Identification of the SPG15 gene, encoding spastizin, as a frequent cause of complicated autosomal-recessive spastic paraplegia, including Kjellin syndrome

Abstract

Hereditary spastic paraplegias (HSPs) are genetically and phenotypically heterogeneous disorders. Both "uncomplicated" and "complicated" forms have been described with various modes of inheritance. Sixteen loci for autosomal-recessive "complicated" HSP have been mapped. The SPG15 locus was first reported to account for a rare form of spastic paraplegia variably associated with mental impairment, pigmented maculopathy, dysarthria, cerebellar signs, and distal amyotrophy, sometimes designated as Kjellin syndrome. Here, we report the refinement of SPG15 to a 2.64 Mb genetic interval on chromosome 14q23.3-q24.2 and the identification of ZFYVE26, which encodes a zinc-finger protein with a FYVE domain that we named spastizin, as the cause of SPG15. Six different truncating mutations were found to segregate with the disease in eight families with a phenotype that included variable clinical features of Kjellin syndrome. ZFYVE26 mRNA was widely distributed in human tissues, as well as in rat embryos, suggesting a possible role of this gene during embryonic development. In the adult rodent brain, its expression profile closely resembled that of SPG11, another gene responsible for complicated HSP. In cultured cells, spastizin colocalized partially with markers of endoplasmic reticulum and endosomes, suggesting a role in intracellular trafficking.

Figures

Figure 1

Critical Region of the SPG15 Locus, Structure of the ZFYVE26 Gene, and Mutations Identified in Eight SPG15 Families (A) Physical and genetic map of human chromosome 14q23.3-q24.2, with markers defining the reduced SPG15 candidate interval in bold. Location and direction of transcription (arrow) of the known genes are schematically represented. The candidate genes analyzed in this study and in a previous report [indicated with “#”] are indicated by black boxes. Distances on chromosome 14 are indicated according to the Ensembl and UCSC Genome Browser databases. (B) Structure of the ZFYVE26 gene (GenBank NM_015346) and location of the six different disease-causing mutations. The gene, located on chromosome 14q24.1, is transcribed from telomere to centromere and consists of 42 exons covering a genomic region of 70,063 bp. The full-length transcript is 9,688 bp long, with a coding sequence (exons 2–42) of 7,620 bp (mRNA NM_015346.2). The coding region is indicated in gray, and untranscribed regions (UTRs) (5′ and 3′) are indicated in white. The mutations are numbered according to the nomenclature of the Human Genome Variation Society, such that +1 is the A of the start codon (ATG) of the cDNA sequence. (C) Putative functional domains (boxes) present in spastizin (according to Predictprotein).

Figure 2

Pedigrees Showing Segregation of Disease-Causing Mutations in ZFYVE26 in Eight Families Linked to SPG15 Square symbols represent men, circles represent women. Affected subjects are represented with filled symbols. The numbers are an internal reference for each sampled individual. The genotypes are indicated below the analyzed individuals. “+” indicates wild-type allele. For the detection of the genomic rearrangement in family 671, primers 21aF and 23R (Table S1) were used, and they generated a 238bp-fragment in mutation carriers only.

Figure 3

Expression Profile of ZFYVE26 Comparison of ZFYVE26 (SPG15) and KIAA1840 (SPG11) mRNA expression in the adult rat brain (P68) via in situ hybridization with a pool of three sense or antisense probes. Both ZFYVE26 and KIAA1840 expression resembled expression of the neuronal marker NeuN more than expression of the glial marker GFAP labeled on adjacent slices. No specific staining was observed with the sense probes. Expression of ZFYVE26, like KIAA1840, was low throughout the brain except in the following structures: HIP, hippocampus; PG, pineal gland; GrC, granular cell layer of the cerebellum; and the edges of the ventricles (DV3, third ventricle; LV, lateral ventricles). Similar results for both genes were obtained on adjacent slices with use of the pool of three probes or each probe independently.

Figure 4

Cellular Expression of Spastizin A spastizin-HIS-V5 fusion protein was overexpressed in COS-7 cells and labeled 48 hr after transfection with an antibody against the V5 tag (green). The Pearson coefficient (Rr) was calculated for estimation of the degree of colocalization between spastizin-HIS-V5 and the organelle markers (in red). The values range from −1.0 (not colocalized) to +1.0 (fully colocalized). Nuclei are counterstained with DAPI (blue). Images were obtained with a Leica SP1 confocal microscope (objective ×63, scale bar represents 10 μm). Spastizin partially colocalized with the endosomal marker EEA1 and the endoplasmic-reticulum marker calreticulin but did not show any significant colocalization with Golgi (anti-giantin), mitochondria (anti-Cox2), or lysosomes (anti-Lamp2). Expression of a V5-tagged fusion protein of the expected size was verified on Western blots of cell extracts with a V5 antibody (data not shown).