Recombinant Alleles Research Articles

Unusual Rearrangement of the α-Globin Gene Cluster Containing Both the −α3.7 and αααanti-4.2 Crossover Junctions: Clinical Diagnostic Implications and Possible Mechanisms

Misalignment of the homologous regions of the α-globin gene cluster and unequal crossover during meiosis produce single α-globin gene deletions (−α) and reciprocal α-globin gene triplications (ααα). Further unequal crossover of such recombinant alleles with wild-type alleles may produce more complex derivative alleles, such as quadruplicated alleles (1)(2)(3). Complex crossover events producing “patchwork” genes have also been reported at the human α- and β-globin gene cluster (4)(5)(6). In this report, we describe the identification of a novel rearrangement of the α-globin gene cluster containing both the −α3.7 and αααanti-4.2 crossover junctions. This allele was identified in 2 unrelated individuals and a parent in the course of screening by Southern analysis of patients with β-thalassemia major and minor for α-globin gene deletions (Table 1⇓ ). For patient 1, a routine α-globin gene configuration Southern analysis was performed to screen for the presence of the −\-SEA α-thalassemia deletion, a common amelioration factor of severe β-thalassemia (7). In the case of patient 2, Southern analysis was performed to rule out the presence of the −\-SEA α-thalassemia deletion, because hemoglobin H inclusion bodies typically present in α-thalassemia are absent when there is concurrent β-thalassemia (8). Southern analysis was performed by hybridizing [32P]dATP-labeled α- or ζ-globin gene probes to Bam HI- or Bgl II-digested genomic DNA. With an α-globin probe, an αααanti-4.2 triplication contributes an 18.2-kb hybridizing Bam HI band and 16.8- and 7.4-kb Bgl II bands, whereas a −α3.7 deletion contributes a 10.3-kb Bam HI band and a 16.3-kb Bgl II band. With a ζ-globin probe, both the αααanti-4.2 and −α3.7 alleles contribute 5.9-kb and 10.8/11.3-kb Bam HI bands, whereas αααanti-4.2 contributes 11.3/12.6-kb and 16.8-kb Bgl II bands, and −α3.7 contributes 11.3/12.6-kb …

Mutant Analysis Reveals a Specific Requirement for Protein P30 inMycoplasma pneumoniaeGliding Motility

The cell-wall-less prokaryote Mycoplasma pneumoniae, long considered among the smallest and simplest cells capable of self-replication, has a distinct cellular polarity characterized by the presence of a differentiated terminal organelle which functions in adherence to human respiratory epithelium, gliding motility, and cell division. Characterization of hemadsorption (HA)-negative mutants has resulted in identification of several terminal organelle proteins, including P30, the loss of which results in developmental defects and decreased adherence to host cells, but their impact on M. pneumoniae gliding has not been investigated. Here we examined the contribution of P30 to gliding motility on the basis of satellite growth and cell gliding velocity and frequency. M. pneumoniae HA mutant II-3 lacking P30 was nonmotile, but HA mutant II-7 producing a truncated P30 was motile, albeit at a velocity 50-fold less than that of the wild type. HA-positive revertant II-3R producing an altered P30 was unexpectedly not fully wild type with respect to gliding. Complementation of mutant II-3 with recombinant wild-type and mutant alleles confirmed the correlation between gliding defect and loss or alteration in P30. Surprisingly, fusion of yellow fluorescent protein to the C terminus of P30 had little impact on cell gliding velocity and significantly enhanced HA. Finally, while quantitative examination of HA revealed clear distinctions among these mutant strains, gliding defects did not correlate strictly with the HA phenotype, and all strains attached to glass at wild-type levels. Taken together, these findings suggest a role for P30 in gliding motility that is distinct from its requirement in adherence.

Na,K-ATPase from Mice Lacking the γ Subunit (FXYD2) Exhibits Altered Na+ Affinity and Decreased Thermal Stability

The gamma subunit of the Na,K-ATPase, a 7-kDa single-span membrane protein, is a member of the FXYD gene family. Several FXYD proteins have been shown to bind to Na,K-ATPase and modulate its properties, and each FXYD protein appears to alter enzyme kinetics differently. Different results have sometimes been obtained with different experimental systems, however. To test for effects of gamma in a native tissue environment, mice lacking a functional gamma subunit gene (Fxyd2) were generated. These mice were viable and without observable pathology. Prior work in the mouse embryo showed that gamma is expressed at the blastocyst stage. However, there was no delay in blastocele formation, and the expected Mendelian ratios of offspring were obtained even with Fxyd2-/- dams. In adult Fxyd2-/- mouse kidney, splice variants of gamma that have different nephron segment-specific expression patterns were absent. Purified gamma-deficient renal Na,K-ATPase displayed higher apparent affinity for Na+ without significant change in apparent affinity for K+. Affinity for ATP, which was expected to be decreased, was instead slightly increased. The results suggest that regulation of Na+ sensitivity is a major functional role for this protein, whereas regulation of ATP affinity may be context-specific. Most importantly, this implies that gamma and other FXYD proteins have their effects by local and not global conformation change. Na,K-ATPase lacking the gamma subunit had increased thermal lability. Combined with other evidence that gamma participates in an early step of thermal denaturation, this indicates that FXYD proteins may play an important structural role in the enzyme complex.

Inducible Nitric-oxide Synthase and NO Donor Induce Insulin Receptor Substrate-1 Degradation in Skeletal Muscle Cells

Chronic inflammation plays an important role in insulin resistance. Inducible nitric-oxide synthase (iNOS), a mediator of inflammation, has been implicated in many human diseases including insulin resistance. However, the molecular mechanisms by which iNOS mediates insulin resistance remain largely unknown. Here we demonstrate that exposure to NO donor or iNOS transfection reduced insulin receptor substrate (IRS)-1 protein expression without altering the mRNA level in cultured skeletal muscle cells. NO donor increased IRS-1 ubiquitination, and proteasome inhibitors blocked NO donor-induced reduction in IRS-1 expression in cultured skeletal muscle cells. The effect of NO donor on IRS-1 expression was cGMP-independent and accentuated by concomitant oxidative stress, suggesting an involvement of nitrosative stress. Inhibitors for phosphatidylinositol-3 kinase, mammalian target of rapamycin, and c-Jun amino-terminal kinase failed to block NO donor-induced IRS-1 reduction, whereas these inhibitors prevented insulin-stimulated IRS-1 decrease. Moreover iNOS expression was increased in skeletal muscle of diabetic (ob/ob) mice compared with lean wild-type mice. iNOS gene disruption or treatment with iNOS inhibitor ameliorated depressed IRS-1 expression in skeletal muscle of diabetic (ob/ob) mice. These findings indicate that iNOS reduces IRS-1 expression in skeletal muscle via proteasome-mediated degradation and thereby may contribute to obesity-related insulin resistance.

Rapid Evolution of the MH Class I Locus Results in Different Allelic Compositions in Recently Diverged Populations of Atlantic Salmon

We compared major histocompatibility class I allelic diversity in two currently reproductively isolated Atlantic salmon (Salmo salar) populations (Irish and Norwegian) with a common postglacial origin in order to test for among-population differences in allelic composition and patterns of recombination and point mutation. We also examined the evidence for adaptive molecular divergence at this locus by analyzing the rate of amino acid replacement in relation to a neutral expectation. Contrary to our prediction, and in contrast to the situation for other genetic markers, the two populations have almost nonoverlapping sets of major histocompatibility class I alleles. Although there is a strong signal of point mutation that predates population divergence, recent recombination, acting in similar, but not identical, ways in both populations appears to be a significant force in creating new alleles. Moreover, selection acting on peptide-binding residues seems to favor new recombinant alleles and is likely to be responsible for the rapid divergence between populations.

Identification and Complementation of a Mutation Associated with Loss of Mycoplasma pneumoniae Virulence-Specific Proteins B and C

A mutation in gene MPN142 (orf6) was identified in the Mycoplasma pneumoniae cytadherence mutant III-4. MPN142 encodes virulence-specific proteins P90 and P40 (proteins B and C, respectively). Analysis of MPN142 in a cytadhering revertant and complementation using a recombinant wild-type allele confirmed the role of this mutation in the cytadherence defect.

PERINATAL LETHAL GAUCHER DISEASE: A DISTINCT PHENOTYPE ALONG THE NEURONOPATHIC CONTINUUM

Gaucher disease, the inherited deficiency of glucocerebrosidase, is characterized by significant genetic and phenotypic heterogeneity. At the extreme end of the phenotypic continuum is the perinatal lethal variant, typically presenting in utero or during the neonatal period as hydrops and/orcongenital ichthyosis, with severe and progressive neurological involvement. Insights from the null-allele Gaucher mouse model contributed to the identification of this distinct phenotype, which has unique epidermal involvement. While multiple mutations are encountered, many affected infants are homozygous for recombinant alleles. The diagnosis is often missed due to the early lethality and the failure to recognize the association between lysosomal disorders and hydrops fetalis. The incidence of severe perinatal Gaucher disease may prove more common than currently appreciated with greater physician awareness of the disorder.

The silent KIR3DP1 gene (CD158c) is transcribed and might encode a secreted receptor in a minority of humans, in whom the KIR3DP1, KIR2DL4 and KIR3DL1/KIR3DS1 genes are duplicated

Killer-cell Ig-like receptors (KIR) are structurally and functionally diverse, and enable human NK cells to survey the expression of individual HLA class I molecules, often altered in infections and tumors. Multiple events of non-reciprocal recombination have contributed to the rapid diversification of KIR. We show that approximately 4.5% of the individuals of a Caucasoid population bear a recombinant allele of KIR3DP1, officially designed KIR3DP1*004, that associates tightly with gene duplications of KIR3DP1, KIR2DL4 and KIR3DL1/KIR3DS1. The KIR3DP1 gene is normally silent, but the recombinant allele carries a novel promoter sequence and, as a consequence, is transcribed in all tested individuals. Messenger RNA of KIR3DP1*004 is made up of six exons; of these, exons 1-5 are similar to, and spliced like, those encoding the leader peptide and Ig-domains of KIR3D. By contrast, exon 6 is homologous to no other human KIR sequence, but only to possible homologs in chimpanzees and rhesus macaques, and encodes a short hydrophilic tail. The putative KIR3DP1*004 product, like those of the related genes LAIR-2 and LILRA3/ILT6/LIR4, is predicted to be secreted to the extracellular medium rather than anchored to the cell membrane.

Genetic characterisation of Helicobacter pylori isolates from an Argentinean adult population based on cag pathogenicity island right-end motifs, lspA-glmM polymorphism and iceA and vacA genotypes

Isolates of Helicobacter pylori from 88 patients were characterised by cagA status, cagA pathogenicity island (PAI) right-end motifs, iceA, vacA and lspA-glmM genotypes, primarily by PCR-based analysis, to investigate whether Argentinean isolates differed from those recovered in southern Europe or other Latin American countries. PCR-based analysis of vacA alleles was confirmed by reverse hybridisation in 56 cases, while sequence analysis was performed either when iceA and vacA genotypes could not be determined by PCR, or to investigate PCR and reverse hybridisation vacA genotyping discordance. Typing by lspA-glmM restriction fragment length polymorphism was performed with HhaI and AluI. The pattern of cag PAI right-end motifs and the prevalence of type Ia were similar to those in isolates from southern European countries, with cagA(+)/iceA1/vacA-s1 m1 being the commonest genotype. Reverse hybridisation identified a vacA-s1a/s1b recombinant allele, confirmed by sequencing analysis. Analysis of lspA-glmM genotypes identified at least 73 unrelated strains. Few mixed infections were identified, but in one case, isolates from a single biopsy exhibiting two vacA alleles were shown by lspA-glmM fingerprints to be two unrelated strains. No associated effect on ulcer disease risk was demonstrated by analysis of cagA, vacA and iceA status. Overall, the isolates of H. pylori from Argentina were similar to isolates from southern Europe or Latin American countries, and infections were associated mainly with single H. pylori strains.

Production of Functionally Active Recombinant HLA Molecules Representing the Supertypes B62 and B58 and the Generation of Corresponding Peptide‐Binding Assays and Tetramers

Rationale: Major histocompatibility complex class I (MHC I) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic CD8+ T cells. The goal of the human MHC project is to predict the binding strength of any given peptide/MHC complex. This prediction allows the design of peptide‐based vaccines. The prediction requires representative binding data from MHC alleles from all the nine HLA supertypes. Here, we describe the genetic construction, protein production and purification as well as the establishment‐binding assays for two recombinant MHC supertype alleles, HLA‐B*1501 and HLA‐B*5801. Methods: Using the Quikchange Multisite Directed Mutagenesis Kit (Stratagene), codon‐optimized genes encoding HLA‐B*1501 and HLA‐B*5801 are created. The two MHC I molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. The binding (KD) of natural T‐cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (RIAs) and enzyme‐linked immunosorbent assays (ELISAs). The MHC molecules are biotinylated during expression. Results: The expression of MHC I resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of KD for various peptide ligands ranging from strong binders (KD < 50 nM) to low binders (KD > 5 µM). Tetramerization is visualized by SDS‐PAGE. Conclusion: An effective method for the production of highly pure MHC I molecules has been applied to HLA‐B*1501 and HLA‐B*5801, and RIA and ELISA binding assays for those alleles have been established, showing the binding of various peptide ligands to the MHC I molecules.

RyR2 and Calpain-10 Delineate a Novel Apoptosis Pathway in Pancreatic Islets

Cells are programmed to die when critical signaling and metabolic pathways are disrupted. Inhibiting the type 2 ryanodine receptor (RyR2) in human and mouse pancreatic beta-cells markedly increased apoptosis. This mode of programmed cell death was not associated with robust caspase-3 activation prompting a search for an alternative mechanism. Increased calpain activity and calpain gene expression suggested a role for a calpain-dependent death pathway. Using a combination of pharmacological and genetic approaches, we demonstrated that the calpain-10 isoform mediated ryanodine-induced apoptosis. Apoptosis induced by the fatty acid palmitate and by low glucose also required calpain-10. Ryanodine-induced calpain activation and apoptosis were reversed by glucagon-like peptide or short-term exposure to high glucose. Thus RyR2 activity seems to play an essential role in beta-cell survival in vitro by suppressing a death pathway mediated by calpain-10, a type 2 diabetes susceptibility gene with previously unknown function.

Evolution of the O alleles of the human ABO blood group gene.

To date, at least 40 different alleles O have been characterized on the basis of exon 6 and exon 7 sequences but not always for intron 6. Among 415 individuals, from four continents (Africa, Europe, South America, and Asia), studied for exon 6 and exon 7 sequences, we selected 46 individuals (of respective phenotypes O [39], AB [3], B [3], or A [1]) for sequencing 1800-bp amplicons spanning exon 6, intron 6, and exon 7. The amplicons were characterized either by direct sequencing or after cloning when required. We defined 14 new intron 6 O allele sequences, including four recombinant alleles. Based on sequence comparison, a phylogenetic network was constructed. It confirmed recombinant allele origins and that most O alleles are derived by point mutations from the two worldwide distributed alleles O01 and O02. Allele O phylogenetic analysis suggests that the most frequent silencing mutation (deletion of a G in exon 6) appeared once in human evolution in the ancient O02 allele lineage and that allele O01 resulted from an interallele exchange between O02 and A101. Assuming constancy of evolutionary rate, diversification of the representative alleles of the three human ABO lineages (A101, B101, and O02) was estimated at 4.5 to 6 million years ago.

Identification and functional characterization of five novel mutant alleles in 58 Italian patients with Gaucher disease type 1

Gaucher disease (GD) is the most frequent lysosomal glycolipid storage disorder due to an autosomal recessive deficiency of acid beta-glucosidase characterized by the accumulation of glucocerebroside. In this work we carried out the molecular analysis of the glucocerebrosidase gene (GBA) in 58 unrelated patients with GD type 1. We identified five novel genetic alterations: three missense changes c.187G>A (p.D63N), c.473T>G (p.I158S), c.689T>A (p.V230E), a gene-pseudogene recombinant allele and a non-pseudogene-derived complex allele [c.1379G>A;c.1469A>G] encoding [p.G460D;p.H490R]. All mutant alleles were present as compound heterozygotes in association with c.1226A>G (p.N409S), the most common mutation in GD1. The missense mutant proteins were expressed in vitro in COS-1 cells and analyzed by enzyme activity, protein processing and intracellular localization. Functional studies also included the c.662C>T (p.P221L) mutation recently reported in the Spanish GD population (Montfort et al., 2004). The missense mutant alleles retained an extremely low residual enzyme activity with respect to wild type; the complex allele expressed no activity. Processing of the mutant proteins was unaltered except for c.473T>G which was differently glycosylated due to the exposition of an additional glycosylation site. Immunofluorescence studies showed that protein trafficking into the lysosomes was unaffected in all cases. Finally, the characterization of the novel recombinant allele identified a crossover involving the GBA gene and pseudogene between intron 5 and exon 7.

Adeno-associated viral vector-mediated ApoE expression in Alzheimer's disease mice: low CNS immune response, long-term expression, and astrocyte specificity.

Recombinant Adenovirus and Adeno-associated virus (AAV) are highly effective vehicles for gene transfer into CNS cells. However, the duration of gene expression and the cytotoxicity to cells are quite different between these viral approaches. We initially investigated these distinctions by stereotaxically injecting both Adenovirus vector and AAV vectors expressing reporter genes into mouse hippocampus. The adenovirus vector induced a pronounced immune response with a marked increase in CD45 and MHC class I protein expression and transgene expression was shorter than six weeks. In contrast, with the AAV vector there was lower expression of CD45 and MHC class I immune activation markers, and longer expression of reporter gene (up to 12 months). To study the roles of human Apolipoprotein E (ApoE) alleles in the pathogenesis of Alzheimer's disease and other CNS diseases, we generated recombinant AAV-apoE alleles driven by the GFAP promoter and expressed them in the mouse brain of Alzheimer's disease mouse. High level ApoE expressions in mouse brain lasted for 12 months, and ApoE was specifically expressed in astrocytes. We demonstrate that AAV-GFAP-ApoE is valuable in studying the pathogenesis and in gene therapy for Alzheimer's disease and other CNS diseases.

Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants.

Homologous recombination can be used to generate recombinants on episomes or directly on the Escherichia coli chromosome with PCR products or synthetic single-stranded DNA (ssDNA) oligonucleotides (oligos). Such recombination is possible because bacteriophage lambda-encoded functions, called Red, efficiently recombine linear DNA with homologies as short as 20-70 bases. This technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homologous recombination mechanisms. The Red Beta function, which binds and anneals ssDNA to complementary ssDNA, is able to recombine 70-base oligos with the chromosome. In E. coli, methyl-directed mismatch repair (MMR) can affect these ssDNA recombination events by eliminating the recombinant allele and restoring the original sequence. In so doing, MMR can reduce the apparent recombination frequency by >100-fold. In the absence of MMR, Red-mediated oligo recombination can incorporate a single base change into the chromosome in an unprecedented 25% of cells surviving electroporation. Our results show that Beta is the only bacteriophage function required for this level of recombination and suggest that Beta directs the ssDNA to the replication fork as it passes the target sequence.

Molecular genetic characterization of the distal NKC recombination hotspot and putative murine CMV resistance control locus.

The NK gene complex (NKC) controls murine cytomegalovirus (MCMV) immunity through Cmv1-dependent natural killer (NK) cell responses. Ly49H expression correlates with Cmv1 phenotypes in different inbred strains, is required for MCMV resistance in C57BL/6 (B6) mice, and its interaction with the MCMV encoded m157 protein leads to NK cell-mediated destruction of virus-infected cells. However, genetic mapping studies have previously indicated that Cmv1 should reside in the D6Wum9-16 NKC interval, distal to Ly49h. Since these data suggested that multiple NKC-linked loci could regulate viral immunity, a putative MCMV resistance control ( Mrc) locus was pinpointed to within the D6Wum9-16 interval on a NKC-aligned bacterial artificial chromosome (BAC). Sequence analysis of BAC 151 revealed several novel G-protein coupled receptor genes, an HMG-1 remnant and many additional polymorphic microsatellites that were useful in determining the minimal genetic interval for the Mrc locus. Moreover, comparison of B6, BALB/c, A/J and recombinant Mrc alleles restricted the genetic interval to approximately 470 bp and showed that it was also a hotspot for recombination. MCMV challenge of novel NKC recombinant mice demonstrated that Mrc(B6) was not required for MCMV resistance nor could it directly complement the Ly49(BALB) haplotype to rescue MCMV susceptibility. Taken together, these data show that while Mrc apparently guides recombination, Ly49H expression is sufficient for MCMV resistance in B6 mice. A direct role for Mrc(B6) in virus resistance is excluded in the novel mice.

Killer‐cell immunoglobulin‐like receptor (KIR) nomenclature report, 2002

Killer‐cell immunoglobulin‐like receptor (KIR) nomenclature report, 2002

Reciprocal and Nonreciprocal Recombination at the Glucocerebrosidase Gene Region: Implications for Complexity in Gaucher Disease

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.

Myoclonic epilepsy in Gaucher disease: genotype-phenotype insights from a rare patient subgroup.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide spectrum of manifestations. Although Gaucher disease has been divided into three clinical types, patients with atypical presentations continue to be recognized. A careful phenotypic and genotypic assessment of patients with unusual symptoms may help define factors that modify phenotype in this disorder. One such example is a rare subgroup of patients with type 3 Gaucher disease who develop progressive myoclonic epilepsy. We evaluated 16 patients with myoclonic epilepsy, nine of whom were diagnosed by age 4 y with severe visceral involvement and myoclonus, and seven with a more chronic course, who were studied between ages 22 and 40. All of the patients had abnormal horizontal saccadic eye movements. Fourteen different genotypes were encountered, yet there were several shared alleles, including V394L (seen on two alleles), G377S (seen on three alleles), and L444P, N188S, and recombinant alleles (each found on four alleles). V394L, G377S, and N188S are mutations that have previously been associated with non-neuronopathic Gaucher disease. The spectrum of genotypes differed significantly from other patients with type 3 Gaucher disease, where genotypes L444P/L444P and R463C/null allele predominated. Northern blot studies revealed a normal glucocerebrosidase transcript, whereas Western studies showed that the patients studied lacked the processed 56 kD isoform of the enzyme, consistent with neuronopathic Gaucher disease. Brain autopsy samples from two patients demonstrated elevated levels of glucosylsphingosine, a toxic glycolipid, which could contribute to the development of myoclonus. Thus, although there were certain shared mutant alleles found in these patients, both the lack of a shared genotype and the variability in clinical presentations suggest that other modifiers must contribute to this rare phenotype.

Gene rearrangements in the glucocerebrosidase-metaxin region giving rise to disease-causing mutations and polymorphisms. Analysis of 25 Rec NciI alleles in Gaucher disease patients.

The glucocerebrosidase and metaxin genes lie in a gene-rich region that also includes two corresponding pseudogenes. This gives rise to recombinant alleles. We analysed two groups of patients from Argentina and Spain: 25 bearing the Rec NciI allele and 36 carrying L444P. The mutational mechanism is described and the crossover site precisely defined. Most of the Rec NciI alleles were generated by gene conversion. Rearranged alleles involving the metaxin gene were also identified. The high frequency of Rec NciI alleles associated with a polymorphic rearrangement at the metaxin level is probably due to a founder effect.