De novo duplication and deletions at 7q in a three‐generation family
Bertrand IsidorOlaya VillaOlivier PichonAnnaïg BriandDamien PoulainPierre BoisseauLuis Alberto Pérez‐JuradoCédric Le Caignec
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Chromosomal imbalances are a major cause of intellectual disability and multiple congenital anomalies (MR/MCA). With the advent of the array-based comparative genomic hybridization (array CGH) method, increasingly smaller and more complex rearrangements are detected in patients with MR/MCA. Among the numerous copy-number variants (CNVs) of various sizes identified when testing such patients, de novo large (i.e., >1 Mb) CNVs are relatively rare events [Miller et al., 2010]. When identified in a patient, such events have a high likelihood for being responsible for the abnormal phenotype. In the next generation, the genomic imbalance is transmitted following the classical Mendelian rules and, based on the current literature, is not supposed to increase the likelihood of the occurrence of a second de novo event. Here, we observed three de novo chromosomal rearrangements on the same chromosome arm in a three-generation family. The proposita (Patient III-1) presented with craniofacial anomalies (Fig. 1), moderate intellectual disability, and behavior disturbance. Her father (Patient II-2) had no physical anomalies but expressive language delay which required speech therapy. He could not complete normal school and was the only member of the family who had language delay and school difficulties. Supplementary clinical information is available online (See Supporting Information). A: Pedigree of the present family. Normal: no imbalance identified by array CGH. The arrow indicates the proposita. B: Photographs showing facial dysmorphic features with prominent forehead, high frontal hairline, low-set, and posteriorly rotated ears in Patient III-1. The karyotypes performed on the child (III-1) and her father (II-2) were both normal: 46,XX and 46,XY at ISCN +550 bands, respectively. A 44K oligoarray CGH performed on Patient III-1 led to the identification of a ∼1.43 Mb duplication in the 7q11.23 region and a ∼1.92 Mb deletion in the 7q22 region (see Methods in Supporting Information online). No other imbalance was identified. To fine-map the aberrations, 400K oligoarray was performed on Patient III-1. The 7q11.23 duplication was confirmed while the 7q22 deletion was more complex than previously thought, with two close deleted regions (Fig. 2A–C). A 1.34 Mb deletion was followed by a 361 kb two-copies (diploid) region and a 214 kb deletion. The parents' DNAs of Patient III-1 were analyzed with 44K arrays and showed normal results in the mother (II-3) while the father (II-2) only carried the duplication. These parental analyses demonstrated that the two 7q22 deletions occurred de novo in Patient III-1 while the 7q11.23 duplication was paternally inherited. We next performed 44K arrays in the phenotypically normal grandparents (individuals I-1 and I-2). Both grandparents were normal by array CGH, demonstrating that the 7q11.23 occurred de novo in Patient II-2. The Agilent 400K array-derived profile of chromosomes 7 and FISH analyses performed on Patient III-1. Black, red, and green dots indicate the log2-transformed fluorescence intensity ratios for normal, duplicated, or deleted (respectively) oligonucleotide probe falling outside the threshold setting used (ADM-2 algorithm at a threshold of 6.0 using CGH Analytics version 4.0). A: The ideogram and the fluorescence intensity ratio for each oligonucleotide probe of the entire chromosome 7. B: Detailed view of the duplication. C: Detailed view of the complex deletion. The horizontal axis indicates the log2-transformed fluorescence intensity ratio for each oligonucleotide probe and the vertical axis indicates their physical position on chromosome 7. The aberrant area is indicated by grey rectangle. Italic symbols indicate gene names. D–F: Dual-color FISH analyses performed on metaphases and interphase nuclei of Patient III-1. FISH with RP11-193P05 BAC clone located in the centromeric 7q22 deletion and RP11-667P12 BAC clone located in the 7q11.23 duplication showed strong red signals on metaphases and two close red signals on interphase nuclei and no green signal suggesting that the duplication and the centromeric 7q22 deletion were located on the same chromosome (probes were labeled with SpectrumGreen and SpectrumOrange, respectively). G: FISH with RP11-193P05 BAC clone located in the centromeric 7q22 deletion and G248P88412A8 fosmid located in the telomeric 7q22 deletion showed no red and green signals on the same chromosome demonstrating that both 7q22 deletions were located on the same chromosome (probes were labeled with SpectrumGreen and SpectrumOrange, respectively). A subtelomeric 7p probe labeled in green was used as control probe. The arrows mark the deletions observed on metaphases. FISH performed in Patients II-2 and III-1 and individuals I-1 and I-2 confirmed the array CGH results (Fig. 2D–F). In addition, interphase and metaphase FISH performed in Patient III-1 with a combination of probes located in the deleted and in the duplicated regions showed that the two deletions and the duplication occurred on the same haplotype (Fig. 2E–G). A target 60K custom oligoarray CGH experiment was performed on Patient III-1 to refine the locations of the breakpoints of the 7q22 deletions (data not shown). Next, by long-range PCR (TaKaRa LA Taq, TAKARA) and direct sequencing (primer sequences are available upon request), we characterized the breakpoints at a molecular level for the two 7q22 deletions. Sequence analyses showed microhomologies at the breakpoint junctions for both deletions (see Supplementary Fig. 1 in Supporting Information online). However, BLAST comparison with regions of 5 kb flanking and within the two 7q22 deletions as template did not reveal significant homologies among them or with sequences within or surrounding the WBS locus. Analysis of single-copy STR markers within the WBS locus revealed that Patient II-2 carried three distinct alleles at loci AFMb055xe5, D7S489B, CR16T, and D7S1870, two of them of paternal origin in all informative positions. Thus, a de novo 7q11.23 duplication in Patient II-2 was confirmed, generated by interchromosomal exchange in meiosis I in grandfather's (I-1) germ cells. Patient III-1 inherited the same alleles from her father and paternal grandfather. The analysis of two multi-copy STRs (BASTR1 and BBSTR1), which are present in the different blocks of segmental duplications, revealed a gain of one block type in Patients II-2 and III-1, with normal copy number of the other blocks, and normal results in all other family members (see Supplementary Fig. 2 in Supporting Information online). These data indicate that the duplication is ∼1.55 Mb in size, the exact reciprocal of the most common WBS deletion, generated by non-allelic homologous recombination (NAHR) between specific blocks of segmental duplications. We then used indirect assays based on site-specific nucleotide dosage analysis to infer the location of the breakpoint and the relative orientation of the WBS locus duplication [Somerville et al., 2005]. Comparing Patient II-2 with respect to his parents, relative gains of pseudogene-type NCF1 copy and medial-type copy at the second site were observed, suggesting that the site of exchange between chromosomes had occurred between the medial centromeric blocks B, in a position proximal to the NCF1 gene (see Supplementary Fig. 2 in Supporting Information online). Therefore, the WBS locus duplication is most likely located in tandem and was not mediated by a parental inversion polymorphism. Although we were not able to confirm the orientation of the duplication in Patients II-2 and III-1, three color interphase FISH (using BACs RP11-552B12, RP11-329B05, and RP11-746H03 as probes) confirmed that the WBS locus was not inverted in individual I-1 (data not shown). Lack of paternal inheritance in Patient III-1 at STR loci D7S501 and D7S2453 further confirmed that both deletions were de novo, of paternal origin, and occurring on the same grandpaternal chromosome 7 carrying the duplication. By haplotyping, no recombination was observed in the paternal chromosome 7 inherited by PIII-1, indicating an intrachromosomal origin for both 7q22 deletions (see Supplementary Fig. 3 in Supporting Information online). Since all rearrangements (WBS duplication and complex 7q22 deletions) had originated in the same chromosome from the grandfather (I-1), we further searched for putative inversion polymorphism at the 7q22 edge of the rearranged intervals. We performed multicolor interphase FISH with BAC probes located within the centromeric deletion (RP11-193P05) and in normal copy-number regions: between the two deletions (RP11-768J05) and telomeric to the deletions (RP11-698M09). Multicolor interphase FISH did not reveal any abnormal probe order in the grandparental (I-1) or paternal (II-2) samples, ruling out inversions, while confirmed the deletion of RP11-193P05 in Patient III-1 (see Supplementary Fig. 4 in Supporting Information online). Interstitial deletions of 7q11.23 cause Williams–Beuren syndrome (OMIM #194050) while the reciprocal duplication is responsible for 7q11.23 duplication syndrome (OMIM #609757) [Somerville et al., 2005]. A series of 14 patients with the latter condition showed that cognitive abilities ranged from normal to moderate intellectual disability but variable speech delay was a constant finding [Van der Aa et al., 2009]. Autism was also associated with this duplication [Sanders et al., 2011]. These data are consistent with Patient II-2's phenotype who carried a 7q11.23 duplication and only presented with speech delay and learning disability. In addition to the 7q11.23 duplication, Patient III-1 carried two close 7q22 deletions. Few patients with intellectual disability and a cytogenetically visible deletion including the 7q22 region have been reported [Franceschini et al., 1978; Serup, 1980; Abuelo and Padre-Mendoza, 1982]. However, these deletions were much larger and cannot be compared with the interstitial deletions that we describe. An additional patient carrying a de novo 7q22 deletion (chr7:103,887,195-106,790,044; hg18) has been uploaded in the DECIPHER database (Patient #4470; http://decipher.sanger.ac.uk/). The deletion of our patient is smaller (1.9 vs. 2.9 Mb) and entirely included in the deletion of the DECIPHER's patient. This patient shared intellectual disability with our patient. He also presented with overgrowth features (tall stature, macrocephaly, large tongue, and high birth weight) while Patient III-1 of our study presented with measurements relatively above the mean [aged 7 years and 1 month, her weight, height, and OFC were 31 kg (+3 SD), 126 cm (+1.5 SD), 54 cm (+2.25 SD), respectively]. Several genomic variants of small size located in the 7q22 region have been identified in individuals from the general population (http://projects.tcag.ca/variation/) but none of them significantly overlapped with the deletion observed in Patient III-1. A two-hit model for severe developmental delay has been recently proposed [Girirajan et al., 2010]. Within this model, a parent carries a microdeletion/microduplication which predisposes to learning disability and/or neuropsychiatric phenotypes but is not sufficient for being responsible for severe developmental delay. In the next generation, a child carries the parental event and a second event. Such co-occurrence is responsible for severe developmental delay. The present family illustrates such a two-hit model. The father only presented with learning disability and a single event (i.e., a 7q11.23 duplication). In contrast, his intellectually disabled child presented with two CNVs: the paternally inherited 7q11.23 duplication and a complex 7q22 deletion. The chromosome 7q11.23 microdeletion/duplication occurs because of the unique genomic architecture in this region. This region is flanked by highly homologous clusters of genes and pseudogenes organized into low-copy-repeat blocks known as duplicons. The high degree of sequence homology among these flanking duplicons, as well as their proximity to each other, predisposes to recurrent chromosomal rearrangements through NAHR mechanism during meiosis [Lupski, 1998] Although genomic structural polymorphisms, including paracentric inversions and copy number variation in the flanking segmental duplications, have been reported as susceptibility alleles for germline rearrangements at the WBS locus [Bayés et al., 2003; Cuscó et al., 2008], we did not find them in the grandfather's sample (I-1). In addition, molecular testing and breakpoint mapping suggested a tandem orientation of the duplicated interval, being the exact reciprocal of the WBS most common deletion and mediated by interchromosomal NAHR. In contrast, several mechanisms, including non-homologous end-joining (NHEJ) [Lieber, 2008] or the recently described replication-based mechanisms of fork stalling and template switching/microhomology-mediated break induced replication (FoSTeS) [Lee et al., 2007] can be responsible for non-recurrent rearrangements without the need for a homologous template or only requiring microhomology [Zhang et al., 2009]. Sequence analyses performed in Patient III-1 showed microhomologies at the breakpoint junctions for both 7q22 deletions. These data suggest that the deletions may have been mediated by NHEJ or FoSTeS mechanisms, and both through intrachromosomal rearrangements on the same chromosome arm harboring the WBS locus duplication, as shown by haplotype analysis. Chromosomes rearrangements (i.e., deletions and duplications) are thought to be stable through generations. This assumption is fundamental to current guidelines for parental follow-up studies for cytogenetically visible alterations, FISH and array CGH abnormalities. However, this assumption is not always true. Size expansions of a deletion from a normal parent to his affected child have been reported in two independent families [Faravelli et al., 2007; South et al., 2008]. The awareness that inherited deletions are not always stable challenges current algorithms for family studies. Furthermore, it is assumed that a genomic imbalance does not increase the likelihood of the occurrence of a second de novo event elsewhere in the genome. Therefore, the two chromosomal rearrangements observed in the intellectually disabled child of the present family may have occurred by chance. However, since de novo large CNVs are relatively rare events and that both duplication and deletions occurred on the same chromosome 7, we speculate that the duplication may have predisposed to the occurrence of the second de novo events. In theory, the duplication could alter chromosome territories in the nuclei, prompting to the formation of abnormal chromatin loops than could facilitate additional rearrangements [Cremer and Cremer, 2001]. Proximity of chromosomes or chromosomal loci during cell division, the so called chromosome kissing, has been shown to correlate with the frequency of recurrent translocations and other rearrangements [Cavalli, 2007]. If such predisposition was further confirmed, this finding would challenge our current algorithms for family studies. Indeed, in this family, if the father carrying a 7q11.23 duplication had asked for a prenatal diagnosis during a pregnancy, only a specific FISH probe targeting the 7q11.23 region would have been used. In such case, the de novo 7q22 deletions would have been missed in the child. Additional observations and further studies are needed to determine whether the occurrence of these abnormalities is coincidental or whether the presence of an initial CNV predisposes to the recurrence of additional de novo genomic imbalances. We are grateful to the family who participated in this study and to Rémi Houlgatte and Catherine Chevalier from Biogenouest de Nantes, France. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.A bstract Segmental duplications (SDs) are long genomic duplications fixed in a genome. SDs play an important evolutionary role: entire genes together with regulatory sequences can be duplicated. Ancestral segmental duplications gave rise to genes involved in human brain development, as well as provided sites for further genomic rearrangements. Some duplicated loci were extensively studied, however, universal principles or biological factors of SDs propagation are not fully described yet. Segmental duplications can be arranged into a network where edges correspond to real duplication events, while nodes to affected genomic sites. This gave us an opportunity to estimate how many duplications happened in each locus. We studied genomic features associated with increased duplication rates with especial interest in high-copy repeats distribution relative to duplicated regions breakpoints. Our comprehensive study of genomic features associated with duplications and those associated with increased duplication rates allowed us to identify several biological factors affecting a segmental duplication process. We found genomic features associated with increased duplication rates, three signatures of duplication process and associations of SDs with different classes of high-copy repeats.
Segmental duplication
Breakpoint
Comparative genomic hybridization
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Objective To study the routine methods that can be easily used in clinics to detect the Charcot Marie Tooth (CMT) disease gene duplication. Method Polymerase chain reaction(PCR) combined with restriction enzyme digestion and amplification of short tandem repeat (STR) sequence were used to detect gene duplication on chromosome 17p11.2～12 in 30 CMT1 patients and 10 CMT2 patients coming from unrelated families. 40 controls were also detected. Results 46.7%(14/30) of CMT1 patients were identified to have specific junction fragments. 53.3%(16/30)of them were identified as duplication by STR analysis. 70.0%(21/30) of CMT1 patients were identified to have gene duplication using both methods. Duplication was not identified in 10 unrelated CMT2 patients and 40 controls. Conclusion The PCR combined with restriction enzyme digestion represented a relatively sensitive and accurate method for detecting gene duplication in CMT1A cases for clinical diagnosis. The detecting rate of duplication can be increased using both restriction enzyme digestion of PCR products and STR methods.
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Aspergillus nidulans
Chromosome instability
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Abstract Genomic scale duplication of genes generates raw genetic material, which may facilitate new adaptations for the organism. Previous studies on eels have reported specific gene duplications, however a species-specific large-scale gene duplication has never before been proposed. In this study, we have assembled a de novo European eel transcriptome and the data show more than a thousand gene duplications that happened, according to a 4dTv analysis, after the teleost specific 3R whole genome duplication (WGD). The European eel has a complex and peculiar life cycle, which involves extensive migration, drastic habitat changes and metamorphoses, all of which could have been facilitated by the genes derived from this large-scale gene duplication. Of the paralogs created, those with a lower genetic distance are mostly found in tandem repeats, indicating that they are young segmental duplications. The older eel paralogs showed a different pattern, with more extensive synteny suggesting that a Whole Genome Duplication (WGD) event may have happened in the eel lineage. Furthermore, an enrichment analysis of eel specific paralogs further revealed GO-terms typically enriched after a WGD. Thus, this study, to the best of our knowledge, is the first to present evidence indicating an Anguillidae family specific large-scale gene duplication, which may include a 4R WGD.
Synteny
Segmental duplication
Lineage (genetic)
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Abstract Duplication of all genes associated with X‐linked intellectual disability (XLID) have been reported but the majority of the duplications include more than one XLID gene. It is exceptional for whole XLID gene duplications to cause the same phenotype as sequence variants or deletions of the same gene. Duplication of PLP1 , the gene associated with Pelizaeus‐Merzbacher syndrome, is the most notable duplication of this type. More commonly, duplication of XLID genes results in very different phenotypes than sequence alterations or deletions. Duplication of MECP2 is widely recognized as a duplication of this type, but a number of others exist. The phenotypes associated with gene duplications are often milder than those caused by deletions and sequence variants. Among some duplications that are clinically significant, marked skewing of X‐inactivation in female carriers has been observed. This report describes the phenotypic consequences of duplication of 22 individual XLID genes, of which 10 are described for the first time.
Segmental duplication
Sequence (biology)
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ABSTRACT Salmonella typhimurium strains containing a duplication of nearly a third of the genome have been isolated by a simple procedure involving selection for improved utilization of L-malate as sole carbon source. The duplication occurs at a very high spontaneous frequency. Strains containing the duplication can be isolated selectively on malate medium, or by a non-selective procedure involving Hfr conjugation. When strains containing the duplication are maintained on non-selective medium, the duplication is readily lost. Genetic evidence suggests that the duplication is chromosomal and tandem. The fact that the recA gene is included in the duplication has been used to obtain evidence that the recA1 marker is recessive to its wild-type allele. Unlike tandem duplications previously described in E. coli, the duplication described in this report appears to have unique endpoints
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The C4 gene duplication has been studied among 78 families with 366 members in Chinese.
This study allowed the detection of 7 groups of C4 gene duplication or triplication in the family
material rlpresenting an overall frequency of 9.0%.The total members with duplicated of tripli-
cated C4 genes were 17,which representsa frequency of 6.4%.The duplicated C4 gnes all be-
long to the C4B genes.The kinds of C4B gene duplication and the number each are:①)C4B(1,
2)×2,②B(1,12)×6,③B(1,1)×5,④B(1,96,96)×2 and ⑤B(2,2)×2.
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Abstract Background In the evolutionary study of gene families, exploring the duplication mechanisms of gene families helps researchers understand their evolutionary history. The tubby-like protein (TLP) family is essential for growth and development in plants and animals. Much research has been done on its function; however, limited information is available with regard to the evolution of the TLP gene family. Herein, we systematically investigated the evolution of TLP genes in seven representative Poaceae lineages. Results Our research showed that the evolution of TLP genes was influenced not only by whole-genome duplication (WGD) and dispersed duplication (DSD) but also by transposed duplication (TRD), which has been neglected in previous research. For TLP family size, we found an evolutionary pattern of progressive shrinking in the grass family. Furthermore, the evolution of the TLP gene family was at least affected by evolutionary driving forces such as duplication, purifying selection, and base mutations. Conclusions This study presents the first comprehensive evolutionary analysis of the TLP gene family in grasses. We demonstrated that the TLP gene family is also influenced by a transposed duplication mechanism. Several new insights into the evolution of the TLP gene family are presented. This work provides a good reference for studying gene evolution and the origin of duplication.
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Duplication is thought to be one of the main processes providing a substrate on which the effects of evolution are visible. The mechanisms underlying this chromosomal rearrangement were investigated here in the yeast Saccharomyces cerevisiae. Spontaneous revertants containing a duplication event were selected and analyzed. In addition to the single gene duplication described in a previous study, we demonstrated here that direct tandem duplicated regions ranging from 5 to 90 kb in size can also occur spontaneously. To further investigate the mechanisms in the duplication events, we examined whether homologous recombination contributes to these processes. The results obtained show that the mechanisms involved in segmental duplication are RAD52-independent, contrary to those involved in single gene duplication. Moreover, this study shows that the duplication of a given gene can occur in S.cerevisiae haploid strains via at least two ways: single gene or segmental duplication.
RAD52
Gene conversion
Gene dosage
Segmental duplication
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