Inverted Low‐Copy Repeats and Genome Instability—A Genome‐Wide Analysis

Inverse paralogous low‐copy repeats (IP‐LCRs) can cause genome instability by nonallelic homologous recombination (NAHR)‐mediated balanced inversions. When disrupting a dosage‐sensitive gene(s), balanced inversions can lead to abnormal phenotypes. We delineated the genome‐wide distribution of IP‐LCRs >1 kB in size with >95% sequence identity and mapped the genes, potentially intersected by an inversion, that overlap at least one of the IP‐LCRs. Remarkably, our results show that 12.0% of the human genome is potentially susceptible to such inversions and 942 genes, 99 of which are on the X chromosome, are predicted to be disrupted secondary to such an inversion! In addition, IP‐LCRs larger than 800 bp with at least 98% sequence identity (duplication/triplication facilitating IP‐LCRs, DTIP‐LCRs) were recently implicated in the formation of complex genomic rearrangements with a duplication‐inverted triplication–duplication (DUP‐TRP/INV‐DUP) structure by a replication‐based mechanism involving a template switch between such inverted repeats. We identified 1,551 DTIP‐LCRs that could facilitate DUP‐TRP/INV‐DUP formation. Remarkably, 1,445 disease‐associated genes are at risk of undergoing copy‐number gain as they map to genomic intervals susceptible to the formation of DUP‐TRP/INV‐DUP complex rearrangements. We implicate inverted LCRs as a human genome architectural feature that could potentially be responsible for genomic instability associated with many human disease traits.     

Disclosing the Hidden Structure and Underlying Mutational Mechanism of a Novel Type of Duplication CNV Responsible for Hereditary Multiple Osteochondromas

The additional mutational complexity associated with copy number variation (CNV) can provide important clues as to the underlying mechanisms of CNV formation. Correct annotation of the additional mutational complexity is, however, a prerequisite for establishing the mutational mechanism. We illustrate this point through the characterization of a novel ～230 kb EXT1 duplication CNV causing autosomal dominant hereditary multiple osteochondromas. Whole‐genome sequencing initially identified the CNV as having a 22‐bp insertion at the breakpoint junction and, unprecedentedly, multiple breakpoint‐flanking micromutations on both sides of the duplication. Further investigation revealed that this genomic rearrangement had a duplication‐inverted triplication–duplication structure, the inverted triplication being a 41‐bp sequence synthesized from a nearby template. This permitted the identification of the sequence determinants of both the initiation (an inverted Alu repeat) and termination (a triplex‐forming sequence) of break‐induced replication and suggested a possible model for the repair of replication‐associated double‐strand breaks.     

Exonic rearrangements in DMD in Chinese Han individuals affected with Duchenne and Becker muscular dystrophies

Exonic deletions and duplications within DMD are the main pathogenic variants in Duchenne and Becker muscular dystrophies (DMD/BMD). However, few studies have profiled the flanking sequences of breakpoints and the potential mechanism underlying the breakpoints in different fragile regions of DMD. In this study, 896 Chinese male probands afflicted with DMD/BMD were selected from unrelated families and analyzed using multiplex ligation‐dependent probe amplification of the DMD gene, in which we identified exon deletions in 784 subjects and duplications in 112 subjects. Deletions occurred most frequently in the genomic region encompassing exons 45–55, accounting for 73% of all deletion patterns. Furthermore, to unravel the potential mechanism that induced breaks, DMD gene capture and sequencing were performed to identify the breakpoints in 37 subjects with deletions encompassing exons 45–55 of DMD; we found that DMD instability did not arise from a single cause; instead, long‐sequence motifs, nonconsensus microhomologies, low‐copy repeats, and microindels were embedded around the breakpoints, which may predispose DMD to instability. In summary, this study highlights the heterogeneous characteristics of the flanking sequences around the breakpoints and helps us to understand the mechanism underlying DMD gene instability.     

Pure intronic rearrangements leading to aberrant pseudoexon inclusion in dystrophinopathy: a new class of mutations?

We report on two unprecedented cases of pseudoexon (PE) activation in the DMD gene resulting from pure intronic double‐deletion events that possibly involve microhomology‐mediated mechanisms. Array comparative genomic hybridization analysis and direct genomic sequencing allowed us to elucidate the causes of the pathological PE inclusion detected in the RNA of the patients. In the first case (Duchenne phenotype), we showed that the inserted 387‐bp PE was originated from an inverted ～57 kb genomic region of intron 44 flanked by two deleted ～52 kb and ～1 kb segments. In the second case (Becker phenotype), we identified in intron 56 two small deletions of 592 bp (del 1) and 29 bp (del 2) directly flanking a 166‐bp PE located in very close proximity (134 bp) to exon 57. The key role of del 1 in PE activation was established by using splicing reporter minigenes. However, the analysis of mutant constructs failed to identify cis elements that regulate the inclusion of the PE and suggested that other splicing regulatory factors may be involved such as RNA structure. Our study introduces a new class of mutations in the DMD gene and emphasizes the potential role of underdetected intronic rearrangements in human diseases. Hum Mutat 32:1–9, 2011. © 2011 Wiley‐Liss, Inc.     

Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort

Mutations in the DMD gene, encoding the dystrophin protein, are responsible for the dystrophinopathies Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), and X-linked Dilated Cardiomyopathy (XLDC). Mutation analysis has traditionally been challenging, due to the large gene size (79 exons over 2.2 Mb of genomic DNA). We report a very large aggregate data set comprised of DMD mutations detected in samples from patients enrolled in the United Dystrophinopathy Project, a multicenter research consortium, and in referral samples submitted for mutation analysis with a diagnosis of dystrophinopathy. We report 1,111 mutations in the DMD gene, including 891 mutations with associated phenotypes. These results encompass 506 point mutations (including 294 nonsense mutations) and significantly expand the number of mutations associated with the dystrophinopathies, highlighting the utility of modern diagnostic techniques. Our data supports the uniform hypermutability of CGA>TGA mutations, establishes the frequency of polymorphic muscle (Dp427m) protein isoforms and reveals unique genomic haplotypes associated with “private” mutations. We note that 60% of these patients would be predicted to benefit from skipping of a single DMD exon using antisense oligonucleotide therapy, and 62% would be predicted to benefit from an inclusive multiexonskipping approach directed toward exons 45 through 55. Hum Mutat 30:1657–1666, 2009. © 2009 Wiley-Liss, Inc.     

Two classes of low-copy repeats comediate a new recurrent rearrangement consisting of duplication at 8p23.1 and triplication at 8p23.2

We describe a new type of rearrangement consisting of the duplication of 8p23.1 and the triplication of 8p23.2 [dup trp(8p)] in two patients affected by mental retardation and minor facial dysmorphisms. Array-comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and genotyping of polymorphic loci allowed us to demonstrate that this rearrangement is mediated by the combined effects of two unrelated low-copy repeats (LCRs). The first set of LCRs consists of the two clusters of olfactory receptor genes (OR-REPs) lying at 8p23.1. The second type of LCRs consists of a 15-kb segmental duplication, lying in inverted orientation at 8p23.2 and enclosing a nonrepeated sequence of approximately 130 kb, named MYOM2-REP because of its proximity to the MYOM2 gene. The molecular characterization of a third case with a dicentric chromosome 8 demonstrated that the rearrangement had been generated by nonallelic homologous recombination between the two MYOM2-REPs. Based on our findings, we propose a model showing that a second recombination event at the level of the OR-REPs leads to the formation of the dup trp(8p) chromosome. This rearrangement can only arise during meiosis in heterozygous carriers of the polymorphic 8p23.1 inversion, whereas in subjects with noninverted chromosomes 8 or homozygous for the inversion only the dicentric chromosome can be formed. Our study demonstrates that nonallelic homologous recombination involving multiple LCRs can generate more complex rearrangements and cause a greater variety of genomic diseases.     

DMD exon 1 truncating point mutations: Amelioration of phenotype by alternative translation initiation in exon 6

Mutations in the DMD gene result in two common phenotypes associated with progressive muscle weakness: the more severe Duchenne muscular dystrophy (DMD) and the milder Becker muscular dystrophy (BMD). We have previously identified a nonsense mutation (c.9G>A; p.Trp3X) within the first exon of the DMD gene, encoding the unique N‐terminus of the 427‐kDa muscle isoform of the dystrophin protein. Although this mutation would be expected to result in severe disease, the clinical phenotype is very mild BMD, with ambulation preserved into the seventh decade. We identify the molecular mechanism responsible for the amelioration of disease severity to be initiation of translation at two proximate AUG codons within exon 6. Analysis of large mutational data sets suggests that this may be a general mechanism of phenotypic rescue for point mutations within at least the first two exons of the DMD gene. Our results directly demonstrate, for the first time, the use of alternate translational initiation codons within the DMD gene, and suggest that dystrophin protein lacking amino acids encoded by the first five exons retains significant function. Hum Mutat 0:1–8, 2009. © 2009 Wiley‐Liss, Inc.     

Intronic deletion and duplication proximal of the EXT1 gene: A novel causative mechanism for multiple osteochondromas

Multiple osteochondromas (MO) is a syndrome in which benign cartilage‐capped neoplasms develop at the surface of the long bones. Most cases are caused by exonic changes in EXT1 or EXT2, but 15% are negative for these changes. Here we report for the first time a family of MO patients with germline genomic alterations at the EXT1 locus without detectable mutations or copy number alterations of EXT exonic sequences. Array‐CGH showed an 80.7 kb deletion of Intron 1 of EXT1 and a 68.9 kb duplication proximal of EXT1. We identified a breakpoint between the distal end of the duplicated region and a sequence distal of the deleted region in the first intron. This breakpoint was absent in non‐affected family members. The configuration of the breakpoint indicates a direct insertion of the duplicated region into the deletion. However, no other breakpoint was found, which suggests a more complex genomic rearrangement has occurred within the duplicated region. Our results reveal intronic deletion and duplication as a new causative mechanism for MO not detected by conventional diagnostic methods. © 2013 Wiley Periodicals, Inc.     

Two Non-Contiguous Duplications in the DMD Gene in a Spanish Family

DMD and BMD are X-linked myopathy diseases in most cases caused by intragenic deletions, but duplications also appear in a significant number of cases. We present a complex duplication pattern detected by MLPA, a recently formulated method applied here to amplify the 79 exons of the DMD gene. We found a double-duplication in two DMD-affected brothers and in their carrier mother, which consist of two non-contiguous duplications encompassing exons 2 to 7 and exons 50 to 55. Different models are presented to explain formation of this genetic variant.     

Occurrence of a non deleterious gene conversion event in the BRCA1 gene

The duplication in the primate lineage of a portion of the breast and ovarian cancer susceptibility gene BRCA1 has created a BRCA1 pseudogene 45 kb away. Non‐allelic homologous recombination (NAHR) between BRCA1 and BRCA1P1 has generated recurrent deleterious germ‐line 37‐kb deletions encompassing the first two exons of BRCA1, accounting for several breast and ovarian cancer families in various populations. In principle, NAHR intermediates resolution could also lead through a non‐crossover configuration to interlocus gene conversion (IGC), but none had been described as yet. Here, we report for the first time an IGC event identified in a breast and ovarian cancer family involving exactly the same segment as that involved in the 37‐kb deletions. Close examination of the consequences of this IGC event showed that it does not impact BRCA1 expression. Detailed analysis of the regions of homology between BRCA1 and its pseudogene revealed the specificity of the segment where recombination systematically occurs. © 2015 Wiley Periodicals, Inc.     

PLP1 duplication at the breakpoint regions of an apparently balanced t(X;22) translocation causes Pelizaeus–Merzbacher disease in a girl

Fonseca ACS, Bonaldi A, Costa SS, Freitas MR, Kok F, Vianna‐Morgante AM. PLP1 duplication at the breakpoint regions of an apparently balanced t(X;22) translocation causes Pelizaeus–Merzbacher disease in a girl. PLP1 (proteolipid protein1 gene) mutations cause Pelizaeus–Merzbacher disease (PMD), characterized by hypomyelination of the central nervous system, and affecting almost exclusively males. We report on a girl with classical PMD who carries an apparently balanced translocation t(X;22)(q22;q13). By applying array‐based comparative genomic hybridization (a‐CGH), we detected duplications at 22q13 and Xq22, encompassing 487–546 kb and 543–611 kb, respectively. The additional copies were mapped by fluorescent in situ hybridization to the breakpoint regions, on the derivative X chromosome (22q13 duplicated segment) and on the derivative 22 chromosome (Xq22 duplicated segment). One of the 14 duplicated X‐chromosome genes was PLP1.The normal X chromosome was the inactive one in the majority of peripheral blood leukocytes, a pattern of inactivation that makes cells functionally balanced for the translocated segments. However, a copy of the PLP1 gene on the derivative chromosome 22, in addition to those on the X and der(X) chromosomes, resulted in two active copies of the gene, irrespective of the X‐inactivation pattern, thus causing PMD. This t(X;22) is the first constitutional human apparently balanced translocation with duplications from both involved chromosomes detected at the breakpoint regions.     

An unusual cause for Coffin–Lowry syndrome: Three brothers with a novel microduplication in RPS6KA3

Coffin–Lowry syndrome (CLS) is a rare X‐linked disorder characterized by moderate to severe intellectual disability, hypotonia, craniofacial features, tapering digits, short stature, and skeletal deformities. Using whole exome sequencing and high‐resolution targeted comparative genomic hybridization array analysis, we identified a novel microduplication encompassing exons five through nine of RPS6KA3 in three full brothers. Each brother presented with intellectual disability and clinical and radiographic features consistent with CLS. qRT‐PCR analyses performed on mRNA from the peripheral blood of the three siblings revealed a marked reduction of RPS6KA3 levels suggesting a loss‐of‐function mechanism. PCR analysis of the patients’ cDNA detected a band greater than expected for an exon 4–10 amplicon, suggesting this was likely a direct duplication that lies between exons 4 through 10, which was later confirmed by Sanger sequencing. This microduplication is only the third intragenic duplication of RPS6KA3, and the second and smallest reported to date thought to cause CLS. Our study further supports the clinical utility of methods such as next‐generation sequencing and high‐resolution genomic arrays to detect small intragenic duplications. These methods, coupled with expression studies and cDNA structural analysis have the capacity to confirm the diagnosis of CLS in these rare cases.     

Clinical application of 2.7M Cytogenetics array for CNV detection in subjects with idiopathic autism and/or intellectual disability

Qiao Y, Tyson C, Hrynchak M, Lopez‐Rangel E, Hildebrand J, Martell S, Fawcett C, Kasmara L, Calli K, Harvard C, Liu X, Holden JJA, Lewis SME, Rajcan‐Separovic E. Clinical application of 2.7M Cytogenetics array for CNV detection in subjects with idiopathic autism and/or intellectual disability. Higher resolution whole‐genome arrays facilitate the identification of smaller copy number variations (CNVs) and their integral genes contributing to autism and/or intellectual disability (ASD/ID). Our study describes the use of one of the highest resolution arrays, the Affymetrix^® Cytogenetics 2.7M array, coupled with quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF) for detection and validation of small CNVs. We studied 82 subjects with ASD and ID in total (30 in the validation and 52 in the application cohort) and detected putatively pathogenic CNVs in 6/52 cases from the application cohort. This included a 130‐kb maternal duplication spanning exons 64–79 of the DMD gene which was found in a 3‐year‐old boy manifesting autism and mild neuromotor delays. Other pathogenic CNVs involved 4p14, 12q24.31, 14q32.31, 15q13.2‐13.3, and 17p13.3. We established the optimal experimental conditions which, when applied to select small CNVs for QMPSF confirmation, reduced the false positive rate from 60% to 25%. Our work suggests that selection of small CNVs based on the function of integral genes, followed by review of array experimental parameters resulting in highest confirmation rate using multiplex PCR, may enhance the usefulness of higher resolution platforms for ASD and ID gene discovery.     

CHRNA7 triplication associated with cognitive impairment and neuropsychiatric phenotypes in a three-generation pedigree

Although deletions of CHRNA7 have been associated with intellectual disability (ID), seizures and neuropsychiatric phenotypes, the pathogenicity of CHRNA7 duplications has been uncertain. We present the first report of CHRNA7 triplication. Three generations of a family affected with various neuropsychiatric phenotypes, including anxiety, bipolar disorder, developmental delay and ID, were studied with array comparative genomic hybridization (aCGH). High-resolution aCGH revealed a 650-kb triplication at chromosome 15q13.3 encompassing the CHRNA7 gene, which encodes the alpha7 subunit of the neuronal nicotinic acetylcholine receptor. A small duplication precedes the triplication at the proximal breakpoint junction, and analysis of the breakpoint indicates that the triplicated segment is in an inverted orientation with respect to the duplication. CHRNA7 triplication appears to occur by a replication-based mechanism that produces inverted triplications embedded within duplications. Co-segregation of the CHRNA7 triplication with neuropsychiatric and cognitive phenotypes provides further evidence for dosage sensitivity of CHRNA7.     

Complete ascertainment of intragenic copy number mutations (CNMs) in the CFTR gene and its implications for CNM formation at other autosomal loci

Over the last 20 years since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, more than 1,600 different putatively pathological CFTR mutations have been identified. Until now, however, copy number mutations (CNMs) involving the CFTR gene have not been methodically analyzed, resulting almost certainly in the underascertainment of CFTR gene duplications compared with deletions. Here, high‐resolution array comparative genomic hybridization (averaging one interrogating probe every 95 bp) was used to analyze the entire length of the CFTR gene (189 kb) in 233 cystic fibrosis chromosomes lacking conventional mutations. We succeeded in identifying five duplication CNMs that would otherwise have been refractory to analysis. Based upon findings from this and other studies, we propose that deletion and duplication CNMs in the human autosomal genome are likely to be generated in the proportion of approximately 2–3:1. We further postulate that intragenic gene duplication CNMs in other disease loci may have been routinely underascertained. Finally, our analysis of ±20 bp flanking each of the 40 CFTR breakpoints characterized at the DNA sequence level provide support for the emerging concept that non‐B DNA conformations in combination with specific sequence motifs predispose to both recurring and nonrecurring genomic rearrangements. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

Microdeletion of Xq28 involving the AFF2 (FMR2) gene in two unrelated males with developmental delay

Fragile X E (FRAXE) is an X‐linked form of intellectual disability characterized by mild to moderate cognitive impairment, speech delay, hyperactivity, and autistic behavior. The folate‐sensitive fragile site FRAXE is located in Xq28 approximately 600 kb distal to the fragile X syndrome fragile site (FRAXA) and harbors an unstable GCC (CCG) triplet repeat adjacent to a CpG island in the 5′ untranslated region of the AFF2 (FMR2) gene. The disorder results from amplification and methylation of the GCC repeat and resultant silencing of AFF2. Although chromosome abnormalities that disrupt AFF2 have been reported in two individuals with mild‐moderate intellectual disability, microdeletions of Xq28 that delete only AFF2 have not been described as a potential cause of FRAXE‐intellectual disability. We performed clinical and molecular characterization of two males with 240 and 499 kb deletions, respectively, at Xq28, both of which encompassed only one gene, AFF2. The 240 kb deletion in Patient 1 was intragenic and lead to the loss of 5′ exons 2–4 of AFF2; the 499 kb deletion in Patient 2 removed the 5′ exons 1–2 of AFF2 including approximately 350 kb upstream of the gene. Both individuals had developmental and speech delay, and one had mild dysmorphism. We predict disruption of AFF2 in these two patients is likely the cause of their overlapping phenotypes. © 2011 Wiley Periodicals, Inc.     

Novel intragenic deletion in OPHN1 in a family causing XLMR with cerebellar hypoplasia and distinctive facial appearance

Al‐Owain M, Kaya N, Al‐Zaidan H, Al‐Hashmi N, Al‐Bakheet A, Al‐Muhaizea M, Chedrawi A, Basran RK, Milunsky A. Novel intragenic deletion in OPHN1 in a family causing XLMR with cerebellar hypoplasia and distinctive facial appearance. X‐linked mental retardation (XLMR) is notably a heterogeneous condition and often poses a diagnostic challenge. The oligophrenin 1 gene (OPHN1) is a protein with a Rho‐GTPase‐activating domain required in the regulation of the G‐protein cycle. Mutations in the OPHN1 cause XLMR with cerebellar hypoplasia and distinctive facial appearance. We report a large Saudi family of four boys and one girl affected with XLMR. The boys had moderate MR, seizure disorder, facial dysmorphism, and cerebellar vermis hypoplasia. The girl had mild MR, seizures, and mild cerebellar hypoplasia. A novel deletion of at least exons 7–15 was identified by polymerase chain reaction analysis and multiple ligation probe amplification of the OPHN1 gene. The array comparative genomic hybridization further delineated approximately 68 kb deletion of the 7–15 exons and nearly half of intron 15. In addition, the X‐inactivation confirmed random pattern in the girl. Although the affected boys have remarkably similar phenotype, there was some variability in the severity of the seizure disorder and the cerebellar hypoplasia. The report confirms the previous findings that carrier females may be symptomatic.