Megabase-sized, complex, repetitive regions of genomes are poorly studied, due to the technical and computational challenges inherent to both assembling precise reference sequences and accurately assessing structural variation across contiguous megabase DNA regions. Here we describe a strategy to overcome these challenges, CISMR (CRISPR-mediated isolation of specific megabase-sized regions of the genome), which enables us to perform targeted isolation of contiguous megabase-sized segments of the genome. Direct sequencing of the purified DNA segments can have >100-fold enrichment of the target region, thus enabling the exploration of both DNA sequence and structural diversity of complex genomic regions in any species.

A variety of distinct phenotypes has been associated with supernumerary inv dup(15) chromosomes. Although different cytogenetic rearrangements have been associated with distinguishable clinical syndromes, precise genotype‐phenotype correlations have not been determined. However, the availability of chromosome 15 DNA markers provides a means to characterize inv dup(15) chromosomes in detail to facilitate the determination of specific genotype‐phenotype associations. We describe 2 patients with an autistic disorder, mental retardation, developmental delay, seizures, and supernumerary inv dup(15) chromosomes. Conventional and molecular cytogenetic studies confirmed the chromosomal origin of the supernumerary chromosomes and showed that the duplicated region extended to at least band 15q13. An analysis of chromosome 15 microsatellite CA polymorphisms suggested a maternal origin of the inv dup(15) chromosomes and biparental inheritance of the two intact chromosome 15 homologs. The results of this study add to the existing literature which suggests that the clinical phenotype of patients with a supernumerary inv dup(15) chromosome is determined not only by the extent of the duplicated region, but by the dosage of genes located within band 15q13 and the origin of the normal chromosomes 15. © 1996 Wiley‐Liss, Inc.

Impaired replication progression leads to de novo copy number variant (CNV) formation at common fragile sites (CFSs). We previously showed that these hotspots for genome instability reside in late-replicating domains associated with large transcribed genes and provided indirect evidence that transcription is a factor in their instability. Here, we compared aphidicolin (APH)-induced CNV and CFS frequency between wild-type and isogenic cells in which FHIT gene transcription was ablated by promoter deletion. Two promoter-deletion cell lines showed reduced or absent CNV formation and CFS expression at FHIT despite continued instability at the NLGN1 control locus. APH treatment led to critical replication delays that remained unresolved in G2/M in the body of many, but not all, large transcribed genes, an effect that was reversed at FHIT by the promoter deletion. Altering RNase H1 expression did not change CNV induction frequency and DRIP-seq showed a paucity of R-loop formation in the central regions of large genes, suggesting that R-loops are not the primary mediator of the transcription effect. These results demonstrate that large gene transcription is a determining factor in replication stress-induced genomic instability and support models that CNV hotspots mainly result from the transcription-dependent passage of unreplicated DNA into mitosis.

Epigenetic control of gene expression is a consistent feature of differentiated mammalian cell types. Epigenetic expression patterns are mitotically heritable and are stably maintained in adult cells. However, unlike somatic DNA mutation, little is known about the occurrence of epigenetic change, or epimutation, during normal adult life. We have monitored the age-associated maintenance of two epigenetic systems—X inactivation and genomic imprinting—using the genes Atp7a and Igf2, respectively. Quantitative measurements of RNA transcripts from the inactive and active alleles were performed in mice from 2 to 24 months of age. For both genes, older animal cohorts showed reproducible increases in transcripts expressed from the silenced alleles. Loss of X chromosome silencing showed cohort mean increases of up to 2.2%, while imprinted-gene activation increased up to 6.7%. The results support the hypothesis that epigenetic loss of gene repression occurs in normal tissues and may be a contributing factor in progressive physiological dysfunction seen during mammalian aging. Quantitatively, the loss of epigenetic control may be one to two orders of magnitude greater than previously determined somatic DNA mutation.