Neurogenetics

, Volume 8, Issue 3, pp 231–233

A de novo SPAST mutation leading to somatic mosaicism is associated with a later age at onset in HSP

Authors

  • Christel Depienne
    • INSERM, UMR 679 (formerly U289), GH Pitié-Salpêtrière
    • Département de GénétiqueCytogénétique et Embryologie
    • Université Pierre et Marie Curie
  • Estelle Fedirko
    • Département de GénétiqueCytogénétique et Embryologie
  • Jean-Marc Faucheux
    • Centre hospitalier
  • Sylvie Forlani
    • INSERM, UMR 679 (formerly U289), GH Pitié-Salpêtrière
  • Bernard Bricka
    • Département de GénétiqueCytogénétique et Embryologie
  • Cyril Goizet
    • Hôpital Pellegrin
  • Sylvie Lesourd
    • Département de GénétiqueCytogénétique et Embryologie
    • Université Pierre et Marie Curie
  • Giovanni Stevanin
    • INSERM, UMR 679 (formerly U289), GH Pitié-Salpêtrière
    • Département de GénétiqueCytogénétique et Embryologie
    • Université Pierre et Marie Curie
  • Merle Ruberg
    • INSERM, UMR 679 (formerly U289), GH Pitié-Salpêtrière
  • Alexandra Durr
    • INSERM, UMR 679 (formerly U289), GH Pitié-Salpêtrière
    • Département de GénétiqueCytogénétique et Embryologie
    • INSERM, UMR 679 (formerly U289), GH Pitié-Salpêtrière
    • Département de GénétiqueCytogénétique et Embryologie
    • Université Pierre et Marie Curie
Letter to the Editors

DOI: 10.1007/s10048-007-0090-4

Cite this article as:
Depienne, C., Fedirko, E., Faucheux, J. et al. Neurogenetics (2007) 8: 231. doi:10.1007/s10048-007-0090-4
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Abstract

SPG4/SPAST, the gene-encoding spastin, is responsible for the most frequent form of autosomal dominant hereditary spastic paraplegia (HSP). SPG4-HSP is a heterogeneous disorder characterized by both interfamilial and intrafamilial variation, especially regarding the severity and the age at onset. In this study, we investigated the origin of the mutation and the factors involved in intra-familial heterogeneity in a family with a SPG4 mutation. We demonstrated that the mutation occurred de novo and show evidence of somatic mosaicism in the grandfather, who was the only affected member of six siblings. His disease began at age 55, much later than in his daughter, who had onset at age 18, and his grandson, in whom onset was at age 5. These observations indicate that de novo mutations can occur in SPG4, and that somatic mosaicism might account for intra-familial variation in SPG4-linked HSP.

Mutations in SPG4/SPAST, encoding spastin, are responsible for the most frequent form of autosomal dominant hereditary spastic paraplegia (HSP), a group of disorders characterized by progressive spasticity and weakness in the lower limbs (MIM#182601). SPG4-HSP is heterogeneous and characterized by inter- and intrafamilial variation in severity and age at onset, which ranges from 1 to 80 years [1].

We investigated the origin of the mutation and the factors involved in intrafamilial heterogeneity in a previously described family with the c.1684C>T/p.Arg562X mutation [2, 3]. The proband (patient 1), the first generation to be affected, had an affected daughter (patient 4) and grandson (patient 6); all three had pure HSP and the mutation (Fig. 1a). The disease started with stiffness in the legs for patient 1 and his daughter at age 55 and 18, respectively. Patient 6 was known to be affected since age 5, but was only mildly impaired at age 17 (Table 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs10048-007-0090-4/MediaObjects/10048_2007_90_Fig1_HTML.gif
Fig. 1

a Segregation of the SPG4 mutation in the family. Nine microsatellite markers located inside and on both sides of the SPG4 gene were genotyped in patient 1, his siblings, and his offspring. The same haplotype (boxed in red) was transmitted with the 1684T mutation to patients 1, 4, and 6 and without the mutation to an unaffected brother (7) and two unaffected half-sisters (8 and 10). Parentage was confirmed by analysis of a large panels of X, Y and autosomal markers (not shown). b DHPLC analysis of the c.1684C>T/p.Arg562X mutation in patient 1, his siblings, and his offspring. The elution profiles of five members of the family are shown. The peaks reflect the amount of homoduplex (white arrow) and heteroduplex double-stranded PCR products. The profiles of the unaffected half-sister and brother of patient 1 show only homoduplexes; those of patients 1, 4, and 6 have both homo- and heteroduplexes. Note that patient 1 has more homoduplexes and fewer heteroduplexes than his daughter, patient 4, reflecting a lower level of mutant alleles in his blood cells. c DNA sequencing confirms that patient 1 has lower levels of the mutant allele than the normal allele (red arrow), whereas in patients 4 and 6, the levels are the same. d Quantification of the c.1684C>T mutation relative to a control gene (RNAseP) in blood cells of patients 1, 4, 6, and unaffected relatives 7, 9, and 10. An allele-specific real time PCR assay was developed: the reverse primer has the c.1684C>T mutation at the 3′ end preceded by a mismatch at position N-1, preventing selectively the amplification of the normal allele without affecting the quantitative amplification of the mutated allele. Relative ratios (y-axis) were calculated from three independent experiments using the formula \( r = 2^{{ - \Delta \Delta Ct}} \) with \( \Delta \Delta Ct = {\left( {Ct_{{{\text{mutation}}}} - Ct_{{{\text{RNAseP}}}} } \right)}_{{\,{\text{tested individual}}}} - {\left( {Ct_{{{\text{mutation}}}} - Ct_{{{\text{RNAseP}}}} } \right)}_{{{\text{control}}}} \). Linear amplification of the mutated allele was validated using a series of dilutions of DNA from patient 4 in control DNA (75, 50, 25, and 12.5%). e Quantification of the c.1684C>T mutation in skin fibroblasts from patients 1, 4, and 6 and a control (Ctrl) and a series of dilutions of DNA from patient 4 in control DNA (75, 50, 25, and 12.5%)

Table 1

Clinical features of patients 1, 4, and 6

 

Patient 1

Patient 4

Patient 6

HSP type

Pure

Pure

Pure

Age at onset

55 years

18 years

5 years

Signs at beginning

Stiffness in the legs

Stiffness in the legs

Falls

Disease severity

Needs help walking at 59 years; walks with a cane at 74 years

Help walking at 30 years

Walking still unlimited at age 17 years

Increased reflexes

+ in all limbs

+ in lower limbs

ND

Proximal weakness

Moderate

Moderate

ND

Spasticity

Severe

Moderate

ND

ND not determined

We followed the transmission of the alleles using microsatellite markers located within and flanking the SPAST gene. This analysis revealed that the haplotype segregating with the disease and the mutation in patients 1, 4, and 6 was transmitted by the mother, but was also present in the unaffected brother and two unaffected half-sisters of patient 1 who did not have the mutation (Fig. 1a). This strongly suggests that the mutation occurred de novo in patient 1. Another striking feature was the difference in the DHPLC elution profile of patient 1 compared to that of his affected offspring: it showed more homoduplexes and less heteroduplexes, indicating a different ratio between the normal and mutated alleles (Fig. 1b). Sequencing confirmed that he had a lower level of mutant allele in his blood cells (Fig. 1c). A quantitative PCR assay that specifically amplifies the c.1684C>T mutation [4] showed that patient 1 had 25–30% less of the mutated allele in his blood cells and fibroblasts than his daughter and grandson (p < 0.01, Fig. 1d and e), providing evidence of somatic mosaicism in this patient.

The high proportion of the mutation in both lymphoblasts and fibroblasts suggests that the mutation occurred at an early stage of embryogenesis. Whether patient 1 had mosaicism in the central nervous system could not be investigated. However, this patient had later onset than his offspring (see clinical features and ages at onset in Table 1), which is compatible with a smaller number of motor neurons containing mutated spastin. It has been shown that reduced intracellular levels of functional spastin are not well tolerated since leaky splice site mutations (creating both normal and aberrant mRNA) were reported to be pathogenic [5]. Our results now suggest that reducing the proportion of neurons containing functional spastin is sufficient to cause HSP, although with a later onset. The proportion of de novo mutations and somatic mosaicism in an adult-onset disease is probably low and not the only cause of intrafamilial disease variability, but this possibility should be considered. The histopathologic analysis of such cases would help to understand the pathophysiological mechanisms of HSP.

Acknowledgment

The authors thank the family for its participation and the DNA and cell bank of IFR70 for DNA extraction. This work was supported by the VERUM foundation and the Programme Hospitalier de Recherche Clinique AP-HP (n°AOM03059, to AD).

Copyright information

© Springer-Verlag 2007