Targeted high-throughput sequencing identifies mutations in atlastin-1 as a cause of hereditary sensory neuropathy type I

Abstract

Hereditary sensory neuropathy type I (HSN I) is an axonal form of autosomal-dominant hereditary motor and sensory neuropathy distinguished by prominent sensory loss that leads to painless injuries. Unrecognized, these can result in delayed wound healing and osteomyelitis, necessitating distal amputations. To elucidate the genetic basis of an HSN I subtype in a family in which mutations in the few known HSN I genes had been excluded, we employed massive parallel exon sequencing of the 14.3 Mb disease interval on chromosome 14q. We detected a missense mutation (c.1065C>A, p.Asn355Lys) in atlastin-1 (ATL1), a gene that is known to be mutated in early-onset hereditary spastic paraplegia SPG3A and that encodes the large dynamin-related GTPase atlastin-1. The mutant protein exhibited reduced GTPase activity and prominently disrupted ER network morphology when expressed in COS7 cells, strongly supporting pathogenicity. An expanded screen in 115 additional HSN I patients identified two further dominant ATL1 mutations (c.196G>C [p.Glu66Gln] and c.976 delG [p.Val326TrpfsX8]). This study highlights an unexpected major role for atlastin-1 in the function of sensory neurons and identifies HSN I and SPG3A as allelic disorders.

Figure 1

Partial Pedigree of a Family Affected by HSN I The pedigree shows all individuals included in the linkage study (disease status at time of diagnosis is indicated). Additional unaffected family members and individuals not available for this study are not depicted for privacy. Individual II/3 was neurologically normal at the age of 69, but NCS could not be carried out. Also, the children of this individual did not have a history of gait disturbances or foot ulcerations. Medical history indicated that individuals II/9 and III/11 were unaffected, but they refused neurological and neurophysiological examination. However, individual III/11 agreed to participate in the genetic analysis. Filled symbols indicate affected individuals; empty symbols define unaffected individuals. U indicates patients with severe sensory neuropathy and foot ulcerations and/or amputations; patients with a + also presented with upper-motor-neuron signs. Neg indicates that an individual tested negative for the p.Asn355Lys mutation.

Figure 2

Clinical Findings in Patients Carrying the Asn355Lys Atlastin-1 Variant Images of the feet of patients (III/2-U, III/8-U, and III/9-U) with prominent axonal sensory neuropathy, trophic skin and nail changes, distal muscle atrophy, mild pes cavus, and amputation of the great toe are shown.

Figure 3

Detection of ATL1 Mutations by Direct Sequencing Representative electropherograms derived from ATL1 sequencing of genomic DNA from patients (upper panel) compared to unaffected individuals (wild-type, lower panel). The positions of the heterozygous mutations are indicated with an arrow above the sequence. Effects of the mutations on amino acid sequence are shown on the right.

Figure 4

Asn355Lys Atlastin-1 Exhibits Altered GTPase Activity and Disrupts ER Morphology when It Is Expressed in Cells (A) Myc-tagged wild-type atlastin-1 or the indicated missense mutants were immunopurified from COS7 cells, and GTP hydrolysis was plotted as a function of time (top). Representative thin-layer chromatography plates show conversion of GTP to GDP (bottom). Error bars represent means ± standard deviation. (B) COS7 cells were transfected with Myc-tagged wild-type or mutant atlastin-1 and immunostained for Myc-epitope (red) and β-tubulin (green). Merged images are at the right. DAPI nuclear staining is in blue. The scale bar represents 20 μm. (C) Quantification of ER disruption (three trials of n = 100 cells per condition, means ± SD); ^∗p < 0.01. (D) COS7 cells transfected with Myc-tagged wild-type or Asn355Lys atlastin-1 were immunoblotted for Myc-epitope. Actin levels were monitored as a control for protein loading.

Figure 5

Schematic Model of Atlastin-1 Mutations in SPG3A and HSN I The model shows the distribution of autosomal-dominant, SPG3A-associated mutations and their amino acid residue changes, as well as the novel ATL1 mutations (bold letters) associated with HSN I identified in this study. Mutations known to be associated with peripheral neuropathies are underlined. Exons 1–14 are indicated.