REPORT

De Novo Variants in the F-Box Protein FBXO11

in 20 Individuals with a Variable
Neurodevelopmental Disorder
Anne Gregor,1 Lynette G. Sadleir,2 Reza Asadollahi,3,4 Silvia Azzarello-Burri,3,4 Agatino Battaglia,5
Lilian Bomme Ousager,6 Paranchai Boonsawat,3,4 Ange-Line Bruel,7 Rebecca Buchert,8
Eduardo Calpena,9 Benjamin Cogné,10,11 Bruno Dallapiccola,12 Felix Distelmaier,13 Frances Elmslie,14
Laurence Faivre,7,15 Tobias B. Haack,8 Victoria Harrison,16 Alex Henderson,17 David Hunt,16
Bertrand Isidor,10 Pascal Joset,3,4 Satoko Kumada,18 Augusta M.A. Lachmeijer,19 Melissa Lees,20
Sally Ann Lynch,21 Francisco Martinez,22 Naomichi Matsumoto,23 Carey McDougall,24
Heather C. Mefford,25 Noriko Miyake,23 Candace T. Myers,25 Sébastien Moutton,7,15 Addie Nesbitt,26
Antonio Novelli,12 Carmen Orellana,22 Anita Rauch,3,4 Monica Rosello,22 Ken Saida,23
Avni B. Santani,26,27 Ajoy Sarkar,28 Ingrid E. Scheffer,29 Marwan Shinawi,30 Katharina Steindl,3,4
Joseph D. Symonds,31 Elaine H. Zackai,24 University of Washington Center for Mendelian Genomics,
DDD Study,32 André Reis,1 Heinrich Sticht,33 and Christiane Zweier1,*

Next-generation sequencing combined with international data sharing has enormously facilitated identification of new disease-associ-
ated genes and mutations. This is particularly true for genetically extremely heterogeneous entities such as neurodevelopmental disor-
ders (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 con-
trolling 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 intellec- and genetically extremely heterogeneous. Currently, path-
tual disability (ID), autism spectrum disorders, and devel- ogenic variants in more than 1,000 genes have been linked
opmental and epileptic encephalopathies, are clinically to NDDs (SysID database).1 In non-consanguineous
1
Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany; 2Department of Paediatrics and Child
Health, University of Otago, Wellington 6242, New Zealand; 3Institute of Medical Genetics, University of Zurich, 8952 Schlieren-Zurich, Switzerland;
4
radiz – ‘‘Rare Disease Initiative Zurich, Clinical Research Priority Program for Rare Diseases University of Zurich,’’ 8032 Zurich, Switzerland; 5Stella Maris
Clinical Research Institute for Child and Adolescent Neurology and Psychiatry, 56128 Calambrone, Pisa, Italy; 6Department of Clinical Genetics, Odense
University Hospital, 5000 Odense, Denmark; 7INSERM U1231, LNC UMR1231 GAD, Burgundy University, 21079 Dijon, France; 8Institute of Medical
Genetics and Applied Genomics, University of Tübingen, 72076 Tübingen, Germany; 9Clinical Genetics Group, MRC Weatherall Institute of Molecular
Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK; 10Department of Medical Genetics, CHU Nantes, 44093 Nantes, France;
11
l’Institut du Thorax, INSERM, CNRS, UNIV Nantes, 44007 Nantes, France; 12Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino
Gesù, IRCCS, Rome 00146, Italy; 13Department of General Pediatrics, Neonatology and Pediatric Cardiology, University Children’s Hospital Düsseldorf,
Medical Faculty, Heinrich Heine University, 40225 Düsseldorf, Germany; 14South West Thames Regional Genetics Service, St. George’s, University of
London, London SW17 0RE, UK; 15Reference Center for Developmental Anomalies, Department of Medical Genetics, Dijon University Hospital, 21000
Dijon, France; 16Wessex Clinical Genetics Service, Princess Anne Hospital, Southampton SO16 5YA, UK; 17Northern Genetics Service, Newcastle upon
Tyne Hospitals, Newcastle upon Tyne NE1 3BZ, UK; 18Department of Neuropediatrics, Tokyo Metropolitan Neurological Hospital, Tokyo 183-0042, Japan;
19
Department of Genetics, University Medical Center Utrecht, PO Box 85090, 3508 AB Utrecht, the Netherlands; 20North East Thames Regional Genetics
Service, Great Ormond Street Hospital for Children NHS Foundation Trust, Great Ormond Street Hospital, London WC1N 3JH, UK; 21Dept of Clinical Ge-
netics, Temple Street Children’s Hospital Dublin 1, D12 V004 Dublin, Ireland; 22Unidad de Genética, Hospital Universitario y Politécnico La Fe, Avda Fer-
nando Abril Martorell 106, 46026 Valencia, Spain; 23Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama
236-0004, Japan; 24Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA; 25Division of Genetic Medicine,
Department of Pediatrics, University of Washington, Seattle, WA 98195, USA; 26Division of Genomic Diagnostics, Children’s Hospital of Philadelphia, Phil-
adelphia, PA 19104, USA; 27Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
19104, USA; 28Nottingham Regional Genetics Service, City Hospital Campus, Nottingham University Hospitals NHS Trust, The Gables, Hucknall Road,
Nottingham NG5 1PB, UK; 29Departments of Medicine and Paediatrics, Austin Health and Royal Children’s Hospital, The University of Melbourne, Park-
ville, VIC 3050, Australia; 30Division of Genetics and Genomic Medicine, Department of Pediatrics, Washington University School of Medicine, St. Louis,
MO 63110, USA; 31Paediatric Neurosciences Research Group, Fraser of Allander Neurosciences Unit, Royal Hospital for Children, Glasgow G51 4TF, UK;
32
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK; 33Institute of Biochemistry, Emil-Fischer Center,
Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany
*Correspondence: christiane.zweier@uk-erlangen.de
https://doi.org/10.1016/j.ajhg.2018.07.003.
Ó 2018 American Society of Human Genetics.

The American Journal of Human Genetics 103, 305–316, August 2, 2018 305
 from the parents or legal guardians of the affected individ-
Table 1. Summary of Clinical Details
uals. In two individuals, chromosomal microarray analysis
Missense Del/LGD Total
(n ¼ 8) (n ¼ 12) (n ¼ 20) was performed. In 13 individuals, trio exome sequencing
with filtering for de novo variants was performed, and in
Developmental delay/ID mild DD, mild DD, 100%
profound ID severe ID four individuals, affected-only exome sequencing with
filtering against lists including confirmed and candidate
Walking age 12 mo–5 y 13 mo–6 y 12 mo–6 y
genes such as SysID1 was carried out. Panel sequencing
Age first words delay–no delay–no delay–no in individual 18 has been described previously6 (for details
speech speech speech
see Table S1). De novo occurrence of the FBXO11 variant
Short stature 3 1 20% was shown for all case subjects by trio exome and/or
Tall stature 1 1 10% Sanger sequencing (see also Table S1). Clinical information
Obesity 1 4 25%
is summarized in Tables 1 and 2. All identified individuals
had developmental delay manifested by delayed motor
Microcephaly 1 4 25%
and/or speech milestones and a variable degree of ID
Macrocephaly 2 0 10% ranging from normal IQ with difficulties in specific areas
Seizures 4 (þ1) 1 25% to severe or profound ID. Individuals of school age were
attending either special or mainstream schools with addi-
Behavioral anomalies 7 10 85%
tional support. Behavioral abnormalities including autistic
Sleeping difficulties 5 4 45% features and attention deficits as well as sleeping diffi-
Cleft lip/palate/ bifid uvula 2 1 15% culties were reported in most of the individuals. Addition-
Skeletal/hand/feet 5 7 60%
ally, variable aspects such as growth and vision anomalies,
anomalies hypotonia, and skeletal anomalies occurred. Epilepsy in at
Vision anomalies 5 6 55%
least five individuals ranged from developmental and
epileptic encephalopathy in individual 7 to a single seizure
Recurrent infections/otitis 2 4 30%
media
in individual 15. Facial dysmorphisms were frequently
noted. Though certain individuals resembled each other
Abbreviations: mo, months; y, years
(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,
populations, de novo mutations have been highlighted as overall no distinctive facial gestalt could be delineated
an important contributor.2–4 World-wide data sharing ap- (Figure 1A). De novo FBXO11 variants are therefore associ-
proaches facilitated by various platforms have further ated with a variable neurodevelopmental disorder and a
increased the rate of novel disease-gene discovery and range of additional anomalies but without a distinct,
the delineation of associated phenotypes. recognizable presentation.
By trio exome sequencing on an Illumina HiSeq 2500 FBXO11 encodes a 927 amino acid F-Box protein which
platform and data analysis with an in-house analysis pipe- is highly conserved in evolution, reflected in a 100%
line as described previously,5 we identified a de novo amino acid identity with Pan troglodytes, 99.5% with
missense variant in FBXO11 (MIM: 607871) in a non-ver- mice, and 60% with C. elegans. According to GTEX, it is
bal individual with severe ID and short stature. So far, broadly expressed across various tissues with particularly
only two variants in FBXO11 have been reported as associ- high levels in the brain. FBXO11 contains an F-Box
ated with NDDs, a likely gene-disrupting (LGD) de novo domain, three carbohydrate binding and hydrolase
variant (c.2738_2739delAT [p.Tyr913*]) in an individual (CASH) domains, and a zinc finger-UBR domain (Fig-
with developmental delay, microcephaly, and other fea- ure 1B). The F-Box motif is shared by more than 60 human
tures6 and a homozygous missense variant (c.2596G>A F-Box proteins, with FBXL denoting proteins additionally
[p.Val866Met]) in another individual with severe ID and containing leucine-rich repeats, FBXW denoting proteins
muscular hypotonia.7 Searching the DECIPHER Data- with additional WD repeats, and FBXO denoting F-Box
base,3,8 using GeneMatcher9 and personal communication proteins with either another or no other motif.11 Several
with colleagues enabled us to assemble clinical and muta- other genes encoding F-Box proteins have been implicated
tional details on a total of 20 individuals with de novo var- in NDDs so far (Figure S1). Recessive mutations in FBXL4
iants in FBXO11, including the previously published (MIM: 605654) cause an encephalomyopathic, mitochon-
variant in individual 18.6 Testing in collaborating centers drial DNA depletion syndrome (MIM: 615471), a homozy-
was performed either in the setting of routine diagnostic gous LGD variant in FBXO31 (MIM: 609102) was identified
testing without the requirement for institutional ethics in one family with non-syndromic mild to moderate ID
approval or within research settings approved by the (MIM: 615979),12 and a homozygous missense variant in
ethical review boards of the respective institutions (e.g., FBXO47 (MIM: 609498) has been found in four affected
University Erlangen-Nuremberg, Yokohama City Univer- members of one family with non-syndromic ID.13 The
sity School of Medicine). Informed consent was obtained only dominant variant in another F-box gene reported so

306 The American Journal of Human Genetics 103, 305–316, August 2, 2018
far is a de novo missense variant in FBXO10 (MIM: 609092)
identified in a single individual with autism-spectrum dis-
order and an IQ of 67.14 Interestingly, FBXO10 is the pro-
tein 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
18,6 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 dele-
tions of FBXO11 in individuals with a phenotype of devel-
opmental 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,15 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 (Fig-
ure 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 vari-
ants on protein stability and function, we performed
structural modeling. Variants were modeled with Swiss-
Model,23 and RasMol24 was used for structure analysis
and visualization. Missense variants for which structural
information was available are likely to lead to a destabiliza-
tion 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 carbo-
hydrate binding proteins and hydrolases and are impor-
tant for substrate recognition in F-Box proteins.25,26 Dele-
tion 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 muta-
tions.25 CASH domains exhibit an elongated structure
that consists of right-handed b helices.27 Ile538 points to
the interior of this b-helical structure and forms hydropho-
The American Journal of Human Genetics 103, 305–316, August 2, 2018 307
bic interactions with Val551 (Figure 2B). These interactions
cannot be formed by the shorter sidechain in the
p.Ile538Val variant (Figure 2C), thereby probably destabi-
lizing the first CASH domain. The Thr623 sidechain points
to the outside of the b-helical structure and forms polar in-
teractions with the sidechains of Tyr598 and His600
(Figure 2D). Due to the different length, the Arg623 side-
chain 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 degrada-
tion signals in substrate proteins.22 The Ser840 stabilizes
the N terminus of an a helix. Its sidechain interacts with
the backbone amide groups of residues 839 and 840 via a
bridging water molecule (Figure 2G). This bridging is no
longer possible in the p.Ser840Pro variant, because proline
lacks both the sidechain hydroxyl group and the backbone
amide hydrogen present in the wild-type serine. In addi-
tion, the cyclic proline sidechain results in steric clashes
with the bridging water, thus leading to an entire
disruption of this helix-stabilizing structural element
(Figure 2G). Ala892 forms hydrophobic interactions with
Phe887 (Figure 2H), which stabilizes the orientation of
the connecting loop that contains two cysteines (Cys888,
Cys890) coordinating a zinc ion. The p.Ala892Asp ex-
change places the negatively charged aspartate sidechain
in the immediate vicinity of the hydrophobic Phe887
sidechain (Figure 2I). This unfavorable interaction is
expected to cause a conformational rearrangement of
the entire sequence stretch, which will also affect the cys-
teines required for zinc coordination, consequently desta-
bilizing the domain structure. Variants p.Pro905Arg and
p.Asp910Gly are located downstream of the zf-UBR
domain (aa 833–904), thereby preventing structural
modeling. Interestingly, this C-terminal region seems to
be a hotspot for variants with two LGD and three missense
variants.
Two missense variants, p.Arg138Ser and p.Gln156Arg,
are located upstream or within the F-Box like domain
(aa 153–201), respectively. F-Boxes are interaction sites
for SKP1 (MIM: 601434), and both proteins together
form the substrate specificity module of the E3 ubiquitin
ligase. SKP1 then interacts with CUL1 (MIM: 603134).28
No structural information was available for this region.
One might speculate that mutations in the F-Box domain
could impair interaction with SKP1 and therefore render
this E3 ubiquitin ligase complex non-functional.
Seizures occurred more frequently in individuals with
missense variants, and microcephaly and obesity occurred
more frequently in individuals with LGD variants (Table 1).
Apart from that, the phenotype of individuals with
missense variants is not apparently distinct from that
associated with LGD variants. This is in accordance with
loss of function as the most likely mechanism. However,
Table 2. Clinical Details of Individuals with De Novo FBXO11 Variants
Individual 1 2 3 4 5 6 7 8 9 10

Gender male female male male female female male male male female

Age at last 9 y 3 mo 8y 7 y 5 mo 8y 29 y 10 y 10 mo 15 y 7 y 6 mo 3 y 10 mo 5y
investigation

De novo c.1612A>G c.414A>T c.467A>G c.1868C>G c.2518T>C c.2675C>A c.2714C>G c.2729A>G c.319_320del c.506_507del
variant (p.Ile538Val) (p.Arg138Ser) (p.Gln156Arg); (p.Thr623Arg) (p.Ser840Pro) (p.Ala892Asp) (p.Pro905Arg) (p.Asp910Gly) (p.Leu107Alafs* (p.Ser169Leufs*9)
mosaic (60%) 45)

B. 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) normal NA 2,580 g (2.4) 3,624 g (0.4)

B. length (SD) 51 cm (0.7) NA normal 52 cm (0.2) NA 51 cm (0.3) normal NA 45 cm (3.3) 51 cm (0.3)

B. OFC (SD) 36 cm (0.3) small normal 35 cm (0.5) NA 35 cm (0.1) normal NA 33 cm (2) 34 cm (0.7)

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 119.4 cm (1.6) 105.3 cm (0.5) 115.5 cm (1.1)
parents)

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)

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) NA 48 cm (2.1) 50 cm (0.4)

Walking 17 mo 22 mo 18 mo 5y 16 mo 2 y 2 mo 12 mo 2 y 6 mo 13 mo 15 mo
at age

First words no speech, 2 y/speech mild speech 8 y/2 words, 20 mo 2 y/oral 18 mo/short 3 y: no words/ 23 mo/speech NA
at age/ limit. therapy delay/speech compreh. motor sentences signs, good therapy,
speech compreh. therapy better dyspraxia non-verbal rhinolalia
abilities commun. aperta

DD/ID severe moderate global DD, profound mild-moderate moderate mild- moderate uneven profile mild-moderate
(tested) (22 mo: WPPSI-III-NL: moderate (IQ 75-88)
12 mo delay) TIQ 98,
VIQ 106,
PIQ 96

Seizures no yes (7 y) no yes (3 y) yes (4 y) possibly yes (2 y no no no

(onset) (focal, (GTCS, (atonic, GTCS) 1 (14 mo) 10 mo)
(type) nightly) absence) (myoclonic
atonic,
myoclonic,
GTCS,
absence)

Regression no no no no no NA yes (3 y) NA no no

MRI no reduced no no craniosynostosis NA parenchymal NA NA NA

anomalies white cysts right
matter perioccipital
bulk white matter

Hypotonia yes yes yes severe yes mild yes severe in infancy

Sleeping needs little sleep sleep awakes each no sleep initiation no sleep no frequent sleep initiation
difficulties initiation night difficulty initiation awakenings difficulty
difficulty difficulty

Behavioral autistic feat., autistim short 4 y: little autism, anxiety no autistic poor emotional short attention
anomalies little social spectrum concentration social features concentration, immaturity, span, ‘‘social and
interaction, disorder, span, attention interaction, sociable, some low attention emotional
no eye anxiety, panic deficit, limited intermittent behavioral span delays’’,
contact, attacks expression, hand flapping issues hand wringing
(auto-) lacks social
aggressivity intuition

(Continued on next page)

308 The American Journal of Human Genetics 103, 305–316, August 2, 2018
 6
11 12 13 14 15 16 17 18 19 20

male female male male male female male male male female

14 y 1 y 6 mo 8 y 4 mo 9 y 10 mo 11 y 5 mo 19 y 2 y 4 mo 9 y 2 mo 13 y 6 mo 15 y 4 mo

c.10421G> c.1260þ1G> c.1825_ c.2084 c.20846_ c.2700_2703dup c.2709dup c.2738_ deletiona deletionb
C (p.?) C (p.?) 1829del 1G>A (p.?) 2085dup (p.Ala902Ilefs*4) (p.Glu904*) 2739del exons plus 3
(p.Glu609*) (p.Gly697*) (p.Tyr913*) 1–18 genes

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)

52 cm (0.2) 40 cm (5.3) 47 cm (2) 52 cm (0.2) NA NA 45.0 cm (2.6) 48 cm (2) 47 cm (2.4) NA

36 cm (0.3) 28.5 cm (4.9) 32 cm (2.3) 33.5 cm (1.6) NA NA 32.0 cm (1.1) 32 cm (2.8) 32.4 cm (2.5) (0.66)

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) 172 cm (1.1) 164.9 cm (0)
at 5 y

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) 80 kg (1.7) 71.85 kg (1.2)
at 5 y

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) 54 cm (0.7) 55 cm (0.1)
at 5 y

24 mo not yet 18 mo 4y 14 mo 2–3 y 2 y 4 mo 6y 18 mo 2 y 2 mo

speech delay 13 mo/ 2–3 y/limit. no speech limit. vocabulary, sentences no words, 28 mo/only 12 mo/long speech delay
stagnation, vocabulary, short sentences, several signs disyllabic stagnation,
2 words saccadic speech single words words, limit. mild oral
compreh. motor
dyspraxia

mild-moderate moderate DD, no ID in severe moderate moderate moderate severe mild- mild-
WISC-V, reduced moderate moderate
comprehension
and speed,
learning
difficulties

no no no no yes (single no no no no no
seizure 5 y)

no NA no no yes (1 y) no NA no no NA

no no NA, CT normal NA NA prominent no no right NA

posterior opercular
cerebral sulci, focal
low brainstem dysplasia
volume

NA yes mild yes NA yes yes yes facial yes

NA sleep no no sleep initiation NA no no no no

initiation difficulty, awakes
difficulty, each night
frequent
awakenings

hyperactivity, hyperactivity no anxiety, autism, ADHD, disruptive no autistic feat., repetitive autism,
mood disorder agressive anxiety, behavior stereotypies and ritual anxiety
behavior aggressive disorder, sentences,
outbursts, picking difficulties
difficulty with behaviors in social
social interaction, interaction
food seeking

(Continued on next page)

The American Journal of Human Genetics 103, 305–316, August 2, 2018 309
Table 2. Continued
Individual 1 2 3 4 5 6 7 8 9 10

Facial triangular deep set eyes periorbital brachy- and long facies, downslanting no plagiocephallus, large eyes, deep-set,
dysmorphism face, broad fullness, mild plagiocephalus, micrognathia palpebral narrow palpebral arched hooded eyes,
forehead, mild hypertelorism deep set eyes, fissures, long fissures, eyebrows, long broad nasal
exophthalmos, broad eyebrows, philtrum, low blepharochalasis, eyelashes, bridge, ear
long eyelashes, long eyelashes, set ears, large full cheeks, downslanting pit on left,
mild narrow eyebrows pointed chin palpebral high arched
hypertelorism, palpebral fissures, palate
large incisors fissures, small hypertelorism,
nose, bulbous prominent
nasal tip, nasal bridge,
anteverted broad nasal tip,
nares, smooth, smooth, short
long philtrum, philtrum, thin
narrow mouth, upper lip,
thin upper and pointed chin,
everted lower prominent
vermillion, lower
pointed chin, vermillion,
large pinnae large pinnae,
rotated ears,
prominent
tragus

Abnormalities fetal fingerpads intoeing mild small hands, arachnodactyly, NA no bilateral small hands, no
of hands and clinodactyly V, broad feet, pes planus camptodactyly broad distal
feet mild fetal short finger V, sandal phalanges,
finger pads fingersþtoes, gap, broad fetal finger
narrow end thumbsþhalluces, pads, broad
phalanges, unilat. single feet, sandal gap
clinodactyly V, palmar crease
pits on the
back of hands,
hyperconvex
nails

Cleft lip or no no no uvula bifida no short uvula no cleft palate no no

palate

Skeletal joint laxity no hypermobility slender long sagittal no no no NA no

anomalies bones, craniosynostosis
2 fractures,
osteopenia,
delayed
bone age

Recurrent no no respiratory respiratory no no no no NA no

infections/ (early tract (early
otitis media childhood childhood)
adeno
tonsillectomy)

Vision strabismus right no convergent NA strabismus no convergent convergent no

anomalies divergent strabismus squint, strabismus
squint alternans hypermetropia

Other hairy elbows, severe NA feeding tube syringomyelia, NA no atrial septal pectus tongue tie
anomalies constipation hyperemesis until age 8 y increased defect, dry excavatum (repaired), 1
gravidarum, sweating, feet with fine of inferior hypopigmented
low APGARs, pneumothorax, creases sternum spot on right
brief marfanoid shoulder
resuscitation features
required

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.
a
hg19:chr2:g.48045596-48418922; 373,327 bp
b
hg19:chr2:g.48032122-48698515; 666,394 bp

a broader spectrum of mutational consequences including
dominant-negative or gain-of-function effects that might
explain clinical characteristics unique to certain individ-
uals is possible. In four individuals, exome sequencing
revealed additional (de novo) variants of unknown signifi-
cance (Table S1). To our knowledge, neither TCTEX1D4
(MIM: 611713), TP73 (MIM: 601990), nor SMARCAD1
(MIM: 612761) have been linked to NDDs and GPT2 (MIM:
138210) has been linked only to recessive ID (MRT49
[MIM: 616281]). A contributory effect to the phenotype of
these individuals, however, cannot be excluded. Of note,
the deletion in I20 also affects the last five exons of MSH6
(MIM: 600678), leading to a possible additional diagnosis
of Lynch syndrome (MIM: 120435). Generally, additional
modifying factors have to be considered in regard to the
large phenotypic variability observed in this cohort.
All variants described here occurred de novo. Except for
an unclear, maternally inherited variant in I7 with a low
frequency in ExAC and which was not phased, there was
no indication of additional, likely pathogenic variants on

310 The American Journal of Human Genetics 103, 305–316, August 2, 2018
 6
11 12 13 14 15 16 17 18 19 20

long face, low-set ears, sharp, tapered long angular eyebrows, large nose, frontal bossing, epicanthus, downslanting flat philtrum,
epicanthic large eye corners, palpebral broad mouth large lips, curled hair, sparse palpebral cowlick
folds, eyebrows, long eyelashes, fissures, and philtrum hooded highly arched eyebrows, fissures,
retrognathia, downslanting supernumerary simple with full lower eyelids, and sparse large everted everted lower
mandibular palpebral left superior prominent lip, full cheeks, coarse facies eyebrows, long ears, thick lips lip, mild
hypoplasia fissures incisor ears slightly uplifted eyelashes, prognathism
ear lobes downslanting
palpebral fissures,
depressed nasal
ridge,
tented upper lip

tapering no fetal NAD clinodactyly V bilateral deep plantar small and cold no toe
fingers, flat feet, fingerpads, morton’s toe, creases handsþfeet, syndactyly
sandal gap short hands small left 3rd polysyndactyly II/III, sandal
and fingers toe, broad digit V left foot gaps,
fingers with ingrown toe
hyperconvex nails
nails

NA no cleft lip and no no no no no no no

alveolar ridge

metopic no no no no no no no no no
craniosynostosis,
joint
hypermobility

yes no no no recurrent recurrent otitis 2 mo–1 y: no no NA

otitis media media, low- several otitis
frequency media
hearing loss

hypermetropia, normal hypermetropia normal bilateral bilateral no convergent normal normal

strabismus Duane degenerative starbismus
anomaly progressive
high myopia,
strabismus

cubitus valgus, recurrent NA constipation GORD in asthma, feeding cryptorchidism, no constipation,

striae, mild vomiting infancy primary difficulities renal ectasia hirsutism
aortic dilatation, amenorrhea
luxation of patella,
trichonocephaly

the second allele in the herewith reported individuals.
Given the report of a homozygous missense variant,7
FBXO11 might belong to the genes in which both domi-
nant and recessive variants can be associated with NDDs,
as has been reported for other genes, for example NALCN
(MIM: 611549) (causing congenital contractures of the
limbs and face, hypotonia, and developmental delay
[MIM: 616266] and hypotonia, infantile, with psychomo-
tor retardation and characteristic facies 1 [MIM: 615419])29
or ATAD3A (MIM: 612316) (causing Harel-Yoon syndrome
[MIM: 617183]).30 However, the evidence for autosomal-
recessive inheritance of FBXO11 variants is based on a sin-
gle case subject and therefore limited so far.
Initially, FBXO11 (or PRMT9) was identified as a type II
protein arginine methyltransferase, but with a structure
different to other protein arginine methyltransferases.31
F-Box proteins constitute a subunit of the SKP1-Cullin-
F-Box (SCF) complex, an E3-ubiquitin ligase complex
involved in phosphorylation-dependent ubiquitina-
tion.28 Ubiquitin-mediated proteasomal degradation is a

The American Journal of Human Genetics 103, 305–316, August 2, 2018 311
Figure 1. De Novo Variants Identified in FBXO11
(A) Clinical pictures of individuals with pathogenic variants in FBXO11, displaying minor and nonspecific dysmorphic features such as
epicanthal folds, down-slanting palpebral fissures, and a prominent nasal tip.
(B) Schematic drawing of genomic and protein structure of FBXO11 (isoform GenBank: NM_001190274.1, genomic coordinates
FBXO11 (hg19): chr2:48,034,059–48,132,932). Domains are color-coded according to InterPro.10 Genomic deletions are depicted
with black arrows, missense variants are highlighted in black, and likely gene-disrupting (LGD) variants are shown in red. The
herewith published variants are spread out across the protein. Corresponding positions of mutations in FBXO11 mutant mice are shown
in green.

conserved cellular mechanism that controls critical biolog-
ical processes by regulating protein turnover. FBXO11
therefore also adds to the number of NDD-associated genes
involved in ubiquitination and protein degradation such
as UBE3A32 (MIM: 601623) (causing Angelman syndrome
[MIM: 105830]), UBE3B33 (MIM: 608047) (causing Kauf-
man oculocerebrofacial syndrome [MIM: 244450]),
HUWE134 (MIM: 300697) (causing mental retardation,
X-linked syndromic, Turner type [MIM: 300706]),
USP9X35 (MIM: 300072) (causing mental retardation,
X-linked 99 [MIM: 300919] and mental retardation,
X-linked 99, syndromic, female restricted [MIM:
300968]), CUL4B36 (MIM: 300304) (causing mental retar-
dation, X-linked, syndromic (Cabezas type) [MIM:
300354]), PSMD1237 (MIM: 604450) (causing Stankie-
wicz-Isidor syndrome [MIM: 617516]), and very recently
RHOBTB238 (MIM: 607352) (causing epileptic encephalop-
athy, early infantile, 64 [MIM: 618004]). These disorders
are associated with variable ID and syndromic or non-syn-
dromic features.
So far, the role of FBXO11 has been studied mainly in the
context of tumorigenesis. Somatic inactivating mutations
in FBXO11 have been identified in various B cell malig-
nancies, where its loss resulted in increased stability
of proto-oncogene BCL6.25 In gastric cancer, overexpres-
sion of FBXO11 was found to result in increased prolifera-
tion, migration, and invasion through suppression of
PTEN (MIM: 601728) and activation of the PI3K/AKT
pathway.39 Additionally, FBXO11 promotes neddylation,
another post-translational modification analogous to ubiq-
uitination.40
Intronic variants in FBXO11 have been associated with
otitis media susceptibility in humans.41–43 Similarly, a het-
erozygous mouse model, Jeff (Jf), carrying a semi-dominant

312 The American Journal of Human Genetics 103, 305–316, August 2, 2018
Figure 2. Conservation and Structural Modeling of FBXO11 Missense Variants
(A) Conservation of amino acids affected by FBXO11 missense variants depicted with clustal omega.16,17 All herewith reported missense
variants affect highly conserved amino acid residues.
(B–E) Structural effect of FBXO11 missense variants. To evaluate the effect of the p.Ile538Val and p.Thr623Arg variants, the first and sec-
ond CASH domain of FBXO11 were modeled based on the crystal structure of the alginate epimerase AlgG (PDB: 4OZZ, 4NK818). The
template for modeling was identified using LOMETS19 and HHpred,20 and modeling was performed with Modeler 9.16.21
(B) Ile538 is located in the first CASH domain and forms hydrophobic interactions with Val551 (green arrow).
(C) These interactions cannot be formed by the shorter sidechain in the p.Ile538Val variant.
(D) Thr623 is located in the second CASH domain and forms polar interactions (green lines) with the Tyr598 and His600 sidechains.
(E) Arg623 can form only weak polar interactions with Tyr598 and instead an electrostatic repulsion between Arg623 and His600 is
observed (magenta arrow).
(F–I) The effects of the variants p.Ser840Pro and p.Ala892Asp were evaluated based on the crystal structure of the FBXO11 Zf-UBR
domain (PDB: 5VMD22).
(F) Ser840 is located in the UBR domain and coordinates a bridging water molecule (blue ball) that forms a total of three hydrogen bonds
(green lines).
(G) In the p.Ser840Pro variant, two of these hydrogen bonds are lost due to the lack of the respective hydrogens in proline and a steric
clash (magenta arrow) is caused by the cyclic proline sidechain.
(H and I) Ala892 (H) is located in the UBR domain and forms stabilizing hydrophobic interactions with Phe887 (green line), whereas in
the p.Ala892Asp variant (I), an unfavorable interaction is observed between the negatively charged Asp892 and the hydrophobic Phe887
(magenta arrow).

The American Journal of Human Genetics 103, 305–316, August 2, 2018 313
missense variant located between the two CASH domains
of Fbxo11,44 had chronic otitis media and conductive hear-
ing loss. Additionally, heterozygous mice were smaller
than wild-type littermates and displayed mild craniofacial
abnormalities,44,45 in agreement with growth anomalies
and facial dysmorphisms found in several of the herewith
reported individuals. Otitis media, however, was described
in only three individuals of the herewith reported group.
Another mouse model, Mutt, carrying a missense variant
between F-box and CASH domain (Figure 1B), showed
mild craniofacial anomalies but did not develop otitis me-
dia, thus pointing to a milder hypomorphic effect of that
mutation.44 Interestingly, both mouse models in a homo-
zygous state showed various degrees of perinatal lethality
and displayed facial clefting and cleft palate.44 Cleft palate
or a bifid uvula were observed in three of the individuals
reported here. These findings in humans support a role
of FBXO11 in the closure of the palatal shelves.46
To conclude, we identified de novo missense and
likely gene-disrupting variants in the E3-ubiquitin ligase
FBXO11 in individuals with a neurodevelopmental disor-
der comprising a variable degree of intellectual disability
and various other features.
Accession Numbers
Variants were submitted to LOVD (accession numbers: 00165141-
158).
Supplemental Data
Supplemental Data include one figure, one table, and Supple-
mental Acknowledgements and can be found with this article on-
line at https://doi.org/10.1016/j.ajhg.2018.07.003.
Acknowledgments
We thank all affected individuals and their families for participating
in this study. We thank Laila Distel for excellent technical assis-
tance. We thank Christian T. Thiel for the in-house NGS tool and
Arif Ekici and Steffen Uebe from the NGS facility in the Institute
of Human Genetics, Erlangen. This study makes use of data gener-
ated by the DECIPHER community (for additional information see
Supplemental Acknowledgements). Studies regarding individuals
18 and 11 were supported by grant PI14/00350 (Instituto de Salud
Carlos III -Acción Estratégica en Salud 2013–2016; FEDER -Fondo
Europeo de Desarrollo Regional) and by PARI 2012 from Regional
Council of Burgundy / Dijon University Hospital, respectively.
Exome sequencing for individual 7 was performed at the University
of Washington Center for Mendelian Genomics (UW-CMG) and
was funded by the NHRGI and the NHLBI (grant HG006493).
L.G.S. is supported by grants from the Health Research Council
and Cure Kids New Zealand. A. Rauch was supported by the ERA-
NET grant ‘‘Euromicro’’ (SNF 31ER30_154238). A. Reis was sup-
ported by the German Ministry of Education and Research
(01GS08160) and the Interdisciplinary Center for Clinical Research
(IZKF) Erlangen (E16). F.D. was supported by a grant of the German
Research Foundation (DFG) (DI 1731/2-1). T.B.H. is supported by
the German Federal Ministry of Education and Research (BMBF)
314 The American Journal of Human Genetics 103, 305–316, August 2, 2018
through the Juniorverbund in der Systemmedizin ‘‘mitOmics’’
(01ZX1405C). N. Matsumoto is supported by grants from AMED
(JP18ek0109280, JP18dm0107090, and JP18ek0109301). C.Z. is
supported by grants from the DFG (ZW184/1-2, ZW184/3-1, and
GRK2162) and by the IZKF Erlangen (E26). E.C. was supported by
the National Institute for Health Research (NIHR) Oxford Biomed-
ical Research Centre Programme.
Declaration of Interests
I.E.S. has served on scientific advisory boards for UCB, Eisai,
GlaxoSmithKline, Biomarin, and Nutricia; editorial boards of the
Annals of Neurology, Neurology, and Epileptic Disorders; may
accrue future revenue on pending patent WO61/010176 (filed:
2008): Therapeutic Compound; has received speaker honoraria
from GlaxoSmithKline, Athena Diagnostics, UCB, Eisai, and
Transgenomics; has received funding for travel from Athena Diag-
nostics, UCB, Biocodex, GlaxoSmithKline, Biomarin, and Eisai;
and receives/has received research support from the National
Health and Medical Research Council of Australia, National Insti-
tutes of Health, Australian Research Council, Health Research
Council of New Zealand, CURE, and March of Dimes.
All other authors declare no conflicts of interest.
Received: April 9, 2018
Accepted: June 29, 2018
Published: July 26, 2018
Web Resources
ExAC Browser, http://exac.broadinstitute.org/
GenBank, https://www.ncbi.nlm.nih.gov/genbank/
gnomAD Browser, http://gnomad.broadinstitute.org/
GTEx Portal, https://www.gtexportal.org/home/
LOVD, http://www.lovd.nl/3.0/home
OMIM, http://www.omim.org/
RCSB Protein Data Bank, http://www.rcsb.org/pdb/home/
home.do
UCSC Genome Browser, https://genome.ucsc.edu
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