An intronic variant in BRAT1 creates a cryptic splice site, causing epileptic encephalopathy without prominent rigidity

Abstract

BRAT1-related neurodevelopmental disorders are characterized by heterogeneous phenotypes with varying levels of clinical severity. Since the discovery of BRAT1 variants as the molecular etiology of lethal neonatal rigidity and multifocal seizure syndrome (RMFSL, OMIM 614498), these variants have also been identified in patients with milder clinical forms including neurodevelopmental disorder with cerebellar atrophy and with or without seizures (NEDCAS, OMIM 618056), epilepsy of infancy with migrating focal seizures (EIMFS), and congenital ataxia (CA). This study aims to examine the consequences and pathogenicity of a novel homozygous splice site variant in BRAT1 in a patient presenting with migrating focal seizures since birth without prominent rigidity. The patient was born from a consanguineous marriage and has had seizures since the neonatal period. He presented with dysmorphic features, pontocerebellar hypoplasia, and migrating focal seizures. Despite supportive treatment, his symptoms rapidly progressed to intractable myoclonic seizures, bouts of apnea and bradycardia, and arrest of head growth, with no acquisition of developmental milestones. Clinical exome sequencing yielded a novel homozygous splice variant in BRAT1. Genetic analysis based on reverse transcription of the patient's RNA followed by PCR amplifications performed on synthesized cDNA and Sanger sequencing was undertaken, and the functional effect of a BRAT1 variant on splicing machinery was demonstrated for the first time. The severe clinical presentation of migrating focal seizures and pontocerebellar hypoplasia in the absence of rigidity further expands the genotypic and phenotypic spectrum of BRAT1-related neurodevelopmental disorders.

Compound heterozygous loss‐of‐function variants in BRAT1 cause lethal neonatal rigidity and multifocal seizure syndrome

“…Since the BRAT1 was first reported as the causative gene of RMFSL (Puffenberger et al, 2012), 45 mutations in the BRAT1 have been reported to date according to the Human Gene Mutation Database HGMD (http://www.hgmd.org/; Professional 2022.1) and the existing literature, including 20 missense/nonsense mutations, 8 splice mutations, 9 small deletions, 5 small insertions/duplications, and 3 gross deletions mutations (Figure 5, Table 1) (Capalbo et al, 2019;Colak et al, 2020;Fernández-Jaén et al, 2016;Hanes et al, 2015;Heide et al, 2020;Li et al, 2021;Na et al, 2020;Qi et al, 2022;Scheffer et al, 2020;Szymańska et al, 2018;Wu et al, 2021). In this study, we found a sixth splice site mutation c.431-2A>G (Colak et al, 2020;Horn et al, 2016;Srivastava et al, 2014;Stödberg et al, 2020;Rudolf et al, 2020). To determine the effect of this splicing variant, Sanger sequencing analysis of the cDNA covering exons 2-8 of the BRAT1 showed that the 3′-splice site acceptor c.431-2A>G variant activated a cryptic acceptor splice site, which resulted in the loss of 29 nucleotides and generation of a premature stop codon at code 180, producing a truncated BRAT1 (c.432_460del; p.Ala145Argfs*36).…”

“…Since the BRAT1 was first reported as the causative gene of RMFSL (Puffenberger et al, 2012), 45 mutations in the BRAT1 have been reported to date according to the Human Gene Mutation Database HGMD (http://www.hgmd.org/; Professional 2022.1) and the existing literature, including 20 missense/nonsense mutations, 8 splice mutations, 9 small deletions, 5 small insertions/duplications, and 3 gross deletions mutations (Figure 5, Table 1) (Capalbo et al, 2019; Colak et al, 2020; Fernández‐Jaén et al, 2016; Hanes et al, 2015; Heide et al, 2020; Li et al, 2021; Na et al, 2020; Qi et al, 2022; Scheffer et al, 2020; Szymańska et al, 2018; Wu et al, 2021). In this study, we found a sixth splice site mutation c.431‐2A>G (Colak et al, 2020; Horn et al, 2016; Srivastava et al, 2014; Stödberg et al, 2020; Rudolf et al, 2020). To determine the effect of this splicing variant, Sanger sequencing analysis of the cDNA covering exons 2–8 of the BRAT1 showed that the 3′‐splice site acceptor c.431‐2A>G variant activated a cryptic acceptor splice site, which resulted in the loss of 29 nucleotides and generation of a premature stop codon at code 180, producing a truncated BRAT1 (c.432_460del; p.Ala145Argfs*36).…”

“…In 2012, Puffenberger et al reported the first RMFSL case with a homozygous variant in the BRAT1 (Puffenberger et al, 2012). Subsequently, BRAT1 variants were found in patients with epilepsy of infancy with migrating focal seizures (EIMFS), neurodevelopmental disorder cerebellar atrophy and with or without seizures (NEDCAS), brain malformation and congenital ataxia (CA) as well as milder non‐lethal clinical symptoms (Celik et al, 2017; Colak et al, 2020; Mundy et al, 2016; Oatts et al, 2017; Rim et al, 2018; Smith et al, 2016; Srivastava et al, 2016; Valence et al, 2019).…”

Clinical variability at the mild end of BRAT1 ‐related spectrum: Evidence from two families with genotype–phenotype discordance

“…Moreover, it is involved in p53‐mediated apoptosis, cellular growth signaling, and mitochondrial homeostasis (Aglipay et al, 2006; So & Ouchi, 2014). Recessive BRAT1 mutations were originally reported to cause rigidity and multifocal seizure syndrome, lethal neonatal (RMFSL, MIM# 614498), a rare congenital disorder characterized by microcephaly, hypertonia, autonomic instability, and intractable seizure, leading to death in the first 2 years of life (Burgess et al, 2019; Colak et al, 2020; Pourahmadiyan et al, 2020; Scheffer et al, 2020; Valence et al, 2019; Van Ommeren et al, 2018). Brain imaging was highly variable, ranging from normal findings to global atrophy (Srivastava et al, 2016).…”

A review of the clinical spectrum of BRAT1 disorders and case of developmental and epileptic encephalopathy surviving into adulthood

“… 12 months Van Ommeren, 2018 [27] 1 Homozygous c.1395G > C, p.(Thr465Thr) Day 1 Myoclonic RMFSL Thinning of corpus callosum Diffuse encephalopathy, with frequent ictal activity from multiple cortical areas. 2 months Mahjoub, 2019 [28] 1 Homozygous c.185 T > A, p.(Val62Glu) No seizures reported None NEDCAS Cerebellar atrophy NA Alive at 24 years 2 No seizures reported None NEDCAS Cerebellar atrophy NA Alive at 7 years Colak, 2020 [29] 2 Homozygous c.1395G > C, p.(Thr465Thr) No seizures reported None NEDCAS Cerebellar atrophy Generalized epileptiform activity, migrating focal epileptiform activity, background deceleration. Alive at 7 years Pourahmadiyan, 2020 [30] 1 Homozygous c.2041G > T, p.(E681*) NK Focal seizures RMFSL NA NA 6 days Scheffer, 2020 [10] 1 Compound heterozygous c.964C > T, p.(Gln322*); c.2284C > T, p.(Gln762*) Day 1 Focal motor migrating between hemispheres EIMFS Underopercularization of the Sylvian fissure Multifocal epileptiform discharges, discontinuous background Ictal: focal seizure migrating from left central region to right hemisphere.…”