A genomewide linkage-disequilibrium scan localizes the Saguenay-Lac-Saint-Jean cytochrome oxidase deficiency to 2p16

Abstract

Leigh syndrome (LS) affects 1/40,000 newborn infants in the worldwide population and is characterized by the presence of developmental delay and lactic acidosis and by a mean life expectancy variously estimated at 3-5 years. Saguenay-Lac-Saint-Jean (SLSJ) cytochrome oxidase (COX) deficiency (LS French-Canadian type [LSFC] [MIM 220111]), an autosomal recessive form of congenital lactic acidosis, presents with developmental delay and hypotonia. It is an LS variant that is found in a geographically isolated region of Quebec and that occurs in 1/2,178 live births. Patients with LSFC show a phenotype similar to that of patients with LS, but the two groups differ in clinical presentation. We studied DNA samples from 14 patients with LSFC and from their parents, representing a total of 13 families. Because of founder effects in the SLSJ region, considerable linkage disequilibrium (LD) was expected to surround the LSFC mutation. We therefore performed a genomewide screen for LD, using 290 autosomal microsatellite markers. A single marker, D2S1356, located on 2p16, showed significant (P < 10(-5)) genomewide LD. Using high-resolution genetic mapping with additional markers and four additional families with LSFC, we were able to identify a common ancestral haplotype and to limit the critical region to approximately 2 cM between D2S119 and D2S2174. COX7AR, a gene encoding a COX7a-related protein, had previously been mapped to this region. We determined the genomic structure and resequenced this gene in patients with LSFC and in controls but found no functional mutations. Although the LSFC gene remains to be elucidated, the present study demonstrates the feasibility of using a genomewide LD strategy to localize the critical region for a rare genetic disease in a founder population.

Figure 1

Haplotypes of patients with LSFC, for the microsatellite markers that gave significant P_excess values. A, Ancestral haplotype, as defined by the markers (shared by 26 [72%] of 36 individuals). B, P_excess values for all the markers, with their Généthon map distances.

Figure 2

Pedigree and haplotypes of members of family COX007, for some of the markers used in the high-density mapping of LSFC. The inherited paternal chromosome is framed in the right column, for father and both daughters. Straight lines indicate the maternal chromosomes, and the points of recombination are depicted in the maternal genotype. The thicker vertical lines indicate the maternal alleles inherited by patient 7-12, and the thinner vertical lines indicate the maternal alleles inherited by patient 7-13. The two patients share alleles only for D2S119 and D2S2298.

Figure 3

Genomic structure of COX7AR. The genomic sequence was determined for a region of 7934 bp. This region contained the entire COX7AR gene, consisting of three exons (of 111, 132, and 817 bp, respectively), two introns (of 3753 and 1898 bp, respectively), and the 5′ upstream region of 1007 bp. The four SNPs found in the SLSJ population are referred to here as SNP1, SNP2, SNP3, and SNP4.

Figure 4

Immunoblotting of mitochondria from fibroblast cultures. Mitochondria (100 μg) were prepared from skin fibroblast cultures, were subjected to PAGE separation, and were transferred to nitrocellulose. Parallel prepared blots were then probed with antibodies to beef heart COX (top), SURF1 (middle), and COX7RP (7aRP) (bottom). The anti-COX antibody recognizes subunits II, IV, Vb, and VIa of the human COX complex. Lane 1, Control. Lanes 2, 3, and 5, Patients with LS and SURF1 mutation. Lane 4, Patients with LS COX deficiency and normal SURF1. Lanes 6, 7, and 8, Patients with LSFC-COX.