Biallelic mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.


Introduction
The nucleoporin NUP88 [MIM 602552] is a constituent of the nuclear pore complex (NPC), the gate for all trafficking between the nucleus and the cytoplasm [1]. NUP88 resides on both the cytoplasmic and the nuclear side of NPCs [2] and it is found in distinct sub-complexes: on the cytoplasmic face it associates with NUP214 [ [2][3][4][5]. The NUP88-NUP214 complex plays an important role in the nuclear export of a subset of proteins and pre-ribosomes, which is mediated by the nuclear export receptor CRM1 (Required for chromosome maintenance, alias exportin 1, XPO1 [MIM 602559]) [6][7][8]. Depletion of NUP88 alters the intracellular localization of NF-κB proteins [9][10][11]. Moreover, NUP88 is frequently overexpressed in a variety of human cancers and its role therein appears linked to the deregulation of the anaphase promoting complex [12,13] and its binding to vimentin [14].
Fetal movement is a prerequisite for normal fetal development and growth. Intrauterine movement restrictions cause a broad spectrum of disorders characterized by one or more of the following features: contractures of the major joints (arthrogryposis), pulmonary hypoplasia, facial abnormalities, hydrops fetalis, pterygia, polyhydramnios and in utero growth restriction [15]. The unifying feature is a reduction or lack of fetal movement, giving rise to the term fetal akinesia deformations sequence (FADS [OMIM 208150]) [16]. FADS is a clinically and genetically heterogeneous condition of which the traditionally named Pena-Shokeir subtype is characterized by multiple joint contractures, facial abnormalities, and lung hypoplasia resulting from the decreased in utero movement of the fetuses [15]. Affected fetuses are often lost as spontaneous abortions (in utero fetal demise) or stillborn. Many of those born alive are premature and die shortly after birth. In the past, the genetic basis for these disorders was frequently unknown, but due to the recent availability of next generation sequencing, the molecular etiology is becoming increasingly understood. Many cases of FADS result from impairment along the neuromuscular axis and from mutations in genes encoding components of the motor neurons, peripheral nervous system, neuromuscular junction and the skeletal muscle. Genes encoding components critical to the neuromuscular junction and acetylcholine receptor (AChR) clustering represent a major class of FADS disease genes, these include RAPSN [MIM 601592] [17,18], DOK7 [MIM 610285] [19], and MUSK [MIM 601296] [20], as well as mutations in the subunits of the muscular nicotinic acetylcholine receptor (AChR) [17,21]. These mutations are expected to affect neuromuscular junctions [22].
Here, we report a Mendelian, lethal developmental human disorder caused by mutations in NUP88. We demonstrate that biallelic mutations in NUP88 are associated with fetal akinesia of the Pena-Shokeir-like subtype. We confirm in zebrafish that loss of Nup88 impairs locomotion behavior and that the mutant alleles are functionally null. We show that loss of NUP88 affects protein levels and localization of rapsyn in cell lines and subject samples. Consistent with altered rapsyn, AChR clustering in zebrafish is abnormal. We propose that defective NUP88 function in FADS impairs neuromuscular junction formation.

Identification of NUP88 mutations in individuals affected by fetal akinesia
We performed exome sequencing and Sanger sequencing on genomic DNA from individuals affected with FADS from two families (Fig 1A). Clinical and genetic findings are summarized in Table 1, pedigrees and gene structure are shown in Fig 1A and 1B. Family A comprises four affected individuals, three male and one female (Fig 1A; A.II. 3,4,5,7), and four healthy siblings born to consanguineous parents of Palestinian origin. Exome sequencing of the last affected fetus A.II.7 revealed a homozygous missense mutation c.1300G>T (p.D434Y) in the NUP88 gene [NM_002532.5] (Fig 1A), absent in relevant databases (dbSNP, Ensembl, UCSC, TGP, ExAC, HGMD, gnomAD). Sanger sequencing revealed identical homozygous missense mutation in the third affected fetus (Fig 1A, A.II.5; S1A Fig). Both parents and unaffected siblings A.II.1, A.II.2 and A.II. 6 are heterozygous carriers of the mutation, unaffected sibling A. II.8 carries two intact alleles of NUP88 after in vitro fertilization and preimplantation diagnostic ( Fig 1A). DNA was unavailable from the first and second miscarriage (A.II.3 and A.II.4), but clinical phenotypes resemble those of the two affected individuals A.II.5 and A.II.7 (Table 1). In Family B, one affected son was born to healthy unrelated parents of European descent. Exome sequencing in the affected individual, his parents and his two unaffected sibs (S1B Fig) revealed that the individual is compound heterozygous for two NUP88 mutations, i.e. a nonsense c.1525C>T (p.R509 � ) and a single amino-acid deletion c.1899_1901del (p. E634del; Fig 1A; B.II.2), absent in relevant databases. Parents and healthy siblings were heterozygous carriers of the one or the other of the mutations, thus confirming correct segregation consistent with recessive inheritance (Fig 1A). The missense substitution p.D434Y and deletion p.E634del affect evolutionary highly conserved NUP88 residues ( Fig 1B) indicating functional relevance. Accordingly, SIFT/Provean, Polyphen-2, and MutationTaster predicted both mutations to be disease causing or potentially pathogenic (S1 Table).
Models obtained (see Methods) predicted the N-terminal domain (NTD) to form a 7-bladed ß-propeller, set up in a (4,4,4,4,4,4,3) arrangement of ß-strands and no Velcro lock as typical for classical ß-propellers (Fig 2A). Around 60 residues precede the ß-propeller and are located at the bottom or side of the propeller thereby shielding 2-4 blades in their vicinity (Fig  2A). The model reveals high similarity to the PDB deposited structures of Nup82 from Baker's yeast and Nup57 from Chaetomium thermophilum (Fig 2B). The most prominent differences are a loop region and a helix-turn-helix (HTH) motif emanating from blades 4 and 5, respectively ( Fig 2B). Models obtained for NUP88's C-terminal domain (CTD) exhibited low reliability, but the CTD, in analogy to its yeast homolog, is likely composed of extended α-helices ( Fig  2C) that form trimeric coiled-coils, either in cis or in trans. In this context, an arrangement NUP88 in fetal akinesia with its complex partners NUP214 and NUP62 in trans is most likely, as described for the yeast counterpart of the complex [23,24]. According to the model structure, the p.D434Y mutation is located in the loop of a HTH motif between the two outermost ß-strands of blade 6 ( Fig 2B,  magnification). The mutation likely leads to a decrease in the interaction with one of the neighboring proteins, thereby leading to a destabilization of the complex. The nonsense mutation c.1525C>T resulting in p.R509 � is located just after the ß-propeller in the linker region to the CTD resulting in a complete loss of all α-helices. Thus, the interaction of NUP88 with its complex partners is likely reduced to only propeller interactions, if the protein is not completely lost due to nonsense mediated decay of the mRNA. The p.E634del mutation is located in the middle of the CTD sequence and predicted to lie in the last fifth of an extended helix. The deletion results in a frame-shift of the remainder of the α-helix, which shifts the following residues by about a third of a helical turn and thus disrupts the interaction pattern of all following residues, which, as a consequence, decreases the overall stability of the interactions within this helix bundle.

Danio rerio nup88 model
FADS is a developmental disorder and to study the function of NUP88 in vertebrate development, we used a zebrafish (D. rerio) model. We first examined the spatial expression of nup88 during embryonic development. The single zebrafish nup88 orthologue (ENSDARG00000003235) encodes a protein of 720 amino acid translated from a single 2410 bp transcript. The predicted translated gene product shares 63% identity and 75% similarity with human NUP88. Whole-mount in situ hybridization (WISH) and RT-PCR analysis in wild-type AB zebrafish showed that nup88 transcripts are maternally deposited early in development (S2A and S2B Fig, four-cell-stage embryos) and then ubiquitously expressed at 5 hours post fertilization (hpf). By 24 hpf, while expressed ubiquitously, particularly high levels of nup88 mRNA were detected in highly proliferative frontal regions of the embryo, i.e. the central nervous system, brain, eye and anterior trunk. At 72 hpf, nup88 transcript levels are decreasing in these frontal regions and only slightly higher than in other regions of the zebrafish larvae. Similar expression patterns in the developing zebrafish have been described for the two NUP88-binding partners, NUP98 and NUP62 [25,26].

Genetic disruption of nup88 affects zebrafish development
To study the impact of nup88 deletion on zebrafish development, we used the nup88 sa2206 allele generated by the Zebrafish Mutation Project [27,28]. Heterozygous nup88 sa2206 carriers were outcrossed for four generations with wild-type AB zebrafish prior to phenotypic analysis. The nup88 sa2206 allele is characterized by a nonsense mutation, c.732T>A (Fig 3A), resulting in a premature stop codon at amino acid 244. nup88 mRNA levels are reduced by about 90% in 5 dpf nup88 mutants (see below), suggesting that the mRNA is subjected to nonsense-mediated decay. For the purpose of this study, nup88 sa2206/sa2206 is therefore referred to as nup88 -/-. During early stages of development and up to 3 dpf, no marked differences in morphological features of nup88 -/compared to nup88 +/+ and nup88 +/siblings were observed. Starting at 4 dpf, phenotypic alterations became visible: smaller head and eyes, lack of a protruding mouth, downwards curvature of the anterior-posterior axis, abnormal gut and aplastic swim-bladder ( Fig 3B). Further analyses of the cranial abnormalities revealed that nup88 -/larvae exhibit severe defects in the ventral viscerocranium formed by seven cartilaginous pharyngeal arches [29,30]. In nup88 -/larvae, the posterior pharyngeal arches 3-7 were dramatically reduced, distorted or even absent ( Fig 3C). The reduced size of head and eyes correlated with an increase in apoptosis in the head of nup88 -/embryos ( Fig 3D). Apoptotic cells, as assessed by acridine orange staining, were readily detected in the eyes, the brain and the anterior trunk of 35 hpf mutant embryos, but not in other parts of the body (S2C Fig). Together, these data indicate that nup88 mutants are phenotypically similar to the large class of jaw and branchial arch zebrafish mutants, designated as the flathead group [31,32]. Disruption of nup88 furthermore led to impaired survival with lethality occurring at or after 9 dpf (Fig 3E).

FADS-related mutations in nup88 lead to a loss-of-function phenotype
To address the question whether NUP88 mutations identified in the familial cases of FADS affect NUP88 function, we performed phenotypic rescue experiments in zebrafish. Two of the three mutated residues in the uncovered FADS cases are conserved between human and zebrafish (Fig 1B), hence we introduced the corresponding mutations on zebrafish expression constructs by site-directed mutagenesis. Human c.1300G>T, p.D434Y corresponds to c.1240G>T, p.D414Y in zebrafish and human c.1899_1901del, p.E634del to zebrafish c.1837_1839del, p. E613del. Human p.R509 is not conserved in zebrafish, therefore we inserted a stop codon at c.1468-1470>TGA, p.H490 � , a residue in a similar position as human R509. Subsequently synthetic mRNA corresponding to each variant was microinjected into one-cell stage nup88 -/mutants and their rescue capacity was assessed by evaluating the eye size as well as the number and morphology of the pharyngeal arches. Injection of wild-type (WT) nup88 mRNA partially rescued the developmental defects of the 5 dpf mutant larvae as indicated by significant restoration of the eye size (Fig 4A and 4B) and a significant increase in the number of pharyngeal arches (Fig 4A and 4C). In addition, the arches resembled the morphologically wild-type structures (Fig 4A and 4D). In contrast to WT nup88 mRNA, injection of nup88 D414Y mRNA, nup88 H490 � mRNA as well as nup88 E613del mRNA failed to suppress the nup88 -/phenotypes ( Fig 4A-4D), indicating that these nup88 mutant transcripts are functionally null.
Although the results suggest that the encoded variant proteins are functionally inactive, the lack of rescue could also be the consequence of transcript or protein instability.

nup88 mutants show strongly impaired locomotor behavior
To further investigate the implication of NUP88 in the etiology of FADS, we next determined whether locomotor function was impaired by loss of Nup88 using locomotion and touchevoked escape assays in nup88 -/zebrafish. Zebrafish embryos develop spontaneous muscle contractions at 18 hpf [33], therefore we first analyzed the coiling behavior of nup88 -/embryos as compared to nup88 +/+ and nup88 +/embryos at 22-24 hpf. We did not detect problems in coiling behavior in nup88 -/embryos at this developmental stage (S3A Fig and S1 Movie). Next, we analyzed spontaneous swimming activity at 4 dpf ( Fig 5A) and found that only about 35% of the nup88 -/larvae showed spontaneous movement as compared to~83% of nup88 +/+ and about 73% of nup88 +/larvae ( Fig 5B). Moreover, those moving nup88 -/larvae displayed drastically reduced motor activity, traveled shorter distance ( Fig 5C) and initiated swim bouts less often ( Fig 5D). In contrast, statistically significant differences in the mean velocity were not observed in nup88 mutant larvae ( Fig 5E). We next performed touch-evoked escape response assays at 3 dpf and 4 dpf. At 3 dpf, percentages of responsive animals and response duration were not significantly different between wild-type and nup88 mutant larvae (S3B Fig). At 4 dpf, percentages of responsive animals were also not significantly different between wild-type and nup88 mutant larvae (Fig 5F), however, the response duration among the larvae that moved was significantly reduced in homozygous nup88 mutants in comparison to wild-type zebrafish. Interestingly also heterozygous nup88 mutants showed a shortened response duration, although less significant (Fig 5G and 5H and S2-S4 Movies).

FADS-related mutations in NUP88 alter NUP88 interactions
Next we wanted to assess whether the NUP88 mutations identified in the individuals with FADS interfere with the recruitment of NUP88 to NPCs. Due to a lack of relevant cell lines and the limited availability of tissue samples from affected fetuses, we performed immunofluorescence microscopy of GFP-tagged NUP88 proteins. Upon expression in HeLa cells, wildtype NUP88 and all mutants were co-localizing with the NPC marker mAb414, although recruitment of the NUP88 p.R509 � and p.E634del mutants to NPCs appeared reduced compared to wild-type NUP88 and the p.D434Y mutant (S4A Fig). Moreover, all forms of NUP88 also localized partially to the cytoplasm, as previously seen for NUP88 overexpression [2,12], and NUP88 p.R509 � to the nucleoplasm (S4A Fig). To define the effect of the mutations in NUP88 on the interface with its binding partners NUP214, NUP98 and NUP62, we employed GFP trap affinity purification in combination with Western blot analysis of lysates from HeLa cells expressing the GFP-NUP88 mutants. We found that NUP88 and the p.D434Y mutant copurified NUP214 and NUP62, while the p.R509 � and the p.E634del mutant did not so ( Fig  6A). Binding of NUP214 to GFP alone was similar as compared to the p.R509 � and the p. E634del mutants, indicating some non-specific binding of the NUP214 and/or the antibodies to GFP. The disrupted interaction between NUP88 and NUP214, however, did not impair NUP214 localization at NPCs (Fig 6B), whereas NUP62 association with NPCs was reduced in cells expressing NUP88 E634del (Fig 6C). Our GFP trap assays further showed that NUP98 associated with NUP88 and all mutant forms ( Fig 6A). Consequently, NUP98 association with NPCs appeared unaffected in cells expressing NUP88 mutants ( Fig 6D). Furthermore, the disease-related mutations in NUP88 did not affect the organization of the nuclear envelope as revealed by immunofluorescence analysis of lamin A/C (LA/C; S4B Fig), Western blots analysis of protein levels of lamin A/C and GST-pull-down down assays with NUP88 and the p.D434Y mutant (S4C and S4D Fig and S1 Methods). Moreover and consistent with data from higher vertebrates, NUP88 appears to not be essential for NPC integrity: immunolabelling of NPCs using a monoclonal antibody (mAb414) recognizing a subset of nucleoporins appears unaffected by the loss of nup88 in D. rerio brain sections (S4E  dpf nup88 homozygous and heterozygous mutants, and wild-type controls during one-minute long spontaneous locomotion recordings. (B) Quantification of percentages of larvae displaying spontaneous movement, also shown as (number of moving larvae/total number of larvae). Beeswarm graphs with individual data points depicting distance travelled/ minute (C), bouts/minute (D), and mean velocity of swimming bouts (E) of nup88 mutants and wild-type controls displaying spontaneous locomotion. (F) Quantification of percentages of larvae displaying touch-induced escape response, also shown as (number of responsive larvae/total number of larvae). (G) Representative examples of touch-induced escape behavior of 4 dpf head-restrained nup88 mutant and wild-type larvae. The first column depicts single frames taken 50 ms before an escape response was induced by touching the trunk of larvae with a pipette tip (red dashed lines). The other columns show superimposed frames of escape responses at consecutive time intervals after touch. The asymmetric tail movement displayed in some of the images (nup88 +/and nup88 -/at 201-400 ms, and all genotypes at 401-600 ms) represents slow motion of the tail toward its original position. The escape response terminated before this phase (see also S2-S4 Movies). (H) Graph showing average durations of escape response in nup88 mutants and wild-types. � p < 0.05, �� p < 0.004, ��� p = 0.001, n.s. = non-significant, two-tailed Fisher exact test (B and G) or two-tailed t-test (C-E, H). Data in C-E and H are shown as mean ± SD. n is number of embryos/larvae analyzed. https://doi.org/10.1371/journal.pgen.1007845.g005

NUP88 in fetal akinesia
As NUP88 is critically involved in CRM1-dependent nuclear export of proteins, we further asked whether the mutations in NUP88 affect nuclear import and/or export, but we observed no defects in general nuclear protein import or export (S6A Fig) or

Loss of NUP88 affects rapsyn levels and localization
Impeded formation of AChR clusters at the neuromuscular junction (NMJ) is one cause of FADS. Given the central role of rapsyn in AChR clustering and in FADS [17,18,21], we therefore asked whether a loss of NUP88 function would negatively affect rapsyn and depleted NUP88 by siRNAs from HeLa and C2C12 cells and monitored protein levels of rapsyn by Western blotting. As shown in Fig 7A, depletion of NUP88 from HeLa cells in fact coincided with a decrease in rapsyn levels. Similarly, reduced rapsyn levels were observed in C2C12 cells depleted for NUP88 using siRNAs (Fig 7B and 7C) and shRNAs (Fig 7C). Quantification revealed that siRNA treatment led to reduction of NUP88 by 80-90% and at the same time a reduction of rapsyn by 40-60% (Fig 7C). In contrast to that, NUP88 downregulation had no effect on MuSK levels, another key player in FADS [20], and no effect on known NUP88 targets, such as CRM1 and NF-κB levels (Fig 7A and 7B). Muscle biopsy of affected individual B. II.2 and immunohistochemistry on paraffin sections furthermore showed weaker staining and irregular distribution of rapsyn in the cytoplasm in comparison to biopsy samples from a control fetus (Fig 7D). Rapsyn is known to not only localize to the plasma membrane, but also to the cytoplasm [34][35][36]. Rapsyn protein levels could not be determined in zebrafish due to a lack of antibodies, but qRT-PCR analyses revealed a reduction of rapsn mRNA by about 20%, while nup88 mRNA levels were reduced by about 90% (Fig 7E).
Consistent with reduced rapsyn levels, we observed impaired AChR clustering in fasttwitch muscle fiber synapses, but not in myoseptal synapses of the 5 dpf zebrafish trunk ( Fig  7G). Quantification of the size of individual AChR cluster in WT and mutant zebrafish revealed that the diameter of the AChR clusters was significantly reduced in nup88 -/larvae as compared to nup88 +/+ larvae (Fig 7H). Interestingly, AChR cluster size was also reduced in nup88 +/larvae, both in comparison to WT and nup88 -/larvae. This reduced size of AChR clusters in the heterozygotes may account for the observed defects in touch-evoke response ( Fig 5H). In accordance with impaired neuromuscular junction formation as a consequence of loss of nup88, muscle organization in zebrafish appeared indistinguishable in electron micrographs from nup88 +/+ and nup88 -/larvae (Fig 7F). Similarly, in affected fetus B.II.2 skeletal muscle structure was, based on the autopsy report, intact. Thus, both in vitro and in vivo evidence support the notion that loss-of-function of NUP88 has a negative effect on rapsyn, which likely affects AChR clustering and proper formation of neuromuscular junctions.

Discussion
Here, we have identified biallelic homozygous and compound heterozygous mutations in NUP88 as a cause of fetal akinesia. We demonstrate that the mutations in NUP88 lead to a loss-of-function phenotype, which coincides with reduced spontaneous motor activity and touch-evoked escape response in zebrafish. Consistent with the fact that the mutations in NUP88 affect different regions of the protein, we observed distinct effects of the mutants on binding to NUP214 and NUP62 in GFP-trap assays, whereas binding to NUP98 appears indistinguishable between wild-type and mutant forms of NUP88 (Fig 6A). This suggests that impaired interaction with partner nucleoporins may contribute, but are unlikely to be causative for NUP88 malfunction in FADS. Our data further suggest that NUP88 malfunction in FADS is at least in part due to dysfunctional rapsyn, a known key player in FADS, and GAPDH was used as loading control. Rapsyn protein levels are reduced in NUP88-depleted cells. Note MuSK has a predicted molecular weight of 97 kDa, but migrates higher [20]. (C) Quantification of the respective expression levels of NUP88 and rapsyn after transfection of HeLa and C2C12 cells with the indicated siRNAs and shRNA-mediated depletion of NUP88 in C2C12 cells. Blots from three independent experiments for each condition were analyzed. Data present mean ± SEM. P-values ���� <0.0001, ��� <0.001; �� <0.01, � <0.05; t-test, one-tailed. (D) Bright-field images of histological muscle sections from individual B.II.2 and a control fetus stained with anti-rapsyn antibodies (brown). Nuclei were visualized by hematoxylin. (E) qRT-PCR analysis of nup88 and rapsn transcripts in 5 dpf wild-type and nup88 -/larvae. (F) Skeletal muscle organization remains unaffected in nup88 -/zebrafish larvae. Larvae were prepared at 5 dpf for transmission electron microscopy. Skeletal muscle of WT and mutant zebrafish show intact myofibril alignment with their regularly stacked Z-line and clearly identifiable H-and I-zones. Shown are representative longitudinal sections of skeletal muscle from nup88 +/+ and nup88 -/larvae, respectively. Scale bar, 1 μm. (G) Anterior trunk regions of wild-type (left) and nup88 heterozygous and homozygous mutant larvae were stained with antibodies against the AChR and secondary Alexa 488 antibodies. AChR cluster size was significantly reduced in nup88 -/larvae as compared to nup88 +/+ and nup88 +/larvae (higher magnification insets). Insets are taken from the marked area in the respective consequently impaired AChR clustering and neuromuscular junction formation. Muscle integrity, in contrast, appears grossly unaffected by a loss of NUP88.
Genetic disruption of nup88 in zebrafish led to pleiotropic morphological defects, including micrognathia, smaller head and eyes, distortion of the body axis, and aplastic swim bladder (Figs 3 and 4), and to impaired locomotor behavior (Fig 5). These phenotypes parallel defects observed in human fetuses affected by FADS, such as reduced fetal movement, micrognathia, joint contractures and lung hypoplasia (for detailed comparison see Table 2). The reduced head and eye size likely originates from increased apoptosis of neuronal cells (Fig 3D). nup88 inactivation affected spontaneous movement of zebrafish from 4 dpf onwards, which resembles the onset of symptoms in the affected fetuses at about week 18 of gestation. The impaired touch-evoked response seen in the mutant zebrafish further matches the absence of reflex response observed in at least some fetuses (Family A, I.2 and B.F., personal communication). Rescue experiments in zebrafish with wild-type or nup88 mutants revealed that none of the mutant mRNA could restore a wild-type-like phenotype (Fig 4), indicating that NUP88 mutations in individuals affected with FADS are loss-of-function variants. The zebrafish model developed here therefore provides a valuable in vivo system to further test nup88 deficiency. This in turn will be key in understanding the role of NUP88 in the etiology of FADS and its function in embryonic development.
Reduced rapsyn levels might partly cause fetal akinesia upon loss of NUP88. Rapsyn is one of the many contributing proteins required for the correct assembly of the AChR and is particularly involved in AChR assembly and localization to the cell membrane [37,38]. We observed reduced rapsyn protein levels in the absence of functional NUP88 in cellular assays using human and mouse cell lines in combination with siRNA and shRNA-mediated depletion of NUP88 (Fig 7). Due to a lack of cell lines derived from affected individuals, we could analyze rapsyn only in histological sections from muscle of one affected fetus, which revealed a weaker staining for and a perturbed intracellular localization of rapsyn (Fig 7D). Consistent with aberrant rapsyn expression, AChR clustering in trunk regions of nup88 +/and nup88 -/zebrafish was impaired (Fig 7G and 7H). Rapsyn protein levels could not be determined in zebrafish due to a lack of antibodies, but qRT-PCR analyses revealed a reduction of its mRNA by about 20% (Fig 7E). Our data therefore suggest that absence of functional NUP88 causes fetal akinesia at least in part through misregulation of rapsyn expression. How loss of NUP88 results in reduced rapsyn levels on a mechanistic level remains to be seen. Moreover, this likely is not overview image. Inset for nup88 -/was rotated by 180˚. Scale bars, 50 μm (overview), 5 μm (insets). (H) Quantification of the AChR size in 4 dpf nup88 +/ + (n = 8), nup88 +/-(n = 14), and nup88 -/-(n = 12) larvae. Per larvae 100 clusters throughout z-stacks of confocal images were manually measured using ImageJ. Data present mean ± SD. P-values ���� <0.0001, ��� <0.001; t-test, one-tailed.
https://doi.org/10.1371/journal.pgen.1007845.g007 the only pathway by which NUP88 acts in NMJs, as effects on only one cellular pathway would be indeed (i) very surprising for a nucleoporin per se, (ii) irreconcilable with the cranial defects observed in human and zebrafish, and (iii) not in line with the broad central nervous system expression pattern of nup88 in zebrafish. Our data, however, demonstrate (i) that the nup88 spectrum of phenotypes indeed include locomotor defects and that therefore NUP88 deficiencies might result in FADS in humans, and (ii) that human alleles are dysfunctional. We further observed that (iii) NMJ defects correlate to this phenotype. Whether this is the primary cause of akinesia is impossible to determine given the pleiotropic effects of Nup88 deficiency, but at least they are sufficient to explain the phenomenon. Further mechanistic details will be subject for future studies.

DNA sequencing family A
Exome sequencing in one affected baby (A.II.7) was performed at the Clinical Exome Sequencing (CES) at University of California, Los Angeles and the sequencing report is on hand. In total 22,843 DNA variants were identified, including 21,625 single nucleotide substitutions and 1,218 small deletions/insertions (1-10 bp): the data were consistent with a high quality genomic sequence and fall within normal human genomic variation quality parameters. Estimated from these data, about 93% of the exome was reliably sequenced with at least 10x coverage. In total, 5 homozygous and 329 rare heterozygous protein-altering variants of uncertain clinical significance were identified across 313 genes. A rare autosomal-recessive model of inheritance with homozygous causative mutations due to consanguinity of the parents was assumed. After applying appropriate filters, a novel homozygous variant c.1300G>T, p.D434Y in the NUP88 gene [NM_002532.5] was identified in the subject's DNA. This variant had not been previously observed in the general population and was predicted to be deleterious/probably damaging by three in silico prediction algorithms (S1 Table). Additionally, the homozygous mutation c.1300G>T, p. D434Y in NUP88 was confirmed in a second affected fetus A.II.5 by Sanger sequencing.

DNA sequencing family B
Exome sequencing was performed on DNA from the proband B.II.2, both healthy sisters and both parents from the European family, as outlined previously [39]. Exome enrichment was performed on DNA using an Ampliseq Whole Exome kit, (Thermo Fisher Scientific). Briefly, a total of 100 ng of DNA was amplified in 12 separate PCR pools, each containing~25,000 primer pairs. After amplification, the individual reactions were pooled and digested to degrade the PCR primers. Next, barcoded sequencing adaptors were ligated and the library was purified using AMPure beads (Beckman Coulter), amplified and purified again and analyzed on a 2100 Bioanalyzer (Agilent Technologies). Libraries were diluted to 18-26 pM and attached to Ion Sphere Particles (ISPs) using an Ion Proton Template 200 V3 kit and sequenced on a P1 sequencing chip for 520 flows on an Ion Proton sequencer (Ion Sequencing 200 kit V3). Two samples were pooled and sequenced on a single chip. Following sequencing, reads were trimmed to remove low quality bases from the 3' end and mapped to the human genome reference sequence (HG19) using tmap (Torrent Suite 4.2). Variant calling was performed using the Torrent Variant Caller with custom settings optimized for whole exomes and the data was annotated using ion Reporter 4.0. Variants were filtered with ANNOVAR [40] against ENCODE GENECODE v.19, 1000genomes (threshold >0.5%), dbSNP138 common databases and against a list of in-house common variants. Genes with variants that fitted an autosomal recessive inheritance pattern and co-segregated with disease in the family were prioritized.
NUP88 was the only candidate gene that harbored variants compatible with an autosomal recessive inheritance pattern in this family (S1B Fig). Individual B.II.2 was compound heterozygous for an in-frame 3 bp deletion in exon 14 (c.1899_1901del, p.E634del) and a nonsense mutation c.1525C>T, p.R509 � in exon 11. Mutation c.1899_1901del mutation was inherited on the maternal allele and the c.1525C>T paternally. Healthy sister B.II.1 is heterozygous for the maternal mutation, healthy sister B.II.3 is a heterozygous carrier of the paternal mutation (Fig 1A, pedigree Family B).

Structure modelling
Human NUP88 protein sequence Q99567 (UniProt database) was used for structural investigation and modelling using an N-terminal and C-terminal part, according to predictions. Database search comparisons using HHPRed [41][42][43][44][45] revealed as best hits for the N-terminal domain (NTD): Nuclear pore complex proteins Nup82 from S. cerevisiae and Chaetomium thermophilum (PDBid: 3pbp A, 3tkn_A and 5cww_B, respectively) and for the C-terminal domain (CTD) Nup57 from Chaetomium thermophilum (PDBid: 5cws E) and Nup54 from Homo sapiens (PDBid: 5ijn F). The high similarity to yeast and Chaetomium Nup82, the structures of which have been solved in part [46][47][48] strongly indicate that the NTD also forms a ßpropeller and predictions suggest that the CTD is in an all helical arrangement (PsiPred and JPred4) [49,50]. In order to gain insight into the putative fold and the location of the diseaserelated residues identified in hsNUP88, four servers were used for structure prediction. Phyre2, Robetta, I-Tasser, RaptorX [51][52][53][54][55][56][57] were supplied with either full length sequence or only the NTD (residues 1-495) or CTD (residues 496-741) in case of residue limitations or implausible models resulting from full length submission. All modelling was performed using standard settings. All figures were generated using Pymol.

Zebrafish husbandry
Zebrafish (Danio rerio) were raised and bred at 28˚C on a 14 h/10 h light/dark cycle. Embryos and larvae were raised in egg water (0.3 g/l Instant Ocean Salt, 75 mg/l CaSO 4 ; 1 mg/l Methylene Blue). The line carrying nup88 sa2206 allele was obtained from Zebrafish Mutation Project, Knockouts for Disease Models (http://www.sanger.ac.uk/sanger/ Zebrafish_Zmpgene/ENSDARG00000003235#sa2206). Heterozygous fish for nup88 were out crossed for four generations with wild-type fish before analysis. All animal experiments were performed in accordance with the rules of the State of Belgium (protocol approval number: CEBEA-IBMM-2017-22:65).

RNA expression constructs
Capped messenger RNA was synthesized using the mMESSAGE mMACHINE kit (Ambion). The following expression plasmids were generated and used in this study: the full-length zebrafish nup88 ORF was cloned from 48 hpf cDNA and recombined into BamHI-XhoI digested pCS2 using In-fusion cloning (TaKaRa). nup88 mutants corresponding to the sequences identified in human fetal akinesia cases were generated by site-directed mutagenesis using Quik-Change Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). All primer sequences are listed in S2 Table. mRNAs (300 pg) were injected at the one-cell stage.

Genotyping and RT-PCR
nup88 sa2206 genotyping was performed by RFLP assay using MseI restriction of a 150 bp PCR product. Real-time PCR was done at various stages of embryonic and larval development (4-cell to 5 dpf). Primer sequences are listed in S2 Table. Staging of embryos was performed according to [58].

Alcian blue staining
Alcian Blue staining and histology were performed as described elsewhere [59].
All images were acquired using an Olympus SZX16 stereomicroscope and an Olympus XC50 camera using the imaging software Cell � after embryo anesthesia with a low dose of tricaine.

Apoptosis assay using acridine orange in live embryos
Live embryos at 24 hpf, 36 hpf, 48 hpf were dechorionated and immersed in egg water containing 5 μg/ml acridine orange. They were incubated at 28.5˚C for 15 min in the dark and then thoroughly washed with egg water. Embryos were mounted in low-melting agarose for positioning and immediately imaged using an Axio Observer Z1-1 microscope. Images were processed using Zeiss Zen software.

Behavioral assays in zebrafish embryos and larvae
Spontaneous tail coiling of 22-24 hpf embryos, placed in the grooves of an agarose chamber, was recorded for 2 min at 30 frames per second. Coiling events were scored manually. To analyze spontaneous locomotion, 4 dpf larvae were placed into 96-well plates (one larva per well). Their behavior was recorded at 30 frames per second for 5 min and quantified using EthoVision XT 8.5 software (Noldus).
Touch-induced escape responses were analyzed in 4 dpf head-restrained larvae. Larvae were first embedded in 2% low melting point agarose, and then the agarose surrounding their tail was removed with a blade. Escape behavior was induced by touching the tail of larvae with a plastic pipette tip and recorded at 300 frames per second. Duration of behavioral responses was quantified with ImageJ.
Quantitative PCR was then performed using SYBR Premix Ex Taq TM II (Tli RNaseH Plus), Bulk kit (RR820L; Takara, Dalian, China). 1 μl of cDNA was used in each 20 μl-PCR well with 300 nM primers pair final concentration. Each sample was assayed in triplicate and samples not reverse-transcribed were used as negative controls. Eight housekeeping genes were tested according to [60] and the most stable combination was determined using qbase + qPCR analysis software (https://www.qbaseplus.com). actb2, gapdh and ybx1 were the best combination of housekeeping genes at this stage of larval development and used for normalization.
Differences in amplification curves between the target genes and housekeeping genes were identified by comparing standard curve slopes. Real-Time PCR was performed using Applied Biosystems StepOnePlus Real-Time PCR System and PCR analyses were performed with qbase+ system software.

Zebrafish immunostaining
For whole-mount immunostaining, zebrafish embryos were fixed in 4% PFA for 3 h at room temperature (RT), washed and permeabilized with proteinase K (40μg/ml) at 37˚C for 1 h. For blocking, larvae were gently shaken in PBS containing 10% of normal goat serum, 1% DMSO and 0.8% Triton X-100 for 1 h and then, incubated with the primary anti-acetylcholine receptor antibody (mouse mAb35 (DSHB, Hybridoma Bank; 1:100) overnight at 4˚C. After washing, larvae were incubated with the secondary antibody (anti-mouse IgG Alexa Fluor 594, 1:1000) for 2 h at RT and washed six times for 15 min in PBST prior to mounting and confocal imaging (Zeiss LSM710). Size of AChR cluster were determined using ImageJ.

Transmission electron microscopy of zebrafish skeletal muscle
Skeletal muscle of 5 dpf nup88 +/+ and nup88 -/zebrafish larvae were analyzed by transmission electron microscopy on longitudinal and transversal ultrathin sections.
Briefly, larvae were fixed in 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer, pH 7.4 overnight at 4˚C and post-fixed in 1% osmium tetroxide, 1.5% ferrocyanide in 0.15 M cacodylate buffer for 1 h at RT. After serial dehydration in increasing ethanol concentrations, samples were embedded in agar 100 (Agar Scientific Ltd., UK) and left to polymerize for 2 days at 60˚C. Ultrathin sections (80 nm thick) were collected using a Leica EM UC6 ultramicrotome and stained with uranyl acetate and lead citrate. Images were recorded on a Tecnai10 electron microscope (FEI) equipped with an Olympus VELETA camera and processed using the AnalySIS software.

Immunohistochemistry-paraffin (IHC-P) labelling of fetal muscle sections
Brain and skeletal muscle paraffin-embedded blocks were obtained from autopsy of individual B. II.2. Control tissues of a fetus of the same age without neuromuscular disorder were used for IHC-P. Written consent form for use of paraffin samples for functional analysis was signed by the parents. This study was performed according to the guidelines of the local ethics committee (Ghent University Hospital), which does not require formal review for the use of autopsy samples.
Blocks were cut with a microtome and 5 μm sections were mounted. Sections were deparaffinized and rehydrated. Heat-induced epitope retrieval was performed using Tris/EDTA, pH 9.0 for all tissues when using antibodies against rapsyn and with sodium citrate, pH 6.0 for antinucleoporin antibody mAb414. 0.3% H 2 O 2 for 20 min was used to block samples endogenous peroxidase followed by washes with PBS. Tissues were permeabilized using PBS containing 0.5% Triton X-100 for 5 min at RT and subsequently blocked with PBS containing 1% BSA for 30 min. Primary antibodies were diluted in blocking buffer and incubated overnight at 4˚C in a humidification chamber. Samples were washed in PBS for 10 min. Biotinylated goat anti-rabbit-Ig (1:400; DAKO-E0432) or biotinylated goat anti-mouse (1:400; DAKO-E0433) were added for 30 min.
After washing in PBS, samples were incubated 30 min with streptavidin-HRP (DAKO-P0397) diluted 1:300 in PBS and thoroughly washed with PBS. DAKO Liquid DAB + Substrate Chromogen System K3468 (1ml substrate + 1 drop DAB) was added on samples and left until a brown coloring appeared (maximum 6 min). After several PBS washes, the samples were counterstained with hematoxylin for 15 seconds, washed thoroughly under running tap water to remove excess staining agent and mounted for subsequent observations with Aquatex. Images were acquired with an Olympus BX41 microscope and were processed using the Olympus cellSense software.

Plasmids for studies in human cell lines
For all constructs, human NUP88 was amplified by PCR. All constructs were verified by DNA sequencing. GFP-NUP88 was produced as described previously [2]. FLAG-NUP88 was cloned into KpnI/XbaI cut pFLAG-CMV2 (Sigma-Aldrich). GFP-NUP88 and FLAG-NUP88 mutants were generated by site-directed mutagenesis using the QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies) following the manufacturer's instructions. Primers are listed in S2 Table.

Antibodies
The following polyclonal antibodies were used in this study for Western blotting (WB), immunofluorescence ( Secondary antibodies for immunofluorescence were the corresponding goat anti-mouse-IgG Alexa 568 (1:1000; Invitrogen), goat anti-rabbit IgG Alexa 568 (1:1000; Invitrogen). Secondary antibodies were either alkaline phosphatase coupled antibodies from Sigma/Aldrich and used at 1:20.000 or HRP coupled antibodies from Cell Signaling Technology at 1:8000.

Immunofluorescence microscopy of HeLa cells
HeLa cells were grown on glass coverslips, transfected, fixed in 4% PFA in PBS for 5 min, permeabilized with 0.5% Triton-X-100 in PBS for 5 min and then fixed again. Blocking was performed with 2% BSA/0.1% Triton-X-100 in PBS for 30 min at RT. Primary antibodies were incubated at 4˚C over-night in a humidified chamber. Secondary antibodies were incubated 1 h at RT in the dark. Excess antibodies after primary and secondary antibody staining were removed by three washing steps using 0.1% Triton-X-100 in PBS for 5 min. Cells were imaged using a Zeiss LSM 710 (Zeiss, Oberkochen, Germany) confocal laser scanning microscope with Zeiss Plan-Apochromat 63x/1.4 oil objective. Images were acquired using the microscope system software and processed using Image J and Adobe Photoshop (Adobe Systems, Mountain View, CA).

GFP-trap assay
HeLa cells, grown in a 10 cm dish, were transfected with GFP and GFP-NUP88 wild-type and mutant variants, respectively. Cells were grown for 48 h at 37˚C in a humidified atmosphere with 5% CO 2 . To harvest cells, growth medium was aspirated off, 1 ml of ice-cold PBS was added to cells and cells were scraped from dish. The cells were transferred to a pre-cooled tube, centrifuged at 500 xg for 3 min at 4˚C and the supernatant was discarded. The cell pellet was washed twice with ice-cold PBS. Pellets were subsequently lysed with 200 μl of ice-cold lysis buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, proteasephosphatase inhibitor) using a syringe before incubating on ice for 30 min. The tubes were centrifuged for 15 min at 16.000 xg at 4˚C and the supernatant was transferred into a new reaction tube.
Bradford assay was used to determine the protein concentration of the lysates and 300 μg of protein lysate adjusted to 500 μl in dilution buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, protease-phosphatase inhibitor) was added to 25 μl of GFP-Trap_MA beads (ChromoTek, Planegg-Martinsried, Germany). Beads were prewashed twice with dilution buffer. The beads and the lysates were incubated 1h at 4˚C on an end-to-end rotor. The magnetic beads were then washed three times in dilution buffer containing 150 mM, 250 mM and 500 mM NaCl following the addition of 20 μl of 2x SDS-sample buffer (120 mM Tris/HCl, pH 6.8, 20% glycerol, 4% SDS, 0.04% bromophenol blue, 10% β-mercaptoethanol) and boiling at 95˚C for 10 min. The eluates were subsequently loaded on to 7% polyacrylamide gels and Western blot was carried out (see below).

Western blotting
HeLa and C2C12 (provided by Vincent Mouly, The Pitié-Salpêtrière Hospital, Institute of Myology, Paris, France) cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1% Nonidet-P40 and protease inhibitor cocktail tablets (Roche, Basel Switzerland)). 20 μg of protein were loaded and separated by sodium dodecyl sulfate-polyacrylamide (5% or 7%) gel electrophoresis (SDS-PAGE). The proteins were transferred onto a PVDF membrane (Immobilon-P, Millipore) and the membranes were blocked with TBS containing 0.1% Tween 20 and 5% non-fat dry milk for 1 h. The membranes were then incubated for 1 h in blocking solution containing a primary antibody followed by washing 3x in TBS containing 0.1% Tween 20 and 5% non-fat dry. The membranes were next incubated with secondary antibodies for 1 h, washed 3x in TBS and developed. X-ray films were scanned and processed using ImageJ. Transcripts of nup88 were analyzed by whole-mount in situ hybridization at the indicated developmental stages. nup88 transcripts are maternally deposited during zebrafish oogenesis (4-cell stage). nup88 is further expressed upon zygotic genome activation subsequently to mid-blastula transition (5 hpf and later). nup88 is ubiquitously expressed up to 16 hpf and from 24 hpf prominent in the eye, brain and anterior trunk. nup88 transcripts in somites are low (30 hpf-72 hpf). (B) The presence of maternally deposited nup88 and zygotic nup88 transcripts was confirmed by RT-PCR at stages 4-cell to 5 dpf. actb2, gapdh and ybx1 were used as controls. (C) Acridine orange staining of the tail region of wild-type and nup88-/-mutants at 36 hpf. No major apoptotic events were detected. Shown are confocal images. Scale bars, 100 μm. Touch response is not impaired in nup88 mutant larvae at 3 dpf stage of development. Quantification of percentages of larvae displaying touch-induced escape response (left) and response duration (right) in nup88+/+, nup88+/-and nup88-/embryos. n.s., not significant, two-tailed t-test (A, B right) or two-tailed Fisher exact test (B left). Data are shown as mean ± SEM. n is number of embryos/larvae analyzed. (C) Quantification of body length after microinjection of wild-type or the respective mutant nup88 were at the one-cell stage. Body length was evaluated at 5 dpf and revealed non-statistically significant differences were detected. Data are shown as mean ± SEM. n is number of embryos/larvae analyzed. (TIFF) S4 Fig. NUP88 binding properties and nuclear envelope organization of NUP88 mutants. (A) All NUP88 mutants co-localize with the NPC-specific mAB414 antibodies in HeLa cells. Wild-type NUP88, NUP88 D434Y, and NUP88 E634del localize to the NE and the cytoplasm, whereas NUP88 R509 � can additionally be found in the nucleus. (B) Nuclear envelope proteins remain unaffected in the presence of mutant NUP88 based on lamin A/C distribution in HeLa cells overexpressing GFP-NUP88 and GFP-NUP88 disease-related mutants. Cells in (A) and (B) were analyzed by indirect immunofluorescence microscopy. Shown are confocal sections on the midplane of the nuclear envelope. Scale bars, 10 μm. (C) Bacterially expressed glutathione-S-transferase (GST), GST-NUP88 and GST-NUP88D434Y were bound to prewashed glutathione sepharose beads and incubated with a total HeLa protein extract. Proteins were eluted using Laemmli buffer and bound and unbound fractions were analyzed by immunoblotting using anti-lamin A, anti-Nup214, and anti-actin antibodies. (D) HeLa cells transiently expressing green-fluorescent protein (GFP), GFP-NUP88 and GFP-NUP88 D434Y were lysed and subjected to Western blot analysis using anti-NUP88, anti-lamin A/C antibodies. Actin served as a loading control. NPCs show normal distribution in (E) the wild-type (WT) and nup88 -/zebrafish as well as in (F) histological muscle sections from individual B.II.2 and a control fetus. Shown are confocal images of sagittal cryo-sections of the diencephalon of 5 dpf zebrafish larvae and bright-field images of paraffin-embedded skeletal muscle section, respectively. NPCs were visualized using the NPC-specific antibody mAB414 (red in (E), brown in (F)).