Introduction

The nucleus is a double membrane-bound organelle that only permits selective transport through the nuclear pore complex (NPC), which together with the lamin and the double nuclear membrane form the nuclear “envelope” (Cronshaw et al. 2002; Rout et al. 2000). NPC is a universal nuclear feature in all eukaryotes (Field et al. 2014). In mammals, NPC comprises around 30 nucleoporins (NUPs), encoded by NUP genes, that form distinct subcomplexes. The 3D structure of the NPC is complex with an eightfold symmetry comprising multiples of eight of each of the subcomplexes, a twofold symmetry comprising pairs of inner and outer ring complexes, and filamentous projections into the cytoplasm and nucleoplasm (Alber et al. 2007; Schwartz 2005). Cryo-electron tomography studies of fully native NPCs have revealed remarkable details about their 3D structure with distinct features observed on the cytoplasmic and nuclear sides (Stoffler et al. 2003). The cytoplasmic side anchors cytoplasmic filaments while the nuclear side anchors the chromatin and has a central ring on top of the central pore, with the latter being partially obstructed by a mobile central plug (Stoffler et al. 2003). This structural conformation endows NPC with the dual function of regulating nucleocytoplasmic transport as well as maintaining structural integrity of the nuclear envelope (Hetzer et al. 2005).

NUP214 forms a subcomplex with NUP88 such that eight subunits of NUP88–NUP214 form the cytoplasmic annual ring that anchors the cytoskeleton to the NPC (Suntharalingam and Wente 2003; Bastos et al. 1997; Kraemer et al. 1994). It was also found to facilitate the export of mRNA through its interaction with DDX19 (Schmitt et al. 1999). Another study demonstrated direct interaction with TTP (Tristetraprolin), a key player in ARE (AU-rich elements)-mediated transcription-level regulation of inflammatory cytokines such as TNFα, and this interaction may regulate TTP subcellular localization (Carman and Nadler 2004). Similarly, NUP214 directly binds SMAD2, SMAD3 and SMAD4 and regulates their nucleocytoplasmic shuttling (Xu et al. 2002, 2003). Knockout of Nup214 in mouse is embryonic lethal (Van Deursen et al. 1996). At the cellular level, NUP214 deficiency results in cell cycle arrest, impaired nucleocytoplasmic transport and accumulation of polyadenylated RNA in the nucleus (Van Deursen et al. 1996). Additionally, nup214 mutant Drosophila larvae display impaired NES nuclear export, most likely as a result of impaired localization of CRM1, a key mediator of NES nuclear export (Sabri et al. 2007; Xylourgidis et al. 2006). Finally, NUP214 is recruited to the mitotic spindles, and its deficiency has been shown to severely impair mitosis, although the latter may also be caused by impaired nucleocytoplasmic shuttling of molecules that are critical for mitosis (Bhattacharjya et al. 2015; Chatel and Fahrenkrog 2011).

Despite the extensive study of NUP214 in cells and in model organisms, our understanding of its medical relevance remains largely limited to the field of cancer. In fact, NUP214 was first identified in the context of cancer where fusion proteins created by translocations involving the N- and C-termini of NUP214 were identified in ALL (acute lymphoblastic leukemia) and AML disease (acute myeloid leukemia), respectively (Graux et al. 2004; Von Lindern et al. 1992). Very recently, an individual with developmental delay and failure to thrive was reported to have deletion of NUP214 in one allele and a deep intronic likely splicing variant in trans (Egloff et al. 2018). In this study, we present data that support NUP214 as a bona fide Mendelian gene, mutation of which causes an autosomal recessive severe form of early infantile encephalopathy.

Materials and methods

Human subjects

The study family was enrolled in an IRB-approved research protocol (KFSHRC RAC#2080006) with informed consent. Venous blood was collected from the patient, healthy siblings and parents. In addition, banked DNA samples were available from two of the three affected siblings. A skin biopsy was also obtained from the proband for functional studies. Phenotypic data were collected from hospital records.

Autozygome and linkage analysis

DNA samples were genotyped on the Axiom SNP Chip platform following the manufacturer’s instructions. Determination of the autozygome was based on regions of homozygosity > 2 Mb as surrogates of autozygosity given the parental consanguinity followed by mapping of the candidate autozygome that is exclusively shared by the affected members using AutoSNPa (http://dna.leeds.ac.uk/autosnpa). Linkage analysis was used to confirm the candidate autozygome and calculate LOD score based on a fully penetrant autosomal recessive model using the EasyLINKAGE package.

Exome sequencing and variant filtering

Exome capture was performed using the TruSeq Exome Enrichment kit (Illumina, San Diego, CA, USA) as per the manufacturer’s instructions. Samples were prepared as an Illumina sequencing library, and in the second step, the sequencing libraries were enriched for the desired target using the Illumina Exome Enrichment protocol. Captured libraries were sequenced using Illumina HiSeq 2000 Sequencer, and the reads mapped against UCSC hg19 (http://genome.ucsc.edu/) by BWA (http://bio-bwa.sourceforge.net/). The SNPs and Indels were detected by SAMTOOLS (http://samtools.sourceforge.net/). The resulting variants were filtered as follows: homozygous → coding/splicing → within candidate autozygome → absent or very rare in Saudi and public exome databases → predicted to be pathogenic by SIFT/PolyPhen/CADD as previously described (Alkuraya 2013).

Cell culture, immunostaining and enveloping surface ratio (ESR) calculation

We followed a standard protocol of establishing a primary fibroblast cell line from a punch skin biopsy and maintained the cells in a standard culture containing MEM + 10% FCS, 1% l-glutamine and 1% penicillin. The following antibodies were used under standard immunofluorescence conditions: Anti-NUP214 antibody (ab70497). Images were taken using Zeiss Axioimager Z2. For ESR (envelope vs surface ratio) calculation, we followed a previously published protocol (Noda et al. 2015). Briefly, exact area of a nucleus was measured as the area surrounded by the contour line, and then a polygon area was divided by the exact area to make the ESR (Noda et al. 2015). Nuclei with a ratio of ≥ 1.05 were defined as dysmorphic.

Results

Clinical report

The proband (IV:8) is a 13-month-old girl with severe hypotonia and profound global developmental delay. She was born at term with normal growth parameters. She was admitted at age 2-weeks because of poor weight gain and cyanotic spells and was found to have severe hypotonia and poor suck, but no gross facial dysmorphism. Her severe hypotonia persisted and evolved into profound global developmental delay where she at age 13 months cannot roll over, vocalize or track visually. Abnormal movements were frequently reported and although her EEG was not conclusive for frank epileptiform discharges, she was diagnosed clinically with epilepsy, which remained poorly controlled with therapy. She had frequent lower respiratory tract infections initially thought to be related to aspiration but they persisted even after cessation of oral feeding, placement of gastrostomy tube and undertaking of fundoplication. She had multiple admissions to the PICU for respiratory failure thought to be due to a combination of severe hypotonia and central apnea. Although her somatic growth showed improvement after gastrostomy tube feeding, she had progressive microcephaly. She had extensive metabolic investigations including CSF metabolites, plasma amino acids, acylcarnitines, urine organic acids and mitochondrial sequence analysis, all of which were negative. Her brain MRI was initially normal but later showed evidence of nonspecific atrophy. Nerve conduction studies were normal. Ophthalmologic examination suggested central vision loss. Clinical whole exome sequencing did not reveal any pathogenic variant that explains her phenotype. She died at age 15 months due to respiratory failure. Family history was notable for first cousin healthy parents, two healthy siblings, two siblings who died with an identical clinical presentation at age 1 year and one sister who died within hours of delivery with severe hypotonia (Fig. 1; Table 1).

Fig. 1
figure 1

a Pedigree of the study family (genotypes for the identified mutation in NUP214 are shown). b Clinical images showing lack of gross facial dysmorphism. c EasyLINKAGE revealed a single locus on chromosome 9 (NUP214 is boxed in red)

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Table 1 Clinical findings
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NUP214 is mutated in a novel recessive form of encephalopathy

By combining the genotypes of the proband, her two deceased affected siblings (no DNA was available from the sister who died shortly after birth), the two healthy siblings and the parents, we were able to identify a candidate autozygous interval chr9:127,113,732–135,288,807 delineated by rs12555646; rs2885495. This was the only autozygous interval exclusively shared by the affected members of the family. Linkage analysis confirmed this interval and assigned a LOD score of 3.2 (Fig. 1c). Although clinical exome sequencing was “negative”, we performed our own exome re-sequencing and analysis since our experience suggested that many such “negative” cases may harbor mutations in novel disease genes (Shamseldin et al. 2017). Indeed, we identified a single novel variant in NUP214 (NM_005085.3:c.461A>G,p.D154G), a gene with no OMIM entry for Mendelian diseases, within the candidate locus, and Sanger sequencing confirmed its full segregation with the phenotype in the family in a strictly autosomal recessive fashion (Fig. 1a). This variant is absent in gnomAD and local database of ethnically matched exomes and is predicted to be pathogenic by SIFT (0.0), PolyPhen (0.9) and CADD (25), and is highly conserved in different species (Fig. 2a, b).

Fig. 2
figure 2

a Sanger chromatogram of the mutation. b Conservation of the mutated residue in different species. c RT-PCR showing expression of NUP214 in different human tissues. d Cartoon for NUP214 transcript and proteins (arrow denotes the position of mutated base)

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p.D154G impairs the function of NUP214 and leads to abnormal nuclear morphology

Multilobulated, even “flower-like” nuclei have been observed when two individual NUPs were knocked down: NUP153 and NUP214 (Bhattacharjya et al. 2015; Hashizume et al. 2010; Lussi et al. 2010). Thus, we asked whether p.D154G recapitulates the knockdown phenotype of NUP214 in the nucleus of the patient fibroblasts, especially after demonstrating that NUP214 is ubiquitously expressed in different human tissues (Fig. 2c). Indeed, dysmorphic nuclei (defined morphologically by the presence of blebs, hernias, invaginations, and/or lobulations) were far more frequently observed in the patient cells vs controls (Fig. 3a, b). In order to quantify this defect, we measured the ESR, and found that (a) number of nuclei with ESR of > 1.05 in the patient cells is nearly three times that in controls (Fig. 4c) and (b) average ESR in the patient cells is higher than that in controls (Fig. 4D). NUP214 has a characteristic rim-staining pattern (Kinoshita et al. 2012). This pattern was clearly displaced to the nucleoplasm in our patient cells (Figure S1). Taken together, these results show that the p.D154G variant is a deleterious variant that recapitulates the induced NUP214 deficiency described previously.

Fig. 3
figure 3

a DAPI staining showing different dysmorphic features in the nucleus: blebs (yellow diamond), invagination (white arrows), micro-nucleoli (yellow arrow), and herniated nuclei (white asterisk). b Bar graph of the percentage of nuclei with dysmorphic morphology in the patient vs control fibroblasts

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Fig. 4
figure 4

a Calculation of ESR using polygon area divided by the exact area to calculate the degree of nuclear dysmorphology (see text). b Correlation of increased ESR with nuclear dysmorphology. c Bar graph of the percentage of nuclei with ESR ≥ 1.05 (p value = 0.007). d Average of ESR values in the patient vs control nuclei (error bars are also shown)

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Discussion

NUP214 was first studied in the context of cancer where fusion proteins created by translocations involving the N- and C-termini of NUP214 were identified in ALL and AML disease, respectively (Graux et al. 2004; Von Lindern et al. 1992). NUPs share the following major domains in an N- to C- orientation: coiled-coils, FG repeats, α-helical solenoids, β-propellers, and zinc fingers (Devos et al. 2006). The N-terminus has a seven-bladed beta propeller with each blade comprising four antiparallel beta strands that together form a sevenfold axis around a central cavity (Napetschnig et al. 2007). Our variant p.D154 constitutes the third residue of the VIDMKW motif in the A strand of the third blade, which is required for the hydrogen bonding with other beta strands (Napetschnig et al. 2007). This may explain why the p.D154G variant we detected in the study family is associated with such a dramatic disruption of NUP214 localization and function.

The nuclear envelope is a dynamic structure as best seen in its dissolution during cell division (nuclear envelope breakdown, NEBD), which is accompanied by the disassembly of NPC into its individual NUP subcomplexes followed by their reassembly as daughter cells reform their nuclear envelopes (Hetzer et al. 2005). Thus, it is possible to speculate that the pathogenesis of abnormal nuclear morphology we observed in the context of NUP214 mutation is related to impaired dynamics of nuclear envelope stability.

The critical role played by NPC in development is readily evident by the fact that complete knockouts of individual NUPs are usually embryonic lethal in mouse or associated with significant phenotypic abnormalities (Sakuma and D’Angelo 2017). Several aspects of the NPC’s relevance to human diseases have emerged in recent years. In Huntington disease, severe mislocalization and aggregation of NUPs with accompanying abnormal nucleocytoplasmic transport have been observed (Grima et al. 2017). More relevant to the Mendelian disorder we describe in this study is the recent identification of NUP mutations in the context of brain developmental disorders. In 2015, we described a family with a homozygous truncating variant in NUP107 and a combination of microcephaly and chronic kidney disease, also known as Mowat–Galloway syndrome (Alazami et al. 2015). Since then, several studies have similarly shown that biallelic mutations in NUP107 and a number of other NUP genes cause varying combination of microcephaly and glomerular renal disease (Braun et al. 2015, 2018). Very recently, a case with a compound heterozygous mutation in NUP214 was described, albeit with very limited clinical information (developmental delay, failure to thrive and some dysmorphic features) (Egloff et al. 2018).

To our knowledge, the phenotype of NUP214-related encephalopathy we describe here is the most severe early infantile brain-related phenotype observed thus far in the context of NUP mutations. Given the fully lethal phenotype of complete Nup214 deficiency in mouse models, it is likely that our mutation despite its profound deleterious effect is not a complete functional null. The specific involvement of certain body parts in the pathogenesis of NUP-related disorders remains poorly understood although it may be related to tissue-specific differences in the composition of NPCs (Raices and D’angelo 2012). The spatial and temporal variation in NPC composition was first demonstrated for NPU210, but has since been shown for several other NUPs (D’Angelo et al. 2012; Lupu et al. 2008). It is also possible that the demand for normal nucleocytoplasmic transport differs among organs, and that the brain may be particularly vulnerable to impairment of such a fundamental cellular function as has been shown for several others.

In conclusion, we propose that while NUP214 complete deficiency may be lethal in humans, partial deficiency results in a novel autosomal recessive disorder characterized by severe encephalopathy and early death. The pathogenesis of this disorder remains to be fully elucidated although it does seem to involve perturbation of the nuclear envelope integrity. Future cases will shed light on the allelic and phenotypic spectrum of NUP214-related encephalopathy syndrome.