Nucleotide sequence analysis of human hypoxanthine phosphoribosyltransferase (HPRT) gene deletions.

We have determined the nucleotide sequences of 10 intragenic human HPRT gene deletion junctions isolated from thioguanine-resistant PSV811 Werner syndrome fibroblasts or from HL60 myeloid leukemia cells. Deletion junctions were located by fine structure blot hybridization mapping and then amplified with flanking oligonucleotide primer pairs for DNA sequence analysis. The junction region sequences from these 10 HPRT mutants contained 13 deletions ranging in size from 57 bp to 19.3 kb. Three DNA inversions of 711, 368, and 20 bp were associated with tandem deletions in two mutants. Each mutant contained the deletion of one or more HPRT exon, thus explaining the thioguanine-resistant cellular phenotype. Deletion junction and donor nucleotide sequence alignments suggest that all of these HPRT gene rearrangements were generated by the nonhomologous recombination of donor DNA duplexes that share little nucleotide sequence identity. This result is surprising, given the potential for homologous recombination between copies of repeated DNA sequences that constitute approximately a third of the human HPRT locus. No difference in deletion structure or complexity was observed between deletions isolated from Werner syndrome or from HL60 mutants. This suggests that the Werner syndrome deletion mutator uses deletion mutagenesis pathway(s) that are similar or identical to those used in other human somatic cells.     

Molecular description of three macro-deletions and an Alu-Alu recombination-mediated duplication in the HPRT gene in four patients with Lesch-Nyhan disease

Mutations in the HPRT gene cause a spectrum of diseases that ranges from hyperuricemia alone to hyperuricemia with profound neurological and behavioral dysfunction. The extreme phenotype is termed Lesch-Nyhan syndrome. In 271 cases in which the germinal HPRT mutation has been characterized, 218 different mutations have been found. Of these, 34 (13%) are large- (macro-) deletions of one exon or greater and four (2%) are partial gene duplications. The deletion breakpoint junctions have been defined for only three of the 34 macro-deletions. The molecular basis of two of the four duplications has been defined. We report here the breakpoint junctions for three new deletion mutations, encompassing exons 4-8 (20033bp), exons 4 and 5 (13307bp) and exons 5 and 6 (9454bp), respectively. The deletion breakpoints were defined by a combination of long polymerase chain reaction (PCR) amplifications, and conventional PCR and DNA sequencing. All three deletions are the result of non-homologous recombinations. A fourth mutation, a duplication of exons 2 and 3, is the result of an Alu-mediated homologous recombination between identical 19bp sequences in introns 3 and 1. In toto, two of three germinal HPRT duplication mutations appear to have been caused by Alu-mediated homologous recombination, while only one of six deletion mutations appears to have resulted from this type of recombination mechanism. The other five deletion mutations resulted from non-homologous recombination. With this admittedly limited number of characterized macro-mutations, Alu-mediated unequal homologous recombinations account for at least 8% (3 of 38) of the macro-alterations and 1% (3 of 271) of the total HPRT germinal mutations.     

Duplication in the hypoxanthine phosphoribosyl-transferase gene caused by Alu-Alu recombination in a patient with Lesch Nyhan syndrome

We have determined the structure, at the nucleotide sequence level, of a duplication in the hprt gene in a patient with Lesch-Nyhan syndrome (LN). The duplication extends over exons 7 and 8 and approximately 1.8 kb of the surrounding hprt sequence. The duplication junction is localized within two Alu sequences and has apparently been generated by unequal homologous recombination. This is the second reported case of a partial duplication of the hprt gene in an LN patient, and the first that involves an Alu-Alu recombination.     

Mechanisms of tandem duplication in the Duchenne muscular dystrophy gene include both homologous and nonhomologous intrachromosomal recombination.

Three tandem duplications were previously identified in patients with Duchenne muscular dystrophy and were shown in each case to have a subset of dystrophin gene exons duplicated. The origin of these duplications was traced to the single X chromosome of the maternal grandfathers, suggesting that an intrachromosomal event (unequal sister chromatid exchange) was involved in the formation of these duplications. In the present study, a DNA segment containing the duplication junction and the normal DNA that corresponds to both ends of the duplicated region have been cloned. Subsequent mapping studies confirmed the tandem arrangement (head to tail) of these duplications and revealed their sizes to be 130 kb, approximately 300 kb, and 35-80 kb, respectively. Sequence analysis of the duplication junctions showed that one duplication was due to homologous recombination between two repetitive elements (Alu sequences) and the other two were due to recombination between unrelated nonhomologous sequences. In the latter cases, the preferred cleavage sites of the eukaryotic type I and II DNA topoisomerases were found at the junctions of these duplications, suggesting a possible role of these enzymes in the chromatid exchange events. This study provides the first insight into the molecular basis of gene duplications formed through unequal sister chromatid exchange in humans.     

Molecular characterization of a deletion in the HPRT1 gene in a patient with Lesch-Nyhan syndrome

Lesch-Nyhan syndrome is caused by a deficiency of hypoxanthine phosphoribosyltransferase (HPRT) encoded by HPRT1. About 20% of patients have a deletion of HPRT1 and large deletions of HPRT1 are not always fully characterized at the molecular level. Here, we report on a case of Lesch-Nyhan syndrome with a 33-kb deletion involving exon 1 of HPRT1. This novel mutation is caused by a nonhomologous recombination between different classes of interspersed repetitive DNA.     

A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders

The prevailing mechanism for recurrent and some nonrecurrent rearrangements causing genomic disorders is nonallelic homologous recombination (NAHR) between region-specific low-copy repeats (LCRs). For other nonrecurrent rearrangements, nonhomologous end joining (NHEJ) is implicated. Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder caused most frequently (60%-70%) by nonrecurrent duplication of the dosage-sensitive proteolipid protein 1 (PLP1) gene but also by nonrecurrent deletion or point mutations. Many PLP1 duplication junctions are refractory to breakpoint sequence analysis, an observation inconsistent with a simple recombination mechanism. Our current analysis of junction sequences in PMD patients confirms the occurrence of simple tandem PLP1 duplications but also uncovers evidence for sequence complexity at some junctions. These data are consistent with a replication-based mechanism that we term FoSTeS, for replication Fork Stalling and Template Switching. We propose that complex duplication and deletion rearrangements associated with PMD, and potentially other nonrecurrent rearrangements, may be explained by this replication-based mechanism.     

Identification of Uncommon Recurrent Potocki-Lupski Syndrome-Associated Duplications and the Distribution of Rearrangement Types and Mechanisms in PTLS

Nonallelic homologous recombination (NAHR) can mediate recurrent rearrangements in the human genome and cause genomic disorders. Smith-Magenis syndrome (SMS) and Potocki-Lupski syndrome (PTLS) are genomic disorders associated with a 3.7 Mb deletion and its reciprocal duplication in 17p11.2, respectively. In addition to these common recurrent rearrangements, an uncommon recurrent 5 Mb SMS-associated deletion has been identified. However, its reciprocal duplication predicted by the NAHR mechanism had not been identified. Here we report the molecular assays on 74 subjects with PTLS-associated duplications, 35 of whom are newly investigated. By both oligonucleotide-based comparative genomic hybridization and recombination hot spot analyses, we identified two cases of the predicted 5 Mb uncommon recurrent PTLS-associated duplication. Interestingly, the crossovers occur in proximity to a recently delineated allelic homologous recombination (AHR) hot spot-associated sequence motif, further documenting the common hot spot features shared between NAHR and AHR. An additional eight subjects with nonrecurrent PTLS duplications were identified. The smallest region of overlap (SRO) for all of the 74 PTLS duplications examined is narrowed to a 125 kb interval containing only RAI1, a gene recently further implicated in autism. Sequence complexities consistent with DNA replication-based mechanisms were identified in four of eight (50%) newly identified nonrecurrent PTLS duplications. Our findings of the uncommon recurrent PTLS-associated duplication at a relative prevalence reflecting the de novo mutation rate and the distribution of 17p11.2 duplication types in PTLS reveal insights into both the contributions of new mutations and the different underlying mechanisms that generate genomic rearrangements causing genomic disorders.     

Heterogeneous duplications in patients with Pelizaeus-Merzbacher disease suggest a mechanism of coupled homologous and nonhomologous recombination

We describe genomic structures of 59 X-chromosome segmental duplications that include the proteolipid protein 1 gene (PLP1) in patients with Pelizaeus-Merzbacher disease. We provide the first report of 13 junction sequences, which gives insight into underlying mechanisms. Although proximal breakpoints were highly variable, distal breakpoints tended to cluster around low-copy repeats (LCRs) (50% of distal breakpoints), and each duplication event appeared to be unique (100 kb to 4.6 Mb in size). Sequence analysis of the junctions revealed no large homologous regions between proximal and distal breakpoints. Most junctions had microhomology of 1-6 bases, and one had a 2-base insertion. Boundaries between single-copy and duplicated DNA were identical to the reference genomic sequence in all patients investigated. Taken together, these data suggest that the tandem duplications are formed by a coupled homologous and nonhomologous recombination mechanism. We suggest repair of a double-stranded break (DSB) by one-sided homologous strand invasion of a sister chromatid, followed by DNA synthesis and nonhomologous end joining with the other end of the break. This is in contrast to other genomic disorders that have recurrent rearrangements formed by nonallelic homologous recombination between LCRs. Interspersed repetitive elements (Alu elements, long interspersed nuclear elements, and long terminal repeats) were found at 18 of the 26 breakpoint sequences studied. No specific motif that may predispose to DSBs was revealed, but single or alternating tracts of purines and pyrimidines that may cause secondary structures were common. Analysis of the 2-Mb region susceptible to duplications identified proximal-specific repeats and distal LCRs in addition to the previously reported ones, suggesting that the unique genomic architecture may have a role in nonrecurrent rearrangements by promoting instability.     

利用Red同源重组系统构建兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶基因打靶载体

利用EL350基因工程菌进行同源重组,成功进行基因敲除已有报道,但利用该系统进行兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶(Hypoxanthine guanine phosphoribosyl transferase,HPRT)基因突变和基因打靶方面的研究还没有报道。本实验首先在已经筛选到含有兔全长HPRT基因BAC克隆（LBNL1-304M19）的基础上,利用Red重组系统,通过Gap-Repair方式从此克隆上将一段47Kb无启动子的HPRT基因组片段（不含有第1个外显子）克隆到pBACLinkSp质粒上,产生pBACLinkSp-rHPRT质粒。然后基于pBACLinkSp-rHPRT质粒,设计不同的同源臂,从而删除了HPRT基因的不同编码区,成功构建了三个不同的HPRT基因打靶载体。同时对利用同源重组技术敲除不同大小的DNA片段的效率进行了研究。基于本实验所构建的三个不同的兔HPRT基因打靶载体,为探索兔成纤维细胞和胚胎干细胞基因打靶的适宜条件,及进一步获得兔HPRT基因敲除动物疾病模型奠定了基础。     

Patterns of segmental duplication in the human genome

We analyzed the completed human genome for recent segmental duplications (size > or = 1 kb and sequence similarity > or = 90%). We found that approximately 4% of the genome is covered by duplications and that the extent of segmental duplication varies from 1% to 14% among the 24 chromosomes. Intrachromosomal duplication is more frequent than interchromosomal duplication in 15 chromosomes. The duplication frequencies in pericentromeric and subtelomeric regions are greater than the genome average by approximately threefold and fourfold. We examined factors that may affect the frequency of duplication in a region. Within individual chromosomes, the duplication frequency shows little correlation with local gene density, repeat density, recombination rate, and GC content, except chromosomes 7 and Y. For the entire genome, the duplication frequency is correlated with each of the above factors. Based on known genes and Ensembl genes, the proportion of duplications containing complete genes is 3.4% and 10.7%, respectively. The proportion of duplications containing genes is higher in intrachromosomal than in interchromosomal duplications, and duplications containing genes have a higher sequence similarity and tend to be longer than duplications containing no genes. Our simulation suggests that many duplications containing genes have been selectively maintained in the genome.     

Uracil incorporation into a gene targeting construct reduces the frequency of homologous and nonhomologous recombinants in human cells

Gene targeting allows the introduction of specific modifications into the eukaryotic genome by homologous recombination, but its efficiency is low in many mammalian systems. We are exploring different ways to increase the efficiency of gene targeting and we report here the effect of uracil incorporation in the targeting construct. Plasmids containing uracil substituting for a fraction of thymine residues are hyperrecombinogenic in some bacterial systems. To test whether a similar stimulation of recombination occurs in mammalian cells, we have prepared a uracil-rich HPRT targeting construct and quantified its homologous and nonhomologous recombination frequencies compared to the same plasmid lacking uracil. The uracil-rich plasmid led to reductions in both homologous and nonhomologous recombination in human cells.     

Gene duplication and inactivation in the HPRT gene family

Hypoxanthine phosphoribosyltransferase (HPRT1) is a key enzyme in the purine salvage pathway, and mutations in HPRT1 cause Lesch-Nyhan disease. The studies described here utilized targeted comparative mapping and sequencing, in conjunction with database searches, to assemble a collection of 53 HPRT1 homologs from 28 vertebrates. Phylogenetic analysis of these homologs revealed that the HPRT gene family expanded as the result of ancient vertebrate-specific duplications and is composed of three groups consisting of HPRT1, phosphoribosyl transferase domain containing protein 1 (PRTFDC1), and HPRT1L genes. All members of the vertebrate HPRT gene family share a common intron-exon structure; however, we have found that the three gene groups have distinct rates of evolution and potentially divergent functions. Finally, we report our finding that PRTFDC1 was recently inactivated in the mouse lineage and propose the loss of function of this gene as a candidate genetic basis for the phenotypic disparity between HPRT-deficient humans and mice.     

EGFR Intron Recombination in Human Gliomas: Inappropriate Diversion of V(D)J Recombination?

The epidermal growth factor receptor (EGFR) is a membrane-bound, 170 kDa, protein tyrosine kinase that plays an important role in tumorigenesis. The EGFR gene, which is composed of over 168 kb of sequence, including a 123-kb first intron, is frequently amplified and rearranged in malignant gliomas leading to the expression of oncogenic deletion (DM) and tandem duplication (TDM) mutants. The most common DM in gliomas is EGFRvIII, which arises from recombination between introns 1 and 7 with deletion of exons 2 through 7 and intervening introns. In addition, some human gliomas express 180- to 190-kDa TDM, which are constitutively active and highly oncogenic. Both DM and TDM arise by recombination of introns that contain sequences with homology to the recombination signal sequence (RSS) heptamers and nonamers present in the V(D)J region of the immunoglobin and T lymphocyte antigen receptor genes. V(D)J RSS have also been identified in certain proto-oncogenes like bcl-2 that are involved in translocations associated with the development of human lymphomas and in other genes such as hypoxanthine-guainine phosphoribosyl transferase (HPRT) in which deletion mutations and intron rearrangements are a common phenomenon. Together with the expression of recombination associated gene (RAG) and nonhomologous end-joining (NHEJ) proteins in gliomas, these observation suggest that aberrant activity of the V(D)J recombinase may be involved in the activation of proto-oncogenes in both liquid and solid tumors.     

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production

Engineering of the methylmalonyl-CoA (mmCoA) metabolite node of the Saccharopolyspora erythraea wild-type strain through duplication of the mmCoA mutase (MCM) operon led to a 50% increase in erythromycin production in a high-performance oil-based fermentation medium. The MCM operon was carried on a 6.8kb DNA fragment in a plasmid which was inserted by homologous recombination into the S. erythraea chromosome. The fragment contained one uncharacterized gene, ORF1; three MCM related genes, mutA, mutB, meaB; and one gntR-family regulatory gene, mutR. Additional strains were constructed containing partial duplications of the MCM operon, as well as a knockout of ORF1. None of these strains showed any significant alteration in their erythromycin production profile. The combined results showed that increased erythromycin production only occurred in a strain containing a duplication of the entire MCM operon including mutR and a predicted stem-loop structure overlapping the 3' terminus of the mutR coding sequence.     

Mechanism,Prevalence, and More Severe Neuropathy Phenotype of the Charcot-Marie-Tooth Type 1A Triplication

Copy-number variations cause genomic disorders. Triplications, unlike deletions and duplications, are poorly understood because of challenges in molecular identification, the choice of a proper model system for study, and awareness of their phenotypic consequences. We investigated the genomic disorder Charcot-Marie-Tooth disease type 1A (CMT1A), a dominant peripheral neuropathy caused by a 1.4 Mb recurrent duplication occurring by nonallelic homologous recombination. We identified CMT1A triplications in families in which the duplication segregates. The triplications arose de novo from maternally transmitted duplications and caused a more severe distal symmetric polyneuropathy phenotype. The recombination that generated the triplication occurred between sister chromatids on the duplication-bearing chromosome and could accompany gene conversions with the homologous chromosome. Diagnostic testing for CMT1A (n = 20,661 individuals) identified 13% (n = 2,752 individuals) with duplication and 0.024% (n = 5 individuals) with segmental tetrasomy, suggesting that triplications emerge from duplications at a rate as high as ∼1:550, which is more frequent than the rate of de novo duplication. We propose that individuals with duplications are predisposed to acquiring triplications and that the population prevalence of triplication is underascertained.     

High Frequency Repeat-Induced Point Mutation (Rip) Is Not Associated with Efficient Recombination in Neurospora

Duplicated DNA sequences in Neurospora crassa are efficiently detected and mutated during the sexual cycle by a process named repeat-induced point mutation (RIP). Linked, direct duplications have previously been shown to undergo both RIP and deletion at high frequency during premeiosis, suggesting a relationship between RIP and homologous recombination. We have investigated the relationship between RIP and recombination for an unlinked duplication and for both inverted and direct, linked duplications. RIP occurred at high frequency (42-100%) with all three types of duplications used in this study, yet recombination was infrequent. For both inverted and direct, linked duplications, recombination was observed, but at frequencies one to two orders of magnitude lower than RIP. For the unlinked duplication, no recombinants were seen in 900 progeny, indicating, at most, a recombination frequency nearly three orders of magnitude lower than the frequency of RIP. In a direct duplication, RIP and recombination were correlated, suggesting that these two processes are mechanistically associated or that one process provokes the other. Mutations due to RIP have previously been shown to occur outside the boundary of a linked, direct duplication, indicating that RIP might be able to inactivate genes located in single-copy sequences adjacent to a duplicated sequence. In this study, a single-copy gene located between elements of linked duplications was inactivated at moderate frequencies (12-14%). Sequence analysis demonstrated that RIP mutations had spread into these single-copy sequences at least 930 base pairs from the boundary of the duplication, and Southern analysis indicated that mutations had occurred at least 4 kilobases from the duplication boundary.     

Nonhomologous recombination in the human genome: deletions in the human factor VIII gene

Four deletions in the human factor VIII gene have been characterized at the sequence level in patients with hemophilia A. Deletion JH 1 extends 57 kb from IVS 10 to IVS 18. Intron 13 and exon 14 are partially deleted in patients JH 7 and JH 37, with a loss of 3.2 and 2.4 kb of DNA, respectively. The 3' deletion breakpoint of the JH 21 event resides in intron 3 and extends 5' into intron 1, resulting in the loss of exons 2 and 3. Seven of the eight breakpoints sequenced (5' and 3' for each of the four deletions) occur in nonrepetitive sequence, while the 3' breakpoint of the JH 1 resides in an Alu repetitive element. All of the deletions are the result of nonhomologous recombination. The 5' and 3' breakpoints of JH 1, JH 7, and JH 37 share 2- to 3-bp homologies at the deletion junctions. In contrast, two nucleotides have been inserted at the JH 21 deletion junction. Short sequence homologies may facilitate end-joining reactions in nonhomologous recombination events.     

Ancient duplications of the human proglucagon gene

The human proglucagon gene (GCG) is encoded within a finished 576-kb DNA sequence generated by the Human Genome Project. GCG is flanked by 18 kb and 65 kb of DNA, 5' and 3', respectively, that do not encode genes. The genomic sequence that includes GCG was found to have a long history of gene duplication events. Some members of the glucagon-like family of genes, GCG on chromosome 2 and GIP on chromosome 17, may be products of ancient genome duplications on the early vertebrate lineage. A large genomic tandem duplication event that included DPP4-like and GCG genes occurred before the amphibian-mammal divergence, but one of the duplicated copies of GCG has been lost on the human lineage. Recently, a processed pseudogene of the X-chromosome-linked gene TIMM8A was inserted downstream of GCG. Some ancient duplicates of GCG may retain physiological functions in other vertebrates.