Mutations in SEPT9 causehereditary neuralgic amyotrophyGregor Kuhlenbaumer1–3,13, Mark C Hannibal4,13, Eva Nelis3,13,Anja Schirmacher1, Nathalie Verpoorten3, Jan Meuleman3,4,Giles D J Watts4, Els De Vriendt3, Peter Young1,Florian Stogbauer1, Hartmut Halfter1, Joy Irobi3, Dirk Goossens3,Jurgen Del-Favero3, Benjamin G Betz4, Hyun Hor1,Gert Kurlemann5, Thomas D Bird6,7, Eila Airaksinen8,Tarja Mononen9, Adolfo Pou Serradell10, Jose M Prats11,Christine Van Broeckhoven3, Peter De Jonghe3,12,Vincent Timmerman3,13, E Bernd Ringelstein1,2,13 &Phillip F Chance4,6,13

Hereditary neuralgic amyotrophy (HNA) is an autosomaldominant recurrent neuropathy affecting the brachial plexus.HNA is triggered by environmental factors such as infection orparturition. We report three mutations in the gene septin 9(SEPT9) in six families with HNA linked to chromosome 17q25.HNA is the first monogenetic disease caused by mutations in agene of the septin family. Septins are implicated in formationof the cytoskeleton, cell division and tumorigenesis.

HNA (OMIM162100) is an autosomal dominant peripheral neuro-pathy with a worldwide distribution1. The clinical hallmarks of HNAare recurrent painful brachial plexus neuropathies with weakness andatrophy of arm muscles and sensory loss. Full or partial recovery occurs

in most affected individuals within weeks to months. A more commonsporadic form of painful brachial plexus neuropathy, called Parsonage-Turner syndrome, is clinically indistinguishable from HNA. Attacks ofbrachial plexus neuritis are often triggered by infections, immuniza-tions, parturition or strenuous use of the affected limb. Inflammatorychanges in the blood and brachial plexus have been shown, suggestinginvolvement of the immune system. The relapsing-remitting courseand the environmental triggering make HNA unique among theinherited neuropathies and might render it a model for more commonsporadic diseases like Parsonage-Turner and Guillain-Barre syndromes.Dysmorphic features such as hypotelorism, epicanthal folds and, rarely,cleft palate have been found in many but not all individuals withHNA1. We previously assigned a major HNA locus to a 3.5-cM(1.8-Mb) candidate region on chromosome 17q25 and found evidencefor a founder effect among some North American families2–5.

In this study, we included ten previously reported multigenerationfamilies with the classical HNA phenotype from different geographicorigins (Table 1). The study was approved by the ethics committee ofthe Universities of Antwerp, Munster and Seattle, and informedconsent was obtained from all participants. All families showed linkageto chromosome 17q25 (refs. 2–5). Segregation analysis of shorttandem repeat (STR) markers in informative recombinants of thesefamilies allowed further reduction of the HNA locus to a B600-kbinterval containing only two known genes, SEC14-like 1 (SEC14L1)and SEPT9 (Fig. 1a and Supplementary Fig. 1 and SupplementaryTable 1 online). In addition, we confirmed allele sharing over at least23 consecutive STRs between families K4004 and K4015 but disprovedallele sharing between these families and family K4018, previously

Table 1 Ethnic origin, genetic findings and presence of dysmorphic features

Family HNA-2 HNA-5 HNA-8 HNA-9 K4000 K4003 K4004 K4007 K4015 K4018

Origin TU FI SP SP AE AE AE AE AE AE

Allele sharing No No No No Yesa No Yesa Yesa Yesa No

SEPT9 mutation �131G-C 262C-T 262C-T 262C-T None 262C-T None None None 278C-T

Amino acid substitution None, 5¢ UTR R88W R88W R88W – R88W – – – S93F

Control individualsb 500 GE, 107 TU 500 GE, 100 AE,

102 FI, 97 SP

500 GE, 100 AE,

102 FI, 97 SP

500 GE, 100 AE,

102 FI, 97 SP

– 500 GE, 100 AE,

102 FI, 97 SP

– – – 500 GE, 100 AE

Average age of onset (y) 17 13 15 11 15 19 9 16 17 12

Dysmorphic features Absent Present Present Present Present Present Present Present Present Present

AE, North American of European descent; FI, Finnish; GE, German; SP, Spanish; TU, Turkish.aDetails regarding allele sharing are given in Supplementary Figure 2 and Supplementary Table 1 online. bNumber and ethnicity of control individuals in whom the respectivemutation was absent.

Received 3 May; accepted 3 August; published online 25 September 2005; doi:10.1038/ng1649

1Department of Neurology and 2Leibniz Institute of Atherosclerosis Research, University of Munster, Domagkstr. 3, D-48149 Munster, Germany. 3Molecular GeneticsDepartment, Flanders Interuniversity Institute for Biotechnology, University of Antwerp, Antwerpen, Belgium. 4Division of Genetics and Developmental Medicine,Department of Pediatrics, University of Washington, Seattle, Washington, USA. 5Department of Pediatric Neurology, University of Munster, Germany. 6Departmentof Neurology, University of Washington, Seattle, Washington, USA. 7Veterans Affairs Puget Sound Health Care System, Seattle, Washington, USA. 8Department ofPaediatrics, University of Kuopio, Kuopio, Finland. 9Department of Clinical Genetics, Kuopio University Hospital, Kuopio, Finland. 10Department of Neurology,Hospital del Mar, Autonome University of Barcelona, Barcelona, Spain. 11Division of Child Neurology, Hospital de Cruces, Barakaldo, Basque Country, Spain.12Division of Neurology, University Hospital, Antwerpen, Belgium. 13These authors contributed equally to this work. Correspondence should be addressed to G.K.(gkuhlen@uni-muenster.de).

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assumed to share alleles with families K4004 and K4015 based on afour-marker haplotype5 (Supplementary Fig. 2 and SupplementaryTable 1 online).

We sequenced the coding region of SEPT9 including its untrans-lated regions (UTRs), multiple splice variants and alternative firstexons (Fig. 1b and Supplementary Table 2 online). In four familieswith HNA, we found a sequence variation (262C-T) in exon 2 ofSEPT9 (Table 1 and Fig. 1c). This transition causes the amino acidchange R88W. These four families do not share a common disease-associated haplotype, suggestive of a mutation hot spot rather than afounder mutation. The genomic variation did occur at a potentialhypermutable CG dinucleotide. In family K4018, we detected atransition (278C-T) leading to a S93F amino acid substitution(Table 1 and Fig. 1c). In family HNA-2, we found a sequencevariation (�131G-C) in the 5¢ UTR of the SEPT9 alpha transcript(Table 1 and Fig. 1c). None of the three sequence variants was foundin ethnically matched control individuals (Table 1 and Supplemen-tary Table 3 online). All three mutation sites showed very highinterspecies conservation (Fig. 1d).

In the six families, the SEPT9 mutations were found in allindividuals with HNA. In a few families, nonpenetrance orincomplete penetrance (indicated by the presence of dysmorphicfacial features but absence of HNA attacks) occurred in individualscarrying a SEPT9 mutation. In four North American familieswith HNA, we could not detect a disease-associated mutationin SEPT9 (Table 1). But the affected individuals in these familiesshared a disease-linked haplotype and can therefore be viewed asone large ancient family in which the mutation might be locatedin a region not covered by our mutation screen5. The STRmarkers MSFtri–GT1 (Supplementary Table 1 online) locatedin SEPT9 did not show triple alleles or hemizygosity in the allele-sharing families, arguing against a large duplication or deletion.Semiquantitative PCR analysis of exons 1–11 from genomicDNA did not detect a duplication or deletion in families K4000,K4004, K4007 and K4015. In addition, PCR amplification andsequencing of somatic cell hybrids containing only the affectedchromosome of affected individual K4000-47 showed no evidencefor a deletion.

CA

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FM FMSEC14L1

Alternative exons 1a 2a 3a 4a 5a 6a 7a119753 8641Exons

alpha

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HumanMouseRatDogChickenClawed frog

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2 10

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SEPT9

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Figure 1 Refined HNA candidate region, genomic organization of SEPT9, SEPT9 mutations in families with HNA and their conservation in different species.

(a) The 600-kb HNA candidate region with locations of known and self-generated STR markers and genomic organization of SEC14L1 and SEPT9. FM,

flanking marker. (b) Genomic organization of the different SEPT9 splice variants, including the reference cDNA SEPT9 alpha. Accession numbers of SEPT9

cDNAs are given in Supplementary Table 4 online. We numbered the exons of the SEPT9 alpha cDNA as exons 1–11 and all other exons according to their

genomic location, adding the suffix ‘a’ to the exon number (1a–7a). (c) Sequence variants found in families with HNA. The –131G-C variant found in

family HNA-2 is located in the 5¢ UTR of the SEPT9 alpha transcript. The 262C-T and the 278C-T transitions are located in exon 2 and lead to the

amino acid changes R88W and S93F, respectively, in the N terminus of SEPT9. (d) Interspecies conservation of the untranslated –131G nucleotide at the

genomic DNA level and of the R88 and S93 amino acid residues at the protein level.

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We analyzed the expression of mouse Sept9 in ventral horns (motorneurons) and dorsal root ganglia (sensory neurons) of mouse embryosat embryonic day 13 and found that Sept9 was expressed in both typesof neurons (data not shown).

In an earlier mutation report of SEPT9, we detected the R88Wmutation in a single family with HNA (HNA-8) but erroneouslyconcluded that this mutation was a rare polymorphism6. Here werepeated the segregation analysis in family HNA-8 in two independentsets of genomic DNA samples and found, in contrast to the firstanalysis, faithful cosegregation of HNA with the SEPT9 mutation.

The septin gene family has been implicated in many functions7. Allhuman septins contain a polybasic domain preceding a central GTP-binding domain. The structural feature distinguishing SEPT9 from allother septins is the long N terminus of unknown function, which doesnot show significant homology to other proteins and does not containany known protein motifs7. Both missense mutations, R88W andS93F, are located in the N terminus and target amino acid residuesthat are located in a stretch of 15 highly conserved amino acids(Fig. 1d). Comparable sequence conservation is not found anywhereelse in the N terminus of SEPT9, suggestive of an important yetunknown function. Recent experimental evidence suggests that theN terminus of SEPT9 might be responsible for binding a Rho guaninenucleotide exchange factor as well as for forming a complex withSEPT7 and SEPT11 (refs. 8,9).

There were no apparent clinical differences between the family withthe S93F mutation and the families with the R88W mutation. Althoughboth missense mutations affect multiple isoforms of SEPT9 (Fig. 1b),the �131G-C mutation found in family HNA-2 is restricted to the 5¢UTR of the SEPT9 alpha transcript. This mutation site and thesurrounding area of the 5¢ UTR show exceptional interspecies con-servation at the genomic level (Fig. 1d). Notably, family HNA-2 is theonly family in which no dysmorphic features are found, suggesting thatdifferent transcripts might have different functions.

SEPT9 has a role in cell division, indicated by the fact that SEPT9-depleted cells often fail to complete cytokinesis10. This functional cluefor SEPT9 has interesting implications for the genesis of dysmorphicfeatures associated with HNA10. The SEPT9 protein forms filamentsand colocalizes with cytoskeletal elements such as actin and tubulin,

suggesting that it has a structural function in the cell10,11. Finally,generalized overexpression of Sept9 was described in mouse models ofhuman breast cancer and in human breast cancer cell lines, indicatingthat septin can also be involved in tumorigenesis12. We conclude thatmutations in SEPT9 are a primary cause of HNA.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSWe thank the affected individuals and their relatives for participating in thisstudy; K. Berger and E. Battologlu for contributing anonymous control samples;the VIB Genetic Service Facility and the Genetics Core of the Center onHuman Development and Disability of the University of Washington forcontributing technically to the genetic analyses; and A. Jacobs and S. Weiserfor technical assistance. This work was supported by grants from the DeutscheForschungsgemeinschaft to G.K., The Neuropathy Association and the USNational Institutes of Health to P.F.C.; by the Veterans Affairs Research Fund toT.D.B.; and by the University of Antwerp, the Fund for Scientific Research, theInteruniversity Attraction Poles program of the Belgian Federal Science PolicyOffice and the Medical Foundation Queen Elisabeth to V.T. J.M. received apostdoctoral fellowship from the Charcot-Marie-Tooth Association; N.V. receiveda PhD fellowship of the Institute of Science and Technology; and E.N. and J.I. arepostdoctoral fellows of the Fund for Scientific Research.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/naturegenetics/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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(2004).9. Nagata, K. & Inagaki, M. Oncogene 24, 65–76 (2005).10. Surka, M.C., Tsang, C.W. & Trimble, W.S. Mol. Biol. Cell 13, 3532–3545 (2002).11. Nagata, K. et al. J. Biol. Chem. 278, 18538–18543 (2003).12. Montagna, C. et al. Cancer Res. 63, 2179–2187 (2003).

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