Abstract

The most frequent causes of autosomal dominant (AD) hereditary spastic paraplegias (HSP) (ADHSP) are mutations in theSPAST gene (SPG4 locus). However, roughly 60% of patients are negative forSPAST mutations, despite their family history being compatible with AD inheritance. A mutation in the gene for an acetyl-CoA transporter (SLC33A1) has recently been reported in one Chinese family to cause ADHSP-type SPG42. In this study, we screened 220 independentSPAST mutation-negative ADHSP samples for mutations in theSLC33A1 gene by high-resolution melting curve analysis. Conspicuous samples were validated by direct sequencing. Moreover, copy number variations affectingSLC33A1 were screened by multiplex ligation-dependent probe amplification assay. We could not identify potentially disease-causing mutations in our patients either by mutation scanning or by gene dosage analysis, as for the latter specific positive controls are not available to date. As our sample represents ADHSP patients for whomSPAST mutations and almost in all casesATL1 andREEP1 mutations had been excluded, we considerSLC33A1 gene mutations as being very rare in a European ADHSP cohort, if present at all. To date, as SPG42 has still not been identified in a second, unrelated family, systematic genetic testing forSLC33A1 mutations is not recommended.

INTRODUCTION

The hereditary spastic paraplegias (HSPs) are a group of neurodegenerative diseases clinically characterized by progressive lower limb spasticity, pyramidal weakness and extensor plantar responses. The main neuropathological feature of these conditions is declining degeneration of the corticospinal tracts. Pure (uncomplicated) and complicated forms of HSP have been described according to the presence of additional neurological features such as cerebellar ataxia, peripheral neuropathy and/or cognitive impairment, in addition to spastic paraparesis. In addition to clinical manifestation, age at onset and genetic background are also highly heterogeneous.^1,2 To date, at least 46 different loci have been mapped, associated with autosomal dominant (AD), autosomal recessive and X-linked mode of inheritance.^1,2,3 A total of 17 responsible genes have been identified according to the HUGO and OMIM databases. Consistent with this high variability, HSP gene products seem to be involved in a wide range of cellular functions and pathogenic mechanisms.⁴ AD inheritance is the most common trait in pure HSP. At present, 18 loci have been linked to ADHSP, and 9 genes have been identified. Mutations in genesSPASTIN (MIM# 604277; SPG4, MIM# 182601),ATL1 (MIM# 606439; SPG3, MIM# 182600),KIF5A (MIM# 602821; SPG10, MIM# 604187) andREEP1 (MIM# 609139; SPG31, MIM# 610250) account for ∼40, ∼10, ∼3 and ∼8% of all ADHSP patients, respectively.^2,5,6,7 The remaining HSP genes seem to be relatively rare.^8,9 Spastic paraplegia type 42 (SPG42, MIM# 612539) was recently mapped to the 3q24–26 chromosomal region in a single Chinese family presenting with pure ADHSP. On the basis of the detection of a serine-to-arginine substitution at codon113 in this family, the authors reportedSLC33A1 (MIM# 603690) to be the responsible gene.¹⁰ The protein product ofSLC33A1 is an acetyl-CoA transporter, which serves as a substrate of acetyltransferases that modify the sialyl residues of gangliosides and glycoproteins.¹¹ It is hypothesized that the modification of gangliosides and glycoproteins by acetylation probably has a critical role in the outgrowth and maintenance of axons of motor neurons.¹⁰

The frequency of SPG42 and its associated phenotypes is not known, as no further families withSLC33A1 mutations have been described so far.¹⁰ The purpose of this study was to determine the frequency of SPG42 by screening 220 European (German, French, Norwegian) ADHSP patients for conventional and gene dosage mutations.

PATIENTS AND METHODS

RESULTS AND DISCUSSION

HRM analysis detected an aberrant melting profile caused by an A>G substitution at position 512 of the coding sequence that causes a p. D171G substitution in 16 index patients (Figure 1). This sequence abnormality represents the known single-nucleotide polymorphism (SNP) rs3804769 according to the SNP database.¹⁶ No other sequence alterations were detected. As HRM analysis is a screening method with ∼99% sensitivity,^17,18 we handled the results with care. Along with this high sensitivity, HRM is a simple, rapid and low-cost method to screen for unknown sequence variants in a high-throughput modality. Compared with other methods, such as denaturing high-performance liquid chromatography, HRM offers at least two additional benefits: (1) the temperature gradient covers all potential melting domains of amplicons, which provides superior detection sensitivity; and (2) the closed-tube method reduces post-PCR manipulations and the risk of pipetting errors and contaminations.^17,18 Moreover, our identification of the rs3804769 SNP represents a positive control and argues for the general validity of our approach.

Similar to all PCR-based techniques, large insertions or deletions are usually not detected during gene scanning by HRM.¹⁷ This mutational class is frequent (∼20%) in SPG4 and is also relevant for SPG31.^7,14,19 Both forms are associated with pure ADHSP. To assess quantitative changes inSLC33A1 copy number, MLPA was used but no rearrangements were identified. As no duplications or deletions ofSLC33A1 have been reported so far, no positive control was available. However, the technique can be regarded as rather robust, and homemade probe sets developed by other groups (ie, Ganesamoorthyet al²⁰), as well as by our groups (unpublished), have been successfully used in unambiguously detecting copy number alterations. Moreover, high overall data quality (data not shown) argues for the applicability of this assay; therefore, we believe that our findings are not likely to be false negative.

Screening for gene dosage seemed especially important in SPG42, as haploinsufficiency is supposed to be the pathogenic disease mechanism.¹⁰ In addition,SLC33A1 has an unusually high content of Alu sequences (38.1% compared with the ∼10% genomic average²¹). In other genes, such high values are associated with an increased susceptibility to genomic deletions (ie, MSH2).²² Nevertheless, MLPA failed to identify this potential pathological mutation class in our study.

The 220 index patients screened in this study represent a cohort of more than 500 consecutive ADHSP index patients, as mutations in common HSP genes (SPASTIN,ATL1 andREEP1), which account for >50% of our ADHSP diagnoses, had already been excluded. On the basis of data published previously by Linet al,¹⁰ the results of this study can be interpreted in two ways. First, the lack ofSLC33A1 mutations in our sample may indicate that SPG42 is an extremely rare form of ADHSP, at least in our European cohort. An alternative explanation for the negative finding of our study may consider theSLC33A1 gene to not be the true disease gene for the SPG42 HSP subtype. As only a single SPG42 family has been described so far, it is difficult to draw any genotype/phenotype correlations. In this Chinese family, 20 affected subjects presented with a ‘pure' form of HSP starting between 4 and 42 years of age; only some had wasting of lower limbs mentioned, apart from pyramidal symptoms.

Identification of additional SPG42 families would help to decide whether SPG42 represents a pure spastic paraplegia or whether a complicated course might be seen in other patients with different mutations. Moreover, a next-generation sequencing approach encompassing the whole locus in the only SPG42 family known so far should help to search for sequence alterations in the nearly 125 genes that have not been screened yet.

Acknowledgments

We thank the patients for participating and the DNA and Cell bank of the Centre de Recherche de l'Institut du Cerveau et de la Moelle épinière (Paris, France). This study was supported by the German Ministry of Education and research (BMBF) funding the German Network for Movement Disorders (GeNeMove) Grant 01GM0603, the Programme Hospitalier de Recherche Clinique (to AD) and by the E-RARE program of the EU funding the European Network of Spastic Paraplegia (EUROSPA) Grant 01GM0807. CB was supported by the Foundation for Pathobiochemistry and Molecular Diagnostics.

The authors declare no conflict of interest.

Footnotes

Supplementary Material

References

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