Introduction

Cardio-facio-cutaneous (CFC) syndrome (OMIM 115150) is a multiple congenital anomaly first described by Reynolds et al. [1]. Typical features of CFC syndrome include facial abnormalities (sparse and friable hair, high forehead with bitemporal constriction, hypoplastic supraorbital ridges, downslanting palpebral fissures, a depressed nasal bridge, posteriorly angulated ears with prominent helices), heart defects (pulmonary stenosis, atrial septal defects, hypertrophic cardiomyopathy), and ectodermal abnormalities (hyperkeratotic skin, ichyosis-like condition). In addition, diverse phenotypic characteristics have been reported, which include perinatal problems (polyhydramnios, prematurity), growth failure, neurological problems (seizures, hypotonia, brain anatomical abnormalities), and gastrointestinal problems [2–3].

KRAS, BRAF, MAP2K1, and MAP2K2, which interact with the RAS/mitogen-activated protein kinase (MAPK) pathway, regulate cell differentiation, proliferation, and apoptosis and have been reported as causative genes of CFC syndrome [4,4,5,6,7]. BRAF is a cytoplasmic serine/threonine kinase that is activated by binding to RAS. Among the CFC syndromes, BRAF variant is the most frequently identified (approximately 43% [8] or 35% [9] of patients studied). However, the genotype–phenotype correlation in BRAF variant is still not clearly defined. Here we report a case with a severe clinical phenotype caused by a novel microdeletion of Leucine-485.

Case report

This 2-year-old Japanese boy was the second child of healthy and nonconsanguineous parents. As he was diagnosed with a polycystic kidney by intrauterine ultrasonography, his mother received amniocentesis to treat polyhydramnios at 28 weeks gestational age. He was born after early rupture of the membrane at a gestational age of 30 weeks and 4 days. His birth weight, body length, and occipito-frontal head circumference (OFC) were 1815 g (+2.0 SD), 37 cm (−1.3 SD), and 29.5 cm (+2.0 SD), respectively.

After birth, endotracheal intubation was performed for respiratory management

He undertook nasal-directional positive airway pressure after extubation, but could not withdraw due to frequent apneic spells. Increased apneic spells and muscle stiffness became evident at day 33. At the same time, irritability, an exaggerated startle response, and involuntary movements appeared. His startle response was accompanied with facial flash and apnea, which was exaggerated by tactile or acoustic stimuli. His involuntary movements consisted of intermittent fragmentary myoclonic movements of the extremities, eyelid myokymia, and jaw closing dystonia. Electroencephalography (EEG) at 2 months of age showed equivocal sharp waves in the right temporal area. Startle response and myoclonic movement were not correlated with epileptic discharges. There was no giant sensory evoked potential. The auditory brainstem response was normal. At the age of 4 months, he developed epileptic seizures, which consisted of facial myoclonic movements and tachycardia, tonic–clonic seizures of the upper limbs, and generalized tonic seizures. The interictal EEG showed multifocal spikes in the frontal, central, and temporal areas independently, and ictal EEG showed secondary generalization from their focal discharges (Fig. 1g). We diagnosed him with peculiar encephalopathy with a mixture of focal epilepsy, hyperekplexia, and involuntary movements of unknown cause. Although several drugs were used (vitamin B6, folic acid, lidocaine, clonazepam, valproic acid, carbamazepine, perampanel, and piracetam), his startle response, involuntary movements, and seizures were poorly controlled. Because of his frequent startle response, involuntary movements, and seizure-disturbed effective ventilation, he had a tracheostomy at 5 months. Since then, he has been cared for under continuous mechanical ventilation. Brain magnetic resonance imaging (MRI) at 1 year showed delayed myelination, reduced volume of the white matter, and hypoplasia of the genu to the tail of the corpus callosum (Fig. 1a–d).

Fig. 1
figure 1

Brain magnetic resonance imaging, axial T2WI (a–c), sagittal T1WI (d) at 1 year, echography of the right kidney (e), facial features (f), and ictal EEG at 1 year (g). Delayed myelination and reduced volume of the white matter as well as severe hypoplasia of corpus callosum body to the splenium are remarkable (a–d). Unclear cortico-medullary junction and multiple cysts are evident (e). Sparse hair and eyebrows, broad forehead, coarse face, depressed nasal bridge, short chin, and short neck are characteristic findings (f). The ictal EEG showed focal spikes bursts in the left occipital area spreading to the left temporal area, when he showed facial myoclonus followed by bilateral tonic seizures. Multifocal spikes are also noted (g)

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At present, he is 2 years and 6 months old, he has severe psychomotor retardation with no head control or social smiles, visual pursuit or voluntary movements of the extremities. His awareness is persistently impaired. He has peripheral hypotonia with joint contractures of the elbow, wrist, and knee, as well as pes equinus. He shows severe mixed quadriplegia, startle response, tremor-like myoclonus of the extremities, lip and eye lid myokymia, exaggerated tendon reflexes, and neck rigidity, as well as jaw closing dystonia. Growth failure is also remarkable; his body weight, height, and OFC are 6 kg (−3.4 SD), 60 cm (−5.6 SD), and 41 cm (−3.3 SD), respectively. He has some dysmorphic features (sparse hair and eyebrows, broad forehead, coarse face, depressed nasal bridge, short chin, and short neck; Fig. 1f) and multiple anomalies (hypoplasia of the thorax, atrial septal defect, supravalvular pulmonary stenosis, polycystic and dysplastic kidney (Fig. 1e), cryptorchidism, and ejaculatory duct obstruction). There are no overt skin abnormalities. His startle response is somewhat better than neonatal period. However, involuntary myoclonic movements and seizures are difficult to control despite anticonvulsants (phenobarbital, lacosamide, gabapentin, clobazam) or a ketogenic diet.

We excluded metabolic abnormalities, e.g., mitochondrial disease, sulfite oxidase deficiency, or peroxisomes diseases, from several laboratory tests or ophthalmologic examination. The analyses of causative genes for hyperekplexia, including GLRA1, SLC6A5, GLRB, SLC32A1, and SLC6A, were unremarkable. To explore the molecular diagnosis to clarify his etiology, we performed trio-based whole-exome sequencing and identified a de novo deletion in the BRAF gene (c.1453_1455del p.Leu485del) (NM_004333), which was confirmed by Sanger sequencing. No other pathogenic mutation was observed.

To analyze the effect of p.Leu485del on the ERK pathway, we conducted reporter gene assays for measuring transactivation of ELK, which is a downstream transcription factor of ERK (see Supplementary Methods). We transfected BRAF expression constructs with pFR-luc trans-reporter vector, pFA2-Elk1, and phRL-null in NIH3T3 cells. Relative luciferase activity (RLA) in cells transfected with BRAF p.Leu485del was significantly higher than that in cells expressing wild-type BRAF (Fig. 2). Compared with the p.V600E mutant, the p.Leu485del and p.Leu485Phe mutants showed intermediate activity as described by Wan et al. [10]. These results suggest that the p.Leu485del, as well as p.Leu485Phe, causes increased ELK transactivation, suggesting activation of the ERK pathway.

Fig. 2
figure 2

Stimulation of ELK transcription in BRAF mutations. Relative luciferase activity (RLA) in cells expressing BRAF p.Leu485del, p.Leu485Phe, and p.Val600Glu was significantly higher than that in cells expressing wild-type BRAF. Representative data from three experiments performed in triplicate are shown. Date are shown as mean ± SE of three wells of cells. RLA relative luciferase activity, WT wild-type, NC negative control. *p < 0.05, **p < 0.0001 versus BRAF wild-type by Tukey–Kramer tests.

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Discussion

The present case provides some outstanding findings: (1) a novel BRAF gene variant, p.Leu485del; (2) a severe clinical phenotype, including severe intellectual disability, impaired awareness, hyperekplexia, involuntary movements, early onset refractory seizures, and delayed myelination on brain MRI; and (3) previously unreported malformation in CFC syndrome or RAS/MAPK syndromes related to BRAF variant, including a polycystic and dysplastic kidney.

BRAF is found predominantly in neural tissues, testis, and melanocytic and hematopoietic cells and is a key regulator of the RAS/MAPK pathway, which is important for cell proliferation, growth, and death [5, 11]. The brain MRI in CFC syndrome caused by BRAF variant was described only in 16 patients among 117 CFC syndrome patients reported [2, 6, 7, 12,13,14,15]. Most details were not described, but among them, delayed myelination was reported in two patients, both had Leucine-485 variant. Including present case, these MRI findings indicate that Leucine-485 plays a critical role in the neurodevelopmental process. Missense variants of Leucine-485 have been reported in five patients [6, 12,13,14] (Table 1). Adachi et al. [13] described a girl with frequent involuntary movements, early onset refractory seizures, and delayed myelination on brain MRI similar to our case. However, based on the frequent involuntary movements, hyperekplexia, associated anomalies, and high needs of nursing care, our case can be described as the severe end of the clinical spectrum of Leucine-485 variant.

Table 1 Detailed clinical summary and findings of the patient and previously reported patients
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Several reports have shown that conditional ablation of BRAF in mice results in deficits in extracellular signal-regulated kinase (ERK) activation, a MAPK signaling pathway, and severe neurological impairments, such as dysmyelination, defective oligodendrocyte differentiation, progressive loss of coordination, tremors, and ataxia [16,17,18]. These loss-of-function phenotypes are partially consistent with the present case. However, contrary to the total ablation of BRAF in mice, one study described BRAF p.Q241R knock in mouse showing a gain-of-function mutation [19], which had a CFC-like phenotype, including craniofacial abnormalities (mandibular hypoplasia), kyphosis and ossification in the interparietal bone, multiple heart defects, liver necrosis, and edema. Another report stated that ERK activation via BRAF signaling plays a critical role in renal cystogenesis in PKD2 transgenic mice (PKD2 is the causative gene of human autosomal dominant polycystic kidney disease) [20].

In this study, we showed that p.Leu485del variant indicated activation of the ERK pathway. The result was identical to our previous report of two BRAF variants (p.L485F and p.K499E) of CFC syndrome [6]. From these findings, we can anticipate that the BRAF gain-of-function variant plays an important role in the phenotype of CFC syndrome, including renal cyst formation. But, as we showed in past research, the severity of phenotypes cannot explain only by the activity of ERK pathway [6]. We need more case accumulation to know the pathomechanism affecting the severity of CFC syndrome.

In conclusion, CFC syndrome, especially caused by BRAF variant, should be included in the differential diagnosis of patients with developmental and epileptic encephalopathies and hyperekplexia. We need to keep in mind that among the BRAF variants, point mutation or the deletion of Leucine-485 may be associated with severe symptoms of CFC syndrome.