Breakpoint Junction Sequences Research Articles

Reclassification of DMD Duplications as Benign: Recommendations for Cautious Interpretation of Variants Identified in Prenatal Screening.

Duplications are the main type of dystrophin gene (DMD) variants, which typically cause dystrophinopathies such as Duchenne muscular dystrophy and Becker muscular dystrophy. Maternally inherited exon duplication in DMD in fetuses is a relatively common finding of genetic screening in clinical practice. However, there is no standard strategy for interpretation of the pathogenicity of DMD duplications during prenatal screening, especially for male fetuses, in which maternally inherited pathogenic DMD variants more frequently cause dystrophinopathies. Here, we report three non-contiguous DMD duplications identified in a woman and her male fetus during prenatal screening. Multiplex ligation probe amplification and long-read sequencing were performed on the woman and her family members to verify the presence of DMD duplications. Structural rearrangements in the DMD gene were mapped by long-read sequencing, and the breakpoint junction sequences were validated using Sanger sequencing. The woman and her father carried three non-contiguous DMD duplications. Long-read and Sanger sequencing revealed that the woman's father carried an intact DMD copy and a complex structural rearrangement of the DMD gene. Therefore, we reclassified these three non-contiguous DMD duplications, one of which is listed as pathogenic, as benign. We postulate that breakpoint analysis should be performed on identified DMD duplication variants, and the pathogenicity of the duplications found during prenatal screening should be interpreted cautiously for clinical prediction and genetic/reproductive counseling.

Identification and characterization of occult human-specific LINE-1 insertions using long-read sequencing technology.

Long Interspersed Element-1 (LINE-1) retrotransposition contributes to inter- and intra-individual genetic variation and occasionally can lead to human genetic disorders. Various strategies have been developed to identify human-specific LINE-1 (L1Hs) insertions from short-read whole genome sequencing (WGS) data; however, they have limitations in detecting insertions in complex repetitive genomic regions. Here, we developed a computational tool (PALMER) and used it to identify 203 non-reference L1Hs insertions in the NA12878 benchmark genome. Using PacBio long-read sequencing data, we identified L1Hs insertions that were absent in previous short-read studies (90/203). Approximately 81% (73/90) of the L1Hs insertions reside within endogenous LINE-1 sequences in the reference assembly and the analysis of unique breakpoint junction sequences revealed 63% (57/90) of these L1Hs insertions could be genotyped in 1000 Genomes Project sequences. Moreover, we observed that amplification biases encountered in single-cell WGS experiments led to a wide variation in L1Hs insertion detection rates between four individual NA12878 cells; under-amplification limited detection to 32% (65/203) of insertions, whereas over-amplification increased false positive calls. In sum, these data indicate that L1Hs insertions are often missed using standard short-read sequencing approaches and long-read sequencing approaches can significantly improve the detection of L1Hs insertions present in individual genomes.

Distinct patterns of complex rearrangements and a mutational signature of microhomeology are frequently observed in PLP1 copy number gain structural variants

BackgroundWe investigated the features of the genomic rearrangements in a cohort of 50 male individuals with proteolipid protein 1 (PLP1) copy number gain events who were ascertained with Pelizaeus-Merzbacher disease (PMD; MIM: 312080). We then compared our new data to previous structural variant mutagenesis studies involving the Xq22 region of the human genome. The aggregate data from 159 sequenced join-points (discontinuous sequences in the reference genome that are joined during the rearrangement process) were studied. Analysis of these data from 150 individuals enabled the spectrum and relative distribution of the underlying genomic mutational signatures to be delineated.MethodsGenomic rearrangements in PMD individuals with PLP1 copy number gain events were investigated by high-density customized array or clinical chromosomal microarray analysis and breakpoint junction sequence analysis.ResultsHigh-density customized array showed that the majority of cases (33/50; ~ 66%) present with single duplications, although complex genomic rearrangements (CGRs) are also frequent (17/50; ~ 34%). Breakpoint mapping to nucleotide resolution revealed further previously unknown structural and sequence complexities, even in single duplications. Meta-analysis of all studied rearrangements that occur at the PLP1 locus showed that single duplications were found in ~ 54% of individuals and that, among all CGR cases, triplication flanked by duplications is the most frequent CGR array CGH pattern observed. Importantly, in ~ 32% of join-points, there is evidence for a mutational signature of microhomeology (highly similar yet imperfect sequence matches).ConclusionsThese data reveal a high frequency of CGRs at the PLP1 locus and support the assertion that replication-based mechanisms are prominent contributors to the formation of CGRs at Xq22. We propose that microhomeology can facilitate template switching, by stabilizing strand annealing of the primer using W-C base complementarity, and is a mutational signature for replicative repair.

A novel gross deletion and breakpoint junction sequence analysis of ATP7B in a Chinese family with Wilson disease using next‑generation sequencing and Sanger sequencing.

Wilson disease (WD) is a rare autosomal recessive genetic disorder that causes abnormal copper metabolism, resulting in pathological accumulation of copper in the liver, brain and other organs. Mutations in the ATPase copper transporter 7B (ATP7B) gene, which encodes a membrane P-type adenosine triphosphatase, have been identified as being responsible for WD. The present study analyzed clinical data and collected DNA samples from a pediatric patient with WD and her healthy parents. Mutation screening for ATP7B was performed using direct sequencing, multiplex ligation-dependent probe amplification(MLPA), next-generation sequencing (NGS) and Sanger sequencing of the breakpoint junction sequence. The patient (age, 2.7 years) presented with early-onset hepatic disease. The present study identified compound heterozygous mutations of ATP7B, including a heterozygous mutation (p.Arg1,041Trp) and a novel heterozygous gross deletion of a 57,771 bp fragment (chr13: 52490972-52548742) (GRCh37) from partial exon2- exon21 to external ATP7B sequence (15.833bp) in the patient. Analysis of the family members of the patient showed that the missense mutation and the gross deletion mutation were inherited from her mother and father, respectively. Microhomology and inverted repeat sequences, which may mediate the deletion mutation, were identified through sequence analysis on both sides of the breakpoints of this deletion. The present study provided additional information on the genotypic spectrum of the ATP7B gene, particularly with regard to early onset hepatic disease, as observed in the present patient with WD. The identification of the precise breakpoint junction sequence warrants further investigation of DNA break and recombination mechanisms. In detecting precise deletions, the NGS associated with Sanger sequencing of breakpoint junction sequence have been found to have more advantages than MLPA.

Xq22 deletions and correlation with distinct neurological disease traits in females: Further evidence for a contiguous gene syndrome

Xq22 deletions that encompass PLP1 (Xq22-PLP1-DEL) are notable for variable expressivity of neurological disease traits in females ranging from a mild late-onset form of spastic paraplegia type 2 (MIM# 312920), sometimes associated with skewed X-inactivation, to an early-onset neurological disease trait (EONDT) of severe developmental delay, intellectual disability, and behavioral abnormalities. Size and gene content of Xq22-PLP1-DEL vary and were proposed as potential molecular etiologies underlying variable expressivity in carrier females where two smallest regions of overlap (SROs) were suggested to influence disease. We ascertained a cohort of eight unrelated patients harboring Xq22-PLP1-DEL and performed high-density array comparative genomic hybridizationand breakpoint-junction sequencing. Molecular characterization of Xq22-PLP1-DEL from 17 cases (eight herein and nine published) revealed an overrepresentation of breakpoints that reside within repeats (11/17, ~65%) and the clustering of ~47% of proximal breakpoints in a genomic instability hotspot with characteristic non-B DNA density. These findings implicate a potential role for genomic architecture in stimulating the formation of Xq22-PLP1-DEL. The correlation of Xq22-PLP1-DEL gene content with neurological disease trait in female cases enabled refinement of the associated SROs to a single genomic interval containing six genes. Our data support the hypothesis that genes contiguous to PLP1 contribute to EONDT.

High-efficiency CRISPR induction of t(9;11) chromosomal translocations and acute leukemias in human blood stem cells

AbstractChromosomal rearrangements involving the mixed lineage leukemia (MLL) gene, also known as KMT2A, are often observed in human leukemias and are generally associated with a poor prognosis. To model these leukemias, we applied clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL chromosomal rearrangements in human hematopoietic stem and progenitor cells purified from umbilical cord blood. Electroporation of ribonucleoprotein complexes containing chemically modified synthetic single guide RNAs and purified Cas9 protein induced translocations between chromosomes 9 and 11 [t(9;11)] at an efficiency >1%. Transplantation of gene-edited cells into immune-compromised mice rapidly induced acute leukemias of different lineages and often with multiclonal origins dictated by the duration of in vitro culture prior to transplantation. Breakpoint junction sequences served as biomarkers to monitor clonal selection and progression in culture and in vivo. High-dimensional cell surface and intracellular protein analysis by mass cytometry (CyTOF) revealed that gene-edited leukemias recapitulated disease-specific protein expression observed in human patients and showed that MLL-rearranged (MLLr) mixed phenotype acute leukemias (MPALs) were more similar to acute myeloid leukemias (AMLs) than to acute lymphoblastic leukemias (ALLs). Therefore, highly efficient generation of MLL chromosomal translocations in primary human blood stem cells using CRISPR/Cas9 reliably models human acute MLLr leukemia and provides an experimental platform for basic and translational studies of leukemia biology and therapeutics.

Nonrecurrent PMP22-RAI1 contiguous gene deletions arise from replication-based mechanisms and result in Smith-Magenis syndrome with evident peripheral neuropathy.

Hereditary neuropathy with liability to pressure palsies (HNPP) and Smith-Magenis syndrome (SMS) are genomic disorders associated with deletion copy number variants involving chromosome 17p12 and 17p11.2, respectively. Nonallelic homologous recombination (NAHR)-mediated recurrent deletions are responsible for the majority of HNPP and SMS cases; the rearrangement products encompass the key dosage-sensitive genes PMP22 and RAI1, respectively, and result in haploinsufficiency for these genes. Less frequently, nonrecurrent genomic rearrangements occur at this locus. Contiguous gene duplications encompassing both PMP22 and RAI1, i.e., PMP22-RAI1 duplications, have been investigated, and replication-based mechanisms rather than NAHR have been proposed for these rearrangements. In the current study, we report molecular and clinical characterizations of six subjects with the reciprocal phenomenon of deletions spanning both genes, i.e., PMP22-RAI1 deletions. Molecular studies utilizing high-resolution array comparative genomic hybridization and breakpoint junction sequencing identified mutational signatures that were suggestive of replication-based mechanisms. Systematic clinical studies revealed features consistent with SMS, including features of intellectual disability, speech and gross motor delays, behavioral problems and ocular abnormalities. Five out of six subjects presented clinical signs and/or objective electrophysiologic studies of peripheral neuropathy. Clinical profiling may improve the clinical management of this unique group of subjects, as the peripheral neuropathy can be more severe or of earlier onset as compared to SMS patients having the common recurrent deletion. Moreover, the current study, in combination with the previous report of PMP22-RAI1 duplications, contributes to the understanding of rare complex phenotypes involving multiple dosage-sensitive genes from a genetic mechanistic standpoint.

Nonrecurrent 17p11.2p12 Rearrangement Events that Result in Two Concomitant Genomic Disorders: The PMP22-RAI1 Contiguous Gene Duplication Syndrome

The genomic duplication associated with Potocki-Lupski syndrome (PTLS) maps in close proximity to the duplication associated with Charcot-Marie-Tooth disease type 1A (CMT1A). PTLS is characterized by hypotonia, failure to thrive, reduced body weight, intellectual disability, and autistic features. CMT1A is a common autosomal dominant distal symmetric peripheral polyneuropathy. The key dosage-sensitive genes RAI1 and PMP22 are respectively associated with PTLS and CMT1A. Recurrent duplications accounting for the majority of subjects with these conditions are mediated by nonallelic homologous recombination between distinct low-copy repeat (LCR) substrates. The LCRs flanking a contiguous genomic interval encompassing both RAI1 and PMP22 do not share extensive homology; thus, duplications encompassing both loci are rare and potentially generated by a different mutational mechanism. We characterized genomic rearrangements that simultaneously duplicate PMP22 and RAI1, including nine potential complex genomic rearrangements, in 23 subjects by high-resolution array comparative genomic hybridization and breakpoint junction sequencing. Insertions and microhomologies were found at the breakpoint junctions, suggesting potential replicative mechanisms for rearrangement formation. At the breakpoint junctions of these nonrecurrent rearrangements, enrichment of repetitive DNA sequences was observed, indicating that they might predispose to genomic instability and rearrangement. Clinical evaluation revealed blended PTLS and CMT1A phenotypes with a potential earlier onset of neuropathy. Moreover, additional clinical findings might be observed due to the extra duplicated material included in the rearrangements. Our genomic analysis suggests replicative mechanisms as a predominant mechanism underlying PMP22-RAI1 contiguous gene duplications and provides further evidence supporting the role of complex genomic architecture in genomic instability.

Alu-mediated diverse and complex pathogenic copy-number variants within human chromosome 17 at p13.3.

Alu repetitive elements are known to be major contributors to genome instability by generating Alu-mediated copy-number variants (CNVs). Most of the reported Alu-mediated CNVs are simple deletions and duplications, and the mechanism underlying Alu-Alu-mediated rearrangement has been attributed to non-allelic homologous recombination (NAHR). Chromosome 17 at the p13.3 genomic region lacks extensive low-copy repeat architecture; however, it is highly enriched for Alu repetitive elements, with a fraction of 30% of total sequence annotated in the human reference genome, compared with the 10% genome-wide and 18% on chromosome 17. We conducted mechanistic studies of the 17p13.3 CNVs by performing high-density oligonucleotide array comparative genomic hybridization, specifically interrogating the 17p13.3 region with ∼150 bp per probe density; CNV breakpoint junctions were mapped to nucleotide resolution by polymerase chain reaction and Sanger sequencing. Studied rearrangements include 5 interstitial deletions, 14 tandem duplications, 7 terminal deletions and 13 complex genomic rearrangements (CGRs). Within the 17p13.3 region, Alu-Alu-mediated rearrangements were identified in 80% of the interstitial deletions, 46% of the tandem duplications and 50% of the CGRs, indicating that this mechanism was a major contributor for formation of breakpoint junctions. Our studies suggest that Alu repetitive elements facilitate formation of non-recurrent CNVs, CGRs and other structural aberrations of chromosome 17 at p13.3. The common observation of Alu-mediated rearrangement in CGRs and breakpoint junction sequences analysis further demonstrates that this type of mechanism is unlikely attributed to NAHR, but rather may be due to a recombination-coupled DNA replicative repair process.

Clustering of genomic breakpoints at the MLL locus in therapy‐related acute leukemia with t(4;11)(q21;q23)

Genomic characterization of translocation breakpoints is relevant to identify possible mechanisms underlying their origin. The consistent association of anthracylines (e.g., epirubicin and idarubicin) in inducing therapy-related acute leukemias (t-AL) with mixed lineage leukemia (MLL) gene rearrangement suggests that MLL translocations are causative events for t-AL. Using asymmetric multiplex PCR strategy followed by direct DNA sequencing, we characterized the genomic breakpoints of the MLL and AFF1 genes in two patients who developed t-AL with t(4;11)(q21;q23). Chemotherapeutic treatment of the primary disease in both patients included topoisomerase II (topo II) targeting agents. In one case, the MLL breakpoint was located in intron 9 at nucleotide position chr11:118354284 while the AFF1 breakpoint was in intron 3 at nucleotide position chr4:87992070. The breakpoint junction sequences revealed an insertion of two nucleotides at the MLL-AFF1 junction. In the other patient, the MLL breakpoint was located in intron 11 at nucleotide position chr11:118359130-32 and the AFF1 break was in intron 3 at nucleotide position chr4:87996215-17. The MLL breakpoint found in the latter patient was identical to that of two previously reported cases, strongly suggesting the presence of a preferential site of DNA cleavage in the presence of topo II inhibitor. In addition, microhomologies at the breakpoint junctions were indicative of DNA repair by the non-homologous end joining (NHEJ) pathway. This study further supports the evidence that MLL breakpoints in therapy-related acute leukemia with MLL-AFF1 are clustered in the telomeric half of the breakpoint cluster region that contains topo II recognition sites.

The role of topoisomerase II beta on breakage and proximity of RUNX1 to partner alleles RUNX1T1 and EVI1

Rearrangements involving the RUNX1 gene account for approximately 15% of balanced translocations in therapy-related acute myeloid leukemia (t-AML) patients and are one of the most common genetic abnormalities observed in t-AML. Drugs targeting the topoisomerase II (TOP2) enzyme are implicated in t-AML; however, the mechanism is not well understood and to date a single RUNX1-RUNX1T1 t-AML breakpoint junction sequence has been published. Here we report an additional five breakpoint junction sequences from t-AML patients with the RUNX1- RUNX1T1 translocation. Using a leukemia cell line model, we show that TOP2 beta (TOP2B) is required for induction of RUNX1 chromosomal breaks by the TOP2 poison etoposide and that, while TOP2 alpha (TOP2A) and TOP2B proteins are both present on RUNX1 and RUNX1T1 chromatin, only the TOP2B enrichment reached significance following etoposide exposure at a region on RUNX1 where translocations occur. Furthermore, we demonstrate that TOP2B influences the separation between RUNX1 and two translocation partners (RUNX1T1 and EVI) in the nucleus of lymphoid cells. Specifically, we identified a TOP2B-dependent increase in the number of nuclei displaying juxtaposed RUNX1 and RUNX1T1 loci following etoposide treatment.

Genomic rearrangements at the FRA2H common fragile site frequently involve non-homologous recombination events across LTR and L1(LINE) repeats

Common fragile sites (cFSs) are non-random chromosomal regions that are prone to breakage under conditions of replication stress. DNA damage and chromosomal alterations at cFSs appear to be critical events in the development of various human diseases, especially carcinogenesis. Despite the growing interest in understanding the nature of cFS instability, only a few cFSs have been molecularly characterised. In this study, we fine-mapped the location of FRA2H using six-colour fluorescence in situ hybridisation and showed that it is one of the most active cFSs in the human genome. FRA2H encompasses approximately 530 kb of a gene-poor region containing a novel large intergenic non-coding RNA gene (AC097500.2). Using custom-designed array comparative genomic hybridisation, we detected gross and submicroscopic chromosomal rearrangements involving FRA2H in a panel of 54 neuroblastoma, colon and breast cancer cell lines. The genomic alterations frequently involved different classes of long terminal repeats and long interspersed nuclear elements. An analysis of breakpoint junction sequence motifs predominantly revealed signatures of microhomology-mediated non-homologous recombination events. Our data provide insight into the molecular structure of cFSs and sequence motifs affected by their activation in cancer. Identifying cFS sequences will accelerate the search for DNA biomarkers and targets for individualised therapies.

NIPBL rearrangements in Cornelia de Lange syndrome: evidence for replicative mechanism and genotype–phenotype correlation

PurposeCornelia de Lange syndrome (CdLS) is a multisystem congenital anomaly disorder characterized by mental retardation, limb abnormalities, distinctive facial features, and hirsutism. Mutations in three genes involved in sister chromatid cohesion, NIPBL, SMC1A, and SMC3, account for ~55% of CdLS cases. The molecular etiology of a significant fraction of CdLS cases remains unknown. We hypothesized that large genomic rearrangements of cohesin complex subunit genes may play a role in the molecular etiology of this disorder. MethodsCustom high-resolution oligonucleotide array comparative genomic hybridization analyses interrogating candidate cohesin genes and breakpoint junction sequencing of identified genomic variants were performed. ResultsOf the 162 patients with CdLS, for whom mutations in known CdLS genes were previously negative by sequencing, deletions containing NIPBL exons were observed in 7 subjects (~5%). Breakpoint sequences in five patients implicated microhomology-mediated replicative mechanisms—such as serial replication slippage and fork stalling and template switching/microhomology-mediated break-induced replication—as a potential predominant contributor to these copy number variations. Most deletions are predicted to result in haploinsufficiency due to heterozygous loss-of-function mutations; such mutations may result in a more severe CdLS phenotype. ConclusionOur findings suggest a potential clinical utility to testing for copy number variations involving NIPBL when clinically diagnosed CdLS cases are mutation-negative by DNA-sequencing studies.Genet Med 2012:14(3):313–322

Mechanisms in Leukemia-Associated Chromosomal Translocations: Novel, High-Throughput Sequencing Method to Detect Topoisomerase II (TOP2) Cleavage Complexes Genome-Wide with Exact Base Resolution,

Abstract 3445Associations of chemotherapeutic TOP2 poisons with secondary leukemia have implicated TOP2-mediated DNA damage in balanced chromosomal translocations underlying many common forms of leukemia. “TOP2 poisons” convert native TOP2 into a cellular toxin by disrupting the cleavage/re-ligation equilibrium, either by decreasing the reverse rate of re-ligation or increasing the forward rate of cleavage, thus increasing cleavage complexes and causing DNA strand breaks that can promote recombination or initiate apoptosis. Besides anticancer chemotherapy, several dietary substances and the benzene metabolite p-benzoquinone are TOP2 poisons. Population and molecular epidemiology, translocation breakpoint junction sequences, temporal origins of translocations, and correlations of TOP2 in vitro cleavage sites with translocation breakpoints have pointed to a model in which TOP2 is the DNA damage mediator and resolution of TOP2 cleavage complexes, whether induced by chemotherapy, dietary substances, environmental toxins or ROS mediated damage, forms translocation breakpoint junctions. Still, the DNA damage mechanism(s) remain controversial. Investigation of cleavage complexes at the DNA sequence level in a human hematopoietic progenitor cell model that approximates target cells for translocations is the critical next step in testing this model. We invented a high-throughput sequencing-based method (Provisional Patent Filed) to address cause-and-effect relationships between TOP2 cleavage complexes and translocation breakpoints in the context of DNA topological structure in the chromatin of human hematopoietic cells.TOP2 relaxes supercoiled DNA by transiently cleaving and re-ligating both strands of the double helix. Each subunit of the TOP2 homodimer forms a phosphodiester bond with the base 3' to the cleavage. This creates a fleeting covalent TOP2-DNA intermediate called the cleavage complex with 4-base staggered DNA ends tethered by the enzyme. We implemented, refined, optimized and validated a novel assay system for immunodepletion of the alpha isoform of TOP2 (TOP2A) including DNA-bound TOP2A, with concomitant isolation and purification of the DNA from cleavage complexes in human hematopoietic cells. By taking advantage of the covalent phosphodiester bonds between TOP2A and DNA, the activity of calf intestinal phosphatase (CIP) (i.e. hydrolysis of phosphodiester bonds via removal of 5' phosphates) was used for a purpose never used before: to release DNA from cleavage complexes at exact sites of cleavage. These steps were performed in CEM cells and, to better mimic target cells for translocations, fresh cord blood mononuclear cells (MNCs) from three newborn infants. Western blot and Q-PCR analyses proved that we achieved: 1) isolation and immunodepletion of TOP2A and TOP2A-bound DNA, 2) CIP release of TOP2A-bound DNA from the cleavage complexes, and 3) quantitative enrichment of DNA amplicons near known MLL translocation breakpoint hotspots over that obtained using a negative control antibody for immunodepletion. By morphology and immunophenotype, the cord blood MNCs contained lymphocyte, monocyte, and minor CMP and GMP populations, and they mainly were in G0/G1 by cell cycle analysis. Even though TOP2A cleavage complex enrichment was evident, on Western blot analysis the TOP2A in the cord blood MNCs appeared predominantly degraded, which is consistent with known TOP2A cell cycle dependence and downregulation in quiescent cells.This validation forms the basis for the next steps in the assay to localize cleavage complexes at single base resolution genome-wide through high-throughput sequencing of DNA ends created by TOP2 and mapping them to the genome. This strategy comprises an entirely novel application of high-throughput sequencing with many possible future uses to define TOP2 cleavage complexes, as well as other adaptations to identify covalent DNA modifications with exact base precision. Secondary leukemias are a growing problem, and the incidence of infant leukemia where TOP2 poisons also have been implicated is increasing. Solving how DNA breaks arise and form chromosomal translocations would have profound implications for anticancer treatment and leukemia prevention. Disclosures:No relevant conflicts of interest to declare.

Unique Familial MLL-rearranged Precursor B Cell Infant Acute Lymphoblastic Leukemia (ALL) in Non-Twin Siblings

Abstract 2417Leukemia is the commonest malignancy in infants, the most frequently occurring form of which is infant ALL. When ALL occurs in infants the disease is clinically aggressive and associated with poor outcome. MLL gene rearrangements producing transforming fusion oncoproteins are found in 75% of infant ALL and they are adverse prognostic factors. Infant ALL has never occurred in families except in monozygous twins, where concordance in leukemia occurrence is nearly 100%. Identical but non-germline genomic breakpoint junction sequences have pointed to an in utero origin of MLL gene rearrangements in these twin cases, where it is believed that metastasis of cells with the rearrangement occurs from one twin to the other via the placental circulation. Here we describe two highly novel siblings both deceased from precursor B cell infant ALL (ages at diagnosis: proband 160 d, sibling 121 d). Remarkably, the second of these two decedents is survived by a now 3 year-old monozygous twin who is as yet unaffected.MLLrearrangements in the leukemia blasts of both affected siblings were characterized by conventional cytogenetics and/or FISH, M-FISH, high resolution Illumina 610K Bead Chip SNP array and Southern blot analysis. MLL genomic breakpoint junction sequences and fusion transcripts were defined using panhandle PCR approaches, PCR with gene-specific primers and reverse transcriptase PCR. The twins were confirmed to be monozygous using the genotype calls from SNP array analysis of the peripheral blood and bone marrow of the unaffected and affected twins, respectively. Quantitative real-time PCR analysis of leukemia DNA was used to determine the allele specific sequences of the NQO1 (NADPH quinone oxidorecutase 1) gene, an inactivating polymorphism in which previously was implicated as a risk factor for MLL-rearranged infant ALL.The complex karyotype in the leukemia cells of the proband was 46, XX, der(2) t(2;3) (q3?;?), der(3) ?t(3;4;11), del(4) (q21), der(11) ?del(11) (p11.2) t(3;11) (?;q23).ish der(3) (5'MLL+), der(11) (3'MLL+) [14]/46, XX[8], suggesting extensive damage to the genome. Consistent with a three-way t(3;4;11) translocation, two alternately spliced 5'-MLL-MLLT2(AF-4)-3' fusion transcripts were identified, indicating disruption of the chromosome band 4q21 partner gene MLLT2. Also consistent with the three-way translocation, reverse panhandle PCR detected a 5'-partner-MLL-3' genomic breakpoint junction fusing 3' MLL to the upstream region of a highly novel chromosome 3 gene encoding a nucleotidyltransferase fold protein C3ORF31 (Accession no. NM_138807; Kuchta, 2009). Unlike in the proband, the ALL of the affected twin exhibited a simple t(4;11)(q21;q23) translocation, the reciprocal genomic breakpoint junctions of which fusing MLL and MLLT2 also have been characterized. Similar to non-familial infant ALL, the genomic breakpoint junctions in both infants contained sequence features of nonhomologous end joining DNA repair. The different MLL gene rearrangements in the leukemia cells in the affected siblings indicate that the translocations were not hereditary. Even though the NQO1 inactivating polymorphism is one genetic risk factor for infant ALL, the NQO1 genotype was wild-type in both affected siblings.Further studies in this uniquely afflicted family with two siblings who succumbed to infant ALL and a monozygous twin of one of the decedents surviving beyond infancy unaffected, will provide a one-time opportunity to capture novel mutations predisposing to the development of, and cooperating with, MLL translocations in infant ALL. The MLLT2 involvement in both cases has even further implications for the knowledge to be gained because the MLL-MLLT2 rearrangement occurs in 50% of infant ALL and adversely impacts outcome. Disclosures:Felix:Children's Hospital of Philadelphia: Methods and Kits for Analysis of Chromosomal Rearrangements Associated with Leukemia - U.S. Patent # 6,368,791 issued April 9, 2002.

Diverse mutational mechanisms cause pathogenic subtelomeric rearrangements

Chromosome rearrangements are a significant cause of intellectual disability and birth defects. Subtelomeric rearrangements, including deletions, duplications and translocations of chromosome ends, were first discovered over 40 years ago and are now recognized as being responsible for several genetic syndromes. Unlike the deletions and duplications that cause some genomic disorders, subtelomeric rearrangements do not typically have recurrent breakpoints and involve many different chromosome ends. To capture the molecular mechanisms responsible for this heterogeneous class of chromosome abnormality, we coupled high-resolution array CGH with breakpoint junction sequencing of a diverse collection of subtelomeric rearrangements. We analyzed 102 breakpoints corresponding to 78 rearrangements involving 28 chromosome ends. Sequencing 21 breakpoint junctions revealed signatures of non-homologous end-joining, non-allelic homologous recombination between interspersed repeats and DNA replication processes. Thus, subtelomeric rearrangements arise from diverse mutational mechanisms. In addition, we find hotspots of subtelomeric breakage at the end of chromosomes 9q and 22q; these sites may correspond to genomic regions that are particularly susceptible to double-strand breaks. Finally, fine-mapping the smallest subtelomeric rearrangements has narrowed the critical regions for some chromosomal disorders.

Evidence for disease penetrance relating to CNV size: Pelizaeus–Merzbacher disease and manifesting carriers with a familial 11 Mb duplication at Xq22

The potential causes for the incomplete penetrance of Pelizaeus-Merzbacher disease (PMD) in female carriers of PLP1 mutations are not well understood. We present a family with a boy having PMD in association with PLP1 duplication and three females who are apparent manifesting carriers. Custom high-resolution oligonucleotide array comparative genomic hybridization (aCGH) and breakpoint junction sequencing were performed and revealed a familial complex duplication consisting of a small duplicated genomic interval (∼56 kb) and a large segmental duplication (∼11 Mb) that resulted in a PLP1 copy number variation gain. Breakpoint junction analysis implicates a replication-based mechanism underlying the rearrangement formation. X-inactivation studies (XCI) showed a random to moderate advantageous skewing pattern in peripheral blood cells but a moderate to extremely skewed (≥90%) pattern in buccal cells. In conclusion, our data show that complex duplications involving PLP1 are not uncommon, can be detected at the level of genome resolution afforded by clinical aCGH and duplication and inversion can be produced in the same event. Furthermore, the observation of three manifesting carriers with a large genomic rearrangement supports the contention that duplication size along with genomic content can be an important factor for penetrance of the PMD phenotype in females.

Duchenne muscular dystrophy caused by a complex rearrangement between intron 43 of the DMD gene and chromosome 4

Deletions/duplications of exons in the DMD gene cause about 70% of all cases of Duchenne muscular dystrophy (DMD). Most remaining mutations are point mutations or small insertion–deletions located mainly in the coding but also in deep intronic regions of the DMD gene. We describe a novel complex rearrangement in a patient affected with DMD that was undetectable using standard molecular diagnostic methods. Analysis of the proband’s mRNA from a muscle biopsy revealed the insertion of an 80bp cryptic exon from chromosome 4 between exons 43 and 44 of the dystrophin gene. Array comparative genomic hybridization and breakpoint junction sequence analysis indicated this cryptic exon originated from a complex genomic 90kb insertion of non-coding chromosome 4 into intron 43 of the dystrophin gene. This rearrangement was also detectable in the patient’s mother. The genomic characterization of this novel complex mutation was essential for accurate carrier and genetic counseling of this family and emphasizes the need for comprehensive molecular diagnosis of patients with clinical signs of DMD and clear pathological changes.

DbCRID: a database of chromosomal rearrangements in human diseases

Chromosomal rearrangement (CR) events result from abnormal breaking and rejoining of the DNA molecules, or from crossing-over between repetitive DNA sequences, and they are involved in many tumor and non-tumor diseases. Investigations of disease-associated CR events can not only lead to important discoveries about DNA breakage and repair mechanisms, but also offer important clues about the pathologic causes and the diagnostic/therapeutic targets of these diseases. We have developed a database of Chromosomal Rearrangements In Diseases (dbCRID, http://dbCRID.biolead.org), a comprehensive database of human CR events and their associated diseases. For each reported CR event, dbCRID documents the type of the event, the disease or symptoms associated, and—when possible—detailed information about the CR event including precise breakpoint positions, junction sequences, genes and gene regions disrupted and experimental techniques applied to discover/analyze the CR event. With 2643 records of disease-associated CR events curated from 1172 original studies, dbCRID is a comprehensive and dynamic resource useful for studying DNA breakage and repair mechanisms, and for analyzing the genetic basis of human tumor and non-tumor diseases.

Translocations used to generate chromosome segment duplications in Neurospora can disrupt genes and create novel open reading frames

In Neurospora crassa, crosses between normal sequence strains and strains bearing some translocations can yield progeny bearing a duplication (Dp) of the translocated chromosome segment. Here, 30 breakpoint junction sequences of 12 Dp-generating translocations were determined. The breakpoints disrupted 13 genes (including predicted genes), and created 10 novel open reading frames. Insertion of sequences from LG III into LG I as translocation T(UK8-18) disrupts the eat-3 gene, which is the ortholog of the Podospora anserine gene ami1. Since ami1-homozygous Podospora crosses were reported to increase the frequency of repeat-induced point mutation (RIP), we performed crosses homozygous for a deficiency in eat-3 to test for a corresponding increase in RIP frequency. However, our results suggested that, unlike in Podospora, the eat-3 gene might be essential for ascus development in Neurospora. Duplication-heterozygous crosses are generally barren in Neurospora; however, by using molecular probes developed in this study, we could identify Dp segregants from two different translocation-heterozygous crosses, and using these we found that the barren phenotype of at least some duplication-heterozygous crosses was incompletely penetrant.