c.202_204del in NUP214 causes late onset form of febrile encephalopathy

Nucleoporins (NUPs) are a group of transporter proteins that maintain homeostasis of nucleocytoplasmic transport of proteins and ribonucleic acids under physiological conditions. Biallelic pathogenic variants in NUP214 are known to cause susceptibility to acute infection‐induced encephalopathy‐9 (IIAE9, MIM#618426), which is characterized by severe and early‐onset febrile encephalopathy causing neuroregression, developmental delay, microcephaly, epilepsy, ataxia, brain atrophy, and early death. NUP214‐related IIAE9 has been reported in eight individuals from four distinct families till date. We identified a novel in‐frame deletion, c.202_204del p.(Leu68del), in NUP214 by exome sequencing in a 20‐year‐old male with episodic ataxia, seizures, and encephalopathy, precipitated by febrile illness. Neuroimaging revealed progressive cerebellar atrophy. In silico predictions show a change in the protein conformation that may alter the downstream protein interactions with the NUP214 N‐terminal region, probably impacting the mRNA export. We report this novel deletion in NUP214 as a cause for a late onset and less severe form of IIAE9.


| INTRODUCTION
Susceptibility to acute infection-induced encephalopathy-9 (IIAE9, MIM# 618426) is a recently described rare autosomal recessive neurodevelopmental disorder caused by biallelic pathogenic variants in NUP214.Eight individuals from four distinct families have been reported with this condition till date (Egloff et al., 2018;Fichtman et al., 2019;Shamseldin et al., 2019).Most of these individuals presented with infantile or early childhood onset encephalopathy precipitated by febrile illness and harbored homozygous or compound heterozygous missense or frameshift variants in NUP214.In addition to IIAE9, somatic mutations in NUP214 are also known to be associated with acute myeloid and lymphoblastic leukemia (Mendes & Fahrenkrog, 2019).
In this report, we describe a 20-year-old male with milder and late-onset manifestations of IIAE9, harboring a novel in-frame deletion in NUP214, thus expanding the phenotypic and genotypic spectrum of this disorder.

| CASE REPORT
We ascertained a 20-year-old male, the second born of a third-degree consanguineous couple (Figure 1a).At 15 years of age, following a febrile illness, he developed seizures, altered sensorium, and ataxia.
Brain magnetic resonance imaging (MRI) done at 19 years of age showed cerebellar atrophy.At 20 years of age, following another febrile episode, he developed seizures and ataxia.On examination, he had dysarthria, peripheral involuntary movements, nystagmus, hypotonia, and brisk deep tendon reflexes.He had mild hyponatremia (131.4 mEq/L, ref range: 135-145 mEq/L).Ultrasound of the abdomen, echocardiography, and electroencephalography showed normal results.Ophthalmologic and hearing evaluation also did not reveal any abnormality.Brain MRI during this episode showed progressive and severe cerebellar atrophy (Figure 1b).After 3 months, following an episode of febrile illness, he again developed ataxia and other signs of cerebellar dysfunction.Since then, there was no further deterioration in his condition.However, he has residual mild dysarthria, past pointing, and dysdiadochokinesia.
Informed consent with approval from the institutional research committee was taken from the family.Genomic DNA extracted (Qiagen, 51104) from patient's blood cells was used for singleton exome sequencing using the NovaSeq platform (Illumina Inc., USA) with a targeted average coverage of 100Â.Raw reads were aligned to the human reference genome (GRCh38).Overall, the process was similar to that outlined in our previous study (Kausthubham et al., 2021).Validation and segregation analysis of this variant was done by our in-house Sanger sequencing facility.Alignment of sequences of NUP214 of different species was done in Clustal Omega (Sievers et al., 2011).We queried and obtained the wild-type NUP214 protein structure from the Protein Data Bank (PDB) (Berman et al., 2000), and the PDB ID 2OIT (Napetschnig et al., 2007) was selected for analysis.In silico mutagenesis was done by obtaining the amino acid sequence of 2OIT and deleting leucine at the 68th position.Homology modeling was done using SWISS-MODEL (Waterhouse et al., 2018) to obtain the mutant protein structure.The template with the highest overall quality score and sequence identity (>30%) was chosen to build the model of the mutant protein.Visualization of the protein structures was done using Chimera 1.16 (Pettersen et al., 2004).

| DISCUSSION
Susceptibility to acute infection-induced encephalopathy-9 (IIAE9) is a recently described rare autosomal recessive neurodevelopmental disorder.It is characterized by episodic acute neurodegeneration and neuroregression associated with infections and febrile illness (Fichtman et al., 2019;Shamseldin et al., 2019).The individual we describe in this report is a 20-year-old male with milder manifestations of IIAE9 as compared to the previously reported individuals, with onset of condition at 15 years of age.In contrast, seven of the previously reported individuals with IIAE9 presented with infantile or early childhood onset encephalopathy precipitated by febrile illness.However, there is lack of information about the age of onset of encephalopathy in the individual reported by Egloff et al. (2018).These individuals presented with recurrent episodes of febrile encephalopathy that led to variable degree of neuroregression, seizures, ataxia, cerebellar atrophy, less commonly cerebral atrophy, and early death.
All these individuals also had recurrent episodes of hyponatremia.Some of them had partial recovery in between the episodes, but the overall disease course was progressive, leading to global developmental delay, microcephaly, seizures, and cerebellar atrophy (Fichtman et al., 2019;Shamseldin et al., 2019).The individual we report also developed seizures, altered sensorium, ataxia, dysarthria, hyponatremia, and cerebellar atrophy following a febrile illness at 15 years of age.After that, he had two more similar occurrences of febrile encephalopathy; last one at 20 years of age.Partial recovery was observed in between the episodes.On evaluation at 21 years of age, he had mild dysarthria, past pointing, dysdiadochokinesia, and ataxia.Since then, there has been no further deterioration in his condition.
Comparison of the phenotypic and genotypic details of the individuals with NUP214 variants is depicted in Table 1.
Nuclear pore complexes (NPCs) are large passages located within the two layers of the nuclear envelope which regulate the molecular traffic in and out of the nucleus.NPCs consist of several copies of approximately 30 distinct proteins collectively known as nucleoporins (Nup) that form different sub-complexes (Kabachinski & Schwartz, 2015).The three-dimensional structure of NPCs is quite complex.The cytoplasmic side of it attaches to the cytoplasmic filaments, and the nuclear side binds to the chromatin and has a central ring on top of the central pore that is partially covered by a mobile plug.This structural conformation allows NPCs to regulate the nucleocytoplasmic transport along with maintaining the structural integrity of the nuclear envelope (Fichtman et al., 2019).NUP214 and NUP88 form a sub-complex that leads to the formation of a cytoplasmic annual ring that attaches the cytoskeleton to the NPC.NUP214 is involved in the transport of mRNA from the nucleus to the cytoplasm, which is achieved by interacting with NXF1 and ATP-dependent DEAD-box helicase 19 (DDX19) proteins, via its N-terminal region.This interaction is necessary for the steady localization of DDX19 to the cytoplasmic periphery of the NPC (Lin et al., 2018;Mikhailova et al., 2017;Napetschnig et al., 2007).NUP214 also binds to SMAD2, SMAD3, and SMAD4 proteins and plays a key role in their nucleocytoplasmic transport (Shamseldin et al., 2019).NUP214 deficiency has also been observed to severely impede mitosis (Chatel & Fahrenkrog, 2011), and the knockout of Nup214 has shown embryonic lethality in mice models (Mendes & Fahrenkrog, 2019).
Seven of the previously reported individuals harbored homozygous or compound heterozygous missense or frameshift variants in NUP214 (Fichtman et al., 2019;Shamseldin et al., 2019).Fichtman et al. (2019) demonstrated that the levels of NUP214 and NUP88 proteins were decreased in primary skin fibroblasts that were obtained from the affected individuals, but the total number as well as the density of NPCs were normal.They also carried out nuclear transport assays to demonstrate deficiency in the classical protein import as well as in the mRNA export pathways in the affected cells.Direct surface imaging of fibroblast nuclei by a scanning electron microscope showed a substantial increase in the appearance of central plugs in the nuclear pore channels of the affected cells as compared to the controls.Shamseldin et al. (2019) performed functional analysis on the patient-derived fibroblasts that revealed dysmorphic phenotypes of nuclei such as blebs, invagination, micronucleoli, and herniated nuclei.They concluded that the pathogenesis of this condition might involve the perturbation of the nuclear envelope integrity.The individual reported by Egloff et al. (2018) harbored a 1.9-Mb deletion in the 9q34.12q34.13region in trans with an intronic single-nucleotide deletion in NUP214.They identified a significant reduction in the expression level of the NUP214 transcript (close to zero) in the leukocytes obtained from the blood sample of the affected individual as compared to the controls.Also, the expression of NUP214 transcripts was decreased by about 50% in the parental samples.
The individual we report harbored a homozygous in-frame deletion in NUP214.We performed an in silico mutagenesis to elucidate the impact of the p.(Leu68del) variant on the NUP214 protein structure.It was observed that leucine in the wild-type protein is involved in alpha helix secondary structure formation, while the mutant protein revealed a loss of this alpha helix structure.As the NUP214 protein plays an important role in the nucleocytoplasmic trafficking as well as in the mRNA transport from the nucleus to the cytoplasm, we hypothesize that the loss of this secondary structure and its subsequent effect on the downstream protein interactions likely impairs the NUP214 function through its impact on mRNA transport from the nucleus to the cytoplasm.

F
I G U R E 1 (a) Three-generation pedigree (b) Brain magnetic resonance imaging (MRI) shows severe cerebellar atrophy and minimal cortical atrophy, (c) Multiple sequence alignment of the NUP214 protein sequence shows leucine at position 68 is conserved in Homo sapiens, Bos taurus, Sus scrofa, Rattus norvegicus, Mus musculus, Cavia porcellus, and Caenorhabditis elegans, (d) In silico mutagenesis analysis showing the (i) wild-type protein with leucine at the 68th position in the alpha helix structure (yellow) and the (ii) mutant protein showing loss of the alpha helix structure (yellow).
To conclude, biallelic variants in NUP214 lead to an autosomal recessive condition characterized by severe and early-onset encephalopathy in most individuals.However, the individual we report T A B L E 1 Comparison of phenotypic and genotypic details of individuals with NUP214 variants.