Alpha-galactosidase A gene rearrangements causing Fabry disease. Identification of short direct repeats at breakpoints in an Alu-rich gene

Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-linked gene encoding the lysosomal enzyme, alpha-galactosidase A (EC 3.2.1.22). Six alpha-galactosidase A gene rearrangements that cause Fabry disease were investigated to assess the role of Alu repetitive elements and short direct and/or inverted repeats in the generation of these germinal mutations. The breakpoints of five partial gene deletions and one partial gene duplication were determined by either cloning and sequencing the mutant gene from an affected hemizygote, or by polymerase chain reaction amplifying and sequencing the genomic region containing the novel junction. Although the alpha-galactosidase A gene contains 12 Alu repetitive elements (representing approximately 30% of the 12-kilobase (kb) gene or approximately 1 Alu/1.0 kb), only one deletion resulted from an Alu-Alu recombination. The remaining five rearrangements involved illegitimate recombinational events between short direct repeats of 2 to 6 base pairs (bp) at the deletion or duplication breakpoints. Of these rearrangements, one had a 3' short direct repeat within an Alu element, while another was unusual having two deletions of 1.7 kb and 14 bp separated by a 151-bp inverted sequence. These findings suggested that slipped mispairing or intrachromosomal exchanges involving short direct repeats were responsible for the generation of most of these gene rearrangements. There were no inverted repeat sequences or alternating purine-pyrimidine regions which may have predisposed the gene to these rearrangements. Intriguingly, the tetranucleotide CCAG and the trinucleotide CAG (or their respective complements, CTGG and CTG) occurred within or adjacent to the direct repeats at the 5' breakpoints in three and four of the five alpha-galactosidase A gene rearrangements, respectively, suggesting a possible functional role in these illegitimate recombinational events. These studies indicate that short direct repeats are important in the formation of gene rearrangements, even in human genes like alpha-galactosidase A that are rich in Alu repetitive elements.     

Characterization of a recent retroposon insertion on mouse Chromosome 2 and localization of the cognate parental gene to Chromosome 11

The genomic sequence on mouse Chromosome (Chr) 2 corresponding to a previously identified novel cDNA has been characterized. The genomic organization of this locus, adjacent to the beta 2 microglobulin gene, has the properties of a processed gene or retroposon including the presence of a short flanking direct repeat, a polyadenylation signal/poly A tract, and the absence of introns. Analysis of inbred and wild-derived Mus DNAs reveals the retroposon to be a feature only of M. m. domesticus subspecies, suggesting that the insertion event is relatively recent. This notion is supported by the presence of an open reading frame and the lack of sequence divergence in the flanking direct repeats. The complex chromatin configuration characteristic of this region in mouse and human is not, therefore, related to this cDNA. The cognate parental gene encoding the cDNA was mapped to Chr 11. A further, more ancient retroposon present in many Mus species localizes to Chr 17. Received: 20 June 1997 / Accepted: 30 September 1997     

Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene.

Mammalian ribonucleotide reductase consists of two non-identical subunits, proteins M1 and M2. M2-related DNA sequences are present on mouse chromosomes 4, 7, 12 and 13. However, M2-overproducing mouse cells show amplification of a chromosome 12-specific, single 13 kb HindIII fragment, which probably represents the active gene. We have isolated this fragment from parental mouse cell DNA and used it to clone and characterize the functional M2 gene. The 5770 bp transcribed M2 sequence contains ten exons separated by nine 95-917 bp introns. The 501 bp of 5' flanking DNA is G + C rich and contains TTTAAA and CCAAT sequences as well as potential Sp1 binding sites. The M2-related sequence on chromosome 13, which contains only the last six exons and several internal rearrangements, is a pseudogene. Transfection of BALB/3T3 cells with the M2 gene resulted in stable transformants with a 10-fold reduction in sensitivity to hydroxyurea, compared to control cells. This confirmed that the cloned M2 genomic DNA represents the functional gene and conclusively establishes the link between hydroxyurea resistance and M2 expression in mammalian cells. M2 genomic DNA should be a valuable dominant, selectable marker for identifying and isolating stable co-transformants.     

Identification and mapping of the gene encoding the glycoprotein complex gp82-gp105 of human herpesvirus 6 and mapping of the neutralizing epitope recognized by monoclonal antibodies.

Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against human herpesvirus 6 (HHV-6) variant A strain GS recognized virion envelope glycoprotein complex gp82-gp105 and neutralized the infectivity of HHV-6 variant A group isolates. A 624-bp genomic fragment (82G) was identified from an HHV-6 strain GS genomic library constructed in the lambda gt11 expression system by immunoscreening with MAb 2D6. Rabbit antibodies against the fusion protein expressed from the genomic insert recognized glycoprotein complex gp82-gp105 from HHV-6-infected cells, thus confirming that the genomic fragment is a portion of the gene(s) that encodes gp82-gp105. This genomic insert hybridized specifically with viral DNAs from HHV-6 variant A strains GS and U1101 under high-stringency conditions but hybridized with HHV-6 variant B strain Z-29 DNA only under low-stringency conditions. DNA sequence analysis of the insert revealed a 167-amino-acid single open reading frame with an open 5' end and a stop codon at the 3' end. Hybridization studies with HHV-6A strain U1102 DNA localized the gp82-gp105-encoding gene to the unique long region near the direct repeat at the right end of the genome. To locate the neutralizing epitope(s) recognized by the MAbs, a series of deletions from the 3' end of the gene were constructed with exonuclease III, and fusion proteins from deletion constructs were tested for reactivity with MAbs in a Western immunoblot assay. Sequencing of deletion constructs at the reactive-nonreactive transition point localized the epitope recognized by the three neutralizing MAbs within or near a repeat amino acid sequence (NIYFNIY) of the putative protein. This repeat sequence region is surrounded on either side by two potential N-glycosylation sites and three cysteine residues.     

A novel homologue of the TIAP/m-survivin gene

The inhibitor of apoptosis (IAP) proteins comprise a highly conserved gene family that prevents cell death in response to a variety of stimuli. TIAP/m-survivin, a murine homologue of human Survivin, is a member of the IAP family. TIAP/m-survivin has one baculovirus IAP repeat (BIR) and lacks a C-terminal RING finger motif. Here we identified the genomic DNA region (TIAP-2) that is homologous to the TIAP/m-survivin gene by a low stringency genomic DNA hybridization. The region is on the chromomsome 9 which is distinct from that (chromosome 11) of the TIAP/m-survivin gene, and contains DNA sequence similar to a part of the BIR and the 3' side of the TIAP/m-survivin gene and the sequence homology between them is 92%. Expression of TIAP-2 mRNA was detected in various murine tissues by RT-PCR. Although expression of TIAP/m-survivin mRNA is upregulated in synchronized cells at S to G2/M phase of the cell cycle, expression of TIAP-2 mRNA was constant in the cell cycle, suggesting the different role of TIAP-2 from that of TIAP/m-survivin.     

Genomic rearrangements of the APC tumor-suppressor gene in familial adenomatous polyposis

Germline mutations of the adenomatous polyposis coli (APC) tumor-suppressor gene result in the hereditary colorectal cancer syndrome familial adenomatous polyposis (FAP). Almost all APC mutations that have been identified are single-nucleotide alterations, small insertions, or small deletions that would truncate the protein product of the gene. No well-characterized intragenic rearrangement of APC has been described, and the prevalence of this type of mutation in FAP patients is not clear. We screened 49 potential FAP families and identified 26 different germline APC mutations in 30 families. Four of these mutations were genomic rearrangements resulting from homologous and nonhomologous recombinations mediated by Alu elements. Two of these four rearrangements were complex, involving deletion and insertion of nucleotides. Of these four rearrangements, one resulted in the deletion of exons 11 and 12 and two others resulted in either complete or partial deletion of exon 14. The fourth rearrangement grossly altered the sequence within intron 14. Although this rearrangement did not affect any coding sequence of APC at the genomic DNA level, it caused inappropriate splicing of exon 14. These rearrangements were initially revealed by analyzing cDNAs and could not have been identified by using mutation detection methods that screened each exon individually. The identification of a rearrangement that did not alter any coding exons yet affected the splicing further underscores the importance of using cDNA for mutation analysis. The identification of four genomic rearrangements among 30 mutations suggests that genomic rearrangements are frequent germline APC mutations.     

Detection of rearrangements in the NF2 gene using semi-quantitative multiplex fluorescent PCR

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder that predisposes to the development of bilateral vestibular schwannomas (sometimes associated with schwannomas at other locations), meningiomas, and ependymomas. Point mutations that inactivate the NF2 tumor suppressor gene, located in 22q12, have been found in 45-85% of NF2 patients; in addition, large genomic deletions can be found. To evaluate the presence of genomic NF2 rearrangements, we have developed a fluorescent semiquantitative multiplex PCR method. Briefly, short fragments corresponding to the 17 exons, the promoter region, and the 3' end of the NF2 gene were co-amplified by PCR using dye primers. An additional fragment, corresponding to another gene used as an internal control, was systematically amplified in each multiplex PCR. Initially, we validated the method by using monosomic 22q and trisomic 22 samples. The fluorescent multiplex PCR method was then used to analyze 21 NF2 individuals in which single-strand conformational polymorphism (SSCP) analysis and/or direct sequencing had revealed no NF2 point mutations; we were able to detect two deletions and one duplication in NF2 in 3 patients. In conclusion, the method we developed could easily be applied in detecting NF2 deletions and duplications. Discovering genomic duplications is invaluable because they are probably the most difficult molecular alterations to detect with conventional methods and, as a consequence, might be an underestimated cause of NF2.     

Mitochondrial gene rearrangements: new paradigm in the evolutionary biology and systematics

Mitochondrial (mt) genomic study may reveal significant insight into the molecular evolution and several other aspects of genome evolution such as gene rearrangements evolution, gene regulation, and replication mechanisms. Other questions such as patterns of gene expression mechanism evolution, genomic variation and its correlation with physiology, and other molecular and biochemical mechanisms can be addressed by the mt genomics. Rare genomic changes have attracted evolutionary biology community for providing homoplasy free evidence of phylogenetic relationships. Gene rearrangements are considered to be rare evolutionary events and are being used to reconstruct the phylogeny of diverse group of organisms. Mt gene rearrangements have been established as a hotspot for the phylogenetic and evolutionary analysis of closely as well as distantly related organisms.     

Structural characterization of the dihydroflavonol 4-reductase B (DFR-B) gene in the sweet potato.

A genomic fragment containing the dihydroflavonol 4-reductase B (DFR-B) gene was cloned from the sweet potato (Ipomoea batatas) and its nucleotide sequence was analyzed. The exons and flanking regions were highly homologous to those of previously reported DFR-B genes of the Japanese morning glory, whereas the introns and the intergenic region were less conserved. In addition to the sequences of three miniature inverted-repeat transposable elements (MITEs) and one direct repeat previously reported in the DFR-B gene of Japanese morning glory, two mobile element-like sequences were newly identified in the sweet potato DFR-B gene. At least four allelic sequences were found to exist by amplification of the DFR-B gene from various sweet potato cultivars. One of these allelic sequences had a 2-kb deletion in the intergenic region and was observed in the cultivars with high anthocyanin content in their storage roots.     

Comparative study of the genomic organization of DNA repeats within the 5′-flanking region of the natural resistance-associated macrophage protein gene (NRAMP1) between humans and great apes

The human NRAMP1 gene located on Chromosome (Chr) region 2q35 is a candidate gene for increased risk of infection by several intracellular macrophage parasites, including M. tuberculosis and M. leprae. In search for a possible mutational hot spot, we have analyzed a 3.5-kb region 5′ to NRAMP1 that is highly enriched for DNA repeat sequences. The repeat sequences could be grouped into one Mer element and six Alu elements, representing five Alu subfamilies, that had integrated in the same DNA region during successive rounds of Alu retropositional activity. Comparative sequence analysis of the Alu cluster region in humans, chimpanzee (Pan paniscus), and gorilla (Gorilla gorilla) revealed only modest sequence variability and failed to detect any evidence for genomic instability of the highly repetitive DNA region. These results show that sequence length variants in the Alu-flanking regions as well as nucleotide substitutions are the most common genomic variations even in a region of extreme Alu-clustering. Moreover, the high degree of sequence conservation among three primate species argues against the Alu cluster being the site of frequent genomic rearrangements or other frequent genetic events that might influence NRAMP1 expression. Received: 20 September 1997 / Accepted: 23 January 1998     

Novel complex genomic rearrangement of the BRCA1 gene

Initial BRCA1 and BRCA2 analyses conducted in breast and ovarian cancer families were focused on identification of mutations in coding sequences and splicing sites of the genes. Large genomic rearrangements as well as mutations in promoter or untranslated regions have been missed by standard detection strategies. Nevertheless, in Western countries, a detailed study of families with strong linkage to BRCA1 identified large genomic deletions and rearrangements in this gene as early as 1997. To date, no such gene alteration has been described in Central and Eastern European populations. In our study of BRCA1/2 genes in the Czech population, we have detected a complex genomic rearrangement in BRCA1 using RNA-based analysis for mutation screening. This rearrangement involves exons 21 and 22 and results in a protein product lacking BRCT domain important for its function.     

Immunoglobulin gene rearrangements in normal mouse B cells.

We have analyzed the structure of rearranged mu heavy-chain genes obtained from the genomic DNA of normal BALB/c mouse spleen cells expressing surface immunoglobulin M. Examples were found of two types of nonproductive rearrangements, which may be responsible for allelic exclusion in normal B cells. In one of these rearrangements, a germ line D gene segment has joined to the JH4 gene segment but no V/D joining has occurred. We present evidence that D gene segments lie as a cluster between V and J gene segments in the germ line. A comparison of conserved sequences in V and D gene segments suggests that the D gene segments, which are found only in the heavy-chain gene family, may have evolved from V gene segments similar to the Vk family.     

Chromosomal aberrations induced by double strand DNA breaks

It has been suggested that introduction of double strand DNA breaks (DSBs) into mammalian chromosomes can lead to gross chromosomal rearrangements through improper DNA repair. To study this phenomenon, we employed a model system in which a double strand DNA break can be produced in human cells in vivo at a predetermined location. The ensuing chromosomal changes flanking the breakage site can then be cloned and characterized. In this system, the recognition site for the I-SceI endonuclease, whose 18 bp recognition sequence is not normally found in the human genome, is placed between a strong constitutive promoter and the Herpes simplex virus thymidine kinase (HSV-tk) gene, which serves as a negative selectable marker. We found that the most common mutation following aberrant DSB repair was an interstitial deletion; these deletions typically showed features of non-homologous end joining (NHEJ), such as microhomologies and insertions of direct or inverted repeat sequences. We also detected more complex rearrangements, including large insertions from adjacent or distant genomic regions. The insertion events that involved distant genomic regions typically represented transcribed sequences, and included both L1 LINE elements and sequences known to be involved in genomic rearrangements. This type of aberrant repair could potentially lead to gene inactivation via deletion of coding or regulatory sequences, or production of oncogenic fusion genes via insertion of coding sequences.     

Characterization of six partial deletions in the low-density-lipoprotein (LDL) receptor gene causing familial hypercholesterolemia (FH).

Two hundred thirty-four unrelated heterozygotes for familial hypercholesterolemia (FH) were screened to detect major rearrangements in the low-density-lipoprotein (LDL) receptor gene. Total genomic DNA was analyzed by Southern blot hybridization to probes encompassing exons 1-18 of the LDL receptor gene. Six different mutations were detected and characterized by the use of exon-specific probes and detailed restriction mapping. Each mutation is unique and suggests that molecular heterogeneity underlies the molecular pathology of FH. There appear to be preferential sites within the LDL receptor gene for major rearrangements resulting in deletions.     

Characterization of the bovine prothrombin gene

The bovine prothrombin gene was characterized by Southern blot analysis of bovine genomic DNA using bovine prothrombin cDNA fragments as hybridization probes. These analyses suggested that the bovine genome contains a single prothrombin gene that is at least 10 kilobase pairs (kbp) in size. To characterize the gene more thoroughly, two bovine genomic phage libraries were screened by using prothrombin cDNAs as hybridization probes. Heteroduplex analysis of the cloned genomic DNA and cDNA showed that the prothrombin gene is 14.9 kbp in size and contains at least 14 exons interrupted by 13 introns. The exons vary in size from 28 to 317 base pairs (bp), while the introns vary in size from less than 100 to 6940 bp. Regions of self-complementarity were observed within some of the introns, suggesting the presence of inverted repeat sequences. The bovine prothrombin gene shows similarities in structure to both the human prothrombin gene and the human factor IX gene.