MECP2 duplications in six patients with complex sex chromosome rearrangements

Duplications of the Xq28 chromosome region resulting in functional disomy are associated with a distinct clinical phenotype characterized by infantile hypotonia, severe developmental delay, progressive neurological impairment, absent speech, and proneness to infections. Increased expression of the dosage-sensitive MECP2 gene is considered responsible for the severe neurological impairments observed in affected individuals. Although cytogenetically visible duplications of Xq28 are well documented in the published literature, recent advances using array comparative genomic hybridization (CGH) led to the detection of an increasing number of microduplications spanning MECP2. In rare cases, duplication results from intrachromosomal rearrangement between the X and Y chromosomes. We report six cases with sex chromosome rearrangements involving duplication of MECP2. Cases 1-4 are unbalanced rearrangements between X and Y, resulting in MECP2 duplication. The additional Xq material was translocated to Yp in three cases (cases 1-3), and to the heterochromatic region of Yq12 in one case (case 4). Cases 5 and 6 were identified by array CGH to have a loss in copy number at Xp and a gain in copy number at Xq28 involving the MECP2 gene. In both cases, fluorescent in situ hybridization (FISH) analysis revealed a recombinant X chromosome containing the duplicated material from Xq28 on Xp, resulting from a maternal pericentric inversion. These cases add to a growing number of MECP2 duplications that have been detected by array CGH, while demonstrating the value of confirmatory chromosome and FISH studies for the localization of the duplicated material and the identification of complex rearrangements.     

Large structural polymorphisms predispose genomic sequences to disease-causing rearrangements

A large and complex structural polymorphism at 16p12.1 underlies microdeletion disease risk Antonacci et al. (2010) Nature Genetics 42:745–751     

Large genomic rearrangements in NIPBL are infrequent in Cornelia de Lange syndrome

Cornelia de Lange Syndrome (CdLS) is a multiple congenital anomaly syndrome characterized by a distinctive facial appearance, malformations of the upper limbs, and delay in growth and development. Mutations in NIPBL are associated with CdLS in 27-56% of cases and have been reported as point mutations, small insertions and deletions in coding regions, regulatory regions and at splice junctions. All previous studies used PCR-based exon-scanning methodologies that do not allow detection of large genomic rearrangements. We studied the relative copy number of NIPBL exons in a series of 50 CdLS probands, negative for NIPBL mutations, by multiplex ligation-dependent probe amplification (MLPA). In a single patient, we found a 5.2 kb deletion encompassing exons 41-42 of NIPBL. Our studies indicate that large NIPBL rearrangements do occur in CdLS but are likely to be infrequent events.     

Three Rett patients with both MECP2 mutation and 15q11-13 rearrangements

Autism and Rett syndrome, a severe neurological disorder with autistic behavior, are classified as separate disorders on clinical and etiological ground. Rett syndrome is a monogenic X-linked dominant condition due to de novo mutations in the MECP2 gene, whereas autism is a neurodevelopmental and behavioral disorder with complex genetic basis. Maternally inherited duplications on 15q11-q13 are found in a fraction of autistic children suggesting that an abnormal dosage of gene(s) within this region might cause susceptibility to autism. Now we show that three Rett patients are carriers of both a MECP2 mutation and a 15q11-q13 rearrangement, suggesting that there might be a relationship between autism-related genes and the MECP2 gene.     

Gross rearrangements of the MECP2 gene are found in both classical and atypical Rett syndrome patients

MECP2 mutations are identifiable in approximately 80% of classic Rett syndrome (RTT), but less frequently in atypical RTT. We recruited 110 patients who fulfilled the diagnostic criteria for Rett syndrome and were referred to Cardiff for molecular analysis, but in whom an MECP2 mutation was not identifiable. Dosage analysis of MECP2 was carried out using multiplex ligation dependent probe amplification or quantitative fluorescent PCR. Large deletions were identified in 37.8% (14/37) of classic and 7.5% (4/53) of atypical RTT patients. Most large deletions contained a breakpoint in the deletion prone region of exon 4. The clinical phenotype was ascertained in all 18 of the deleted cases and in four further cases with large deletions identified in Goettingen. Five patients with large deletions had additional congenital anomalies, which was significantly more than in RTT patients with other MECP2 mutations (2/193; p<0.0001). Quantitative analysis should be included in molecular diagnostic strategies in both classic and atypical RTT.     

Recurrent genomic rearrangements in primary testicular lymphoma

Primary testicular diffuse large B cell lymphoma (PTL) is an aggressive malignancy that occurs in the immune‐privileged anatomical site of the testis. We have previously shown that structural genomic rearrangements involving the MHC class II transactivator CIITA and programmed death ligands (PDLs) 1 and 2 are frequent across multiple B cell lymphoma entities. Specifically in PTL, we found rearrangements in the PDL locus by fluorescence in situ hybridization (FISH). However, breakpoint anatomy and rearrangement partners were undetermined, while CIITA rearrangements had not been reported previously in PTL. Here, we performed bacterial artificial chromosome capture sequencing on three archival, formalin‐fixed, paraffin‐embedded tissue biopsies, interrogating 20 known rearrangement hotspots in B cell lymphomas. We report novel CIITA, FOXP1 and PDL rearrangements involving IGHG4, FLJ45248, RFX3, SMARCA2 and SNX29. Moreover, we present immunohistochemistry data supporting the association between PDL rearrangements and increased protein expression. Finally, using FISH, we show that CIITA (8/82; 10%) and FOXP1 (5/74; 7%) rearrangements are recurrent in PTL. In summary, we describe rearrangement frequencies and novel rearrangement partners of the CIITA, FOXP1 and PDL loci at base‐pair resolution in a rare, aggressive lymphoma. Our data suggest immune‐checkpoint inhibitor therapy as a promising intervention for PTL patients harbouring PDL rearrangements. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Nonrecurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair

Recurrent submicroscopic genomic copy number changes are the result of nonallelic homologous recombination (NAHR). Nonrecurrent aberrations, however, can result from different nonexclusive recombination-repair mechanisms. We previously described small microduplications at Xq28 containing MECP2 in four male patients with a severe neurological phenotype. Here, we report on the fine-mapping and breakpoint analysis of 16 unique microduplications. The size of the overlapping copy number changes varies between 0.3 and 2.3 Mb, and FISH analysis on three patients demonstrated a tandem orientation. Although eight of the 32 breakpoint regions coincide with low-copy repeats, none of the duplications are the result of NAHR. Bioinformatics analysis of the breakpoint regions demonstrated a 2.5-fold higher frequency of Alu interspersed repeats as compared with control regions, as well as a very high GC content (53%). Unexpectedly, we obtained the junction in only one patient by long-range PCR, which revealed nonhomologous end joining as the mechanism. Breakpoint analysis in two other patients by inverse PCR and subsequent array comparative genomic hybridization analysis demonstrated the presence of a second duplicated region more telomeric at Xq28, of which one copy was inserted in between the duplicated MECP2 regions. These data suggest a two-step mechanism in which part of Xq28 is first inserted near the MECP2 locus, followed by breakage-induced replication with strand invasion of the normal sister chromatid. Our results indicate that the mechanism by which copy number changes occur in regions with a complex genomic architecture can yield complex rearrangements.     

MECP2 mutations in males

Rett syndrome (RS; MIM 312750) is a severe neurological disorder affecting exclusively females. Its prevalence is about 1 in 10,000 female births, and it is a prominent cause of profound mental handicap in women. RS is caused by mutations in the X-linked methyl CpG-binding protein 2 (MECP2) gene. These mutations were initially thought to be lethal in males. However, MECP2 mutations are now frequently identified in mentally retarded male patients. The frequency of disease-causing MECP2 mutations in this population is between 1.3% and 1.7%. Surprisingly, MECP2 mutations in males are responsible for a wide spectrum of neurological disorders, ranging from mild mental retardation to severe neonatal encephalopathy. The aim of this review is to describe the nature of the MECP2 mutations identified in male patients to date and their associated phenotypes.     

Subtelomeric rearrangements as neutral genomic polymorphisms

Submicroscopic chromosomal rearrangements affecting telomeres are important aetiological contributors to the development of mental retardation. Results from over 2,500 analysed patients with mental retardation demonstrated that about 5% have a subtelomeric aberration. However, some subtelomeric rearrangements have no phenotypic consequences. Due to the heterogeneity of such rearrangements and to the limited information about which monosomy or trisomy can be tolerated without phenotypic effect, conclusions about the association of a specific aberration and the phenotypical consequences are often hard to draw. We performed a study of subtelomeric aberrations with the aim to provide more insights into the understanding of such rearrangements as neutral genomic polymorphisms. We found two new polymorphisms: a duplication or triplication of the subtelomeric region of the long arm of chromosome 4 and a trisomy of the subtelomeric region of the short arm of chromosome 6 owing to a transposition to chromosome 22. These new data are presented and discussed in the context of the published literature.     

Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome: Report of one case of MECP2 deletion and one case of MECP2 duplication

Mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2) are found in 70-80% of cases of classical Rett syndrome (RTT) and in about 50% of cases of preserved speech variant (PSV). This high percentage of MECP2 mutations, especially in classical RTT cases, suggests that another major RTT locus is unlikely. Missed mutations may be due to the limited sensitivity of the methodology used for mutation scanning and/or the presence of intronic mutations. In a double-copy gene, such as MECP2 in females, current methodologies (e.g., DGGE, SSCP, DHPLC, direct sequencing) are prone to miss gross rearrangements. Three previous reports during 2001-2003 have shown the presence of large deletions in a fraction of MECP2-negative classical RTT patients. We developed a reliable, single tube, quantitative PCR assay for rapid determination of MECP2 gene dosage. This method involves a multiplex reaction using a FAM labeled TaqMan probe with a TAMRA quencher derived from MECP2 exon 4 and two primers derived from the same exon and RNAaseP as an internal reference. The copy number of the MECP2 gene was determined by the comparative threshold cycle method (ddCt). Each sample was run in quadruplicate. We validated this assay through the analysis of 30 healthy controls (15 female and 15 male) and we then applied this method to eight classical RTT and six PSV patients, all negative for MECP2 mutations. We identified gross rearrangements in two patients: a deletion in a classical RTT patient and a duplication in a PSV patient. Our results confirm that a fraction of MECP2-negative RTT cases have MECP2 gross rearrangements and we propose real-time quantitative PCR as a simple and reliable method for routine screening of MECP2 in addition to DHPLC analysis.     

Characterization of six novel patients with MECP2 duplications due to unbalanced rearrangements of the X chromosome

Males with duplication of the Xq28 region, including methyl CpG-binding protein 2 (MECP2), exhibit a characteristic phenotype, including developmental delay, intellectual disability, limited or absent speech, limited or absent ambulation, and recurrent respiratory infections. We report six males with MECP2 duplications identified using array comparative genomic hybridization. The minimal sizes of these duplications range from ～0.08 to 14.13?Mb, which, to the best of our knowledge, are respectively the smallest and largest minimal size duplications molecularly characterized to date. Adjunct metaphase fluorescence in situ hybridization analysis further classified these duplications as tandem or as products of complex chromosomal rearrangements. Specifically, one complex rearrangement was described as a der(12)t(X;12)(q28;q24.33), which is the first report of a translocation involving MECP2 on Xq and chromosome 12. The other complex rearrangement was described as a rec(X)dup(Xq)inv(X)(p22.32q28)mat. Synthesis of the dysmorphic features identified in individuals with rec(X) chromosomes, including deletions in the pseudoautosomal region 1 (PAR1) at Xp22.33/Yp11.3 and duplications of the distal Xq region including MECP2, revealed a high prevalence of undescended testes (7/8) and micropenis (3/8) in this cohort. Given that micropenis is rare in the general population, but present in 38% of individuals in this cohort, a dosage anomaly at one or both loci may be a significant risk factor for this condition. Therefore, we recommend microarray testing for patients with unexplained micropenis, particularly when accompanied by other phenotypic anomalies.     

Intertypic genomic rearrangements of poliovirus strains in vaccinees

In vivo intertypic RNA recombination has previously been observed in excreted type 3 poliovirus isolates from a normal asymptomatic primary vaccinee. This study examines isolates from additional primary vaccinees to determine whether intertypic recombination is a general occurrence in excreted polioviruses. T1 RNase oligonucleotide finger-printing and limited dideoxy primer extension RNA sequencing demonstrated no evidence of intertypic recombination among type 1 or type 2 excreted strains. However, vaccinees excreting type 3 strains for long periods of time eventually produced recombinant strains involving either type 1 or type 2 poliovirus. Moreover, a characteristic time course of appearance of excreted type 3 intertypic recombinant polioviruses was established. Type 3/type 1 and type 3/type 2 recombinant strains appeared at Days 10-11 with a single crossover site in the gene for nonstructural protein 2C. Type 3/type 2/type 3 complex recombinant strains with an additional crossover site in the polymerase gene replaced type 3/type 2 strains at approximately Day 28. A significant portion of the genome of the type 3 Sabin vaccine strain is thus replaced during long-term excretion by vaccinees, and the appearance of some genomic arrangements coincided with a base deletion at the 3' terminus.     

Increased MECP2 gene copy number as the result of genomic duplication in neurodevelopmentally delayed males.

PURPOSE: Mutations in the MECP2 gene are associated with Rett syndrome, an X-linked mental retardation disorder in females. Mutations also cause variable neurodevelopmental phenotypes in rare affected males. Recent clinical testing for MECP2 gene rearrangements revealed that entire MECP2 gene duplication occurs in some males manifesting a progressive neurodevelopmental syndrome. METHODS: Clinical testing through quantitative DNA methods and chromosomal microarray analysis in our laboratories identified seven male patients with increased MECP2 gene copy number. RESULTS: Duplication of the entire MECP2 gene was found in six patients, and MECP2 triplication was found in one patient with the most severe phenotype. The Xq28 duplications observed in these males are unique and vary in size from approximately 200 kb to 2.2 Mb. Three of the mothers who were tested were asymptomatic duplication carriers with skewed X-inactivation. In silico analysis of the Xq28 flanking region showed numerous low-copy repeats with potential roles in recombination. CONCLUSIONS: These collective data suggest that increased MECP2 gene copy number is mainly responsible for the neurodevelopmental phenotypes in these males. These findings underscore the allelic and phenotypic heterogeneity associated with the MECP2 gene and highlight the value of molecular analysis for patient diagnosis, family members at risk, and genetic counseling.     

基因组重排的机制研究

基因组结构变异是人类基因组中常见的一种变异形式,往往涉及染色体或大片段染色体区域的改变,将原来两个分开的DNA序列重新连接在一起,从而形成染色体片段的缺失、重复复制、插入、倒位或者易位.在核苷酸水平上对大量重排断裂点接头序列特征的分析,为研究基因组重排的机制提供了宝贵的资源.本文综述了导致基因组重排的同源重组和非同源修复机制,并提出复制过程在基因组重排中发挥了重要作用,是细胞压力和结构变异的潜在桥梁,对于理解人类基因组进化和人类疾病的发生发展具有重要意义.