GRACILE syndrome, a lethal metabolic disorder with iron overload, is caused by a point mutation in BCS1L

Abstract

GRACILE (growth retardation, aminoaciduria, cholestasis, iron overload, lactacidosis, and early death) syndrome is a recessively inherited lethal disease characterized by fetal growth retardation, lactic acidosis, aminoaciduria, cholestasis, and abnormalities in iron metabolism. We previously localized the causative gene to a 1.5-cM region on chromosome 2q33-37. In the present study, we report the molecular defect causing this metabolic disorder, by identifying a homozygous missense mutation that results in an S78G amino acid change in the BCS1L gene in Finnish patients with GRACILE syndrome, as well as five different mutations in three British infants. BCS1L, a mitochondrial inner-membrane protein, is a chaperone necessary for the assembly of mitochondrial respiratory chain complex III. Pulse-chase experiments performed in COS-1 cells indicated that the S78G amino acid change results in instability of the polypeptide, and yeast complementation studies revealed a functional defect in the mutated BCS1L protein. Four different mutations in the BCS1L gene have been reported elsewhere, in Turkish patients with a distinctly different phenotype. Interestingly, the British and Turkish patients had complex III deficiency, whereas in the Finnish patients with GRACILE syndrome complex III activity was within the normal range, implying that BCS1L has another cellular function that is uncharacterized but essential and is putatively involved in iron metabolism.

Figure 1

A, Critical GRACILE region. The locations of the two functionally and positionally promising candidate genes, BCS1L and ABCB6, are shown in relation to the markers demonstrating linkage disequilibrium in GRACILE chromosomes. The marker order and distances are presented as in the NCBI MapViewer, May 2002. B, Genomic structure of the BCS1L gene. The sizes of the exons and introns are indicated in base pairs. The BCS1L mutations reported here (in Finnish and British patients) are indicated above the figure, and the BCS1L mutations described elsewhere (de Lonlay et al. 2001) are marked below. The sizes of the first exon and the first intron (marked with asterisks) vary because of alternative splicing. C, The BCS1L polypeptide of 419 amino acid residues. The numbers indicate the polypeptide regions encoded by distinct exons of the BCS1L gene. Residues 9–32 correspond to the transmembrane region of the yeast BCS1p protein (Fölsch et al. 1996). Residues 224–344 represent the conserved AAA domain, according to CD-Search (Marchler-Bauer et al. 2002) at the NCBI BLAST service.

Figure 2

Northern blot analysis of BCS1L in Finnish patients with GRACILE syndrome. For the fibroblast filter, each line was loaded with 9 μg poly-A-RNA; for the liver filter, each line was loaded with 6 μg poly-A-RNA. The same filters were rehybridized with β-actin cDNA, to control for the total mRNA quantity.

Figure 3

Subcellular localization of the wild-type and S78G mutant BCS1L polypeptides in transiently transfected COS-1 cells. The BCS1L polypeptides were detected with anti-FLAG antibody (green), and the distribution was compared with mitochondrial staining (red). Colocalization is indicated in yellow. Magnification = 630×.

Figure 4

Expression of the wild-type and S78G mutant BCS1L polypeptides. COS-1 cells transfected with the wild-type and mutant BCS1L constructs were subjected to western blot analysis using anti-FLAG antibody. Each lane was loaded with 25 μg protein. pCMV5 = cells transfected with the vector only.

Figure 5

Stability of the wild-type and S78G mutant BCS1L polypeptides. Transiently transfected COS-1 cells were pulse-labeled for 1 h and were chased for 0, 12, 24, 36, and 48 h. The BCS1L polypeptides were immunoprecipitated, were separated on 10% SDS-PAGE, and were visualized by fluorography. The radioactive bands were quantitated by densitometry. The results are presented as a percentage of values at 0 h chase. The values on the curve are average values of duplicate experiments, with error bars showing the SD.

Figure 6

Yeast complementation experiment with BCS1L. The BCS1 deletion yeast strain was transformed with the wild-type and mutant BCS1L constructs in a high-copy yeast expression plasmid pRS425. The wild-type yeast BCS1 cDNA in the same vector was used as a positive control, and the plain vector was used as a negative control. A, YPEG (ethanol-glycerol) plate incubated at 30°C for 11 d. B, YPEG plate incubated at 37°C for 8 d. C, YPD (glucose) plate incubated at 30°C for 3 d.