Haploinsufficiency predictions without study bias

Any given human individual carries multiple genetic variants that disrupt protein-coding genes, through structural variation, as well as nucleotide variants and indels. Predicting the phenotypic consequences of a gene disruption remains a significant challenge. Current approaches employ information from a range of biological networks to predict which human genes are haploinsufficient (meaning two copies are required for normal function) or essential (meaning at least one copy is required for viability). Using recently available study gene sets, we show that these approaches are strongly biased towards providing accurate predictions for well-studied genes. By contrast, we derive a haploinsufficiency score from a combination of unbiased large-scale high-throughput datasets, including gene co-expression and genetic variation in over 6000 human exomes. Our approach provides a haploinsufficiency prediction for over twice as many genes currently unassociated with papers listed in Pubmed as three commonly-used approaches, and outperforms these approaches for predicting haploinsufficiency for less-studied genes. We also show that fine-tuning the predictor on a set of well-studied ‘gold standard’ haploinsufficient genes does not improve the prediction for less-studied genes. This new score can readily be used to prioritize gene disruptions resulting from any genetic variant, including copy number variants, indels and single-nucleotide variants.     

Characterising and predicting haploinsufficiency in the human genome

Haploinsufficiency, wherein a single functional copy of a gene is insufficient to maintain normal function, is a major cause of dominant disease. Human disease studies have identified several hundred haploinsufficient (HI) genes. We have compiled a map of 1,079 haplosufficient (HS) genes by systematic identification of genes unambiguously and repeatedly compromised by copy number variation among 8,458 apparently healthy individuals and contrasted the genomic, evolutionary, functional, and network properties between these HS genes and known HI genes. We found that HI genes are typically longer and have more conserved coding sequences and promoters than HS genes. HI genes exhibit higher levels of expression during early development and greater tissue specificity. Moreover, within a probabilistic human functional interaction network HI genes have more interaction partners and greater network proximity to other known HI genes. We built a predictive model on the basis of these differences and annotated 12,443 genes with their predicted probability of being haploinsufficient. We validated these predictions of haploinsufficiency by demonstrating that genes with a high predicted probability of exhibiting haploinsufficiency are enriched among genes implicated in human dominant diseases and among genes causing abnormal phenotypes in heterozygous knockout mice. We have transformed these gene-based haploinsufficiency predictions into haploinsufficiency scores for genic deletions, which we demonstrate to better discriminate between pathogenic and benign deletions than consideration of the deletion size or numbers of genes deleted. These robust predictions of haploinsufficiency support clinical interpretation of novel loss-of-function variants and prioritization of variants and genes for follow-up studies.     

Different unequal cross-over events between NCF1 and its pseudogenes in autosomal p47phox-deficient chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot generate superoxide (O₂^?) and other microbicidal oxidants due to mutations in one of the five components of the O₂^?-generating NADPH oxidase complex. The most common autosomal subtype of CGD is caused by mutations in NCF1, encoding the NADPH subunit p47^phox. Usually, these mutations are the result of unequal exchange of chromatid between NCF1 and one of its two pseudogenes. We have now investigated in detail the breakpoints within or between these (pseudo) NCF1 genes in 43 families with p47^phox-deficient CGD by means of multiplex ligase-dependent probe amplification (MLPA). In 24 families the patients totally lacked NCF1 sequences, indicating that in these families the cross-over points are located between NCF1 and its pseudogenes. Six other families were compound heterozygous for a total NCF1 deletion and another mutation in NCF1 on the other allele. In 8 families, the patients lacked NCF1 exons 1–4 but had retained NCF1 exons 6–10, indicating that a cross-over point is located within NCF1 between exons 4 and 6. Similarly, in 4 families a cross-over point was located within NCF1 between exons 2 and 4. Similar cross-overs, in heterozygous form, were observed in family members of the patients. Several patients were compound heterozygous for total and partial NCF1 deletions. Thus, at least three different cross-over points exist within the NCF1 gene cluster, indicating that autosomal p47^phox-deficient CGD is genetically heterogeneous but can be dissected in detail by MLPA.     

Mechanisms of haploinsufficiency revealed by genome-wide profiling in yeast

Haploinsufficiency is defined as a dominant phenotype in diploid organisms that are heterozygous for a loss-of-function allele. Despite its relevance to human disease, neither the extent of haploinsufficiency nor its precise molecular mechanisms are well understood. We used the complete set of Saccharomyces cerevisiae heterozygous deletion strains to survey the genome for haploinsufficiency via fitness profiling in rich (YPD) and minimal media to identify all genes that confer a haploinsufficient growth defect. This assay revealed that approximately 3% of all approximately 5900 genes tested are haploinsufficient for growth in YPD. This class of genes is functionally enriched for metabolic processes carried out by molecular complexes such as the ribosome. Much of the haploinsufficiency in YPD is alleviated by slowing the growth rate of each strain in minimal media, suggesting that certain gene products are rate limiting for growth only in YPD. Overall, our results suggest that the primary mechanism of haploinsufficiency in yeast is due to insufficient protein production. We discuss the relevance of our findings in yeast to human haploinsufficiency disorders.     

Haploinsufficiency for tumour suppressor genes: when you don't need to go all the way

Classical tumour suppressor genes are thought to require mutation or loss of both alleles to facilitate tumour progression. However, it has become clear over the last few years that for some genes, haploinsufficiency, which is loss of only one allele, may contribute to carcinogenesis. These effects can either be directly attributable to the reduction in gene dosage or may act in concert with other oncogenic or haploinsufficient events. Here we describe the genes that undergo this phenomenon and discuss possible mechanisms that allow haploinsufficiency to display a phenotype and facilitate the pathogenesis of cancer.     

Apolipoprotein B mRNA editing: the sequence to the event

Editing of the mRNA coding for apolipoprotein B involves a cytidine to uridine transition at nucleotide 6666 and enables translation of two protein variants. The development of in vitro editing systems has led to the identification of three sequence requirements in this process. The mechanism for C→U editing requires specific sequences for editing site recognition, positioning of the catalytic activity and enhancement of catalytic efficiency. The dependence of in vitro editing on extract proteins has focused future research in this field on the identification of factors involved in apoB mRNA editing and the role of these factors in the assembly of ribonucleoprotein editosomes.     

Germline Variation in Cancer-Susceptibility Genes in a Healthy,Ancestrally Diverse Cohort: Implications for Individual Genome Sequencing

Technological advances coupled with decreasing costs are bringing whole genome and whole exome sequencing closer to routine clinical use. One of the hurdles to clinical implementation is the high number of variants of unknown significance. For cancer-susceptibility genes, the difficulty in interpreting the clinical relevance of the genomic variants is compounded by the fact that most of what is known about these variants comes from the study of highly selected populations, such as cancer patients or individuals with a family history of cancer. The genetic variation in known cancer-susceptibility genes in the general population has not been well characterized to date. To address this gap, we profiled the nonsynonymous genomic variation in 158 genes causally implicated in carcinogenesis using high-quality whole genome sequences from an ancestrally diverse cohort of 681 healthy individuals. We found that all individuals carry multiple variants that may impact cancer susceptibility, with an average of 68 variants per individual. Of the 2,688 allelic variants identified within the cohort, most are very rare, with 75% found in only 1 or 2 individuals in our population. Allele frequencies vary between ancestral groups, and there are 21 variants for which the minor allele in one population is the major allele in another. Detailed analysis of a selected subset of 5 clinically important cancer genes, BRCA1, BRCA2, KRAS, TP53, and PTEN, highlights differences between germline variants and reported somatic mutations. The dataset can serve a resource of genetic variation in cancer-susceptibility genes in 6 ancestry groups, an important foundation for the interpretation of cancer risk from personal genome sequences.     

Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates.

The previously presented consensus sequence for eukaryotic translation initiation sites by Kozak was derived substantially from vertebrate mRNA sequences. Drosophila nuclear genes exhibit a significantly different translation start consensus sequence. These differences probably do not represent mechanistic differences in translation initiation inasmuch as both taxa exhibit identical preferences and restrictions at the crucial -3 position. Using more conservative criteria for the assignment of consensus the following consensus sequences were derived: vertebrate--CANCAUG and Drosophila--CAAAACAUG.     

Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes

Understanding regulatory mechanisms of protein synthesis in eukaryotes is essential for the accurate annotation of genome sequences. Kozak reported that the nucleotide sequence GCCGCC(A/G)CCAUGG (AUG is the initiation codon) was frequently observed in vertebrate genes and that this 'consensus' sequence enhanced translation initiation. However, later studies using invertebrate, fungal and plant genes reported different 'consensus' sequences. In this study, we conducted extensive comparative analyses of nucleotide sequences around the initiation codon by using genomic data from 47 eukaryote species including animals, fungi, plants and protists. The analyses revealed that preferred nucleotide sequences are quite diverse among different species, but differences between patterns of nucleotide bias roughly reflect the evolutionary relationships of the species. We also found strong biases of A/G at position -3, A/C at position -2 and C at position +5 that were commonly observed in all species examined. Genes with higher expression levels showed stronger signals, suggesting that these nucleotides are responsible for the regulation of translation initiation. The diversity of preferred nucleotide sequences around the initiation codon might be explained by differences in relative contributions from two distinct patterns, GCCGCCAUG and AAAAAAAUG, which implies the presence of multiple molecular mechanisms for controlling translation initiation.     

Efficient search,mapping, and optimization of multi‐protein genetic systems in diverse bacteria

Developing predictive models of multi‐protein genetic systems to understand and optimize their behavior remains a combinatorial challenge, particularly when measurement throughput is limited. We developed a computational approach to build predictive models and identify optimal sequences and expression levels, while circumventing combinatorial explosion. Maximally informative genetic system variants were first designed by the RBS Library Calculator, an algorithm to design sequences for efficiently searching a multi‐protein expression space across a > 10,000‐fold range with tailored search parameters and well‐predicted translation rates. We validated the algorithm's predictions by characterizing 646 genetic system variants, encoded in plasmids and genomes, expressed in six gram‐positive and gram‐negative bacterial hosts. We then combined the search algorithm with system‐level kinetic modeling, requiring the construction and characterization of 73 variants to build a sequence‐expression‐activity map (SEAMAP) for a biosynthesis pathway. Using model predictions, we designed and characterized 47 additional pathway variants to navigate its activity space, find optimal expression regions with desired activity response curves, and relieve rate‐limiting steps in metabolism. Creating sequence‐expression‐activity maps accelerates the optimization of many protein systems and allows previous measurements to quantitatively inform future designs.     

The Characteristics of Heterozygous Protein Truncating Variants in the Human Genome

Sequencing projects have identified large numbers of rare stop-gain and frameshift variants in the human genome. As most of these are observed in the heterozygous state, they test a gene’s tolerance to haploinsufficiency and dominant loss of function. We analyzed the distribution of truncating variants across 16,260 autosomal protein coding genes in 11,546 individuals. We observed 39,893 truncating variants affecting 12,062 genes, which significantly differed from an expectation of 12,916 genes under a model of neutral de novo mutation (p<10⁻⁴). Extrapolating this to increasing numbers of sequenced individuals, we estimate that 10.8% of human genes do not tolerate heterozygous truncating variants. An additional 10 to 15% of truncated genes may be rescued by incomplete penetrance or compensatory mutations, or because the truncating variants are of limited functional impact. The study of protein truncating variants delineates the essential genome and, more generally, identifies rare heterozygous variants as an unexplored source of diversity of phenotypic traits and diseases.     

Complementation of Yeast Genes with Human Genes as an Experimental Platform for Functional Testing of Human Genetic Variants

While the pace of discovery of human genetic variants in tumors, patients, and diverse populations has rapidly accelerated, deciphering their functional consequence has become rate-limiting. Using cross-species complementation, model organisms like the budding yeast, Saccharomyces cerevisiae, can be utilized to fill this gap and serve as a platform for testing human genetic variants. To this end, we performed two parallel screens, a one-to-one complementation screen for essential yeast genes implicated in chromosome instability and a pool-to-pool screen that queried all possible essential yeast genes for rescue of lethality by all possible human homologs. Our work identified 65 human cDNAs that can replace the null allele of essential yeast genes, including the nonorthologous pair yRFT1/hSEC61A1. We chose four human cDNAs (hLIG1, hSSRP1, hPPP1CA, and hPPP1CC) for which their yeast gene counterparts function in chromosome stability and assayed in yeast 35 tumor-specific missense mutations for growth defects and sensitivity to DNA-damaging agents. This resulted in a set of human–yeast gene complementation pairs that allow human genetic variants to be readily characterized in yeast, and a prioritized list of somatic mutations that could contribute to chromosome instability in human tumors. These data establish the utility of this cross-species experimental approach.     

Aryl hydrocarbon receptor-dependent induction of the NADPH oxidase subunit NCF1/p47 expression leading to priming of human macrophage oxidative burst

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene (BaP) are toxic environmental contaminants known to regulate gene expression through activation of the aryl hydrocarbon receptor (AhR). In the present study, we demonstrated that acute treatment by BaP markedly increased expression of the NADPH oxidase subunit gene neutrophil cytosolic factor 1 (NCF1)/p47^phox in primary human macrophages; NCF1 was similarly up-regulated in alveolar macrophages from BaP-instilled rats. NCF1 induction in BaP-treated human macrophages was prevented by targeting AhR, through its chemical inhibition or small interference RNA-mediated down-modulation of its expression. BaP moreover induced activity of the NCF1 promoter sequence, containing a consensus AhR-related xenobiotic-responsive element (XRE), and electrophoretic mobility shift assays and chromatin immunoprecipitation experiments indicated that BaP-triggered binding of AhR to this XRE. Finally, we showed that BaP exposure resulted in p47^phox protein translocation to the plasma membrane and in potentiation of phorbol myristate acetate (PMA)-induced superoxide anion production in macrophages. This BaP priming effect toward NADPH oxidase activity was inhibited by the NADPH oxidase specific inhibitor apocynin and the chemical AhR inhibitor α-naphtoflavone. These results indicated that BaP induced NCF1/p47^phox expression and subsequently enhanced superoxide anion production in PMA-treated human macrophages, in an AhR-dependent manner; such an NCF1/NADPH oxidase regulation by polycyclic aromatic hydrocarbons may participate in deleterious effects toward human health triggered by these environmental contaminants, including atherosclerosis and smoking-related diseases.     

An iterative gene-editing strategy broadens eIF4E1 genetic diversity in Solanum lycopersicum and generates resistance to multiple potyvirus isolates

Resistance to potyviruses in plants has been largely provided by the selection of natural variant alleles of eukaryotic translation initiation factors (eIF) 4E in many crops. However, the sources of such variability for breeding can be limited for certain crop species, while new virus isolates continue to emerge. Different methods of mutagenesis have been applied to inactivate the eIF4E genes to generate virus resistance, but with limited success due to the physiological importance of translation factors and their redundancy. Here, we employed genome editing approaches at the base level to induce non-synonymous mutations in the eIF4E1 gene and create genetic diversity in cherry tomato (Solanum lycopersicum var. cerasiforme). We sequentially edited the genomic sequences coding for two regions of eIF4E1 protein, located around the cap-binding pocket and known to be important for susceptibility to potyviruses. We show that the editing of only one of the two regions, by gene knock-in and base editing, respectively, is not sufficient to provide resistance. However, combining amino acid mutations in both regions resulted in resistance to multiple potyviruses without affecting the functionality in translation initiation. Meanwhile, we report that extensive base editing in exonic region can alter RNA splicing pattern, resulting in gene knockout. Altogether our work demonstrates that precision editing allows to design plant factors based on the knowledge on evolutionarily selected alleles and enlarge the gene pool to potentially provide advantageous phenotypes such as pathogen resistance.