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Mutations in SEPT9 cause hereditary neuralgic amyotrophy Gregor Kuhlenba¨umer1–3,13, Mark C Hannibal4,13, Eva Nelis3,13, Anja Schirmacher1, Nathalie Verpoorten3, Jan Meuleman3,4, Giles D J Watts4, Els De Vriendt3, Peter Young1, Florian Sto¨gbauer1, 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 autosomal dominant recurrent neuropathy affecting the brachial plexus. HNA is triggered by environmental factors such as infection or parturition. 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 a gene of the septin family. Septins are implicated in formation of the cytoskeleton, cell division and tumorigenesis. HNA (OMIM162100) is an autosomal dominant peripheral neuropathy with a worldwide distribution1. The clinical hallmarks of HNA are recurrent painful brachial plexus neuropathies with weakness and atrophy of arm muscles and sensory loss. Full or partial recovery occurs
in most affected individuals within weeks to months. A more common sporadic form of painful brachial plexus neuropathy, called ParsonageTurner syndrome, is clinically indistinguishable from HNA. Attacks of brachial plexus neuritis are often triggered by infections, immunizations, parturition or strenuous use of the affected limb. Inflammatory changes in the blood and brachial plexus have been shown, suggesting involvement of the immune system. The relapsing-remitting course and the environmental triggering make HNA unique among the inherited neuropathies and might render it a model for more common sporadic 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 with HNA1. We previously assigned a major HNA locus to a 3.5-cM (1.8-Mb) candidate region on chromosome 17q25 and found evidence for a founder effect among some North American families2–5. In this study, we included ten previously reported multigeneration families with the classical HNA phenotype from different geographic origins (Table 1). The study was approved by the ethics committee of the Universities of Antwerp, Mu¨nster and Seattle, and informed consent was obtained from all participants. All families showed linkage to chromosome 17q25 (refs. 2–5). Segregation analysis of short tandem repeat (STR) markers in informative recombinants of these families allowed further reduction of the HNA locus to a B600-kb interval containing only two known genes, SEC14-like 1 (SEC14L1) and SEPT9 (Fig. 1a and Supplementary Fig. 1 and Supplementary Table 1 online). In addition, we confirmed allele sharing over at least 23 consecutive STRs between families K4004 and K4015 but disproved allele sharing between these families and family K4018, previously
Table 1 Ethnic origin, genetic findings and presence of dysmorphic features Family
HNA-2
Origin
TU
Allele sharing SEPT9 mutation
No 131G-C
HNA-5
HNA-8
HNA-9
Dysmorphic features
K4003
K4004 K4007 K4015
K4018
FI
SP
SP
AE
AE
AE
AE
AE
AE
No 262C-T
No 262C-T
No 262C-T
Yesa None
No 262C-T
Yesa None
Yesa None
Yesa None
No 278C-T
– –
R88W 500 GE, 100 AE,
– –
– –
– –
S93F 500 GE, 100 AE
9
16
17
Amino acid substitution None, 5¢ UTR R88W R88W R88W Control individualsb 500 GE, 107 TU 500 GE, 100 AE, 500 GE, 100 AE, 500 GE, 100 AE, Average age of onset (y)
K4000
17
102 FI, 97 SP 13
102 FI, 97 SP 15
102 FI, 97 SP 11
15
102 FI, 97 SP 19
Absent
Present
Present
Present
Present
Present
Present Present Present
12 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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1
SEC14L1
FM
SEPT9
CA5
617 4 622 88 642 635 606
GT6
573 7 577 07 194
509 730
GT1
D17 S93 9
408 303
380 058
MS MSFtri F 345 pen D17826 S93 7
MS F 300 36296 240
Sec 14p en
301 8 392 5 2 430 0 43 617 48
181 4
a
263 055 72G T1
B R I E F C O M M U N I C AT I O N S
FM
b
SEPT9 Alternative exons 1a
2a
3a
4a 5a
6a
Exons 1 alpha
7a
2
3
456789 1011
zeta epsilon gamma beta
c
delta AK056495
Control
Control
HNA-2
K4003, HNA-5 HNA-8, HNA-9
K4018
–131G→C
262C→T
278C→T Protein
Genomic DNA
d
R88W Human Mouse Rat Dog Chicken
S93F
Human Mouse Rat Dog 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 protein level.
assumed to share alleles with families K4004 and K4015 based on a four-marker haplotype5 (Supplementary Fig. 2 and Supplementary Table 1 online). We sequenced the coding region of SEPT9 including its untranslated regions (UTRs), multiple splice variants and alternative first exons (Fig. 1b and Supplementary Table 2 online). In four families with HNA, we found a sequence variation (262C-T) in exon 2 of SEPT9 (Table 1 and Fig. 1c). This transition causes the amino acid change R88W. These four families do not share a common diseaseassociated haplotype, suggestive of a mutation hot spot rather than a founder mutation. The genomic variation did occur at a potential hypermutable CG dinucleotide. In family K4018, we detected a transition (278C-T) leading to a S93F amino acid substitution (Table 1 and Fig. 1c). In family HNA-2, we found a sequence variation ( 131G-C) in the 5¢ UTR of the SEPT9 alpha transcript (Table 1 and Fig. 1c). None of the three sequence variants was found in ethnically matched control individuals (Table 1 and Supplementary Table 3 online). All three mutation sites showed very high interspecies conservation (Fig. 1d).
2
In the six families, the SEPT9 mutations were found in all individuals with HNA. In a few families, nonpenetrance or incomplete penetrance (indicated by the presence of dysmorphic facial features but absence of HNA attacks) occurred in individuals carrying a SEPT9 mutation. In four North American families with HNA, we could not detect a disease-associated mutation in SEPT9 (Table 1). But the affected individuals in these families shared a disease-linked haplotype and can therefore be viewed as one large ancient family in which the mutation might be located in a region not covered by our mutation screen5. The STR markers MSFtri–GT1 (Supplementary Table 1 online) located in SEPT9 did not show triple alleles or hemizygosity in the allelesharing families, arguing against a large duplication or deletion. Semiquantitative PCR analysis of exons 1–11 from genomic DNA did not detect a duplication or deletion in families K4000, K4004, K4007 and K4015. In addition, PCR amplification and sequencing of somatic cell hybrids containing only the affected chromosome of affected individual K4000-47 showed no evidence for a deletion.
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B R I E F C O M M U N I C AT I O N S We analyzed the expression of mouse Sept9 in ventral horns (motor neurons) and dorsal root ganglia (sensory neurons) of mouse embryos at embryonic day 13 and found that Sept9 was expressed in both types of neurons (data not shown). In an earlier mutation report of SEPT9, we detected the R88W mutation in a single family with HNA (HNA-8) but erroneously concluded that this mutation was a rare polymorphism6. Here we repeated the segregation analysis in family HNA-8 in two independent sets of genomic DNA samples and found, in contrast to the first analysis, faithful cosegregation of HNA with the SEPT9 mutation. The septin gene family has been implicated in many functions7. All human septins contain a polybasic domain preceding a central GTPbinding domain. The structural feature distinguishing SEPT9 from all other septins is the long N terminus of unknown function, which does not show significant homology to other proteins and does not contain any known protein motives7. Both missense mutations, R88W and S93F, are located in the N terminus and target amino acid residues that are located in a stretch of 15 highly conserved amino acids (Fig. 1d). Comparable sequence conservation is not found anywhere else in the N terminus of SEPT9, suggestive of an important yet unknown function. Recent experimental evidence suggests that the N terminus of SEPT9 might be responsible for binding a Rho guanine nucleotide exchange factor as well as for forming a complex with SEPT7 and SEPT11 (refs. 8,9). There were no apparent clinical differences between the family with the S93F mutation and the families with the R88W mutation. Although both 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 the surrounding area of the 5¢ UTR show exceptional interspecies conservation at the genomic level (Fig. 1d). Notably, family HNA-2 is the only family in which no dysmorphic features are found, suggesting that different transcripts might have different functions. SEPT9 has a role in cell division, indicated by the fact that SEPT9depleted cells often fail to complete cytokinesis10. This functional clue for SEPT9 has interesting implications for the genesis of dysmorphic features associated with HNA10. The SEPT9 protein forms filaments and colocalizes with cytoskeletal elements such as actin and tubulin,
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suggesting that it has a structural function in the cell10,11. Finally, generalized overexpression of Sept9 was described in mouse models of human breast cancer and in human breast cancer cell lines, indicating that septin can also be involved in tumorigenesis12. We conclude that mutations in SEPT9 are a primary cause of HNA. Note: Supplementary information is available on the Nature Genetics website. ACKNOWLEDGMENTS We thank the affected individuals and their relatives for participating in this study; K. Berger and E. Battologlu for contributing anonymous control samples; the VIB Genetic Service Facility and the Genetics Core of the Center on Human Development and Disability of the University of Washington for contributing technically to the genetic analyses; and A. Jacobs and S. Weiser for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft to G.K., The Neuropathy Association and the US National Institutes of Health to P.F.C.; by the Veterans Affairs Research Fund to 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 Office and the Medical Foundation Queen Elisabeth to V.T. J.M. received a postdoctoral fellowship from the Charcot-Marie-Tooth Association; N.V. received a PhD fellowship of the Institute of Science and Technology; and E.N. and J.I. are postdoctoral fellows of the Fund for Scientific Research. COMPETING INTERESTS STATEMENT The 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/
1. Windebank, A. in Peripheral Neuropathy vol. 2 (eds. Dyck, P., Thomas, P. & Griffin, J.) 1137–1148 (WB Saunders, Philadelphia, 1993). 2. Pellegrino, J.E., Rebbeck, T.R., Brown, M.J., Bird, T.D. & Chance, P.F. Neurology 46, 1128–1132 (1996). 3. Pellegrino, J. et al. Hum. Genet. 101, 277–283 (1997). 4. Meuleman, J. et al. Eur. J. Hum. Genet. 7, 920–927 (1999). 5. Watts, G.D., O’Briant, K.C. & Chance, P.F. Hum. Genet. 110, 166–172 (2002). 6. Meuleman, J. et al. Hum. Genet. 108, 390–393 (2001). 7. Hall, P.A. & Russell, S.E. J. Pathol. 204, 489–505 (2004). 8. Nagata, K., Asano, T., Nozawa, Y. & Inagaki, M. J. Biol. Chem. 279, 55895–55904 (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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