Cornelis A. Albers

A Systematic Survey of Loss-of-Function Variants in Human Protein-Coding Genes

Defective Gene Detective Identifying genes that give rise to diseases is one of the major goals of sequencing human genomes. However, putative loss-of-function genes, which are often some of the first identified targets of genome and exome sequencing, have often turned out to be sequencing errors rather than true genetic variants. In order to identify the true scope of loss-of-function genes within the human genome, MacArthur et al. (p. 823; see the Perspective by Quintana-Murci) extensively validated the genomes from the 1000 Genomes Project, as well as an additional European individual, and found that the average person has about 100 true loss-of-function alleles of which approximately 20 have two copies within an individual. Because many known disease-causing genes were identified in “normal” individuals, the process of clinical sequencing needs to reassess how to identify likely causative alleles. Validation of predicted nonfunctional alleles in the human genome affects the medical interpretation of genomic analyses. Genome-sequencing studies indicate that all humans carry many genetic variants predicted to cause loss of function (LoF) of protein-coding genes, suggesting unexpected redundancy in the human genome. Here we apply stringent filters to 2951 putative LoF variants obtained from 185 human genomes to determine their true prevalence and properties. We estimate that human genomes typically contain ~100 genuine LoF variants with ~20 genes completely inactivated. We identify rare and likely deleterious LoF alleles, including 26 known and 21 predicted severe disease–causing variants, as well as common LoF variants in nonessential genes. We describe functional and evolutionary differences between LoF-tolerant and recessive disease genes and a method for using these differences to prioritize candidate genes found in clinical sequencing studies.

Dindel: Accurate indel calls from short-read data

Small insertions and deletions (indels) are a common and functionally important type of sequence polymorphism. Most of the focus of studies of sequence variation is on single nucleotide variants (SNVs) and large structural variants. In principle, high-throughput sequencing studies should allow identification of indels just as SNVs. However, inference of indels from next-generation sequence data is challenging, and so far methods for identifying indels lag behind methods for calling SNVs in terms of sensitivity and specificity. We propose a Bayesian method to call indels from short-read sequence data in individuals and populations by realigning reads to candidate haplotypes that represent alternative sequence to the reference. The candidate haplotypes are formed by combining candidate indels and SNVs identified by the read mapper, while allowing for known sequence variants or candidates from other methods to be included. In our probabilistic realignment model we account for base-calling errors, mapping errors, and also, importantly, for increased sequencing error indel rates in long homopolymer runs. We show that our method is sensitive and achieves low false discovery rates on simulated and real data sets, although challenges remain. The algorithm is implemented in the program Dindel, which has been used in the 1000 Genomes Project call sets.

Compound inheritance of a low-frequency regulatory SNP and a rare null mutation in exon-junction complex subunit RBM8A causes TAR syndrome

The exon-junction complex (EJC) performs essential RNA processing tasks. Here, we describe the first human disorder, thrombocytopenia with absent radii (TAR), caused by deficiency in one of the four EJC subunits. Compound inheritance of a rare null allele and one of two low-frequency SNPs in the regulatory regions of RBM8A, encoding the Y14 subunit of EJC, causes TAR. We found that this inheritance mechanism explained 53 of 55 cases (P < 5 × 10−228) of the rare congenital malformation syndrome. Of the 53 cases with this inheritance pattern, 51 carried a submicroscopic deletion of 1q21.1 that has previously been associated with TAR, and two carried a truncation or frameshift null mutation in RBM8A. We show that the two regulatory SNPs result in diminished RBM8A transcription in vitro and that Y14 expression is reduced in platelets from individuals with TAR. Our data implicate Y14 insufficiency and, presumably, an EJC defect as the cause of TAR syndrome.

Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome

Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder that is characterized by mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated individuals and identified NBEAL2 as the causative gene; it has no previously known function but is a member of a gene family that is involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation.

The origin, evolution, and functional impact of short insertion–deletion variants identified in 179 human genomes

Short insertions and deletions (indels) are the second most abundant form of human genetic variation, but our understanding of their origins and functional effects lags behind that of other types of variants. Using population-scale sequencing, we have identified a high-quality set of 1.6 million indels from 179 individuals representing three diverse human populations. We show that rates of indel mutagenesis are highly heterogeneous, with 43%-48% of indels occurring in 4.03% of the genome, whereas in the remaining 96% their prevalence is 16 times lower than SNPs. Polymerase slippage can explain upwards of three-fourths of all indels, with the remainder being mostly simple deletions in complex sequence. However, insertions do occur and are significantly associated with pseudo-palindromic sequence features compatible with the fork stalling and template switching (FoSTeS) mechanism more commonly associated with large structural variations. We introduce a quantitative model of polymerase slippage, which enables us to identify indel-hypermutagenic protein-coding genes, some of which are associated with recurrent mutations leading to disease. Accounting for mutational rate heterogeneity due to sequence context, we find that indels across functional sequence are generally subject to stronger purifying selection than SNPs. We find that indel length modulates selection strength, and that indels affecting multiple functionally constrained nucleotides undergo stronger purifying selection. We further find that indels are enriched in associations with gene expression and find evidence for a contribution of nonsense-mediated decay. Finally, we show that indels can be integrated in existing genome-wide association studies (GWAS); although we do not find direct evidence that potentially causal protein-coding indels are enriched with associations to known disease-associated SNPs, our findings suggest that the causal variant underlying some of these associations may be indels.

Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells

Summary Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14+ monocytes, CD16+ neutrophils, and naive CD4+ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.

SMIM1 underlies the Vel blood group and influences red blood cell traits

The blood group Vel was discovered 60 years ago, but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel. To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 × 10−15). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors.

Dynamics of gene silencing during X inactivation using allele-specific RNA-seq

BackgroundDuring early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq.ResultsInduction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs.ConclusionsThe gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of escape regions with TADs, in mouse and human, suggests that TADs are the primary targets during propagation of XCI over the X chromosome.

Maps of open chromatin highlight cell type–restricted patterns of regulatory sequence variation at hematological trait loci

Nearly three-quarters of the 143 genetic signals associated with platelet and erythrocyte phenotypes identified by meta-analyses of genome-wide association (GWA) studies are located at non-protein-coding regions. Here, we assessed the role of candidate regulatory variants associated with cell type-restricted, closely related hematological quantitative traits in biologically relevant hematopoietic cell types. We used formaldehyde-assisted isolation of regulatory elements followed by next-generation sequencing (FAIRE-seq) to map regions of open chromatin in three primary human blood cells of the myeloid lineage. In the precursors of platelets and erythrocytes, as well as in monocytes, we found that open chromatin signatures reflect the corresponding hematopoietic lineages of the studied cell types and associate with the cell type-specific gene expression patterns. Dependent on their signal strength, open chromatin regions showed correlation with promoter and enhancer histone marks, distance to the transcription start site, and ontology classes of nearby genes. Cell type-restricted regions of open chromatin were enriched in sequence variants associated with hematological indices. The majority (63.6%) of such candidate functional variants at platelet quantitative trait loci (QTLs) coincided with binding sites of five transcription factors key in regulating megakaryopoiesis. We experimentally tested 13 candidate regulatory variants at 10 platelet QTLs and found that 10 (76.9%) affected protein binding, suggesting that this is a frequent mechanism by which regulatory variants influence quantitative trait levels. Our findings demonstrate that combining large-scale GWA data with open chromatin profiles of relevant cell types can be a powerful means of dissecting the genetic architecture of closely related quantitative traits.

New insights into the genetic basis of TAR (thrombocytopenia-absent radii) syndrome.

Thrombocytopenia with absent radii (TAR) syndrome is a rare disorder combining specific skeletal abnormalities with a reduced platelet count. Rare proximal microdeletions of 1q21.1 are found in the majority of patients but are also found in unaffected parents. Recently it was shown that TAR syndrome is caused by the compound inheritance of a low-frequency noncoding SNP and a rare null allele in RBM8A, a gene encoding the exon-junction complex subunit member Y14 located in the deleted region. This finding provides new insight into the complex inheritance pattern and new clues to the molecular mechanisms underlying TAR syndrome. We discuss TAR syndrome in the context of abnormal phenotypes associated with proximal and distal 1q21.1 microdeletion and microduplications with incomplete penetrance and variable expressivity.