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.     

Maternal uniparental isodisomy 20 in a foetus with trisomy 20 mosaicism: clinical,cytogenetic and molecular analysis

The clinical significance of trisomy 20 mosaicism detected prenatally remains uncertain due to the rarity of liveborn cases with inconsistent clinical findings, and lack of long-term follow-up and outcome. We describe a case of true trisomy 20 mosaicism in a liveborn girl with maternal uniparental isodisomy of chromosome 20 in the diploid blood cells. Trisomy 20 mosaicism was originally detected in amniotic fluid (98%) and was confirmed in the term placenta (100%), as well as in the blood (10%) and urine sediment (100%) of the neonate. There was intrauterine and postnatal growth retardation, but otherwise the newborn manifested no gross abnormalities. At 9 months of age moderate psychomotor retardation, central hypotonia with peripheral hypertonia, numerous minor morphogenetic variants, marked kyphosis, and extensive Mongolian spot were observed. To our knowledge this represents the first case of trisomy 20 mosaicism detected prenatally and confirmed in different tissues of the newborn, where uniparental disomy was demonstrated in the diploid cell line. The clinical and laboratory findings in our patient are compared with those of five previously reported cases of UPD20, suggesting that maternal UPD20 might be associated with a characteristic phenotype.     

Mosaic 22q13 deletions: evidence for concurrent mosaic segmental isodisomy and gene conversion

Although 22q terminal deletions are well documented, very few patients with mosaicism have been reported. We describe two new cases with mosaic 22q13.2-qter deletion, detected by karyotype analysis, showing the neurological phenotype of 22q13.3 deletion syndrome. Case 1 represents an exceptional case of mosaicism for maternal 22q13.2-qter deletion (45% of cells) and 22q13.2-qter paternal segmental isodisomy (55% of cells). This complex situation was suspected because cytogenetic, FISH and array-CGH analyses showed the presence of an 8.8 Mb mosaic 22q13.2-qter deletion, whereas microsatellite marker analysis was consistent with maternal deletion without any evidence of mosaic deletion. Molecular analysis led to the definition of very close, but not coincident, deletion and uniparental disomy (UPD) break points. Furthermore, we demonstrated that the segmental UPD arose by gene conversion in the same region. In Case 2, mosaicism for a paternal 8.9 Mb 22q13.2-qter deletion (73% of cells) was detected. In both patients, the level of mosaicism was also verified in saliva samples. We propose possible causative mechanisms for both rearrangements. Although the size of the deletions was quite similar, the phenotype was more severe in Case 2 than in Case 1. As maternal UPD 22 has not been generally associated with any defects and as the size of the deletion is very similar in the two cases, phenotype severity is likely to depend entirely on the degree of mosaicism in each individual.     

Expanding the genotypic spectrum of Jalili syndrome: Novel CNNM4 variants and uniparental isodisomy in a north American patient cohort

Jalili syndrome is a rare multisystem disorder with the most prominent features consisting of cone‐rod dystrophy and amelogenesis imperfecta. Few cases have been reported in the Americas. Here we describe a case series of patients with Jalili syndrome examined at the National Eye Institute's Ophthalmic Genetics clinic between 2016 and 2018. Three unrelated sporadic cases were systematically evaluated for ocular phenotype and determined to have cone‐rod dystrophy with bull's eye maculopathy, photophobia, and nystagmus. All patients had amelogenesis imperfecta. Two of these patients had Guatemalan ancestry and the same novel homozygous CNNM4 variant (p.Arg236Trp c.706C > T) without evidence of consanguinity. This variant met likely pathogenic criteria by the American College of Medical Genetics guidelines. An additional patient had a homozygous deleterious variant in CNNM4 (c.279delC p.Phe93Leufs*31), which resulted from paternal uniparental isodisomy for chromosome 2p22‐2q37. This individual had additional syndromic features including developmental delay and spastic diplegia, likely related to mutations at other loci. Our work highlights the genotypic variability of Jalili syndrome and expands the genotypic spectrum of this condition by describing the first series of patients seen in the United States.     

Engineering translocations with delayed replication: evidence for cis control of chromosome replication timing

Certain chromosome rearrangements, found in cancer cells or in cells exposed to ionizing radiation, exhibit a chromosome-wide delay in replication timing (DRT) that is associated with a delay in mitotic chromosome condensation (DMC). We have developed a chromosome engineering strategy that allows the generation of chromosomes with this DRT/DMC phenotype. We found that approximately 10% of inter-chromosomal translocations induced by two distinct mechanisms, site-specific recombination mediated by Cre or non-homologous end joining of DNA double-strand breaks induced by I-Sce1, result in DRT/DMC. Furthermore, on certain balanced translocations only one of the derivative chromosomes displays the phenotype. Finally, we show that the engineered DRT/DMC chromosomes acquire gross chromosomal rearrangements at an increased rate when compared with non-DRT/DMC chromosomes. These results indicate that the DRT/DMC phenotype is not the result of a stochastic process that could occur at any translocation breakpoint or as an epigenetic response to chromosome damage. Instead, our data indicate that the replication timing of certain derivative chromosomes is regulated by a cis-acting mechanism that delays both initiation and completion of DNA synthesis along the entire length of the chromosome. Because chromosomes with DRT/DMC are common in tumor cells and in cells exposed to ionizing radiation, we propose that DRT/DMC represents a common mechanism responsible for the genomic instability found in cancer cells and for the persistent chromosomal instability associated with cells exposed to ionizing radiation.     

Concurrent neuroborreliosis and Alzheimer's disease: analysis of the evidence

Several recent reports have claimed a possible association between Borrelia burgdorferi infection and Alzheimer's disease (AD). Herein, we describe our search for additional evidence of neuroborreliosis in AD. Brain tissue from neuropathologically confirmed cases of AD was cultured for B burgdorferi using standard microbiologic methods. Material derived from culture was further examined using electron microscopy, direct immunofluorescence and acridine orange fluorescence. Previous studies have shown high titers of antiborrelia antibodies in CSF in all cases of confirmed neuroborreliosis; therefore, we tested CSF from neuropathologically confirmed cases of AD by indirect immunofluorescence and enzyme-linked immunoassay. In addition, imprint preparations from AD and control brain tissues were studied by direct immunofluorescence using a monoclonal antiborrelia antibody. Finally, a Western blot method was used to analyze protein extracts from cultures and AD brain tissue for the presence of borrelia antigen. Contrary to previous studies, our results do not support an association between infection with B burgdorferi and AD.     

NIPBL rearrangements in Cornelia de Lange syndrome: evidence for replicative mechanism and genotype-phenotype correlation

PurposeCornelia de Lange syndrome (CdLS) is a multisystem congenital anomaly disorder characterized by mental retardation, limb abnormalities, distinctive facial features, and hirsutism. Mutations in three genes involved in sister chromatid cohesion, NIPBL, SMC1A, and SMC3, account for ~55% of CdLS cases. The molecular etiology of a significant fraction of CdLS cases remains unknown. We hypothesized that large genomic rearrangements of cohesin complex subunit genes may play a role in the molecular etiology of this disorder.MethodsCustom high-resolution oligonucleotide array comparative genomic hybridization analyses interrogating candidate cohesin genes and breakpoint junction sequencing of identified genomic variants were performed.ResultsOf the 162 patients with CdLS, for whom mutations in known CdLS genes were previously negative by sequencing, deletions containing NIPBL exons were observed in 7 subjects (~5%). Breakpoint sequences in five patients implicated microhomology-mediated replicative mechanisms—such as serial replication slippage and fork stalling and template switching/microhomology-mediated break-induced replication—as a potential predominant contributor to these copy number variations. Most deletions are predicted to result in haploinsufficiency due to heterozygous loss-of-function mutations; such mutations may result in a more severe CdLS phenotype.ConclusionOur findings suggest a potential clinical utility to testing for copy number variations involving NIPBL when clinically diagnosed CdLS cases are mutation-negative by DNA-sequencing studies.Genet Med 2012:14(3):313–322     

CCMG guidelines: prenatal and postnatal diagnostic testing for uniparental disomy

The aim of this statement is to provide clinicians, cytogeneticists and molecular geneticists of the Canadian College of Medical Geneticists (CCMG) a comprehensive review of the role of UPD in constitutional genetic diagnosis and to provide a guideline as to when investigation for UPD is recommended. Members of the CCMG Cytogenetics, Molecular Genetics, Clinical Practice, and Prenatal Diagnosis committees reviewed the relevant literature on uniparental disomy (UPD) in constitutional genetic diagnosis (May 2010). Guidelines were developed for UPD testing in Canada. The guidelines were circulated for comment to the CCMG members at large and following appropriate modification, approved by the CCMG Board of Directors (July 2010).     

Chromosomal analysis of renal angiomyolipoma by comparative genomic hybridization: evidence for clonal origin

Angiomyolipoma has long been considered a hamartomatous polyclonal proliferation. However, recent molecular analyses have indicated that these tumors may be clonal neoplasms rather than polyclonal proliferations. We investigated chromosomal imbalances in angiomyolipoma by comparative genomic hybridization. DNA was extracted from archival paraffin-embedded and frozen tissues of 12 angiomyolipomas (10 usual variant, two epithelioid variant). The 10 angiomyolipomas of the usual variant included bilateral tumors from one tuberous sclerosis patient. Fluorescence ratio distributions from tumor hybridizations were compared with those from control hybridizations to detect changes in DNA copy number with high sensitivity and specificity. We identified 20 chromosomal imbalances in seven sporadic angiomyolipomas, including both tumors of the epithelioid variant. The remaining five tumors, including the two angiomyolipomas from a tuberous sclerosis patient, were devoid of chromosomal imbalances. Seventy-five percent of the imbalances were partial or whole chromosomal deletions involving disparate genomic regions, some of which have previously been associated with tumors of adipose tissue and smooth muscle tumors. Four angiomyolipomas of the usual variant showed 5q deletions with a common region of deletion spanning 5q33 to q34. In two tumors, deletion on 5q was the sole abnormality. One epithelioid angiomyolipoma showed 5q gain encompassing the same region in addition to other alterations. We concluded that (1) Chromosomal imbalances are common in renal angiomyolipomas; (2) Presence of clonal genomic alterations lends further support to the neoplastic pathogenesis of these tumors; (3) The 5q33-q34 region may contain a tumor suppressor gene significant in the histogenesis of some renal angiomyolipomas.     

Concurrent encoding of frequency and amplitude modulation in human auditory cortex: MEG evidence

A natural sound can be described by dynamic changes in envelope (amplitude) and carrier (frequency), corresponding to amplitude modulation (AM) and frequency modulation (FM), respectively. Although the neural responses to both AM and FM sounds are extensively studied in both animals and humans, it is uncertain how they are corepresented when changed simultaneously but independently, as is typical for ecologically natural signals. This study elucidates the neural coding of such sounds in human auditory cortex using magnetoencephalography (MEG). Using stimuli with both sinusoidal modulated envelope (f(AM), 37 Hz) and carrier frequency (f(FM), 0.3-8 Hz), it is demonstrated that AM and FM stimulus dynamics are corepresented in the neural code of human auditory cortex. The stimulus AM dynamics are represented neurally with AM encoding, by the auditory steady-state response (aSSR) at f(AM). For sounds with slowly changing carrier frequency (f(FM) <5 Hz), it is shown that the stimulus FM dynamics are tracked by the phase of the aSSR, demonstrating neural phase modulation (PM) encoding of the stimulus carrier frequency. For sounds with faster carrier frequency change (f(FM) > or = 5 Hz), it is shown that modulation encoding of stimulus FM dynamics persists, but the neural encoding is no longer purely PM. This result is consistent with the recruitment of additional neural AM encoding over and above the original neural PM encoding, indicating that both the amplitude and phase of the aSSR at f(AM) track the stimulus FM dynamics. A neural model is suggested to account for these observations.     

Inhibition of vaccinia virus replication by nicotinamide: evidence for ADP-ribosylation of viral proteins

Replication of vaccinia virus (VV) in monolayers of BSC40 cells was inhibited 99.9% in the presence of 60 mM nicotinamide (NIC), a competitive inhibitor of ADP-ribosylation reactions. Dot-blot hybridization analysis of infected cell extracts utilizing a VV DNA-specific probe indicated that the drug had only minimal effects on viral DNA synthesis. SDS-polyacrylamide gel electrophoresis of newly synthesized VV proteins pulse-labeled at early (2 h) or late (8 h) times post-infection revealed that although the full spectrum of expected viral polypeptides was evident, quantitative differences in the levels of expression of a distinct subset of viral proteins were observed in the presence of the drug. Velocity sedimentation of virus-infected cell lysates established that no mature particles were assembled in drug treated cells. Additional evidence suggesting that VV morphogenesis was abortive in the presence of NIC was obtained by pulse-chase labeling experiments that demonstrated that the two VV major late core polypeptide precursors P94 and P65, whose proteolytic processing to VP62 and VP60 is intimately associated with viral assembly, were not cleaved in the presence of NIC. Interestingly, growth of VV in the presence of [3H]adenosine resulted in the metabolic labeling of eight proteins that were associated with purified virions. These proteins co-migrated with proteins labeled with [3H]adenosine that were present in extracts of VV-infected, but not uninfected, cells. These analyses also revealed that the [3H]adenosine-labeling of a subset of cellular proteins (MW 18-20 kDa, possibly histones) was increased 4-fold by VV infection. The observed induction of either increased synthesis or hyper-modification of these 18-20 kDa proteins was inhibited by NIC. These results are discussed with respect to whether one or more VV polypeptides are subject to obligatory ADP-ribosylation modification reactions in order to attain their active configuration, and if so, whether the enzymes catalyzing these reactions are specified by the virus or host cell.     

Paternal uniparental disomy for chromosome 14: A case report and review

Uniparental disomy (UPD) for several chromosomes has been associated with disease phenotypes. Maternal UPD for chromosome 14 has been described and has a characteristic abnormal phenotype. Paternal UPD14 is rare and only three previous cases have been reported. We describe a new case of paternal UPD for chromosome 14 in an infant with a 45,XX,der(13q;14q) karyotype, which was confirmed by molecular analysis. The proposita had findings similar to those of the previous cases of patUPD14 and we conclude that there is a characteristic patUPD14 syndrome most likely due to imprinting effects. Couples with Robertsonian translocations involving chromosome 14 should be counseled as to the possibility of UPD14 and the option of prenatal diagnosis when indicated. Am. J. Med. Genet. 70:74–79, 1997. © 1997 Wiley-Liss, Inc.