Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia

Abstract

Hereditary spastic paraplegia (HSP) is a progressive upper-motor neurodegenerative disease. The eighth HSP locus, SPG8, is on chromosome 8p24.13. The three families previously linked to the SPG8 locus present with relatively severe, pure spastic paraplegia. We have identified three mutations in the KIAA0196 gene in six families that map to the SPG8 locus. One mutation, V626F, segregated in three large North American families with European ancestry and in one British family. An L619F mutation was found in a Brazilian family. The third mutation, N471D, was identified in a smaller family of European origin and lies in a spectrin domain. None of these mutations were identified in 500 control individuals. Both the L619 and V626 residues are strictly conserved across species and likely have a notable effect on the structure of the protein product strumpellin. Rescue studies with human mRNA injected in zebrafish treated with morpholino oligonucleotides to knock down the endogenous protein showed that mutations at these two residues impaired the normal function of the KIAA0196 gene. However, the function of the 1,159-aa strumpellin protein is relatively unknown. The identification and characterization of the KIAA0196 gene will enable further insight into the pathogenesis of HSP.

Figure 1.

Pedigrees for families with KIAA0196 mutations. A, Family FSP24. B, Family FSP29. C, Family FSP34. D, Family FSP91. Blackened boxes represent affected individuals, and a diagonal line through the symbol means the individual is deceased. A vertical blackened bar indicates an individual with an unconfirmed phenotype. Sex of each individual has been masked to preserve confidentiality. Individuals marked “P” represent proximal recombinants; “D” represents the distal recombinant. An asterisk (*) indicates that DNA and clinical information have been collected for the particular individual. The age at onset of affected individuals is listed below each symbol, although this information is not available for each patient. All studied affected patients are heterozygous for a c.1956C→T mutation (pedigrees A, B, and C) or a c.A1491G mutation (pedigree D) in KIAA0196.

Figure 2.

Region spanning the SPG8 locus. A, Markers defining the borders of each described family with the SPG8 mutation and the scaled marker positions on chromosome 8q24.13. B, Candidate region used to search for the SPG8 gene between markers D8S1804 and D8S1774. Genes in the region are shown in their observed orientation. C, The 28-exon KIAA0196 gene, drawn to scale, with the location of three mutations in exons 11, 14, and 15 highlighted.

Figure 3.

Mutation analysis of the KIAA0196 gene. A–C, Sequence trace of a patient with HSP above the sequence trace of a control individual. Exon 15 (A), 14 (B), and 11 (C) heterozygous point mutations are indicated. D, Multiple-sequence alignment for strumpellin homologues surrounding the two coding changes (boxed). The Probcons (v.1.09) program was used for cluster analysis. E, RT-PCR of multiple brain regions performed using a KIAA0196-specific probe. F, Northern blot of the KIAA0196 transcript performed using 30 μg of total RNA and a 1-kb C-terminal probe.

Figure 4.

Three-dimensional modeling of strumpellin, with d1dn1b as a template, through use of SwissProt database viewer. Two helices from the 1,159-aa protein are shown, including amino acids 614–634 in one α-helix and amino acids 662–672 from a nearby α-helix in the antiparallel direction. A, Residues L619 and V626, in the same orientation in an α-helix opposite a second helix in an antiparallel direction. Only residue side chains that are closest in physical space are shown. B, The L619F mutation adds a bulky phenylalanine side group that likely exceeds the space available between the two α-helices. C, V626F mutation. The ε carbon of the F626 aromatic ring impinges on Q666 and may force apart the two α-helices.

Figure 5.

Zebrafish knockdown and rescue of KIAA0196 function. A, Gross morphological features of normal wild-type zebrafish, depicted at 3 dpf. B, Injection of a 5-bp mismatch morpholino (CTL MO), which results in no obvious disease phenotype. C and D, KIAA MO–injected fish with a severely curly tail (C) or with a slightly curly tail (D). Their heart cavities are also enlarged, which is commonly seen in injected fish. E and F, Fish, injected with both KIAA MO and normal human KIAA0196 mRNA, with partially developed curly tail (F) or no effect at all (E), depending on the injected quantity. G and H, Disease phenotype not alleviated when the KIAA MO is injected with the mutant forms (×14 [panel G] and ×15 [panel H]) of the human mRNA. These fish resemble the KIAA MO fish (C and D).

Figure 6.

Immunohistochemical analysis of zebrafish with the KIAA0196 knockdown phenotype. A, Motor neurons in the ventral roots of wild-type zebrafish, segmented and oriented at 3 dpf. The spinal cord consists of the cell bodies of motor neurons and interneuron bundles. The picture was taken near the gut of the fish. B, Mismatch control has a motor-neuron distribution similar to the wild type. C, E, and F, Zebrafish injected with KIAA MO (C) and fish coinjected with mutant mRNA (×14 [panel E] and ×15 [panel F]), showing shorter, branching motor neurons that are not oriented. D, Wild-type KIAA0196 mRNA coinjections with KIAA MO, which partially rescue the motor-neuron phenotype. The axons are longer and oriented.