Deletion Endpoints Research Articles

Recombineering 101: Making an in-Frame Deletion Mutant.

DNA recombineering uses phage λ Red recombination functions to promote integration of DNA fragments generated by polymerase chain reaction (PCR) into the bacterial chromosome. The PCR primers are designed to have the last 18-22 nt anneal on either side of the donor DNA and to carry 40- to 50-nt 5' extensions homologous to the sequences flanking the chosen insertion site. The simplest application of the method results in knockout mutants of nonessential genes. Deletions can be constructed by replacing a portion or the entirety of a target gene with an antibiotic-resistance cassette. In some commonly used template plasmids, the antibiotic-resistance gene can be coamplified with a pair of flanking FRT (Flp recombinase recognition target) sites that, following insertion of the fragment into the chromosome, allow excision of the antibiotic-resistance cassette via the activity of the site-specific Flp recombinase. The excision step leaves behind a "scar" sequence comprising an FRT site and flanking primer annealing sequences. Removal of the cassette minimizes undesired perturbations on the expression of neighboring genes. Even so, polarity effects can result from the occurrence of stop codons within, or downstream of, the scar sequence. These problems can be avoided by the appropriate choice of the template and by designing primers so that the reading frame of the target gene is maintained past the deletion end point. This protocol is optimized for use with Salmonella enterica and Escherichia coli.

A synergistic arrangement of two unrelated IS elements facilitates adjacent deletion in Micrococcus luteus ATCC49732.

Mutants of Micrococcus luteus strain ATCC49732 lacking the yellow pigment sarcinaxanthin were observed at an unexpectedly high frequency and the molecular basis was investigated. PCR probing revealed complete deletion of the crt biosynthetic operon in 11/14 mutants. Inverse PCR was used to identify a common breakpoint 35kb downstream from crt precisely at the end of the right inverted repeat (IRR) of a partial ISMlu8 element that lies between two inversely oriented full-length ISMlu2. A total of three different breakpoints 5' to crt were found with the sequence CTAG one bp 5' to each novel junction. Analysis of 35 genomic sites with single ISMlu8 insertions showed that ISMlu8 transposase has high specificity for CTAG, implicating its key role in formation of the Δcrt deletions. No downstream deletion endpoints were observed at an immediately adjacent ISMlu8 with a nearly identical IRR in the same orientation and slightly closer to the crt operon, indicating that access of ISMlu8 transposase to the ISMlu2-flanked ISMlu8 IRR is greatly enhanced by the surrounding oppositely oriented ISMlu2s. The association of high frequency genomic rearrangement with this distinctive natural configuration of ISs from two different IS families offers a new insight into IS element evolutionary potential.

The 22q11 low copy repeats are characterized by unprecedented size and structural variability.

Low copy repeats (LCRs) are recognized as a significant source of genomic instability, driving genome variability and evolution. The Chromosome 22 LCRs (LCR22s) mediate nonallelic homologous recombination (NAHR) leading to the 22q11 deletion syndrome (22q11DS). However, LCR22s are among the most complex regions in the genome, and their structure remains unresolved. The difficulty in generating accurate maps of LCR22s has also hindered localization of the deletion end points in 22q11DS patients. Using fiber FISH and Bionano optical mapping, we assembled LCR22 alleles in 187 cell lines. Our analysis uncovered an unprecedented level of variation in LCR22s, including LCR22A alleles ranging in size from 250 to 2000 kb. Further, the incidence of various LCR22 alleles varied within different populations. Additionally, the analysis of LCR22s in 22q11DS patients and their parents enabled further refinement of the rearrangement site within LCR22A and -D, which flank the 22q11 deletion. The NAHR site was localized to a 160-kb paralog shared between the LCR22A and -D in seven 22q11DS patients. Thus, we present the most comprehensive map of LCR22 variation to date. This will greatly facilitate the investigation of the role of LCR variation as a driver of 22q11 rearrangements and the phenotypic variability among 22q11DS patients.

Sequence Analysis of a Hybrid HK022/λ Bacteriophage and the Precise Identification of the λb515 and λb519 Deletion Endpoints.

Bacteriophage O276 is a laboratory-generated hybrid that carries the immunity region of bacteriophage HK022 and all remaining genes from phage λ. Its construction was instrumental in the discovery of RNA-mediated antitermination, an intriguing alternative to the protein-mediated mechanism of transcription antitermination found in most lambdoid phages.

α0-Thalassemia Due to a 90.7 kb Deletion (– –NFLD)

We report an α0-thalassemia (α0-thal) trait in Newfoundlanders caused by a novel 90.7 kb deletion. The deletion, designated the Newfoundland deletion (– –NFLD), removes both the HBA2 and HBA1 genes, while leaving the HBZ gene intact. The 5′ deletion endpoint is within the HBAP1 pseudogene, approximately 3.7 kb upstream of the HBA2 gene. The 3′ deletion endpoint is approximately 82.5 kb downstream of the HBA1 gene, within the second intervening sequence (IVS-II) of the FAM234A gene. This is the second α0-thal deletion reported in Newfoundland families of northern European descent.

Sudanese (δβ)0-Thalassemia: Identification and Characterization of a Novel 9.6 kb Deletion

We report a case of δβ-thalassemia (δβ-thal) trait in an adult male originally from Sudan. Multiplex ligation-dependent probe amplification (MLPA) was used to localize the approximate boundaries of the deletion, followed by polymerase chain reaction (PCR) amplification and sequence analysis of the junction fragment to determine the precise deletion endpoints. The deletion spans 9594 bp, with the 5' deletion endpoint located 1560 bp upstream of the δ-globin gene and the 3' endpoint within the second intervening sequence (IVS-II) of the β-globin gene.

Mapping the deletion endpoints in individuals with 22q11.2 deletion syndrome by droplet digital PCR.

BackgroundChromosome 22q11.2 deletion syndrome (22q11DS) is the most common human microdeletion syndrome and is associated with many cognitive, neurological and psychiatric disorders. The majority of individuals have a 3 Mb deletion while others have a nested 1.5 Mb deletion, but rare atypical deletions have also been described. To date, a study using droplet digital PCR (ddPCR) has not been conducted to systematically map the chromosomal breakpoints in individuals with 22q11DS, which would provide important genotypic insight into the various phenotypes observed in this syndrome.MethodsThis study uses ddPCR to assess copy number (CN) changes within the chromosome 22q11 deletion region and allows the mapping of the deletion endpoints. We used eight TaqMan assays interspersed throughout the deleted region of 22q11.2 to characterize the deleted region of chromosome 22 in 80 individuals known to have 22q11DS by FISH. Ten EvaGreen assays were used for finer mapping of the six identified individuals with 22q11DS atypical deletions and covering different regions of chromosome 22.ResultsddPCR provided non-ambiguous CN measurements across the region, confirmed the presence of the deletion in the individuals screened, and led to the identification of five differently sized and located deletions. The majority of the participants (n = 74) had the large 3 Mb deletions, whereas three had the smaller 1.5 Mb deletions, and the remaining three had an interstitial deletion of different size.ConclusionsThe lower cost, rapid execution and high reliability and specificity provided by ddPCR for CN measurements in the 22q11 region constitutes a significant improvement over the variable CN values generated by other technologies. The ability of the ddPCR approach, to provide a high resolution mapping of deletion endpoints may result in the identification of genes that are haplo-insufficient and play a role in the pathogenesis of 22q11DS. Finally, this methodology can be applied to the characterization of other microdeletions throughout the genome.

Evaluation of Chromosome 11p Imbalances in Aniridia and Wilms Tumor Patients

Newborn sporadic aniridia patients with an 11p13 deletion including the WT1 gene have an increased risk to develop Wilms tumor. At present a risk for Wilms tumor cannot be estimated in patients with deletions not extending into, but ending close to WT1. Therefore, it is important to determine the distance of deletion endpoints from the WT1 gene and survey these patients for a longer follow-up time to obtain a more defined risk estimation. Using molecular methods, such as Multiplex Ligation-dependent Probe Amplification (MLPA), deletion endpoints can be mapped more accurately than with FISH. We describe here the analysis of six aniridia patients, in two of these the deletions extend close to the 3' end of WT1. At the ages of 3.8 and 4 years they have not developed a Wilms tumor, suggesting a low tumor risk in such patients. In addition we have studied 24 non-AN cases with a higher likelihood for WT1 alterations with MLPA and found no deletions. In conclusion newborns with aniridia should be studied with molecular methods that can determine deletion endpoints in 11p13 exactly. For a better Wilms tumor risk estimation cases with deletion endpoints close to WT1 should be followed for at least 4-5 years. Furthermore germ line intragenic deletions affecting WT1 in patients with a higher likelihood for a WT1 association, for example, bilateral tumors, genitourinary aberrations, or nephrotic syndrome, were not found in this study, suggesting that deletions are rare events.

Deletion at the SLC1A1 glutamate transporter gene co‐segregates with schizophrenia and bipolar schizoaffective disorder in a 5‐generation family

Growing evidence for genetic overlap between schizophrenia (SCZ) and bipolar disorder (BPD) suggests that causal variants of large effect on disease risk may cross traditional diagnostic boundaries. Extended multigenerational families with both SCZ and BPD cases can be a valuable resource for discovery of shared biological pathways because they can reveal the natural evolution of the underlying genetic disruptions and their phenotypic expression. We investigated a deletion at the SLC1A1 glutamate transporter gene originally identified as a copy number variant exclusively carried by members of a 5-generation Palauan family. Using an expanded sample of 21 family members, quantitative PCR confirmed the deletion in all seven individuals with psychosis, three "obligate-carrier" parents and one unaffected sibling, while four marry-in parents were non-carriers. Linkage analysis under an autosomal dominant model generated a LOD-score of 3.64, confirming co-segregation of the deletion with psychosis. For more precise localization, we determined the approximate deletion end points using alignment of next-generation sequencing data for one affected deletion-carrier and then designed PCR amplicons to span the entire deletion locus. These probes established that the deletion spans 84,298 bp, thus eliminating the entire promoter, the transcription start site, and the first 59 amino acids of the protein, including the first transmembrane Na(2+)/dicarboxylate symporter domain, one of the domains that perform the glutamate transport action. Discovery of this functionally relevant SLC1A1 mutation and its co-segregation with psychosis in an extended multigenerational pedigree provides further support for the important role played by glutamatergic transmission in the pathophysiology of psychotic disorders.

Identification of glutinous maize landraces and inbred lines with altered transcription of waxy gene

Waxy maize has little or very low content of amylose (<5 %) in grain starch and carries null mutations of the waxy (Wx) gene. With important uses as fresh maize or as an ingredient in food, textile, adhesive, and paper industries, two types of wx allele, wx-D7 and wx-D10, had been identified. In the present study, 10 accessions carrying neither wx-D7 nor wx-D10 allele were identified from a collection of 325 waxy maize accessions. The Wx locus of these 10 accessions was sequenced, but no potential causative mutation was detected. Further sequencing of the full-length complementary DNAs revealed that 6 of the 10 accessions had alternative splicing patterns while the other 4 had wild-type Wx transcripts. Among the six accessions, one generated transcripts identical to the wx-D7 allele, two had the same transcripts as the wx-D10 allele, another two generated transcripts with deletion of the 10th and the 11th exon, yet the other one produced transcripts of various lengths due to deletions from part of the 2nd exon to part of the 12th exon. The wx alleles with the above alternative splicing modes are referred to as wx-tD7, wx-tD10, wx-tD10-11, and wx-tD2-12, respectively. Real-time quantitative reverse-transcription polymerase chain reaction analysis of two waxy accessions that produced Wx transcripts showed significantly decreased expression, having only 47.3 and 3.6 % transcription level compared with B73. Sequence analysis of deletions in the transcripts with comparison with wild one showed short direct repeats at deletion endpoints, similar to reported signatures of DNA deletions. The waxy accessions present unique wx alleles for waxy maize breeding as well as for transcriptional regulation studies in plants.

Mutation of the Variant α-Tubulin TUBA8 Results in Polymicrogyria with Optic Nerve Hypoplasia

The critical importance of cytoskeletal function for correct neuronal migration during development of the cerebral cortex has been underscored by the identities of germline mutations underlying a number of human neurodevelopmental disorders. The proteins affected include TUBA1A, a major alpha-tubulin isoform, and microtubule-associated components such as doublecortin, and LIS1. Mutations in these genes are associated with the anatomical abnormality lissencephaly, which is believed to reflect failure of neuronal migration. An important recent observation has been the dependence of cortical neuronal migration upon acetylation of alpha-tubulin at lysine 40 by the histone acetyltransferase Elongator complex. Here, we describe a recognizable autosomal recessive syndrome, characterized by generalized polymicrogyria in association with optic nerve hypoplasia (PMGOH). By autozygosity mapping, we show that the molecular basis for this condition is mutation of the TUBA8 gene, encoding a variant alpha-tubulin of unknown function that is not susceptible to the lysine 40 acetylation that regulates microtubule function during cortical neuron migration. Together with the unique expression pattern of TUBA8 within the developing cerebral cortex, these observations suggest a role for this atypical microtubule component in regulating mammalian brain development.

Application of dual-genome oligonucleotidearray-based comparative genomic hybridization to the molecular diagnosis of mitochondrial DNA deletion and depletion syndromes

PurposeMitochondrial disorders constitute a group of clinically and genetically heterogeneous diseases for which molecular diagnosis has been a challenge. The current procedures for diagnosis of mitochondrial DNA deletion and depletion syndromes based on Southern analysis and quantitative polymerase chain reaction are particularly inefficient for determining important parameters of deletion endpoints and percent heteroplasmy. We have developed an improved approach for routine analyses of these disorders in a clinical laboratory. MethodsA custom-designed oligonucleotide array-based comparative genomic hybridization platform was developed to provide both tiled coverage of the entire 16.6-kb mitochondrial genome and high-density coverage of nuclear genes involved in mitochondrial biogenesis and function, for quick evaluation of mitochondrial DNA deletion and depletion. ResultsFor initial validation, the performance of this array was characterized in 20 samples with known mitochondrial DNA deletions and 12 with apparent depletions. All previously known deletions were clearly detected and the break points were correctly identified by the oligonucleotide array-based comparative genomic hybridization, within the limits of resolution of the array. The extent of mitochondrial DNA depletion and the percentage of deletion heteroplasmy were estimated using an automated computational approach that gave results comparable to previous methods. Conclusions from subsequent application of this approach with >300 new clinical samples have been in 100% concordance with those from standard methods. Finally, for one sample, we were able to identify an intragenic deletion in a nuclear gene that was responsible for the observed mitochondrial DNA depletion. ConclusionWe conclude that this custom array is capable of reliably detecting mitochondrial DNA deletion with elucidation of the deletion break points and the percentage of heteroplasmy. In addition, simultaneous detection of the copy number changes in both nuclear and mitochondrial genomes makes this dual genome array of tremendous value in the diagnoses of mitochondrial DNA depletion syndromes.

A novel double deletion underscores the importance of characterizing end points of the CFTR large rearrangements

Large genomic rearrangements in patients with cystic fibrosis (CF) account for up to 16-24% of CF alleles negative for point mutations in European populations. Herein, we identified a new large rearrangement removing exon 19 in a young CF patient, who hitherto harbored only the F508del mutation. By using LightCycler technology, we successfully and rapidly delineated the deletion end points by determining the relative copy number of a set CFTR sequence from introns 18 to 19. Fine mapping of the sequences bordering its break points was achieved using direct sequencing. We reported the first complex CFTR rearrangement containing two successive deletion events putatively linked. We evidenced the presence of short direct repeats in the vicinity of the deletions suggesting a possible replication slippage model. In this report, we also discussed the putative molecular mechanism and consequences of this complex gene rearrangement, unprecedented in CF. This complex deletion illustrates the importance of delineating the genomic rearrangement to improve our knowledge of the CFTR mutational spectrum and to better understand the molecular mechanism controlling the CFTR expression.

Characterization of a 7.6-Mb germline deletion encompassing the NF1 locus and about a hundred genes in an NF1 contiguous gene syndrome patient

We describe a large germline deletion removing the NF1 locus, identified by heterozygosity mapping based on microsatellite markers, in an 8-year-old French girl with a particularly severe NF1 contiguous gene syndrome. We used gene-dose mapping with sequence-tagged site real-time PCR to locate the deletion end points, which were precisely characterized by means of long-range PCR and nucleotide sequencing. The deletion is located on chromosome arm 17q and is exactly 7 586 986 bp long. It encompasses the entire NF1 locus and about 100 other genes, including numerous chemokine genes, an attractive in silico-selected cerebrally expressed candidate gene (designated NUFIP2, for nuclear fragile X mental retardation protein interacting protein 2; NM_020772) and four microRNA genes. Interestingly, the centromeric breakpoint is located in intron 4 of the PIPOX gene (pipecolic acid oxidase; NM_016518) and the telomeric breakpoint in intron 5 of the GGNBP2 gene (gametogenetin binding protein 2; NM_024835) coding a transcription factor. As PIPOX and GGNBP2 have the same transcriptional orientation, we postulated, and then confirmed, the existence of a chimeric transcript. This transcript, and/or haploinsufficiency of one or several deleted genes, could explain the clinical severity of the syndrome in this patient.

Screening of deletions and RFLP analysis in Turkish DMD/BMD families by PCR.

We have screened 76 DMD and 5 BMD patients for deletions, using two separate Multiplex gene amplification systems. The use of both systems together revealed deletions in 52% of the cases in the Turkish population. The majority of these deletions (33/37) were found to be localized within the central region of the dystrophin gene. The remaining deletions were mapped to the proximal hotspot. Deletion end-points were identified by PCR and/or by Southern blot analysis with cDNA probes, and exceptions to the Open Reading Frame (ORF) hypothesis are discussed. PCR-based techniques to screen the pERT87.15/XmnI, pERT87.15/BamHI, and pERT87.8/TaqI polymorphisms were used for linkage analysis in the Turkish DMD/BMD families, and approximately 70% of the mothers at risk were found to be informative for at least one of these polymorphisms studied.

22q11.2 Distal Deletion: A Recurrent Genomic Disorder Distinct from DiGeorge Syndrome and Velocardiofacial Syndrome

Microdeletions within chromosome 22q11.2 cause a variable phenotype, including DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS). About 97% of patients with DGS/VCFS have either a common recurrent approximately 3 Mb deletion or a smaller, less common, approximately 1.5 Mb nested deletion. Both deletions apparently occur as a result of homologous recombination between nonallelic flanking low-copy repeat (LCR) sequences located in 22q11.2. Interestingly, although eight different LCRs are located in proximal 22q, only a few cases of atypical deletions utilizing alternative LCRs have been described. Using array-based comparative genomic hybridization (CGH) analysis, we have detected six unrelated cases of deletions that are within 22q11.2 and are located distal to the approximately 3 Mb common deletion region. Further analyses revealed that the rearrangements had clustered breakpoints and either a approximately 1.4 Mb or approximately 2.1 Mb recurrent deletion flanked proximally by LCR22-4 and distally by either LCR22-5 or LCR22-6, respectively. Parental fluorescence in situ hybridization (FISH) analyses revealed that none of the available parents (11 out of 12 were available) had the deletion, indicating de novo events. All patients presented with characteristic facial dysmorphic features. A history of prematurity, prenatal and postnatal growth delay, developmental delay, and mild skeletal abnormalities was prevalent among the patients. Two patients were found to have a cardiovascular malformation, one had truncus arteriosus, and another had a bicuspid aortic valve. A single patient had a cleft palate. We conclude that distal deletions of chromosome 22q11.2 between LCR22-4 and LCR22-6, although they share some characteristic features with DGS/VCFS, represent a novel genomic disorder distinct genomically and clinically from the well-known DGS/VCF deletion syndromes.

Molecular mechanisms and diagnosis of chromosome 22q11.2 rearrangements

Several recurrent, constitutional genomic disorders are present on chromosome 22q. These include the translocations and deletions associated with DiGeorge and velocardiofacial syndrome and the translocations that give rise to the recurrent t(11;22) supernumerary der(22) syndrome (Emanuel syndrome). The rearrangement breakpoints on 22q cluster around the chromosome-specific segmental duplications of proximal 22q11, which are involved in the etiology of these disorders. While the deletions are the result of nonallelic homologous recombination (NAHR) between low copy repeats or segmental duplications within 22q11, the t(11;22) is the result of rearrangement between palindromic AT-rich repeats on 11q and 22q. Here we describe the mechanisms responsible for these recurrent rearrangements, discuss the recurrent deletion endpoints that are the result of NAHR between chromosome 22q specific low copy repeats as well as present current diagnostic approaches to deletion detection.

Detailed analysis of 22q11.2 with a high density MLPA probe set

The presence of chromosome-specific low-copy repeats (LCRs) predisposes chromosome 22 to deletions and duplications. The current diagnostic procedure for detecting aberrations at 22q11.2 is chromosomal analysis coupled with fluorescence in situ hybridization (FISH) or PCR-based multiplex ligation dependent probe amplification (MLPA). However, there are copy number variations (CNVs) in 22q11.2 that are only detected by high-resolution platforms such as array comparative genomic hybridization (aCGH). We report on development of a high-definition MLPA (MLPA-HD) 22q11 kit that detects copy number changes at 37 loci on the long arm of chromosome 22. These include the 3-Mb region commonly deleted in DiGeorge/velocardiofacial syndrome (DGS/VCFS), the cat eye syndrome (CES) region, and more distal regions in 22q11 that have recently been shown to be deleted. We have used this MLPA-HD probe set to analyze 363 previously well-characterized samples with a variety of different rearrangements at 22q11 and demonstrate that it can detect copy number alterations with high sensitivity and specificity. In addition to detection of the common recurrent deletions associated with DGS/VCFS, variant and novel chromosome 22 aberrations have been detected. These include duplications within as well as deletions distal to this region. Further, the MLPA-HD detects deletion endpoint differences between patients with the common 3-Mb deletion. The MLPA-HD kit is proposed as a cost effective alternative to the currently available detection methods for individuals with features of the 22q11 aberrations. In patients with the relevant phenotypic characteristics, this MLPA-HD probe set could replace FISH for the clinical diagnosis of 22q11.2 deletions and duplications.

A linear plasmid truncation induces unidirectional flagellar phase change in H:z66 positive Salmonella Typhi

The process by which bacteria regulate flagellar expression is known as phase variation and in Salmonella enterica this process permits the expression of one of two flagellin genes, fliC or fljB, at any one time. Salmonella Typhi (S. Typhi) is normally not capable of phase variation of flagellar antigen expression as isolates only harbour the fliC gene (H:d) and lacks an equivalent fljB locus. However, some S. Typhi isolates, exclusively from Indonesia, harbour an fljB equivalent encoded on linear plasmid, pBSSB1 that drives the expression of a novel flagellin named H:z66. H:z66+S. Typhi isolates were stimulated to change flagellar phase and genetically analysed for the mechanism of variation. The phase change was demonstrated to be unidirectional, reverting to expression from the resident chromosomal fliC gene. DNA sequencing demonstrated that pBSSB1 linear DNA was still detectable but that these derivatives had undergone deletion and were lacking fljAz66 (encoding a flagellar repressor) and fljBz66. The deletion end-point was found to involve one of the plasmid termini and a palindromic repeat sequence within fljBz66, distinct to that found at the terminus of pBSSB1. These data demonstrate that, like some Streptomyces linear elements, at least one of the terminal inverted repeats of pBSSB1 is non-essential, but that a palindromic repeat sequence may be necessary for replication.

Molecular definition of chromosome arm 5q deletion end points and detection of hidden aberrations in patients with myelodysplastic syndromes and isolated del(5q) using oligonucleotide array CGH

Isolated deletions of the long arm of chromosome 5, del(5q), are observed in 10% of myelodysplastic syndromes (MDS) and are associated with a more favorable prognosis, although the clinical course varies considerably. If one or more additional chromosomal aberrations are present, this correlates with a significantly shorter overall survival. To assess the frequency of hidden abnormalities in cases with an isolated cytogenetic del(5q), we have performed a genome wide high resolution 44 K 60mer oligonucleotide array comparative genomic hybridization (aCGH) study using DNA from bone marrow cells of 12 MDS and one AML patient. In one case a single additional hidden 5.6 Mb deletion of 13q14 and in another case multiple larger aberrations involving many chromosomes were found. Fluorescence in situ hybridization demonstrated that aberrations present in 35% of the bone marrow cells can be detected by aCGH. Furthermore with oligonucleotide aCGH the deletion end points in 5q were mapped precisely, revealing a cluster of proximal breakpoints in band q14.3 (n = 8) and a distal cluster between bands q33.2 and q34 (n = 11). This study shows the high resolution of oligonucleotide CGH arrays for precisely mapping genomic alterations and for refinement of deletion end points. In addition, the high sensitivity of this method enables the study of whole bone marrow cells from MDS patients, a disease with a low blast count.