Genome design of hybrid potato.

Reinventing potato from a clonally propagated tetraploid into a seed-propagated diploid, hybrid potato, is an important innovation in agriculture. Due to deleterious mutations, it has remained a challenge to develop highly homozygous inbred lines, a prerequisite to breed hybrid potato. Here, we employed genome design to develop a generation of pure and fertile potato lines and thereby the uniform, vigorous F1s. The metrics we applied in genome design included the percentage of genome homozygosity and the number of deleterious mutations in the starting material, the number of segregation distortions in the S1 population, the haplotype information to infer the break of tight linkage between beneficial and deleterious alleles, and the genome complementarity of the parental lines. This study transforms potato breeding from a slow, non-accumulative mode into a fast-iterative one, thereby potentiating a broad spectrum of benefits to farmers and consumers.

Ancient and modern genomes unravel the evolutionary history of the rhinoceros family.

Only five species of the once-diverse Rhinocerotidae remain, making the reconstruction of their evolutionary history a challenge to biologists since Darwin. We sequenced genomes from five rhinoceros species (three extinct and two living), which we compared to existing data from the remaining three living species and a range of outgroups. We identify an early divergence between extant African and Eurasian lineages, resolving a key debate regarding the phylogeny of extant rhinoceroses. This early Miocene (∼16 million years ago [mya]) split post-dates the land bridge formation between the Afro-Arabian and Eurasian landmasses. Our analyses also show that while rhinoceros genomes in general exhibit low levels of genome-wide diversity, heterozygosity is lowest and inbreeding is highest in the modern species. These results suggest that while low genetic diversity is a long-term feature of the family, it has been particularly exacerbated recently, likely reflecting recent anthropogenic-driven population declines.

A Therapy for Most with Cystic Fibrosis.

TRIKAFTA is the third drug approved by the FDA that rescues defects caused by the major mutation F508del. It is superior to its predecessors that were approved for patients who are homozygous for F508del because TRIKAFTA is also effective in CF patients who harbor only one copy of this mutation.

Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.

Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.

Recurrent SMARCB1 Mutations Reveal a Nucleosome Acidic Patch Interaction Site That Potentiates mSWI/SNF Complex Chromatin Remodeling.

Mammalian switch/sucrose non-fermentable (mSWI/SNF) complexes are multi-component machines that remodel chromatin architecture. Dissection of the subunit- and domain-specific contributions to complex activities is needed to advance mechanistic understanding. Here, we examine the molecular, structural, and genome-wide regulatory consequences of recurrent, single-residue mutations in the putative coiled-coil C-terminal domain (CTD) of the SMARCB1 (BAF47) subunit, which cause the intellectual disability disorder Coffin-Siris syndrome (CSS), and are recurrently found in cancers. We find that the SMARCB1 CTD contains a basic α helix that binds directly to the nucleosome acidic patch and that all CSS-associated mutations disrupt this binding. Furthermore, these mutations abrogate mSWI/SNF-mediated nucleosome remodeling activity and enhancer DNA accessibility without changes in genome-wide complex localization. Finally, heterozygous CSS-associated SMARCB1 mutations result in dominant gene regulatory and morphologic changes during iPSC-neuronal differentiation. These studies unmask an evolutionarily conserved structural role for the SMARCB1 CTD that is perturbed in human disease.

Aldehydes Pose a Threat to BRCA2 Mutation Carriers.

Humans with inherited heterozygous BRCA2 mutations have an increased risk of developing cancer; however, what triggers carcinogenesis in these individuals is unclear. Tan et al. find that environmental and metabolic aldehydes pose a threat to these individuals by promoting degradation of wild-type BRCA2 protein, thereby predisposing them to genomic instability and perhaps to cancer.

Type 2 Diabetes Variants Disrupt Function of SLC16A11 through Two Distinct Mechanisms.

Type 2 diabetes (T2D) affects Latinos at twice the rate seen in populations of European descent. We recently identified a risk haplotype spanning SLC16A11 that explains ∼20% of the increased T2D prevalence in Mexico. Here, through genetic fine-mapping, we define a set of tightly linked variants likely to contain the causal allele(s). We show that variants on the T2D-associated haplotype have two distinct effects: (1) decreasing SLC16A11 expression in liver and (2) disrupting a key interaction with basigin, thereby reducing cell-surface localization. Both independent mechanisms reduce SLC16A11 function and suggest SLC16A11 is the causal gene at this locus. To gain insight into how SLC16A11 disruption impacts T2D risk, we demonstrate that SLC16A11 is a proton-coupled monocarboxylate transporter and that genetic perturbation of SLC16A11 induces changes in fatty acid and lipid metabolism that are associated with increased T2D risk. Our findings suggest that increasing SLC16A11 function could be therapeutically beneficial for T2D. VIDEO ABSTRACT.

Heterozygous RPA2 variant as a novel genetic cause of telomere biology disorders.

Premature telomere shortening or telomere instability is associated with a group of rare and heterogeneous diseases collectively known as telomere biology disorders (TBDs). Here we identified two unrelated individuals with clinical manifestations of TBDs and short telomeres associated with the identical monoallelic variant c.767A>G; Y256C in RPA2 Although the replication protein A2 (RPA2) mutant did not affect ssDNA binding and G-quadruplex-unfolding properties of RPA, the mutation reduced the affinity of RPA2 with the ubiquitin ligase RFWD3 and reduced RPA ubiquitination. Using engineered knock-in cell lines, we found an accumulation of RPA at telomeres that did not trigger ATR activation but caused short and dysfunctional telomeres. Finally, both patients acquired, in a subset of blood cells, somatic genetic rescue events in either POT1 genes or TERT promoters known to counteract the accelerated telomere shortening. Collectively, our study indicates that variants in RPA2 represent a novel genetic cause of TBDs. Our results further support the fundamental role of the RPA complex in regulating telomere length and stability in humans.

Germline HLA landscape does not predict efficacy of pembrolizumab monotherapy across solid tumor types.

We evaluated the impact of class I and class II human leukocyte antigen (HLA) genotypes, heterozygosity, and diversity on the efficacy of pembrolizumab. Seventeen pembrolizumab clinical trials across eight tumor types and one basket trial in patients with advanced solid tumors were included (n > 3,500 analyzed). Germline DNA was genotyped using a custom genotyping array. HLA diversity (measured by heterozygosity and evolutionary divergence) across class I loci was not associated with improved response to pembrolizumab, either within each tumor type evaluated or across all patients. Similarly, HLA heterozygosity at each class I and class II gene was not associated with response to pembrolizumab after accounting for the number of tests conducted. No conclusive association between HLA genotype and response to pembrolizumab was identified in this dataset. Germline HLA genotype or diversity alone is not an important independent determinant of response to pembrolizumab and should not be used for clinical decision-making in patients treated with pembrolizumab.

A lethal mitonuclear incompatibility in complex I of natural hybrids.

The evolution of reproductive barriers is the first step in the formation of new species and can help us understand the diversification of life on Earth. These reproductive barriers often take the form of hybrid incompatibilities, in which alleles derived from two different species no longer interact properly in hybrids1-3. Theory predicts that hybrid incompatibilities may be more likely to arise at rapidly evolving genes4-6 and that incompatibilities involving multiple genes should be common7,8, but there has been sparse empirical data to evaluate these predictions. Here we describe a mitonuclear incompatibility involving three genes whose protein products are in physical contact within respiratory complex I of naturally hybridizing swordtail fish species. Individuals homozygous for mismatched protein combinations do not complete embryonic development or die as juveniles, whereas those heterozygous for the incompatibility have reduced complex I function and unbalanced representation of parental alleles in the mitochondrial proteome. We find that the effects of different genetic interactions on survival are non-additive, highlighting subtle complexity in the genetic architecture of hybrid incompatibilities. Finally, we document the evolutionary history of the genes involved, showing signals of accelerated evolution and evidence that an incompatibility has been transferred between species via hybridization.

A deep catalogue of protein-coding variation in 983,578 individuals.

Rare coding variants that substantially affect function provide insights into the biology of a gene1-3. However, ascertaining the frequency of such variants requires large sample sizes4-8. Here we present a catalogue of human protein-coding variation, derived from exome sequencing of 983,578 individuals across diverse populations. In total, 23% of the Regeneron Genetics Center Million Exome (RGC-ME) data come from individuals of African, East Asian, Indigenous American, Middle Eastern and South Asian ancestry. The catalogue includes more than 10.4 million missense and 1.1 million predicted loss-of-function (pLOF) variants. We identify individuals with rare biallelic pLOF variants in 4,848 genes, 1,751 of which have not been previously reported. From precise quantitative estimates of selection against heterozygous loss of function (LOF), we identify 3,988 LOF-intolerant genes, including 86 that were previously assessed as tolerant and 1,153 that lack established disease annotation. We also define regions of missense depletion at high resolution. Notably, 1,482 genes have regions that are depleted of missense variants despite being tolerant of pLOF variants. Finally, we estimate that 3% of individuals have a clinically actionable genetic variant, and that 11,773 variants reported in ClinVar with unknown significance are likely to be deleterious cryptic splice sites. To facilitate variant interpretation and genetics-informed precision medicine, we make this resource of coding variation from the RGC-ME dataset publicly accessible through a variant allele frequency browser.

De novo variants in the RNU4-2 snRNA cause a frequent neurodevelopmental syndrome.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 base pair region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals in whom it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologues. Using RNA sequencing, we show how 5' splice-site use is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 base pair region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide.

BACH2 immunodeficiency illustrates an association between super-enhancers and haploinsufficiency.

The transcriptional programs that guide lymphocyte differentiation depend on the precise expression and timing of transcription factors (TFs). The TF BACH2 is essential for T and B lymphocytes and is associated with an archetypal super-enhancer (SE). Single-nucleotide variants in the BACH2 locus are associated with several autoimmune diseases, but BACH2 mutations that cause Mendelian monogenic primary immunodeficiency have not previously been identified. Here we describe a syndrome of BACH2-related immunodeficiency and autoimmunity (BRIDA) that results from BACH2 haploinsufficiency. Affected subjects had lymphocyte-maturation defects that caused immunoglobulin deficiency and intestinal inflammation. The mutations disrupted protein stability by interfering with homodimerization or by causing aggregation. We observed analogous lymphocyte defects in Bach2-heterozygous mice. More generally, we observed that genes that cause monogenic haploinsufficient diseases were substantially enriched for TFs and SE architecture. These findings reveal a previously unrecognized feature of SE architecture in Mendelian diseases of immunity: heterozygous mutations in SE-regulated genes identified by whole-exome/genome sequencing may have greater significance than previously recognized.

Mono- and biallelic variant effects on disease at biobank scale.

Identifying causal factors for Mendelian and common diseases is an ongoing challenge in medical genetics1. Population bottleneck events, such as those that occurred in the history of the Finnish population, enrich some homozygous variants to higher frequencies, which facilitates the identification of variants that cause diseases with recessive inheritance2,3. Here we examine the homozygous and heterozygous effects of 44,370 coding variants on 2,444 disease phenotypes using data from the nationwide electronic health records of 176,899 Finnish individuals. We find associations for homozygous genotypes across a broad spectrum of phenotypes, including known associations with retinal dystrophy and novel associations with adult-onset cataract and female infertility. Of the recessive disease associations that we identify, 13 out of 20 would have been missed by the additive model that is typically used in genome-wide association studies. We use these results to find many known Mendelian variants whose inheritance cannot be adequately described by a conventional definition of dominant or recessive. In particular, we find variants that are known to cause diseases with recessive inheritance with significant heterozygous phenotypic effects. Similarly, we find presumed benign variants with disease effects. Our results show how biobanks, particularly in founder populations, can broaden our understanding of complex dosage effects of Mendelian variants on disease.

Autoantibodies against type I IFNs in humans with alternative NF-κB pathway deficiency.

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs)1,2, conferring a predisposition to life-threatening COVID-19 pneumonia3. Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52LOF/IκBδGOF). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52LOF/IκBδLOF) or gain-of-function of p52 (hereafter, p52GOF/IκBδLOF). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases.

NRF1 association with AUTS2-Polycomb mediates specific gene activation in the brain.

The heterogeneous family of complexes comprising Polycomb repressive complex 1 (PRC1) is instrumental for establishing facultative heterochromatin that is repressive to transcription. However, two PRC1 species, ncPRC1.3 and ncPRC1.5, are known to comprise novel components, AUTS2, P300, and CK2, that convert this repressive function to that of transcription activation. Here, we report that individuals harboring mutations in the HX repeat domain of AUTS2 exhibit defects in AUTS2 and P300 interaction as well as a developmental disorder reflective of Rubinstein-Taybi syndrome, which is mainly associated with a heterozygous pathogenic variant in CREBBP/EP300. Moreover, the absence of AUTS2 or mutation in its HX repeat domain gives rise to misregulation of a subset of developmental genes and curtails motor neuron differentiation of mouse embryonic stem cells. The transcription factor nuclear respiratory factor 1 (NRF1) has a novel and integral role in this neurodevelopmental process, being required for ncPRC1.3 recruitment to chromatin.

Reduced reproductive success is associated with selective constraint on human genes.

Genome-wide sequencing of human populations has revealed substantial variation among genes in the intensity of purifying selection acting on damaging genetic variants1. Although genes under the strongest selective constraint are highly enriched for associations with Mendelian disorders, most of these genes are not associated with disease and therefore the nature of the selection acting on them is not known2. Here we show that genetic variants that damage these genes are associated with markedly reduced reproductive success, primarily owing to increased childlessness, with a stronger effect in males than in females. We present evidence that increased childlessness is probably mediated by genetically associated cognitive and behavioural traits, which may mean that male carriers are less likely to find reproductive partners. This reduction in reproductive success may account for 20% of purifying selection against heterozygous variants that ablate protein-coding genes. Although this genetic association may only account for a very minor fraction of the overall likelihood of being childless (less than 1%), especially when compared to more influential sociodemographic factors, it may influence how genes evolve over time.

APOE4 impairs myelination via cholesterol dysregulation in oligodendrocytes.

APOE4 is the strongest genetic risk factor for Alzheimer's disease1-3. However, the effects of APOE4 on the human brain are not fully understood, limiting opportunities to develop targeted therapeutics for individuals carrying APOE4 and other risk factors for Alzheimer's disease4-8. Here, to gain more comprehensive insights into the impact of APOE4 on the human brain, we performed single-cell transcriptomics profiling of post-mortem human brains from APOE4 carriers compared with non-carriers. This revealed that APOE4 is associated with widespread gene expression changes across all cell types of the human brain. Consistent with the biological function of APOE2-6, APOE4 significantly altered signalling pathways associated with cholesterol homeostasis and transport. Confirming these findings with histological and lipidomic analysis of the post-mortem human brain, induced pluripotent stem-cell-derived cells and targeted-replacement mice, we show that cholesterol is aberrantly deposited in oligodendrocytes-myelinating cells that are responsible for insulating and promoting the electrical activity of neurons. We show that altered cholesterol localization in the APOE4 brain coincides with reduced myelination. Pharmacologically facilitating cholesterol transport increases axonal myelination and improves learning and memory in APOE4 mice. We provide a single-cell atlas describing the transcriptional effects of APOE4 on the aging human brain and establish a functional link between APOE4, cholesterol, myelination and memory, offering therapeutic opportunities for Alzheimer's disease.

Dominance and multi-locus interaction.

Dominance is usually considered a constant value that describes the relative difference in fitness or phenotype between heterozygotes and the average of homozygotes at a focal polymorphic locus. However, the observed dominance can vary with the genetic background of the focal locus. Here, alleles at other loci modify the observed phenotype through position effects or dominance modifiers that are sometimes associated with pathogen resistance, lineage, sex, or mating type. Theoretical models have illustrated how variable dominance appears in the context of multi-locus interaction (epistasis). Here, we review empirical evidence for variable dominance and how the observed patterns may be captured by proposed epistatic models. We highlight how integrating epistasis and dominance is crucial for comprehensively understanding adaptation and speciation.