Mutations disrupting selenocysteine formation cause progressive cerebello-cerebral atrophy

Abstract

The essential micronutrient selenium is found in proteins as selenocysteine (Sec), the only genetically encoded amino acid whose biosynthesis occurs on its cognate tRNA in humans. In the final step of selenocysteine formation, the essential enzyme SepSecS catalyzes the conversion of Sep-tRNA to Sec-tRNA. We demonstrate that SepSecS mutations cause autosomal-recessive progressive cerebellocerebral atrophy (PCCA) in Jews of Iraqi and Moroccan ancestry. Both founder mutations, common in these two populations, disrupt the sole route to the biosynthesis of the 21st amino acid, Sec, and thus to the generation of selenoproteins in humans.

Figure 1

Sequential MRI Studies in PCCA Images are of individual 4 of family A (Figure 2). (A) Age 8 months: a T1 coronal image at the level of the occipital lobes demonstrates a normal-looking brain. (B) Age 18 months: a T1 sagital image showing vermian atrophy with mild cerebral atrophy. (C) Age 3 years: a coronal image at the level of the occipital lobes demonstrates severe cerebellar and cerebral atrophy.

Figure 2

Genetic Mapping of the Disease-Associated Locus Haplotypes of all four families on chromosome 4p15.2 are shown. The disease-associated haplotype is boxed. Additional microsatellite markers were used for fine mapping the candidate region identified by the genome-wide scan with the 10K SNP arrays. Families A and B are of Iraqi origin. Families C and D are of Iraqi and Moroccan origin. The minimum disease-associated haplotype lies between markers D4S425 and D4S391 for the “Iraqi” haplotype and between markers D4S1551 and rs2048506 for the “Moroccan” haplotype. Gray shading indicates the SepSecS position within the haplotype.

Figure 3

SepSecS Mutations in PCCA Families (A–C) The c.1001A>G SepSecS mutation in exon 8. Sequence analysis is shown for an affected individual (A), a carrier (B), and an unaffected individual (C) in families A and B. (D and E) The c.715G>A mutation in exon 6. Sequence analysis is shown for a carrier (D) and unaffected individuals (E) in families C and D. Affected individuals in families C and D were compound heterozygous for the two mutations. The arrows indicate the positions of the mutations.

Figure 4

Alignment of the Mutated Sequence Regions in Some Eukaryotic and Archaeal SepSecS Proteins

Figure 5

Functional Studies of the SepSecS Mutations In vivo assays of human SepSecS mutants. Formation of Sec-tRNA^Sec in vivo is assayed by the ability of the wild-type human SepSecS and the mutant variants p.Lys284Thr, p,Ala239Thr, and p.Tyr334Cys to restore the benzyl-viologen-reducing activity of the selenoprotein FDH_H in the E. coli ΔselA deletion strain. The Methanocaldococcus jannaschii PSTK is cotransformed to generate the Sep-tRNA^Sec intermediate. K284T is a previously described inactive SepSecS mutant.

Figure 6

Structure of Selenocysteine and Its Route of Biosynthesis Seryl-tRNA synthetase attaches serine to tRNA^Sec. (Top) Bacteria use selenocysteine synthase (SelA) to convert Ser-tRNA^Sec directly to Sec-tRNA^Sec. (Bottom) Eukaryotes and archaea employ a two-step conversion; a kinase (PSTK) provides the phosphorylated intermediate Sep-tRNA^Sec, the required substrate of SepSecS (see text).