A Novel Heterozygous Missense Variant in YWHAG Gene Cause Early-Onset Epilepsy in a Chinese Family Zhi Yi The Aliated Hospital of Qingdao University Zhenfeng Song The Aliated Hospital of Qingdao University Jiao Xue The Aliated Hospital of Qingdao University Chengqing Yang The Aliated Hospital of Qingdao University Fei Li The Aliated Hospital of Qingdao University Hua Pan The Aliated Hospital of Qingdao University Xuan Feng The Aliated Hospital of Qingdao University Ying Zhang ( [email protected] ) The Aliated Hospital of Qingdao University https://orcid.org/0000-0002-6360-6566 Hong Pan Peking University First Hospital Case report Keywords: YWHAG, developmental and epileptic encephalopathy 56, intellectual disability, early onset seizures Posted Date: December 31st, 2020 DOI: https://doi.org/10.21203/rs.3.rs-136482/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/10 Abstract Background: Developmental and epileptic encephalopathies (DEE) are a heterogeneous group of severe disorders which are characterized by early-onset, refractory seizures and developmental slowing or regression. Genetic variations are signicant causes for them. De novo variants in an increasing number of candidate genes have been found to be causal. YWHAG gene variants have been reported to cause developmental and epileptic encephalopathy 56 (DEE56). Case presentation: Here, we report a novel heterozygous missense variant c.170G>A (p.R57H) in YWHAG gene cause early-onset epilepsy in a Chinese family. Both the proband and his mother exhibit early onset seizures, intellectual disability, developmental delay. While the proband achieve seizure control with sodium valproate, his mother's seizures were not well controlled. Conclusions: Our report further conrming the haploinsuciency of YWHAG results in developmental and epileptic encephalopathies. Background Developmental and epileptic encephalopathies (DEE) are a heterogeneous group of severe disorders which are characterized by early-onset, refractory seizures and developmental slowing or regression. Genetic variations are signicant causes for them 1,2. With the wide application of whole genome and exome sequencing, an increasing number of candidate genes had been found to be causal, but most of these are only been discovered in a small proportion of cases3. YWHAG (* 605356) is one such gene that had been recently related to DEE. YWHAG gene is located on Chr7q11.23 which encodes for YWHAG, a member of 14-3-3 protein family, is highly expressed in brain, skeletal muscle, and heart4. Interstitial deletion at 7q11.23 which include YWHAG gene cause infantile spasm and cardiomegaly in Williams-Beuren syndrome patient. Subsequently knocking down ywhag1 revealed reduced brain size and increased diameter of the heart tube in zebrash, indicating that the infantile spasms and cardiomegaly seen in the patient was due to haploinsuciency of YWHAG5. Research on mouse models found that both knock down and overexpression of Ywhag results in delays in pyramidal neuron migration, indicating an appropriate level of Ywhag is required for brain development6,7. There have been 10 variants reported in YWHAG gene causing developmental and epileptic encephalopathy 56 (DEE56) (# 617665) or autism3,8−11. Here, we report a novel missense variant c.170G > A (p.R57H) in exon2 of YWHAG which cause early-onset epilepsy in a Chinese family. Case Presentation And Methods Ethical approval Page 2/10 Informed consent for whole-exome sequencing and subsequent Sanger sequencing as a part of the diagnostic process (approved by the Medical Ethical Committee of Aliated Hospital of Qingdao University) was obtained from the proband’s parents and grandparents. The proband The patient (III.1, Fig. 1 A) is a boy who was born in full-term cesarean section. His parents are not in consanguinity. His father is health. His mother (II.2, Fig. 1 A) has seizures and developmental delay. He is the rst child of his parents and his mother had no history of miscarriages. His birth weight was 3200 g, birth length was 50 cm, birth head circumference was 34 cm. There was no history of asphyxia and anoxia. He has no unusual features. His gross motor development was delayed since birth. He can raise his head at age of 4 month, turn over at age of 6 month, sit at age of 8 month and walk without assistance at age of 1 year and 6 months. He is now 3 years and 8 months and cannot jump. His language development is seriously backward. Now, he can only say “papa, mama, no” or express his needs by point to the object. He can understand simple command language. He is very irritable. He had his rst seizure at age of 1 year and 11 months, manifested as generalized tonic-clonic seizure (GTCS). EEG at that time did not catch a seizure and show no abnormalities in background. Neuroimaging was unremarkable. He was start to be treated with sodium valproate, and was seizure free after 4 months of treatment. The proband’s mother The proband’s mother (II.2, Fig. 1 A) had her rst seizure at age of 2 years, and also manifested as GTCS. Her relatives did not report other types of seizures. She was treated with antiepileptic drugs, but it is not known what they were, and she had a 5 to 6 years remission of epilepsy. During pregnancy, she stopped taking drugs without the guidance of a doctor and the seizures returned. Now she is being treated with carbamazepine, phenytoin sodium and sodium valproate, but the seizure was not under complete control. The mother also had global developmental delay. She sat at age of 8 month and walked without support at age of 1 years and 5 months. She didn't do well in school. She can cook, but she cannot shop. She refused to take intelligence tests. Whole-exome sequencing Trio-based whole-exome sequencing was performed on the proband and his parents. Peripheral blood samples (2 ml) were collected from the boy and his families. Then the samples were sent to MyGenostics, Beijing for trio-whole exon sequencing. Genomic DNA was extracted from peripheral blood using the DNA Extraction kit (TIANGEN, Beijing, China) following the manufacturer’s instructions. A minimum of 3 ug DNA was used for the indexed Illumina libraries according to manufacturer’s protocol (MyGenostics, Inc., Beijing, China). DNA fragments with sizes ranging from 350 bp to 450 bp and those including the adapter sequences were selected for the DNA libraries. Next, the target DNA fragments from the DNA libraries were captured using the biotinylated capture probes (P039-Exome) and then amplied through the GenCap custom enrichment kit (MyGenostics, Inc., Beijing, China) following the Page 3/10 manufacturer’s protocol. Enriched DNA samples were sequenced on an Illumina NextSeq 500 (Illumina, San Diego, CA, USA) for paired-end reads of 150 bp. Following sequencing, raw image les were processed using Bcl2Fastq software(Bcl2Fastq 2.18.0.12, Illumina, Inc.) for base calling and raw data generation. Short Oligonucleotide Analysis Package (SOAP) aligner software (SOAP2.21; soap.genomics.org.cn/soapsnp.html) was then used to align the clean reads to the reference human genome (hg19). The nally identied variants were evaluated using the three algorithms, PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), Sorting Intolerant From Tolerant [SIFT; (http://sift.jcvi.org/)], Mutation Taster (http://www.mutationtaster.org/ ) to predict pathogenicity. Variant interpretation was performed according to the American College of Medical Genetics (ACMG) guidelines 12. Sanger sequencing was utilized for further validation of variants and variant detection of other relatives. Results We performed trio-based whole-exome sequencing on the proband and his parents. We identied a heterozygous variant c.170G > A (p.R57H) in exon2 of YWHAG (NM_012479) which is inherited from his mother (Fig. 1 B). Further investigation with sanger sequencing found that the grandmother and grandfather did not carry the variant (Fig. 1 B). This is a novel heterozygous variant which had not ever been reported. This variant causes arginine at position 57 to be replaced by histidine. PolyPhen-2 predicted it to be probably damaging with a score of 1.000. It is also predicted to be disease causing by mutation taster and damaging with a score of 0.000 by SIFT. This site is highly conserved between species and subtypes of the protein family, and the arginine at position 57 is part of the highly conserved triad of two arginines and a tyrosine (Arg132-Arg57-Tyr133) that normally form a positively charged patch within a binding groove for interacting phosphopeptides 3,13. This ability of the protein to bind phosphopeptides is potentially affected by substitution at this site (Fig. 1 C and D). Discussion Functional study of YWHAG YWHAG gene is located on Chr7q11.23 which encodes for YWHAG, a member of 14-3-3 protein family, is highly expressed in brain, skeletal muscle, and heart4. The 14-3-3 proteins function in vital cellular processes, such as metabolism, protein tracking, signal transduction, apoptosis and cell-cycle regulation14. They exist in monomeric and dimeric states as homo- and heterodimers, respectively, but YWHAG is almost entirely dimeric. Each monomer consists of a bundle of nine α-helices (αA to αI), of which, Helices αC, αE, αG, and αI form a conserved peptide-binding groove, which has a positively charged patch on one side and a hydrophobic patch on the other (Fig. 1 C). The positively charged patch is formed by a conserved triad of two arginines and a tyrosine residue (Arg-57, Arg-132, and Tyr-133), which bind the phosphate group of the interacting phosphopeptide/protein3,13,15. In 2010, Komoike et al reported a Williams-Beuren syndrome patient presenting with infantile spasms, which is extremely rare in Williams-Beuren syndrome. The deletion of this patient is larger than the common Williams-Beuren Page 4/10 syndrome deletion and include the telomeric YWHAG gene.