Whole‐Genome Sequencing of Cytogenetically Balanced Chromosome Translocations Identifies Potentially Pathological Gene Disruptions and Highlights the Importance of Microhomology in the Mechanism of Formation

Most balanced translocations are thought to result mechanistically from nonhomologous end joining or, in rare cases of recurrent events, by nonallelic homologous recombination. Here, we use low‐coverage mate pair whole‐genome sequencing to fine map rearrangement breakpoint junctions in both phenotypically normal and affected translocation carriers. In total, 46 junctions from 22 carriers of balanced translocations were characterized. Genes were disrupted in 48% of the breakpoints; recessive genes in four normal carriers and known dominant intellectual disability genes in three affected carriers. Finally, seven candidate disease genes were disrupted in five carriers with neurocognitive disabilities (SVOPL, SUSD1, TOX, NCALD, SLC4A10) and one XX‐male carrier with Tourette syndrome (LYPD6, GPC5). Breakpoint junction analyses revealed microhomology and small templated insertions in a substantive fraction of the analyzed translocations (17.4%; n = 4); an observation that was substantiated by reanalysis of 37 previously published translocation junctions. Microhomology associated with templated insertions is a characteristic seen in the breakpoint junctions of rearrangements mediated by error‐prone replication‐based repair mechanisms. Our data implicate that a mechanism involving template switching might contribute to the formation of at least 15% of the interchromosomal translocation events.

[1]  J. Shendure,et al.  Characterization of apparently balanced chromosomal rearrangements from the developmental genome anatomy project. , 2008, American journal of human genetics.

[2]  Hugo Y. K. Lam,et al.  Analysis of deletion breakpoints from 1,092 humans reveals details of mutation mechanisms , 2015, Nature Communications.

[3]  M. Lieber,et al.  Non-homologous end joining often uses microhomology: implications for alternative end joining. , 2014, DNA repair.

[4]  K. Reichard,et al.  t(9;22)(q34;q11.2) is a recurrent constitutional non-Robertsonian translocation and a rare cytogenetic mimic of chronic myeloid leukemia. , 2011, Cancer genetics.

[5]  Toshiro K. Ohsumi,et al.  Sequencing Chromosomal Abnormalities Reveals Neurodevelopmental Loci that Confer Risk across Diagnostic Boundaries , 2012, Cell.

[6]  Dagmar Wieczorek,et al.  Heterozygous submicroscopic inversions involving olfactory receptor-gene clusters mediate the recurrent t(4;8)(p16;p23) translocation. , 2002, American journal of human genetics.

[7]  Rochelle M. Witt,et al.  Heparan Sulfate Proteoglycans Containing a Glypican 5 Core and 2-O-Sulfo-iduronic Acid Function as Sonic Hedgehog Co-receptors to Promote Proliferation*♦ , 2013, The Journal of Biological Chemistry.

[8]  L. Vissers,et al.  Genome sequencing identifies major causes of severe intellectual disability , 2014, Nature.

[9]  J. Lupski.  Structural variation mutagenesis of the human genome: Impact on disease and evolution , 2015, Environmental and molecular mutagenesis.

[10]  N. Carter,et al.  Breakpoint mapping and array CGH in translocations: comparison of a phenotypically normal and an abnormal cohort. , 2008, American journal of human genetics.

[11]  A. Malkova,et al.  Break-Induced Replication Is Highly Inaccurate , 2011, PLoS biology.

[12]  J. Lupski,et al.  Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome , 2011, Nature Genetics.

[13]  M. Till,et al.  Breakpoint mapping by next generation sequencing reveals causative gene disruption in patients carrying apparently balanced chromosome rearrangements with intellectual deficiency and/or congenital malformations , 2013, Journal of Medical Genetics.

[14]  P. Stankiewicz,et al.  Structural variation in the human genome and its role in disease. , 2010, Annual review of medicine.

[15]  M. Gerstein,et al.  CNVnator: an approach to discover, genotype, and characterize typical and atypical CNVs from family and population genome sequencing. , 2011, Genome research.

[16]  F. Collins,et al.  Directional cloning of DNA fragments at a large distance from an initial probe: a circularization method. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Joakim Lundeberg,et al.  Large Scale Library Generation for High Throughput Sequencing , 2011, PloS one.

[18]  P. Stankiewicz,et al.  The Alu-rich genomic architecture of SPAST predisposes to diverse and functionally distinct disease-associated CNV alleles. , 2014, American journal of human genetics.

[19]  A. Tomkinson,et al.  Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. , 2014, Molecular cell.

[20]  J. Lupski,et al.  Mechanisms underlying structural variant formation in genomic disorders , 2016, Nature Reviews Genetics.

[21]  Z. Ou,et al.  Observation and prediction of recurrent human translocations mediated by NAHR between nonhomologous chromosomes. , 2011, Genome research.

[22]  A. Jauch,et al.  Mosaic deletion of EXOC6B: Further evidence for an important role of the exocyst complex in the pathogenesis of intellectual disability , 2014, American journal of medical genetics. Part A.

[23]  Naomichi Matsumoto,et al.  Precise detection of chromosomal translocation or inversion breakpoints by whole-genome sequencing , 2014, Journal of Human Genetics.

[24]  D. Lilley,et al.  DNA Repair , 1998, Nucleic Acids and Molecular Biology.

[25]  J. Lupski,et al.  Replicative mechanisms for CNV formation are error prone , 2013, Nature Genetics.

[26]  M. Kvarnung,et al.  Intragenic duplication—A novel causative mechanism for SATB2‐associated syndrome , 2014, American journal of medical genetics. Part A.

[27]  J. Lupski,et al.  A DNA Replication Mechanism for Generating Nonrecurrent Rearrangements Associated with Genomic Disorders , 2007, Cell.

[28]  P. Jacobs,et al.  Estimates of the frequency of chromosome abnormalities detectable in unselected newborns using moderate levels of banding. , 1992, Journal of medical genetics.

[29]  D. Jong,et al.  GRM1 is upregulated through gene fusion and promoter swapping in chondromyxoid fibroma , 2014, Nature Genetics.

[30]  Cynthia J. Sakofsky,et al.  Translesion Polymerases Drive Microhomology-Mediated Break-Induced Replication Leading to Complex Chromosomal Rearrangements. , 2015, Molecular cell.

[31]  J. Lupski,et al.  A Microhomology-Mediated Break-Induced Replication Model for the Origin of Human Copy Number Variation , 2009, PLoS genetics.

[32]  E. Blennow,et al.  Improved structural characterization of chromosomal breakpoints using high resolution custom array‐CGH , 2010, Clinical genetics.

[33]  P. Stankiewicz,et al.  Genome architecture, rearrangements and genomic disorders. , 2002, Trends in genetics : TIG.

[34]  David W. Wyatt,et al.  Essential Roles for Polymerase θ-Mediated End Joining in the Repair of Chromosome Breaks. , 2016, Molecular cell.

[35]  Steven J. M. Jones,et al.  Circos: an information aesthetic for comparative genomics. , 2009, Genome research.

[36]  Xun Xu,et al.  A Robust Approach for Blind Detection of Balanced Chromosomal Rearrangements with Whole‐Genome Low‐Coverage Sequencing , 2014, Human mutation.

[37]  Robert Fredriksson,et al.  Identification of six putative human transporters with structural similarity to the drug transporter SLC22 family. , 2007, Genomics.

[38]  D. Warburton,et al.  De novo balanced chromosome rearrangements and extra marker chromosomes identified at prenatal diagnosis: clinical significance and distribution of breakpoints. , 1991, American journal of human genetics.

[39]  J. Lupski,et al.  Alu-specific microhomology-mediated deletion of the final exon of SPAST in three unrelated subjects with hereditary spastic paraplegia , 2011, Genetics in Medicine.

[40]  Markus Wolff,et al.  GRIN2B Mutations in West Syndrome and Intellectual Disability with Focal Epilepsy , 2014, Annals of neurology.

[41]  Michael E Talkowski,et al.  Describing sequencing results of structural chromosome rearrangements with a suggested next-generation cytogenetic nomenclature. , 2014, American Journal of Human Genetics.

[42]  P. Stankiewicz,et al.  Mechanisms for Complex Chromosomal Insertions , 2016, PLoS genetics.

[43]  C. Shaw,et al.  Mus81 and converging forks limit the mutagenicity of replication fork breakage , 2015, Science.

[44]  Benjamin P. Blackburne,et al.  Mutation spectrum revealed by breakpoint sequencing of human germline CNVs , 2010, Nature Genetics.

[45]  B. Emanuel,et al.  Chromosomal translocations and palindromic AT-rich repeats. , 2012, Current opinion in genetics & development.

[46]  W. J. Kent,et al.  BLAT--the BLAST-like alignment tool. , 2002, Genome research.

[47]  A. Piotrowski,et al.  Palindrome‐Mediated and Replication‐Dependent Pathogenic Structural Rearrangements within the NF1 Gene , 2014, Human mutation.

[48]  M. Zollino,et al.  Interstitial deletion of 3p22.3p22.2 encompassing ARPP21 and CLASP2 is a potential pathogenic factor for a syndromic form of intellectual disability: A co‐morbidity model with additional copy number variations in a large family , 2013, American journal of medical genetics. Part A.

[49]  Somasekar Seshagiri,et al.  The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment. , 2003, Genome research.

[50]  Martin Vingron,et al.  Mapping translocation breakpoints by next-generation sequencing. , 2008, Genome research.

[51]  Ira M. Hall,et al.  Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration , 2012, Nature Genetics.

[52]  L. Symington,et al.  Template switching during break-induced replication , 2007, Nature.

[53]  J. Lupski,et al.  Mechanisms of change in gene copy number , 2009, Nature Reviews Genetics.

[54]  P. Stankiewicz,et al.  Position effects due to chromosome breakpoints that map ∼900 Kb upstream and ∼1.3 Mb downstream of SOX9 in two patients with campomelic dysplasia , 2005 .

[55]  Michael E Talkowski,et al.  Clinical diagnosis by whole-genome sequencing of a prenatal sample. , 2012, The New England journal of medicine.

[56]  A. Dahl,et al.  Tox: a multifunctional transcription factor and novel regulator of mammalian corticogenesis , 2015, The EMBO journal.

[57]  P. Stankiewicz,et al.  Alu-mediated diverse and complex pathogenic copy-number variants within human chromosome 17 at p13.3. , 2015, Human molecular genetics.

[58]  J. Kere,et al.  CTNND2—a candidate gene for reading problems and mild intellectual disability , 2014, Journal of Medical Genetics.

[59]  Michael Kühl,et al.  Lypd6 enhances Wnt/β-catenin signaling by promoting Lrp6 phosphorylation in raft plasma membrane domains. , 2013, Developmental cell.

[60]  Björn Usadel,et al.  Trimmomatic: a flexible trimmer for Illumina sequence data , 2014, Bioinform..

[61]  P. Stankiewicz,et al.  Position effects due to chromosome breakpoints that map approximately 900 Kb upstream and approximately 1.3 Mb downstream of SOX9 in two patients with campomelic dysplasia. , 2005, American journal of human genetics.

[62]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[63]  P. Stankiewicz,et al.  Deletions of recessive disease genes: CNV contribution to carrier states and disease-causing alleles , 2013, Genome research.

[64]  H. Ropers,et al.  Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes , 2010, Nature Genetics.

[65]  M. Hurles,et al.  Absence of heterozygosity due to template switching during replicative rearrangements. , 2015, American journal of human genetics.

[66]  V. Beneš,et al.  Disruption of EXOC6B in a patient with developmental delay, epilepsy, and a de novo balanced t(2;8) translocation , 2013, European Journal of Human Genetics.