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Point mutations in SPG4, the gene encoding spastin, are a frequent cause of autosomal dominant hereditary spastic paraplegia (AD‐HSP). However, standard methods for genetic analyses fail to detect exonic microdeletions.
121 mutation‐negative probands were screened for rearrangements in SPG4 by multiplex ligation‐dependent probe amplification.
24 patients with 16 different heterozygotic exon deletions in SPG4 (20%) were identified, ranging from one exon to the whole coding sequence. Comparison with 78 patients with point mutations showed a similar clinical picture but an earlier age at onset.
Exon deletions in SPG4 are as frequent as point mutations, and SPG4 is responsible for 40% of AD‐HSP.
Hereditary spastic paraplegias (HSPs) are a genetically heterogeneous group of neurodegenerative disorders clinically characterised by progressive stiffness and weakness of the lower limbs that result from axonal neurodegeneration in the corticospinal tract. Pure and complicated forms of HSP have been described depending on whether spasticity occurs in the absence or presence of other clinical features such as cerebellar ataxia, neuropathy, retinal degeneration, cognitive impairment, dementia or epilepsy. The clinical heterogeneity of HSP is partly explained by its genetic heterogeneity. To date, 35 loci have been identified, associated with autosomal dominant, autosomal recessive and X linked modes of inheritance (for additional information, see the HUGO site at http://www.gene.ucl.ac.uk/nomenclature/).
The most common form of autosomal dominant HSP (AD‐HSP) is caused by mutations in the SPG4/SPAST gene (MIM 604277), encoding spastin, a member of the AAA family of ATPases.1SPG4 has been shown to account for 15–40% of all AD‐HSP families, depending on the population.2,3,4,5 HSP due to SPG4 mutations (MIM 182601) is generally described as a pure form of the disease—that is, as spastic paraparesis often associated with a decreased sense of vibration in the lower limbs and urinary problems—but has also occasionally been described in association with additional features, including cognitive impairment, peripheral neuropathy, cerebellar signs or epilepsy.6,7,8 Age at onset is highly variable, ranging from early infancy up to the eighth decade. More than 150 different mutations have been identified in all exons of the SPG4 gene except for exon 4, which is alternatively spliced. All types of DNA alterations are observed, including missense, non‐sense, splice‐site mutations, and small insertions or deletions.9 This wide spectrum of mutations, combined with the observation that many mutations reduce the abundance of the normal full‐length transcript and/or functionally normal spastin protein, suggests that the pathogenic mechanism is haploinsufficiency—that is, the disease occurs once the level of functional spastin falls below a critical level.10,11 Although larger deletions in SPG4 have been described in two cases5,12 and should theoretically lead to HSP, they are missed by standard screening methods such as direct sequencing and denaturing high‐performance liquid chromatography and have not yet been systematically sought. In this study, we screened 121 patients for micro‐rearrangements in the SPG4 gene by multiplex ligation‐dependent probe amplification (MLPA) to assess their frequency in families with HSP.
We selected 121 European, mostly French (105/121), probands with HSP, in whom mutations in the SPG4 gene were not detected by denaturing high‐performance liquid chromatography. Dominant transmission was observed in 120 families. The remaining family had two affected sibs. Most of the probands presented pure HSP (n=114). The complicated forms (n=7) included signs of peripheral neuropathy (n=3), and one each with cerebellar signs, mental retardation, cognitive impairment and parkinsonism. Informed written consent was obtained from each individual before blood sampling. This study was approved by the ethical committee Paris‐Necker (CCPPRB, number 03‐12‐07, 2/10/2004).
The autosomal dominant spastic paraplegia MLPA kit (P165) designed to search for deletions or duplications in SPG4 was purchased from MRC‐Holland (Amsterdam, The Netherlands). MLPA reactions were carried out according to the manufacturer's instructions. Electrophoresis of polymerase chain reaction products was performed using an ABI 3730 sequencer and MLPA data were analysed using the GeneMapper V.4.0 software (PE Applied Biosystems, Foster City, California, USA). Relative ratios were calculated for each peak using the formula r = mean (peak areapatient/control areapatient)/(peak areacontrol individual/control areacontrol).
Frequencies were compared with the χ2 test or Fisher's exact test when appropriate. Quantitative variables were compared with the Mann–Whitney U test. Statistical analysis was performed using SPSS software (v 13.0). Values are expressed as mean (SD).
We identified 24 index patients (22 French, 1 Portuguese and 1 Spanish) with microdeletions in the SPG4 gene. This corresponds to a frequency of 20% (24/121). Sixteen different exon deletions were detected, spanning a single exon to the whole coding sequence of the gene (ie, exons 1–17). Five of the deletions (exon 1, exons 1–17, exons 8–17, exon 13 and exon 16) were detected in more than one family, but the remaining 11 were private rearrangements. Segregation of the microdeletion with the disease was analysed when DNA from other affected individuals was available (table 11).). All affected relatives (n=32) of probands from 12 families also had the corresponding exon deletion, confirming that these rearrangements are responsible for the disease. In addition, we tested six asymptomatic relatives and examined one deletion carrier who was still asymptomatic at age 46 years. These observations confirm that the penetrance is age dependent and incomplete in patients with SPG4 deletions, as described previously for SPG4 point mutations.
Age at onset in patients with SPG4 deletions, including the 24 index cases and 32 affected relatives, ranged from 1 to 77 years (mean (SD) age at onset 28.7 (17.4) years, n=44). Age at onset could not be determined for 12 patients, including 3 who presented minor signs only detected on examination. All index cases with exon deletions had pure HSP. However, cognitive impairment was present in a single affected relative. One index patient (FSP‐774) had cerebellar atrophy on cerebral MRI but no cerebellar signs at examination. The age at onset for the four index patients with the largest deletion spanning exons 1–17 was between 4 and 35 years, similar to the mean age at onset of the whole group.
Comparison of probands with microdeletions (n=24) and probands with point mutations (n=78, unpublished data) showed that the former were significantly younger at onset (25.4 (12) vs 31.8 (16.8) years, p=0.033; table 22).). All other features were strikingly similar in both groups, except for a tendency towards less severe walking disability in the deleted group compared with the other group (9% vs 14%), despite similar disease durations.
We identified 24 patients with partial exonic deletions in SPG4 among 121 patients who were negative for point mutations. These findings demonstrate that a large proportion (20%) of patients with mutation‐negative HSP in fact carry SPG4 microdeletions and confirm that haploinsufficiency of SPG4 is a major cause of AD‐HSP.
Previous estimates of the proportion of patients with SPG4 mutations who had family histories of the disease ranged between 15% and 40%. The percentage of patients with point mutations in SPG4 in our large series of French HSP families (which include patients with unknown linkage status) is, however, approximately 25% (A Durr, C Depienne and A Brice, personal communication), a proportion also confirmed in another large European series.13 When taking into account only the French families with autosomal dominant inheritance and excluding families with SPG4 point mutations in this study, the proportion of cases with exon deletions reaches 16% of AD‐HSP. These results indicate that SPG4 could indeed be responsible for 40% of HSP families in the French population. This proportion might even be greater considering that mutations or deletions in the SPG4 promoter have not yet been systematically sought.
Many different combinations of exon deletions are observed, indicating that dosage should be applied to all SPG4 exons. This can easily be performed in a single reaction using MLPA. A few deletions were found more than once, and it will be interesting to determine whether they descend from a common founder or are recurrent mutations.
Globally, the clinical picture of the patients with exon deletions is similar to that of patients with point mutations: most patients present pure HSP which includes, in addition to spasticity in the lower limbs, brisk reflexes, decreased sense of vibration at ankles, proximal or generalised weakness in the lower limbs, as well as frequent sphincter disturbances as part of the “SPG4 phenotype”. However, the age at onset seemed to be significantly earlier in patients with microdeletions who tended to be less severely affected than patients with point mutations. Disease progression had indeed been previously shown to be more rapid in late‐onset than in early‐onset cases.3 Patients with a deletion of the whole gene also had a clinical phenotype and an age at onset similar to patients with other deletion types or point mutations. The functional consequence of SPG4 mutation is therefore the loss of spastin function.
This study confirms that HSP is caused by SPG4 haploinsufficiency, attested by the similar phenotypes associated with different types of mutations including whole‐gene deletions. The fact that exon deletions are almost as frequent as point mutations in this large series of families with AD‐HSP justifies the inclusion of gene dosage experiments in future genetic tests for SPG4 so that appropriate genetic counselling can be given.
We thank the bank of IFR70 for DNA extraction, especially Christiane Penet for her kind participation. We thank the families for participating, the SPATAX network and the clinicians who referred their patients: Perrine Charles, Bertrand Fontaine, Richard Levy, Olivier Lyon‐Caen, Alain Lagueny, Robert‐Thierry Ghnassia, Jean‐Marc Visy, Valérie Drouin Garraud, Jean‐Pierre Salles, Christine Tranchant, Tanya Stojkovic, Pierre Labauge, Laurent Magy, Isabelle Penisson‐Besnier and Isabelle Desguerre. This work was financially supported by the VERUM Foundation and the Programme Hospitalier de Recherche Clinique AP‐HP (number AOM03059, to AD).
AD‐HSP - autosomal dominant hereditary spastic paraplegia
HSP - hereditary spastic paraplegia
MLPA - multiplex ligation‐dependent probe amplification
Competing interests: None declared.