Alternative splicing plays a critical role in mouse neurodevelopment, regulating neurogenesis, cortical lamination, and synaptogenesis, yet few human neurodevelopmental disorders are known to result from pathogenic variation in splicing regulator genes. Nuclear Speckle Splicing Regulator Protein 1 (NSRP1) is a ubiquitously expressed splicing regulator not known to underlie a Mendelian disorder.
Exome sequencing and rare variant family-based genomics was performed as a part of the Baylor-Hopkins Center for Mendelian Genomics Initiative. Additional families were identified via GeneMatcher.
We identified six patients from three unrelated families with homozygous loss-of-function variants in NSRP1. Clinical features include developmental delay, epilepsy, variable microcephaly (Z-scores −0.95 to −5.60), hypotonia, and spastic cerebral palsy. Brain abnormalities included simplified gyral pattern, underopercularization, and/or vermian hypoplasia. Molecular analysis identified three pathogenic NSRP1 predicted loss-of-function variant alleles: c.1359_1362delAAAG (p.Glu455AlafsTer20), c.1272dupG (p.Lys425GlufsTer5), and c.52C>T (p.Gln18Ter). The two frameshift variants result in a premature termination codon in the last exon, and the mutant transcripts are predicted to escape nonsense mediated decay and cause loss of a C-terminal nuclear localization signal required for NSRP1 function.
We establish NSRP1 as a gene for a severe autosomal recessive neurodevelopmental disease trait characterized by developmental delay, epilepsy, microcephaly, and spastic cerebral palsy.
Alternative splicing is a major contributor to transcriptome diversity . This diversity is most apparent in the brain, which exhibits the highest messenger RNA (mRNA) isoform complexity of all tissues [2, 3]. Splicing patterns vary considerably throughout development and are orchestrated by RNA-binding proteins . These splicing factors recognize specific RNA motifs and influence spliceosome assembly at nearby splice sites. Through selective inclusion or exclusion of exons, alternative splicing generates multiple mRNA and protein isoforms with different functional properties from single genes . Splicing factors shape mouse neurodevelopment, directing neurogenesis, cell migration, and synaptogenesis . They also regulate neuronal excitability and thereby play a pivotal role in brain homeostasis . Despite these observations, few splicing factors have been shown to underlie specific Mendelian disease traits .
Nuclear speckles are punctate membraneless subnuclear organelles rich in splicing factors, including small nuclear ribonucleoproteins, spliceosomal subunits, and arginine/serine-rich splicing factors [6, 7]. One widely expressed, speckle-associated splicing factor is Nuclear Speckle Splicing Regulatory Protein 1 [8, 9]. In humans, it is encoded by the seven exon NSRP1 gene, which maps to chromosome 17q11.2. Mice heterozygous for a Nsrp1 null allele had no overt phenotype, but Nsrp−/− mice were not detected from heterozygous crosses as early as embryonic day 6.5, demonstrating null associated embryonic lethality and a surmised essential role for NSRP1 in development . Yet, NSRP1 has not been previously implicated in human Mendelian disorders and no NSRP1 variant alleles have been investigated.
Here, we identify six individuals from three unrelated families with severe neurodevelopmental disorders (NDDs) with brain abnormalities and biallelic loss-of-function (LoF) variants in NSRP1, implicating Nuclear Speckle Splicing Regulatory Protein 1 as a key splicing factor in human brain development.
MATERIALS AND METHODS
All individuals in this study provided informed consent, including consent to publish photographs. Collaborators were connected through GeneMatcher . Exome sequencing (ES) was performed either through research institutions or clinical diagnostic labs. Additional experimental details, including absence of heterozygosity (AOH), as a surrogate measure of runs of homozygosity (ROH) and genomic intervals identical by descent, and inbreeding coefficient calculations can be found in the Supplemental Material. All identified variants were deposited in ClinVar under SCV001547247, SCV001547247, and SCV001547249.
Using ES and family-based rare variant analysis, we identified six individuals from three unrelated families with distinct homozygous LoF variants in NSRP1 (Fig. 1a–c) . After VCF file parsing/filtering, analysis, and testing Mendelian expectations for either dominant or recessive disease trait models, no other variants in known disease genes or novel genes could parsimoniously explain the clinical synopsis of observed rare phenotypes. Two families had a known family history of consanguinity. Inbreeding coefficients calculated from ES data were higher than expected for each family (Tables S1,S2), with family 3’s calculated inbreeding coefficients (F = 0.1093, 0.1257) approaching that of an uncle–niece marriage (F = 0.125) rather than the value expected based on their third cousin marriage (F = 0.003906) . Similarly, AOH calculations demonstrated large AOH blocks (3.1 Mb, 7.7 Mb, and 6.9 Mb) surrounding each NSRP1 trait-associated variant consistent with clan genomics identity-by-descent (IBD) (Fig. 1a–c) [13, 14].
Deep phenotyping was performed, and detailed clinical data are available in the Supplemental Material (Table S3). Core clinical findings include developmental delay (DD, 6/6), epilepsy (6/6), hypotonia (6/6), appendicular spasticity (6/6), microcephaly (5/6, Z-scores −0.95 to −5.60), dysphagia (4/6), and dysmorphic facies (4/6) (Fig. 1d–h). Most individuals were nonverbal (5/6) and 3/6 were nonambulatory. Seizures began in infancy and were often drug-resistant (3/6). Brain abnormalities included underopercularization (3/4), simplified gyral pattern (3/4), superior and/or inferior cerebellar vermian hypoplasia (3/4), corpus callosum dysgenesis (1/4), and thin brainstem (1/4) (Fig. 1i–t). All patients had abnormal electroencephalography with epileptiform discharges being the most common finding (5/6) (Table S3, Fig. S1).
NSRP1 (NM_032141.4) contains 7 exons and encodes a 558 amino acid (AA) protein (Fig. 2a, b) . The NSRP1 protein contains two RNA recognition motif (RRM) domains and an arginine-serine (RS)-like domain (Fig. 2b) . There are two coiled-coil domains that overlap with the RRM and RS-like domains and are involved in self-oligomerization and splicing activity (Fig. 2b) . Finally, a C-terminal nuclear localization signal (AA 531–540) lies within the RS-like domain and is required for nuclear localization and splicing activity (Fig. 2b). There is also a major alternate transcript, ENST00000612959.4, which lacks the second exon of NM_032141.4 (Fig. 2a).
All variants were orthogonally studied and segregation in accordance with Mendelian expectations was confirmed by Sanger sequencing (Fig. S2). Variant allele details are summarized in Fig. 2 and Supplemental Table 4. All variants are ultrarare (minor allele frequency [MAF] < 1/10,000) . Two are private variants (c.1359_1362delAAAG and c.52C>T) found neither in gnomAD nor our internal database of >13,000 exomes. The third variant, c.1272dupG, which occurs in a 13-base polypurine stretch after a GG dinucleotide, is found in gnomAD only once in the heterozygous state. Both frameshift variants occur in the last exon and are predicted to disrupt the critical nuclear localization signal required for NSRP1-mediated alternative splicing (Fig. 2b) . c.52C>T (p.Gln18Ter) results in a premature termination codon (PTC) within the second exon of NM_032141.4 and has a CADD score (GRCh37-v1.6) of 37. This variant is noncoding (c.−49 + 1248C>T) in ENST00000612959.4 (Fig. 2a). Homozygous LoF variants are absent from gnomAD, and biallelic LoF variants are otherwise absent in our internal database.
NSRP1 is a widely expressed nuclear speckle protein . In splicing assays, human NSRP1 modulated splice site selection, resulting in exon inclusion or exclusion in a gene-specific fashion [8, 9]. NSRP1 interacts with splicing factors SRSF1 and SRSF2 and counteracts their alternative splicing activities . Mice heterozygous for an Nsrp1 null allele had no obvious deficits, but homozygous Nsrp1 null mice exhibit embryonic lethality as early as embryonic day 6.5 . Similarly, homozygosity for a null allele in C. elegans ortholog ccdc-55 resulted in early larval arrest, whereas RNAi allowed larval development but resulted in abnormal distal tip cell migration . Finally, homozygosity for a null allele in D. melanogaster ortholog CG15747 (also known as nito) caused larval lethality . While early lethality has limited investigation of NSRP1’s role in the brain, nito knockdown in D. melanogaster CCAP/buriscon neurons reduced axon outgrowth during the transition from larval to adult form .
Here, we report biallelic LoF variants in NSRP1 in six individuals from three unrelated families. A clinical synopsis of the key phenotypic features defining this autosomal recessive (AR) disease trait are DD, epilepsy, hypotonia, appendicular spasticity, microcephaly, dysphagia, and dysmorphic facies. As half of the cohort has drug-resistant epilepsy, electrographic abnormalities, and DD, the NSRP1 associated disease trait fulfills clinical criteria for developmental and epileptic encephalopathies . While dysmorphic features are common, a consistent clinically recognizable pattern was not discernible. The cause of early death, seen in two of the six patients and an additional affected sibling who was not genotyped, is unclear. Brain abnormalities included simplified gyral pattern, underopercularization, and superior and/or inferior vermian hypoplasia. These abnormalities were more striking in individuals who underwent imaging in infancy rather than later in childhood. This may represent an early delay in brain maturation that subsequently improves with age. Further identification of patients with biallelic pathogenic NSRP1 variants and serial imaging will clarify the associated imaging spectrum.
Only two families in this report are consanguineous based on historical report. However, analysis of AOH regions and inbreeding coefficient (F) calculation using unphased ES data demonstrated higher than expected degrees of consanguinity (Table S1,S2). This was particularly striking for family 3 who would be regarded as nonconsanguineous based on clinical history (third cousins) yet exhibited calculated inbreeding coefficients (F = 0.1093, 0.1257) in excess of the clinical genetics definition for a consanguineous union (F = 0.0156, second cousins). Such discrepancies from expectations can result from high consanguinity rates in the underlying population or from unknown loops of consanguinity within consecutive generations. Indeed, the calculated inbreeding coefficients in a study of 1,020 healthy individuals from Pakistan, family 3’s country of origin, ranged from 0.029 to 0.091 . Thus, it is critical to utilize ES-derived AOH data and inbreeding coefficients in personal genome analysis to assess the likelihood of a recessive disorder and to identify regions of homozygosity harboring pathogenic alleles regardless of known family structure recorded by clinical history.
Three homozygous LoF NSRP1 variants were identified: c.1359_1362delAAAG (family 1), c.1272dupG (family 2), and c.52C>T (family 3) in families from Egypt, Iran, and Pakistan, respectively. While c.1359_1362delAAAG and c.1272dupG mRNA result in PTCs in the final exon and likely escape nonsense mediated decay (NMD), both are predicted to result in a mutant protein with loss of the nuclear localization signal (Fig. 2b) . Prior studies of human NSRP1 demonstrated this signal’s deletion results in abnormal cytoplasmic localization and loss of alternative splicing activity (Fig. 2c) . Both variants are therefore regarded as LoF variant alleles. c.52C>T results in a PTC in exon 2, and the mutant mRNA transcript is predicted to be unstable and subject to NMD. Thus, the association of human disease with biallelic LoF NSRP1 variants is congruent with studies in model organisms showing tolerance of haploinsufficiency but not biallelic null alleles [8, 16, 17]. As homozygous null NSRP1 alleles are incompatible with life in M. musculus, D. melanogaster, and C. elegans, the survival of these patients suggests these disease associated LoF variants may not be null alleles but rather hypomorphic LoF [8, 16, 17]. Alternatively, there may be reduced dependence on NSRP1 or greater redundancy during early embryonic development in humans.
It is important to acknowledge that NSRP1 itself undergoes alternative splicing. There are three major transcripts expressed within the adult human brain: ENST00000247026.9, ENST00000612959.4, and ENST00000394826.8 (Fig. S3, https://www.gtexportal.org/home/). ENST00000247026.9 is equivalent to NM_032141.4 and encodes the full-length 558-AA protein (Fig. 2a, S3). ENST00000612959.4 results in the N-terminal truncation of the first 54 AA, resulting in a 504-AA protein. Finally, ENST00000394826.8 has an alternative second exon that introduces a PTC in exon 3 and is therefore predicted to undergo NMD. The relative abundance of ENST00000247026.9 and ENST00000612959.4 varies between brain regions, with ENST00000612959.4 being the dominant transcript in some areas (e.g., the substantia nigra) and ENST00000247026.9 in others (e.g., the cerebellar hemispheres) (Fig. S3). As the variant found in family 3 is coding in ENST00000247026.9 (c.52C>T) and noncoding in ENST00000394826.8, it is possible this variant does not cause complete LoF, with the important caveat that GTEx reflects the adult rather than developmental brain transcriptome. In fact, this may help explain the phenotypic spectrum of the cohort. While the phenotypes highly overlap and form a consistent syndrome, the phenotype of family 3 with the c.52C>T variant allele is slightly milder. For example, the three siblings have less severe epilepsy and achieved more developmental milestones than families 1 and 2 (Table S3). This is congruent with model organism data showing milder phenotypic manifestations from incomplete loss of NSRP1 in C. elegans and X. laevis [16, 21].
There is presently little evidence for a neurodegenerative course in NSRP1-related disease. Considering the static nature of the syndrome and the occurrence of spastic quadriparesis, NSRP1 could be potentially considered as a cerebral palsy (CP) gene. While CP was traditionally attributed to perinatal insults, many patients lack such history, and genetic etiologies are increasingly recognized . In a large CP cohort, an enrichment of de novo variants (DNV) was detected . This enrichment is consistent with the epidemiology of CP, with most cases occurring sporadically . However, biallelic damaging variants in several hereditary spastic paraplegia genes were also identified, demonstrating both dominant and recessive disease traits contribute to CP genetics . Network analysis revealed enrichment of damaging variants in genes involved in neuritogenesis including extracellular matrix, focal adhesions, cytoskeleton, and Rho GTPases . Given NSRP1’s role in splicing regulation, it will be important to examine how genes downstream of NSRP1 intersect with genes previously associated with CP, intellectual disability (ID), or epilepsy.
Splicing dysfunction is a major cause of Mendelian disorders. The proportion of human pathogenic variants affecting cis-acting elements is estimated between 15% and 60% . In contrast, few Mendelian genetic diseases result from pathogenic variation in splicing factors . Until recently, Mendelian disorders involving spliceosomal components or splicing factors fit into two categories: craniofacial–skeletal disorders or isolated retinitis pigmentosa with rare overlap (Fig. S4) . It is unclear why ubiquitously expressed splicing factors segregate into these disease categories but may reflect differential tissue sensitivities. Many splicing factors involved in mouse brain development (e.g. PTBP1, PTBP2, NOVA1) exhibit LoF intolerance (pLI 0.98-1) yet presently lack human disease associations, suggesting many Mendelian splicing factor disorders remain to be discovered . Supporting this contention, of 28 novel candidate genes identified in a statistical analysis of 31,058 parent–offspring trios of individuals with developmental disorders, 3 encode splicing factors or spliceosomal components (SRRM2, U2AF2, and HNRNPD) . However, they may also cause human disease through biallelic recessive trait mechanisms as seen with NSRP1 and therefore should be prioritized in gene discovery efforts concentrated on consanguineous families.
In conclusion, these data establish that biallelic pathogenic variants in the splicing regulator NSRP1 cause an AR NDD trait characterized by DD, epilepsy, microcephaly, and spastic cerebral palsy and implicate NSRP1 as a key splicing factor in human brain development. Neuron-specific knockout of NSRP1 in model organisms, and studies of an NSRP1 allelic series during development, will provide further insight into its role. Such studies may also provide insights into the downstream gene conformers involved in mammalian brain development.
All data described in this study are provided within the article and Supplementary Material. Raw sequencing data and de-identified clinical data is available from the corresponding authors upon request.
Ule J, Blencowe BJ. Alternative splicing regulatory networks: functions, mechanisms, and evolution. Mol Cell. 2019;76:329–45.
Yeo G, Holste D, Kreiman G, Burge CB. Variation in alternative splicing across human tissues. Genome Biol. 2004;5:R74.
Raj B, Blencowe BJ. Alternative splicing in the mammalian nervous system: recent insights into mechanisms and functional roles. Neuron. 2015;87:14–27.
Vuong CK, Black DL, Zheng S. The neurogenetics of alternative splicing. Nat Rev Neurosci. 2016;17:265–81.
Griffin C, Saint-Jeannet JP. Spliceosomopathies: diseases and mechanisms. Dev Dyn. 2020;249:1038–46.
Chen Y, Belmont AS. Genome organization around nuclear speckles. Curr Opin Genet Dev. 2019;55:91–9.
Galganski L, Urbanek MO, Krzyzosiak WJ. Nuclear speckles: molecular organization, biological function and role in disease. Nucleic Acids Res. 2017;45:10350–68.
Kim YD, Lee JY, Oh KM, Araki M, Araki K, Yamamura K, et al. NSrp70 is a novel nuclear speckle-related protein that modulates alternative pre-mRNA splicing in vivo. Nucleic Acids Res. 2011;39:4300–14.
Kim CH, Kim YD, Choi EK, Kim HR, Na BR, Im SH, et al. Nuclear speckle-related protein 70 binds to serine/arginine-rich splicing factors 1 and 2 via an arginine/serine-like region and counteracts their alternative splicing activity. J Biol Chem. 2016;291:6169–81.
Sobreira N, Schiettecatte F, Valle D, Hamosh A. GeneMatcher: a matching tool for connecting investigators with an interest in the same gene. Hum Mutat. 2015;36:928–30.
Eldomery MK, Coban-Akdemir Z, Harel T, Rosenfeld JA, Gambin T, Stray-Pedersen A, et al. Lessons learned from additional research analyses of unsolved clinical exome cases. Genome Med. 2017;9:26.
Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. 2007;81:559–75.
Karaca E, Posey JE, Coban Akdemir Z, Pehlivan D, Harel T, Jhangiani SN, et al. Phenotypic expansion illuminates multilocus pathogenic variation. Genet Med. 2018;20:1528–37.
Gonzaga-Jauregui C, Yesil G, Nistala H, Gezdirici A, Bayram Y, Nannuru KC, et al. Functional biology of the Steel syndrome founder allele and evidence for clan genomics derivation of COL27A1 pathogenic alleles worldwide. Eur J Hum Genet. 2020;28:1243–64.
Hansen AW, Murugan M, Li H, Khayat MM, Wang L, Rosenfeld J, et al. A genocentric approach to discovery of Mendelian disorders. Am J Hum Genet. 2019;105:974–86.
Kovacevic I, Ho R, Cram EJ. CCDC-55 is required for larval development and distal tip cell migration in Caenorhabditis elegans. Mech Dev. 2012;128:548–59.
Yan D, Perrimon N. spenito is required for sex determination in Drosophila melanogaster. Proc Natl Acad Sci USA. 2015;112:11606–11.
Gu T, Zhao T, Kohli U, Hewes RS. The large and small SPEN family proteins stimulate axon outgrowth during neurosecretory cell remodeling in Drosophila. Dev Biol. 2017;431:226–38.
Scheffer IE, Berkovic S, Capovilla G, Connolly MB, French J, Guilhoto L, et al. ILAE classification of the epilepsies: position paper of the ILAE Commission for Classification and Terminology. Epilepsia. 2017;58:512–21.
Anwar I, Taroni F. Genetic peopling of Pakistan: Influence of consanguinity on population structure and forensic evaluation of traces. Forensic Sci Int Genet Suppl Ser. 2019;7:232–3.
Lee SH, Kim C, Lee HK, Kim YK, Ismail T, Jeong Y, et al. NSrp70 is significant for embryonic growth and development, being a crucial factor for gastrulation and mesoderm induction. Biochem Biophys Res Commun. 2016;479:238–44.
Jin SC, Lewis SA, Bakhtiari S, Zeng X, Sierant MC, Shetty S, et al. Mutations disrupting neuritogenesis genes confer risk for cerebral palsy. Nat Genet. 2020;52:1046–56.
Park E, Pan Z, Zhang Z, Lin L, Xing Y. The expanding landscape of alternative splicing variation in human populations. Am J Hum Genet. 2018;102:11–26.
Kaplanis J, Samocha KE, Wiel L, Zhang Z, Arvai KJ, Eberhardt RY, et al. Evidence for 28 genetic disorders discovered by combining healthcare and research data. Nature. 2020;586:757–62.
This study was supported in part by the US National Human Genome Research Institute (NHGRI) and National Heart Lung and Blood Institute (NHLBI) to the Baylor-Hopkins Center for Mendelian Genomics (BHCMG, UM1 HG006542, J.R.L); NHGRI grant to Baylor College of Medicine Human Genome Sequencing Center (U54HG003273 to R.A.G.), NHGRI grant to the Yale-NIH Center for Mendelian Genomics (U54 HG006504-01); U.S. National Institute of Neurological Disorders and Stroke (NINDS) (R35NS105078 to J.R.L. and R01NS106298 to M.C.K.), Muscular Dystrophy Association (MDA) (512848 to J.R.L.), and Spastic Paraplegia Foundation Research Grant to J.R.L. S.B. is supported by a Cerebral Palsy Alliance Research Foundation Career Development Award (#CDG01318). D.M. is supported by a Medical Genetics Research Fellowship Program through the United States National Institute of Health (T32 GM007526-42). T.M. is supported by the Uehara Memorial Foundation. D.P. is supported by a fellowship award from International Rett Syndrome Foundation (IRSF grant #3701‐1). J.E.P. was supported by NHGRI K08 HG008986. D.G.C. is supported by NIH - Brain Disorders and Development Training Grant (T32 NS043124-19) and MDA Development Grant (873841).
This study adheres to the principles in the Declaration of Helsinki. The study was reviewed by Baylor College of Medicine Institutional Review Board (IRB) protocol H-29697. Written informed consent was obtained from all participants including consent for publication of photographs as required by the IRB. Consent forms are archived and available upon request.
J.R.L. has stock ownership in 23andMe, is a paid consultant for Regeneron Genetics Center, and is a co-inventor on multiple United States and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, and bacterial genomic fingerprinting. The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing conducted at Baylor Genetics (BG) Laboratories. M.C.K. is a paid consultant for PTC Therapeutics and Aeglea. R.P. and R.E.S. are employees of GeneDx. The other authors have no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Calame, D.G., Bakhtiari, S., Logan, R. et al. Biallelic loss-of-function variants in the splicing regulator NSRP1 cause a severe neurodevelopmental disorder with spastic cerebral palsy and epilepsy. Genet Med 23, 2455–2460 (2021). https://doi.org/10.1038/s41436-021-01291-x