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1.  The Rate of Nonallelic Homologous Recombination in Males Is Highly Variable, Correlated between Monozygotic Twins and Independent of Age 
PLoS Genetics  2014;10(3):e1004195.
Nonallelic homologous recombination (NAHR) between highly similar duplicated sequences generates chromosomal deletions, duplications and inversions, which can cause diverse genetic disorders. Little is known about interindividual variation in NAHR rates and the factors that influence this. We estimated the rate of deletion at the CMT1A-REP NAHR hotspot in sperm DNA from 34 male donors, including 16 monozygotic (MZ) co-twins (8 twin pairs) aged 24 to 67 years old. The average NAHR rate was 3.5×10−5 with a seven-fold variation across individuals. Despite good statistical power to detect even a subtle correlation, we observed no relationship between age of unrelated individuals and the rate of NAHR in their sperm, likely reflecting the meiotic-specific origin of these events. We then estimated the heritability of deletion rate by calculating the intraclass correlation (ICC) within MZ co-twins, revealing a significant correlation between MZ co-twins (ICC = 0.784, p = 0.0039), with MZ co-twins being significantly more correlated than unrelated pairs. We showed that this heritability cannot be explained by variation in PRDM9, a known regulator of NAHR, or variation within the NAHR hotspot itself. We also did not detect any correlation between Body Mass Index (BMI), smoking status or alcohol intake and rate of NAHR. Our results suggest that other, as yet unidentified, genetic or environmental factors play a significant role in the regulation of NAHR and are responsible for the extensive variation in the population for the probability of fathering a child with a genomic disorder resulting from a pathogenic deletion.
Author Summary
Many genetic disorders are caused by deletions of specific regions of DNA in sperm or egg cells that go on to produce a child. This can occur through ectopic homologous recombination between highly similar segments of DNA at different positions within the genome. Little is known about the differences in rates of deletion between individuals or the factors that influence this. We analysed the rate of deletion at one such section of DNA in sperm DNA from 34 male donors, including 16 monozygotic co-twins. We observed a seven-fold variation in deletion rate across individuals. Deletion rate is significantly correlated between monozygote co-twins, indicating that deletion rate is heritable. This heritability cannot be explained by age, any known genetic regulator of deletion rate, Body Mass Index, smoking status or alcohol intake. Our results suggest that other, as yet unidentified, genetic or environmental factors play a significant role in the regulation of deletion. These factors are responsible for the extensive variation in the population for the probability of fathering a child with a genomic disorder resulting from a pathogenic deletion.
PMCID: PMC3945173  PMID: 24603440
2.  Fine Characterisation of a Recombination Hotspot at the DPY19L2 Locus and Resolution of the Paradoxical Excess of Duplications over Deletions in the General Population 
PLoS Genetics  2013;9(3):e1003363.
We demonstrated previously that 75% of infertile men with round, acrosomeless spermatozoa (globozoospermia) had a homozygous 200-Kb deletion removing the totality of DPY19L2. We showed that this deletion occurred by Non-Allelic Homologous Recombination (NAHR) between two homologous 28-Kb Low Copy Repeats (LCRs) located on each side of the gene. The accepted NAHR model predicts that inter-chromatid and inter-chromosome NAHR create a deleted and a duplicated recombined allele, while intra-chromatid events only generate deletions. Therefore more deletions are expected to be produced de novo. Surprisingly, array CGH data show that, in the general population, DPY19L2 duplicated alleles are approximately three times as frequent as deleted alleles. In order to shed light on this paradox, we developed a sperm-based assay to measure the de novo rates of deletions and duplications at this locus. As predicted by the NAHR model, we identified an excess of de novo deletions over duplications. We calculated that the excess of de novo deletion was compensated by evolutionary loss, whereas duplications, not subjected to selection, increased gradually. Purifying selection against sterile, homozygous deleted men may be sufficient for this compensation, but heterozygously deleted men might also suffer a small fitness penalty. The recombined alleles were sequenced to pinpoint the localisation of the breakpoints. We analysed a total of 15 homozygous deleted patients and 17 heterozygous individuals carrying either a deletion (n = 4) or a duplication (n = 13). All but two alleles fell within a 1.2-Kb region central to the 28-Kb LCR, indicating that >90% of the NAHR took place in that region. We showed that a PRDM9 13-mer recognition sequence is located right in the centre of that region. Our results therefore strengthen the link between this consensus sequence and the occurrence of NAHR.
Author Summary
We demonstrated previously that most men with globozoospermia, who produce only round acrosomeless spermatozoa and are 100% infertile, had a homozygous deletion removing the totality of DPY19L2. We also showed that this deletion occurred by Non-Allelic Homologous Recombination (NAHR). NAHR results in the production of deletions and duplications of regions encompassed by two homologous sequences, normally with a higher occurrence of deletions over duplications. Analysis of public databases at the DPY19L2 locus paradoxically revealed that, in the general population, duplications were approximately three times as frequent as deletions. Analysis of sperm DNA permits us to quantify de novo events that take place during male meiosis. We therefore measured the rates of de novo deletion and duplication in the sperm of three healthy donors. As predicted by the NAHR theoretical model and contrary to the allelic frequency observed in the general population, we identified an approximate 2-fold excess of deletions over duplications. We calculated that the measured rate of de novo deletion was compensated by evolutionary loss, whereas duplications, not subjected to selection, increased gradually. Purifying selection against infertile homozygous deleted men may be sufficient for this compensation, or heterozygously deleted men may also suffer a small fitness penalty.
PMCID: PMC3605140  PMID: 23555282
3.  Genome-Wide Signatures of ‘Rearrangement Hotspots’ within Segmental Duplications in Humans 
PLoS ONE  2011;6(12):e28853.
The primary objective of this study was to create a genome-wide high resolution map (i.e., >100 bp) of ‘rearrangement hotspots’ which can facilitate the identification of regions capable of mediating de novo deletions or duplications in humans. A hierarchical method was employed to fragment segmental duplications (SDs) into multiple smaller SD units. Combining an end space free pairwise alignment algorithm with a ‘seed and extend’ approach, we have exhaustively searched 409 million alignments to detect complex structural rearrangements within the reference-guided assembly of the NA18507 human genome (18× coverage), including the previously identified novel 4.8 Mb sequence from de novo assembly within this genome. We have identified 1,963 rearrangement hotspots within SDs which encompass 166 genes and display an enrichment of duplicated gene nucleotide variants (DNVs). These regions are correlated with increased non-allelic homologous recombination (NAHR) event frequency which presumably represents the origin of copy number variations (CNVs) and pathogenic duplications/deletions. Analysis revealed that 20% of the detected hotspots are clustered within the proximal and distal SD breakpoints flanked by the pathogenic deletions/duplications that have been mapped for 24 NAHR-mediated genomic disorders. FISH Validation of selected complex regions revealed 94% concordance with in silico localization of the highly homologous derivatives. Other results from this study indicate that intra-chromosomal recombination is enhanced in genic compared with agenic duplicated regions, and that gene desert regions comprising SDs may represent reservoirs for creation of novel genes. The generation of genome-wide signatures of ‘rearrangement hotspots’, which likely serve as templates for NAHR, may provide a powerful approach towards understanding the underlying mutational mechanism(s) for development of constitutional and acquired diseases.
PMCID: PMC3237539  PMID: 22194928
4.  Human endogenous retroviral elements promote genome instability via non-allelic homologous recombination 
BMC Biology  2014;12:74.
Recurrent rearrangements of the human genome resulting in disease or variation are mainly mediated by non-allelic homologous recombination (NAHR) between low-copy repeats. However, other genomic structures, including AT-rich palindromes and retroviruses, have also been reported to underlie recurrent structural rearrangements. Notably, recurrent deletions of Yq12 conveying azoospermia, as well as non-pathogenic reciprocal duplications, are mediated by human endogenous retroviral elements (HERVs). We hypothesized that HERV elements throughout the genome can serve as substrates for genomic instability and result in human copy-number variation (CNV).
We developed parameters to identify HERV elements similar to those that mediate Yq12 rearrangements as well as recurrent deletions of 3q13.2q13.31. We used these parameters to identify HERV pairs genome-wide that may cause instability. Our analysis highlighted 170 pairs, flanking 12.1% of the genome. We cross-referenced these predicted susceptibility regions with CNVs from our clinical databases for potentially HERV-mediated rearrangements and identified 78 CNVs. We subsequently molecularly confirmed recurrent deletion and duplication rearrangements at four loci in ten individuals, including reciprocal rearrangements at two loci. Breakpoint sequencing revealed clustering in regions of high sequence identity enriched in PRDM9-mediated recombination hotspot motifs.
The presence of deletions and reciprocal duplications suggests NAHR as the causative mechanism of HERV-mediated CNV, even though the length and the sequence homology of the HERV elements are less than currently thought to be required for NAHR. We propose that in addition to HERVs, other repetitive elements, such as long interspersed elements, may also be responsible for the formation of recurrent CNVs via NAHR.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-014-0074-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4195946  PMID: 25246103
HERV; copy number variation; genome instability; non-allelic homologous recombination
5.  Analysis of meiotic recombination in 22q11.2, a region that frequently undergoes deletions and duplications 
BMC Medical Genetics  2007;8:14.
The 22q11.2 deletion syndrome is the most frequent genomic disorder with an estimated frequency of 1/4000 live births. The majority of patients (90%) have the same deletion of 3 Mb (Typically Deleted Region, TDR) that results from aberrant recombination at meiosis between region specific low-copy repeats (LCRs).
As a first step towards the characterization of recombination rates and breakpoints within the 22q11.2 region we have constructed a high resolution recombination breakpoint map based on pedigree analysis and a population-based historical recombination map based on LD analysis.
Our pedigree map allows the location of recombination breakpoints with a high resolution (potential recombination hotspots), and this approach has led to the identification of 5 breakpoint segments of 50 kb or less (8.6 kb the smallest), that coincide with historical hotspots. It has been suggested that aberrant recombination leading to deletion (and duplication) is caused by low rates of Allelic Homologous Recombination (AHR) within the affected region. However, recombination rate estimates for 22q11.2 region show that neither average recombination rates in the 22q11.2 region or within LCR22-2 (the LCR implicated in most deletions and duplications), are significantly below chromosome 22 averages. Furthermore, LCR22-2, the repeat most frequently implicated in rearrangements, is also the LCR22 with the highest levels of AHR. In addition, we find recombination events in the 22q11.2 region to cluster within families. Within this context, the same chromosome recombines twice in one family; first by AHR and in the next generation by NAHR resulting in an individual affected with the del22q11.2 syndrome.
We show in the context of a first high resolution pedigree map of the 22q11.2 region that NAHR within LCR22 leading to duplications and deletions cannot be explained exclusively under a hypothesis of low AHR rates. In addition, we find that AHR recombination events cluster within families. If normal and aberrant recombination are mechanistically related, the fact that LCR22s undergo frequent AHR and that we find familial differences in recombination rates within the 22q11.2 region would have obvious health-related implications.
PMCID: PMC1855045  PMID: 17397557
6.  Generation of Tandem Direct Duplications by Reversed-Ends Transposition of Maize Ac Elements 
PLoS Genetics  2013;9(8):e1003691.
Tandem direct duplications are a common feature of the genomes of eukaryotes ranging from yeast to human, where they comprise a significant fraction of copy number variations. The prevailing model for the formation of tandem direct duplications is non-allelic homologous recombination (NAHR). Here we report the isolation of a series of duplications and reciprocal deletions isolated de novo from a maize allele containing two Class II Ac/Ds transposons. The duplication/deletion structures suggest that they were generated by alternative transposition reactions involving the termini of two nearby transposable elements. The deletion/duplication breakpoint junctions contain 8 bp target site duplications characteristic of Ac/Ds transposition events, confirming their formation directly by an alternative transposition mechanism. Tandem direct duplications and reciprocal deletions were generated at a relatively high frequency (∼0.5 to 1%) in the materials examined here in which transposons are positioned nearby each other in appropriate orientation; frequencies would likely be much lower in other genotypes. To test whether this mechanism may have contributed to maize genome evolution, we analyzed sequences flanking Ac/Ds and other hAT family transposons and identified three small tandem direct duplications with the structural features predicted by the alternative transposition mechanism. Together these results show that some class II transposons are capable of directly inducing tandem sequence duplications, and that this activity has contributed to the evolution of the maize genome.
Author Summary
The recent explosion of genome sequence data has greatly increased the need to understand the forces that shape eukaryotic genomes. A common feature of higher plant genomes is the presence of large numbers of duplications, often occurring as tandem repeats of thousands of base pairs. Despite the importance of gene duplications in evolution and disease, the precise mechanism(s) that generate tandem duplications are still unclear. In this study we identified nine new spontaneous duplications that arose flanking elements of the Ac transposon system. These duplications range in size from 8 kbp to >5,000 kbp, and all cases exhibit features characteristic of Ac transposition. Using similar criteria in a bioinformatics search, we identified three smaller duplications adjacent to other hAT family transposons in the maize B73 reference genome sequence. Our results show that transposable elements can directly generate tandem duplications via alternative transposition, and that this mechanism is responsible for at least some of the duplications present in the maize B73 genome. This work extends the significance of Barbara McClintock's discovery of transposable elements by demonstrating how they can act as agents of genome expansion.
PMCID: PMC3744419  PMID: 23966872
7.  Recurrent reciprocal deletions and duplications of 16p13.11: the deletion is a risk factor for MR/MCA while the duplication may be a rare benign variant 
Journal of Medical Genetics  2008;46(4):223-232.
Genomic disorders are often caused by non-allelic homologous recombination between segmental duplications. Chromosome 16 is especially rich in a chromosome-specific low copy repeat, termed LCR16.
Methods and Results:
A bacterial artificial chromosome (BAC) array comparative genome hybridisation (CGH) screen of 1027 patients with mental retardation and/or multiple congenital anomalies (MR/MCA) was performed. The BAC array CGH screen identified five patients with deletions and five with apparently reciprocal duplications of 16p13 covering 1.65 Mb, including 15 RefSeq genes. In addition, three atypical rearrangements overlapping or flanking this region were found. Fine mapping by high-resolution oligonucleotide arrays suggests that these deletions and duplications result from non-allelic homologous recombination (NAHR) between distinct LCR16 subunits with >99% sequence identity. Deletions and duplications were either de novo or inherited from unaffected parents. To determine whether these imbalances are associated with the MR/MCA phenotype or whether they might be benign variants, a population of 2014 normal controls was screened. The absence of deletions in the control population showed that 16p13.11 deletions are significantly associated with MR/MCA (p = 0.0048). Despite phenotypic variability, common features were identified: three patients with deletions presented with MR, microcephaly and epilepsy (two of these had also short stature), and two other deletion carriers ascertained prenatally presented with cleft lip and midline defects. In contrast to its previous association with autism, the duplication seems to be a common variant in the population (5/1682, 0.29%).
These findings indicate that deletions inherited from clinically normal parents are likely to be causal for the patients’ phenotype whereas the role of duplications (de novo or inherited) in the phenotype remains uncertain. This difference in knowledge regarding the clinical relevance of the deletion and the duplication causes a paradigm shift in (cyto)genetic counselling.
PMCID: PMC2658752  PMID: 18550696
8.  Competitive Repair by Naturally Dispersed Repetitive DNA during Non-Allelic Homologous Recombination 
PLoS Genetics  2010;6(12):e1001228.
Genome rearrangements often result from non-allelic homologous recombination (NAHR) between repetitive DNA elements dispersed throughout the genome. Here we systematically analyze NAHR between Ty retrotransposons using a genome-wide approach that exploits unique features of Saccharomyces cerevisiae purebred and Saccharomyces cerevisiae/Saccharomyces bayanus hybrid diploids. We find that DNA double-strand breaks (DSBs) induce NAHR–dependent rearrangements using Ty elements located 12 to 48 kilobases distal to the break site. This break-distal recombination (BDR) occurs frequently, even when allelic recombination can repair the break using the homolog. Robust BDR–dependent NAHR demonstrates that sequences very distal to DSBs can effectively compete with proximal sequences for repair of the break. In addition, our analysis of NAHR partner choice between Ty repeats shows that intrachromosomal Ty partners are preferred despite the abundance of potential interchromosomal Ty partners that share higher sequence identity. This competitive advantage of intrachromosomal Tys results from the relative efficiencies of different NAHR repair pathways. Finally, NAHR generates deleterious rearrangements more frequently when DSBs occur outside rather than within a Ty repeat. These findings yield insights into mechanisms of repeat-mediated genome rearrangements associated with evolution and cancer.
Author Summary
The human genome is structurally dynamic, frequently undergoing loss, duplication, and rearrangement of large chromosome segments. These structural changes occur both in normal and in cancerous cells and are thought to cause both benign and deleterious changes in cell function. Many of these structural alterations are generated when two dispersed repeated DNA sequences at non-allelic sites recombine during non-allelic homologous recombination (NAHR). Here we study NAHR on a genome-wide scale using the experimentally tractable budding yeast as a eukaryotic model genome with its fully sequenced family of repeated DNA elements, the Ty retrotransposons. With our novel system, we simultaneously measure the effects of known recombination parameters on the frequency of NAHR to understand which parameters most influence the occurrence of rearrangements between repetitive sequences. These findings provide a basic framework for interpreting how structural changes observed in the human genome may have arisen.
PMCID: PMC2996329  PMID: 21151956
9.  Genome destabilization by homologous recombination in the germline 
Meiotic recombination, which promotes proper homologous chromosome segregation at the first meiotic division, normally occurs between allelic sequences on homologues. However, recombination can also take place between non-allelic DNA segments that share high sequence identity. Such non-allelic homologous recombination (NAHR) can markedly alter genome architecture during gametogenesis by generating chromosomal rearrangements. Indeed, NAHR-mediated deletions, duplications, inversions and other alterations have been implicated in numerous human genetic disorders. Studies in yeast have revealed insights into the molecular mechanisms of meiotic NAHR as well as the cellular strategies that limit NAHR.
PMCID: PMC3073813  PMID: 20164840
10.  Mechanisms for human genomic rearrangements 
PathoGenetics  2008;1:4.
Genomic rearrangements describe gross DNA changes of the size ranging from a couple of hundred base pairs, the size of an average exon, to megabases (Mb). When greater than 3 to 5 Mb, such changes are usually visible microscopically by chromosome studies. Human diseases that result from genomic rearrangements have been called genomic disorders. Three major mechanisms have been proposed for genomic rearrangements in the human genome. Non-allelic homologous recombination (NAHR) is mostly mediated by low-copy repeats (LCRs) with recombination hotspots, gene conversion and apparent minimal efficient processing segments. NAHR accounts for most of the recurrent rearrangements: those that share a common size, show clustering of breakpoints, and recur in multiple individuals. Non-recurrent rearrangements are of different sizes in each patient, but may share a smallest region of overlap whose change in copy number may result in shared clinical features among different patients. LCRs do not mediate, but may stimulate non-recurrent events. Some rare NAHRs can also be mediated by highly homologous repetitive sequences (for example, Alu, LINE); these NAHRs account for some of the non-recurrent rearrangements. Other non-recurrent rearrangements can be explained by non-homologous end-joining (NHEJ) and the Fork Stalling and Template Switching (FoSTeS) models. These mechanisms occur both in germ cells, where the rearrangements can be associated with genomic disorders, and in somatic cells in which such genomic rearrangements can cause disorders such as cancer. NAHR, NHEJ and FoSTeS probably account for the majority of genomic rearrangements in our genome and the frequency distribution of the three at a given locus may partially reflect the genomic architecture in proximity to that locus. We provide a review of the current understanding of these three models.
PMCID: PMC2583991  PMID: 19014668
11.  Common AZFc structure may possess the optimal spermatogenesis efficiency relative to the rearranged structures mediated by non-allele homologous recombination 
Scientific Reports  2015;5:10551.
The azoopsermia factor c (AZFc) region of human Y-chromosome is an essential genomic segment for spermatogenesis with frequent non-allele homologous recombination (NAHR). Recent case-control studies on the association of the NAHR-based AZFc structural mutations with spermatogenic failure produced inconsistent results. To more precisely evaluate their spermatogenesis effects, we investigated the correlation between the subdivided AZFc mutations and sperm production in 3,439 Han Chinese males. Our results showed that both partial AZFc deletion-only and primary duplication mutation presented a significant risk for decreased sperm production. Restoration of the reduced dosage of the AZFc content to the normal level had a milder effect, whereas an overdose of the AZFc content arising from multiple duplications of a partial AZFc-deleted structure produced a more serious consequence compared to the partial deletion-only mutation. Additionally, the AZFc-mutated structures with excessive NAHR-substrate showed a notably negative effect on spermatogenesis. These results suggest that the recurrent NAHR-based AZFc mutations may be associated with decreased spermatogenesis efficiency in present population. More significantly, our finding implies that the overdose of AZFc NAHR-substrate may produce an additional risk for the massive AZFbc deletions during the multi-stage division process of germ cells and thus impair the global spermatogenesis efficiency in the carriers.
PMCID: PMC4441132  PMID: 26000765
12.  Genomic Hypomethylation in the Human Germline Associates with Selective Structural Mutability in the Human Genome 
PLoS Genetics  2012;8(5):e1002692.
The hotspots of structural polymorphisms and structural mutability in the human genome remain to be explained mechanistically. We examine associations of structural mutability with germline DNA methylation and with non-allelic homologous recombination (NAHR) mediated by low-copy repeats (LCRs). Combined evidence from four human sperm methylome maps, human genome evolution, structural polymorphisms in the human population, and previous genomic and disease studies consistently points to a strong association of germline hypomethylation and genomic instability. Specifically, methylation deserts, the ∼1% fraction of the human genome with the lowest methylation in the germline, show a tenfold enrichment for structural rearrangements that occurred in the human genome since the branching of chimpanzee and are highly enriched for fast-evolving loci that regulate tissue-specific gene expression. Analysis of copy number variants (CNVs) from 400 human samples identified using a custom-designed array comparative genomic hybridization (aCGH) chip, combined with publicly available structural variation data, indicates that association of structural mutability with germline hypomethylation is comparable in magnitude to the association of structural mutability with LCR–mediated NAHR. Moreover, rare CNVs occurring in the genomes of individuals diagnosed with schizophrenia, bipolar disorder, and developmental delay and de novo CNVs occurring in those diagnosed with autism are significantly more concentrated within hypomethylated regions. These findings suggest a new connection between the epigenome, selective mutability, evolution, and human disease.
Author Summary
The human genome contains many loci with high incidence of structural mutations, including insertions and deletions of chromosomal segments. This excessive mutability has accelerated evolution and contributed to human disease but has yet to be explained. Segments of DNA repeated in low-copy numbers (LCRs) have been previously implicated in promoting structural mutability in specific disease-associated loci. Lack of methylation (hypomethylation) of genomic DNA has been previously associated with high structural mutability in gibbons and in human cancer cells, but the association with structural mutability in the human germline has not been explored prior to this study. Our analyses confirm the role of LCRs in promoting structural mutability on the genome scale but also reveal a surprisingly strong association of genomic instability with hypomethylation. Specifically, evolutionary analyses reveal that methylation deserts, the ∼1% fraction of the human genome with the lowest methylation in human sperm, harbor a tenfold higher number of structural mutations than genome-wide average. Moreover, the structural mutations in individuals diagnosed with schizophrenia, bipolar disorder, developmental delay, and autism are significantly more concentrated within hypomethylated regions. Our findings suggest a new connection between methylation of genomic DNA, selective structural mutability, evolution, and human disease.
PMCID: PMC3355074  PMID: 22615578
13.  What drives recombination hotspots to repeat DNA in humans? 
Recombination between homologous, but non-allelic, stretches of DNA such as gene families, segmental duplications and repeat elements is an important source of mutation. In humans, recent studies have identified short DNA motifs that both determine the location of 40 per cent of meiotic cross-over hotspots and are significantly enriched at the breakpoints of recurrent non-allelic homologous recombination (NAHR) syndromes. Unexpectedly, the most highly penetrant form of the motif occurs on the background of an inactive repeat element family (THE1 elements) and the motif also has strong recombinogenic activity on currently active element families including Alu and LINE2 elements. Analysis of genetic variation among members of these repeat families indicates an important role for NAHR in their evolution. Given the potential for double-strand breaks within repeat DNA to cause pathological rearrangement, the association between repeats and hotspots is surprising. Here we consider possible explanations for why selection acting against NAHR has not eliminated hotspots from repeat DNA including mechanistic constraints, possible benefits to repeat DNA from recruiting hotspots and rapid evolution of the recombination machinery. I suggest that rapid evolution of hotspot motifs may, surprisingly, tend to favour sequences present in repeat DNA and outline the data required to differentiate between hypotheses.
PMCID: PMC2871820  PMID: 20308096
recombination hotspot; mutation; repeat element
14.  Reciprocal duplication of the Williams-Beuren syndrome deletion on chromosome 7q11.23 is associated with schizophrenia 
Biological psychiatry  2013;75(5):371-377.
Several copy number variants (CNVs) have been implicated as susceptibility factors for schizophrenia (SZ). Some of these same CNV also increase risk for autism spectrum disorders (ASD), suggesting an etiologic overlap between these conditions. Recently, de novo duplications of a region on chromosome 7q11.23 were associated with ASD. The reciprocal deletion of this region causes Williams-Beuren syndrome (WBS).
We assayed an Ashkenazi Jewish cohort of 554 SZ cases and 1014 controls for copy number variation (CNV), using a high-density genome-wide array. An excess of large rare and de novo CNV were observed, including a 1.4 Mb duplication on chromosome 7q11.23 identified in two unrelated patients. To test whether this 7q11.23 duplication is also associated with SZ, we obtained data for 14,387 SZ cases and 28,139 controls from seven additional studies with high-resolution genome-wide CNV detection. We performed a meta-analysis, correcting for study population of origin, to assess whether the 7q11.23 duplication is associated with SZ.
We find duplications at 7q11.23 in 11 of 14,387 SZ cases with only 1 in 28,139 controls (unadjusted odds ratio, 21.52, 95% CI: 3.13-922.6, p-value 5.5×10-5; adjusted odds ratio 10.8, 95% CI: 1.46-79.62, p-value 0.007). Of three SZ duplication carriers with available detailed retrospective data, all show social anxiety and language delay premorbid to SZ onset, consistent with both human studies and animal models of the 7q11.23 duplication.
We have identified a new CNV associated with SZ. Reciprocal duplication of the Williams syndrome deletion at chromosome 7q11.23 confers an approximately 10-fold increase in risk for SZ.
PMCID: PMC3838485  PMID: 23871472
Schizophrenia; 7q11.23 duplication syndrome; Psychiatric genetics; Schizophrenia genetics; Autism; Williams-Beuren syndrome
15.  Characterization of the past and current duplication activities in the human 22q11.2 region 
BMC Genomics  2011;12:71.
Segmental duplications (SDs) on 22q11.2 (LCR22), serve as substrates for meiotic non-allelic homologous recombination (NAHR) events resulting in several clinically significant genomic disorders.
To understand the duplication activity leading to the complicated SD structure of this region, we have applied the A-Bruijn graph algorithm to decompose the 22q11.2 SDs to 523 fundamental duplication sequences, termed subunits. Cross-species syntenic analysis of primate genomes demonstrates that many of these LCR22 subunits emerged very recently, especially those implicated in human genomic disorders. Some subunits have expanded more actively than others, and young Alu SINEs, are associated much more frequently with duplicated sequences that have undergone active expansion, confirming their role in mediating recombination events. Many copy number variations (CNVs) exist on 22q11.2, some flanked by SDs. Interestingly, two chromosome breakpoints for 13 CNVs (mean length 65 kb) are located in paralogous subunits, providing direct evidence that SD subunits could contribute to CNV formation. Sequence analysis of PACs or BACs identified extra CNVs, specifically, 10 insertions and 18 deletions within 22q11.2; four were more than 10 kb in size and most contained young AluYs at their breakpoints.
Our study indicates that AluYs are implicated in the past and current duplication events, and moreover suggests that DNA rearrangements in 22q11.2 genomic disorders perhaps do not occur randomly but involve both actively expanded duplication subunits and Alu elements.
PMCID: PMC3040729  PMID: 21269513
16.  Screening of copy number variants in the 22q11.2 region of congenital heart disease patients from the São Miguel Island, Azores, revealed the second patient with a triplication 
BMC Genetics  2014;15:115.
The rearrangements in the 22q11.2 chromosomal region, responsible for the 22q11.2 deletion and microduplication syndromes, are frequently associated with congenital heart disease (CHD). The present work aimed to identify the genetic basis of CHD in 87 patients from the São Miguel Island, Azores, through the detection of copy number variants (CNVs) in the 22q11.2 region. These structural variants were searched using multiplex ligation-dependent probe amplification (MLPA). In patients with CNVs, we additionally performed fluorescent in situ hybridization (FISH) for the assessment of the exact number of 22q11.2 copies among each chromosome, and array comparative genomic hybridization (array-CGH) for the determination of the exact length of CNVs.
We found that four patients (4.6%; A to D) carried CNVs. Patients A and D, both affected with a ventricular septal defect, carried a de novo 2.5 Mb deletion of the 22q11.2 region, which was probably originated by inter-chromosomal (inter-chromatid) non-allelic homologous recombination (NAHR) events in the regions containing low-copy repeats (LCRs). Patient C, with an atrial septal defect, carried a de novo 2.5 Mb duplication of 22q11.2 region, which could have been probably generated during gametogenesis by NAHR or by unequal crossing-over; additionally, this patient presented a benign 288 Kb duplication, which included the TOP3B gene inherited from her healthy mother. Finally, patient B showed a 3 Mb triplication associated with dysmorphic facial features, cognitive deficit and heart defects, a clinical feature not reported in the only case described so far in the literature. The evaluation of patient B’s parents revealed a 2.5 Mb duplication in her father, suggesting a paternal inheritance with an extra copy.
This report allowed the identification of rare deletion and microduplication syndromes in Azorean CHD patients. Moreover, we report the second patient with a 22q11.2 triplication, and we suggest that patients with triplications of chromosome 22q11.2, although they share some characteristic features with the deletion and microduplication syndromes, present a more severe phenotype probably due to the major dosage of implicated genes.
PMCID: PMC4228150  PMID: 25376777
Congenital heart disease; 22q11.2 deletion; 22q11.2 microduplication; 22q11.2 triplication
17.  Structures and Molecular Mechanisms for Common 15q13.3 Microduplications Involving CHRNA7: Benign or Pathological? 
Human mutation  2010;31(7):840-850.
We have investigated four ~1.6-Mb microduplications and 55 smaller 350–680-kb microduplications at 15q13.2–q13.3 involving the CHRNA7 gene that were detected by clinical microarray analysis. Applying high-resolution array-CGH, we mapped all 118 chromosomal breakpoints of these microduplications. We also sequenced 26 small microduplication breakpoints that were clustering at hotspots of nonallelic homologous recombination (NAHR). All four large microduplications likely arose by NAHR between BP4 and BP5 LCRs, and 54 small microduplications arose by NAHR between two CHRNA7-LCR copies. We identified two classes of ~1.6-Mb microduplications and five classes of small microduplications differing in duplication size, and show that they duplicate the entire CHRNA7. We propose that size differences among small microduplications result from preexisting heterogeneity of the common BP4–BP5 inversion. Clinical data and family histories of 11 patients with small microduplications involving CHRNA7 suggest that these microduplications might be associated with developmental delay/mental retardation, muscular hypotonia, and a variety of neuropsychiatric disorders. However, we conclude that these microduplications and their associated potential for increased dosage of the CHRNA7-encoded α7 subunit of nicotinic acetylcholine receptors are of uncertain clinical significance at present. Nevertheless, if they prove to have a pathological effects, their high frequency could make them a common risk factor for many neurobehavioral disorders.
PMCID: PMC3162316  PMID: 20506139
microduplication; CHRNA7; NAHR; hypotonia; autism spectrum disorder
18.  Detecting non-allelic homologous recombination from high-throughput sequencing data 
Genome Biology  2015;16(1):72.
Non-allelic homologous recombination (NAHR) is a common mechanism for generating genome rearrangements and is implicated in numerous genetic disorders, but its detection in high-throughput sequencing data poses a serious challenge. We present a probabilistic model of NAHR and demonstrate its ability to find NAHR in low-coverage sequencing data from 44 individuals. We identify NAHR-mediated deletions or duplications in 109 of 324 potential NAHR loci in at least one of the individuals. These calls segregate by ancestry, are more common in closely spaced repeats, often result in duplicated genes or pseudogenes, and affect highly studied genes such as GBA and CYP2E1.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0633-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4425883  PMID: 25886137
19.  Drosophila Duplication Hotspots Are Associated with Late-Replicating Regions of the Genome 
PLoS Genetics  2011;7(11):e1002340.
Duplications play a significant role in both extremes of the phenotypic spectrum of newly arising mutations: they can have severe deleterious effects (e.g. duplications underlie a variety of diseases) but can also be highly advantageous. The phenotypic potential of newly arisen duplications has stimulated wide interest in both the mutational and selective processes shaping these variants in the genome. Here we take advantage of the Drosophila simulans–Drosophila melanogaster genetic system to further our understanding of both processes. Regarding mutational processes, the study of two closely related species allows investigation of the potential existence of shared duplication hotspots, and the similarities and differences between the two genomes can be used to dissect its underlying causes. Regarding selection, the difference in the effective population size between the two species can be leveraged to ask questions about the strength of selection acting on different classes of duplications. In this study, we conducted a survey of duplication polymorphisms in 14 different lines of D. simulans using tiling microarrays and combined it with an analogous survey for the D. melanogaster genome. By integrating the two datasets, we identified duplication hotspots conserved between the two species. However, unlike the duplication hotspots identified in mammalian genomes, Drosophila duplication hotspots are not associated with sequences of high sequence identity capable of mediating non-allelic homologous recombination. Instead, Drosophila duplication hotspots are associated with late-replicating regions of the genome, suggesting a link between DNA replication and duplication rates. We also found evidence supporting a higher effectiveness of selection on duplications in D. simulans than in D. melanogaster. This is also true for duplications segregating at high frequency, where we find evidence in D. simulans that a sizeable fraction of these mutations is being driven to fixation by positive selection.
Author Summary
DNA duplications are important contributors to the phenotypic differences observed between individuals. These mutations can disrupt the normal functioning of genes and so are often associated with disease. But because they can add genetic information they can also lead to evolutionary change. Understanding how selection and non-random mutation processes shape the distribution of duplications throughout the genome is important to elucidate both the medical and evolutionary impacts of these mutations. Here, we examined the roles of selection and mutation in shaping patterns of duplication polymorphisms across the genomes of the fruit fly Drosophila melanogaster and its sister species, D. simulans. We found that selection is pervasive in both genomes but is more efficient in D. simulans than in D. melanogaster. We also found that these two species have shared duplication hotspots, i.e. orthologous regions experiencing high rates of duplication in the two genomes. After excluding the hypothesis that Drosophila duplication hotspots are associated with regions of the genome rich in segmental duplications (as observed for mammalian genomes), we show that they are associated with late-replicating regions of the genome. Our work therefore proposes a link between DNA replication and rates of duplication across the genome.
PMCID: PMC3207856  PMID: 22072977
20.  A Novel Hybrid CFHR1/CFH Gene Causes Atypical Hemolytic Uremic Syndrome 
Pediatric nephrology (Berlin, Germany)  2013;28(11):2221-2225.
Mutations in complement factor H (CFH) are associated with complement dysregulation and the development of an aggressive form of atypical hemolytic uremic syndrome (aHUS) that progresses to end-stage renal disease and in most patients has a high rate of recurrence following transplantation. Sequence analysis of CFH and its downstream complement factor H-related genes (CFHR1-5) reveals several macrohomologous blocks caused by large genomic duplications. This high degree of sequence identity renders this area susceptible to nonallelic homologous recombination (NAHR) events, resulting in large-scale deletions, duplications, and the generation of hybrid CFH genes.
Here, we report the finding of a novel CFHR1/CFH hybrid gene created by a de novo NAHR event in a 14-year-old girl with aHUS. The resulting fusion protein contains the first three short consensus repeats (SCRs) of CFHR1 and the terminal two SCRs of CFH.
This finding demonstrates a novel pathogenic mechanism for the development of aHUS. Additionally, since standard Sanger sequencing is unable to detect such rearrangements, all aHUS patients should receive comprehensive genetic screening that includes analysis of copy number variation in order to identify patients with poor clinical prognoses.
PMCID: PMC4433496  PMID: 23880784
Atypical hemolytic uremic syndrome; alternative pathway of complement; nonallelic homologous recombination; factor H hybrid genes
21.  Identical repeated backbone of the human genome 
BMC Genomics  2010;11:60.
Identical sequences with a minimal length of about 300 base pairs (bp) have been involved in the generation of various meiotic/mitotic genomic rearrangements through non-allelic homologous recombination (NAHR) events. Genomic disorders and structural variation, together with gene remodelling processes have been associated with many of these rearrangements. Based on these observations, we identified and integrated all the 100% identical repeats of at least 300 bp in the NCBI version 36.2 human genome reference assembly into non-overlapping regions, thus defining the Identical Repeated Backbone (IRB) of the reference human genome.
The IRB sequences are distributed all over the genome in 66,600 regions, which correspond to ~2% of the total NCBI human genome reference assembly. Important structural and functional elements such as common repeats, segmental duplications, and genes are contained in the IRB. About 80% of the IRB bp overlap with known copy-number variants (CNVs). By analyzing the genes embedded in the IRB, we were able to detect some identical genes not previously included in the Ensembl release 50 annotation of human genes. In addition, we found evidence of IRB gene copy-number polymorphisms in raw sequence reads of two diploid sequenced genomes.
In general, the IRB offers new insight into the complex organization of the identical repeated sequences of the human genome. It provides an accurate map of potential NAHR sites which could be used in targeting the study of novel CNVs, predicting DNA copy-number variation in newly sequenced genomes, and improve genome annotation.
PMCID: PMC2845111  PMID: 20096123
22.  The role of retrotransposons in gene family expansions: insights from the mouse Abp gene family 
Retrotransposons have been suggested to provide a substrate for non-allelic homologous recombination (NAHR) and thereby promote gene family expansion. Their precise role, however, is controversial. Here we ask whether retrotransposons contributed to the recent expansions of the Androgen-binding protein (Abp) gene families that occurred independently in the mouse and rat genomes.
Using dot plot analysis, we found that the most recent duplication in the Abp region of the mouse genome is flanked by L1Md_T elements. Analysis of the sequence of these elements revealed breakpoints that are the relicts of the recombination that caused the duplication, confirming that the duplication arose as a result of NAHR using L1 elements as substrates. L1 and ERVII retrotransposons are considerably denser in the Abp regions than in one Mb flanking regions, while other repeat types are depleted in the Abp regions compared to flanking regions. L1 retrotransposons preferentially accumulated in the Abp gene regions after lineage separation and roughly followed the pattern of Abp gene expansion. By contrast, the proportion of shared vs. lineage-specific ERVII repeats in the Abp region resembles the rest of the genome.
We confirmed the role of L1 repeats in Abp gene duplication with the identification of recombinant L1Md_T elements at the edges of the most recent mouse Abp gene duplication. High densities of L1 and ERVII repeats were found in the Abp gene region with abrupt transitions at the region boundaries, suggesting that their higher densities are tightly associated with Abp gene duplication. We observed that the major accumulation of L1 elements occurred after the split of the mouse and rat lineages and that there is a striking overlap between the timing of L1 accumulation and expansion of the Abp gene family in the mouse genome. Establishing a link between the accumulation of L1 elements and the expansion of the Abp gene family and identification of an NAHR-related breakpoint in the most recent duplication are the main contributions of our study.
PMCID: PMC3669608  PMID: 23718880
House mouse; Gene duplication; Androgen-binding protein; LINE1; ERVII; NAHR
23.  Recurrent Deletions and Reciprocal Duplications of 10q11.21q11.23 Including CHAT and SLC18A3 are Likely Mediated by Complex Low-Copy Repeats 
Human mutation  2011;33(1):165-179.
We report 24 unrelated individuals with deletions and 17 additional cases with duplications at 10q11.21q21.1 identified by chromosomal microarray analysis. The rearrangements range in size from 0.3 to 12 Mb. Nineteen of the deletions and eight duplications are flanked by large, directly oriented segmental duplications of >98% sequence identity, suggesting that nonallelic homologous recombination (NAHR) caused these genomic rearrangements. Nine individuals with deletions and five with duplications have additional copy number changes. Detailed clinical evaluation of 20 patients with deletions revealed variable clinical features, with developmental delay (DD) and/or intellectual disability (ID) as the only features common to a majority of individuals. We suggest that some of the other features present in more than one patient with deletion, including hypotonia, sleep apnea, chronic constipation, gastroesophageal and vesicoureteral refluxes, epilepsy, ataxia, dysphagia, nystagmus, and ptosis may result from deletion of the CHAT gene, encoding choline acetyltransferase, and the SLC18A3 gene, mapping in the first intron of CHAT and encoding vesicular acetylcholine transporter. The phenotypic diversity and presence of the deletion in apparently normal carrier parents suggest that subjects carrying 10q11.21q11.23 deletions may exhibit variable phenotypic expressivity and incomplete penetrance influenced by additional genetic and nongenetic modifiers.
PMCID: PMC3655525  PMID: 21948486
CHAT; SLC18A3; genomic rearrangement; array CGH
24.  Segmental duplications mediate novel, clinically relevant chromosome rearrangements 
Human Molecular Genetics  2009;18(16):2957-2962.
Copy number studies have led to an explosion in the discovery of new segmental duplication-mediated deletions and duplications. We have analyzed copy number changes in 2419 patients referred for clinical array comparative genomic hybridization studies. Twenty-three percent of the abnormal copy number changes we found are immediately flanked by segmental duplications ≥10 kb in size and ≥95% identical in direct orientation, consistent with deletions and duplications generated by non-allelic homologous recombination. Here, we describe copy number changes in five previously unreported loci with genomic organization characteristic of NAHR-mediated gains and losses; namely, 2q11.2, 7q36.1, 17q23, 2q13 and 7q11.21. Deletions and duplications of 2q11.2, deletions of 7q36.1 and deletions of 17q23 are interpreted as pathogenic based on their genomic size, gene content, de novo inheritance and absence from control populations. The clinical significance of 2q13 deletions and duplications is still emerging, as these imbalances are also found in phenotypically normal family members and control individuals. Deletion of 7q11.21 is a benign copy number change well represented in control populations and copy number variation databases. Here, we discuss the genetic factors that can modify the phenotypic expression of such gains and losses, which likely play a role in these and other recurrent genomic disorders.
PMCID: PMC2714723  PMID: 19443486
25.  Genome-wide analyses of LINE–LINE-mediated nonallelic homologous recombination 
Nucleic Acids Research  2015;43(4):2188-2198.
Nonallelic homologous recombination (NAHR), occurring between low-copy repeats (LCRs) >10 kb in size and sharing >97% DNA sequence identity, is responsible for the majority of recurrent genomic rearrangements in the human genome. Recent studies have shown that transposable elements (TEs) can also mediate recurrent deletions and translocations, indicating the features of substrates that mediate NAHR may be significantly less stringent than previously believed. Using >4 kb length and >95% sequence identity criteria, we analyzed of the genome-wide distribution of long interspersed element (LINE) retrotransposon and their potential to mediate NAHR. We identified 17 005 directly oriented LINE pairs located <10 Mbp from each other as potential NAHR substrates, placing 82.8% of the human genome at risk of LINE–LINE-mediated instability. Cross-referencing these regions with CNVs in the Baylor College of Medicine clinical chromosomal microarray database of 36 285 patients, we identified 516 CNVs potentially mediated by LINEs. Using long-range PCR of five different genomic regions in a total of 44 patients, we confirmed that the CNV breakpoints in each patient map within the LINE elements. To additionally assess the scale of LINE–LINE/NAHR phenomenon in the human genome, we tested DNA samples from six healthy individuals on a custom aCGH microarray targeting LINE elements predicted to mediate CNVs and identified 25 LINE–LINE rearrangements. Our data indicate that LINE–LINE-mediated NAHR is widespread and under-recognized, and is an important mechanism of structural rearrangement contributing to human genomic variability.
PMCID: PMC4344489  PMID: 25613453

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