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in most affected individuals within weeks to months. A more common Mutations in SEPT9 cause sporadic form of painful brachial plexus neuropathy, called Parsonage- Turner syndrome, is clinically indistinguishable from HNA. Attacks of hereditary neuralgic amyotrophy brachial plexus neuritis are often triggered by infections, immuniza- Gregor Kuhlenba¨umer1–3,13, Mark C Hannibal4,13, Eva Nelis3,13, tions, parturition or strenuous use of the affected limb. Inflammatory Anja Schirmacher1, Nathalie Verpoorten3, Jan Meuleman3,4, changes in the blood and brachial plexus have been shown, suggesting Giles D J Watts4, Els De Vriendt3, Peter Young1, involvement of the immune system. The relapsing-remitting course Florian Sto¨gbauer1, Hartmut Halfter1, Joy Irobi3, Dirk Goossens3, and the environmental triggering make HNA unique among the Jurgen Del-Favero3, Benjamin G Betz4, Hyun Hor1, inherited neuropathies and might render it a model for more common Gert Kurlemann5, Thomas D Bird6,7, Eila Airaksinen8, sporadic diseases like Parsonage-Turner and Guillain-Barre´ syndromes. Tarja Mononen9, Adolfo Pou Serradell10, Jose´ M Prats11, Dysmorphic features such as hypotelorism, epicanthal folds and, rarely, Christine Van Broeckhoven3, Peter De Jonghe3,12, cleft palate have been found in many but not all individuals with 1 Vincent Timmerman3,13, E Bernd Ringelstein1,2,13 & HNA . We previously assigned a major HNA locus to a 3.5-cM Phillip F Chance4,6,13 (1.8-Mb) candidate region on 17q25 and found evidence for a founder effect among some North American families2–5. Hereditary neuralgic amyotrophy (HNA) is an autosomal In this study, we included ten previously reported multigeneration dominant recurrent neuropathy affecting the brachial plexus. families with the classical HNA phenotype from different geographic HNA is triggered by environmental factors such as infection or origins (Table 1). The study was approved by the ethics committee of parturition. We report three mutations in the 9 the Universities of Antwerp, Mu¨nster and Seattle, and informed (SEPT9) in six families with HNA linked to chromosome 17q25. consent was obtained from all participants. All families showed linkage HNA is the first monogenetic disease caused by mutations in a to chromosome 17q25 (refs. 2–5). Segregation analysis of short gene of the septin family. are implicated in formation tandem repeat (STR) markers in informative recombinants of these of the cytoskeleton, cell division and tumorigenesis. families allowed further reduction of the HNA locus to a B600-kb interval containing only two known , SEC14-like 1 (SEC14L1) HNA (OMIM162100) is an autosomal dominant peripheral neuro- and SEPT9 (Fig. 1a and Supplementary Fig. 1 and Supplementary pathy with a worldwide distribution1. The clinical hallmarks of HNA Table 1 online). In addition, we confirmed allele sharing over at least are recurrent painful brachial plexus neuropathies with weakness and 23 consecutive STRs between families K4004 and K4015 but disproved atrophy of arm muscles and sensory loss. Full or partial recovery occurs allele 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 À131G-C262C-T262C-T262C-TNone262C-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, 500 GE, 100 AE, 500 GE, 100 AE, – 500 GE, 100 AE, – – – 500 GE, 100 AE 102 FI, 97 SP 102 FI, 97 SP 102 FI, 97 SP 102 FI, 97 SP 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 respective mutation was absent.

1Department of Neurology and 2Leibniz Institute of Atherosclerosis Research, University of Mu¨ nster, Domagkstr. 3, D-48149 Mu¨ nster, Germany. 3Molecular Genetics Department, 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 Mu¨ nster, Germany. 6Department of Neurology, University of Washington, Seattle, Washington, USA. 7Veterans Affairs Puget Sound Health Care System, Seattle, Washington, USA. 8Department of Paediatrics, 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. ([email protected]). Received 3 May; accepted 3 August; published online 25 September 2005; doi:10.1038/ng1649

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a

1814 30185 39220 43043 61748 Sec14pen 263055 72GT1 MSF36296300240 MSFtriMSFpen345826D17S937 380058 408303 D17S939 GT1 509730 GT6 573707577194 617488622642 635606 CA5

SEC14L1 SEPT9 FM FM

b SEPT9 Alternative exons 1a 2a 3a 4a 5a 6a 7a Exons 1 2 3 456789 1011 alpha zeta epsilon gamma beta c delta AK056495

Control Control

K4003, HNA-5 HNA-2 HNA-8, HNA-9

K4018

–131G→C 262C→T 278C→T

Protein Genomic DNA d R88W S93F Human Human Mouse Mouse Rat Rat Dog Dog Chicken Chicken Clawed frog Zebrafish

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 level. assumed to share alleles with families K4004 and K4015 based on a In the six families, the SEPT9 mutations were found in all four-marker haplotype5 (Supplementary Fig. 2 and Supplementary individuals with HNA. In a few families, nonpenetrance or Table 1 online). incomplete penetrance (indicated by the presence of dysmorphic We sequenced the coding region of SEPT9 including its untrans- facial features but absence of HNA attacks) occurred in individuals lated regions (UTRs), multiple splice variants and alternative first carrying a SEPT9 mutation. In four North American families exons (Fig. 1b and Supplementary Table 2 online). In four families with HNA, we could not detect a disease-associated mutation with HNA, we found a sequence variation (262C-T) in exon 2 of in SEPT9 (Table 1). But the affected individuals in these families SEPT9 (Table 1 and Fig. 1c). This transition causes the amino acid shared a disease-linked haplotype and can therefore be viewed as change R88W. These four families do not share a common disease- one large ancient family in which the mutation might be located associated haplotype, suggestive of a mutation hot spot rather than a in a region not covered by our mutation screen5. The STR founder mutation. The genomic variation did occur at a potential markers MSFtri–GT1 (Supplementary Table 1 online) located hypermutable CG dinucleotide. In family K4018, we detected a in SEPT9 did not show triple alleles or hemizygosity in the allele- transition (278C-T) leading to a S93F amino acid substitution sharing families, arguing against a large duplication or deletion. (Table 1 and Fig. 1c). In family HNA-2, we found a sequence Semiquantitative PCR analysis of exons 1–11 from genomic variation (À131G-C) in the 5¢ UTR of the SEPT9 alpha transcript DNA did not detect a duplication or deletion in families K4000, (Table 1 and Fig. 1c). None of the three sequence variants was found K4004, K4007 and K4015. In addition, PCR amplification and in ethnically matched control individuals (Table 1 and Supplemen- sequencing of somatic cell hybrids containing only the affected tary Table 3 online). All three mutation sites showed very high chromosome of affected individual K4000-47 showed no evidence interspecies conservation (Fig. 1d). for a deletion.

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We analyzed the expression of mouse Sept9 in ventral horns (motor suggesting that it has a structural function in the cell10,11.Finally, neurons) and dorsal root ganglia (sensory neurons) of mouse embryos generalized overexpression of Sept9 was described in mouse models of at embryonic day 13 and found that Sept9 was expressed in both types human breast cancer and in human breast cancer cell lines, indicating of neurons (data not shown). that septin can also be involved in tumorigenesis12. We conclude that In an earlier mutation report of SEPT9,wedetectedtheR88W mutations in SEPT9 are a primary cause of HNA. mutation in a single family with HNA (HNA-8) but erroneously concluded that this mutation was a rare polymorphism6.Herewe Note: Supplementary information is available on the Nature Genetics website. repeated the segregation analysis in family HNA-8 in two independent ACKNOWLEDGMENTS sets of genomic DNA samples and found, in contrast to the first We thank the affected individuals and their relatives for participating in this analysis, faithful cosegregation of HNA with the SEPT9 mutation. study; K. Berger and E. Battologlu for contributing anonymous control samples; The septin gene family has been implicated in many functions7.All the VIB Genetic Service Facility and the Genetics Core of the Center on Human Development and Disability of the University of Washington for human septins contain a polybasic domain preceding a central GTP- contributing technically to the genetic analyses; and A. Jacobs and S. Weiser binding domain. The structural feature distinguishing SEPT9 from all for technical assistance. This work was supported by grants from the Deutsche other septins is the long N terminus of unknown function, which does Forschungsgemeinschaft to G.K., The Neuropathy Association and the US not show significant homology to other and does not contain National Institutes of Health to P.F.C.; by the Veterans Affairs Research Fund to any known protein motives7.Bothmissensemutations,R88Wand T.D.B.; and by the University of Antwerp, the Fund for Scientific Research, the Interuniversity Attraction Poles program of the Belgian Federal Science Policy S93F, are located in the N terminus and target amino acid residues Office and the Medical Foundation Queen Elisabeth to V.T. J.M. received a that are located in a stretch of 15 highly conserved amino acids postdoctoral fellowship from the Charcot-Marie-Tooth Association; N.V. received (Fig. 1d). Comparable sequence conservation is not found anywhere a PhD fellowship of the Institute of Science and Technology; and E.N. and J.I. are else in the N terminus of SEPT9, suggestive of an important yet postdoctoral fellows of the Fund for Scientific Research. unknown function. Recent experimental evidence suggests that the COMPETING INTERESTS STATEMENT N terminus of SEPT9 might be responsible for binding a Rho guanine The authors declare that they have no competing financial interests. nucleotide exchange factor as well as for forming a complex with SEPT7 and SEPT11 (refs. 8,9). Published online at http://www.nature.com/naturegenetics/ There were no apparent clinical differences between the family with Reprints and permissions information is available online at http://npg.nature.com/ the S93F mutation and the families with the R88Wmutation. Although reprintsandpermissions/ both missense mutations affect multiple isoforms of SEPT9 (Fig. 1b), the À131G-C mutation found in family HNA-2 is restricted to the 5¢ 1. Windebank, A. in Peripheral Neuropathy vol. 2 (eds. Dyck, P., Thomas, P. & Griffin, J.) 1137–1148 (WB Saunders, Philadelphia, 1993). UTR of the SEPT9 alpha transcript. This mutation site and the 2. Pellegrino, J.E., Rebbeck, T.R., Brown, M.J., Bird, T.D. & Chance, P.F. Neurology 46, surrounding area of the 5¢ UTR show exceptional interspecies con- 1128–1132 (1996). servation at the genomic level (Fig. 1d). Notably, family HNA-2 is the 3. Pellegrino, J. et al. Hum. Genet. 101, 277–283 (1997). 4. Meuleman, J. et al. Eur. J. Hum. Genet. 7, 920–927 (1999). only family in which no dysmorphic features are found, suggesting that 5. Watts, G.D., O’Briant, K.C. & Chance, P.F. Hum. Genet. 110, 166–172 (2002). different transcripts might have different functions. 6. Meuleman, J. et al. Hum. Genet. 108, 390–393 (2001). 7. Hall, P.A. & Russell, S.E. J. Pathol. 204, 489–505 (2004). SEPT9 has a role in cell division, indicated by the fact that SEPT9- 8. Nagata, K., Asano, T., Nozawa, Y. & Inagaki, M. J. Biol. Chem. 279, 55895–55904 10 depleted cells often fail to complete cytokinesis .Thisfunctionalclue (2004). for SEPT9 has interesting implications for the genesis of dysmorphic 9. Nagata, K. & Inagaki, M. Oncogene 24, 65–76 (2005). 10 10. Surka, M.C., Tsang, C.W. & Trimble, W.S. Mol. Biol. Cell 13, 3532–3545 (2002). features associated with HNA . The SEPT9 protein forms filaments 11. Nagata, K. et al. J. Biol. Chem. 278, 18538–18543 (2003). and colocalizes with cytoskeletal elements such as actin and tubulin, 12. Montagna, C. et al. Cancer Res. 63, 2179–2187 (2003).

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