De novo and inherited TCF20 pathogenic variants are associated with intellectual disability, dysmorphic features, hypotonia, and neurological impairments with similarities to Smith–Magenis syndrome

Background Neurodevelopmental disorders are genetically and phenotypically heterogeneous encompassing developmental delay (DD), intellectual disability (ID), autism spectrum disorders (ASDs), structural brain abnormalities, and neurological manifestations with variants in a large number of genes (hundreds) associated. To date, a few de novo mutations potentially disrupting TCF20 function in patients with ID, ASD, and hypotonia have been reported. TCF20 encodes a transcriptional co-regulator structurally related to RAI1, the dosage-sensitive gene responsible for Smith–Magenis syndrome (deletion/haploinsufficiency) and Potocki–Lupski syndrome (duplication/triplosensitivity). Methods Genome-wide analyses by exome sequencing (ES) and chromosomal microarray analysis (CMA) identified individuals with heterozygous, likely damaging, loss-of-function alleles in TCF20. We implemented further molecular and clinical analyses to determine the inheritance of the pathogenic variant alleles and studied the spectrum of phenotypes. Results We report 25 unique inactivating single nucleotide variants/indels (1 missense, 1 canonical splice-site variant, 18 frameshift, and 5 nonsense) and 4 deletions of TCF20. The pathogenic variants were detected in 32 patients and 4 affected parents from 31 unrelated families. Among cases with available parental samples, the variants were de novo in 20 instances and inherited from 4 symptomatic parents in 5, including in one set of monozygotic twins. Two pathogenic loss-of-function variants were recurrent in unrelated families. Patients presented with a phenotype characterized by developmental delay, intellectual disability, hypotonia, variable dysmorphic features, movement disorders, and sleep disturbances. Conclusions TCF20 pathogenic variants are associated with a novel syndrome manifesting clinical characteristics similar to those observed in Smith–Magenis syndrome. Together with previously described cases, the clinical entity of TCF20-associated neurodevelopmental disorders (TAND) emerges from a genotype-driven perspective. Electronic supplementary material The online version of this article (10.1186/s13073-019-0623-0) contains supplementary material, which is available to authorized users.

tissue showed evidence of an extra copy of the MYCN gene, consistent with aneuploidy of chromosome 2. No evidence of MYCN gene amplification was observed in any cells examined.
Subject #2 (DDD_ 269612) is a 14-year-old female with global developmental delay, hypotonia, intellectual disability, severe language disorder. She had a large build and mild dysmorphic features including frontal bossing, full cheeks, long fingers with a contracture of 5th finger, long toes, a wide carrying angle and joint hypermobility.
Subject #3 is a 20-year-old male presenting with congenital hypotonia and global developmental delay; he walked and started talking around 3 years of age. He has been diagnosed with high functioning ASD, anxiety, dyspraxia and ADHD requiring medication. Head circumference is around the 97 th percentile with an IQ of 79-100. He has been noted to be clumsy with mild kyphosis. MRI and metabolic tests were normal. Subject #4 is a 3.25-year-old male with ASD diagnosed at the age of 20 months, hypotonia, sensory processing disorder, visuo spatial perception difficulty, dyspraxia, history of sleep problems, mild dysmorphic features including triangular face, frontal bossing, with head circumference of 51 cm (around 99 th percentile).
Subject #5 (DDD_277986) is a 8.5-year-old male with history of severe developmental delay, hypotonia, and feeding difficulties. Now at 8.5 -year-old he is presenting with moderate intellectual disability, possible ADHD with no formal diagnosis, and attends a special needs school. He had feeding problems previously which has improved. His current head circumference is 50.5cm (0.4 th -2 nd ), weight is 20.1kg (2 nd ) and height is 106.4 (<0.4 th ). His symptomatic mother is reported to have had a history of feeding difficulties during early childhood and mild-moderate learning difficulties.
Subject #6 is a 10.2-year-old male with developmental delays, hypotonia and dysmorphic features.
Detailed neurological examination revealed decreased strength in upper and lower extremities, distal and proximal hypotonia, also poor coordination. Furthermore, has been diagnosed with epileptic syndrome with complex partial seizures with intractable epilepsy. Developmental delays include expressive language, fine motor, and to lesser degree receptive language and gross motor skills. He also has poor growth velocity and present with remarkable short stature but with proportional weight. He has a history of pneumonia.
Subject #7 is a 10-year-old male with global developmental delay, autistic spectrum disorder, self-harming behaviors, significant anxiety, attention deficit hyperactivity disorder, poor muscular tone, refractory generalized non-convulsive epilepsy, mild dysmorphic features, tapered 5 th fingers with minor cutaneous 2-3 to syndactyly. Most recent EEG in 2017 showed epileptic disorder with multifocal origin and EEG in 2013 showed atypical absence seizures. The family history indicates that the mother and maternal half-sister both have intellectual functioning disability the maternal half-sister also has attention deficit disorder. His height, weight, and FOC were within normal range.
Subject #10 (DDD_261665) is a 11.5-year-old female with global developmental delay, mild-tosevere intellectual disability, severe delay in receptive and expressive language, obsessive-compulsive and food-seeking behavior, altered sleep pattern with frequent awakening, gross motor and fine skills delay, obesity, and joint hypermobility. Subject #11 is a 3.9-year-old male diagnosed with congenital hypotonia with moderate distal spasticity, autistic spectrum disorder (ASD) diagnosed by Autism Diagnostic Observation Schedule (ADOS) and intellectual disability, failure to thrive and short stature. He is developmentally delayed including gross and fine motor and severe expressive/receptive language disorder. He has recurrent otitis media, frequent infections with monthly episodic fever, and recurrent pneumonia. In addition, he suffers from dysphagia. Facial dysmorphism include relative macrocephaly, deep-set eyes, mild coarsening, long philtrum, wide mouth with full lips, mildly tented upper lip, and hypertelorism. Lab studies identified platelet dysfunction. Previous MRI and other metabolic testing resulted normal; genetic testing, including chromosomal microarray, karyotype, and fragile X molecular analysis, were unrevealing. Subject #12 (DDD_305239) is a 3-year-old female presenting with global developmental delay, ASD, language delay, sleep disturbance, and joint hypermobility. She had dysmorphic features including upslanted palpebral fissures, epicanthus, short nose, depressed nasal bridge, and short lingual frenulum.
Subject #13 is a 14-month old male with global developmental delay, laryngeal cleft, plagiocephaly, prominent mildly over folded ears with bilateral epicanthal folds and left-sided fifth finger clinodactyly.
Subject #16 (DDD_285718) is a 6.5-year-old male with moderate developmental delay, hypotonia, mild intellectual disability, ASD, ADHD and strabismus. Subject #17 is a 5.4-year-old girl with significant expressive/receptive speech delay with developmental delay and history of mild gross motor delay and hypotonia with mild facial dysmorphic features (midface hypoplasia). At the age of 5 year she had a vocabulary or no more than 10 poorly articulated words with a normal ABR by report. She could sign about 30 single words as well. She can follow single step commands and can hold a pen and scribble. Her mother also reports that she is "clumsy" and easily trips, her right foot "turns inward" when walking, and her right eye sometimes turns inward. She has some aggressive behaviors. She has trouble staying asleep despite long-term use of melatonin. She has also food-seeking behavior and lack of satiety.
Her height, weight, and FOC are within normal range.
Subject #18 (DDD_286061) is a 9 -year-old female with intellectual disability, hypotonia, poor concentration, and hyperactivity. She sat at 18 months and walked at 2 ½-year-old. Other features include strabismus, joint laxity, dysmorphic features (high anterior hairline, large and tall forehead, low-set posteriorly rotated ears, narrow mouth and long philtrum).
Subject #19 (DDD_266071) is 10.2-year-old male with global delay, autism, seizures, clumsiness and hyperactivity (requiring Risperidone treatment) as well as faecal soiling as a young child. He presents with episodes of paroxysmal, possible dystonic movements of upper limbs, however the tone and reflexes are recorded as normal. In addition, he has tall stature, brachycephaly, high pitched voice, 5 th finger clinodactyly, confirmed X-linked ichthyosis with a deletion of the STS locus on Xthis is familial, and several normal family members are also affected.
Subject #20 is an 8-year-old male born to a consanguineous family with intellectual disability, motor delay and insomnia. He walked at 1.5-year-old and started talking at 2-year-old.
Examination at 3.5 years showed no dysmorphic features and no abnormal neurological signs. His weight was on the 90th centile till the age of 3 years when it dropped to the 50th and by the age of 8 years it dropped to <25th centile. His height and head circumference have been on the 75th centile throughout. At the age of 8 years he was reported to have slow movements, difficulty in swallowing, excessive salivation and started having blurred speech. Neurological examination revealed slightly increased tone at the elbows, normal deep tendon reflexes and impaired coordination.
Subject #22 (DDD_299802) is a 9.9-year-old female with severe intellectual disability, hypotonia, jerky movements, macrocephaly and overgrowth. At 12 years 3 months her height was 174.5 cm Subjects #27 and #28 (DDD_294521) are two identical male twins with moderate intellectual disability, speech delay, hypotonia, motor coordination difficulties, plagiocephaly, bilateral ptosis, horizontal crus of helix, malar flattening, narrow mouth, short, tapering fingers with incurved 5th fingers, mild bilateral hallux valgus. In addition, they both present with inverted nipples, truncal obesity, and subject #28 has high arched palate. The family history indicates that the father presents with similar phenotype.
Subject #29 was initially evaluated at 4-year-old. She presented with congenital generalized hypotonia, repetitive behavior, and severe mixed expressive/receptive language disorder. She was adopted. Her prenatal history indicates that she was diagnosed with "brain damage syndrome" but no other information was provided. At the time of her adoption, mother noted scars on her legs that appeared to be surgical in nature. In addition, she showed stereotypy as a 6-year-old: hand flapping, walking in circles, hitting her head when frustrated. She was diagnosed with autism spectrum disorder, intellectual disability and inattentive type ADHD. Her height, weight, and FOC were within normal range.
Subject #30 is a 14-year-old male with global psychomotor delay, autism spectrum disorder, stereotypical behavior, severe language delay, macrocephaly, congenital hypotonia, and scoliosis.
He walked when he was older than 2 years of age and started talking in single words when he was 4 years old. He has had disrupted sleep patterns since infancy. Cranial imaging studies included a head CT that showed stable prominence of extra axial fluid spaces and MRI showed a slight asymmetry of the cerebral hemisphere. The picture in Fig. 2L was taken at 22 years old.
Subject #31 is a 5-year-old male with global developmental delay, intellectual disability, seizure and balance disorder. Macrocephaly 37 cm (+2SD) was noted at birth with. At 4 years, the height at +2SD. At 6 years, overgrowth was noted with a height of 120 cm (+2.5DS), a weight of 23.7 Kg (+2SD), and an OFC of 52.5cm (+1SD).
Subject #32 is a globally delayed 13-month-old female with severe receptive/expressive language delay, hypotonia, mild spasticity in ankle dorsiflexors, and mild proximal weakness in shoulders/hips. Her facial features include macrocephaly, midface hypoplasia, long eyelashes, thick lips and an occipital groove. At the last medical examination, she had marked language delay and emerging autistic traits. She displays food-seeking behavior and has sleep disturbance.
Brain MRI showed mildly enlarged extra-axial spaces, lateral ventricles and large cavum septum pellucidum and vergae. Additional clinical findings include a renal cyst, GERD, dysphagia and delayed visual maturation.   S1. Schematic representation of key conserved domains between TCF20 and RAI1. Functional domains of TCF20 (shared with RAI1) include TAD, Glutamine-rich stretches (Q), NLS1 and 2, F-BOX and ePHD/ADD. Additional unique domains present in TCF20 are LZ, NLS3 and DBD domains with the AT-hook region. The relative amino acid position in TCF20 is shown below the structure. . TCF20 alleles with premature termination codon variants escape from nonsense mediated decay (NMD). The mRNA of mutant TCF20 are subject to reverse transcription and PCR. The PCR primers are designed so that the amplicons all span the intron 2 and 3 of TCF20 (~30 kb and ~10 kb, respectively). This facilitates to minimize interference of genomic DNA from PCR. The locations of the primers are indicated on the genomic sequence of TCF20. The primer sequence are as follows. Patient #11 forward 5'-CAGTGGGGACTTACCATGACCC-3', reverse 5'-CGAGAAGTTCTCCTCATGTAGCAAAC-3'. Patient #25 forward 5'-AGCTGGAGTTACAAATCCCTGA-3', reverse 5'-TAGGGCACCTCACCGAGAA-3'. Patient #7 forward 5'-TTGAAATAGAGCCTCAGTCATCAGCAC-3', reverse 5'-GTGCTTAGGGCACCTCACCGAGAA-3'. The expected amplicon sizes from cDNA amplification are indicated in the figure. The sizes of the amplified segments are verified to ensure that no genomic DNA is present. Sanger sequencing chromatograms are illustrated. Individuals with a premature termination codon variant, including patient #11, #25, #7, and the mother and sister of patient #7, are expected to be subject to NMD by the '50 bp penultimate exon' rule. However, they all show a pattern that the mutant allele is of similar intensity to the wildtype allele, suggesting that the mutant mRNA escaped from NMD. Sequencing results from individuals without a TCF20 variant, including the mother of patient #11, mother of patient #25, and an unrelated individual, are included as negative controls.