Identification of novel SNPs of ABCD1, ABCD2, ABCD3, and ABCD4 genes in patients with X-linked adrenoleukodystrophy (ALD) based on comprehensive resequencing and association studies with ALD phenotypes

Abstract

Adrenoleukodystrophy (ALD) is an X-linked disorder affecting primarily the white matter of the central nervous system occasionally accompanied by adrenal insufficiency. Despite the discovery of the causative gene, ABCD1, no clear genotype-phenotype correlations have been established. Association studies based on single nucleotide polymorphisms (SNPs) identified by comprehensive resequencing of genes related to ABCD1 may reveal genes modifying ALD phenotypes. We analyzed 40 Japanese patients with ALD. ABCD1 and ABCD2 were analyzed using a newly developed microarray-based resequencing system. ABCD3 and ABCD4 were analyzed by direct nucleotide sequence analysis. Replication studies were conducted on an independent French ALD cohort with extreme phenotypes. All the mutations of ABCD1 were identified, and there was no correlation between the genotypes and phenotypes of ALD. SNPs identified by the comprehensive resequencing of ABCD2, ABCD3, and ABCD4 were used for association studies. There were no significant associations between these SNPs and ALD phenotypes, except for the five SNPs of ABCD4, which are in complete disequilibrium in the Japanese population. These five SNPs were significantly less frequently represented in patients with adrenomyeloneuropathy (AMN) than in controls in the Japanese population (p=0.0468), whereas there were no significant differences in patients with childhood cerebral ALD (CCALD). The replication study employing these five SNPs on an independent French ALD cohort, however, showed no significant associations with CCALD or pure AMN. This study showed that ABCD2, ABCD3, and ABCD4 are less likely the disease-modifying genes, necessitating further studies to identify genes modifying ALD phenotypes.

Fig. 1

Primer design for ABCD1. All the exons of ABCD1 were amplified using six primer pairs. There were pseudogenes at 2p11, 10p11, 16p11, and 22q11, which were similar in sequence to exons 7–10 of ABCD1 gene (92–96%). The forward primer for exons 8–10 was designed to avoid amplification of the related homologous genes. We could design a specific reverse primer for exons 8–10

Fig. 2

Scan data of the resequencing DNA microarray and sequence data of the direct nucleotide sequence analysis (upper panel: patient, lower panel: control). Each column shows a base position, and each row shows a base call DNA in the scan data of the resequencing DNA microarray. Here, a mutation (G277R) was detected, and the signal intensities around the mutation were reduced because of the mismatch of the mutation site. The sequence data of the direct nucleotide sequence analysis confirmed the scan data of the resequencing DNA microarray

Fig. 3

Identified mutations of ABCD1. Mutations of ABCD1 gene were widely scattered in the entire region of ABCD1 gene. All types of ABCD1 mutations were distributed among all the phenotypes of adrenoleukodystrophy. TM transmembrane domain, EAA-like EAA-like protein motif, Walker A Walker A motif, C sequence nucleotide binding fold conserved sequence, Walker B Walker B motif, fs frameshift

Fig. 4

Identified single nucleotide polymorphisms (SNPs) of ABCD2, ABCD3, and ABCD4 (upper panel). Comprehensive resequencing of ABCD2, ABCD3, and ABCD4 genes of the 40 patients with adrenoleukodystrophy (ALD) revealed two novel SNPs, nine SNPs (six known and three novel SNPs), and 13 SNPs (seven known and six novel SNPs), respectively. Red characters indicate the novel SNPs, blue characters indicate the SNPs identified in the coding region, and black characters indicate the SNPs identified in the noncoding region. Linkage disequilibrium (LD) map of SNPs of ABCD4 in Japanese patients with ALD and the controls using the Haploview version 4.1 (lower panel). The five known SNPs (rs17782508, rs2301345, rs4148077, rs4148078, and rs3742801) were in complete disequilibrium in Japanese patients with ALD and the controls (LOD = 43.97, r ² = 1.0, D′ = 1.0). Novel SNP7 and the five known SNPs (rs17782508, rs2301345, rs4148077, rs4148078, and rs3742801) were not in strong disequilibrium in Japanese patients with ALD and the controls (LOD = 1.15, r ² = 0.037, D′ = 0.706), although novel SNP7 and the five known SNPs (rs17782508, rs2301345, rs4148077, rs4148078, and rs3742801) were strong disequilibrium only in Japanese patients with ALD (LOD = 2.02, r ² = 0.221, D′ = 1.0). The number in the box indicates the data of D′. The color of the box is determined from the LOD score and D′. The block was determined using a confidence interval algorithm [33]