Fork Stalling And Template Switching Research Articles

EP402: Partial trisomy of chromosome 22 mediated by chromoanasynthesis in an 8-month-old male

Chromoanasynthesis is a subtype of chromoanagenesis, a term used to describe the single-step processes by which complex chromosomal rearrangements lead to localized structural genomic reorganization of one or a few chromosomes. During chromoanasynthesis, complex chromosomal rearrangements are mediated by replication-based mechanisms including fork stalling and template switching (FoSTeS) or by microhomology-mediated break-induced replication (MMBIR). As a result, highly reorganized chromosomes with multiple copy number changes including deletions, duplications, and triplications are generated.

Complete characterisation of two new large Xq28 duplications involving F8 using whole genome sequencing in patients without haemophilia A.

Depending on the location of insertion of the gained region, F8 duplications can have variable clinical impacts from benign impact to severe haemophilia A phenotype. To characterize two large Xq28 duplications involving F8 incidentally detected by chromosome microarray analysis (CMA) in two patients presenting severe intellectual disability but no history of bleeding disorder. Whole genome sequencing (WGS) was performed in order to characterize the two large Xq28 duplications at nucleotide level. In patient 1, a 60-73kb gained region encompassing the exons 23-26 of F8 and SMIM9 was inserted at the int22h-2 locus following a non-homologous recombination between int22h-1 and int22h-2. We hypothesized that two independent events, micro-homology-mediated break-induced replication (MMBIR) and break-induced replication (BIR), could be involved in this rearrangement. In patient 2, the CMA found duplication from 101 to 116-kb long encompassing the exons 16-26 of F8 and SMIM9. The WGS analysis identified a more complex rearrangement with the presence of three genomic junctions. Due to the multiple micro-homologies observed at breakpoints, a replication-based mechanism such as fork stalling and template switching (FoSTeS) was greatly suspected. In both cases, these complex rearrangements preserved an intact copy of the F8. This study highlights the value of WGS to characterize the genomic junction at the nucleotide level and ultimately better describe the molecular mechanisms involved in Xq28 structural variations. It also emphasizes the importance of specifying the structure of the genomic gain in order to improve genotype-phenotype correlation and genetic counselling.

Molecular basis of SERPINC1 mutations in Japanese patients with antithrombin deficiency

BackgroundCongenital antithrombin (AT) deficiency, which arises from various SERPINC1 defects, is an autosomal-dominant thrombophilic disorder associated with a high risk of recurrent venous thromboembolism. Patients/methodsWe investigated SERPINC1 defects in Japanese patients with congenital AT deficiency who developed venous thromboembolism or had a family history of deep vein thrombosis. We analyzed the full DNA sequences of SERPINC1 exons and exon-intron junctions by PCR-mediated direct sequencing. If no mutation was found, multiplex ligation-dependent probe amplification (MLPA) was conducted for the relative quantification of the copy number of all exons in SERPINC1. If splice-site mutations were detected, mRNA splicing abnormalities were further investigated using an in vitro cell-based exontrap assay. ResultsWe identified 19 different SERPINC1 abnormalities, including 8 novel mutations, in 21 Japanese patients with AT deficiency. These abnormalities were distributed as follows: 9 missense mutations (42.9%), 3 nonsense mutations (14.3%), 1 splice-site mutation (4.8%), 2 small insertions (9.5%), 2 deletion mutations (9.5%) and 4 large deletions (19.0%). Cases with large deletions of SERPINC1 included Alu-mediated gene rearrangements and non-Alu-mediated complex gene rearrangements; the latter could conceivably be explained using the fork stalling and template switching (FoSTeS) model. ConclusionsWe identified a variety of SERPINC1 defects in Japanese patients with AT deficiency. The SERPINC1 mutations detected in patients with type I AT deficiency included single nucleotide missense or nonsense mutations, small intragenic insertions or deletions, and large genomic structural deletions. Large deletions of SERPINC1 were caused by various recurrent or non-recurrent complex genomic rearrangement mutations.

PMP22 exon 4 deletion causes ER retention of PMP22 and a gain-of-function allele in CMT1E.

ObjectiveTo determine whether predicted fork stalling and template switching (FoSTeS) during mitosis deletes exon 4 in peripheral myelin protein 22 KD (PMP22) and causes gain‐of‐function mutation associated with peripheral neuropathy in a family with Charcot–Marie–Tooth disease type 1E.MethodsTwo siblings previously reported to have genomic rearrangements predicted to involve exon 4 of PMP22 were evaluated clinically and by electrophysiology. Skin biopsies from the proband were studied by RT‐PCR to determine the effects of the exon 4 rearrangements on exon 4 mRNA expression in myelinating Schwann cells. Transient transfection studies with wild‐type and mutant PMP22 were performed in Cos7 and RT4 cells to determine the fate of the resultant mutant protein.ResultsBoth affected siblings had a sensorimotor dysmyelinating neuropathy with severely slow nerve conduction velocities (<10 m/sec). RT‐PCR studies of Schwann cell RNA from one of the siblings demonstrated a complete in‐frame deletion of PMP22 exon 4 (PMP22Δ4). Transfection studies demonstrated that PMP22Δ4 protein is retained within the endoplasmic reticulum and not transported to the plasma membrane.ConclusionsOur results confirm that that FoSTeS‐mediated genomic rearrangement produced a deletion of exon 4 of PMP22, resulting in expression of both PMP22 mRNA and protein lacking this sequence. In addition, we provide experimental evidence for endoplasmic reticulum retention of the mutant protein suggesting a gain‐of‐function mutational mechanism consistent with the observed CMT1E in this family. PMP22Δ4 is another example of a mutated myelin protein that is misfolded and contributes to the pathogenesis of the neuropathy.

Distinct X chromosomal rearrangements in four haemophilia B patients with entire F9 deletion.

Haemophilia B is an X-linked bleeding disorder caused by a coagulation factor IX gene (F9) abnormality. Numerous F9 defects have been identified to date; however, only a few with an entire F9 deletion have been reported in detail. To elucidate the cause of severe haemophilia B, we investigated the precise X chromosome abnormalities in four Japanese patients who did not show all amplifications in F9-specific PCR. We analysed the patient's genomic DNA using Multiplex ligation-dependent probe amplification (MLPA). To assess the extent of any deletions, we further performed mapping PCRs, inverse PCRs or long-range PCRs and direct sequencing analyses of the X chromosome. We detected entire F9 deletions in four haemophilia B patients and identified the precise deleted regions of the X chromosome including F9. Patient 1 had a 149-kb deletion with breakpoints 90-kb upstream and 30-kb downstream from F9. Patients 2 and 3 showed 273-kb and 1.19-Mb deletions respectively. Patient 4 had two deleted regions: a 1663-bp deletion 1.34-Mb upstream from F9 and a 7.2-Mb deletion including F9. These distinct breakpoints found in four different patients suggest that the mechanism of X chromosome deletion may be different between individuals. Non-allelic homologous recombination (NAHR), microhomology-mediated break-induced replication (MMBIR) or fork stalling and template switching (FoSTeS) may occur in respective X chromosomes of the four haemophilia B patients analysed. We identified diverse X chromosomal rearrangements in four haemophilia B patients, which might be caused by distinct mechanisms of genomic rearrangement.

Epigenetic Regulation in Amyloid Precursor Protein with Genomic Rearrangements and the Lesch-Nyhan Syndrome

Recently, epigenetic regulation of alternative APP pre-mRNA splicing in the Lesch-Nyhan syndrome (LNS) has been studied (see Ref. 7) and showed for the first time, the presence of several APP-mRNA isoforms encoding divers APP protein isoforms ranging from 120 to 770 amino acids (with or without mutations and/or deletions). Here, by continuing on this work, I identified, for the first time new APP-mRNA isoforms with a deletion followed by an insertion (INDELS) in LNS and LNVs patients:c.19_2295delinsG166TT…GAGTCC…CTTAGTC…TCT489,p.Leu7Valfs*2;c.19_2295 delinsG169TT…GAGACC…CTTGGTC…TCT492,p.Leu7Valfs*2;and c.16_2313delinsG84CC…CAT616,p.Leu7Hisfs*45. A role of genomic rearrangements of APP gene via the Fork Stalling and Template Switching (FoSTeS) mechanism leading to INDELS was suggested. Epistasis between mutated HPRT1 and APP genes could be one of the factors of epigenetic modifications responsible for genomic rearrangements of APP gene. My findings accounted for epigenetic mechanism in the regulation of alternative APP pre-mRNA splicing as well as for epigenetic control of genomic rearrangements of APP gene may provide therefore new directions not only for investigating the role of APP in neuropathology associated with HGprt-deficiency in LNS and LNVs patients but also for the research in neurodevelopmental and neurodegenerative disorders by which APP gene involved in the pathogenesis of the diseases such as autism, fragile X syndrome (FXS), and Alzheimer's disease (AD) with its diversity and complexity, especially for sporadic form of AD (SAD). An accurate quantification of various APP-mRNA isoforms in brain tissues for detection of initial pathological changes or pathology development is needed and antisense drugs are the potential treatments.

Decoding NF1 Intragenic Copy-Number Variations

Genomic rearrangements can cause both Mendelian and complex disorders. Currently, several major mechanisms causing genomic rearrangements, such as non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ), fork stalling and template switching (FoSTeS), and microhomology-mediated break-induced replication (MMBIR), have been proposed. However, to what extent these mechanisms contribute to gene-specific pathogenic copy-number variations (CNVs) remains understudied. Furthermore, few studies have resolved these pathogenic alterations at the nucleotide-level. Accordingly, our aim was to explore which mechanisms contribute to a large, unique set of locus-specific non-recurrent genomic rearrangements causing the genetic neurocutaneous disorder neurofibromatosis type 1 (NF1). Through breakpoint-spanning PCR as well as array comparative genomic hybridization, we have identified the breakpoints in 85 unrelated individuals carrying an NF1 intragenic CNV. Furthermore, we characterized the likely rearrangement mechanisms of these 85 CNVs, along with those of two additional previously published NF1 intragenic CNVs. Unlike the most typical recurrent rearrangements mediated by flanking low-copy repeats (LCRs), NF1 intragenic rearrangements vary in size, location, and rearrangement mechanisms. We propose the DNA-replication-based mechanisms comprising both FoSTeS and/or MMBIR and serial replication stalling to be the predominant mechanisms leading to NF1 intragenic CNVs. In addition to the loop within a 197-bp palindrome located in intron 40, four Alu elements located in introns 1, 2, 3, and 50 were also identified as intragenic-rearrangement hotspots within NF1.

Advance in research on MECP2 [corrected] duplication syndrome

Methyl-CpG-binding protein 2 gene (MECP2; OMIM 300005) is located at chromosome Xq28. Mutations of the gene including point mutation, duplication and deletion can lead to severe neurodevelopmental disorders. The disease caused by duplication of the entire MECP2 gene, named as MECP2 duplication syndrome, is mostly seen in males. The clinical manifestation of this syndrome include mental retardation, hypotonia, poor speech development, recurrent infection, progressive spasticity, epilepsy, autism or autistic features with or without midface hypoplasia. Most patients have inherited the duplication from their unaffected mothers, with only a few cases having de novo mutation. Females with duplicated MECP2 gene are typically asymptomatic because of a skewed X chromosome inactivation (XCI) pattern. Proposed mechanisms of this genomic rearrangement include fork stalling and template switching (FoSTeS) and microhomology mediated break-induced replication (MMBIR). Since no effective treatment is available for this disease, proper genetic counseling and prenatal diagnosis for the high risk families are crucial.

Genomic instability in the PARK2 locus is associated with Parkinson's disease.

Parkinson’s disease (PD) is a common neurodegenerative disorder affecting mostly elderly people, although there is a group of patients developing so-called early-onset PD (EOPD). Mutations in the PARK2 gene are a common cause of autosomal recessive EOPD. PARK2 belongs to the family of extremely large human genes which are often localised in genomic common fragile sites (CFSs) and exhibit gross instability. PARK2 is located in the centre of FRA6E, the third most mutation-susceptible CFS of the human genome. The gene encompasses a region of 1.3 Mbp and, among its mutations, large rearrangements of single or multiple exons account for around 50 %. We performed an analysis of the PARK2 gene in a group of 344 PD patients with EOPD and classical form of the disease. Copy number changes were first identified using multiplex ligation probe amplification (MLPA), with their ranges characterised by array comparative genomic hybridisation (aCGH). Exact breakpoints were mapped using direct sequencing. Rearrangements were found in eight subjects, including five deletions and three duplications. Rearrangements were mostly non-recurrent and no repetitive sequences or extended homologies were identified in the regions flanking breakpoint junctions. However, in most cases, 1–3 bp microhomologies were present, strongly suggesting that microhomology-mediated mechanisms, specifically non-homologous end joining (NHEJ) and fork stalling and template switching (FoSTeS)/microhomology-mediated break-induced replication (MMBIR), are predominantly involved in the rearrangement processes in this genomic region.

Complex mosaic CDKL5 deletion with two distinct mutant alleles in a 4‐year‐old girl

Mutations of the CDKL5 gene cause early epileptic encephalopathy. Patients manifest refractory epilepsy, beginning before the age of 3 months, which is associated with severe psychomotor delay and features that overlap with Rett syndrome. We report here a patient with mosaicism for CDKL5 exonic deletion, with the presence of two mutant alleles. The affected 4-year-old girl presented with infantile spasms, beginning at the age of 9 months, but subsequent progression of the disease was consistent with the classical CDKL5-related phenotype. A deletion of exons 17 and 18 was suspected on the basis of Multiplex Ligation Probe Amplification analysis, but unexpected results for cDNA analysis, which showed the presence of an abnormal transcript with the deletion of exon 18 only, led us to suspect that two distinct events might have occurred. We used custom array-CGH to determine the size and breakpoints of these deletions. Exon 18 was deleted from one of the abnormal alleles, and exon 17 was deleted from the other. A Fork Stalling and Template Switching (FoSTeS) mechanism was proposed to explain the two events, given the presence of regions of microhomology at the breakpoints. We propose here an original involvement of the FoSTeS mechanism to explain the co-occurrence of these two events in the CDKL5 gene in a single patient. This patient highlights the difficulties involved in the detection of such abnormalities, particularly when they occur in a mosaic state and involve two distinct mutational events in a single gene.

Evidence for replicative mechanism in a CHD7 rearrangement in a patient with CHARGE syndrome

Haploinsufficiency of CHD7 (OMIM# 608892) is known to cause CHARGE syndrome (OMIM# 214800). Molecular testing supports a definitive diagnosis in approximately 65-70% of cases. Most CHD7 mutations arise de novo, and no mutations affecting exon-7 have been reported to date. We report on an 8-year-old girl diagnosed with CHARGE syndrome that was referred to our laboratory for comprehensive CHD7 gene screening. Genomic DNA from the subject with a suspected diagnosis of CHARGE was isolated from peripheral blood lymphocytes and comprehensive Sanger sequencing, along with deletion/duplication analysis of the CHD7 gene using multiplex ligation-dependent probe amplification (MLPA), was performed. MLPA analysis identified a reduced single probe signal for exon-7 of the CHD7 gene consistent with potential heterozygous deletion. Long-range PCR breakpoint analysis identified a complex genomic rearrangement (CGR) leading to the deletion of exon-7 and breakpoints consistent with a replicative mechanism such as fork stalling and template switching (FoSTeS) or microhomology-mediated break-induced replication (MMBIR). Taken together this represents the first evidence for a CHD7 intragenic CGR in a patient with CHARGE syndrome leading to what appears to be also the first report of a mutation specifically disrupting exon-7. Although likely rare, CGR may represent an overlooked mechanism in subjects with CHARGE syndrome that can be missed by current sequencing and dosage assays.

Investigating the properties of a repair replication‐fork in the budding yeast Saccharomyces cerevisiae

Genome instability is the hallmark of several human diseases including cancer. The FoSTeS (Fork Stalling and Template Switching) model – also termed Microhomology‐Mediated Break‐induced Replication (MM‐BIR) – posits that the fork originating from a stalled or collapsed fork can trigger genome instability by means of template switching of the repair‐fork. In order to examine MM‐BIR in detail, we created a system in S. cerevisiae in which a single double‐strand break induced by HO endonuclease can be repaired by a two step process that involve 1) strand invasion with a donor sequence followed by 2) template switch located at about 50 kb away generating a functional URA3 gene as part of a complex nonreciprocal translocation. Our results show that when the sequences are 100 % homologous, these events occur at a frequency of about 2 × 10−3. The presence of sequence heterology had distinct affect on the two steps; heterology severely restricted strand invasion by about four orders of magnitude, while template switch was less affected. For the first time, our results reveal two important features distinguishing canonical strand invasions from template jumps: 1) template jumps were less restrained by heterologies than strand invasions providing evidence for their distinct nature and 2) invasions preferred the use of the very 3′ end whereas template jumps showed no preferred sequences, suggesting repeated strand dissociation and reformation as a hallmark of repair forks. Sequencing of junctions of recombined sequences that shared no significant homologies showed that such events utilized sequences ranging from 3 bp micro‐homologies to no homologies.

Microhomology-Mediated Mechanisms Underlie Non-Recurrent Disease-Causing Microdeletions of the FOXL2 Gene or Its Regulatory Domain

Genomic disorders are often caused by recurrent copy number variations (CNVs), with nonallelic homologous recombination (NAHR) as the underlying mechanism. Recently, several microhomology-mediated repair mechanisms—such as microhomology-mediated end-joining (MMEJ), fork stalling and template switching (FoSTeS), microhomology-mediated break-induced replication (MMBIR), serial replication slippage (SRS), and break-induced SRS (BISRS)—were described in the etiology of non-recurrent CNVs in human disease. In addition, their formation may be stimulated by genomic architectural features. It is, however, largely unexplored to what extent these mechanisms contribute to rare, locus-specific pathogenic CNVs. Here, fine-mapping of 42 microdeletions of the FOXL2 locus, encompassing FOXL2 (32) or its regulatory domain (10), serves as a model for rare, locus-specific CNVs implicated in genetic disease. These deletions lead to blepharophimosis syndrome (BPES), a developmental condition affecting the eyelids and the ovary. For breakpoint mapping we used targeted array-based comparative genomic hybridization (aCGH), quantitative PCR (qPCR), long-range PCR, and Sanger sequencing of the junction products. Microhomology, ranging from 1 bp to 66 bp, was found in 91.7% of 24 characterized breakpoint junctions, being significantly enriched in comparison with a random control sample. Our results show that microhomology-mediated repair mechanisms underlie at least 50% of these microdeletions. Moreover, genomic architectural features, like sequence motifs, non-B DNA conformations, and repetitive elements, were found in all breakpoint regions. In conclusion, the majority of these microdeletions result from microhomology-mediated mechanisms like MMEJ, FoSTeS, MMBIR, SRS, or BISRS. Moreover, we hypothesize that the genomic architecture might drive their formation by increasing the susceptibility for DNA breakage or promote replication fork stalling. Finally, our locus-centered study, elucidating the etiology of a large set of rare microdeletions involved in a monogenic disorder, can serve as a model for other clustered, non-recurrent microdeletions in genetic disease.

The origin, evolution, and functional impact of short insertion–deletion variants identified in 179 human genomes

Short insertions and deletions (indels) are the second most abundant form of human genetic variation, but our understanding of their origins and functional effects lags behind that of other types of variants. Using population-scale sequencing, we have identified a high-quality set of 1.6 million indels from 179 individuals representing three diverse human populations. We show that rates of indel mutagenesis are highly heterogeneous, with 43%–48% of indels occurring in 4.03% of the genome, whereas in the remaining 96% their prevalence is 16 times lower than SNPs. Polymerase slippage can explain upwards of three-fourths of all indels, with the remainder being mostly simple deletions in complex sequence. However, insertions do occur and are significantly associated with pseudo-palindromic sequence features compatible with the fork stalling and template switching (FoSTeS) mechanism more commonly associated with large structural variations. We introduce a quantitative model of polymerase slippage, which enables us to identify indel-hypermutagenic protein-coding genes, some of which are associated with recurrent mutations leading to disease. Accounting for mutational rate heterogeneity due to sequence context, we find that indels across functional sequence are generally subject to stronger purifying selection than SNPs. We find that indel length modulates selection strength, and that indels affecting multiple functionally constrained nucleotides undergo stronger purifying selection. We further find that indels are enriched in associations with gene expression and find evidence for a contribution of nonsense-mediated decay. Finally, we show that indels can be integrated in existing genome-wide association studies (GWAS); although we do not find direct evidence that potentially causal protein-coding indels are enriched with associations to known disease-associated SNPs, our findings suggest that the causal variant underlying some of these associations may be indels.

Two new large deletions of the AVPR2 gene causing nephrogenic diabetes insipidus and a review of previously published deletions

In this paper, we report two new original deletions and present an extended review of the previously characterized AVPR2 gene deletions to better understand the underlying deletion mechanisms. The two novel deletions were defined using polymerase chain reaction mapping and junction fragment sequencing. Bioinformatic analysis was performed on both the previously mapped deletions and the novel ones through several web tools. In our two patients with nephrogenic diabetes insipidus, we found a 23 755 bp deletion and a 9264 bp deletion both comprising the entire AVPR2 gene and part of the ARHGAP4 gene. Through bioinformatic studies, the smallest overlapping region as well as several motifs and repeats that are known to promote rearrangements were confirmed. Through this study, it was determined that the deletion mechanisms in the AVPR2 region do not follow the rules of non-allelic homologous recombination. Two of the 13 deletions can be attributed to the fork stalling and template switching (FoSTeS) mechanism, whereas the remaining 11 deletions could be caused either by non-homologous end joining or by the FoSTeS mechanism. Although no recurrence was found, several groupings of deletion breakpoints were identified.

Neurodevelopmental features in 2q23.1 microdeletion syndrome: Report of a new patient with intractable seizures and review of literature

2q23.1 microdeletion syndrome is a recently characterized chromosomal aberration disorder uncovered through array comparative genomic hybridization (array CGH). Although the cardinal feature is intellectual disability (ID), neurodevelopmental features of the syndrome have not been systematically reviewed. We present a 5-year-old boy with severe psychomotor developmental delay/ID, progressive microcephaly with brain atrophy, growth retardation, and several external anomalies. He manifested intractable epilepsy, effectively treated with combined antiepileptic drug therapy including topiramate. Array CGH demonstrated a de novo interstitial deletion of approximately 1 Mb at 2q23.1-q23.2, involving four genes including MBD5. Nineteen patients have been reported to have the syndrome, including present patient. All patients whose data were available had ID, 17 patients (89%) had seizures, and microcephaly was evident in 9 of 18 patients (50%). Deletion sizes ranged from 200 kb to 5.5 Mb, comprising 1-15 genes. MBD5, the only gene deleted in all patients, is considered to be responsible for ID and epilepsy. Furthermore, the deletion junction was sequenced for the first time in a patient with the syndrome; and homology of three nucleotides, identified at the distal and proximal breakpoints, suggested that the deletion might have been mediated by recently-delineated genomic rearrangement mechanism Fork Stalling and Template Switching (FoSTeS)/microhomology-mediated break-induced replication (MMBIR).

FoSTeS, MMBIR and NAHR at the human proximal Xp region and the mechanisms of human Xq isochromosome formation

The recently described DNA replication-based mechanisms of fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) were previously shown to catalyze complex exonic, genic and genomic rearrangements. By analyzing a large number of isochromosomes of the long arm of chromosome X (i(Xq)), using whole-genome tiling path array comparative genomic hybridization (aCGH), ultra-high resolution targeted aCGH and sequencing, we provide evidence that the FoSTeS and MMBIR mechanisms can generate large-scale gross chromosomal rearrangements leading to the deletion and duplication of entire chromosome arms, thus suggesting an important role for DNA replication-based mechanisms in both the development of genomic disorders and cancer. Furthermore, we elucidate the mechanisms of dicentric i(Xq) (idic(Xq)) formation and show that most idic(Xq) chromosomes result from non-allelic homologous recombination between palindromic low copy repeats and highly homologous palindromic LINE elements. We also show that non-recurrent-breakpoint idic(Xq) chromosomes have microhomology-associated breakpoint junctions and are likely catalyzed by microhomology-mediated replication-dependent recombination mechanisms such as FoSTeS and MMBIR. Finally, we stress the role of the proximal Xp region as a chromosomal rearrangement hotspot.

Genomic and clinical characteristics of microduplications in chromosome 17

Genomic disorders have been increasingly recognized as a significant source of clinically relevant phenotypes largely fostered by advances in technologies for genome-wide analyses. Molecular and clinical studies of copy number variants involving chromosome 17 began with locus-specific studies of Charcot-Marie-Tooth disease type 1A (CMT1A, OMIM #118220) and hereditary neuropathy with liability to pressure palsies (HNPP, OMIM #162500), which laid the foundation for the paradigm of duplication/deletion and gene-dosage for our understanding of genomic disorders. With the clinical introduction of high-resolution array comparative genomic hybridization (aCGH) the number of recognized genomic disorders including microduplications has been increasing rapidly. A relatively high proportion of disease-associated copy number variants map to chromosome 17. This may result from its unique structural features, such as relative abundance of segmental duplications and interspersed repetitive elements, high gene content, and the presence of dosage-sensitive genes. These genomic rearrangements are mediated by diverse mechanisms including Non-Allelic Homologous Recombination (NAHR), Non-Homologous End-Joining (NHEJ), and Fork Stalling and Template Switching (FoSTeS). We provide specific examples of chromosome 17 microduplications with the emphasis on their phenotype, specific clinical features aiding in their diagnosis, and counseling.

Four unrelated patients with lubs X-linked mental retardation syndrome and different Xq28 duplications

The Lubs X-linked mental retardation syndrome (MRXSL) is caused by small interstitial duplications at distal Xq28 including the MECP2 gene. Here we report on four novel male patients with MRXSL and different Xq28 duplications delineated by microarray-based chromosome analysis. All mothers were healthy carriers of the duplications. Consistent with an earlier report [Bauters et al. (2008); Genome Res 18: 847-858], the distal breakpoints of all four Xq28 duplications were located in regions containing low-copy repeats (LCRs; J, K, and L groups), which may facilitate chromosome breakage and reunion events. The proximal breakpoint regions did not contain known LCRs. Interestingly, we identified apparent recurrent breakage sites in the proximal and distal breakpoint regions. Two of the four patients displayed more complex rearrangements. Patient 2 was endowed with a quadruplicated segment and a small triplication within the duplication, whereas patient 3 displayed two triplicated segments within the duplication, supporting that the Fork Stalling and Template Switching (FoSTeS) model may explain a subset of the structural rearrangements in Xq28. Clinically, muscular hypertonia and contractures of large joints may present a major problem in children with MRXSL. Because injection of botulinum toxin (BT-A; Botox) proved to be extremely helpful for patient 1, we recommend consideration of Botox treatment in other patients with MRXSL and severe joint contractures.

Base-Pair Resolution of Somatic and Germline-Derived Genome Rearrangement Breakpoints in Follicular Lymphoma.

Abstract 439 Introduction:Follicular lymphoma (FL) is the most common indolent lymphoid malignancy in North America with approximately 20,000 new cases of this incurable cancer diagnosed each year. In approximately 85% of patients, FL is associated with the reciprocal translocation t(14;18)(q32;q21), which results in a fusion between IGH and BCL2 genes and consequent over-expression of the anti-apoptotic protein BCL2. This translocation likely represents an initiating event for FL, requiring additional mutational events for the onset of clinical disease. To investigate the relationship between genome rearrangements and FL we identified rearrangement locations in the genome followed by detailed, fine-structure analysis of the rearrangements to ascertain their effects on genes and other features of biological interest. Patients and Methods:We used a whole-genome bacterial artificial chromosome (BAC) fingerprint-based approach, termed Fingerprint Profiling (FPP, Krzywinski, M. et al. 2007), to detect genome rearrangements relative to the reference human genome in neoplastic B cells purified from 24 FL patient biopsies. Analysis of 2,640,707 BAC fingerprints revealed 721 candidate genomic rearrangements. To validate these observations and provide base-pair resolution of the rearrangement breakpoints we performed paired-end massively parallel sequencing, on the Illumina Genome Analyzer II platform, of the breakpoint-containing regions captured in the BAC clones. Sequence reads were assembled into contigs using our in-house de novo assembly algorithm ABySS (Assembly By Short Sequences, Simpson, J. et al. 2009) then aligned to the reference human genome. Following manual annotation of the breakpoint junctions PCR primers were designed to assay patient tumour and matched constitutional DNA and thus determine whether the observed genome rearrangements were somatic (acquired) or germline in origin. Results:727 BACs with apparent large-scale genome rearrangements, representing 354 distinct genome rearrangements across 20 patients, were sequenced in 95 pools, generating 72 Gbp of sequence. The 354 distinct events include 163 deletions, 71 inversions, 27 insertions, 83 translocations and 10 duplications, ranging in size from 3 kb to 67 Mb. PCR assays for 194 of the distinct events have been performed thus far identifying 80 distinct somatic and 114 germline-derived structural variations at base-pair resolution. Of the somatic events 5 are present in two or more of the 20 patients analyzed including a 720 kb inversion of 3q27.3 that results in expression of a BCL6-ST6GAL1 fusion transcript. Identification at base-pair resolution of breakpoint sequences enabled a detailed study of breakpoint and fusion mechanisms. We classified breakpoint junctions into 4 groups; those with microhomology (48%), those with sequence additions (28%), those with blunt fusions (20%) and those with flanking low copy repeats (4%). We were particularly interested in establishing the origin of the observed nucleotide sequence additions in 97 breakpoint junctions. The sequence additions ranged in size from a single nucleotide to 454 bp. In one case we have unambiguously mapped a 53 bp sequence, lying within one of the 3q27.3 inversion breakpoints, to chromosome 5q12.3. This finding is consistent with the recently proposed fork stalling and template switching (FoSTeS) DNA replication-based mechanism and thus represents a novel mechanism in FL lymphomagenesis. Conclusions:We have successfully employed high-throughput clone fingerprinting and sequencing to identify numerous novel somatic and germline genome rearrangements from FL primary tumour samples. Furthermore, base-pair resolution of rearrangement breakpoints provides mechanistic insights. With the complete inventory of somatic and germline events in hand we will be able to propose recurrent structurally altered genes in FL patients for validation in independent datasets and improve our understanding of FL biology. Pathway analyses to identify emerging themes from somatic mutations are also being performed. The PCR assays we have developed will also be of utility in identifying germline predisposition alleles in larger FL patient cohorts. Disclosures:No relevant conflicts of interest to declare.