Missense variant in U2AF2 leads to systemic dysmorphism and epilepsy: Report of two cases and literature review

Analyzed the clinical characteristics and genetic characteristics of two patients with the U2AF2 gene variant causing systemic malformation and epilepsy. Methods The clinical data and genetic test results of two patients with systemic deformity and epilepsy were retrospectively analyzed, and the literature was reviewed. Results All two patients presented with Missense variant in U2AF2 leads to systemic dysmorphism and epilepsy: Report of two cases and literature review Shiqin Huang 1†, Mei Li 2†, Yunli Han 1, Hai Yuan 1, Xiaolan Chen 1, Yunxuan Su 1, Xing Li 1 * 1 Departments of Pediatrics, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China 2 Departments of Pediatrics, Nanning Maternal and Child Health Care Hospital, Nanning 530015, Guangxi Zhuang Autonomous Region, China *Correspondence: Xing Li: [email protected] † These authors contributed equally. Key Clinical Message U2AF2 is an essential splicing factor, which could lead to unique neurodevelopmental disorder. We report two patients with neurodevelopmental disorder who presented systemic malformation and status epilepticus, genetically characterized by a heterozygous variant in U2AF2 (c. 445C>T (p. Arg149Trp). The two patients achieved control with antiepileptic drugs treatment.

U2AF2, missense variant, epilepsy 1 INTRODUCTION U2 auxiliary factor (U2AF) is a non-snRNP protein required for the binding of U2 snRNP to the pre-mRNA branch site, which also an essential pre-mRNA splicing factor in an early step of splicing (Sickmier, Frato et al. 2006, Glasser, Maji et al. 2022). Mutations or dysregulation of many genes causing aberrant splicing can leads to neurodevelopmental disorders (Marchetto, Carromeu et al. 2010, van Bokhoven 2011, Lalli, Avey et al. 2020). U2AF2 is an essential pre-mRNA splicing factor in an early step of splicing (Sickmier, Frato et al. 2006), while missense variant in this gene have been reported causing systemic dysmorphism, developmental delay and epilepsy (Kaplanis, Samocha et al. 2020, Hiraide, Tanaka et al. 2021, Kittock, Saifeddine et al. 2023, Kuroda, Matsufuji et al. 2023, Wang, You et al. 2023). Previously, a large trio WES study concerning patients with developmental disorders identified U2AF2 as a potential pathogenic gene, of which ten patients with seven variant types were identified but without detailed clinical information fallowed (Kaplanis, Samocha et al. 2020). For now, there have several case reports on patients with developmental disorders that identified U2AF2 as a pathogenic gene (Hiraide, Tanaka et al. 2021, Kittock, Saifeddine et al. 2023, Kuroda, Matsufuji et al. 2023, Wang, You et al. 2023). Recently, U2AF2 and other two splicing factors establish a genetic network underlying human brain development and function (Li, Wang et al. 2024), it confirms that this gene plays an irreplaceable role in neural development. Herein, two Chinese patients harboring the same variant in U2AF2, showing typical global developmental delay, dysmorphic facial features and epilepsy, the detailed clinical features of these two cases provide additional evidence that U2AF2 playing an indispensable role in neural development. 2 METHODS Written informed consent were obtained from all participants and their parents. This study was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University. Electroencephalography (EEG) monitoring was performed using a 64-channel electroencephalograph manufactured by EEG-1200C (Nihon Kohden Copporation, Japan). Cranial Magnetic Resonance imagines (MRI) was performed using the 1.5 T MR Systems (Siemens MAGENTOM Altea, Germany). Genomic DNA was isolated from the peripheral blood samples of the proband and their parents. Trio whole exome sequencing was performed using the NextSeq500 (Illumina, USA). The mean coverage and depth of all the genes were 98% and 10×, respectively. The variants were validated by Sanger sequencing. The pathogenicity of variants was predicted by PolyPhen-2, SIFT, CADD, and Mutation Taster. Variants were evaluated according to the population in the 1000 Genomes, ExAC, and gnomAD databases. The pathogenicity of the variant was evaluated according to the American College of Medical Genetics (ACMG) standard guidelines. 3 CASE REPORT Clinical features of the proband The first patient was an 8-year-old girl, the Zhuang nationality. She was born to nonconsanguineous parents after 39 weeks of gestation without asphyxia, with birth weight 2690 g, body length 48.0 cm, and head circumference 32 cm. She has one older, unaffected sibling. Her parents are healthy and have no family history of neurodevelopmental disorders. After birth, due to delayed motor development compared to peers, continuous rehabilitation treatment has been carried out, and she repeatedly experienced respiratory infections. At the age of 1, her vision was tested for hyperopia. When at 3 year and ten months old, she had her first epileptic seizure without fever, presenting as a persistent state of generalized tonics and clonus, lasting for up to 30 minutes. Abnormal physical features included microcephaly, global developmental delay. The dysmorphic facial features including large ears, frontal bossing, wide eye distance, bilateral ptosis, wide and sparse eyebrows, and underdeveloped teeth (Fig.1A). This girl has been experiencing recurrent seizures for over 3 years, resulting in focal seizures and a persistent state of multiple seizures. She started valproic treatment at 3 years old. At the age of 6 years and 11 months, this girl was admitted to the hospital due to recurrent convulsions, accompanied by the fever of 40 ℃. Brain MRI shows abnormalities, characterized by small frontal and temporal gyri on both sides, corresponding widening of the subdural space, and small size of the corpus callosum (Fig.1B). Abnormal electroencephalogram with slow background activity. Bilateral anterior slow wave emission during wakefulness, significant on the left side, and bilateral central and parietal spike slow wave emission during sleep (Fig.1C). The patient presented with GTCS (double eyed gaze, cyanosis of the lips, closed teeth, stiffness and trembling of the limbs, inability to respond, and self relief after about 3-4 minutes). Visual evoked potential showed prolonged latency of P100 waves in the full field and central damage. The Wechsler Children’s Intelligence Scale indicated that she was intellectual disability and moderate social maladaptation. Fig.1 Clinical information of the first patient. A: The dysmorphic facial features; B: The results of Cranial Magnetic Resonance imagines (MRI) map; C: The electroencephalogram The second patient was a 6-year-old boy, the Zhuang nationality. He was born after a cesarean section at 36 weeks because of slow fetal heart rate, without history of suffocation rescue, with birth weight 2000 g, body length 44 cm, and head circumference 30 cm. Postnatal examination revealed cardiac murmurs by echocardiography. At 3 months of age, echocardiography revealed a 5 mm ventricular septal defect and multiple atrial septal defects (3 in total, with sizes of 5× 7mm, 5 × 5mm, and 3 × 3mm, respectively). At 1 and 3 years old, follow-up echocardiography revealed multiple atrial septal defects (secondary orifice type) and natural closure of the ventricular septal defect. Motor development lags behind peers by 2-3 months. After birth, neutropenia was found to be reduced, and vitamin B4 treatment was given. Multiple re examinations showed neutrophils ranging from 0.79 to 1.35 × 109 /L, recurrent respiratory infections, and multiple pneumonia. The boy has a similar special face to the previous girl: protruding forehead, wide eye distance, large ears, wide and sparse eyebrows (Fig.2A). He had his first epileptic seizure at the age of 1 year and 6 months, presenting as a persistent state of focal seizures: characterized by loss of consciousness, with eyes turned upwards and staring to the right, accompanied or not accompanied with right corner of mouth and right limb tremor, without fever, lasting for 1 hour. Midazolam medication infusion alleviates symptoms. At the age of 2, he had another seizure and was admitted to the hospital for oral administration of levetiracetam. By the age of 4 and 6 months, physical development was at a lower level, with a weight of 13 kg (<3 standard deviation (SD)) and a height of 95cm (<3 standard deviation (SD)). Development scale results: 46 points for speech, 57 points for operation, 47 points for total scale, and 8 points for social adaptability. Head MRI: an enlarged cavity of the septum pellucidum (6mm), inflammation in the right maxillary and ethmoid sinuses, and adenoid hypertrophy (Fig.2B). Electroencephalogram: Background θ Increased activity, with spikes/sharp waves, sharp slow waves, sharp slow waves, and irregular slow waves emanating from the right posterior head during sleep (Fig.2C). Blood routine and biochemistry showed no abnormalities, while blood gas, lactate, blood sugar, and electrolytes were normal. Fig.2 Clinical information of the second patient. A: The dysmorphic facial features; B: The results of Cranial Magnetic Resonance imagines (MRI) map; C: The electroencephalogram 4 DISCUSSION Disordered RNA splicing can lead to the occurrence of various human diseases, including spinocerebellar ataxias, spinal muscular atrophy, myotonic dystrophy, retinitis pigmentosa, and autoimmune disease (Corrionero, Raker et al. 2011, Poulos, Batra et al. 2011). U2 auxiliary factor (U2AF) is a non-snRNP protein required for the binding of U2 snRNP to the pre-mRNA branch site, and it can guide the early stages of splice-site choice by recognizing polypyrimidine tract consensus sequences near the 3-prime splice site (Sickmier, Frato et al. 2006, Glasser, Maji et al. 2022). The removal of introns from the pre-mRNA via splicing is a crucial component of RNA metabolism, and provides proteomic diversity from a relatively small set of genes (Hoffman and Grabowski 1992). In recent years, de novo mutations in U2AF2 has been reported associated with neurodevelopmental diseases (Kaplanis, Samocha et al. 2020, Hiraide, Tanaka et al. 2021, Kittock, Saifeddine et al. 2023, Wang, You et al. 2023). A large trio WES study concerning patients with developmental disorders identified U2AF2 as a potential pathogenic gene, of which ten patients with seven variant types were identified but without detailed clinical information fallowed (Kaplanis, Samocha et al. 2020). For now, there have several case reports on patients with developmental disorders that identified U2AF2 as a pathogenic gene. The U2AF2 variant c.445C>T, p.R149W has been reported in two cases that having neurodevelopmental disorders (Hiraide, Tanaka et al. 2021, Kittock, Saifeddine et al. 2023, Wang, You et al. 2023). The first case was a 7.5-year-old Japanese girl with developmental delay, dysmorphic facies, and brain anomalies (Hiraide, Tanaka et al. 2021). She was identified having a de novo heterozygous (c.445C>T) in the U2AF2 gene, resulting in an Arg149-to-Trp (R149W) substitution at a conserved residue in the RNA recognition motif 1 (RRM1). This girl showed global developmental delay, intellectual disability, epilepsy, short stature, microcephaly, facial dysmorphism, intermittent exotropia, bilateral ptosis, muscle hypotonia and thin corpus callosum, indicating that U2AF2 -related disorder could include systemic dysmorphisms, epilepsy and brain malformation along with global developmental delay. The mutation, which was found by trio-based whole-exome sequencing and confirmed by Sanger sequencing, was not present in the gnomAD database. By trio-based whole-exome sequencing, the same variant in the U2AF2 gene was identified in a 4-year-old Caucasian male (Kittock, Saifeddine et al. 2023). He has similar symptoms of global developmental delay, seizures, and short stature. These provide strong evidences that the U2AF2 is pathogenic gene and the variant of c.445C>T can leads to unique neurodevelopmental disorder. Very recently, there exist other reports supports the association between U2AF2 and neurodevelopment. A 6-year-old Chinese boy was identified has a de novo heterozygous c.603G-T transversion (c.603G-T, NM_007279.3) in the last nucleotide of exon 6 of the U2AF2 gene, who has a similar clinical phenotype as the patient reported before, including epilepsy, intellectual disability, language delay, microcephaly, and hypoplastic corpus callosum (Wang, You et al. 2023). RT-PCR analysis of patient cells showed that the mutation caused abnormal splicing and skipping of exon 6, resulting in a 39-residue in-frame deletion (E163_E201del). Patient cells showed a 50% reduction in wild type U2AF2 mRNA and protein expression (65-kD) compared to controls. A smaller 55-kD band corresponding to expression of the abnormal protein with the in-frame deletion was detected. The mutation significantly inhibited the proliferation of patient immortalized lymphocytes, with a slightly increased proportion of cells in the G1/G0 phase and a slightly less proportion of cells in the G2/M phase compared to controls. In addition, a de novo heterozygous variant in U2AF2 (c.470C>T, p.Pro157Leu) was identified by trio exome sequencing in a 2-year-old Japanese girl, who was judged as hypomyelinating leukodystrophy with global developmental delay (Kuroda, Matsufuji et al. 2023). In conclusion, our case suggested that U2AF2 -related disorder could include systemic dysmorphisms, epilepsy and brain malformation along with DD and ID. ACKNOWLEDGMENTS We wish to thank the patient family for giving their consent for this publication. The parents gave their consent for the publication of photographs of their child. They were informed that anonymity would be-strictly respected. CONFLICT OF INTEREST STATEMENT The authors report no conflict of interest. DATA AVAILABILITY STATEMENT Additional data supporting the findings of this study are available in this article’s supporting information. Individual-level data are not publicly available due to privacy or ethical restrictions. FUNDING This research was funded by the The First Affiliated Hospital of Guangxi Medical University Clinical Research Climbing Program Youth Science and Technology Start Star Program (YYZS2020019). AUTHOR CONTRIBUTIONS Xing Li and Shiqin Huang contributed to clinical data collection and the determination of the article framework. Mei Li contributed to bioinformatics analysis and original draft preparation. Yunli Han and Hai Yuan contributed to the collection of relevant literature. Yunxuan Su contributed to the collection of relevant case (of illness). Xiaolan Chen contributed to the supplementary revision of the article as well as the submission. ORCID Xing Li https://orcid.org/0000-0002-6375-7655

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Authors Metrics & Citations Metrics Article Usage 546views 266downloads Citations Download citation Shiqin Huang, Mei Li, Yunli Han, et al. Missense variant in U2AF2 leads to systemic dysmorphism and epilepsy: Report of two cases and literature review. Authorea. 20 July 2024. DOI: https://doi.org/10.22541/au.172151769.91930243/v1 DOI: https://doi.org/10.22541/au.172151769.91930243/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.