Pagano M. FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas.Nature. 2012; 481: 90-93Crossref PubMed Scopus (215) Google Scholar, 26Jin J. Cardozo T. Lovering R.C. Elledge S.J. Pagano M. Harper J.W. Systematic analysis and nomenclature of mammalian F-box proteins.Genes Dev. 2004; 18: 2573-2580Crossref PubMed Scopus (543) Google Scholar Deletion of the CASH domain in FBXO11 leads to impaired interaction with proto-oncogene BCL6 (MIM: 109565). Therefore, BCL6 is aberrantly stabilized in cells lacking FBXO11 and in cells with FBXO11 CASH domain mutations.25Duan S. Cermak L. Pagan J.K. Rossi M. Martinengo C. di Celle P.F. Chapuy B. Shipp M. Chiarle R. Pagano M. FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas.Nature. 2012; 481: 90-93Crossref PubMed Scopus (215) Google Scholar CASH domains exhibit an elongated structure that consists of right-handed β helices.27Ciccarelli F.D. Copley R.R. Doerks T. Russell R.B. Bork P. CASH--a beta-helix domain widespread among carbohydrate-binding proteins.Trends Biochem. Sci. 2002; 27: 59-62Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Ile538 points to the interior of this β-helical structure and forms hydrophobic interactions with Val551 (Figure 2B). These interactions cannot be formed by the shorter sidechain in the p.Ile538Val variant (Figure 2C), thereby probably destabilizing the first CASH domain. The Thr623 sidechain points to the outside of the β-helical structure and forms polar interactions with the sidechains of Tyr598 and His600 (Figure 2D). Due to the different length, the Arg623 sidechain can form only a weak interaction with Tyr598, whereas electrostatic repulsion emerges between the two positively charged residues Arg623 and His600 (Figure 2E), which destabilizes the second CASH domain. The missense variants p.Ser840Pro and p.Ala892Asp are located in the UBR-domain, which is characteristic for E3 ubiquitin ligases and binds directly to N-terminal degradation signals in substrate proteins.22Muñoz-Escobar J. Kozlov G. Gehring K. Crystal structure of the UBR-box from UBR6/FBXO11 reveals domain swapping mediated by zinc binding.Protein Sci. 2017; 26: 2092-2097Crossref PubMed Scopus (6) Google Scholar The Ser840 stabilizes the N terminus of an
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