Gene rearrangement on 1q21 introducing a duplication of the glucocerebrosidase pseudogene and a metaxin fusion gene

The genes for glucocerebrosidase and metaxin, both located on chromosome 1q21, each have a highly homologous pseudogene sequence nearby. We describe a novel recombinant allele consisting of a duplication of the glucocerebrosidase pseudogene and a fusion between the metaxin gene and its pseudogene, resulting from a crossover between metaxin and pseudometaxin in the region downstream of the glucocerebrosidase gene. We also show that certain individuals have a metaxin-pseudometaxin fusion gene without a duplication, resulting from the same crossover. DNA from patients with Gaucher disease and normal controls were screened for recombinant alleles by Southern blot analyses prepared with the restriction enzymes SspI and HincII and by direct sequencing. Downstream alterations were identified in eight of the 398 patient alleles studied and in seven of the 200 normal control alleles examined, and were encountered more frequently among patients and controls of African-American ancestry. This is the first recognition of a duplicated allele in the glucocerebrosidase gene region, and its presence may contribute to genotype-phenotype studies in Gaucher disease.     

Infection-associated FUT2 (Fucosyltransferase 2) genetic variation and impact on functionality assessed by in vivo studies

The secretor (Se)/nonsecretor (se) histo-blood group variation depends on the action of the FUT2 enzyme and has major implications for human susceptibility to infections. To characterize the functionality of FUT2 variants, we assessed the correlation between saliva phenotypes and sequence variation at the FUT2 gene in sixty seven individuals from northern Portugal. While most non-secretor haplotypes were found to carry the 428G > A nonsense mutation in association with a 739G > A missense substitution, we have also identified a recombinant haplotype carrying the 739*A allele together with the efficient 428*G variant in individuals with the Se phenotype. This finding suggested, in contrast to previous results, that the 739*A allele encodes an efficient Se allele. To test this hypothesis we evaluated the in vivo enzyme activity of full coding expression constructs in transient transfection of CHO-K1 cells using FACS (fluorescence-activated cell sorting) analysis and expression of type 2 and type 3 chain H structures as read out. We detected FUT2 activity for the 739*A expression construct, demonstrating that the 739G > A substitution is indeed not inactivating. In accordance with the hypothesis that FUT2 is under long standing balancing selection, we estimated that the time depth of FUT2 global genetic variation is as old as 3 million years. Age estimates of specific variants suggest that the 428G > A mutation occurred at least 1.87 million years ago while the 739G > A substitution is about 816,000 years old. The 385A > T missense mutation underlying the non-secretor phenotype in East Asians appears to be more recent and is likely to have occurred about 256,000 years ago.     

Evolution of alpha 2-fucosyltransferase genes in primates: relation between an intronic Alu-Y element and red cell expression of ABH antigens

Coding sequences of the paralogous FUT1 (H), FUT2 (Se), and Sec1 alpha 2-fucosyltransferase genes were obtained from different primate species. Analysis of the primate FUT1-like and FUT2-like sequences revealed the absence of the known human inactivating mutations giving rise to the h null alleles of FUT1 and the se null alleles of FUT2. Therefore, most primate FUT1-like and FUT2-like genes potentially code for functional enzymes. The Sec1-like gene encodes for a potentially functional alpha 2-fucosyltransferase enzyme in nonprimate mammals, New World monkeys, and Old World monkeys, but it has been inactivated by a nonsense mutation at codon 325 in the ancestor of humans and African apes (gorillas, chimpanzees). Human and gorilla Sec1's have, in addition, two deletions and one insertion, respectively, 5' of the nonsense mutation leading to proteins shorter than chimpanzee Sec1. Phylogenetic analysis of the available H, Se, and Sec1 mammalian protein sequences demonstrates the existence of three clusters which correspond to the three genes. This suggests that the differentiation of the three genes is rather old and predates the great mammalian radiation. The phylogenetic analysis also suggests that Sec1 has a higher evolutionary rate than FUT2 and FUT1. Finally, we show that an Alu-Y element was inserted in intron 1 of the FUT1 ancestor of humans and apes (chimpanzees, gorillas, orangutans, and gibbons); this Alu-Y element has not been found in monkeys or nonprimate mammals, which lack ABH antigens on red cells. A potential mechanism leading to the red cell expression of the H enzyme in primates, related to the insertion of this Alu-Y sequence, is proposed.     

Extensive polymorphism of the FUT2 gene in an African (Xhosa) population of South Africa

The human secretor type α(1,2)fucosyltrans-ferase gene (FUT2) polymorphism was investigated in Xhosa and Caucasian populations of South Africa by polymerase chain reaction–restriction fragment length polymorphism and DNA sequencing. Six new base substitutions were found in the coding region of FUT2. A single base (C) deletion at nucleotide 778, which led to a frame shift and produced a stop codon at codon 275, was responsible for the enzyme inactivation. Three nonsynonymous base substitutions, A40G (Ile¹⁴Val), C379T (Arg¹²⁷Cys), and G481A (Asp¹⁶¹Asn), and two synonymous base substitutions, A375G (Glu¹²⁵) and C480T (His¹⁶⁰), were also identified in functional alleles. As a result, seven new alleles, Se ⁴⁰ , Se ⁴⁸¹ , Se ^40,481 , Se ^357,480 , Se ^357,379,480 , Se ³⁷⁵ , and se ^357,480,778 were identified. Population studies revealed that an allele containing a nonsense mutation G428A (Trp¹⁴³stop) (se ⁴²⁸ ) was the common null allele in both Xhosa and Caucasian populations, whereas an allele containing a missense A385T (Ile¹²⁹Phe) mutation (se ^357,385 ), which is the common null allele in Orientals, was found to be absent from both populations. The heterozygosity rates of FUT2 genotypes were as high as 0.75 in the Xhosa population and 0.65 in the Caucasian population. Therefore, the extensive polymorphism and race specificity of the FUT2 gene make it suitable for application as a new tool in genetic studies of modern human evolutionary history. Received: 23 March 1998 / Accepted: 9 May 1998     

Systematic search for single nucleotide polymorphisms in the insulin gene: evidence for a high frequency of -23T-->A in Japanese subjects

It has recently been shown that the A/A genotype at g.-23 of the insulin gene correlates with impaired insulin secretion in response to body weight gain in subjects of European descent. To examine whether there are single nucleotide polymorphisms (SNPs) in the insulin gene associated with type 2 diabetes, all exons with their flanking sequences for 113 Japanese type 2 diabetic patients and 99 nondiabetic control subjects were analyzed using PCR direct sequencing. We have only found g.-23T --> A, 806G --> C, 1128T --> C, and 1141A --> C, which have previously been reported in alpha (A-C-C-C) and beta (T-G-T-A) alleles. The allele frequency of -23T --> A in control Japanese subjects was 97.4%, whereas that in Europeans is about 30%. The A/A genotype was found in 94 of 99 Japanese subjects (94.9%) and the allele frequencies of 806G --> C, 1128T --> C, and 1141A --> C were all 96.5%. The estimated haplotype frequencies were (A-C-C-C) (96.0%), (T-G-T-A) (2.0%), (A-G-T-A) (1.5%), and (T-C-C-C) (0.5%). No association of these SNPs or haplotypes with type 2 diabetes was evident. Thus, the A/A genotype at the g.-23 of insulin gene was generally high in Japanese subjects, which could account for the fact that they typically secrete lower levels of insulin.     

Allele frequencies of single nucleotide polymorphisms in the second exon of the myoglobin gene among the Japanese

The difference in the allele frequencies of two single nucleotide polymorphisms (SNPs) in the second exon of the myoglobin gene between Japanese and other populations is reported. These SNPs are the substitutions of (A79G) and (T109C), and they were investigated by a single polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis followed by direct sequencing. The substitutions were always linked and two alleles were found in the samples used: the A-T allele with no substitution at positions (79A) and (109T) and the G-C allele with substitutions of (79G) and (109C). The frequencies of these alleles were 0.755 and 0.245, respectively, and they were found to be in Hardy-Weinberg equilibrium. The distribution of alleles in the Japanese population was significantly different from that reported among whites, blacks, and Hispanics (p < 0.0001).     

A homozygous nonsense mutation (428G-->A) in the human secretor (FUT2) gene provides resistance to symptomatic norovirus (GGII) infections

Noroviruses (formerly Norwalk-like viruses) are a major cause of acute gastroenteritis worldwide and are associated with a significant number of nosocomial and food-borne outbreaks. In this study we show that the human secretor FUT2 gene, which codes for an alpha(1,2)-fucosyltransferase synthesizing the H-type 1 antigen in saliva and mucosa, is associated with susceptibility to norovirus infections. Allelic polymorphism characterization at nucleotide 428 for symptomatic (n = 53) and asymptomatic (n = 62) individuals associated with nosocomial and sporadic norovirus outbreaks revealed that homozygous nonsense mutation (428G-->A) in FUT2 segregated with complete resistance for the disease. Of all symptomatic individuals, 49% were homozygous (SeSe) and 51% heterozygous (Sese428) secretors, and none were secretor negative (se428se428), in contrast to 20% nonsecretors (se428se428) among Swedish blood donors (n = 104) (P < 0.0002) and 29% for asymptomatic individuals associated with nosocomial outbreaks (P < 0.00001). Furthermore, saliva from secretor-positive and symptomatic patients but not from secretor-negative and asymptomatic individuals bound the norovirus strain responsible for that particular outbreak. This is the first report showing that the FUT2 nonsecretor (se428se428) genotype is associated with resistance to nosocomial and sporadic outbreaks with norovirus.     

ApoB gene nonsense and splicing mutations in a compound heterozygote for familial hypobetalipoproteinemia.

Two novel apoB gene mutations were identified in a patient (CM) with phenotypic homozygous hypobetalipoproteinemia. Haplotype analysis of the apoB alleles from this patient and his family members revealed him to be a genetic compound for the disease. In contrast to previous studies of other hypobetalipoproteinemic patients, no clues existed as to where in the apoB gene the molecular defects resided. Therefore, it was necessary to characterize the apoB genes of the patient by sequence analysis. The apoB gene contains 29 exons and is 43 kb in length. The gene encodes a 14.1 kb mRNA and a 4563 amino acid protein. Both apoB alleles from the patient were cloned via 26 sets of polymerase chain reactions (PCR). These clones contained a total of approximately 24 kb of apoB gene sequence, including regions 5' and 3' to the coding region, 29 exons, and the intron/exon junctions. Complete DNA sequence analysis of these clones showed that each apoB allele had a mutation. In the paternal apoB allele, there was a splicing mutation. The first base of the dinucleotide consensus sequence (GT) in the 5' splice donor site in intron 5 was replaced by a T. It is likely that this base substitution interferes with proper splicing and results in the observed absence of plasma apoB. In the maternal apoB allele, there was a nonsense mutation. The first base of the Arg codon (CGA) at residue 412 in exon 10 was replaced by a T, resulting in a termination codon (TGA). The nonsense mutation is likely to terminate translation after residue 411 resulting in a severely truncated protein only 9% of the length of B-100.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a missense mutation (G329A;Arg(110)--> GLN) in the human FUT7 gene

The human FUT7 gene codes for the alpha1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLe(x)) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLe(x) and SLe(x)-related epitopes. One individual showed lower levels of SLe(x) expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg(110) --> Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. The FUT7 Arg(110) is conserved in all previously cloned vertebrate alpha 1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of < or =1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7 alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLe(x) and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sj?gren's syndrome but had no history of recurrent bacterial infections or leukocytosis.     

Organization of the bovine alpha 2-fucosyltransferase gene cluster suggests that the Sec1 gene might have been shaped through a nonautonomous L1-retrotransposition event within the same locus

By referring to the split coding sequence of the highly conserved alpha 6-fucosyltransferase gene family (assumed to be representative of the common alpha 2 and alpha 6 fucosyltransferase gene ancestor), we have hypothesized that the monoexonic coding sequences of the present alpha 2-fucosyltransferase genes have been shaped in mammals by several events of retrotransposition and/or duplication. In order to test our hypothesis, we determined the structure of the three bovine alpha 2-fucosyltransferase genes (bfut1, bfut2, and sec1) and analyzed their characteristics compared with their human counterparts (FUT1, FUT2, and Sec1). We show that in mammals, a complex nonautonomous L1-retrotransposition event occurred within the locus of the alpha 2-fucosyltransferase ancestor gene itself. A consequence of this event was the processing in Catarrhini of a Sec1 pseudogene via several point mutations.     

Organization of the SUC gene family in Saccharomyces.

The SUC gene family of yeast (Saccharomyces) includes six structural genes for invertase (SUC1 through SUC5 and SUC7) found at unlinked chromosomal loci. A given yeast strain does not usually carry SUC+ alleles at all six loci; the natural negative alleles are called suc0 alleles. Cloned SUC2 DNA probes were used to investigate the physical structure of the SUC gene family in laboratory strains, commercial wine strains, and different Saccharomyces species. The active SUC+ genes are homologous. The suc0 allele at the SUC2 locus (suc2(0) in some strains is a silent gene or pseudogene. Other SUC loci carrying suc0 alleles appear to lack SUC DNA sequences. These findings imply that SUC genes have transposed to different chromosomal locations in closely related Saccharomyces strains.     

Germ line maintenance of the pseudogene donor pool for somatic immunoglobulin gene conversion in chickens.

Somatic immunoglobulin diversity is generated in avian species by sequential gene conversion of variable (V) gene segments of the immunoglobulin heavy- and light-chain loci during B-cell development. The germ line pools of donor sequence information for somatic V-region gene conversion are found in families of V pseudogenes, located 5' of the single functional V gene of each locus. The sequence relationships among the pseudogenes (psi VL) and functional VL1 gene of the chicken light-chain alleles in three inbred strains were compared to determine the extent of diversity within the germ line pseudogene cluster. Numerous differences were observed. For example, compared with the previously reported CB allele and the G4 allele, the S3 allele contains two intact pseudogenes between psi VL16 and psi VL18. These two adjacent psi VL gene segments (psi VL17a and psi VL17b) could have given rise to the psi VL17 segment of the G4 and CB alleles by homologous recombination. The majority of other sequence polymorphisms among the psi VL alleles appear to be the result of meiotic gene conversion. The incidence of untemplated mutations within psi VL segments is significantly lower than the incidence of mutation within the pseudogene flanking regions. Together with the observations that most psi VL segments have open reading frames and lack stop codons, these data support the hypothesis that the psi VL cluster resembles a functional multigene family maintained by evolutionary selection for its functional role in generating somatic antibody diversity. Meiotic gene conversion events within the psi VL cluster serve both to introduce diversity by the exchange of short segments between family members and to prevent the accumulation of random mutations.     

Recessive black is allelic to the yellow plumage locus in Japanese quail and associated with a frameshift deletion in the ASIP gene

The recessive black plumage mutation in the Japanese quail (Coturnix japonica) is controlled by an autosomal recessive gene (rb) and displays a blackish-brown phenotype in the recessive homozygous state (rb/rb). A similar black coat color phenotype in nonagouti mice is caused by an autosomal recessive mutation at the agouti locus. An allelism test showed that wild type and mutations for yellow, fawn-2, and recessive black in Japanese quail were multiple alleles (*N, *Y, *F2, and *RB) at the same locus Y and that the dominance relationship was Y*F2 > Y*Y > Y*N > Y*RB. A deletion of 8 bases was found in the ASIP gene in the Y*RB allele, causing a frameshift that changed the last six amino acids, including a cysteine residue, and removed the normal stop codon. Since the cysteine residues at the C terminus are important for disulphide bond formation and tertiary structure of the agouti signaling protein, the deletion is expected to cause a dysfunction of ASIP as an antagonist of alpha-MSH in the Y*RB allele. This is the first evidence that the ASIP gene, known to be involved in coat color variation in mammals, is functional and has a similar effect on plumage color in birds.     

Complex alleles of the acid beta-glucosidase gene in Gaucher disease.

Gaucher disease is inherited in an autosomal recessive manner and is the most prevalent lysosomal storage disease. Gaucher disease has marked phenotypic variation and molecular heterogeneity, and seven point mutations in the acid beta-glucosidase (beta-Glc) gene have been identified. By means of sequence-specific oligonucleotides (SSO), mutation 6433C has been detected homozygously in neuronopathic type 2 (acute) and type 3 (subacute) patients, as well as in children with severe visceral involvement who are apparently free of neuronopathic disease. To investigate the molecular basis for this puzzling finding, amplified beta-Glc cDNAs from 6433C homozygous type 2 and type 3 Gaucher disease patients were cloned and sequenced. The Swedish type 3 Gaucher disease patient was truly homozygous for alleles only containing the 6433C mutation. In comparison, the type 2 patient contained a singly mutated 6433C allele and a "complex" allele with multiple discrete point mutations (6433C, 6468C, and 6482C). Each of the mutations in the complex allele also was present in the beta-Glc pseudogene. SSO hybridization of 6433C homozygotes revealed that both type 2 patients contained additional mutations in one allele, whereas the 6433C alone was detected in both type 3 and in young severe type 1 Gaucher disease patients. These results suggest that the presence of the complex allele influences the severity of neuronopathic disease in 6433C homozygotes and reveal the central role played by the pseudogene in the formation of mutant alleles of the beta-Glc gene. Analysis of additional cDNA clones also identified two new alleles in a type 3 patient, emphasizing the molecular heterogeneity of neuronopathic Gaucher disease.     

Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels

Previously, we have shown that alleles of the BM1500 microsatellite, located 3.6 kb downstream of the leptin gene in cattle, were associated with carcass fat measures in a population of 154 unrelated beef bulls. Subsequently, a cytosine (C) to thymine (T) transition that encoded an amino acid change of an arginine to a cysteine was identified in exon 2 of the leptin gene. A PCR-RFLP was designed and allele frequencies in four beef breeds were correlated with levels of carcass fat. The T allele was associated with fatter carcasses and the C allele with leaner carcasses. The frequencies of the SNP alleles among breeds indicated that British breeds have a higher frequency of the T allele whereas the continental breeds have a higher occurrence of the C allele. A ribonuclease protection assay was developed to quantify leptin mRNA in a separate group of animals selected by genotype. Animals homozygous for thymine expressed higher levels of leptin mRNA. This may suggest that the T allele, which adds an extra cysteine to the protein, imparts a partial loss of biological function and hence could be the causative mutation.     

Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.     

Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic.

Mutant alleles of SEC4, an essential gene required for the final stage of secretion in yeast, have been generated by in vitro mutagenesis. Deletion of the two cysteine residues at the C terminus of the protein results in a soluble non-functional protein, indicating that those two residues are required for normal localization of Sec4p to secretory vesicles and the plasma membrane. A mutant allele of SEC4 generated to mimic an activated, transforming allele of H-ras, as predicted, does not bind GTP. The presence of this allele in cells containing wild-type SEC4 causes a secretory defect and the accumulation of secretory vesicles. The results of genetic studies indicate that this allele behaves as a dominant loss of function mutant and as such prevents wild-type protein from functioning properly. We propose a model in which Sec4p cycles between an active and an inactive state in order to mediate the fusion of vesicles to the plasma membrane.     

A newly identified null allelic mutation in the human lipoprotein lipase (LPL) gene of a compound heterozygote with familial LPL deficiency.

In a Japanese patient with familial LPL deficiency, a new null allelic mutation, one base pair deletion at nucleotide position 916 was identified in exon 5 of one allele. In exon 3 of the other allele, we found the same nonsense mutation as we described previously in other Japanese kindreds. For the deletional mutant allele, we developed a simple detection method and constructed the DNA haplotype.     

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.