Reciprocal and nonreciprocal recombination at the glucocerebrosidase gene region: implications for complexity in Gaucher disease

Abstract

Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocerebrosidase. The glucocerebrosidase gene is located in a gene-rich region of 1q21 that contains six genes and two pseudogenes within 75 kb. The presence of contiguous, highly homologous pseudogenes for both glucocerebrosidase and metaxin at the locus increases the likelihood of DNA rearrangements in this region. These recombinations can complicate genotyping in patients with Gaucher disease and contribute to the difficulty in interpreting genotype-phenotype correlations in this disorder. In the present study, DNA samples from 240 patients with Gaucher disease were examined using several complementary approaches to identify and characterize recombinant alleles, including direct sequencing, long-template polymerase chain reaction, polymorphic microsatellite repeats, and Southern blots. Among the 480 alleles studied, 59 recombinant alleles were identified, including 34 gene conversions, 18 fusions, and 7 downstream duplications. Twenty-two percent of the patients evaluated had at least one recombinant allele. Twenty-six recombinant alleles were found among 310 alleles from patients with type 1 disease, 18 among 74 alleles from patients with type 2 disease, and 15 among 96 alleles from patients with type 3 disease. Several patients carried two recombinations or mutations on the same allele. Generally, alleles resulting from nonreciprocal recombination (gene conversion) could be distinguished from those arising by reciprocal recombination (crossover and exchange), and the length of the converted sequence was determined. Homozygosity for a recombinant allele was associated with early lethality. Ten different sites of crossover and a shared pentamer motif sequence (CACCA) that could be a hotspot for recombination were identified. These findings contribute to a better understanding of genotype-phenotype relationships in Gaucher disease and may provide insights into the mechanisms of DNA rearrangement in other disorders.

Figure 1

Recombination initiation sites and allele frequencies. The initiation regions for recombination events were determined by comparing mismatches between the GBA functional gene and pseudogene sequences. The region where a recombination starts is defined as the sequence between the last mismatch containing functional gene sequence and the first mismatch carrying pseudogene sequence. The schematics show the exonic structure of GBA, with the different initiation regions indicated in gray, with arrows below. The number of alleles found in patients with type 1, 2, or 3 Gaucher disease with either reciprocal or nonreciprocal recombinations starting within the given region are shown next to each schematic map. The abbreviations for each initiation region are used in tables 1, 2, and 3. a, Int3; the recombination starts within 26 bp in intron 3. b, Int4; the recombination starts within 98 bp in intron 4. c, Int8-Ex9; the recombination starts within 288 bp encompassing the intron 8/exon 9 junction. d, Ex9; the recombination starts within 23 bp of exon 9 following the 55 bp that are deleted in pseudogene. e, Ex9-Int9; the recombination starts within 186 bp encompassing the exon 9/intron 9 junction. f, Int9; the recombination starts within 30 bp at the end of intron 9. g, Int9-Ex10; the recombination starts within 126 bp encompassing the intron 9/exon 10 junction. h, Int10-Ex11; the recombination starts within 300 bp encompassing the intron 10/exon 11 junction. i, 3′ UTR; the recombination starts within 304 bp in the 3′ UTR. j, MTX (not shown); the recombination starts within the MTX pseudogene.

Figure 2

Southern blots of the GBA gene and pseudogene. Genomic DNAs were digested using four different restriction enzymes and were hybridized to a human GBA cDNA probe. A representative selection of patients with different recombinant alleles is shown in each panel. Included are schematic maps showing the normal restriction sites. Samples in each lane are identified by the table, patient number, and the recombinant allele they demonstrate (i.e., T2-2, Rec 7b is table 2, patient 2 with Rec 7b). The approximate sizes of the abnormal bands are shown to the right of each blot. A, SstII-digested DNA samples: lane 1, normal control demonstrating a single 46-kb band; lane 2, T3-2, Rec 7b; lane 3, T3-20, Rec 7b; lane 4, T3-13, Rec 4b; and lane 5, T3-14, Rec 1b. B, SspI-digested DNA samples: lane 1, normal control with 17- and 12-kb bands; lane 2, T3-11, Rec 1b; lane 3, T3-1, Rec 2b; lane 4, T3-20, Rec 7b; lane 5, T2-20, c.1263-1317del-a; and lane 6, T3-17, Rec 5b. C, EcoRI-digested DNA samples: lane 1, normal control with 14- and 12.7-kb bands; lane 2, T3-24, Rec 7b; lane 3, T3-12, Rec 6b; lane 4, T3-13, Rec 4b; and lane 5, T3-14, Rec1b. D, HincII-digested DNA samples: lanes 1, 6, and 8, normal controls showing 21-, 18-, 5.2-, 2.7-, and 0.9-kb bands; lane 2, T3-16, Rec7b; lane 3, T3-11, Rec 1b; lane 4, T2-5, Rec 1a; lane 5, T3-13, Rec 4a; lane 7, T2-20, c.1263-1317del-a; lane 9, T3-2, Rec 7b; and lane 10, T3-5, Rec 6b.

Figure 3

Illustrations of proposed mechanisms for recombination events occurring between the GBA gene and its pseudogene. A, Allelic gene conversion. This is a nonreciprocal sequence exchange, facilitated by sequence homology between allelic genes. The donor sequence is unaltered, but the acceptor sequence is changed by the incorporation of regions copied from the donor sequence. B, Reciprocal crossover between homologous regions. This event results in two possible gene rearrangements. One is a fusion between the gene and its pseudogene and a deletion of the intergenic region shown in (1). The second is a partial duplication of the pseudogene and gene sequences, which are fused together (2). C, Holliday model. This is a mechanism of recombination that includes nicking and reunion between two homologous sequences. Nicking occurs in the same location in both homologous sequences (i.e., in the GBA gene and pseudogene). Crossover, exchanges, and sealing nicks create chi structures. Branch migration and breaking in some base pairs in the four strands increase the chance of bending, rotation, and exchange between strands, resulting in two possible gene conversion–like alleles (1) and two possible fusion-like alleles (2). Figure adapted from Weaver and Hedrick (1989). D, Intramolecular crossover. This is another proposed model for recombination between the GBA gene and pseudogene on the same chromosome, resulting in a fusion allele. In this case, the intergenic region and portions of the gene and pseudogene are removed as an extrachromosomal fragment and are lost.