Author Correction: Exome sequencing of Finnish isolates enhances rare-variant association power.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Exome sequencing of Finnish isolates enhances rare-variant association power.

Exome-sequencing studies have generally been underpowered to identify deleterious alleles with a large effect on complex traits as such alleles are mostly rare. Because the population of northern and eastern Finland has expanded considerably and in isolation following a series of bottlenecks, individuals of these populations have numerous deleterious alleles at a relatively high frequency. Here, using exome sequencing of nearly 20,000 individuals from these regions, we investigate the role of rare coding variants in clinically relevant quantitative cardiometabolic traits. Exome-wide association studies for 64 quantitative traits identified 26 newly associated deleterious alleles. Of these 26 alleles, 19 are either unique to or more than 20 times more frequent in Finnish individuals than in other Europeans and show geographical clustering comparable to Mendelian disease mutations that are characteristic of the Finnish population. We estimate that sequencing studies of populations without this unique history would require hundreds of thousands to millions of participants to achieve comparable association power.

Applicability of Diagnostic Criteria and High Prevalence of Familial Dysbetalipoproteinemia in Russia: A Pilot Study.

Familial dysbetalipoproteinemia (FD) is a highly atherogenic genetically based lipid disorder with an underestimated actual prevalence. In recent years, several biochemical algorithms have been developed to diagnose FD using available laboratory tests. The practical applicability of FD diagnostic criteria and the prevalence of FD in Russia have not been previously assessed. We demonstrated that the diagnostic algorithms of FD, including the diagnostic apoB levels, require correction, taking into account the distribution of apoB levels in the population. At the same time, a triglycerides cutoff ≥ 1.5 mmol/L may be a useful tool in identifying subjects with FD. In this study, a high prevalence of FD was detected: 0.67% (one in 150) based on the ε2ε2 haplotype and triglycerides levels ≥ 1.5 mmol/L. We also analyzed the presence and pathogenicity of APOE variants associated with autosomal dominant FD in a large research sample.

MYB bi-allelic targeting abrogates primitive clonogenic progenitors while the emergence of primitive blood cells is not affected.

MYB is a key regulator of definitive hematopoiesis and it is dispensable for the development of primitive hematopoietic cells in vertebrates. To delineate definitive versus primitive hematopoiesis during differentiation of human embryonic stem cells, we have introduced reporters into the MYB locus and inactivated the gene by bi-allelic targeting. To recapitulate the early developmental events more adequately, the mutant and wild type human embryonic stem cell lines were differentiated in defined culture conditions without the addition of hematopoietic cytokines. The differentiation of the reporter cell lines demonstrated that MYB is specifically expressed throughout emerging hematopoietic cell populations. Here we show that the disruption of the MYB gene leads to severe defects in the development and proliferation of primitive hematopoietic progenitors while the emergence of primitive blood cells is not affected. We also provide evidence that MYB is essential for neutrophil and T cell development and the upregulation of innate immunity genes during hematopoietic differentiation. Our results suggest that the endothelial origin of primitive blood cells is direct and does not include the intermediate step of primitive hematopoietic progenitors.

CpG traffic lights are markers of regulatory regions in human genome.

BACKGROUND: DNA methylation is involved in the regulation of gene expression. Although bisulfite-sequencing based methods profile DNA methylation at a single CpG resolution, methylation levels are usually averaged over genomic regions in the downstream bioinformatic analysis. RESULTS: We demonstrate that on the genome level a single CpG methylation can serve as a more accurate predictor of gene expression than an average promoter / gene body methylation. We define CpG traffic lights (CpG TL) as CpG dinucleotides with a significant correlation between methylation and expression of a gene nearby. CpG TL are enriched in all regulatory regions. Among all promoters, CpG TL are especially enriched in poised ones, suggesting involvement of DNA methylation in their regulation. Yet, binding of only a handful of transcription factors, such as NRF1, ETS, STAT and IRF-family members, could be regulated by direct methylation of transcription factor binding sites (TFBS) or its close proximity. For the majority of TF, an alternative scenario is more likely: methylation and inactivation of the whole regulatory element indirectly represses functional TF binding with a CpG TL being a reliable marker of such inactivation. CONCLUSIONS: CpG TL provide a promising insight into mechanisms of enhancer activity and gene regulation linking methylation of single CpG to gene expression. CpG TL methylation can be used as reliable markers of enhancer activity and gene expression in applications, e.g. in clinic where measuring DNA methylation is easier compared to directly measuring gene expression due to more stable nature of DNA.

The genome of the vervet (Chlorocebus aethiops sabaeus).

We describe a genome reference of the African green monkey or vervet (Chlorocebus aethiops). This member of the Old World monkey (OWM) superfamily is uniquely valuable for genetic investigations of simian immunodeficiency virus (SIV), for which it is the most abundant natural host species, and of a wide range of health-related phenotypes assessed in Caribbean vervets (C. a. sabaeus), whose numbers have expanded dramatically since Europeans introduced small numbers of their ancestors from West Africa during the colonial era. We use the reference to characterize the genomic relationship between vervets and other primates, the intra-generic phylogeny of vervet subspecies, and genome-wide structural variations of a pedigreed C. a. sabaeus population. Through comparative analyses with human and rhesus macaque, we characterize at high resolution the unique chromosomal fission events that differentiate the vervets and their close relatives from most other catarrhine primates, in whom karyotype is highly conserved. We also provide a summary of transposable elements and contrast these with the rhesus macaque and human. Analysis of sequenced genomes representing each of the main vervet subspecies supports previously hypothesized relationships between these populations, which range across most of sub-Saharan Africa, while uncovering high levels of genetic diversity within each. Sequence-based analyses of major histocompatibility complex (MHC) polymorphisms reveal extremely low diversity in Caribbean C. a. sabaeus vervets, compared to vervets from putatively ancestral West African regions. In the C. a. sabaeus research population, we discover the first structural variations that are, in some cases, predicted to have a deleterious effect; future studies will determine the phenotypic impact of these variations.

StructMAn: annotation of single-nucleotide polymorphisms in the structural context.

The next generation sequencing technologies produce unprecedented amounts of data on the genetic sequence of individual organisms. These sequences carry a substantial amount of variation that may or may be not related to a phenotype. Phenotypically important part of this variation often comes in form of protein-sequence altering (non-synonymous) single nucleotide variants (nsSNVs). Here we present StructMAn, a Web-based tool for annotation of human and non-human nsSNVs in the structural context. StructMAn analyzes the spatial location of the amino acid residue corresponding to nsSNVs in the three-dimensional (3D) protein structure relative to other proteins, nucleic acids and low molecular-weight ligands. We make use of all experimentally available 3D structures of query proteins, and also, unlike other tools in the field, of structures of proteins with detectable sequence identity to them. This allows us to provide a structural context for around 20% of all nsSNVs in a typical human sequencing sample, for up to 60% of nsSNVs in genes related to human diseases and for around 35% of nsSNVs in a typical bacterial sample. Each nsSNV can be visualized and inspected by the user in the corresponding 3D structure of a protein or protein complex. The StructMAn server is available at http://structman.mpi-inf.mpg.de.

Re-sequencing expands our understanding of the phenotypic impact of variants at GWAS loci.

Genome-wide association studies (GWAS) have identified >500 common variants associated with quantitative metabolic traits, but in aggregate such variants explain at most 20-30% of the heritable component of population variation in these traits. To further investigate the impact of genotypic variation on metabolic traits, we conducted re-sequencing studies in >6,000 members of a Finnish population cohort (The Northern Finland Birth Cohort of 1966 [NFBC]) and a type 2 diabetes case-control sample (The Finland-United States Investigation of NIDDM Genetics [FUSION] study). By sequencing the coding sequence and 5' and 3' untranslated regions of 78 genes at 17 GWAS loci associated with one or more of six metabolic traits (serum levels of fasting HDL-C, LDL-C, total cholesterol, triglycerides, plasma glucose, and insulin), and conducting both single-variant and gene-level association tests, we obtained a more complete understanding of phenotype-genotype associations at eight of these loci. At all eight of these loci, the identification of new associations provides significant evidence for multiple genetic signals to one or more phenotypes, and at two loci, in the genes ABCA1 and CETP, we found significant gene-level evidence of association to non-synonymous variants with MAF<1%. Additionally, two potentially deleterious variants that demonstrated significant associations (rs138726309, a missense variant in G6PC2, and rs28933094, a missense variant in LIPC) were considerably more common in these Finnish samples than in European reference populations, supporting our prior hypothesis that deleterious variants could attain high frequencies in this isolated population, likely due to the effects of population bottlenecks. Our results highlight the value of large, well-phenotyped samples for rare-variant association analysis, and the challenge of evaluating the phenotypic impact of such variants.

Sequencing strategies and characterization of 721 vervet monkey genomes for future genetic analyses of medically relevant traits.

BACKGROUND: We report here the first genome-wide high-resolution polymorphism resource for non-human primate (NHP) association and linkage studies, constructed for the Caribbean-origin vervet monkey, or African green monkey (Chlorocebus aethiops sabaeus), one of the most widely used NHPs in biomedical research. We generated this resource by whole genome sequencing (WGS) of monkeys from the Vervet Research Colony (VRC), an NIH-supported research resource for which extensive phenotypic data are available. RESULTS: We identified genome-wide single nucleotide polymorphisms (SNPs) by WGS of 721 members of an extended pedigree from the VRC. From high-depth WGS data we identified more than 4 million polymorphic unequivocal segregating sites; by pruning these SNPs based on heterozygosity, quality control filters, and the degree of linkage disequilibrium (LD) between SNPs, we constructed genome-wide panels suitable for genetic association (about 500,000 SNPs) and linkage analysis (about 150,000 SNPs). To further enhance the utility of these resources for linkage analysis, we used a further pruned subset of the linkage panel to generate multipoint identity by descent matrices. CONCLUSIONS: The genetic and phenotypic resources now available for the VRC and other Caribbean-origin vervets enable their use for genetic investigation of traits relevant to human diseases.

Genetic landscape in Russian patients with familial left ventricular noncompaction.

Background: Left ventricular noncompaction (LVNC) cardiomyopathy is a disorder that can be complicated by heart failure, arrhythmias, thromboembolism, and sudden cardiac death. The aim of this study is to clarify the genetic landscape of LVNC in a large cohort of well-phenotyped Russian patients with LVNC, including 48 families (n=214). Methods: All index patients underwent clinical examination and genetic analysis, as well as family members who agreed to participate in the clinical study and/or in the genetic testing. The genetic testing included next generation sequencing and genetic classification according to ACMG guidelines. Results: A total of 55 alleles of 54 pathogenic and likely pathogenic variants in 24 genes were identified, with the largest number in the MYH7 and TTN genes. A significant proportion of variants -8 of 54 (14.8%) -have not been described earlier in other populations and may be specific to LVNC patients in Russia. In LVNC patients, the presence of each subsequent variant is associated with increased odds of having more severe LVNC subtypes than isolated LVNC with preserved ejection fraction. The corresponding odds ratio is 2.77 (1.37 -7.37; p <0.001) per variant after adjustment for sex, age, and family. Conclusion: Overall, the genetic analysis of LVNC patients, accompanied by cardiomyopathy-related family history analysis, resulted in a high diagnostic yield of 89.6%. These results suggest that genetic screening should be applied to the diagnosis and prognosis of LVNC patients.

Intragenic compensation through the lens of deep mutational scanning.

A significant fraction of mutations in proteins are deleterious and result in adverse consequences for protein function, stability, or interaction with other molecules. Intragenic compensation is a specific case of positive epistasis when a neutral missense mutation cancels effect of a deleterious mutation in the same protein. Permissive compensatory mutations facilitate protein evolution, since without them all sequences would be extremely conserved. Understanding compensatory mechanisms is an important scientific challenge at the intersection of protein biophysics and evolution. In human genetics, intragenic compensatory interactions are important since they may result in variable penetrance of pathogenic mutations or fixation of pathogenic human alleles in orthologous proteins from related species. The latter phenomenon complicates computational and clinical inference of an allele's pathogenicity. Deep mutational scanning is a relatively new technique that enables experimental studies of functional effects of thousands of mutations in proteins. We review the important aspects of the field and discuss existing limitations of current datasets. We reviewed ten published DMS datasets with quantified functional effects of single and double mutations and described rates and patterns of intragenic compensation in eight of them. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-022-01005-w.

d-StructMAn: Containerized structural annotation on the scale from genetic variants to whole proteomes.

BACKGROUND: Structural annotation of genetic variants in the context of intermolecular interactions and protein stability can shed light onto mechanisms of disease-related phenotypes. Three-dimensional structures of related proteins in complexes with other proteins, nucleic acids, or ligands enrich such functional interpretation, since intermolecular interactions are well conserved in evolution. RESULTS: We present d-StructMAn, a novel computational method that enables structural annotation of local genetic variants, such as single-nucleotide variants and in-frame indels, and implements it in a highly efficient and user-friendly tool provided as a Docker container. Using d-StructMAn, we annotated several very large sets of human genetic variants, including all variants from ClinVar and all amino acid positions in the human proteome. We were able to provide annotation for more than 46% of positions in the human proteome representing over 60% proteins. CONCLUSIONS: d-StructMAn is the first of its kind and a highly efficient tool for structural annotation of protein-coding genetic variation in the context of observed and potential intermolecular interactions. d-StructMAn is readily applicable to proteome-scale datasets and can be an instrumental building machine-learning tool for predicting genotype-to-phenotype relationships.

Phenotypic vs. genetic cascade screening for familial hypercholesterolemia: A case report.

One of the most common autosomal dominant disorders is familial hypercholesterolemia (FH), causing premature atherosclerotic cardiovascular diseases and a high risk of death due to lifelong exposure to elevated low-density lipoprotein cholesterol (LDL-C) levels. FH has a proven arsenal of treatments and the opportunity for genetic diagnosis. Despite this, FH remains largely underdiagnosed worldwide. Cascade screening is a cost-effective method for the identification of new patients with FH and the prevention of cardiovascular diseases. It is usually based only on clinical data. We describe a 48-year-old index patient with a very high LDL-C level without controlled guidelines-based medication, premature atherosclerosis, and a rare variant in the low-density lipoprotein receptor (LDLR) gene. Phenotypic cascade screening identified three additional FH relatives, namely the proband's daughter, and two young grandsons. The genetic screening made it possible to rule out FH in the proband's younger grandson. This clinical case demonstrates that genetic cascade screening is the most effective way of identifying new FH cases. We also first described in detail the phenotype of patients with a likely pathogenic variant LDLR-p.K223_D227dup.

Identification of Pathogenic Variant Burden and Selection of Optimal Diagnostic Method Is a Way to Improve Carrier Screening for Autosomal Recessive Diseases.

Cystic fibrosis, phenylketonuria, alpha-1 antitrypsin deficiency, and sensorineural hearing loss are among the most common autosomal recessive diseases, which require carrier screening. The evaluation of population allele frequencies (AF) of pathogenic variants in genes associated with these conditions and the choice of the best genotyping method are the necessary steps toward development and practical implementation of carrier-screening programs. We performed custom panel genotyping of 3821 unrelated participants from two Russian population representative samples and three patient groups using real-time polymerase chain reaction (PCR) and next generation sequencing (NGS). The custom panel included 115 known pathogenic variants in the CFTR, PAH, SERPINA1, and GJB2 genes. Overall, 38 variants were detected. The comparison of genotyping platforms revealed the following advantages of real-time PCR: relatively low cost, simple genotyping data analysis, and easier detection of large indels, while NGS showed better accuracy of variants identification and capability for detection of additional pathogenic variants in adjacent regions. A total of 23 variants had significant differences in estimated AF comparing with non-Finnish Europeans from gnomAD. This study provides new AF data for variants associated with the studied disorders and the comparison of genotyping methods for carrier screening.

A Case of Severe Left-Ventricular Noncompaction Associated with Splicing Altering Variant in the FHOD3 Gene.

Left ventricular noncompaction (LVNC) is a highly heterogeneous primary disorder of the myocardium. Its clinical features and genetic spectrum strongly overlap with other types of primary cardiomyopathies, in particular, hypertrophic cardiomyopathy. Study and the accumulation of genotype-phenotype correlations are the way to improve the precision of our diagnostics. We present a familial case of LVNC with arrhythmic and thrombotic complications, myocardial fibrosis and heart failure, cosegregating with the splicing variant in the FHOD3 gene. This is the first description of FHOD3-dependent LVNC to our knowledge. We also revise the assumed mechanism of pathogenesis in the case of FHOD3 splicing alterations.

Positive selection in alternatively spliced exons of human genes.

Alternative splicing is a well-recognized mechanism of accelerated genome evolution. We have studied single-nucleotide polymorphisms and human-chimpanzee divergence in the exons of 6672 alternatively spliced human genes, with the aim of understanding the forces driving the evolution of alternatively spliced sequences. Here, we show that alternatively spliced exons and exon fragments (alternative exons) from minor isoforms experience lower selective pressure at the amino acid level, accompanied by selection against synonymous sequence variation. The results of the McDonald-Kreitman test suggest that alternatively spliced exons, unlike exons constitutively included in the mRNA, are also subject to positive selection, with up to 27% of amino acids fixed by positive selection.

Targeted Sequencing of 242 Clinically Important Genes in the Russian Population From the Ivanovo Region.

We performed a targeted sequencing of 242 clinically important genes mostly associated with cardiovascular diseases in a representative population sample of 1,658 individuals from the Ivanovo region northeast of Moscow. Approximately 11% of 11,876 detected variants were not found in the Single Nucleotide Polymorphism Database (dbSNP) or reported earlier in the Russian population. Most novel variants were singletons and doubletons in our sample, and virtually no novel alleles presumably specific for the Russian population were able to reach the frequencies above 0.1-0.2%. The overwhelming majority (99.3%) of variants detected in this study in three or more copies were shared with other populations. We found two dominant and seven recessive known pathogenic variants with allele frequencies significantly increased compared to those in the gnomAD non-Finnish Europeans. Of the 242 targeted genes, 28 were in the list of 59 genes for which the American College of Medical Genetics and Genomics (ACMG) recommended the reporting of incidental findings. Based on the number of variants detected in the sequenced subset of ACMG59 genes, we approximated the prevalence of known pathogenic and novel or rare protein-truncating variants in the complete set of ACMG59 genes in the Ivanovo population at 1.4 and 2.8%, respectively. We analyzed the available clinical data and observed the incomplete penetrance of known pathogenic variants in the 28 ACMG59 genes: only 1 individual out of 12 with such variants had the phenotype most likely related to the variant. When known pathogenic and novel or rare protein-truncating variants were considered together, the overall rate of confirmed phenotypes was about 19%, with maximum in the subset of novel protein-truncating variants. We report three novel protein truncating variants in APOB and one in MYH7 observed in individuals with hypobetalipoproteinemia and hypertrophic cardiomyopathy, respectively. Our results provide a valuable reference for the clinical interpretation of gene sequencing in Russian and other populations.

The LDLR, APOB, and PCSK9 Variants of Index Patients with Familial Hypercholesterolemia in Russia.

Familial hypercholesterolemia (FH) is a common autosomal codominant disorder, characterized by elevated low-density lipoprotein cholesterol levels causing premature atherosclerotic cardiovascular disease. About 2900 variants of LDLR, APOB, and PCSK9 genes potentially associated with FH have been described earlier. Nevertheless, the genetics of FH in a Russian population is poorly understood. The aim of this study is to present data on the spectrum of LDLR, APOB, and PCSK9 gene variants in a cohort of 595 index Russian patients with FH, as well as an additional systematic analysis of the literature for the period of 1995-2020 on LDLR, APOB and PCSK9 gene variants described in Russian patients with FH. We used targeted and whole genome sequencing to search for variants. Accordingly, when combining our novel data and the data of a systematic literature review, we described 224 variants: 187 variants in LDLR, 14 variants in APOB, and 23 variants in PCSK9. A significant proportion of variants, 81 of 224 (36.1%), were not described earlier in FH patients in other populations and may be specific for Russia. Thus, this study significantly supplements knowledge about the spectrum of variants causing FH in Russia and may contribute to a wider implementation of genetic diagnostics in FH patients in Russia.

ACE2 and TMPRSS2 variation in savanna monkeys (Chlorocebus spp.): Potential risk for zoonotic/anthroponotic transmission of SARS-CoV-2 and a potential model for functional studies.

The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, has devastated health infrastructure around the world. Both ACE2 (an entry receptor) and TMPRSS2 (used by the virus for spike protein priming) are key proteins to SARS-CoV-2 cell entry, enabling progression to COVID-19 in humans. Comparative genomic research into critical ACE2 binding sites, associated with the spike receptor binding domain, has suggested that African and Asian primates may also be susceptible to disease from SARS-CoV-2 infection. Savanna monkeys (Chlorocebus spp.) are a widespread non-human primate with well-established potential as a bi-directional zoonotic/anthroponotic agent due to high levels of human interaction throughout their range in sub-Saharan Africa and the Caribbean. To characterize potential functional variation in savanna monkey ACE2 and TMPRSS2, we inspected recently published genomic data from 245 savanna monkeys, including 163 wild monkeys from Africa and the Caribbean and 82 captive monkeys from the Vervet Research Colony (VRC). We found several missense variants. One missense variant in ACE2 (X:14,077,550; Asp30Gly), common in Ch. sabaeus, causes a change in amino acid residue that has been inferred to reduce binding efficiency of SARS-CoV-2, suggesting potentially reduced susceptibility. The remaining populations appear as susceptible as humans, based on these criteria for receptor usage. All missense variants observed in wild Ch. sabaeus populations are also present in the VRC, along with two splice acceptor variants (at X:14,065,076) not observed in the wild sample that are potentially disruptive to ACE2 function. The presence of these variants in the VRC suggests a promising model for SARS-CoV-2 infection and vaccine and therapy development. In keeping with a One Health approach, characterizing actual susceptibility and potential for bi-directional zoonotic/anthroponotic transfer in savanna monkey populations may be an important consideration for controlling COVID-19 epidemics in communities with frequent human/non-human primate interactions that, in many cases, may have limited health infrastructure.

Contribution of common and rare variants to bipolar disorder susceptibility in extended pedigrees from population isolates.

Current evidence from case/control studies indicates that genetic risk for psychiatric disorders derives primarily from numerous common variants, each with a small phenotypic impact. The literature describing apparent segregation of bipolar disorder (BP) in numerous multigenerational pedigrees suggests that, in such families, large-effect inherited variants might play a greater role. To identify roles of rare and common variants on BP, we conducted genetic analyses in 26 Colombia and Costa Rica pedigrees ascertained for bipolar disorder 1 (BP1), the most severe and heritable form of BP. In these pedigrees, we performed microarray SNP genotyping of 838 individuals and high-coverage whole-genome sequencing of 449 individuals. We compared polygenic risk scores (PRS), estimated using the latest BP1 genome-wide association study (GWAS) summary statistics, between BP1 individuals and related controls. We also evaluated whether BP1 individuals had a higher burden of rare deleterious single-nucleotide variants (SNVs) and rare copy number variants (CNVs) in a set of genes related to BP1. We found that compared with unaffected relatives, BP1 individuals had higher PRS estimated from BP1 GWAS statistics (P = 0.001 ~ 0.007) and displayed modest increase in burdens of rare deleterious SNVs (P = 0.047) and rare CNVs (P = 0.002 ~ 0.033) in genes related to BP1. We did not observe rare variants segregating in the pedigrees. These results suggest that small-to-moderate effect rare and common variants are more likely to contribute to BP1 risk in these extended pedigrees than a few large-effect rare variants.