Targeted High-Throughput Sequencing Identifies Mutations in atlastin-1 as a Cause of Hereditary Sensory Neuropathy Type I

Center for Medical Research, Medical University of Graz, Graz 8010, Austria.
The American Journal of Human Genetics (Impact Factor: 10.93). 01/2011; 88(1):99-105. DOI: 10.1016/j.ajhg.2010.12.003
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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.

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    • "Expression of either enhanced yellow fluorescent protein (eYFP)–tagged Drosophila atlastin (Figure 2, C and inset; quantified in Figure 2, E and F) or hemagglutinin (HA)-tagged atlastin1 (Figure 2, D and inset; quantified in Figure 2, E and F), maintained normal network branch point density after endogenous atlastin depletion, FIGURE 1: Atlastin1/SPG3A mutations. The positions of known atlastin1/SPG3A mutations as of a recent report (Guelly et al., 2011) and further updated ( are indicated on an atlastin1 primary sequence map. Mutations that caused insolubility when expressed in the context of the atlastin1 soluble domain are underlined (Byrnes et al., 2011); mutations that greatly reduced GTPase activity or dimer formation are italicized; mutations that inhibited fusion activity when transferred to Drosophila atlastin are in red (Bian et al., 2011); mutations occurring in the families ADHSP-P, ADHSP-T, and ADHSP-S are indicated by an asterisk (Zhao et al., 2001), and the mutations analyzed in this study are in green. "
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    ABSTRACT: At least 38 distinct missense mutations in the neuronal atlastin1/SPG3A GTPase are implicated in an autosomal dominant form of hereditary spastic paraplegia (HSP), a motor-neurological disorder manifested by lower limb weakness and spasticity and length dependent axonopathy of corticospinal motor neurons. Because the atlastin GTPase is sufficient to catalyze membrane fusion and required to form the ER network, at least in non-neuronal cells, it is logically assumed that defects in ER membrane morphogenesis due to impaired fusion activity are the primary drivers of SPG3A-associated HSP. Here we analyzed a subset of established atlastin1/SPG3A disease variants using cell-based assays for atlastin-mediated ER network formation and biochemical assays for atlastin-catalyzed GTP hydrolysis, dimer formation and membrane fusion. As anticipated, some variants exhibited clear deficits. Surprisingly however, at least two disease variants, one of which represents that most frequently identified in SPG3A HSP patients, displayed wild type levels of activity in all assays. The same variants were also capable of coredistributing ER-localized REEP1, a recently identified function of atlastins that requires its catalytic activity. Altogether, these findings indicate that a deficit in the membrane fusion activity of atlastin1 may be a key contributor, but not required, for HSP causation. © 2015 by The American Society for Cell Biology.
    Molecular Biology of the Cell 03/2015; 26(9). DOI:10.1091/mbc.E14-10-1447 · 4.47 Impact Factor
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    • "Hereditary sensory and autonomic neuropathy type I (HSAN1) is an autosomal dominant ulceromutilating neuropathy with variable motor involvement caused by mutations in six genes: SPTLC1 (Bejaoui et al. 2001; Dawkins et al. 2001), SPTLC2 (Rotthier et al. 2010), ATL1 (Guelly et al. 2011), RAB7 (Verhoeven et al. 2003), DNMT1 (Klein et al. 2011) and ATL3 (Kornak et al. 2014). SPTLC1 and SPTLC2 encode for two of three subunits of the enzyme serine palmitoyltransferase (SPT) which catalyzes the condensation of palmitoyl-CoA with L-serine—the first and rate-limiting step in ceramide de novo synthesis (Hanada 2003). "
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    ABSTRACT: 1-Deoxysphingolipids (1-deoxySL) are atypical sphingolipids that are formed by the enzyme serine palmitoyltransferase (SPT) due to a promiscuous use of l-alanine over its canonical substrate l-serine. Several mutations in SPT are associated with the hereditary sensory and autonomic neuropathy type I (HSAN1). The current hypothesis is that these mutations induce a permanent shift in the affinity from l-serine toward l-alanine which results in a pathologically increased 1-deoxySL formation in HSAN1 patients. Also, wild-type SPT forms 1-deoxySL under certain conditions, and elevated levels were found in individuals with the metabolic syndrome and diabetes. However, the molecular mechanisms which control the substrate shift of the wild-type enzyme are not understood. Here, we report a novel SPTLC2–S384F variant in two unrelated HSAN1 families. Affected patients showed elevated plasma 1-deoxySL levels and expression of the S384F mutant in HEK293 cells increased 1-deoxySL formation. Previously, S384 has been reported as one of the two (S384 and Y387) putative phosphorylation sites in SPTLC2. The phosphorylation of wild-type SPTLC2 was confirmed by isoelectric focusing. The impact of an S384 phosphorylation on SPT activity was tested by creating mutants mimicking either a constitutively phosphorylated (S384D, S384E) or non-phosphorylated (S384A, Y387F, Y387F+S384A) protein. The S384D but not the S384E variant was associated with increased 1-deoxySL formation. The other mutations had no influence on activity and substrate affinity. In summary, our data show that S384F is a novel mutation in HSAN1 and that the substrate specificity of wild-type SPT might by dynamically regulated by a phosphorylation at this position. Electronic supplementary material The online version of this article (doi:10.1007/s12017-014-8339-1) contains supplementary material, which is available to authorized users.
    NeuroMolecular Medicine 01/2015; 17(1). DOI:10.1007/s12017-014-8339-1 · 3.68 Impact Factor
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    • "Direct sequence analyses of whole coding regions of SPAST, ATL1, and REEP1 were performed in order to identify mutations. Sequence-specific primer pairs covering the entire coding region of all three genes were used, as described elsewhere16,17,18 with minor modifications (available upon request). Polymerase chain reaction (PCR) was performed in a Gene Atlas Thermal Cycler (ASTEC, Seoul, Korea). "
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    ABSTRACT: Background and Purpose Hereditary spastic paraplegia (HSP) is a genetically heterogeneous group of neurodegenerative disorders that are characterized by progressive spasticity and weakness of the lower limbs. Mutations in the spastin gene (SPAST) are the most common causes of HSP, accounting for 40-67% of autosomal dominant HSP (AD-HSP) and 12-18% of sporadic cases. Mutations in the atlastin-1 gene (ATL1) and receptor expression-enhancing protein 1 gene (REEP1) are the second and third most common causes of AD-HSP, respectively. Methods Direct sequence analysis was used to screen mutations in SPAST, ATL1, and REEP1 in 27 unrelated Korean patients with pure and complicated HSP. Multiplex ligation-dependent probe amplification was also performed to detect copy-number variations of the three genes. Results Ten different SPAST mutations were identified in 11 probands, of which the following 6 were novel: c.760A>T, c.131C>A, c.1351_1353delAGA, c.376_377dupTA, c.1114A>G, and c.1372A>C. Most patients with SPAST mutations had AD-HSP (10/11, 91%), and the frequency of SPAST mutations accounted for 66.7% (10/15) of the AD-HSP patients. No significant correlation was found between the presence of the SPAST mutation and any of the various clinical parameters of pure HSP. No ATL1 and REEP1 mutations were detected. Conclusions We conclude that SPAST mutations are responsible for most Korean cases of genetically confirmed AD-HSP. Our observation of the absence of ATL1 and REEP1 mutations needs to be confirmed in larger series.
    Journal of Clinical Neurology 07/2014; 10(3):257-61. DOI:10.3988/jcn.2014.10.3.257 · 1.70 Impact Factor
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