Atypical clinical course in two patients with GNB1 variants who developed acute encephalopathy

Introduction: Variants in the GNB1 gene, which encodes the b 1 subunit of a trimeric G protein, can cause moderate to severe psychomotor retardation. Acute encephalopathies have also been observed in patients with central nervous system abnormalities; however, severe neurological sequelae have not previously been reported. Case presentations: Patient 1 was a Japanese female with a de novo GNB1 variant (c.284 T > C). At 8 months old she contracted inﬂuenza A and developed generalized convulsions. In the acute phase, brain magnetic resonance imaging (MRI) ﬁndings indicated acute encephalopathy; diﬀuse cerebral atrophy was present 1 month later. Although multidisciplinary treatment was administered, she had severe neurological sequelae including spastic tetraplegia, severe intellectual disabilities, and refractory epilepsy. Patient 2 was a Japanese male with a de novo GNB1 variant (c.239 T > C). He experienced an unexplained respiratory arrest aged 17 years; refractory convulsions developed. Brain MRI at 1 month showed bilateral basal ganglia high intensities; at 3 months, diﬀuse cerebral cortex and white matter atrophy was observed. Despite multidisciplinary treatment, he developed severe spastic tetraplegia and mental regression. Discussion: We report two patients with GNB1 variants who had acute lesions on brain MRI and unexpected disease courses. In such patients with acute neurological deterioration, multidisciplinary treatment is required; patients should also be carefully observed for progression to acute encephalopathy. (cid:1) 2023 Published by Elsevier B


Introduction
The GNB1 gene encodes the b1 subunit of a trimeric G protein (Gb1). The clinical features of psychomotor retardation caused by GNB1 variants have become clear in recent years [1], and more than 60 cases have been reported to date. The core symptoms are moderate to severe intellectual disability, epilepsy, dystonia, and  anomalies. Nonetheless, acute encephalopathies have been reported in patients with underlying diseases associated with various central nervous system abnormalities [2]. However, there is no known association between patients with GNB1 variants and acute encephalopathies with subsequent severe neurological regression. We report two cases with GNB1 variants who developed acute encephalopathies at different ages with severe neurological sequelae. Magnetic resonance imaging (MRI) (Fig. 1) and electroencephalograms (EEG) (Fig. 2) were performed both before and after illness onset in each patient.
This study was approved by the ethical review board of Kanagawa Children's Medical Center (approval number 145-1). Informed consent was obtained from the parents of each patient for whole-exome sequencing [3] and publication.

Patient 1
The patient was a Japanese female carrying a GNB1 variant (NM_002074.4: c.284 T > C: p.Leu95Pro, de novo). She was the second child of healthy parents. There was no consanguinity or family history of neurological disorders. Prenatal fetal hydronephrosis and oligohydramnios were noted. She was born by cesarean section at 36 weeks with a weight of 2437 g (À1.4 standard deviation [SD]), head circumference 33.0 cm (±0 SD), and 1-and 5-minute Apgar scores of 3 and 6, respectively. She received continuous positive airway pressure treatment until day 3 for transient tachypnea. She had multiple arthrogryposes and muscle hypotonia, including limited extension of the fingers and marked varus deformities of both feet. Brain MRI at 2 months indicated the possibility of hypoplasia of the pons but was otherwise normal (Fig. 1a, b). Bilateral hydronephrosis and left duplicated ureter were observed. No seizures were noted during early infancy and an EEG at 40 weeks of gestation showed a normal neonatal pattern (Fig. 2a).
At 8 months of age, shortly after the onset of influenza A, the patient developed generalized convulsions lasting for 80 min that required intravenous midazolam. On admission, her eyes were open and fixed in the primary position with some limb movements, accompanied by a positive Babinski reflex. On day 2 of illness, lethargy developed, but EEG did not reveal highvoltage slow activities or epileptiform discharges. However, elevated serum enzymes-including aspartate aminotransferase (506 IU/L), alanine aminotransferase (285 IU/L) and lactate dehydrogenase (658 IU/L)-ap peared without cerebrospinal fluid pleocytosis. Because we suspected acute encephalopathy, methylprednisolone pulse therapy and edaravone were introduced. On day 3, EEG showed generalized periodic discharges (Fig. 2b). She was treated with additional combination therapy of immunoglobulin, cyclosporine and 8 days of induced hypothermia because prolonged cerebral edema was indicated on sequential MRI. Despite multidisciplinary treatment, she had severe neurological sequelae including spastic tetraplegia (Gross Motor Function Classification System [GMFCS] level V), severe intellectual disabilities (intelligence quotient < 20), and refractory epilepsy requiring multiple medications (valproate and levetiracetam) with daily tonic seizures; she subsequently needed a tracheostomy. The brain MRI from the acute phase showed symmetrical high-signal abnormalities in the diffuse subcortical white matter in diffusion-weighted imaging (DWI) resembling acute encephalopathy with biphasic seizures and late reduced diffusion (AESD) (Fig. 1b, e). One month later, diffuse cerebral atrophy became evident ( Fig. 1c, f). EEG in the chronic stage showed frequent multifocal spikes without posterior dominant rhythm with diffuse background slowing (Fig. 2c).

Patient 2
The patient was a Japanese male with a GNB1 variant (NM_002074.4: c.239 T > C: p.Ile80Thr, de novo) who was the first child of healthy parents. He was born full term with normal delivery and had a body weight of 3054 g (+0.1 SD) and head circumference of 35.0 cm (+1.2 SD); cryptorchidism was noted. There was no consanguinity or family history of neurological disorders. Immediately after birth, he developed pneumothorax that required 4 days of oxygen therapy. At 1 month of age, he was diagnosed with left muscular torticollis. At 5 months, muscular hypotonia was noted. Generalized tonic seizures developed at age 2. Because the EEG showed multifocal spikes with generalized features (Fig. 2d), he was diagnosed with symptomatic epilepsy and treated with valproate, vitamin B 6 , and zonisamide. Gastrostomy was performed at the age of 16 because of decreased oral intake. In adolescence, the patient was able to follow moving objects with his eyes and perform simple purposeful movements of the upper limbs although they were dystonic. He was non-ambulatory as a result of lower limb spasticity and weakness, with marked distal arthrogryposes.
Aged 17, the patient had an unexplained respiratory arrest. Despite prompt resuscitation by his parents, refractory convulsions developed. On admission, his Glasgow Coma Scale score was 6 (E1V1M4). Because the seizure cluster was not controlled with midazolam, fosphenytoin, levetiracetam, and topiramate were added and valproate dose was increased. Therapeutic normothermia was induced. On day 6 after the acute event, he was weaned off the ventilator. Although multidisciplinary therapy was provided, severe spastic tetraplegia at GMFCS level V and intellectual regression (intelligence quotient < 20) occurred. One month after the acute event, brain MRI (DWI) showed prolonged high intensities in the bilateral basal ganglia with spared cerebral cortex and white matter (Fig. 1h,k), indicating a dif-ferent course from hypoxic encephalopathy. Three months after the event, diffuse atrophy of the cerebral cortex and white matter became apparent, and the abnormal signal in the basal ganglia disappeared (Fig. 1i,l). Along with his neurological worsening, Fig. 1. Axial sections of brain magnetic resonance imaging (MRI) of Patients 1 (a-f) and 2 (g-l). T1-weighted images (a-c, g-i), T2-weighted images (d, j), and diffusion-weighted images (DWI) (e, f, k, l) are shown at the basal ganglia level. Images were taken before the onset of acute encephalopathy (Patient 1: 2 months old, a, d; Patient 2: 6 years old, g, j), and at 5 days (b, e), 1 month (c, f, h, k), and 3 months (i, l) of illness. Note that the diffuse high-signal area in subcortical white matter (Patient 1) and the bilateral basal ganglia (Patient 2) on diffusion-weighted imaging (DWI) were followed by delayed diffuse cortical atrophy.
EEG findings developed into a low-voltage pattern accompanied by generalized slowing (Fig. 2e). Antiseizure medications were tapered off as clinical seizures resolved during the chronic phase.

Discussion
Patients with the GNB1 variants have moderate to severe neurological symptoms; more than half have difficulty walking as well as speech and language delays [4]. The most common variant site is the Ile80 in exon 6, which accounts for nearly 25% of all cases. This variant is thought to have genotype-phenotype correlations because many carriers present with dystonia and growth disturbances [4], as seen in Patient 2 (although his growth disturbance was unremarkable). The Leu95Pro variant in exon 7 has also been previously reported as pathogenic and represents approximately 8% of all cases; it has no known specific genotype-phenotype relationships.
Other than neurological symptoms, ocular abnormalities such as strabismus occur in 58% of patients; 61% of patients have gastrointestinal abnormalities such as constipation, cyclic vomiting, gastroesophageal reflux, and abdominal distension; and 50% of male patients have genitourinary abnormalities [4]. Mastocytosis has been identified in five patients, with a significantly higher prevalence than that of the general population, suggesting an association with gene variants [4][5][6]. Our patients had feeding difficulties as well as genitourinary malformations such as hydronephrosis, a left duplicated ureter in Patient 1, and cryptorchidism in Patient 2.
Although neurological regression has been noted in some cases with GNB1 variants [7], all such cases had a loss of acquired language and are thus considered to have a distinct pathology from the present cases. Hemati et al. [4] reported monozygotic twins with acquired abnormal brain MRI findings caused by ischemia/inflammation; however, there were no episodes of acute disease in the clinical course after birth, thus suggesting the possibility of a perinatal complication. Influenzaassociated encephalopathy is relatively prevalent in East Asia and develops predominantly as a form of cerebral cytokine storm, such as acute necrotizing encephalopathy, hemorrhagic shock and encephalopathy syndrome, or Rye syndrome, although any type of acute encephalopathies may occur [8]. The brain MRI in Patient 1 had similar signal changes to the findings seen in AESD [9,10]. AESD is a type of acute encephalopathy in which delayed clusters of seizures develop after a few days of lucid interval following status epilepticus, resulting in various degrees of neurological deficits. Notably, our patient had a different course to that of AESD in that she did not have a period of lucid interval or second seizures. In Patient 2, DWI abnormalities were observed for more than 1 month, which is unusual for hypoxic encephalopathy. There are few reports of DWI changes after hypoxic events post-adolescence, and acute-phase distribution patterns of abnormalities are highly variable. However, global hypoxic cerebral injury-induced DWI changes evolve slowly and peak around 2-4.5 days after the event [11]. The prolonged bilateral basal ganglia lesions in this patient appear similar to those of metabolic encephalopathies such as mitochondrial diseases [12] or Creutzfeldt-Jakob disease [13]. In a retrospective study, Hirayama et al. [2] reported that 25.4% of patients with acute encephalopathies had underlying diseases associated with central nervous system abnormalities; they speculated that prolonged neurotoxicity as the result of hyperexcitation of cortical neurons may be the underlying cause. The molecular mechanism of the pathogenicity of GNB1 variants has recently been elucidated. In a study that focused on seizure susceptibility in relation to the dysfunction of G protein-gated inwardly rectifying K + channels (GIRK), which are responsible for membrane hyperpolarization, Reddy et al. [14] demonstrated that Gb1 proteins increased or decreased in a mutation-specific manner when GIRK channels were co-expressed with mutated Gb1 proteins in Xenopus oocytes. They reported that the Ile80 mutation of Gb1 proteins especially reduced GIRK channel function via the partial loss of Gb1 expression. It may therefore be that, although the manifestations in the two patients of different ages were not the same, the excitatory-inhibitory instability of neuronal networks triggers acute brain inflammation when damage occurs as the result of an infection or other factors.
In conclusion, we have reported two cases of psychomotor retardation with GNB1 variants, each of whom presented with acute lesions on brain MRI and had an unexpected disease course with severe neurological sequelae. Careful observation is necessary to ensure the prompt introduction of multidisciplinary treatment in the event of acute neurological deterioration, such as prolonged seizures or unexplained respiratory arrest, in such patients. Moreover, the possibility of progression to acute encephalopathy should always be considered.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.