Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort.

Long-read HiFi genome sequencing allows for accurate detection and direct phasing of single nucleotide variants, indels, and structural variants. Recent algorithmic development enables simultaneous detection of CpG methylation for analysis of regulatory element activity directly in HiFi reads. We present a comprehensive haplotype resolved 5-base HiFi genome sequencing dataset from a rare disease cohort of 276 samples in 152 families to identify rare (~0.5%) hypermethylation events. We find that 80% of these events are allele-specific and predicted to cause loss of regulatory element activity. We demonstrate heritability of extreme hypermethylation including rare cis variants associated with short (~200 bp) and large hypermethylation events (>1 kb), respectively. We identify repeat expansions in proximal promoters predicting allelic gene silencing via hypermethylation and demonstrate allelic transcriptional events downstream. On average 30-40 rare hypermethylation tiles overlap rare disease genes per patient, providing indications for variation prioritization including a previously undiagnosed pathogenic allele in DIP2B causing global developmental delay. We propose that use of HiFi genome sequencing in unsolved rare disease cases will allow detection of unconventional diseases alleles due to loss of regulatory element activity.

KaryoCreate: A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres.

Aneuploidy, the presence of chromosome gains or losses, is a hallmark of cancer. Here, we describe KaryoCreate (karyotype CRISPR-engineered aneuploidy technology), a system that enables the generation of chromosome-specific aneuploidies by co-expression of an sgRNA targeting chromosome-specific CENPA-binding ɑ-satellite repeats together with dCas9 fused to mutant KNL1. We design unique and highly specific sgRNAs for 19 of the 24 chromosomes. Expression of these constructs leads to missegregation and induction of gains or losses of the targeted chromosome in cellular progeny, with an average efficiency of 8% for gains and 12% for losses (up to 20%) validated across 10 chromosomes. Using KaryoCreate in colon epithelial cells, we show that chromosome 18q loss, frequent in gastrointestinal cancers, promotes resistance to TGF-ß, likely due to synergistic hemizygous deletion of multiple genes. Altogether, we describe an innovative technology to create and study chromosome missegregation and aneuploidy in the context of cancer and beyond.

Magnitude of modulation of gene expression in aneuploid maize depends on the extent of genomic imbalance.

Aneuploidy has profound effects on an organism, typically more so than polyploidy, and the basis of this contrast is not fully understood. A dosage series of the maize long arm of chromosome 1 (1L) was used to compare relative global gene expression in different types and degrees of aneuploidy to gain insights into how the magnitude of genomic imbalance as well as hypoploidy affects global gene expression. While previously available methods require a selective examination of specific genes, RNA sequencing provides a whole-genome view of gene expression in aneuploids. Most studies of global aneuploidy effects have concentrated on individual types of aneuploids because multiple dose aneuploidies of the same genomic region are difficult to produce in most model genetic organisms. The genetic toolkit of maize allows the examination of multiple ploidies and 1-4 doses of chromosome arms. Thus, a detailed examination of expression changes both on the varied chromosome arms and elsewhere in the genome is possible, in both hypoploids and hyperploids, compared with euploid controls. Previous studies observed the inverse trans effect, in which genes not varied in DNA dosage were expressed in a negative relationship to the varied chromosomal region. This response was also the major type of changes found globally in this study. Many genes varied in dosage showed proportional expression changes, though some were seen to be partly or fully dosage compensated. It was also found that the effects of aneuploidy were progressive, with more severe aneuploids producing effects of greater magnitude.

Causes and effects of haploinsufficiency.

Haploinsufficiency is a form of genetic dominance and is the underlying mechanism of numerous human inherited conditions in which the causal genes are sensitive to altered dosage. This review examines the poorly understood relationships between haploinsufficiency, dosage sensitivity and genetic dominance, whose common theme is the existence of nonlinear relationships between genotype and phenotype. We present an up-to-date account of the bases of haploinsufficiency from the perspective of theoretical and experimental models. We also discuss human conditions caused by haploinsufficiency, including developmental syndromes and cancer. Connections between the understanding of these conditions' genetic mechanisms and advances in treatments are also described.

Global impacts of chromosomal imbalance on gene expression in Arabidopsis and other taxa.

Changes in dosage of part of the genome (aneuploidy) have long been known to produce much more severe phenotypic consequences than changes in the number of whole genomes (ploidy). To examine the basis of these differences, global gene expression in mature leaf tissue for all five trisomies and in diploids, triploids, and tetraploids of Arabidopsis thaliana was studied. The trisomies displayed a greater spread of expression modulation than the ploidy series. In general, expression of genes on the varied chromosome ranged from compensation to dosage effect, whereas genes from the remainder of the genome ranged from no effect to reduced expression approaching the inverse level of chromosomal imbalance (2/3). Genome-wide DNA methylation was examined in each genotype and found to shift most prominently with trisomy 4 but otherwise exhibited little change, indicating that genetic imbalance is generally mechanistically unrelated to DNA methylation. Independent analysis of gene functional classes demonstrated that ribosomal, proteasomal, and gene body methylated genes were less modulated compared with all classes of genes, whereas transcription factors, signal transduction components, and organelle-targeted protein genes were more tightly inversely affected. Comparing transcription factors and their targets in the trisomies and in expression networks revealed considerable discordance, illustrating that altered regulatory stoichiometry is a major contributor to genetic imbalance. Reanalysis of published data on gene expression in disomic yeast and trisomic mouse cells detected similar stoichiometric effects across broad phylogenetic taxa, and indicated that these effects reflect normal gene regulatory processes.

Kinetics genetics: Incorporating the concept of genomic balance into an understanding of quantitative traits.

While most mutations are recessive, variants that affect quantitative traits are largely semi-dominant in their action making hybrids between divergent genotypes intermediate. In parallel, changes in chromosomal dosage (aneuploidy) for multiple regions of the genome modulate quantitative characters. We have previously argued that these observations are a reflection of a common process, originating from the more or less subtle effects of changes in dosage on the action of multi-subunit regulatory machineries. Kinetic analyses that vary the amount of one subunit of a complex while holding others constant do not always predict a linear response for the production of the whole. Indeed, in many instances, strong non-linear effects are expected. Here, we advocate that these kinetic observations and predictions should be incorporated into quantitative genetics thought.

Differential effect of aneuploidy on the X chromosome and genes with sex-biased expression in Drosophila.

Global analysis of gene expression via RNA sequencing was conducted for trisomics for the left arm of chromosome 2 (2L) and compared with the normal genotype. The predominant response of genes on 2L was dosage compensation in that similar expression occurred in the trisomic compared with the diploid control. However, the male and female trisomic/normal expression ratio distributions for 2L genes differed in that females also showed a strong peak of genes with increased expression and males showed a peak of reduced expression relative to the opposite sex. For genes in other autosomal regions, the predominant response to trisomy was reduced expression to the inverse of the altered chromosomal dosage (2/3), but a minor peak of increased expression in females and further reduced expression in males were also found, illustrating a sexual dimorphism for the response to aneuploidy. Moreover, genes with sex-biased expression as revealed by comparing amounts in normal males and females showed responses of greater magnitude to trisomy 2L, suggesting that the genes involved in dosage-sensitive aneuploid effects also influence sex-biased expression. Each autosomal chromosome arm responded to 2L trisomy similarly, but the ratio distributions for X-linked genes were distinct in both sexes, illustrating an X chromosome-specific response to aneuploidy.

Dosage compensation and inverse effects in triple X metafemales of Drosophila.

Dosage compensation, the equalized X chromosome gene expression between males and females in Drosophila, has also been found in triple X metafemales. Inverse dosage effects, produced by genomic imbalance, are believed to account for this modulated expression, but they have not been studied on a global level. Here, we show a global expression comparison of metafemales (XXX; AA) with normal females (XX; AA) with high-throughput RNA-sequencing. We found that the majority of the X-linked genes in metafemales exhibit dosage compensation with an expression level similar to that of normal diploid females. In parallel, most of the autosomal genes were expressed at about two-thirds the level of normal females, the ratio of inverse dosage effects produced by the extra X chromosome. Both compensation and inverse effects were further confirmed by combination of X-linked and autosomally located miniwhite reporter genes in metafemales and relative quantitative PCR of selected genes. These data provide evidence for an inverse dosage component to X chromosome compensation.