A novel variant in UBE3A in a family with multigenerational intellectual disability and developmental delay

Abstract Background Angelman syndrome (AS) is a rare neurodevelopmental disorder and is characterized by severe cognitive disability, motor dysfunction, speech impairment, hyperactivity, and frequent seizures. Although the maternal chromosomal region 15q11.2‐q13 deletion is the most common mechanism of AS, ~10% of individuals with AS are caused by the intragenic variants in the maternally inherited UBE3A, which encodes an E3 ubiquitin ligase. Methods Clinical diagnoses were based on detailed clinical findings. Trio‐based exome sequencing was performed on the proband and her parents to identify the underlying genetic variants. The candidate variants were confirmed by Sanger sequencing following PCR amplification. In silico analyses were conducted to predict the effect of the identified variant on the function of UBE3A protein. Results We identified a novel variant c.2029G>C (p. Gly677Arg) in UBE3A as the most promising candidate. In silico analyses showed that p.Gly677Arg in the UBE3A affects a highly conserved residue. Her mother had the variant at this locus. Sanger sequencing results showed that II‐2, II‐5, II‐7, IV‐1, III‐5, III‐7, III‐8, and III‐9 have the variant c.2029G>C, and all patients inherited maternally variant in UBE3A, while the offsprings of the male carrier were unaffected. Conclusions We identified a novel variant (c.2029G>C) in the UBE3A in a Chinese family with multigenerational intellectual disability and developmental delay. Our findings expanded the genotypic spectrum of AS and provided important information for genetic counseling.


| INTRODUCTION
AS is a unique neurodevelopmental disorder caused by the functional loss of the ubiquitin-protein ligase E6-AP (E6-associated Protein) encoded by the UBE3A gene (OMIM:#601623). This syndrome was characterized by intellectual disability, speech impairment, dysmorphic facial features (such as microcephaly with flat occiput and occipital groove, wide mouth), gait ataxia, and/or tremulousness of the limbs, hypopigmentation and seizures with specific EEG pattern abnormalities. The behavioral phenotype includes happy disposition (frequent laughing, smiling), hyperactivity, attention deficit, and frequent disruption of sleep cycles. The incidence of AS is estimated to be 1/15,000 births (Williams et al., 2006;Williams, Angelman, et al., 1995;Williams, Zori, et al., 1995).
The UBE3A gene is located on chromosome 15q11.2-q13 region. In this region, there is a cluster of imprinted genes that show differential expression depending on the parental origin, in a tissue-specific manner. The UBE3A is an imprinted maternally expressed gene in neurons while the paternal allele is silenced by an antisense transcript from the SNURF-SNPRN gene. Loss of functional UBE3A protein and therefore the absence of UBE3A protein in the brain causes AS (Buiting, 2010). Genetic mechanisms of functional loss of the specific maternal expression of UBE3A include 15q11.2-q13 deletion, variants in the UBE3A gene, paternal uniparental disomy, and genomic imprinting defects (Buiting, 2010;Ramsden et al., 2010).
The UBE3A gene consists of 10 exons (NM_130838.1) and encodes the ubiquitin-protein ligase E6-AP, belonging to the HECT (homologous to E6-AP C-terminus) class of E3 enzymes which is necessary for the ubiquitination of proteins targeted for degradation. Exons 3-10 of the UBE3A gene encode the 40 kDa COOH-terminal catalytic domain (Dagli et al., 2012).
Variants in the UBE3A gene are widely distributed and there is an absence of variant hot spots (Bird, 2014;Sadikovic et al., 2014). Most cases are sporadic, although familial occurrence is not uncommon (Sadikovic et al., 2014). According to the Human Gene Variant Database (HGMD: http://hgmd.cf.ac.uk, 2020.2), 253 different variants of the UBE3A gene have been reported, 95 of which are missense and nonsense variants. Here, we report six patients from a Chinese family diagnosed with AS due to a missense variant c.2029G>C (p.Gly677Arg) of the UBE3A gene. Our findings expanded the genotypic spectrum of AS and provided important information for genetic counseling.

| Samples and DNA extraction
Genomic DNA was extracted from EDTA peripheral blood samples using a Lab-Aid® 824 DNA Extraction Kit according to the manufacturer's protocol (ZEESAN).

| Trio-based exome sequencing
Trio-based exome sequencing was performed in the proband (IV-2) and her parents (III-1 and III-2). On average, 99% of the exome was covered at least 30-fold. Details of the methodology have been described previously (Zhao et al., 2020). Variants with minor allele frequencies (MAF) <1% in the exonic region or with splicing impact were taken for deep interpretation considering ACMG category, evidence of pathogenicity, and clinical synopsis and inheritance model of the associated disease. The variants were classified into five categories-"pathogenic," "likely pathogenic," "uncertain significance," "likely benign," and "benign"according to the American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation of genetic variants (Richards et al., 2015).

| Sanger sequencing
Sanger sequencing was performed for the validation of NGS results in the entire family. DNA was amplified using a standard Polymerase Chain Reaction (PCR). Primers of exon 6 of the UBE3A gene (reference sequence NM_130838.1) were as follows: F: 5′-TGGTATGGGGTTTCTCAGCA-3′; R: 5′-TCCTGCTTCATGTCCTCTTTCT-3′. Amplification was tested by agarose gel electrophoresis and DNA was sequenced by Sangon Biotech.

| Case report
The proband (IV-2) was a 3-year-old girl born uneventfully at a gestational age of 37 weeks. Pregnancy was otherwise uncomplicated. She is the second-born child of healthy non-consanguineous parents. Family history was remarkable. The child exhibited a delay in her gross motor milestones, starting to walk at 15 months of age, and was prone to falls. Regarding communicative behavior, the child utters only two words: "BaBa," and "MaMa," and does not seem to understand simple orders. Neurological examination results presented severe intellectual disability, higher receptive and non-verbal communication abilities than verbal ones, hyperreflexia of the lower extremities, and drooling frequently. She presented with attention deficit, irritability, excessive laughter, and being fascinated with water. But she never had clinical seizures and sleep disorders.
Her brother (IV-1) was an 8-year-old boy born at a gestational age of 36 weeks. He had global developmental delays, particularly in expressive language, though he can understand what others are saying. The child needed help getting dressed, and can only eat with a spoon, not chopsticks. In addition, he presented strabismus in the right eye, hyperreflexia and sleep disorder. Other clinical symptoms were similar to the proband's (Table 1). Patient III-5 presented with a more severe clinical phenotype, such as the later age of starting to walk (40 months), absence of speech, feeding difficulties. The symptoms of patients III-7, III-8, and III-9 in this family are similar to those of the proband. Detailed clinical characteristics are enlisted in Table 1.

| Trio-based exome sequencing revealed a novel variant c.2029G>C (p.Gly677Arg) in UBE3A
The proband (IV-2) was referred to our hospital with developmental delay and intellectual disability. First, chromosomal microarray analysis (CMA) was performed and the result was normal. Then, Trio-based exome sequencing was performed in the proband (IV-2) and her parents (III-1 and III-2), identifying a novel heterozygous missense variant in the UBE3A gene (NM_130838.1) c.2029G>C (p. Gly677Arg). Sanger sequencing confirmed    (Figure 1b). We screened the entire family, and the results showed that the unaffected family members II-2, II-5, and II-7 and the affected members IV-1, III-5, III-7, III-8, and III-9 all carried the heterozygous variant, and the other members unaffected in this family were wild type in the UBE3A gene.

| DISCUSSIONS
AS is characterized by severe developmental delay and classic characteristics of happy demeanor (frequent smiling and laughter), hyperactivity and absent or minimal use of words. Seizures with abnormal EEG are observed in up to 80% of AS patients, and the onset age of seizures usually begins before 3 years old and persists into adulthood. Other common features include hypotonia, feeding problems in infancy, protruding tongue, frequent disruption of sleep cycles, as well as attention deficit (Sadikovic et al., 2014). There are multiple molecular mechanisms by which UBE3A can be disrupted including deletions of the 15q11.2-q13 region, which account for 65%-70% of the cases; paternal uniparental disomy of chromosome 15 in 3%-7% of the cases; variants in the UBE3A gene in 10-15% of the patients and genomic imprinting defects in 2%-5% of patients (Buiting, 2010;Richards et al., 2015). Approximately 10% of patients presenting clinical features of AS have no identified molecular defect (Sadikovic et al., 2014). Genotype-phenotype correlations among molecular subclasses have shown that AS patients with deletions show the most severe and classical features of AS compared with other genotypes, including the worse expressive language skills, higher prevalence of seizures and microcephaly, earlier onset of seizures, higher incidence of relative hypopigmentation and motor difficulties (Gentile et al., 2010;Lossie et al., 2001;Moncla et al., 1999;Sahoo et al., 2007;Varela et al., 2004).
According to the clinical manifestations, genetic testing report, and the typical pedigree, the proband in this family was diagnosed as AS resulting from a UBE3A c.2029G>C variant. In this family, the six patients all show neurodevelopmental disorders such as intellectual disability, speech impairment, attention deficit, frequent laughter, and drooling, and never had any clinical seizures, while the patient III-5 displayed more severe clinical phenotypes with later onset age of walk, absence of speech and cannot take care of himself. Thus, our results indicate that patients with the same UBE3A variant may show intrafamilial phenotypic variability. Because of the atypical genetic pattern of AS, gene diagnosis and genetic testing play an important role in the diagnosis. The proband's mother, almost 35, plans to perform the preimplantation genetic diagnosis after genetic counseling.
In conclusion, we identified a novel variant (c.2029G>C) in the UBE3A in a Chinese family with six AS patients. Our findings expanded the genotypic spectrum of AS and provided important information for genetic counseling.