Introduction

Gaucher disease, the inherited deficiency of glucocerebrosidase (EC 3.2.1.45), manifests with vast phenotypic variation. Studies of genotype/phenotype correlation in this disorder increasingly implicate the contribution of genetic modifiers (Dipple and McCabe 2000; Koprivica et al. 2000; Park et al. 2003). The region of chromosome 1q21 encompassing the human glucocerebrosidase gene (GBA) is particularly gene rich, with seven genes and two pseudogenes located within 85 kb of sequence (Long et al. 1996; Winfield et al. 1997). The closest gene to GBA is metaxin 1 (MTX1), a convergently transcribed novel gene contiguous to the 3′ end of the GBA pseudogene (Fig. 1). Metaxin 1 has been localized to the outer mitochondrial membrane and is hypothesized to be part of the preprotein-import complex (Armstrong et al. 1997; Abdul et al. 2000). A pseudogene for metaxin 1 is located in the 16-kb region between GBA and its pseudogene. A mouse knockout for Mtx1 resulted in an embryonic lethal phenotype, suggesting that the gene is essential in development (Bornstein et al. 1995). Because of its close proximity and potentially interesting function, we explored whether the gene for metaxin 1 was altered in patients with Gaucher disease with diverse manifestations and whether it might be a genetic modifier in this disorder.

Fig. 1
figure 1

The glucocerebrosidase locus, showing the relative positions of the GBA N370S and MTX1 F202L mutations. Polymorphic loci involved in the N370S haplotype are indicated by a downward-pointing arrowhead

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Materials and methods

Genomic DNA was isolated from white blood cells, fibroblasts, or lymphoblasts obtained with informed consent from patients with Gaucher disease and from normal controls, under an Institute Review Board-approved NIH protocol. Mutations in the gene for glucocerebrosidase were identified as previously described (Koprivica et al. 2000).

Exonic and partial intronic sequences of MTX1 were amplified using PCR and sequenced using a BigDye dye terminator kit (Applied Biosystems) on an ABI 310 or 3100 automated capillary DNA sequencer. A 2.4-kb fragment comprising exons 4–8 was amplified using the forward primer 5′-GCATTGGGCGCTGGACCTTC-3′ and the reverse primer 5′-GCGTGAGCTTACAGCGACGTA-3′. Exon 7 was sequenced in both the forward and reverse direction with the forward primer 5′-GCCTGCTGCCCAGAGCCC-3′ and the reverse primer 5′-AACTAGTTCTTGGATCCCTG-3′.

Genotype results were analyzed to determine whether the data fit Hardy-Weinberg expected proportions and whether there was genetic disequilibrium between the GBA N370S and MTX1 F202L sites using the Genetic Data Analysis software package (Lewis and Zaykin 2001).

Results and discussion

Initial sequencing of the complete exonic sequence of MTX1 in eight patients identified a T→C alteration in six patients at cDNA position 628 in exon 7 (Long et al. 1996), resulting in the amino acid change F202L. It was noted that the MTX1 F202L change occurred only in those patients with a GBA N370S allele, and was homozygous in two N370S homozygotes. To determine the frequency of MTX1 F202L, DNA from 76 control individuals of either Ashkenazi or non-Ashkenazi backgrounds were screened using PCR amplification and direct sequencing of exon 7 of MTX1. Seven of 152 alleles (4.6%) were identified with MTX1 F202L (Table 1). None of the seven carried the GBA N370S mutation. Screening of genomic DNA from 90 patients with Gaucher disease (including the original eight) showed that in 25 N370S homozygotes, the MTX1 F202L was coallelic in 49 of 50 alleles (98.0%) (Table 1). Moreover, in 53 N370S heterozygotes, MTX1 F202L was present on 51 of 106 alleles (48.0%). Although it was not determined if the MTX1 F202L was on the same chromosome as the GBA N370S in the heterozygotes, the relative frequency is consistent with the two mutations being co-allelic. Twelve additional patients without N370S all lacked MTX1 F202L. Clinical records for the four patients carrying GBA N370S alleles without MTX1 F202L were reviewed. All four were females with mild to moderately severe manifestations of type 1 Gaucher disease, but without atypical symptoms or disease progression.

Table 1 Frequency of the MTX1 F202L mutation in individuals with Gaucher disease and in normal controls. wt Wild type
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Tests for genetic disequilibrium between the N370S and F202L loci using unphased genotype data demonstrated strong deviations from random assortment with a P-value<0.00000. The disequilibrium was not the result of Hardy-Weinberg deviations.

Twelve polymorphic sites in glucocerebrosidase have been reported, although only two major haplotypes, Pvl.1 + and Pv1.1 −, have been described in Caucasians (Beutler et al. 1992). Linkage disequilibrium between mutation N370S and the Pvl.1 − haplotype and two other two polymorphic repeat sequences in the glucocerebrosidase gene region, 5GC 3.2 (CT)40 and ITCG.2 (AAAT)9, have been reported (Zimran et al. 1990; Lau et al. 1999). In the 26 patients in the present study for whom polymorphism data at the 5GC 3.2, Pv1.1, and ITCG.2 loci were available (Lau et al. 1999), 11 who were homozygous for the reported N370S haplotype were also homozygous for MTX1 F202L, and 13 N370S haplotype heterozygotes also carried an F202L allele. Two patients with N370S alleles had haplotype variations. One, an N370S homozygote, lacked the F202L on one N370S allele and was also heterozygous at the ITCG.2 polymorphic site. The other, an N370S heterozygote, carried the F202L alteration on both alleles but was homozygous for a variant form of the 5GC 3.2 polymorphism. Previous studies report that the N370S mutation has a conserved haplotype on 1q21 in the Ashkenazi Jewish population, supporting the origin of this mutation from a single founder (Diaz et al. 2000). The finding that the majority of N370S alleles are also associated with MTX1 F202L suggests that this alteration may have occurred on the same ancestral allele. Of note, neither the N370S nor the F202L mutations are found in their respective pseudogenes.

Although F202L in metaxin 1 is a polymorphism, there could be possible functional consequences of this finding. All patients carrying the Gaucher N370S mutation are known to have type 1, non-neuronopathic Gaucher disease (Beutler and Grabowski 2001). The mechanisms for the neuroprotective effect of N370S are not altogether clear, since the factors determining whether neuronopathic Gaucher disease will develop are not fully understood. Moreover, attempts to generate a point-mutation mouse model with mutation N370S have unexpectedly led to a neonatal lethal phenotype (Zhao and Grabowski 2002). Murine Mtx1 does not contain the F202L alteration and also lacks the glucocerebrosidase and metaxin pseudogenes found in humans. One intriguing possibility is that the presence of MTX1 F202L or interactions with the adjacent pseudogenes might somehow modify the burden of disease in patients carrying the N370S Gaucher mutation. A mouse model homozygous for both the Gba N370S and Mtx1 F202L mutations may elucidate interactions between the contiguous genes and may determine whether metaxin has a role as a modifier of Gaucher disease.