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Expanding SPTAN1 monoallelic variant associated disorders: From epileptic encephalopathy to pure spastic paraplegia and ataxia

Open AccessPublished:November 03, 2022DOI:https://doi.org/10.1016/j.gim.2022.09.013

      Abstract

      Purpose

      Nonerythrocytic αII-spectrin (SPTAN1) variants have been previously associated with intellectual disability and epilepsy. We conducted this study to delineate the phenotypic spectrum of SPTAN1 variants.

      Methods

      We carried out SPTAN1 gene enrichment analysis in the rare disease component of the 100,000 Genomes Project and screened 100,000 Genomes Project, DECIPHER database, and GeneMatcher to identify individuals with SPTAN1 variants. Functional studies were performed on fibroblasts from 2 patients.

      Results

      Statistically significant enrichment of rare (minor allele frequency < 1 × 10–5) probably damaging SPTAN1 variants was identified in families with hereditary ataxia (HA) or hereditary spastic paraplegia (HSP) (12/1142 cases vs 52/23,847 controls, p = 2.8 × 10–5). We identified 31 individuals carrying SPTAN1 heterozygous variants or deletions. A total of 10 patients presented with pure or complex HSP/HA. The remaining 21 patients had developmental delay and seizures. Irregular αII-spectrin aggregation was noted in fibroblasts derived from 2 patients with p.(Arg19Trp) and p.(Glu2207del) variants.

      Conclusion

      We found that SPTAN1 is a genetic cause of neurodevelopmental disorder, which we classified into 3 distinct subgroups. The first comprises developmental epileptic encephalopathy. The second group exhibits milder phenotypes of developmental delay with or without seizures. The final group accounts for patients with pure or complex HSP/HA.

      Keywords

      Introduction

      The αII-spectrin gene, SPTAN1 (OMIM 182810), encodes a membrane scaffolding protein that plays an important role in the maintenance of integrity of myelinated axons, axonal development, and synaptogenesis.
      • Morrow J.S.
      • Stankewich M.C.
      The spread of spectrin in ataxia and neurodegenerative disease.
      Heterozygous SPTAN1 pathogenic variants have been previously reported with variable phenotypes, most frequently causing mild to severe developmental epileptic encephalopathy (DEE) and developmental delay (DD)
      • Syrbe S.
      • Harms F.L.
      • Parrini E.
      • et al.
      Delineating SPTAN1 associated phenotypes: from isolated epilepsy to encephalopathy with progressive brain atrophy.
      and rarely with hereditary motor neuropathy and autosomal recessive hereditary spastic paraplegia (HSP).
      • Beijer D.
      • Deconinck T.
      • De Bleecker J.L.
      • et al.
      Nonsense mutations in alpha-II spectrin in three families with juvenile onset hereditary motor neuropathy.
      ,
      • Leveille E.
      • Estiar M.A.
      • Krohn L.
      • et al.
      SPTAN1 variants as a potential cause for autosomal recessive hereditary spastic paraplegia.
      A mouse model harboring αII-spectrin missense variant (p.Arg1098Gln) was reported to develop progressive ataxia with global neurodegeneration and seizures.
      • Miazek A.
      • Zalas M.
      • Skrzymowska J.
      • et al.
      Age-dependent ataxia and neurodegeneration caused by an αII spectrin mutation with impaired regulation of its calpain sensitivity.
      On the basis of these findings, we carried out a SPTAN1 gene enrichment analysis in the 100,000 Genomes Project (100K GP)
      • Davies M.D.
      • Elbahy L.
      • Fowler T.
      • et al.
      Data from: The national genomics research and healthcare knowledgebase.
      and identified a statistically significant enrichment for rare probably damaging variants in hereditary ataxia (HA) and HSP groups. In this study, we present an extended phenotypic spectrum of neurologic syndromes caused by pathogenic variations of SPTAN1 gene.

      Materials and Methods

      Patients

      Our initial cohort comprised 100K GP neurology patients.
      • Davies M.D.
      • Elbahy L.
      • Fowler T.
      • et al.
      Data from: The national genomics research and healthcare knowledgebase.
      All 100K GP genomes were previously screened for single nucleotide variants, small insertions/deletions, structural variants (SVs) or copy number variants (CNVs), and short tandem repeats in relevant genes from the PanelApp virtual gene panels (Genomics England).
      • Martin A.R.
      • Williams E.
      • Foulger R.E.
      • et al.
      PanelApp crowdsources expert knowledge to establish consensus diagnostic gene panels.
      We then screened DECIPHER database cohort for patients carrying single nucleotide variants and/or SVs/CNVs in SPTAN1 gene.
      • Firth H.V.
      • Richards S.M.
      • Bevan A.P.
      • et al.
      DECIPHER: database of chromosomal imbalance and phenotype in humans using Ensembl resources.
      Additional families were subsequently recruited through GeneMatcher.
      • Sobreira N.
      • Schiettecatte F.
      • Valle D.
      • Hamosh A.
      GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.
      All coding variants reported in this article are with reference to SPTAN1 RefSeq: NM_001130438.3 transcript. All procedures adhered to the principles set out in the Declaration of Helsinki and all patients/their guardians included in the study consented to participation according to ethical approval of the recruiting center.

      Gene enrichment analysis

      Case-control gene enrichment analysis was performed within the rare disease component of the 100K GP. Cases were defined as all 100K GP probands recruited under HA/HSP, whereas controls were all remaining probands recruited into the 100K GP except for those with neurologic and neurodevelopmental disorders or metabolic disorders. Enrichment of SPTAN1 rare, probably damaging variants in cases compared with controls was assessed via a two-sided Fisher exact test. The contributing variants were defined as rare (minor allele frequency < 1 × 10–5) and either protein-truncating variants or missense variants predicted to be pathogenic by 2 in silico tools (Combined Annotation Dependent Depletion [CADD]
      • Kircher M.
      • Witten D.M.
      • Jain P.
      • O’Roak B.J.
      • Cooper G.M.
      • Shendure J.
      A general framework for estimating the relative pathogenicity of human genetic variants.
      and Polymorphism Phenotyping [PolyPhen]
      • Adzhubei I.A.
      • Schmidt S.
      • Peshkin L.
      • et al.
      A method and server for predicting damaging missense mutations.
      ).

      Functional studies

      Fibroblasts derived from patient 1 (p. Arg19Trp) and patient 29 (p. Glu2207del) were used to test the functional effects of SPTAN1 variants on protein expression compared with that of healthy unrelated controls. Western blot analysis, immunocytochemistry, and confocal microscopy were performed as previously described.
      • Beijer D.
      • Deconinck T.
      • De Bleecker J.L.
      • et al.
      Nonsense mutations in alpha-II spectrin in three families with juvenile onset hereditary motor neuropathy.

      Structural modeling of SPTAN1 missense variants

      Three-dimensional protein modeling was used to evaluate the effect of reported SPTAN1 missense variants. Although the crystal structure for full-length αII-spectrin is unknown, crystal structures of the N-terminal tetramerization site and 2 spectrin repeat unit of chicken brain αII-spectrin have been solved (Protein Data Bank: 3F31 and 3Fb2).
      • Mehboob S.
      • Song Y.
      • Witek M.
      • et al.
      Crystal structure of the nonerythroid alpha-spectrin tetramerization site reveals differences between erythroid and nonerythroid spectrin tetramer formation.
      ,
      • Evans R.J.
      • Davies D.R.
      • Bullard J.M.
      • et al.
      Structure of PolC reveals unique DNA binding and fidelity determinants.
      We used the Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) predicted models for C-terminal and spectrin repeats 13 to 20 of αII-spectrin protein.
      • Kelley L.A.
      • Mezulis S.
      • Yates C.M.
      • Wass M.N.
      • Sternberg M.J.
      The Phyre2 web portal for protein modeling, prediction and analysis.
      DynaMut software was used to predict variant effect.
      • Rodrigues C.H.
      • Pires D.E.
      • Ascher D.B.
      DynaMut: predicting the impact of mutations on protein conformation, flexibility and stability.
      For simulating amino acid substitutions and visualization, UCSF Chimera built-in tools were used.
      • Pettersen E.F.
      • Goddard T.D.
      • Huang C.C.
      • et al.
      UCSF Chimera—a visualization system for exploratory research and analysis.
      In addition, in silico pathogenicity prediction analysis of all missense variants identified in the study and those previously reported in literature was conducted.

      Results

      SPTAN1 heterozygous damaging variants are enriched in families with HSP or HA

      SPTAN1 was investigated as a candidate gene for HA or HSP using gene enrichment analysis in the rare disease component of the 100K GP, which has a total of 35,422 rare disease families, including 1142 HA/HSP probands as cases and 23,847 non-neurologic/non-metabolic unrelated individuals as controls. A case-control analysis revealed a statistically significant enrichment of rare probably damaging heterozygous variants of SPTAN1 in probands with HA or HSP (12/1142 cases vs 52/23,847 controls, p = .00002846, odds ratio = 4.8594, 95% CI = 2.5867-9.1290) (Supplemental Table 1). None of the SPTAN1 variants found in controls were protein-truncating variants and none overlapped with any of the missense variants described in the study.
      Subsequently, we screened 100K GP neurology cohort (16,014 individuals with neurodevelopmental disorders) for probably damaging SPTAN1 variants in families with spasticity and ataxia in addition to the previously described phenotypes of seizures and/or intellectual disability (ID). We identified 11 patients from 9 families (Table 1, Figures 1 and 2). Patients 1 to 4 had pure HSP phenotype and shared the same SPTAN1 variant, p.(Arg19Trp). Later-onset and a more complex phenotype was noted in patient 8 who harbored p.(Ser2448Phe) variant. Although patient 7 was recruited under early-onset dystonia phenotype, she presented with abnormal eye movements, ataxia, myoclonus, and dyspraxia and had SPTAN1 variant, p.(Arg2124Cys). Patient 10, who had pure ataxia, harbored a heterozygous splice alteration in SPTAN1 (NC_000009.12[SPTAN1_v001]:c.3519+2T>G). SPTAN1 gene was also screened for CNVs/SVs in the 100K GP and 1 deletion was identified. Patient 9 carried a large heterozygous in-frame deletion (DEL1), encompassing exons 25 to 27 (Supplemental Figure 1) and presented with pure HA. Both patients 9 and 10 carried sporadic SPTAN1 variants because the de novo nature could not be confirmed owing to the unavailability of family members. Additional 3 probands with SPTAN1 variants presenting with seizures, ID, and ataxia/spasticity were identified.
      Table 1Clinical and genetic findings of 31 patients reported in this study
      General InformationFamily 1Family 2Family 3Family 4Family 5Family 6Family 7Family 8
      Patient 1Patient 2Patient 3Patient 4Patient 5Patient 6Patient 7Patient 8Patient 9Patient 10
      Sex/ethnicityF/AsiaF/EuropeF/EuropeM/EuropeF/EuropeM/EuropeF/NAM/AsiaF/NAM/Europe
      Age at last examination, y40252550171532507254
      Genetic findings
       c.DNAc.55C>Tc.55C>Tc.55C>Tc.55C>Tc.6370C>Tc.7343C>Tarr{hg38}9q34.11(128,597,905-128,602,892)x1NC_000009.12(SPTAN1_v001):c.3519+2T>G exon 25
       Proteinp.Arg19Trpp.Arg19Trpp.Arg19Trpp.Arg19Trpp.Arg2124Cysp.Ser2448Phep.(Asp1139_Lys1193)del

      (DEL 1)
      p.?
       InheritanceSporadicADSporadicDe novoSporadicUnknownSporadicSporadic
      Phenotypic categoryPure HSP/ataxiaPure HSP/ataxiaComplex HSPPure HSPPure HSP/ataxiaComplex HSPPure HAPure HA
      Initial symptomsSpastic gaitSpastic gaitSpastic gaitSpastic gaitLearning disability/ spastic ataxiaSpastic gaitSpastic gaitSpastic gaitAtaxiaAtaxia
      Age of onset, y8NANANA108NANA3635
      SZ (age of onset)Generalized (10 y)
      Response to therapy////VAL; controlled/////
      EEGNANANANANormalNANANANANA
      Developmental history
       ID+
       Learning disability++
       Motor delay
       Language delay
       Microcephaly
      Neurologic findings
       Ataxia++++++++
       Spasticity++++++++
       Extensor planter reflex++
       Abnormal eye movement+++Nystagmus+NystagmusNystagmus, esotropia
       UL weakness++++
       LL weakness++++++
       LL hyperreflexia++++++
       Ankle clonus+++
       Bladder dysfunction+++++
       Amyotrophy+++
       Myoclonus++
       Brain MRINormalNANANASubcortical white mater hyper intensitiesNormalNACerebellar atrophyNACerebellar atrophy marked in vermis
       Other clinical featuresImpaired vibratory sensationMild hearing loss, atrophic left kidneyHead tremors, dyspraxia, postural tremorsFasciculations, impaired proprioception, pes cavus, macular dystrophyApraxia, adult-onset sensorineural hearing lossAreflexia and decreased vibration sense in LL, vertical ophthalmoparesis
      General InformationFamily 9Family 10Family 11Family 12Family 13Family 14Family 15
      Patient 11Patient 12Patient 13Patient 14Patient 15Patient 16Patient 17Patient 18Patient 19Patient 20
      Sex/ethnicityM/EuropeM/EuropeM/EuropeF/EuropeM/EuropeM/NAM/EuropeF/EuropeF/EuropeM/Europe
      Age at last examination, y19972613223226
      Genetic findings
       c.DNAarr{hg38}9q34.11 (128,609,213-128,613,675)x1c.4458delAc.2197C>Tc.4936C>Tc.1879C>Tarr{hg38}9q34.11 (128591376-128600369)x1c.2612del
       Proteinp.(Ile1563_?)del (DEL2)p.Lys1486Asnfs51p.Arg733p.Gln1646p.Arg627p.(Asp1003_Lys1193)del

      (DEL3)
      p.Lys871Serfs5
       InheritanceDe novoADsporadicDe novoADDe novoDe novo
      Phenotypic categoryDD/ataxiaDDDDDDDDDDDD
      Initial symptomsAtaxiaSpeech delaySpeech delayIDIDDDAxial hypotoniaAxial hypotoniaDDID
      Age of onset15 mo2 y2 y3 yNABirth5 mo3 mo9 mo1 y
      SZ (age of onset)Febrile SZOne febrile generalized tonic-clonic SZ (6 y)
      Response to therapy/////////No therapy, controlled
      EEGNANANANANAIncreased excitabilityNANormalNANormal
      Developmental history
       ID+MildMildMild++?
       Learning disability++++++++?Mild
       Motor delay+++++Mild
       Speech delay+++++NA+Moderate
       Microcephaly+
      Neurologic findings
       AtaxiaSevere++
       Spasticity
       Extensor planter reflex
       Abnormal eye movementConvergent strabismusNystagmusNystagmusStrabismus
       UL weakness
       LL weakness++
       LL hyperreflexia
       Ankle clonus
       Bladder dysfunction+
       AmyotrophyNA
       Myoclonus
       Brain MRISevere cerebellar atrophy and slightly dilated fourth ventricleNANANANANonspecific small gliosis in the right anterior border zoneCerebellar atrophySevere cerebellar atrophyNANA
      Other clinical featuresPneumococcal meningitis aged 15 months, hypermetropiaAttention deficit Hyperactivity disorderBasedow disease in remissionobesity; dyslipidemia; testicular hypoplasia (MC4R disease)epicanthus, low-set ears, high philtrum, finger pads, sickle feet, sandal furrow16p11.2 microdeletion involving PRRT2hypotonia, poor motor planning and body awareness, tongue tieobesity, constipation, mild myopia
      General InformationFamily 16Family 17Family 18Family 19Family 20Family 21
      Patient 21Patient 22Patient 23Patient 24Patient 25Patient 26
      Sex/EthnicityF/EuropeM/NAF/EuropeM/EuropeM/NAM/Europe
      Age at last examination, y8951098
      Genetic findings
       c.DNAc.1127G>Ac.4390C>Tarr{hg38}9q34.11 (128,582,754-128,587,726)x1c.6611G>Ac.4476delarr{hg38}9q34.11 (128,587,422-128,600,316)x1
       Proteinp.Trp376p.Arg1464Trpp.(Asn571_?)del (DEL4)p.Arg2204Glnp.Ala1493Argfs44p.(Ala927_Lys1193) del

      (DEL5)
       InheritanceDe novoDe novoDe novoADSporadicDe novo
      Phenotypic categoryDDDD/SZDD/SZDD/SZDD/SZDD/SZ
      Initial symptomsSpeech delaySZHypotoniaDDIDSZ
      Age of onset2 yNA2 mo2 y2 y3 mo
      SZ (age of onset)Febrile and generalized myoclonic (3 y)+ (2 y)Myoclonic absence (5 y)Absence and generalized tonic-clonic (6 y)Infantile spasms (3 mo)
      Response to therapy/NACBZ, controlledNARefractoryPartially controlled
      EEGNANANANATypical absence, continuous spikes and waves during sleep aspectVertex sharp waves, right and left independent parietal waves during sleep
      Developmental history
       ID+++++
       Learning disability++++++
       Motor delay++++
       Speech delay++++++
       Microcephaly+++
      Neurologic findings
      Ataxia++
      SpasticityNA
      Extensor planter reflex
      Abnormal eye movementStrabismus
      UL weaknessNA
      LL weaknessNA
      LL hyperreflexia
      Ankle clonus
      Bladder dysfunction
      Amyotrophy
      Myoclonus
      Brain MRINormalNAMild cerebral atrophyNormalNormalMild delay in myelination
      Other clinical featuresDyspraxia, hyperlaxityHypotonia, sleep problems, constipationADHDPoor proprioception, hypotonia
      General InformationFamily 22Family 23Family 24Family 25Family 26
      Patient 27Patient 28Patient 29Patient 30Patient 31
      Sex/ethnicityF/EuropeM/EuropeF/AfricaF/EuropeM/India
      Age at last examination, y16122071
      Genetic findings
       c.DNAc.6247_6249delc.6811G>Ac.6619_6621delc.4344G>Ac.6908_6916dup
       Proteinp.Lys2083delp.Glu2271Lysp.Glu2207delp.Gln1448= (splice)

      exon 38
      p.Asp2303_Leu2305dup
       InheritanceUnknownDe novoDe novoUnknownDe novo
      Phenotypic categoryDEEDEEDEEDEEDEE
      Initial symptomsSZSZHypotoniaAxial hypotoniaSZ
      Age of onset2 mo4 mo8 moBirthBirth
      SZ (age of onset)Absence SZ (2 mo)Myoclonic jerks, dystonic spasms (4 mo)Tonic, oral automatisms, upward deviation of gazeWest syndrome (4 mo)Generalized tonic-clonic (birth)
      Response to therapyNARefractoryControlledRefractoryRefractory
      EEGNASlow-wave activity, loss of normal rhythms, temporal spike, sharp wave dischargesDiffuse slow activity, irregular low to medium amplitudeHypsarrhythmiaMultifocal epilepsy
      Developmental history
       ID+++severeNA
       Learning disability+++severeNA
       Motor delay+++severesevere
       Speech delay+++severeNA
       Microcephaly+++severe
      Neurologic findings
      Ataxia+NANA
      Spasticity+NA+
      Extensor planter reflex++
      Abnormal eye movementStrabismusStrabismusNystagmusNystagmus
      UL weakness+++
      LL weakness+++
      LL hyperreflexia++
      Ankle clonus+
      Bladder dysfunction
      Amyotrophy+
      Myoclonus
      Brain MRINACerebellar atrophy, delayed myelination, thin corpus callosumNADelayed myelination, progressive brain and pontocerebellar atrophyDelayed myelination, cerebral atrophy, thin corpus callosum
      Other clinical featuresRod cone retinal dystrophy, GERD, scoliosis, dislocated hipVisual impairment, dysmorphism, scoliosis
      All variants are reported with reference to RefSeq NM_001130438.3.
      AD, autosomal dominant; ADHD, attention deficit hyperactivity disorder; CBZ, carbamazepine; DD, developmental delay; DEE, developmental epileptic encephalopathy; EEG, electroencephalography; F, female; GERD, gastroesophageal reflux; HA, hereditary ataxia; HSP, hereditary spastic paraplegia; ID, intellectual disability; LL, lower limbs; M, male; MRI, magnetic resonance imaging; NA, not available; SZ, seizures; UL, upper limbs; VAL, valproic acid; /, not applicable; –, absence of manifestation; +, presence of manifestation.
      Figure thumbnail gr1
      Figure 1Pedigrees of reported families with SPTAN1 variants showing disease segregation.
      Figure thumbnail gr2
      Figure 2Schematic structure of SPTAN1 gene and its coding protein highlighting variants identified in this study. Coding exon numbers (NM_001130438.3) are reported on the gray bar. Truncating variants are indicated on the top. Missenses, in-frame deletion/insertion, and splice variants are on the bottom. Deletion 1, 3, and 5 (green) remain in frame, whereas predictions for deletions 2 and 4 (orange) are not available. p.Gln1448= (c.4344G>A) is predicted to affect exon 33 donor splice site, based on maxENTScan (predicting splice sites using ‘Maximum Entropy Principle’) (maxENT score wild-type 6.99 → 3.84 mutant). The splice altering variant (NC_000009.12(SPTAN1_v001):c.3519+2T>G) predicted to alter exon 25 canonical donor splice site (maxENT score wild-type 10.28 → 2.63 mutant). Variants identified in patients presenting with HSP/HA are highlighted in red. All other variants are represented in black.
      By screening DECIPHER database,
      • Firth H.V.
      • Richards S.M.
      • Bevan A.P.
      • et al.
      DECIPHER: database of chromosomal imbalance and phenotype in humans using Ensembl resources.
      4 additional variants were identified; 1 missense variant in patient 22 and 3 de novo microdeletions in the SPTAN1 gene. DEL2 in patient 11 is a 4.46-kilobase deletion that removes exons 36 to 40. This patient presented with ataxia and severe DD. Patients 23 and 26, who carried DEL4 (exons 14-20) and DEL5 (exons 20-27), respectively, presented with DD and seizures.
      A total of 13 additional SPTAN1 families were identified through GeneMatcher.
      • Sobreira N.
      • Schiettecatte F.
      • Valle D.
      • Hamosh A.
      GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.
      Patients 5 and 6 shared the same de novo missense variant, p.(Arg19Trp), and HSP phenotype as patients 1 to 4. Nevertheless, patient 5 presented with complex HSP, learning disability, and seizures. Three frameshift variants were identified in 5 patients (patients 12-14, 20, and 25) with DD/Seizures. Dominant inheritance was noted in 3 patients (patients 12-14). Five patients (patients 15-18 and 21) with ID/seizures carried nonsense SPTAN1 variants. The last variant was an in-frame deletion (DEL3), which was identified in patient 19, a 2-year-old presented with DD. This deletion is almost 9 kilobase, encompasses exons 22 to 27, and overlaps with DEL5. All variants reported in this study were classified according to the guidelines of the American College of Medical Genetics and Genomics and Association for Molecular Pathology.
      • Richards S.
      • Aziz N.
      • Bale S.
      • et al.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      (Supplemental Table 2).

      Clinical phenotypes

      Detailed clinical information was collated for 31 individuals from 26 unrelated families carrying heterozygous variants in SPTAN1 (Table 1). Common phenotypes including ID/learning disability and motor delay were reported in 73.5% and 58.8% of our cohort, respectively. Remarkably, around half of the patients (15/31) manifested ataxia, and seizures were reported in almost one-third of the cases (12/31).
      Recently reported phenotypes of HSP/HA were identified in a subgroup of our patients. A total of 8 individuals from 6 families presented with pure HSP/HA and further 2 families with complex HSP (Supplemental Video 1). All patients with HSP showed typical features. Most of them had lower limb hyperreflexia and/or ataxia (6/8) whereas none had sensory abnormalities. In contrast, 2 patients with HA showed a pure phenotype. Abnormal eye movement, a common condition in patients with ataxia, was noted in 70% of this HSP/HA group. Severe phenotype of DEE was reported in 5 patients. All had seizures in early months of life and had severe DD. The remaining 16 patients from 13 families presented with varying degrees of DD.

      Functional consequences of SPTAN1 variants in patient-derived fibroblasts

      We analyzed fibroblasts derived from 2 patients with the missense variants p.(Arg19Trp) and p.(Glu2207del) (Figure 3). Although western blot did not show a quantitative difference in protein expression between patient 1 and controls, a quantitative reduction of protein expression in patient 29 was noted. Nevertheless, a high immunofluorescence brightness and intense immunoreactivity of αII-spectrin was observed in both the studied patients. In patient 1, αII-spectrin was ubiquitously expressed throughout the fibroblast cells, whereas there was a more localized aggregation of αII-spectrin in the plasma membrane with a relatively higher immunofluorescence brightness observed in the fibroblasts of patient 29.
      Figure thumbnail gr3
      Figure 3Representative images of αII-spectrin protein expression and staining pattern in fibroblast cells derived from 2 patients and unrelated controls. A. Western blot. 1. Western blotting of protein extracted from fibroblast cell lines of patients 1 and 29 and 3 wild-type age-matched controls. 2. Densitometric analysis of western blot using BioRad Image Lab software after relative normalization to actin as a housekeeping protein. The analysis showed no change in protein expression in patient 1 but showed a quantitative reduction of protein expression in patient 29. B. Immunocytochemical staining of αII-spectrin expression in primary fibroblasts of patients 1 and 29 and unrelated control individual with Alexa Fluor 488 conjugated secondary antibody (green) and Hoescht 33342 nuclear staining (blue). Scale bars represent 50 μm. Immunocytochemical staining showed high immunofluorescence brightness and intense immunoreactivity and aggregation of αII-spectrin in both studied patients compared with the healthy unrelated control. Ctr, control.

      Structural modeling of SPTAN1 missense variants

      The effect of missense variants on αII-spectrin protein structure (Q13813-1) was investigated using homology modeling of experimentally validated models
      • Mehboob S.
      • Song Y.
      • Witek M.
      • et al.
      Crystal structure of the nonerythroid alpha-spectrin tetramerization site reveals differences between erythroid and nonerythroid spectrin tetramer formation.
      • Evans R.J.
      • Davies D.R.
      • Bullard J.M.
      • et al.
      Structure of PolC reveals unique DNA binding and fidelity determinants.
      • Kelley L.A.
      • Mezulis S.
      • Yates C.M.
      • Wass M.N.
      • Sternberg M.J.
      The Phyre2 web portal for protein modeling, prediction and analysis.
      (Supplemental Figure 2). Variant p.(Arg19Trp) had the most deleterious effect on protein structure. This variant is located within the N-terminal tetramerization domain and results in steric clashes with 2 leucine residues in the beta chain. Less severe effect was noted in modeling of p.(Arg1464Trp) and p.(Arg2204Gln) variants. Structural modeling showed no structural effect for p.(Glu2271Lys), p.(Arg2124Cys), and p.(Ser2448Phe); however these would likely affect αII-spectrin heterodimerization with its partner, β-spectrin. Overall, most (4/6) of the reported missense variants led to protein destabilization (Supplemental Table 3). In addition, according to in silico pathogenicity prediction, almost all missense variants identified in this study are potentially pathogenic (Supplemental Table 4).

      Discussion

      Identification of an enrichment of SPTAN1 heterozygous variants in patients presenting with HA and HSP confirms SPTAN1 involvement in a wide phenotypic spectrum. We suggest that SPTAN1 is a genetic cause of neurodevelopmental disorders, with 3 phenotypic subgroups (Table 1). The first group comprises patients with DEE presenting with severe phenotype (OMIM 613477). DEE was identified in 5 families, consistent with previous reports.
      • Syrbe S.
      • Harms F.L.
      • Parrini E.
      • et al.
      Delineating SPTAN1 associated phenotypes: from isolated epilepsy to encephalopathy with progressive brain atrophy.
      A total of 16 patients manifested milder phenotype of DD with or without childhood-onset seizures forming the second phenotypic group. The final group consists of patients (n = 10) with pure or complex HSP/HA.
      Involvement of SPTAN1 variants in peripheral nervous system abnormalities was previously reported for heterozygous variants causing hereditary motor sensory neuropathy
      • Beijer D.
      • Deconinck T.
      • De Bleecker J.L.
      • et al.
      Nonsense mutations in alpha-II spectrin in three families with juvenile onset hereditary motor neuropathy.
      and biallelic variants associated with autosomal recessive HSP.
      • Leveille E.
      • Estiar M.A.
      • Krohn L.
      • et al.
      SPTAN1 variants as a potential cause for autosomal recessive hereditary spastic paraplegia.
      The phenotype of HA and HSP is further supported by a previously reported SPTAN1 mouse model presenting with unsteady gait and spasticity
      • Miazek A.
      • Zalas M.
      • Skrzymowska J.
      • et al.
      Age-dependent ataxia and neurodegeneration caused by an αII spectrin mutation with impaired regulation of its calpain sensitivity.
      (Supplemental Video 2) and the recently published study reporting de novo and dominant variants of SPTAN1 in patients with ataxia and patients with spastic paraplegia.
      • Van de Vondel L.
      • De Winter J.
      • Beijer D.
      • et al.
      De novo and dominantly inherited SPTAN1 mutations cause spastic paraplegia and cerebellar ataxia.
      Phenotypic heterogeneity may be explained by the involvement of SPTAN1 pathogenic variants in different mechanisms of pathogenicity as it was described with other structural proteins.
      • Veitia R.A.
      • Caburet S.
      • Birchler J.A.
      Mechanisms of Mendelian dominance.
      Syrbe et al
      • Syrbe S.
      • Harms F.L.
      • Parrini E.
      • et al.
      Delineating SPTAN1 associated phenotypes: from isolated epilepsy to encephalopathy with progressive brain atrophy.
      concluded that variants in the last 2 αII-spectrin repeats are associated with severe phenotype due to protein aggregation with dominant negative effect. This mechanism is supported by our observation in αII-spectrin immunocytochemistry experiment performed on fibroblast of patient 29. However, further experiments on multiple cell lines would be imperative to support this hypothesis.
      We noted that the excess of truncating variants in the milder category of our cohort (DD +/– seizures) is in agreement with the proposed mechanism of quantitative defect of αII-spectrin protein leading to a milder phenotype.
      • Gartner V.
      • Markello T.C.
      • Macnamara E.
      • et al.
      Novel variants in SPTAN1 without epilepsy: an expansion of the phenotype.
      We suggest that truncating variants are responsible for a mild DD with or without epilepsy.
      In our HSP/HA group, we have detected accumulation of αII-spectrin in fibroblasts of patient 1 with the recurrent p.(Arg19Trp) variant, indicating abnormal protein function. Such interesting finding adds further evidence to Van de Vondel et al
      • Van de Vondel L.
      • De Winter J.
      • Beijer D.
      • et al.
      De novo and dominantly inherited SPTAN1 mutations cause spastic paraplegia and cerebellar ataxia.
      report of the recurrent p.(Arg19Trp) variant detected in 7 families with spastic paraplegia. All other identified variants in this group of our patients were missense variants except for 1 splice altering variant. All are predicted to have a moderate protein effect except for p.(Arg19Trp), which is localized at an essential position of the N-terminal tetramerization domain.
      • Morrow J.S.
      • Stankewich M.C.
      The spread of spectrin in ataxia and neurodegenerative disease.
      An arginine to tryptophan change, p.(Arg35Trp), at the N-terminus has been reported previously in erythrocytic α-spectrin gene (SPTA1), where it prevented N-terminal domain of α-spectrin to form heterotetramers with its beta partner. We suggest a similar mechanism for SPTAN1 variant, p.(Arg19Trp).
      • Morlé L.
      • Morlé F.
      • Roux A.F.
      • et al.
      Spectrin Tunis (Sp alpha I/78), an elliptocytogenic variant, is due to the CGG----TGG codon change (Arg----Trp) at position 35 of the alpha I domain.
      In a previous report, we showed that ataxia and/or HSP cases may be accounted by hypomorphic pathogenic variants in genes known to manifest with severe phenotypes when mutated.
      • Benkirane M.
      • Marelli C.
      • Guissart C.
      • et al.
      High rate of hypomorphic variants as the cause of inherited ataxia and related diseases: study of a cohort of 366 families.
      The p.(Arg2124Cys) and p.(Ser2448Phe) variants identified in families 5 and 6 are missense variants that are predicted to introduce mild structural alterations in the αII-spectrin protein potentially explaining the milder neurologic impairment. In family 8, with mild and late-onset ataxia, a splice altering variant (c.3519+2T>G) was detected. The resulting predicted in-frame deletion rather than a loss of function might explain the mild neurologic phenotype in this family.
      It is interesting to notice that αII-spectrin forms heterotetramers with each of the 4 nonerythrocytic β-spectrins 1 to 4 at precise membrane domains.
      • Leterrier C.
      A dual role for βII-spectrin in axons.
      The first 3 β-spectrin genes (SPTBN1, SPTBN2, and SPTBN4) are responsible of multiple neurologic disorders, depending on the gene and inheritance pattern.
      • Veitia R.A.
      • Caburet S.
      • Birchler J.A.
      Mechanisms of Mendelian dominance.
      Particularly, βIII spectrin (SPTBN2) is specific to Purkinje cells and is involved in a relatively pure late-onset ataxia phenotype.
      • Ikeda Y.
      • Dick K.A.
      • Weatherspoon M.R.
      • et al.
      Spectrin mutations cause spinocerebellar ataxia type 5.
      αII-Spectrin broad distribution in the neurons is therefore a plausible explanation of the pleiotropic consequences of SPTAN1 variants.
      Molecular diagnosis of patients with neurodevelopmental disorders is often challenging owing to both phenotypic and genetic heterogenicity. We were able to expand the phenotypic and genetic spectrum of SPTAN1 variants, shedding light on the critical role that αII-spectrin has in maintaining brain health.

      Data Availability

      De-identified data are available upon request. Data requests can be made via email to the corresponding author and will be pending data use agreements.

      Conflict of Interest

      The authors declare no conflicts of interest.

      Acknowledgments

      H.M., J.V., and H.H. are supported by an Medical Research Council strategic award, MR/S005021/1, to establish International Centre for Genomic Medicine in Neuromuscular Diseases. H.M. is supported by Wellcome Trust grant 220906/Z/20/Z. H.H. is funded by the Medical Research Council (MR/S01165X/1, MR/S005021/1, G0601943), NIHR University College London Hospitals Biomedical Research Centre , Rosetree Trust UK , Ataxia UK , Multiple System Atrophy Trust, Brain Research UK , Sparks GOSH Charity, Muscular Dystrophy UK , and Multiple System Atrophy Trust . R.But. is supported by the Penelope Rare and Undiagnosed Disease Program at the University of Utah with funding from the Center for Genomic Medicine and with support from Matt Velinder (Department of Human Genetics, University of Utah ) and Rong Mao and Pinar Bayrak-Toydemir (ARUP Laboratories). B.M. is supported by NKFIH K138669. This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health and Care Research and NHS England. The Wellcome Trust , Cancer Research UK , and the Medical Research Council have also funded the research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support. This study makes use of data generated by the DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources (DECIPHER) community. A full list of centers which contributed to the generation of the data is available at https://deciphergenomics.org/about/stats and via email from [email protected] . Funding for the DECIPHER project was provided by Wellcome . We are thankful to the Deciphering Developmental Disorders Study for the invaluable collaboration. The Deciphering Developmental Disorders Study (Cambridge South Research Ethics Committee approval 10/H0305/83 and the Republic of Ireland Research Ethics Committee GEN/284/12) presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and Department of Health and the Wellcome Trust Sanger Institute (grant number WT098051). We also thank Lise Larrieu and Morgane Pointaux (Molecular Genetics’ Laboratory, Institut Universitaire de Recherche Clinique, CHU de Montpellier and PhyMedExp Univ Montpellier, Montpellier, France) for their excellent technical assistance. We appreciate the help of Grenenko Cecile (Service de Neurologie Pédiatrique, Centre Hospitalier Universitaire Amiens-Picardie, Amiens, France) and Jedraszak Guillaume new (Laboratoire de Génétique Constitutionnelle, Centre Hospitalier Universitaire Amiens-Picardie, Amiens, France) in the study. We are thankful to Béatrice Desnous (Reference Center for Inherited Metabolic Diseases, Marseille, France) for his contribution to clinical investigation. Finally, we are grateful to all the patients and their families.

      Author Information

      Conceptualization: H.M., H.H.; Data Curation: H.M., M.B., M.St.; Funding Acquisition: M.Ha., H.H.; Investigation: R.M., S.E., M.O., M.Su., S.Ban., J.C.-S., T.W., M.R., J.A.B., M.N., B.Co., R.A.J., T.B., M.He., J.G.-A., I.K., D.W., F.G., M.M., A.G., G.B.-L., H.G.-M., P.G., B.Ch., S.Bac., R.Bui., V.M., M.Z., R.But., D.H., G.B.-L., A.S., B.M., K.S., M.S., V.K.G., V.M.S.; Methodology: K.Z., E.C., V.C., C.R., E.G.; Software: D.M.; Resources: M.S.; Supervision: M.St., M.K., J.V., H.H.; Writing-original draft: H.M., M.B.; Writing-review and editing: R.M., S.E., M.O., M.Su., S.Ban., J.C., T.W., R.M., J.A.B., M.N., B.Co., R.A.J., T.B., H.M., J.G.-A., I.K., D.W., F.G., M.M., A.G., G.-L., H.G.-M., P.G., B.Ch., S.Bac., R.Bui., M.He., V.M., M.Z., R.But., D.H., G.B.-L., A.S., B.M., K.S., M.S., V.K.G., V.M.S., A.G., K.Z., E.C., V.C., C.R., E.G., D.M., M.Ha., M.St., M.K., J.V., H.H.

      Ethics Declaration

      The study was carried out as per the ethical approval from the University College London Hospital Research Ethics Committee, reference: 07/Q0512/26. All institutions involved in this research received local ethical approval. All patients’ data were de-identified. Informed consent was obtained from all participants, their parents, or legal representatives. The authors have received and archived written patient consent for publishing the video of the individual included in this study. Experiments involving mice were approved by the local Ethical Committee in Wroclaw (Poland) under permission number 78/2018 and by the Institutional Animal Care and Use Committee at Yale University, which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care.

      Additional Information

      Members of The Genomics England Research Consortium

      J.C. Ambrose, P. Arumugam, E.L. Baple, M. Bleda, F. Boardman-Pretty, J.M. Boissiere, C.R. Boustred, H. Brittain, M.J. Caulfield, G.C. Chan, C.E.H. Craig, L.C. Daugherty, A. de Burca, A. Devereau, G. Elgar, R.E. Foulger, T. Fowler, P. Furió-Tarí, J.M. Hackett, D. Halai, A. Hamblin, S. Henderson, J.E. Holman, T.J.P. Hubbard, K. Ibáñez, R. Jackson, L.J. Jones, D. Kasperaviciute, M. Kayikci, L. Lahnstein, K. Lawson, S.E.A. Leigh, I.U.S. Leong, F.J. Lopez, F. Maleady-Crowe, J. Mason, E.M. McDonagh, L. Moutsianas, M. Mueller, N. Murugaesu, A.C. Need, C.A. Odhams, C. Patch, D. Perez-Gil, D. Polychronopoulos, J. Pullinger, T. Rahim, A. Rendon, P. Riesgo-Ferreiro, T. Rogers, M. Ryten, K. Savage, K. Sawant, R.H. Scott, A. Siddiq, A. Sieghart, D. Smedley, K.R. Smith, A. Sosinsky, W. Spooner, H.E. Stevens, A. Stuckey, R. Sultana, E.R.A. Thomas, S.R. Thompson, C. Tregidgo, A. Tucci, E. Walsh, S.A. Watters, M.J. Welland, E. Williams, K. Witkowska, S.M. Wood, M. Zarowiecki

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