Hum Genet DOI 10.1007/s00439-014-1498-1

ORIGINAL INVESTIGATION

De novo mutations in beta-catenin (CTNNB1) appear to be a frequent cause of intellectual disability: expanding the mutational and clinical spectrum Alma Kuechler · Marjolein H. Willemsen · Beate Albrecht · Carlos A. Bacino · Dennis W. Bartholomew · Hans van Bokhoven · Marie Jose H. van den Boogaard · Nuria Bramswig · Christian Büttner · Kirsten Cremer · Johanna Christina Czeschik · Hartmut Engels · Koen van Gassen · Elisabeth Graf · Mieke van Haelst · Weimin He · Jacob S. Hogue · Marlies Kempers · David Koolen · Glen Monroe · Sonja de Munnik · Matthew Pastore · André Reis · Miriam S. Reuter · David H. Tegay · Joris Veltman · Gepke Visser · Peter van Hasselt · Eric E. J. Smeets · Lisenka Vissers · Thomas Wieland · Willemijn Wissink · Helger Yntema · Alexander Michael Zink · Tim M. Strom · Hermann-Josef Lüdecke · Tjitske Kleefstra · Dagmar Wieczorek  Received: 15 August 2014 / Accepted: 3 October 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Recently, de novo heterozygous loss-of-function mutations in beta-catenin (CTNNB1) were described for the first time in four individuals with intellectual disability (ID), microcephaly, limited speech and (progressive) spasticity, and functional consequences of CTNNB1 deficiency were characterized in a mouse model. Beta-catenin is a key downstream component of the canonical Wnt signaling pathway. Somatic gain-of-function mutations have already been found in various tumor types, whereas germline loss-of-function T. Kleefstra and D. Wieczorek contributed equally. Electronic supplementary material  The online version of this article (doi:10.1007/s00439-014-1498-1) contains supplementary material, which is available to authorized users. A. Kuechler (*) · B. Albrecht · N. Bramswig · J. C. Czeschik · H.-J. Lüdecke · D. Wieczorek  Institut für Humangenetik, Universitätsklinikum Essen, Universität Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany e-mail: [email protected] M. H. Willemsen · H. van Bokhoven · M. Kempers · D. Koolen · S. de Munnik · J. Veltman · L. Vissers · W. Wissink · H. Yntema · T. Kleefstra  Department of Human Genetics, Radboud University Medical Centre, Nijmegen, The Netherlands C. A. Bacino · W. He  Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA D. W. Bartholomew · M. Pastore  Division of Molecular and Human Genetics, Department of Pediatrics, The Ohio State University/Nationwide Children’s Hospital, Columbus, OH, USA

mutations in animal models have been shown to influence neuronal development and maturation. We report on 16 additional individuals from 15 families in whom we newly identified de novo loss-of-function CTNNB1 mutations (six nonsense, five frameshift, one missense, two splice mutation, and one whole gene deletion). All patients have ID, motor delay and speech impairment (both mostly severe) and abnormal muscle tone (truncal hypotonia and distal hypertonia/spasticity). The craniofacial phenotype comprised microcephaly (typically −2 to −4 SD) in 12 of 16 and some overlapping facial features in all individuals (broad nasal tip, small alae nasi, long and/or flat philtrum, thin upper lip vermillion). With this detailed phenotypic characterization of 16 additional individuals, we expand and further establish the M. J. H. van den Boogaard · K. van Gassen · M. van Haelst · G. Monroe  Department of Medical Genetics, Utrecht Medical Centre, Utrecht, The Netherlands C. Büttner · A. Reis · M. S. Reuter  Institut für Humangenetik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany K. Cremer · H. Engels · A. M. Zink  Institute of Human Genetics, University of Bonn, Bonn, Germany E. Graf · T. Wieland · T. M. Strom  Institut für Humangenetik, Helmholtz Zentrum München, Neuherberg, Germany J. S. Hogue  Department of Pediatrics, Madigan Army Medical Center, Tacoma, WA, USA

13



clinical and mutational spectrum of inactivating CTNNB1 mutations and thereby clinically delineate this new CTNNB1 haploinsufficiency syndrome.

Introduction Intellectual disability (ID, IQ 100×. Data analysis and interpretation were performed using Mercury 1.0, SIFT and PolyPhen-2. For patients 12 and 14, enrichment was performed using Agilent Sureselect XT All Exon V5 kit and sequenced on an Illumina HiSeq 2500 instrument at the Utrecht DNA Sequencing Facility (Utrecht, Netherlands). Reads were aligned using BWA and data were processed with GATK v3.1.1 (McKenna et al. 2010), according to best practice guidelines (Van der Auwera et al. 2013). Obtained coverage was 107× with 92.9 % covered >20×. Variant analysis was performed using the commercially available tool Cartagenia Bench Lab, filtering upon Mendelian violations to find de novo variants and prioritizing the variants using a classification tree, according to presence in public and clinical relevant variant databases, location, coding effect, and corresponding literature. We used PolyPhen-2 (http://genetics.bwh.harvard.edu/ pph2/), SIFT (http://sift.bii.a-star.edu.sg) and MutationTaster (http://www.mutationtaster.org/) to predict the possible impact of a missense mutation on the structure and function of the protein. Sanger sequencing Sanger sequencing of CTNNB1 was performed on whole blood genomic DNA in the patients and their parents to verify the mutations and their de novo status. Primer sequences are available upon request. CTNNB1 reference sequence was NM_001904.3. For patient 16, a whole genome array analysis was performed at a commercial laboratory (Quest Diagnostics, Nichols Institute, Chantilly, Virginia, USA) using an Affymetrix 6.0 array according to standard protocols. De novo occurrence of the detected aberration was investigated by parental array analysis.

Results Clinical reports and identification of CTNNB1 mutations The main clinical findings of the 13 novel patients are summarized in the following case reports; additional detailed information and comparison with so far published patients (de Ligt et al. 2012; Tucci et al. 2014; Dubruc et al. 2014) are provided in Supplementary Table S1. Mutational data are summarized in Table 2. Patient 1 (see Fig. 1a, b) is the second son of healthy non-consanguineous parents. During pregnancy, a single umbilical artery was diagnosed by ultrasonography. He was born by primary Cesarean section with normal weight and length but primary microcephaly (−2.79 SD, see Supplementary Table S1). After a normal neonatal period, hypotonia and delayed psychomotor development became obvious (unsupported walking from approximately 8 years on). At 6 months of age, he was diagnosed with strabismus and later on with hyperopia (+7/+6.5 dpt). He suffered from frequent respiratory infections. Brain MRI was normal. Because of hypertonia of the legs, an MRI scan of the spine was performed at age 3¾ years that revealed a syringomyelia (TH8L1). Upon clinical examination at age 4 years, height and weight were normal; but OFC had decreased to −4.0 SD. Apart from microcephaly, he presented with some craniofacial dysmorphism (hypotelorism, long and flat philtrum, thin upper lip vermillion and strabismus). His hands were broad with short distal phalanges; his feet displayed a pes cavus with a discrete 2–4 soft tissue syndactyly. At re-evaluation at age 8½ years he was able to walk without support (broad-based gait) and was toilet trained during the day. He could speak only few single words but had better speech comprehension and communicated using gestures. WES detected a de novo frameshift mutation in exon 12 [c.1923dupA, p.(Glu642Argfs*6)]. Patient 2 (see Fig. 1c, d) is a 5-year-old girl, first child of healthy non-consanguineous parents from Bangladesh with unremarkable family history. She was born spontaneously with normal birth weight and length but small OFC (−2.1 SD; see Supplementary Table S1). Postnatal course was normal. Neonatal jaundice required phototherapy for 2 days. She developed hypotonia and feeding difficulties (poor sucking, no breast feeding possible), and a severe psychomotor delay soon became obvious. She gained head control at age 8–10 months, started sitting at 18 months, crawling at 23 months and pulling herself to stand at 33 months. Upon several clinical followup examinations her length and weight remained in the low normal range but her OFC dropped down further to about −3 SD. She had no obvious craniofacial dysmorphism (see Fig. 1c, d) apart from a broad nasal tip and a flat occiput. Fingers were tapered. At age 53/12 years, she

13



Hum Genet

Fig. 1  Facial phenotypes of individuals with CTNNB1 mutation. Patient 1 at the age of 9 months (a) and 8½ years (b), patient 2 at 1 year (c) and at 3 years (d), patient 3 at 2½ years (e), patient 4 at 6 years (f) and at 14 years (g), patient 5 at 5 years (h), patients 6 and 7 (siblings) at 4 and 2 years (i, j), patient 9 at 14 months (k) and at 2 years (l), patient 10 at 4 years (m), patient 11 at 4 years (n), patient 12 at 13 years (o), patient 13 at 6 years (p), patient 14 at 14 months

(q) and 3 years (r), patient 15 at 2 years (s), and patient 16 at 3 years of age (t). Photographs of patients 8 were unavailable for publication. All individuals share some craniofacial features—a broad nasal tip with small alae nasi, a long or flat philtrum and a thin upper lip vermillion. In older individuals, the nose appeared longer and the columella more prominent

still could not walk without support, had hypotonia of the trunk and hypertonia of the legs. She showed some stereotypic movements (smacking her lips, protruding her tongue), had lost the ability to speak few single words or syllables, and had poor speech comprehension. Feeding problems had remained; she hardly felt hungry, did not chew or drink properly, had frequent vomiting and suffered from constipation. She had no contact to other children and no interest in toys. She suffered from intermittent sleep disturbances. Her mood was generally friendly but she had occasional outbursts of temper tantrums and crying that were difficult to interrupt. She had no seizures; EEGs were normal as well as a brain MRI (at age 3 years), visual-evoked potentials (VEP), echocardiography, and

otoacoustic emissions (OAEs). She wears glasses because of a hyperopia (+7/+6 dpt). WES identified a de novo heterozygous CTNNB1 nonsense mutation c.1420C>T, p.(Arg474*) in Exon 9. Patient 3 (see Fig. 1e) is the second child of healthy non-consanguineous Turkish parents. During pregnancy, a cardiac “white spot” was detected by ultrasonography. The girl was born with normal measurements (see Supplementary Table S1); postnatal course was normal. Normal breastfeeding was possible in spite of hypotonia. Starting at age 2 months she suffered from skin problems (diaper rash and later eczema). At age 5–6 months, a motor delay became obvious and physiotherapy was started. She was able to turn over at 1½ years but could not sit or walk

13

Hum Genet

without support at age 25/12 years. Speech development was also delayed (no words, only syllables). She showed behavioral abnormalities with outburst of temper tantrums or crying and self-biting. A hip dysplasia and peripheral hypertonia with a spastic component especially in the legs were diagnosed. MRI scans of the brain and the spine (at age 1½ years) were normal apart from a lumbosacral epidural lipoma as an additional finding. EEG, EMG, NCV, ECG, echocardiography and metabolic screening gave normal results. Clinical examination at age 25/12 years revealed microcephaly (−2.2 SD), but normal length and weight (see Supplementary Table S1). She had a slender build and a pectus excavatum. There was little eye contact. Craniofacial findings were small alae nasi, a broad nasal tip, a long philtrum, small upper lip vermillion, deep-set ears, and micrognathia (see Fig. 1e). WES identified a de novo heterozygous CTNNB1 splice mutation (c.1683+1G>A, Intron 10). Patient 4 (see Fig. 1f, g) is the third child of healthy nonconsanguineous Vietnamese parents. She was born after an uneventful pregnancy with normal birth measurements (see Supplementary Table S1). At 3 months of age, motor delay and at 1 year, speech delay were noticed. She had hypotonia of the trunk and hypertonia and dystonic movements of the extremities. Talipes deformities were treated with orthopedic shoes. She learned to walk independently at age 10 years, but could only walk short distances on her tiptoes. She used 2 words and no signs. She developed autoaggressive behavior with self-injuries (biting her hands), showed some stereotypic movements and held only short eye contact. Sleep disturbances were treated with Risperidone. Clinical examination at the age of 28/12 years showed mild microcephaly (−2.1 SD) that was no longer present at reevaluations at the age of 15 years. The patient had strabismus, a flat midface, small alae and broad tip of the nose, a long, flat philtrum (see Fig. 1f, g), a high arched palate and small, low set ears. Metabolic screenings, neurotransmitters, EEG, MRI and CDG investigations were normal. WES revealed a heterozygous de novo CTNNB1 nonsense mutation c.755T>AAC, p.(Leu251*) in Exon 6. Patient 5 (see Fig. 1h) is the third child of healthy, nonconsanguineous German parents. Pregnancy and birth were normal (see Supplementary Table S1). Microcephaly developed within the first year of life. Motor delay was diagnosed at 1 month and speech impairment at 1 year of age. Assisted walking and first words were achieved at age 4 years. She performed repetitive movements and had sleep disturbances in early childhood. Clinical examination at the age of 57/12 years showed microcephaly (−2.94 SD), a long face, small alae nasi, a broad nasal tip, long and smooth philtrum, a thin upper lip (see Fig. 1h) and diastema. She was hypotonic, had an ataxic gait, wore orthopedic shoes and ambulated with assistance of a walker frame. She

communicated by sign language and used about 30 words. MRI of the brain and spinal cord (at age 4 years), EEGs and metabolic screening were normal. WES showed a de novo frameshift mutation c.423_424insG, p.(Tyr142Valfs*4). Patient 6 (see Fig. 1i, sister of patient 7), a 13-year-old girl, is the first child of healthy non-consanguineous parents of mixed European (Italian/Irish) and Puerto Rican ancestry. The family history was unremarkable. She was born by Caesarian section with normal measurements by report (length and OFC not exactly documented). There were no significant perinatal or neonatal issues. She was sitting independently by 12 months, walking but not using any words by 18 months at which time she was diagnosed with global developmental delay, and speech, physical and occupational therapies were initiated. At the age of 3 years she was diagnosed with cerebral palsy due to mixed central hypotonia and peripheral hypertonia with heel cord tightness and toe walking and was referred for initial Genetics evaluation. Her height, weight and OFC were all normal. She had mild dysmorphic features including upslanting palpebral fissures, a boxy nasal tip, thin upper lip, long philtrum, narrow palate, small chin and prominent fingertip pads. At 6 years of age she was diagnosed with ADHD and anxiety and placed on a number of stimulant medications and serotonin-reuptake inhibitors with minimal improvement. At 7 years of age she was diagnosed with scoliosis and a tethered spinal cord and underwent a dorsal spine rhizotomy with some improvement of spasticity in her lower extremities. At last evaluation she was 13-year-old and carried diagnoses of mild ID (with full-scale IQ scores ranging from 46 to 66), autism, ADHD, anxiety, strabismus, myopia and tics. She had initiated normal menarche at 12 years of age and had normal pubertal development. Her parents noted increased behavioral difficulties including frustration, aggression, opposition and occasional self-injury but she was communicative and social. Her facial phenotype remained essentially unchanged. Her height and OFC were in the normal range and BMI above the 97th percentile (see Supplementary Table S1). Metabolic testing (lactic acid, ammonia, acylcarnitine profile and VLCFAs) and brain imaging by CT and MRI were normal. Patient 7 (see Fig. 1j), a 10-year-old boy, is the full sibling of patient 6 and the second and only other child of these parents. He was born by vacuum-assisted vaginal delivery with normal measurements (exact birth length and OFC not documented). He remained in the NICU for one day for respiratory distress which resolved spontaneously. He was diagnosed shortly after birth with a PDA. A bilateral esotropia was treated surgically at 7 months of age. He was sitting independently and using words by 12 months, crawling at 17 months, but not walking until 24 months and carried a diagnosis of cerebral palsy since 12 months of

13



age due to mixed central hypotonia and peripheral hypertonia with tight heel cords at which time he began receiving physical therapy. He was referred for initial genetics evaluation at the age of 13 months along with his sister. His height and weight were normal and his OFC mildly microcephalic (−2.2 SD). He had mild dysmorphic features including dolichocephaly, upslanting palpebral fissures, a boxy nasal tip, thin upper lip, long philtrum, and a small chin (see Fig. 1j). Subsequently at the age of 3 years he was diagnosed with ADHD and placed on a number of stimulant medications with some improvement. He was also diagnosed with tethered spinal cord and underwent dorsal spine rhizotomy at 3 years of age but had little improvement of his lower extremity spasticity. At last evaluation, he was 10 years of age and carried diagnoses of borderline ID, cerebral palsy, ADHD, microcephaly and hypo-/oligodontia. His parents noted increased difficulty with progressive lower extremity spasticity despite treatment with Botox, bracing and an intrathecal Baclofen pump. He was communicative and social. His weight and height remained normal and his OFC microcephalic (−3.2 SD, see Supplementary Table S1). CPK and cholesterol levels, metabolic testing (lactic acid, ammonia, acylcarnitine profile, uric acid level, glycosylated transferrin levels and VLCFAs) and brain imaging by MRI were normal. Both siblings, clearly borderline to mildly mentally impaired, were able to speak in sentences enjoying social/ verbal interaction with somewhat simple speech display but good receptive understanding. WES revealed that both siblings carried a heterozygous CTNNB1 nonsense mutation c.2038_2041dupAGCT, p.(Ser681*). Neither parent was found to carry this mutation (in blood), suggesting parental germline mosaicism. In addition, Patient 7 was found to carry a maternally inherited, heterozygous non-synonymous WNT10A mutation, known to cause ectodermal dysplasia and believed to account for this patient’s hypo-/oligodontia (OMIM*606268). Patient 8 (no photograph shown) is a 15-month-old male, first child of healthy non-consanguineous parents, originating from Italy and the Philippines, respectively. In the 5th month of pregnancy, reduced head growth and enlarged ventricles were detected by ultrasound. Amniocentesis revealed a normal male karyotype. The boy was born spontaneously with normal weight and length but mild microcephaly (−2.23 SD, see Supplementary Table S1). In the neonatal period, feeding difficulties and hypotonia emerged. At age 2 months, neonatal seizures were noted, but postictal EEG examinations were normal. He was evaluated at age 3.5 months because of developmental delay, progressive microcephaly (OFC −3.3 SD), hypotonia of the trunk, hypertonia of the arms and legs, and convergent strabismus. Hearing was normal. A

13

Hum Genet

brain MRI (shown in Supplementary Figure S1) revealed enlarged lateral ventricles, dysgenesis of the corpus callosum, abnormal gyration of the temporal lobe, absence of the right fornix and a hypoplastic brain stem. When last seen at age 15 months, his OFC had dropped to −4.8 SD. He vocalized only a few syllables, did not show sufficient head control, nor could he grasp objects or sit freely. Mild craniofacial dysmorphisms (hypotelorism, upslanting palpebral fissures, epicanthus, long, flat philtrum, thin upper lip, and prominent occiput) were noted. Metabolic screening was normal. Molecular panel analysis resulted in the identification of a single mutation in a gene matching the patient’s phenotype, a c.1251_1252insACGTG, p.(Cys419*) de novo nonsense mutation in Exon 9 of the CTNNB1 gene. Patient 9 (see Fig. 1k, l) was born as the fourth child of healthy non-consanguineous parents with normal birth weight and length; OFC was not reported. Since birth he showed a high muscle tone in his arms and legs. Upon neurological examination at the age of 13 months he had a significant hypertonia of his upper and lower extremities and axial hypotonia. Deep tendon reflexes were increased. He cried a lot and the parents noticed that his development was delayed as compared to his three healthy sisters. During the first year of life, he had mild feeding problems, including difficulties to swallow, chew and eat solid food. At the age of 8 months he started to roll over from his abdomen to his back. He did not crawl. Until the age of 18 months he was not able to sit independently. Since the age of 19/12 years he was able to walk a few steps with a walking aid. His comprehensive language developed much better than his expressive language. He mainly used non-verbal communication. At the age of 2 years, he was able to use a few simple words. Social interaction and eye contact were adequate. He had sleeping problems, mainly including difficulties to fall asleep. His general medical condition was good. During a period of fever he possibly had one seizure. Brain MRI showed delayed myelination in the frontal lobes. At the age of 2 years he had normal height, weight and head circumference (see Supplementary Table S1). He had mild facial dysmorphism including a prominent metopic ridge, upslanting palpebral fissures, full nasal tip, long philtrum, thin upper lip, high palate and pointed chin. In addition, he had deep palmar creases, a mild clinodactyly of the fifth fingers and a medial fatpad on his feet. A metabolic screen in blood and urine revealed mild unspecific abnormalities. These findings could not be related to his phenotype and he was included in family-based whole exome sequencing studies, which revealed a de novo nonsense mutation in CTNNB1: c.1420C>T; p.(Arg474*). Patient 10 (see Fig. 1m) was first seen at 11 months of age. At delivery, a single umbilical artery was noted. Birth measurements were normal. At 6 months of age, a

Hum Genet

low head circumference was reported. At age 12 months, she could army crawl but not sit up, and had no language. Parents described her as being “stiff”. She showed early fisting, right esotropia, cutis marmorata and a somewhat hoarse voice. She began walking with a walker device at about 20 months of age, but always on her toes with ankles almost fixed in extension. She started unsupported walking at age 4 years but falls a lot and has a very abnormal toe-walking gait. Eventually, tizanidine was tried but was withdrawn due to a seizure. She is not particularly dysmorphic but remains speech delayed (~20 words). There was a history of some self-destructive behavior (poking with fingers, biting, head-banging) that has now improved. EEGs were normal as were two cranial MRI scans (a subtle area of abnormal signal in the inferior right basal ganglia/medial right temporal lobe was of unknown significance and finally interpreted as normal). Biochemical studies were normal. WES revealed a nonsense mutation c.283C>T p.(Arg95*) in CTNNB1. Patient 11 (see Fig. 1n) is an 8-year-old male who presented initially to the genetics consult at 28 months of age. He has a long-standing history of global developmental retardation with delayed acquisition of speech and delayed motor development involving fine and gross motor areas. He sat up at 13 months, crawled at 18 months and walked at 30 months. He also had a history of swallowing issues probably due to texture aversions. His food needed to be blended early on although it improved with time and therapy. At the time of the first exam his head was microcephalic (−3.6 SD). A follow-up at 4 years continued to show microcephaly (−3.3 SD). On exam, his ears appeared prominent and protuberant but measured at the 50th centile for length so likely due to the microcephaly. He had a triangular face appearance, deep-set eyes, small nares and prominent columella. He never had seizures but an EEG at age 7 years was abnormal (with normal alpha waves but presence of high voltage slow waves in clumps over the right hemisphere with tendency to paroxysms of 1–2 s) which was interpreted as epileptiform activity with tendency to spread. WES detected a de novo missense mutation in CTNNB1, c.1163T>C, p.(Leu388Pro), which was predicted to be “disease causing” with a score of 1 (MutationTaster), “probably damaging” with a score of 1.000 (PolyPhen-2), and to affect protein function with a score of 0.00 (SIFT). Patient 12 (see Fig. 1o) is a now 13 year-old girl, first child of healthy, non-consanguineous Dutch parents. Family history is unremarkable and a younger sister is healthy. Pregnancy and birth were normal. Her motor and intellectual development is delayed since birth. At the age of 1 year she had generalized pyramidal symptoms which were most prominent on the distal legs, and strabismus

alternans. Assisted walking, first words and toilet training were achieved at around 6 years. She spoke first words at age 6 years and now speaks in sentences with a rather hoarse voice. She never had seizures and generally sleeps well, but has sometimes laughing periods at night. From the age of 8 years she had behavioral problems with rages/ tantrums. Clinical examination at the age of 118/12 years showed a normal head circumference. She had amblyopia of her right eye (probably due to congenital strabismus), a short philtrum and widely spaced teeth. She had long, slender fingers, long toes with a sandal gap, showed pyramidal symptoms of her legs, wore orthopedic shoes and used a walker frame. She had a very friendly personality, a very short attention span and made poor eye contact. She was recently diagnosed with autism by a psychiatrist. Cranial MRIs (at 13 months and 12 years) showed no abnormalities. Metabolic investigations were all normal, apart from a transient elevation of phytanic acid at the age of 4–5 years (22.3 µmol/l, normal reference C in Intron7 of the CTNNB1. Patient 15 (see Fig. 1s) is the first child of healthy parents with unremarkable family history. During pregnancy, an intrauterine growth retardation and breech position were diagnosed. She was born by Caesarian section with low birth weight and normal length; OFC was not reported. Development was initially normal, but from 2 to 3 months of age, she showed a motor delay and later on a stagnation and regression including loss of words and few two-word sentences. She was not hypotonic and never crawled. She had truncal ataxia, dysmetric eye–hand coordination and hypertonia and spasticity of the lower limbs with abnormal postural development but slowly progressing at her own pace until now. At 2 years of age she had a social adaptation of 1 year and a motor developmental level of 7 months. She was microcephalic (−2.5 SD), had thin hair, a square face, deep-set eyes, a stubby nose with broad nasal tip, and a thin upper lip vermillion Her eye movements were not always well coordinated with intermittent strabismus. She babbles now (aged 33/12 years) constantly and some words

13

Hum Genet

are understandable. She is very happy and friendly, but can show a low frustration tolerance. Extensive metabolic screening of urine and plasma was normal. WES analysis revealed a de novo frameshift mutation in CTNNB1:c.1272_1275delTTCT, p.(Ser425Thrfs*11). Patient 16 (see Fig. 1t) was born after complicated pregnancy (chronic hypertension and recognition of intrauterine growth retardation during the third trimester) with normal length but low weight and OFC (see Supplementary Table S1). At about 3 months he was noted to have persistently clenched hands and poor head control. Around 1 year of life he was first identified as having increased tone in all extremities. This has not worsened since that time but he has been noted to have dystonic posturing of the upper extremities as well. He started rolling over around 2 years and standing with assistance shortly thereafter. He did not sit on his own, but walked with assistance up on his toes at 2½ years. He started to babble and say “mama” and “dada” specifically shortly before age 3 years. He had no other intelligible words but could follow simple commands and point to a few body parts. He had a generally happy demeanor. His general health was otherwise good without any history of major illnesses, hospitalizations, or surgeries. On physical exam at 3 years, he was mildly short and had a significant microcephaly (−4.09 SD, see Supplementary Table S1). He made good eye contact and smiled responsively. He had right esotropia, a right frontal hair upsweep and a double posterior hair whorl. His ears were low set, posteriorly rotated, and had a hypoplastic upper crus of the inner helix. His philtrum was mildly/slightly flat and his upper vermillion was thin. His thumbs were adducted at rest and he occasionally displayed dystonic posturing of the arms with attempts to reach for objects. He had mildly decreased tone in the trunk and increased tone that is more notable in the lower extremities with scissoring of the legs. Previous studies included plasma amino acids, acylcarnitine profile, uric acid, and alpha-fetoprotein. Brain MRI showed mild thinning of the corpus callosum and was otherwise normal. Chromosome microarray analysis revealed a 505 kb deletion at 3p22.1 (arr[hg19] 3p22.1(41,209,868-41,714,118)×1) including the entire CTNNB1 and part of ULK4 genes. Parental array analysis was normal confirming that the deletion was de novo.

Discussion Clinical characterization of CTNNB1-related syndromic ID With the availability of next generation sequencing techniques, inactivating de novo CTNNB1 (beta-catenin 1,

Hum Genet

OMIM *116806) mutations have been recognized for the first time as cause of syndromic ID (de Ligt et al. 2012). In this study, we present 16 additional individuals (eight males, eight females) from 15 families with previously unexplained ID plus additional findings, in whom we identified heterozygous de novo inactivating CTNNB1 mutations. When comparing the clinical findings of all 21 patients with CTNNB1 mutations (19 with mutations, 2 with deletions) known so far, consistent phenotypic features emerge (see Table 1 and Supplementary Table S1). The neurological phenotype was the most striking consistent finding in all 21 patients. Hypotonia and delayed motor milestones were observed from early infancy. While hypotonia of the trunk remained, a distal hypertonia (arms and legs) with spasticity especially of the legs developed, leading to an impaired walking capability. Eight out of 20 patients (one is still too young) achieved the ability to walk independently, but often severely delayed (at about 4–5 years or even at 10 years of age); only one patient started unsupported walking at age 18 months. Gait was frequently described as unsteady, ataxic or spastic. Another consistent finding was severe speech impairment (no or only single words) in eleven out of 20 patients (55 %); nine patients (45 %) had a delayed but mild-tomoderately affected speech being able to communicate in sentences. ID was present in all patients, ranging from mild to severe. Some kind of regression (e.g., loss of some already acquired words or fine motor skills) was noticed in about one-third of individuals. Seven out of 14 patients (50 %) had a primary microcephaly (ranging from −2.1 to −4.1 SD). Postnatal microcephaly was a frequent finding (present in about 81 % (17 of 21), ranging from −2.2 to −4.8 SD). In addition, all 21 affected individuals had some overlapping craniofacial features, including a broad nasal tip with small alae nasi, a long or flat philtrum and a thin upper lip vermillion. In older individuals, the nose appeared longer and the columella more prominent. It is of note that no seizures were observed so far (apart from neonatal seizure in patient 8) although beta-catenin has been implicated in epilepsy (Campos et al. 2004). The majority of patients (15/20; 75 %) had some abnormalities of vision—strabismus and/or hyperopia or myopia. Twelve out of 21 patients (57 %) showed behavioral abnormalities; temper tantrums, autistic features, aggressive or auto-aggressive behavior or sleep disturbances were the most frequent findings. Cranial MRI scans were performed in 18 out of 21 patients showing normal results in 13 individuals (72 %). Minor changes such as corpus callosum thinning or mildly enlarged ventricles were observed in three (17 %), delayed myelination of frontal lobes in one and structural

abnormalities in another patient (patient 8, see text, Supplementary Table S1 and Figure S1). In three patients, spinal MRI abnormalities (syringomyelia or tethered cord) were described. Mutational spectrum All 18 intragenic mutations (our study, de Ligt et al. 2012, Tucci et al. 2014, summarized in Table 2) were inactivating mutations—including eight nonsense mutations, seven frameshift mutations, two splice mutation, and one missense mutation predicted to be probably damaging. For two of the initial patients, Tucci et al. could show that the mutations lead to nonsense-mediated mRNA decay. The finding that CTNNB1 haploinsufficiency is responsible for the phenotype is supported by two individuals with different overlapping deletions affecting the entire CTNNB1 gene (our patient 16 and the patient published by Dubruc et al. 2014), representing a clinically similar phenotype to patients with intragenic mutations. A third deletion of 437 kb in size, affecting the entire CTNNB1 gene, is listed in the DECIPHER database (Firth et al. 2009; Decipher ID #275677), and was identified in a patient with abnormalities of higher mental function, behavioral/psychiatric abnormalities, and muscular dystrophy. In all three individuals, the deletions (see Fig. 2a) also comprised parts of the neighboring gene ULK4 (unc51 like kinase 4), one of five members of the unc-51-like serine/threonine kinase (STK) family. Little is known so far about ULK4 function and there is no OMIM entry until now. Null mice with targeted deletion of Ulk4 were recently described to develop congenital hydrocephalus, and their respiratory epithelia and ependymal cells had shorter cilia than normal, indicating ciliopathies (Vogel et al. 2012). Neither our patient 16 nor the patient described by Dubruc et al. (2014), have clinical manifestations consistent with a ciliopathy. In a recent study, ULK4 was reported as a rare susceptibility gene for schizophrenia, based on the finding of recurrent rare intragenic ULK4 deletions in patients with schizophrenia and other psychiatric disorders (Lang et al. 2014). Since Ulk4−/− mice were found to have partial agenesis of the corpus callosum, this gene might be crucial to brain development (Lang et al. 2014). It remains unclear whether the heterozygous partial ULK4 deletions contribute to the clinical phenotype which is dominated by CTNNB1 haploinsufficiency. The characteristic structural components of CTNNB1 are 12 armadillo repeats (ARMs 1–12) facilitating interactions with multiple protein partners such as cadherin (Xing et al. 2008). The mutations identified so far are scattered throughout the entire coding region of CTNNB1, most of them localized in ARM 1–ARM 12 (see Fig. 2b).

13



Hum Genet

Table 1  Summary of the main clinical features in patients with CTNNB1 mutation Clinical features in patients with CTNNB1 mutations (not every feature is documented for all patients)

16 Novel patients (from 15 families)

5 Previously published patients (de Ligt et al., Tucci et al., Dubruc et al.)

Summary (n = 21)

Gender

8 females 8 males 13/12–154/12

4 females 1 males

12 females 9 males

Age range (years) Birth weight Birth length Primary microcephaly Height Weight/BMI

Microcephaly Craniofacial dysmorphism Truncal hypotonia Peripheral hypertonia/spasticity Motor delay

Free walking ability Speech impairment Basic speech comprehension Intellectual disability Regression Behavioral anomalies Seizures Brain MRI anomalies Hearing loss Visual defects CTNNB1 mutation

14 normal 2 C chr3:g.41,277,961_41,277,962delAG

c.2038_2041dupAGCT c.1251_1252insACGTG c.1420C>T c.283C>T c.1163T>C c.1925_1926delAG

Pat 13

chr3:g.41,266,102_41,266,103delTG

c.99_100delTG

Pat 14 Pat 15

chr3:g.41,268,844G>C chr3:d.41,275,106_41,275,109del

c.1081+1G>C c.1272_1275delTTCT

Pat 16 Publ. pat. 1 (“3rd p”)a, b Publ. pat. 2 (P70)a, b

chr3:g.41,209,868_41,714,118del whole gene deletion chr3:g.41,275,648C>T chr3:g.41,275,106_41,275,109delTTCT

c.1543C>T c.1272_1275delTTCT

Publ. pat. 3 (“2nd p”)a, b Publ. pat. 4b

chr3:g.41,267,341C>T chr3:g.41,267,034dupA

c.925C>T c.705dupA

Publ. pat. 5c

chr3:g.41,104,508_41,437,397del whole gene deletion

p.(Tyr142Valfs*4) p.(Ser681*) p.(Cys419*) p.(Arg474*) p.(Arg95*) p.(Leu388Pro) p.(Glu642Valfs*5) p.(Gly34Asnfs*15) splice mutation p.(Ser425Thrfs*11) p.(Arg515*) p.(Ser425Thrfs*11) p.(Gln309*) p.(Gly236Argfs*35)

9 Intron 10 6 4 13 9 9 4 8 12 3 Intron 7 9 10 9 6 5

Genomic, cDNA and protein positions are given as well as affected exons. All genomic positions are based on GRCh37/hg19; CTNNB1 reference sequence is NM_001904.3 a

  Published by de Ligt et al. (2012)

b

  Published by Tucci et al. (2014)

c

  Published by Dubruc et al. (2014)

seven out of 21 individuals also show some autistic features (see Supplementary Table S1). Further functional characterization of a CTNNB1 mutation was performed by Tucci et al. (2014) who used a mouse mutant (‘batface’, Bfc) carrying a heterozygous Thr653Lys mutation in the C-terminal armadillo repeat of the protein (see Fig. 2). They carried out morphological, behavioral, molecular and physiological investigations and compared the murine with the human phenotype. Mutant mice showed craniofacial abnormalities (shortened anteroposterior axis, broad face, and shortened nasal length), as well as morphologic brain changes, such as reduced cerebellar and olfactory bulb size and underdeveloped corpus callosum which was also observed in some of the affected human individuals. Mutant mice also demonstrated behavioral and cognitive abnormalities, motor deficits, and decreased vocalization complexity, the latter possibly being comparable to impaired speech ability in affected humans. Beta-catenin was also found to be critical for dendritic morphogenesis. Yu and Malenka (2003) showed that increasing intracellular levels of beta-catenin and other members of the cadherin/catenin complex enhance dendritic arborization in rat hippocampal neurons.

Beta-catenin is a key downstream component of the canonical Wnt signaling pathway. Somatic gain-offunction mutations in CTNNB1 have already been identified in various tumor types (e.g., colorectal cancer, hepatocellular carcinoma, ovarian cancer and pilomatricoma). In none of the patients with inactivating mutations, tumor manifestations have been observed and are unlikely to be expected due to the converse mechanism of haploinsufficiency.

Conclusion The clinical features of individuals with inactivating CTNNB1 mutations constitute a distinct syndromic phenotype that is characterized by ID, significant motor delay with hypotonia of the trunk and (progressive) distal hypertonia/spasticity of the legs, speech impairment, behavioral anomalies, frequent microcephaly and overlapping facial features. With this detailed clinical characterization of 16 newly identified individuals we delineated and further characterized the clinical features of the novel CTNNB1-related ID syndrome, thereby quadrupling the number of reported

13



Hum Genet

Fig. 2  Localization of deletions and mutations affecting CTNNB1. a Localization of the CTNNB1 deletion detected in our patient 16 compared to the deletions in the patient published by Dubruc et al. (2014), and the DECIPHER patient 275677. All three deletions affect the entire CTNNB1 gene. b Schematic structure of CTNNB1 showing the approximate positions of the mutations in relation to the 12 arma-

dillo repeats (gray boxes). Above the scheme, the newly identified mutations are marked in black and the published mutations in gray (pP1-4 correspond to patients 1–4 published by Tucci et al. 2014). Below the scheme, the mouse mutation investigated by Tucci et al. is depicted as well as the two mutations published in 2012 by O’Roak et al. in patients with autism without further clinical data

patients. Based on the number of patients that we identified in our cohorts we assume that this new CTNNB1 haploinsufficiency syndrome might actually be a relatively frequent cause of ID.

de Ligt J, Willemsen MH, van Bon BW, Kleefstra T, Yntema HG, Kroes T, Vulto-van Silfhout AT, Koolen DA, de Vries P, Gilissen C, del Rosario M, Hoischen A, Scheffer H, de Vries BB, Brunner HG, Veltman JA, Vissers LE (2012) Diagnostic exome sequencing in persons with severe intellectual disability. N Engl J Med 367:1921–1929 Dubruc E, Putoux A, Labalme A, Rougeot C, Sanlaville D, Edery P (2014) A new intellectual disability syndrome caused by CTNNB1 haploinsufficiency. Am J Med Genet A 164:1571–1575 Firth HV, Richards SM, Bevan AP, Clayton S, Corpas M, Rajan D, Van Vooren S, Moreau Y, Pettett RM, Carter NP (2009) DECIPHER: database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources. Am J Hum Genet 84:524–533 Flore LA, Milunsky JM (2012) Updates in the genetic evaluation of the child with global developmental delay or intellectual disability. Semin Pediatr Neurol 19:173–180 Kuechler A, Zink AM, Wieland T, Lüdecke H-J, Cremer K, Salviati L, Magini P, Najafi K, Zweier C, Czeschik JC, Aretz S, Endele S, Tamburrino F, Pinato C, Clementi M, Gundlach J, Maylahn C, Mazzanti L, Wohlleber E, Schwarzmayr T, Kariminejad R, Schlessinger A, Wieczorek D, Strom TM, Novarino G, Engels H (2014) Loss-of-function variants of SETD5 cause intellectual disability and the core phenotype of microdeletion 3p25.3 syndrome. Eur J Hum Genet. doi:10.1038/ejhg.2014.165 Lang B, Pu J, Hunter I, Liu M, Martin-Granados C, Reilly TJ, Gao GD, Guan ZL, Li WD, Shi YY, He G, He L, Stefánsson H, St Clair D, Blackwood DH, McCaig CD, Shen S (2014) Recurrent

Acknowledgments  We are grateful to the patients and their families for participating in this study and for giving consent to publish data and photographs. We thank Sabine Kaya and Daniela Falkenstein for excellent technical assistance. This work was supported by grants of The Netherlands Organization for Health Research and Development (ZonMw grant 907-00-365 to TK) and the European Union under the 7th framework program (Gencodys HEALTH-F4-2010-241995 to HvB and TK). Conflict of interest  The authors declare that they have no competing interests. The views expressed are those of the author and do not reflect the official policy of the Department of the Army, the Department of Defense or the U. S. Government.

References Campos VE, Du M, Li Y (2004) Increased seizure susceptibility and cortical malformation in β-catenin mutant mice. Biochem Biophys Res Commun 320(2):606–614

13

Hum Genet deletions of ULK4 in schizophrenia: a gene crucial for neuritogenesis and neuronal motility. J Cell Sci 127:630–640 Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics 25:1754–1760 McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20(9):1297–1303 O’Roak BJ et al (2012a) Multiplex targeted sequencing identifies recurrently mutated genes in autism spectrum disorders. Science 338(6114):1619–1622 O’Roak BJ et al (2012b) Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. Nature 485(7397):246–250 Rauch A, Wieczorek D, Graf E et al (2012) Range of genetic mutations associated with severe non-syndromic sporadic intellectual disability: an exome sequencing study. Lancet 380:1674–1682 Ropers HH (2010) Genetics of early onset cognitive impairment. Annu Rev Genomics Hum Genet 11:161–187 Tucci V, Kleefstra T, Hardy A, Heise I, Maggi S, Willemsen MH, Hilton H, Esapa C, Simon M, Buenavista MT, McGuffin LJ, Vizor L, Dodero L, Tsaftaris S, Romero R, Nillesen WN, Vissers LE, Kempers MJ, Vulto-van Silfhout AT, Iqbal Z, Orlando

M, Maccione A, Lassi G, Farisello P, Contestabile A, Tinarelli F, Nieus T, Raimondi A, Greco B, Cantatore D, Gasparini L, Berdondini L, Bifone A, Gozzi A, Wells S, Nolan PM (2014) Dominant β-catenin mutations cause intellectual disability with recognizable syndromic features. J Clin Invest 124(4):1468–1482. doi:10.1172/JCI70372 Van der Auwera GA, Carneiro M, Hartl C, Poplin R, del Angel G, Levy-Moonshine A, Jordan T, Shakir K, Roazen D, Thibault J, Banks E, Garimella K, Altshuler D, Gabriel S, DePristo M (2013) From FastQ data to high-confidence variant calls: the genome analysis toolkit best practices pipeline. Current Protoc Bioinform 43:11.10.1–11.10.33 Vogel P, Read RW, Hansen GM, Payne BJ, Small D, Sands AT, Zambrowicz BP (2012) Congenital hydrocephalus in genetically engineered mice. Vet Pathol 49:166–181 Wang K, Li M, Hakonarson H (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38(16):e164 Xing Y, Takemaru K, Liu J, Berndt JD, Zheng JJ, Moon RT, Xu W (2008) Crystal structure of a full-length beta-catenin. Structure 16:478–487 Yu X, Malenka RC (2003) Beta-catenin is critical for dendritic morphogenesis. Nat Neurosci 6:1169–1177

13