Spastic paraplegia 3A-causing mutations in atlastin1 complementary DNA. Schematic representations of the complimentary DNA where darker gray bands present the motifs characteristic of the large guanidin triphosphatases (GTPase)family active site. Exons are numbered from 1 to 14. The initiation and the stop codons are shown. Nucleotide and amino acid changes (in parentheses) are indicated. The mutations shown above were found in our series of patients including 7 new mutations in bold. The previously reported atlastin1 mutations shown below the schematic representation were not detected in our series of patients. Nucleotide changes are numbered according to Zhao et al.12
Partial pedigree of family FSP-1627 with the V253I mutation and incomplete penetrance. For confidentiality, individuals are represented by diamonds. Filled diamonds are clinically affected individuals whether symptomatic or not. The genotype is indicated at nucleotide 925; G is the wild-type and A is the mutated allele. Individual 029, still asymptomatic at age 20 years, was already clinically affected when examined at age 12 years. The number below each diamond identifies the individual according to our internal code. AE indicates age at examination; AO, age at onset of symptoms. Both of these are given when available.
Dürr A, Camuzat A, Colin E, Tallaksen C, Hannequin D, Coutinho P, Fontaine B, Rossi A, Gil R, Rousselle C, Ruberg M, Stevanin G, Brice A. Atlastin1 Mutations Are Frequent in Young-Onset Autosomal Dominant Spastic Paraplegia. Arch Neurol. 2004;61(12):1867-1872. doi:10.1001/archneur.61.12.1867
Copyright 2004 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.2004
Hereditary spastic paraplegias are disorders that are very heterogeneous, both clinically and genetically. The atlastin1 gene has recently been implicated in SPG3A, a form of autosomal dominant pure spastic paraplegia. Atlastin1 mutations have been identified in 8 families so far.
To determine the relative frequency, phenotype, and mutation spectrum of SPG3A in patients with pure autosomal dominant spastic paraplegia and onset before age 20 years.
Patients and Methods
We sequenced the atlastin1 gene in a large series of patients (31 families) in which mutations in the spastin gene, corresponding to the frequent SPG4 locus, had previously been excluded. The phenotype was compared with 126 SPG4 patients.
We identified 12 families (39%) including 34 patients with 9 different missense atlastin1 mutations, 7 of which are newly described. The main clinical characteristic of these SPG3A patients was pure spasticity with very young onset of symptoms (mean age, 4.6 ± 3.9 years) and slow progression. However, additional signs such as decreased vibration sense and wasting in lower limbs, sphincter disturbances, and scoliosis were found in a minority of patients. In addition, several gene carriers were clinically affected but still asymptomatic (n = 5) or had no clinical signs (n = 2), indicating incomplete penetrance. Compared with patients from other families meeting the same diagnostic criteria (43 patients) and families with SPG4 (126 patients), the major form of autosomal dominant spastic paraplegia, SPG3A patients had earlier symptom onset, less frequently increased reflexes in the upper limbs, decreased vibration sense in the lower limbs, and fewer sphincter disturbances, but more frequently observed wasting in the lower limbs and scoliosis. These particularities, as well as frequent abnormal motor evoked potentials, could help identify patients to be screened for atlastin1 gene mutations.
This study enables us to estimate the frequency of the SPG3A mutations in France at 39% in families with young-onset autosomal dominant spastic paraplegia after exclusion of SPG4 cases. So far, most mutations have been private, although they were all found in exons 7, 8, 12, and 13. These exons should be given priority when performing molecular diagnoses for SPG3A.
Hereditary spastic paraplegias (HSP) are characterized by progressive lower limb spasticity. It is useful for clinicians to distinguish pure from complex forms. Pure forms are characterized by lower limb spasticity and weakness that are sometimes associated, later in the evolution of the disease, with sphincter disturbances, minor vibration sense loss, and minimal cerebellar ataxia of the upper limbs, whereas complex forms include 1 or more neurological or extraneurological signs that are highly variable.1 Recent genetic studies revealed that these neurodegenerative disorders are genetically very heterogeneous. At least 24 loci have been associated with autosomal dominant or recessive, or X chromosome–linked transmission.2,3 Only 10 of the responsible genes have been identified. Recent studies have demonstrated that the functions of several of these genes are related to axonal transport or intracellular trafficking. This could account for the selective degeneration of neurons with long axons, such as the pyramidal tract.3,4
The first autosomal gene responsible for a pure form of HSP was assigned to chromosome 14q11-q21 (SPG3A) in a French family,5 before the mapping and identification of SPG4 (spastin) that turned out to be the major locus for pure autosomal dominant forms of the disease.6 Linkage analyses revealed that SPG3A was responsible for HSP in 4 other large families from North America,7,8 Germany,9 Italy,10 and Tibet.11 There was a trend in these families toward early symptom onset, usually within the first or second decades of life. The recently identified SPG3A gene contains 14 exons that span approximately 69 kilobases (kb) and is predominantly expressed in the central nervous system.12 The gene product, atlastin1, is a 558–amino acid protein that belongs to the dynamin family of large guanidin triphosphatases (GTPases) and contains 3 conserved motifs (P-loop, DxxG and RD), which are characteristic of guanylate binding/GTPase active sites. A recent study showed that it is an integral membrane protein and may be involved in Golgi membrane dynamics or vesicle trafficking and, because of its localization, in brain specific axonal growth.13
Six mutations have been reported so far in the atlastin1 gene, including 1 frameshift and 5 missense mutations, all in families with young-onset autosomal dominant HSP.12,14- 17 The frequency of SPG3A mutations in HSP families remains, however, unknown. In the initial study of Zhao et al,12 2 of 10 families with symptom onset before the age of 10 years carried an atlastin1 gene mutation. A recent study showed that only 1 of the 8 families without spastin gene alterations carried an atlastin1 mutation.15 We have screened a large series of 31 HSP families to determine the frequency of SPG3A and to establish phenotype-genotype correlations.
Patients were assessed through the European Network on Spastic Paraplegia and Cerebellar Ataxias (SPATAX) or sampled by the neurogenetics team at the Salpêtrière Hospital in Paris, France. The inclusion criteria for screening were the presence of pure HSP transmitted as an autosomal dominant trait, at least 1 affected member with onset of symptoms before age 20 years, and exclusion of SPG4 mutations. None of these families had been tested for linkage to the SPG3A locus prior to this study. Most families came from France (n = 27), but also from Morocco (n = 1), Portugal (n = 1), Italy (n = 1), and Israel (n = 1). All patients were examined using a standardized diagnostic sheet, and blood samples were taken with their written consent. Additional investigations were conducted in a subset of patients with atlastin1 gene mutations. Electrophysiological examinations and conduction velocities, cerebral magnetic resonance imaging (MRI), and cervical cord and lumbar spine MRI were performed in 5 patients. For several patients, brainstem (n = 3), visual (n = 3), and somatosensory evoked (n = 4) potentials, and central motor conduction was recorded (n = 4). Two patients had ophthalmologic examinations and 1 had neuropsychologic testing for memory loss.
For phenotype-genotype correlations, we compared the clinical features of the 12 newly identified SPG3A families (34 patients) with the 19 non-SPG3A families (43 patients) of the 31 we screened and 31 SPG4 families (126 patients).
Genomic DNA was extracted from blood using the phenol-chloroform technique. All coding exons of SPG3A were amplified by the polymerase chain reaction with the primers listed in Table 1. Conditions for DNA amplification were 200 ng of genomic DNA, 0.4 μM of each primer, 1X buffer (Qiagen Inc, Valencia, Calif), 1X solution Q (Qiagen Inc), 0.2 mM of each dinucleotide diphosphate, and 1 U of Taq polymerase (Qiagen Inc) in a reaction volume of 25 μL. Amplification was performed for 35 cycles of 30 seconds at 96°C, 30 seconds at 50°C to 60°C, and 45 seconds at 72°C, preceded by a 10-minute denaturation at 96°C. This was followed by a final extension of 7 minutes at 72°C. After treatment with 1 U of shrimp alkaline phosphatase (Roche Molecular Biochemicals, Meylan Cédex, France) and 1 U of exonuclease I (New England Biolabs, Beverly, Mass), the polymerase chain reaction products were sequenced using the Abi Prism BigDye Terminator v 3.0 Ready Reaction Cycle Sequencing Kit on an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif). Analysis of all exons and their exon-intron boundaries was performed using the SeqScape software (Applied Biosystems).
Eighty healthy white controls from France were tested for the absence of the newly identified mutations by DNA sequencing as described. Means and proportions were compared by ANOVA followed by the Fisher exact test using SPSS software (SPSS Inc, Chicago, Ill).
Nine different mutations in the atlastin1 gene were detected in 12 families, 11 from France and 1 from Portugal (Table 2). The R239C and S259Y mutations were reported by others.12,17 The novel R495W mutation was found in 3 French families and 1 Portuguese family. The other novel mutations (Q251K, V253I, S398Y, F413L, N440T, and N519N) were detected in one family each. All of the new mutations were located in exons 8, 12, and 13 (Figure 1). The 7 newly identified mutations were considered to be causative since the mutations were present in all affected members of families FSP-114, FSP-007, FSP-1631, FSP-1627, FSP-424, FSP-016, FSP-034, and FSP-092; they were absent in 80 healthy white French controls; the amino acid changes were not conservative except for the V253I mutation; and the mutations affected an amino acid conserved in the other 2 atlastin1 homologues (NM022374, AK097588), except for S398Y and S519N. Several polymorphisms in the coding and noncoding regions of the atlastin1 gene were also detected during this screen (Table 3).
Atlastin1 gene mutations were identified in 12 (39%) of 31 families with early onset autosomal dominant HSP. There were 34 SPG3A patients, including 15 women and 19 men for whom detailed clinical information was available (Table 4). Mean age at symptom onset was 4.6 ± 3.9 years, ranging from birth to 14 years. Age at symptom onset could not be precisely determined in 6 patients, either because they were asymptomatic, although they showed evident pyramidal signs on examination at ages 4, 12, 34, 50, and 53 years (n = 5), or in 1 case because the patient did not know the exact age at which his first symptoms appeared during childhood. Overall, age at symptom onset varied little, since all affected individuals but 1 had manifested the disease by age 10 years. Walking was delayed in all 4 patients in whom the onset of the disease was at birth.
The overall clinical picture was that of a pure spastic gait disorder without involvement of the upper limbs. Onset of symptoms manifested with stiffness of the legs (n = 23) or instability (n = 6). Pyramidal signs consisted of brisk reflexes in the lower limbs (100%), a Babinski sign (94%), and spastic gait (91%). There was no spasticity in the upper limbs, and brisk reflexes were observed in the upper limbs in only 10% of the patients. Spastic gait was absent in 9%, mild in 27%, and moderate or severe in 32%. This is reflected in the functional handicap rated on a scale of 0 to 5: 0 (no symptoms), 6%; 1 (slight decrease in endurance walking), 18%; 2 (mild), 29%; 3 (unable to run), 23%; 4 (needs assistance walking), 21%; 5 (needs a wheelchair), 3%. Spasticity at rest, evaluated by passive mobilization of the legs when supine, was severe in only 12.5%. Scoliosis was found in 22%, and mild pes cavus in 15%. Sensation was not impaired, and vibration sense decreased in the ankles in only 13% of the patients (aged 33-77 years). Additional neurological signs were rare. Two patients had postural tremor in the upper limbs. Tongue fasciculations, hypersomnia, and decreased visual acuity were observed in one patient each. None had cerebellar or bulbar signs.
Stiffness of the legs evolved slowly in all patients and 8 adults needed assistance walking, 1 of whom required a wheelchair at age 63 years. There was a correlation between disease duration and spasticity during gait (P = .01) or at rest (P = .001), as well as with the frequency of decreased vibration sense at the ankles (P = .01) and sphincter disturbances (P = .001). In contrast, weakness (P = .06) and wasting (P = .79) were not significantly correlated with disease duration in our sample.
The clinical picture was not affected by the sex of the patient or the transmitting parent (data not shown). Paraclinical investigations were not very informative. No spinal cord or cerebellar atrophy was observed by brain and spine MRI performed in 5 patients. An arachnoidal cyst was observed in 1 patient. An electromyograph was performed in 5 patients. No peripheral alterations were observed except for a bilateral ulnar compression in 1 patient. Visual and somatosensory evoked potentials were normal in 3 and 4 patients, respectively. Increased central conduction time was observed with auditory brainstem (2 of 3) and motor evoked (3 of 4) potentials. Visual examination results were normal in 2 patients. Neuropsychologic testing revealed attention and concentration deficits in a 34-year-old patient with a memory complaint.
Comparison of SPG3A patients with those recruited on the basis of the same diagnostic criteria, but without atlastin1 gene mutations, revealed several differences (Table 4). Disease durations were similar in both groups, allowing comparison of the clinical pictures. The mean age at symptom onset was significantly earlier in SPG3A patients (4.6 ± 3.9 years vs 12.4 ± 13.6 years; P = .005). Brisk reflexes in the upper limbs were significantly less frequent in these patients (10% vs 32%; P = .04) as was decreased vibration sense at ankles (13% vs 45%, P = .008). However, increased reflexes in lower limbs were significantly more frequent (100% vs 86%; P = .04). There was also a tendency for sphincter disturbances to be less frequent in SPG3A than in other patients (25% vs 49%; P = .053). However, the frequency of severe forms was similar in both groups.
In addition to the differences observed between SPG3A and non-SPG3 patients, wasting in lower limbs was significantly less frequent in SPG4 (5% vs 28%; P = .001), as was scoliosis (6% vs 22%; P = .01), but after a significantly shorter disease duration (21.7 ± 15.6 years vs 32 ± 21.7 years; P = .007).
Five patients had evident pyramidal signs on examination but had no functional complaints. There was also evidence from mutation analysis for reduced penetrance. Mutation analysis was performed in 10 at-risk, but asymptomatic, individuals from 4 families. Two individuals from the same family (FSP-1627) carried the V253I mutation, an 80-year-old obligate carrier (individual 006) and his 18-year-old grandchild (individual 030). Both had normal neurologic examination results (Figure 2). There was also a 20-year-old carrier in this family who was clinically affected but asymptomatic, and only 1 symptomatic patient in whom onset of symptoms occurred at age 2 years.
Atlastin1 is predicted to have 3 different domains. Nineteen patients had mutations in exons 7, 8, and part of exon 12, corresponding to the GTP binding region in the cytoplasm. Fourteen had mutations in the region of exon 12, corresponding to the transmembrane domain. One patient had a mutation in exon 13, the C-terminal cytoplasmic part of the protein. We compared patients with mutations in both cytoplasmic domains to those with mutations in the transmembrane domain. Both groups were of similar age at onset of syptoms and disease durations. However, sphincter disturbances were only present in the group with mutations in the cytoplasmic domains of the protein (42% vs 0%; P = .03).
We have analyzed the largest series of families with autosomal dominant spastic paraplegia for atlastin1 mutations to date. Among families with young onset of symptoms (onset <20 years in at least 1 patient) and after exclusion of SPG4 mutations in the atlastin1, symptoms were more frequent (39%) than in previous studies where the inclusion criteria were different.12,15 Previous linkage studies and mutation analyses identified SPG3A families in France, Germany, Italy, North America, and Tibet. We now add Portugal, confirming that the disease is not restricted to a single geographic region.
This study enabled us to define the SPG3A phenotype. It is characterized by a particularly early onset of symptoms, since in all of our families most symptomatic patients had symptom onset before or at the age of 10 years, with the mean age at 4.6 ± 3.9 years. It is also characteristic that pyramidal signs are almost always restricted to the lower limbs, giving a phenotype of pure spastic paraplegia in accordance with previous reports.5,9- 12,15- 17 However, in contrast to these previous studies, we show that additional signs, such as sphincter disturbances, wasting, and decreased vibration sense in lower limbs, as well as scoliosis, can be observed in a minority of SPG3A patients. Not surprisingly, the severity of spasticity at rest or during gait increased with disease duration as well as the frequency of decreased vibration sense. Despite this progressive worsening, disease severity remains mild. After a mean disease duration of 32 years, less than 25% of the patients require a walking aid or use of a wheelchair.
Patients with SPG3A differ from non-SPG3A patients recruited on the basis of the same diagnostic criteria. Those with SPG4, because of a significantly earlier age at onset of symptoms, show less frequently increased reflexes in upper limbs and decreased vibration sense but have a tendency toward more frequently observed wasting in lower limbs. The presence of scoliosis might be related to the very early onset of SPG3A, which is analogous with Friedreich ataxia where the frequency of this anomaly decreases as the age at onset of symptoms increases.18 There was also a tendency toward less frequent sphincter disturbances in the SPG3A group than in the other groups. Despite the very early onset of SPG3A and the shorter disease duration in our SPG4 sample, the proportion of cases with severe forms is less than in the SPG4 cases (wheelchair use in 3% of patients with SPG3A vs 14% of patients with SPG4).
Paraclinical explorations were found to be of limited use for the diagnosis of SPG3A. Increased central conduction times were often found for auditory and motor symptoms, but not for visual and somatosensory evoked potentials. In SPG4 patients, motor evoked potentials were almost normal.19 These differences, in addition to those found clinically, could help distinguish SPG3A from SPG4.
This study greatly enlarges the spectrum of mutations in the atlastin1 gene by adding 7 new mutations to the 6 mutations already known.12,14- 17 All new mutations were validated by cosegregation analysis in families when possible, and by excluding the hypothesis of a common polymorphism in a sample of 160 white French control chromosomes. Like most of the previously described mutations, all of the new mutations were amino acid substitutions. All but 1 of the mutations were located in both cytoplasmic domains of the protein interfering with its putative oligomerization or in interactions with other proteins. The remaining R495W mutation was found in 4 different families and its location in the transmembrane region may induce an important change in structure.13 Phenotype-genotype correlations established only 1 difference between patients with mutations in the cytoplasmic domains and those patients with mutations in the transmembrane domain, with the presence of sphincter disturbances in the former but not in the latter (8 of 19 vs 0 of 13; P = .031).
It should also be noted that the only family in which evidence for incomplete penetrance was found had the conservative V253I mutation. There was also a 20-year-old carrier in this family who was clinically affected but without symptoms, and only a single symptomatic patient with onset of symptoms at age 2 years. Interestingly, the V253I mutation, located in exon 8, is the only one found in this study involving a conservative amino acid change. We could speculate that this substitution is less deleterious than nonconservative missense mutations, accounting for its incomplete penetrance. Incomplete penetrance was already suspected in a family with a frameshift mutation14 as well as in a family with evidence for linkage to SPG3A.9
Three mutations were detected in more than 1 family; the R239C and S259Y mutations were found in 2 French families in addition to the families previously described. The newly described R495W mutation was present in 3 French and 1 Portuguese kindred. It is not known if these mutations descend from a common ancestor or result from recurrent events. Most of the mutations in the atlastin1 gene have been private. Nonetheless, they all affect the same 4 exons (7, 8, 12, and 13).
In conclusion, this large series of SPG3A patients allowed us to estimate the frequency of the disease in France at 39% in families with young onset autosomal dominant spastic paraplegia after exclusion of SPG4 cases. The phenotype is essentially a very young onset of pure spastic paraplegia with slow progression, but other signs such as deep sensory loss, sphincter disturbances, or scoliosis can also be encountered, as in SPG4 or other types of familial spastic paraplegias. The spectrum of mutations in the atlastin1 gene is larger than previously thought and most mutations are private, although all have been found in exons 7, 8, 12, and 13. These exons should therefore be given priority when performing molecular diagnoses.
Correspondence: Alexandra Dürr, MD, PhD, INSERM U289, Département de Génétique, Cytogénétique et Embryologie Hôpital de la Salpêtrière, 47 boulevard de l’Hôpital, 75013 Paris, France (email@example.com).
Accepted for Publication: March 16, 2004.
Author Contributions:Study concept and design: Dürr, Camuzat, Fontaine, Ruberg, Stevanin, and Brice. Acquisition of data: Dürr, Camuzat, Colin, Tallaksen, Hannequin, Coutinho, Fontaine, Rossi, Gil, Rousselle, Stevanin, and Brice. Analysis and interpretation of data: Dürr, Camuzat, Fontaine, Stevanin, and Brice. Drafting of the manuscript: Dürr, Camuzat, Colin, Tallaksen, Hannequin, Fontaine, Rousselle, and Brice. Critical revision of the manuscript for important intellectual content: Dürr, Colin, Coutinho, Rossi, Gil, Ruberg, Stevanin, and Brice. Obtaining funding: Dürr and Brice. Administrative, technical, and material support: Camuzat, Colin, Hannequin, Fontaine, and Gil. Study supervision: Dürr, Ruberg, Stevanin, and Brice.
Funding/Support: This work was supported by grants from the VERUM foundation, INSERM/AFM (4MR12F/A00044DS) and GIS-Institut des maladies rares/INSERM (GIS AAE02014DSA/A02191DS), and the French Association Strümpell-Lorrain (ASL) to the SPATAX network.
Acknowledgments: We are very grateful to all the patients and their families for participating. We would like to thank Drs Dominique Bonneau, Brigitte Gilbert, Dominique Boggio, Sylvia Cogilnicean, and Frances and Hubert Journel for referring patients; Dr Xinping Zhao and John Fink for information regarding the atlastin1 gene sequence. The DNA and Cell Bank of IFR 070 is acknowledged for the preparation of the DNA samples. Note: Both Drs Camuzat and Dürr contributed equally to this study.