Genome structural variation in human evolution.

Structural variation (SV) is a large difference (typically >100 bp) in the genomic structure of two genomes and includes both copy number variation and variation that does not change copy number of a genomic region, such as an inversion. Improved reference genomes, combined with widespread genome sequencing using short-read sequencing technology, and increasingly using long-read sequencing, have reignited interest in SV. Recent large-scale studies and functional focused analyses have highlighted the role of SV in human evolution. In this review, we highlight human-specific SVs involved in changes in the brain, population-specific SVs that affect response to the environment, including adaptation to diet and infectious diseases, and summarise the contribution of archaic hominin admixture to present-day human SV.

Structural variation of the malaria-associated human glycophorin A-B-E region.

BACKGROUND: Approximately 5% of the human genome shows common structural variation, which is enriched for genes involved in the immune response and cell-cell interactions. A well-established region of extensive structural variation is the glycophorin gene cluster, comprising three tandemly-repeated regions about 120 kb in length and carrying the highly homologous genes GYPA, GYPB and GYPE. Glycophorin A (encoded by GYPA) and glycophorin B (encoded by GYPB) are glycoproteins present at high levels on the surface of erythrocytes, and they have been suggested to act as decoy receptors for viral pathogens. They are receptors for the invasion of the protist parasite Plasmodium falciparum, a causative agent of malaria. A particular complex structural variant, called DUP4, creates a GYPB-GYPA fusion gene known to confer resistance to malaria. Many other structural variants exist across the glycophorin gene cluster, and they remain poorly characterised. RESULTS: Here, we analyse sequences from 3234 diploid genomes from across the world for structural variation at the glycophorin locus, confirming 15 variants in the 1000 Genomes project cohort, discovering 9 new variants, and characterising a selection of these variants using fibre-FISH and breakpoint mapping at the sequence level. We identify variants predicted to create novel fusion genes and a common inversion duplication variant at appreciable frequencies in West Africans. We show that almost all variants can be explained by non-allelic homologous recombination and by comparing the structural variant breakpoints with recombination hotspot maps, confirm the importance of a particular meiotic recombination hotspot on structural variant formation in this region. CONCLUSIONS: We identify and validate large structural variants in the human glycophorin A-B-E gene cluster which may be associated with different clinical aspects of malaria.

Comparative transcriptome profiling of virulent and non-virulent Trypanosoma cruzi underlines the role of surface proteins during infection.

Trypanosoma cruzi, the protozoan that causes Chagas disease, has a complex life cycle involving several morphologically and biochemically distinct stages that establish intricate interactions with various insect and mammalian hosts. It has also a heterogeneous population structure comprising strains with distinct properties such as virulence, sensitivity to drugs, antigenic profile and tissue tropism. We present a comparative transcriptome analysis of two cloned T. cruzi strains that display contrasting virulence phenotypes in animal models of infection: CL Brener is a virulent clone and CL-14 is a clone that is neither infective nor pathogenic in in vivo models of infection. Gene expression analysis of trypomastigotes and intracellular amastigotes harvested at 60 and 96 hours post-infection (hpi) of human fibroblasts revealed large differences that reflect the parasite's adaptation to distinct environments during the infection of mammalian cells, including changes in energy sources, oxidative stress responses, cell cycle control and cell surface components. While extensive transcriptome remodeling was observed when trypomastigotes of both strains were compared to 60 hpi amastigotes, differences in gene expression were much less pronounced when 96 hpi amastigotes and trypomastigotes of CL Brener were compared. In contrast, the differentiation of the avirulent CL-14 from 96 hpi amastigotes to extracellular trypomastigotes was associated with considerable changes in gene expression, particularly in gene families encoding surface proteins such as trans-sialidases, mucins and the mucin associated surface proteins (MASPs). Thus, our comparative transcriptome analysis indicates that the avirulent phenotype of CL-14 may be due, at least in part, to a reduced or delayed expression of genes encoding surface proteins that are associated with the transition of amastigotes to trypomastigotes, an essential step in the establishment of the infection in the mammalian host. Confirming the role of members of the trans-sialidase family of surface proteins for parasite differentiation, transfected CL-14 constitutively expressing a trans-sialidase gene displayed faster kinetics of trypomastigote release in the supernatant of infected cells compared to wild type CL-14.

Absence of heterozygosity due to template switching during replicative rearrangements.

We investigated complex genomic rearrangements (CGRs) consisting of triplication copy-number variants (CNVs) that were accompanied by extended regions of copy-number-neutral absence of heterozygosity (AOH) in subjects with multiple congenital abnormalities. Molecular analyses provided observational evidence that in humans, post-zygotically generated CGRs can lead to regional uniparental disomy (UPD) due to template switches between homologs versus sister chromatids by using microhomology to prime DNA replication-a prediction of the replicative repair model, MMBIR. Our findings suggest that replication-based mechanisms might underlie the formation of diverse types of genomic alterations (CGRs and AOH) implicated in constitutional disorders.

Understanding the Genomic Structure of Copy-Number Variation of the Low-Affinity Fcγ Receptor Region Allows Confirmation of the Association of FCGR3B Deletion with Rheumatoid Arthritis.

Fcγ receptors are a family of cell-surface receptors that are expressed by a host of different innate and adaptive immune cells, and mediate inflammatory responses by binding the Fc portion of immunoglobulin G. In humans, five low-affinity receptors are encoded by the genes FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B, which are located in an 82.5-kb segmental tandem duplication on chromosome 1q23.3, which shows extensive copy-number variation (CNV). Deletions of FCGR3B have been suggested to increase the risk of inflammatory diseases such as systemic lupus erythematosus and rheumatoid arthritis (RA). In this study, we identify the deletion breakpoints of FCGR3B deletion alleles in the UK population and endogamous native American population, and show that some but not all alleles are likely to be identical-by-descent. We also localize a duplication breakpoint, confirming that the mechanism of CNV generation is nonallelic homologous recombination, and identify several alleles with gene conversion events using fosmid sequencing data. We use information on the structure of the deletion alleles to distinguish FCGR3B deletions from FCGR3A deletions in whole-genome array comparative genomic hybridization (aCGH) data. Reanalysis of published aCGH data using this approach supports association of FCGR3B deletion with increased risk of RA in a large cohort of 1,982 cases and 3,271 controls (odds ratio 1.61, P = 2.9×10-3 ).

Chimeric transcripts resulting from complex duplications in chromosome Xq28.

Gene fusions have been observed in somatic alterations in cancer and in schizophrenia. However, the underlying mechanism(s) for their formation are poorly understood. We experimentally demonstrated the expression of splicing variants of in silico predicted chimeric genes F8/CSAG1 and BCAP31/TEX28 in two individuals with de novo complex genomic rearrangements of Xq28; F8/CSAG1 includes exonization of an ERVL-MaLR intronic repetitive element. We provide evidence that replicative repair may contribute to exon shuffling processes and diversify the repertoire of expressed transcripts.

Structural variation and missense mutation in SBDS associated with Shwachman-Diamond syndrome.

BACKGROUND: Shwachman-Diamond syndrome (SDS) is an autosomal recessive ribosomopathy caused mainly by compound heterozygous mutations in SBDS. Structural variation (SV) involving the SBDS locus has been rarely reported in association with the disease. We aimed to determine whether an SV contributed to the pathogenesis of a case lacking biallelic SBDS point mutations. CASE PRESENTATION: Whole exome sequencing was performed in a patient with SDS lacking biallelic SBDS point mutations. Array comparative genomic hybridization and Southern blotting were used to seek SVs across the SBDS locus. Locus-specific polymerase chain reaction (PCR) encompassing flanking intronic sequence was also performed to investigate mutation within the locus. RNA expression and Western blotting were performed to analyze allele and protein expression. We found the child harbored a single missense mutation in SBDS (c.98A > C; p.K33T), inherited from the mother, and an SV in the SBDS locus, inherited from the father. The missense allele and SV segregated in accordance with Mendelian expectations for autosomal recessive SDS. Complementary DNA and western blotting analysis and locus specific PCR support the contention that the SV perturbed SBDS protein expression in the father and child. CONCLUSION: Our findings implicate genomic rearrangements in the pathogenesis of some cases of SDS and support patients lacking biallelic SBDS point mutations be tested for SV within the SBDS locus.

Linking tumor immune infiltrate and systemic immune mediators to treatment response and prognosis in advanced cervical cancer.

Cervical cancer (CC) poses a significant burden on individuals in developing regions, exhibiting heterogeneous responses to standard chemoradiation therapy, and contributing to substantial mortality rates. Unraveling host immune dynamics holds promise for innovative therapies and discovery of clinically relevant biomarkers. We studied prospectively locally advanced CC patients pre-treatment, stratifying them as responders (R) or non-responders (NR). R patients had increased tumor-infiltrating lymphocytes (TILs), while NR patients showed elevated PD-1 scores, CD8+ and PD-L2+ TILs, and PD-L1 immune reactivity. NR patients exhibited higher systemic soluble mediators correlating with TIL immune markers. R patients demonstrated functional polarization of CD4 T cells (Th1, Th2, Th17, and Treg), while CD8+ T cells and CD68+ macrophages predominated in the NR group. Receiver operating characteristic analysis identified potential CC response predictors, including PD-L1-immunoreactive (IR) area, PD-L2, CD8, FGF-basic, IL-7, IL-8, IL-12p40, IL-15, and TNF-alpha. Dysfunctional TILs and imbalanced immune mediators contribute to therapeutic insufficiency, shedding light on local and systemic immune interplay. Our study informs immunological signatures for treatment prediction and CC prognosis.

A worldwide analysis of beta-defensin copy number variation suggests recent selection of a high-expressing DEFB103 gene copy in East Asia.

Beta-defensins are a family of multifunctional genes with roles in defense against pathogens, reproduction, and pigmentation. In humans, six beta-defensin genes are clustered in a repeated region which is copy-number variable (CNV) as a block, with a diploid copy number between 1 and 12. The role in host defense makes the evolutionary history of this CNV particularly interesting, because morbidity due to infectious disease is likely to have been an important selective force in human evolution, and to have varied between geographical locations. Here, we show CNV of the beta-defensin region in chimpanzees, and identify a beta-defensin block in the human lineage that contains rapidly evolving noncoding regulatory sequences. We also show that variation at one of these rapidly evolving sequences affects expression levels and cytokine responsiveness of DEFB103, a key inhibitor of influenza virus fusion at the cell surface. A worldwide analysis of beta-defensin CNV in 67 populations shows an unusually high frequency of high-DEFB103-expressing copies in East Asia, the geographical origin of historical and modern influenza epidemics, possibly as a result of selection for increased resistance to influenza in this region.

Cervical Cancer Stem-Like Cell Transcriptome Profiles Predict Response to Chemoradiotherapy.

Cervical cancer (CC) represents a major global health issue, particularly impacting women from resource constrained regions worldwide. Treatment refractoriness to standard chemoradiotheraphy has identified cancer stem cells as critical coordinators behind the biological mechanisms of resistance, contributing to CC recurrence. In this work, we evaluated differential gene expression in cervical cancer stem-like cells (CCSC) as biomarkers related to intrinsic chemoradioresistance in CC. A total of 31 patients with locally advanced CC and referred to Mário Penna Institute (Belo Horizonte, Brazil) from August 2017 to May 2018 were recruited for the study. Fluorescence-activated cell sorting was used to enrich CD34+/CD45- CCSC from tumor biopsies. Transcriptome was performed using ultra-low input RNA sequencing and differentially expressed genes (DEGs) using Log2 fold differences and adjusted p-value < 0.05 were determined. The analysis returned 1050 DEGs when comparing the Non-Responder (NR) (n=10) and Responder (R) (n=21) groups to chemoradiotherapy. These included a wide-ranging pattern of underexpressed coding genes in the NR vs. R patients and a panel of lncRNAs and miRNAs with implications for CC tumorigenesis. A panel of biomarkers was selected using the rank-based AUC (Area Under the ROC Curve) and pAUC (partial AUC) measurements for diagnostic sensitivity and specificity. Genes overlapping between the 21 highest AUC and pAUC loci revealed seven genes with a strong capacity for identifying NR vs. R patients (ILF2, RBM22P2, ACO16722.1, AL360175.1 and AC092354.1), of which four also returned significant survival Hazard Ratios. This study identifies DEG signatures that provide potential biomarkers in CC prognosis and treatment outcome, as well as identifies potential alternative targets for cancer therapy.

Population genetics of immune-related multilocus copy number variation in Native Americans.

While multiallelic copy number variation (mCNV) loci are a major component of genomic variation, quantifying the individual copy number of a locus and defining genotypes is challenging. Few methods exist to study how mCNV genetic diversity is apportioned within and between populations (i.e. to define the population genetic structure of mCNV). These inferences are critical in populations with a small effective size, such as Amerindians, that may not fit the Hardy-Weinberg model due to inbreeding, assortative mating, population subdivision, natural selection or a combination of these evolutionary factors. We propose a likelihood-based method that simultaneously infers mCNV allele frequencies and the population structure parameter f, which quantifies the departure of homozygosity from the Hardy-Weinberg expectation. This method is implemented in the freely available software CNVice, which also infers individual genotypes using information from both the population and from trios, if available. We studied the population genetics of five immune-related mCNV loci associated with complex diseases (beta-defensins, CCL3L1/CCL4L1, FCGR3A, FCGR3B and FCGR2C) in 12 traditional Native American populations and found that the population structure parameters inferred for these mCNVs are comparable to but lower than those for single nucleotide polymorphisms studied in the same populations.

Characterization of molecular and cellular phenotypes associated with a heterozygous CNTNAP2 deletion using patient-derived hiPSC neural cells.

Neurodevelopmental disorders, such as autism spectrum disorders (ASD) and schizophrenia (SZ), are complex disorders with a high degree of heritability. Genetic studies have identified several candidate genes associated with these disorders, including contactin-associated protein-like 2 (CNTNAP2). Traditionally, in animal models or in vitro, the function of CNTNAP2 has been studied by genetic deletion or transcriptional knockdown, which reduce the expression of the entire gene; however, it remains unclear whether the mutations identified in clinical settings are sufficient to alter CNTNAP2 expression in human neurons. Here, using human induced pluripotent stem cells (hiPSCs) derived from two individuals with a large (289kb) and heterozygous deletion in CNTNAP2 (affecting exons 14-15) and discordant clinical outcomes, we have characterized CNTNAP2 expression patterns in hiPSC neural progenitor cells (NPCs), two independent populations of hiPSC-derived neurons and hiPSC-derived oligodendrocyte precursor cells (OPCs). First, we observed exon-specific changes in CNTNAP2 expression in both carriers; although the expression of exons 14-15 is significantly decreased, the expression of other exons is upregulated. Second, we observed significant differences in patterns of allele-specific expression in CNTNAP2 carriers that were consistent with clinical outcome. Third, we observed a robust neural migration phenotype that correlated with diagnosis and exon- and allele-specific CNTNAP2 expression patterns, but not with genotype. In all, our data highlight the importance of considering the nature, location and regulation of mutated alleles when attempting to connect GWAS studies to gene function.

Identification of secreted virulence factors of Chromobacterium violaceum.

Chromobacterium violaceum, a component of tropical soil microbiota, is an opportunistic pathogenic bacterium that can infect humans and other animals. In addition to identifying a large number of genes that demonstrate the vast biotechnological potential of this bacterium, genome sequencing revealed several virulence factors, including different cytolysins, which can be related to its pathogenicity. Here we confirmed these predictions from genomic analyses by identifying, through mass spectrometry, proteins present in the culture supernatant of C. violaceum that may constitute secreted virulence factors. Among them, we identified a secreted collagenase and the product of a gene with sequence similarity to previously characterized bacterial porins.

Population genetics of GYPB and association study between GYPB*S/s polymorphism and susceptibility to P. falciparum infection in the Brazilian Amazon.

BACKGROUND: Merozoites of Plasmodium falciparum invade through several pathways using different RBC receptors. Field isolates appear to use a greater variability of these receptors than laboratory isolates. Brazilian field isolates were shown to mostly utilize glycophorin A-independent invasion pathways via glycophorin B (GPB) and/or other receptors. The Brazilian population exhibits extensive polymorphism in blood group antigens, however, no studies have been done to relate the prevalence of the antigens that function as receptors for P. falciparum and the ability of the parasite to invade. Our study aimed to establish whether variation in the GYPB*S/s alleles influences susceptibility to infection with P. falciparum in the admixed population of Brazil. METHODS: Two groups of Brazilian Amazonians from Porto Velho were studied: P. falciparum infected individuals (cases); and uninfected individuals who were born and/or have lived in the same endemic region for over ten years, were exposed to infection but have not had malaria over the study period (controls). The GPB Ss phenotype and GYPB*S/s alleles were determined by standard methods. Sixty two Ancestry Informative Markers were genotyped on each individual to estimate admixture and control its potential effect on the association between frequency of GYPB*S and malaria infection. RESULTS: GYPB*S is associated with host susceptibility to infection with P. falciparum; GYPB*S/GYPB*S and GYPB*S/GYPB*s were significantly more prevalent in the in the P. falciparum infected individuals than in the controls (69.87% vs. 49.75%; P<0.02). Moreover, population genetics tests applied on the GYPB exon sequencing data suggest that natural selection shaped the observed pattern of nucleotide diversity. CONCLUSION: Epidemiological and evolutionary approaches suggest an important role for the GPB receptor in RBC invasion by P. falciparum in Brazilian Amazons. Moreover, an increased susceptibility to infection by this parasite is associated with the GPB S+ variant in this population.

No association found between gr/gr deletions and infertility in Brazilian males.

The Y chromosome carries several spermatogenesis genes distributed in three regions: AZFa, AZFb and AZFc. Microdeletions in these regions have been seen in 10% of sterile males with azoospermia or oligozoospermia, the most frequent of them being characterized by a complete deletion of AZFc region. A partial AZFc deletion named gr/gr has been singled out as a risk factor for spermatogenic failure. However, other authors have diagnosed it as a polymorphic deletion with no clinical relevance. We decided to investigate the association of gr/gr deletion and infertility in Brazilian males. We analysed 350 individuals (110 azoospermic, 122 fertile and 118 presumably fertile) and observed 12 g/gr deletions: five in infertile men (4.5%), three among fertile males (2.5%) and four in probably fertile individuals (3.4%). These differences were not statistically significant. Then, we decided to ascertain whether the clinical impact of the gr/gr deletion was associated with the type of Y chromosome. We have identified Y-chromosome haplogroups using 22 unique event polymorphisms (UEPs). Among the individuals with the gr/gr deletion, we found haplogroups R, K*, F*, E1, E3b2 and E3b*, all of which are common in white Brazilian males, and none revealed a particular association with infertility. Taken together, these results show no evidence of association between the occurrence of gr/gr deletion and male infertility.

Color and genomic ancestry in Brazilians: a study with forensic microsatellites.

The population of Brazil, formed by extensive admixture between Amerindians, Europeans and Africans, is one of the most variable in the world. We have recently published a study that used ancestry-informative markers to conclude that in Brazil, at an individual level, color, as determined by physical evaluation, was a poor predictor of genomic ancestry, estimated by molecular markers. To corroborate these findings we undertook the present investigation based on data from 12 commercially available forensic microsatellites that were utilized to estimate the personal genomic origin for each of 752 individuals from the city of São Paulo, belonging to different Brazilian color categories (275 Whites, 192 Intermediates and 285 Blacks). The genotypes permitted the calculation of a personal likelihood-ratio estimator of African or European ancestry. Although the 12 marker set proved capable of discriminating between European and African individuals, we observed very significant overlaps among the three color categories of Brazilians. This was confirmed quantitatively using a Bayesian analysis of population structure that did not demonstrate significant genetic differentiation between the three color groups. These results corroborate and validate our previous conclusions using ancestry-informative markers that in Brazil at the individual level there is significant dissociation of color and genomic ancestry.