A missense mutation in the 3-ketodihydrosphingosine reductase FVT1 as candidate causal mutation for bovine spinal muscular atrophy

Abstract

The bovine form of the autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) shows striking similarity to the human form of the disease. It has, however, been mapped to a genomic region not harboring the bovine orthologue of the SMN gene, mutation of which causes human SMA. After refinement of the mapping results we analyzed positional and functional candidate genes. One of three candidate genes, FVT1, encoding 3-ketodihydrosphingosine reductase, which catalyzes a crucial step in the glycosphingolipid metabolism, showed a G-to-A missense mutation that changes Ala-175 to Thr. The identified mutation is limited to SMA-affected animals and carriers and always appears in context of the founder haplotype. The Ala variant found in healthy animals showed the expected 3-ketodihydrosphingosine reductase activity in an in vitro enzyme assay. Importantly, the Thr variant found in SMA animals showed no detectable activity. Surprisingly, in an in vivo assay the mutated gene complements the growth defect of a homologous yeast knockout strain as well as the healthy variant. This finding explains the viability of affected newborn calves and the later neuron-specific onset of the disease, which might be due to the high sensitivity of these neurons to changes in housekeeping functions. Taken together, the described mutation in FVT1 is a strong candidate for causality of SMA in cattle. This result provides an animal model for understanding the underlying mechanisms of the development of SMA and will allow efficient selection against the disease in cattle.

Fig. 1.

Three-point LOD scores for two alternative analyses by FASTLINK. The thin line shows results for all sampled animals; the analysis represented by the bold line excludes two presumptive phenocopies by setting their disease status to unknown. Only the chromosomal segment that yielded LOD scores above 10 is shown.

Fig. 2.

Fine-mapping and positional candidate gene analysis for bovine SMA. (A) Detail of the fine-mapping results showing only the peak interval. The positions of the used markers are shown relative to the candidate genes, illustrating exclusion of BCL2. The result of FASTLINK three-point analysis with its corresponding three-unit support level is shown in bold. Results of SimWalk 18-point analysis with its corresponding one-unit support level is shown in thin lines and italic letters. (B) Structure of the bovine SMA locus and of the FVT1 gene. Vertical arrows show the position of the markers used for mapping. Relative position of introns and exons is shown for FVT1 with introns in white, coding exons in black, and the 5′ and 3′ UTRs in gray. For exon 7 the nucleotide exchange and the resulting codon change are shown. (C) PCR-RFLP with MvaI analyzed on a 2% agarose gel showing segregation of SMA and the FVT1 genotype. The PCR product for the Ala variant is cut by MvaI producing fragments of 130 and 50 bp, whereas the Thr variant causing SMA remains uncut at 180 bp. SMA carriers and affected and disease-free animals are represented by half-filled, filled, and open symbols, respectively. The white numbers and arrows on the left show the size of the DNA marker bands (M).

Fig. 3.

Position of the evolutionarily conserved Ala that is changed to Thr in animals with SMA. The BLAST alignment with KDS reductases from other species shows the vicinity around the mutated position. The mutant position is in bold, and the catalytic motif with YXXK and the substrate binding Ser-13 residues upstream are underlined. In addition, the sequence of the S. cerevisiae homologue TSC10 is shown.

Fig. 4.

The A175T variant lacks KDS reductase activity. (A) Expression of FVT1 and fvt1-A175T in E. coli; SDS/PAGE of uninduced (−) and induced (+) whole-cell lysates. (B) Purification of the His₆ fusion protein. Both the Thr-175 variant from an affected calf and the normal Ala-175 are expressed and purified in parallel under the same conditions with comparable results. (C) In vitro KDS reductase assay of the protein fractions shown in B.

Fig. 5.

The FVT1 and the fvt1-A175T mutation show an intermediate complementation of the S. cerevisiae Δtsc10 knockout. The Δtsc10 strain was transformed with plasmid-based S.c. TSC10, bovine FVT1, fvt1-A175T, and the empty expression plasmid (−). The functionality of the proteins was assessed by restreaking yeast transformants onto plates lacking phytosphingosine.