A Missense Mutation in the Coiled-Coil Domain of the KIF5A Gene and Late-Onset Hereditary Spastic Paraplegia

OBSERVATION A Missense Mutation in the Coiled-Coil Domain of the KIF5A Gene and Late-Onset Hereditary Spastic Paraplegia

Mariangela Lo Giudice, BS; Marcella Neri, MD; Michele Falco, BS; Maurizio Sturnio, BS; Elisa Calzolari, MD; Daniela Di Benedetto, PhD; Marco Fichera, PhD

Background: To our knowledge, up to now, only 2 Results: The history of the family was consistent with mutations in the KIF5A gene, a member of the kinesin ADHSP characterized by late onset of the disease. superfamily, have been identified as the molecular cause Mutational analysis results were negative for the spas- of early-onset autosomal dominant hereditary spastic para- tin, atlastin, and NIPA1 genes but identified a missense paresis (ADHSP). mutation (c.1082CϾT) in the coiled-coil coding region of the KIF5A gene. Objective: To assess the genetic defect in a family with late-onset ADHSP. Conclusions: This finding enlarges the phenotypic spec- trum of ADHSP linked to KIF5A and enhances the role Patients and Methods: Only the proband agreed to of that gene in the epidemiology of this disease. We pro- undergo complete neurological testing and mutational pose that the KIF5A gene should be routinely analyzed analysis. The proband was screened for mutations in the in patients with hereditary spastic paraplegia negative for spastin, atlastin, NIPA1, and KIF5A genes, either by spastin and atlastin mutations. denaturing high-performance liquid chromatography or sequence analysis. Arch Neurol. 2006;63:284-287

EREDITARY SPASTIC METHODS paraplegia (HSP) is a het- erogeneous group of neu- PATIENTS rodegenerative diseases characterized by progres- This Italian family (Figure 1) was identified siveH weakness, spasticity, and loss of through the neurological examination of vibratory sense in the lower limbs. Clini- the proband (3-9). Nine additional relatives cally, HSPs are divided into “uncompli- were reported as having difficulties in gait, but cated” (symptoms confined to lower ex- only the proband agreed to give DNA for ge- tremity weakness, bladder disturbance, netic testing. and, to a lesser extent, impaired position sense in the legs) or “complicated” GENETIC STUDIES (when additional neurological deficits are present).1 Genomic DNA was extracted from peripheral blood samples by standard protocols after writ- Recent genetic studies revealed that at ten informed consent. Mutational analysis was least 28 loci of HSP have been associated performed on the spastin and KIF5A genes by with autosomal dominant (ADHSP), re- denaturing high-performance liquid chroma- Author Affiliations: Genetic cessive, or X chromosome–linked trans- tography (DHPLC) as previously described4,5 Diagnostic Laboratory, Istituto mission, but only 9 of the responsible genes while the atlastin and NIPA1 genes were ana- di Ricovero e Cura a Carattere have been identified. Among them, spas- lyzed by direct sequencing using polymerase Scientifico (IRCCS) Oasi Maria tin (SPG4), atlastin (SPG3A), KIF5A chain reaction (PCR) primers and protocols 6 SS, Troina, Italy (Mss Lo (SPG10), HSP60 (SPG13),2 and, more re- either previously described (atlastin) or avail- Giudice and Falco, Mr Sturnio, cently, NIPA1 (SPG6)3 have been associ- able on request (NIPA1). The PCR products and Drs Di Benedetto and ated with pure ADHSP. The functions of were purified using the QIAquick PCR puri- Fichera); Department of fication kit (Qiagen, Hilden, Germany) and bi- Experimental Medicine and several of these genes are related to axo- directionally sequenced using the BigDye Ter- Diagnostics, Medical Genetics nal transport or intracellular trafficking. minator v1.1 Cycle Sequencing Kit (Applied Service, University of Ferrara, Herein we report on a new mutation in the Biosystems, Foster City, Calif). Samples were Ferrara, Italy (Drs Neri and KIF5A gene found in the index case of a analyzed on an ABI PRISM 310 automated se- Calzolari). pedigree with ADHSP. quencer (Applied Biosystems).

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The effect of the A361V mutation on the coiled-coil structure 1 of KIF5A was predicted using the COILS program.7 The nor- 12345 mal and A361V mutant sequences were analyzed using the MTIDK matrix,8 with window lengths of 14, 21, and 28. The hypothetical activation by the mutation of a cryptic splice site 2 123 45 was tested using SpliceView software.9

RESULTS 3 12345678 9

PHENOTYPIC DESCRIPTION 4 The proband (3-9) was a 48-year-old man who experi- 12 3 4 5 enced difficulty in gait and urinary urgency since the age of 35 years. Neurological examination revealed corticospi- Figure 1. Pedigree of the studied family. Arrow indicates the proband. Filled symbols represent affected individuals and slash marks, deceased nal tract deficits affecting the lower extremities with an individuals. asymmetry in the physiological range. Major findings were spastic gait and marked hyperreflexia, and extensor plan- tar response muscle tone was increased during gait as well Successive sequence analysis (Figure 2) identified a heterozygous single-nucleotide variant at position 1082 as at passive mobilization, associated with weakness and Ͼ pes cavus. No sensory loss or decreased vibration sense were (c.1082C T) predicted to result in alanine-to-valine sub- recorded. There was no upper limb involvement. Nega- stitution at codon 361 (A361V) in the coiled-coil tail re- tive findings in the clinical evaluation were absence of ex- gion of KIF5A. The affected amino acid is conserved in trapyramidal signs, ataxia, retinal involvement, amyotro- the KIF5A family of several vertebrate species (Figure 3); phy, mental retardation, dementia, deafness, or epilepsy. moreover, this mutation was not found in a group of 750 Neurophysiological examination revealed slight ab- unrelated chromosomes from healthy individuals of the normalities in evoked motor potential of the lower limbs same ethnic origin tested by DHPLC, indicating that this in the presence of a normal central conduction time. The was unlikely to be a rare polymorphism and most prob- upper extremities’ responses were in the normal range. ably represented the mutation underlying the pheno- The evoked sensitive potentials in the lower and upper type observed in the patient studied. extremities and the examination results of the optic trait were normal. Peripheric conduction velocities and F waves BIOINFORMATIC ANALYSIS of the lower extremities were in the normal range, ex- cluding a neuromuscular involvement. Normal mag- The mutation did not show any significant effect either netic resonance imaging results of the brain and spinal on the coiled-coil structure or on the activation of cryp- cord excluded structural abnormalities. The differential tic splice sites (data not shown). diagnosis with multiple sclerosis was supported by normal results of cytochemical analysis of cerebrospinal fluid COMMENT and by the absence of oligoclonal bands. All hemato- chemical analysis results were in the normal range, in par- Neurons are an example of extremely polarized cells ticular the inflammatory index and the folate and vita- with axons that reach up to1minlength; because min B12 levels. axons have little or no protein synthesis machinery, The familial tree (Figure 1) showed recurrence of gait axonal proteins must be synthesized in the cell body. disturbance in this family in 4 generations. The precise Neurons require an appropriate system of intracellular age of disease onset was reported only for the father of traffic and an intracellular transport system, and this our proband, who had clinical symptoms at the age of requirement could be the “Achilles heel” of these 50 years, while other affected members were reported to large, complex cells. have had an adult onset of spastic gait. No clinical data The KIF5A protein belongs to the superfamily of ki- were available for other family members. nesins, which are molecular motor proteins responsible for many of the major microtubule-dependent transport GENETIC ANALYSIS pathways in neuronal and nonneuronal cells.10 They typi- cally consist of 2 identical, approximately 110- to 120-kD DNA analysis was only performed on the proband (3-9) heavy chains and 2 identical, approximately 60- to 70-kD because of the unavailability of the other pedigree mem- light chains. The heavy chains have a 3-domain struc- bers. No mutations were found in the SPG4, SPG3A, and ture. The globular N-terminal motor domain, which con- NIPA1 genes, although we cannot exclude either the pres- tains the adenosine triphosphate and microtubule- ence of cryptic intronic mutations in these genes or that binding sites, is about 340 amino acids long. The heavy mutations may have been missed by DHPLC screening, chains of kinesin dimerize through an ␣-helical coiled- despite its very high sensitivity. Further DHPLC analy- coil region, the so-called stalk domain. The third domain sis of the KIF5A gene revealed a clear heterozygous elu- consists of a C-terminal globular tail that is thought to be tion pattern of the exon 11–related PCR product. involved in light-chain and cargo binding.

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Figure 2. Electropherogram of the sequence flanking the c.1082CϾT mutation.

Homo sapiens KIF5A A E Q W K K K Y E K E K E K T K A Q K E T I A K L E A E L S R W R N G E N V Rattus norvegicus KIF5A A E Q W K K K Y E K E K E K T K A Q K E T I A K L E A E L S R W R N G E N V Pan troglodytes KIF5A A E Q W K K K Y E K E K E K T K A Q K E T I A K L E A E L S R W R N G E N V Mus musculus KIF5A A E Q W K K K Y E K E K E K T K A Q K E T I A K L E A E L S R W R N G E N V Canis familiaris KIF5A A E Q W K K K Y E K E K E K T K A Q K E T I A K L E A E L S R W R N G E N V

A361V

Figure 3. Comparative analysis of the amino acid sequence (339-376) of the KIF5A protein. Arrow shows the alanine residue affected by the A361V mutation.

To our knowledge, to date, only 2 disease-causing mu- destabilize the protein. In addition, the coiled coil may tations in the KIF5A gene have been described in the lit- be further stabilized by salt bridges formed between erature,5,11 localized in exons 10 (R280C) and 8 (N256S), charged side chains in the “g” and “e” positions of respectively. These 2 lesions belong to the region of the opposing helices. The mechanism by which the A361V kinesin devoted to microtubule binding, known to affect mutation results in a pathogenic alteration of KIF5A is the adenosine triphosphatase activity of the motor do- not clear because this mutation occurs in the “b” posi- 12,13 main. tion of the heptad repeat in a residue that is exposed at In this study, we identified a novel missense muta- the surface of the coiled-coil domain, and the replace- tion in the KIF5A gene in the proband of a large pedi- ment of the alanine by the valine does not change the gree with ADHSP. The absence of this mutation was tested hydrophobic character of the structure. in 375 normal controls. Nevertheless, because we were Interestingly, at least the propositus and his father unable to analyze the other members of the family, we in this family are characterized by a late onset of dis- cannot exclude that it is a very rare polymorphism that ease, whereas the patients previously described5,11 showed segregates independently from the disease. This defect occurs in a region adjacent to the globu- a prevalent juvenile onset of the symptoms, probably lar motor domain (termed the neck region) that permits reflecting a different pathologic mechanism of this mu- dimerization and contains a sequence that is predicted tation. to form an ␣-helical coiled-coil domain.14 Furthermore, Recent reports suggest that about 50% of mutations this domain appears to be structurally important for co- involved in ADHSP are related to the spastin (40%) 17 ordinating the activities of the 2 kinesin heads during pro- and atlastin (10%) genes. Among 10 families show- cessive movement along microtubules.14,15 ing a pure ADHSP who were recently screened for the The heptad repeat motif (a, b, c, d, e, f, and g) spastin, atlastin, and KIF5A genes in our laboratory, we forms the structural basis of the ␣-helical coiled coil found 3,5 1 (unpublished result), and 2 mutations,5 and is found in a wide variety of proteins.16 The “a” respectively. Although these figures are too small to be and “d” positions are usually represented by small significant, we propose that, in the absence of linkage nonpolar residues that stabilize the coil. Therefore, information, the KIF5A gene should be routinely substitution of amino acids at these positions may tested in ADHSP.

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 Accepted for Publication: August 12, 2005. (SPG4) found by DHPLC mutation analysis. Neuromuscul Disord. 2004;14: Correspondence: Marco Fichera, PhD, Laboratorio di 750-753. 5. Fichera M, Lo Giudice M, Falco M, et al. Evidence of kinesin heavy chain (KIF5A) Diagnosi Genetica, IRCCS Oasi Maria SS, via Conte involvement in pure hereditary spastic paraplegia. Neurology. 2004;63: Ruggero 73, 94018 Troina (EN), Italy (mfichera@oasi 1108-1110. .en.it). 6. Du¨rr A, Camuzat A, Colin E, et al. Atlastin 1 mutations are frequent in young- Author Contributions: Study concept and design: onset autosomal dominant spastic paraplegia. Arch Neurol. 2004;61: 1867-1872. Lo Giudice. Acquisition of data: Falco and Di Benedetto. 7. Lupas A, Van Dyke M, Stock J. Predicting coiled coils from protein sequences. Analysis and interpretation of data: Neri, Sturnio, Calzolari, Science. 1991;252:1162-1164. and Fichera. Drafting of the manuscript: Lo Giudice, Neri, 8. Coils documentation. Available at: ftp.ebi.ac.uk/pub/software/unix/coils-2.2. Falco, Calzolari, Di Benedetto, and Fichera. Critical re- 9. Webgene SpliceView. The National Research Council Institute for Biomedical Tech- vision of the manuscript for important intellectual content: nologies Web site. Available at: http://l25.itba.mi.cnr.it/~webgene/wwwspliceview .html. Sturnio. Obtained funding: Lo Giudice and Neri. Admin- 10. Hirokawa N. Kinesin and dynein superfamily proteins and the mechanism of or- istrative, technical, and material support: Falco, Sturnio, ganelle transport. Science. 1998;279:519-526. and Di Benedetto. Study supervision: Calzolari and Fichera. 11. Reid E, Kloos M, Ashley-Koch A, et al. A kinesin heavy chain (KIF5A) mutation in Funding/Support: This study was supported by the Ital- hereditary spastic paraplegia (SPG10). Am J Hum Genet. 2002;71:1189-1194. 12. Kikkawa M, Sablin EP, Okada Y, et al. Switch-based mechanism of kinesin motors. ian Ministry of Health, Rome. Nature. 2001;411:439-445. 13. Hoenger A, Thormahlen M, Diaz-Avalos R, et al. A new look at the microtubule binding patterns of dimeric kinesins. J Mol Biol. 2000;297:1087-1103. REFERENCES 14. Tripet B, Vale RD, Hodges S. Demonstration of coiled-coil interactions within the kinesin neck region using synthetic peptides. J Biol Chem. 1997;272: 1. McDermott C, White K, Bushby K, et al. Hereditary spastic paraparesis: a review 8946-8956. of new developments. J Neurol Neurosurg Psychiatry. 2000;69:150-160. 15. Kozielski F, Sack S, Marx A, et al. The crystal structure of dimeric kinesin and 2. Reid E. Science in motion: common molecular pathological themes emerge in implications for microtubule-dependent motility. Cell. 1997;91:985-994. the hereditary spastic paraplegias. J Med Genet. 2003;40:81-86. 16. Adamson JG, Zhou NE, Hodges RS. Structure, function and application of the 3. Rainier S, Chai JH, Tokarz D, et al. NIPA1 gene mutations cause autosomal domi- coiled-coil protein folding motif. Curr Opin Biotechnol. 1993;4:428-437. nant hereditary spastic paraplegia (SPG6). Am J Hum Genet. 2003;73: 17. Park SY, Ki CS, Kim HJ, et al. Mutation analysis of SPG4 and SPG3A genes and 967-971. its implication in molecular diagnosis of Korean patients with hereditary spastic 4. Falco M, Scuderi C, Musumeci S, et al. Two novel mutations in the spastin gene paraplegia. Arch Neurol. 2005;62:1118-1121.

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