Experience with the Ketogenic Diet in a Boy with CLCN4 Related Neurodevelopmental Disorder

Raynaud-Claes syndrome is a rare syndrome linked to the X chromosome. People with this syndrome exhibit facial dysmorphism (long face, prominent chin, flat midface, downslanting palpebral fissures, strabismus), hypotonia, mild to severe intellectual disability, epilepsy, epileptic encephalopathy, behavioral problems, and cerebral atrophy [1]. Some heterozygous females are unaffected; however, mild to severe intellectual disability can be seen in some heterozygous girls. As expected, male patients exhibit a more severe clinical picture [2].

The chloride channel (CLC) gene family comprises nine different channel proteins in mammals, four of which encode plasma membrane CLCs (ClC-1, ClC-2, ClC-Ka, ClC-Kb) and the other five encode intracellular 2Cl−/H+ exchangers (ClC-3–7). The ClC-4 channel protein encoded by the CLCN4 gene in chromosome Xp22.2 is a voltage-dependent 2Cl−/H+ exchanger. Pathogenic variations in the CLCN4 gene cause Raynaud-Claes syndrome (MIM:#300114) listed in the Online Mendelian Inheritance in Man database. ClC-4 is expressed in the brain as well as in striped muscle tissue, heart, intestine, and kidney. CIC-4 is probably involved in the ion homeostasis of endosomes and intracellular trafficking, but its physiological function is still unknown [3].

A study conducted with a meta-analysis revealed that neurodevelopmental disorders associated with CLCN4 have been identified in 122 individuals from 67 families so far [4].

The traditional ketogenic diet is characterized by its high-fat, adequate-protein (1 gram/kg), and low-carbohydrate composition, inducing metabolic alterations reminiscent of a state of starvation. Shifts in plasma ketones, insulin, glucose, glucagon, and free fatty acids may manifest within hours of initiating the diet, exhibiting significant and rapid changes [5].

The effectiveness of ketogenic diet therapy extends to patients with epilepsy across various ages and seizure types, solidifying its status as a beneficial treatment option [6].

Here, we report on three cases of Raynaud-Claes syndrome in a family with a missense variant in the CLCN4 gene.

CLCN4 variants have been associated with X-linked dominant intellectual disability and epilepsy phenotype. Epilepsy is seen in 56% of these patients, and 25% of patients suffer from epilepsy-related deaths. Reported epilepsies are mostly drug resistant and range from absence to epileptic encephalopathy [1,7]. Our patient also has drug-resistant epilepsy accompanied by various types of seizures, such as atypical absence and tonic seizures, and exhibited characteristics of Lennox-Gastaut epilepsy phenotype from both an EEG and clinical perspective [8].

The CLCN4 family of voltage-dependent CLC genes comprises nine members (CLCN-1–7, Ka, and Kb), which demonstrate quite diverse functional characteristics while sharing significant sequence homology. CLCN4 is a voltage-dependent 2Cl−/H+ exchanger. Its precise physiological function is unclear, but ClC-4 is probably involved in the ion homeostasis of endosomes and intracellular trafficking. Additionally, CLCN4 has a significant effect on neuronal differentiation. It was reported that the number and length of dendritic branches decreased significantly in primary hippocampal neurons of CLCN4-null mice and hippocampal or cortical neurons of CLCN4 knock down mice. However, the epilepsy mechanism of CLCN4 variants is still unclear [1,9,10].

So far, 18 missense, 2 frameshift, 1 splice-site, and 1 exonic deletion mutations have been detected in the CLCN4 gene in the literature [1,2,11]. The NM_001830.4: c.1597G>A (p.V533M) variant in the CLCN4 gene detected in this family was previously reported by Fernandez-Marmiesse et al. in a 14-year-old male with Dravet syndrome-like phenotype whose seizures were taken under control by topiramate. This variation in the exon 11 of the CLCN4 gene is known to be located at helical-intra-membrane domains, which play an important role in CLC activity of the CLCN4 protein. This variation was shown to be co-segregated with the disease in our family. This variation is not currently available in population databases (ExAC, gnomAD, 1000 Genomes Project) and was predicted as disease-causing in in-silico analyses (Mutation Taster, Polyphen2, SIFT, CADD). Comparative amino acid sequence alignment of CLCN4 across different species at https://www.ncbi.nlm.nih.gov/homologene revealed that the glycine at position 533 is highly conserved. Considering these data, this variation is thought to be responsible for phenotype [12].

Literature evidence shows that missense variants are more severe than frameshift and intragenic deletions in terms of epilepsy. Our patient also carried a missense variant and had polytherapy-resistant epilepsy. Two separate studies reported that one patient benefited from carbamazepine and one patient benefited from levetiracetam treatment. However, atypical absence seizures were predominant in our patient; therefore, carbamazepine treatment was not initiated. Lamotrigine was reported as beneficial in the literature and treatment could not be continued due to an allergic reaction in our patient. Studies have reported that the effect of valproic acid is limited [1]. Atypical absence status developed under the use of 30 mg/kg/day valproic acid in our patient. It can be concluded that anti-epileptic treatment as first-line therapy is unsuccessful in severe cases. Our patient developed a benzodiazepine-resistant tonic status. There are reports of some Lennox–Gastaut patients developing tonic status with benzodiazepine and the molecular etiopathogenesis of this condition is unclear [13]. This case can lead us to believe that the chlorine channels encoded by CLCN4 caused this. Our patient prominently benefited from ketogenic diet treatment and showed improvement in interictal discharges on EEG. The patient had no seizures for 8 months.

All benzodiazepines enhance the binding of gamma-aminobutyric acid (GABA) to the (GABA) receptor and increase the threat of CLC conductance triggered by the GABA-GABAa receptor interaction following greater chloride influx mediated by an increased frequency of CLC opening [14]. Interestingly, while benzodiazepines do not directly activate channels but only modify GABA binding affinity, phenobarbital can directly promote channel opening in the presence and absence of GABA [15]. The shift in seizure characteristics into tonic status with benzodiazepine use may give us an opportunity to explain the mechanisms of action of CLNC4 on the nervous system.

The male patient ad moderate to severe ID. He had no seizures in the past 8 months under a ketogenic diet. Improvement in social interaction skills and gait were observed. The sister of the patient, who carried the same mutation as heterozygous, has mild ID and her clinical picture is significantly better than her brother. The sister has never had epilepsy. Studies in the literature report normal–moderate ID in female cases and epileptic EEG disorders in some cases. EEG was normal in the sister. The patient’s mother also had mild ID and did not finish primary school. The mother had no history of epilepsy or febrile seizures [2].

Cranial MRI revealed a thin corpus callosum, ventriculomegaly, and white matter atrophy in our patient. Among the reported cases, ventriculomegaly, cortical atrophy, and white matter lesions were reported in 9 patients and no correlation was found between epilepsy severity and cranial abnormality [16].

Ketogenic dietary therapy emerges as a viable treatment option for patients who have not responded to at least two antiseizure medications. There are several conditions in which ketogenic dietary therapy shows notable effectiveness, and it can be considered early in the treatment process. These conditions encompass Doose syndrome, Dravet syndrome, glucose transporter 1 (GLUT-1) deficiency, infantile spasms, pyruvate dehydrogenase deficiency, and tuberous sclerosis complex. Moreover, ketogenic dietary therapy may prove particularly beneficial for individuals with drug-resistant epilepsy who rely on a gastrostomy tube or formula for nutrition.

According to the existing evidence, a consensus panel of experts in 2018 recommended the consideration of ketogenic dietary therapy for children facing drug-resistant epilepsy when two antiseizure medication trials have proven unsuccessful.

Our case is the first one in which a ketogenic diet was applied and yielded successful results in epilepsy cases associated with CLCN4.

In conclusion, many types of seizures can be seen in Raynaud-Claes syndrome, some of which can be life-threatening. CLCN4 variants can be investigated in patients who exhibit an increase in tonic seizures with benzodiazepine treatment. However, ketogenic dietary therapy as the first-line treatment can be lifesaving in resistant epilepsy cases caused by the CLCN4 gene mutations.