Meta-analysis of 46,000 germline de novo mutations linked to human inherited disease.

BACKGROUND: De novo mutations (DNMs) are variants that occur anew in the offspring of noncarrier parents. They are not inherited from either parent but rather result from endogenous mutational processes involving errors of DNA repair/replication. These spontaneous errors play a significant role in the causation of genetic disorders, and their importance in the context of molecular diagnostic medicine has become steadily more apparent as more DNMs have been reported in the literature. In this study, we examined 46,489 disease-associated DNMs annotated by the Human Gene Mutation Database (HGMD) to ascertain their distribution across gene and disease categories. RESULTS: Most disease-associated DNMs reported to date are found to be associated with developmental and psychiatric disorders, a reflection of the focus of sequencing efforts over the last decade. Of the 13,277 human genes in which DNMs have so far been found, the top-10 genes with the highest proportions of DNM relative to gene size were H3-3 A, DDX3X, CSNK2B, PURA, ZC4H2, STXBP1, SCN1A, SATB2, H3-3B and TUBA1A. The distribution of CADD and REVEL scores for both disease-associated DNMs and those mutations not reported to be de novo revealed a trend towards higher deleteriousness for DNMs, consistent with the likely lower selection pressure impacting them. This contrasts with the non-DNMs, which are presumed to have been subject to continuous negative selection over multiple generations. CONCLUSION: This meta-analysis provides important information on the occurrence and distribution of disease-associated DNMs in association with heritable disease and should make a significant contribution to our understanding of this major type of mutation.

Naturally occurring genetic diseases caused by de novo variants in domestic animals.

With the advent of next-generation sequencing, an increasing number of cases of de novo variants in domestic animals have been reported in scientific literature primarily associated with clinically severe phenotypes. The emergence of new variants at each generation is a crucial aspect in understanding the pathology of early-onset diseases in animals and can provide valuable insights into similar diseases in humans. With the aim of collecting deleterious de novo variants in domestic animals, we searched the scientific literature and compiled reports on 42 de novo variants in 31 genes in domestic animals. No clear disease-associated phenotype has been established in humans for three of these genes (NUMB, ANKRD28 and KCNG1). For the remaining 28 genes, a strong similarity between animal and human phenotypes was recognized from available information in OMIM and OMIA, revealing the importance of comparative studies and supporting the use of domestic animals as natural models for human diseases, in line with the One Health approach.

PseudoChecker: an integrated online platform for gene inactivation inference.

The rapid expansion of high-quality genome assemblies, exemplified by ongoing initiatives such as the Genome-10K and i5k, demands novel automated methods to approach comparative genomics. Of these, the study of inactivating mutations in the coding region of genes, or pseudogenization, as a source of evolutionary novelty is mostly overlooked. Thus, to address such evolutionary/genomic events, a systematic, accurate and computationally automated approach is required. Here, we present PseudoChecker, the first integrated online platform for gene inactivation inference. Unlike the few existing methods, our comparative genomics-based approach displays full automation, a built-in graphical user interface and a novel index, PseudoIndex, for an empirical evaluation of the gene coding status. As a multi-platform online service, PseudoChecker simplifies access and usability, allowing a fast identification of disruptive mutations. An analysis of 30 genes previously reported to be eroded in mammals, and 30 viable genes from the same lineages, demonstrated that PseudoChecker was able to correctly infer 97% of loss events and 95% of functional genes, confirming its reliability. PseudoChecker is freely available, without login required, at http://pseudochecker.ciimar.up.pt.

Common polymorphic OTC variants can act as genetic modifiers of enzymatic activity.

Understanding the role of common polymorphisms in modulating the clinical phenotype when they co-occur with a disease-causing lesion is of critical importance in medical genetics. We explored the impact of apparently neutral common polymorphisms, using the gene encoding the urea cycle enzyme, ornithine transcarbamylase (OTC), as a model system. Distinct combinations of genetic backgrounds embracing two missense polymorphisms were created in cis with the pathogenic p.Arg40His replacement. In vitro enzymatic assays revealed that the polymorphic variants were able to modulate OTC activity both in the presence or absence of the pathogenic lesion. First, we found that the combination of the minor alleles of polymorphisms p.Lys46Arg and p.Gln270Arg significantly enhanced enzymatic activity in the wild-type protein. Second, enzymatic assays revealed that the minor allele of the p.Gln270Arg polymorphism was capable of ameliorating OTC activity when combined in cis with the pathogenic p.Arg40His replacement. Structural analysis predicted that the minor allele of the p.Gln270Arg polymorphism would serve to stabilize the OTC wild-type protein, thereby corroborating the results of the experimental assays. Our findings demonstrate the potential importance of cis-interactions between common polymorphic variants and pathogenic missense mutations and illustrate how standing genetic variation can modulate protein function.

Compensatory epistasis explored by molecular dynamics simulations.

A non-negligible proportion of human pathogenic variants are known to be present as wild type in at least some non-human mammalian species. The standard explanation for this finding is that molecular mechanisms of compensatory epistasis can alleviate the mutations' otherwise pathogenic effects. Examples of compensated variants have been described in the literature but the interacting residue(s) postulated to play a compensatory role have rarely been ascertained. In this study, the examination of five human X-chromosomally encoded proteins (FIX, GLA, HPRT1, NDP and OTC) allowed us to identify several candidate compensated variants. Strong evidence for a compensated/compensatory pair of amino acids in the coagulation FIXa protein (involving residues 270 and 271) was found in a variety of mammalian species. Both amino acid residues are located within the 60-loop, spatially close to the 39-loop that performs a key role in coagulation serine proteases. To understand the nature of the underlying interactions, molecular dynamics simulations were performed. The predicted conformational change in the 39-loop consequent to the Glu270Lys substitution (associated with hemophilia B) appears to impair the protein's interaction with its substrate but, importantly, such steric hindrance is largely mitigated in those proteins that carry the compensatory residue (Pro271) at the neighboring amino acid position.

Complete Inactivation of Sebum-Producing Genes Parallels the Loss of Sebaceous Glands in Cetacea.

Genomes are dynamic biological units, with processes of gene duplication and loss triggering evolutionary novelty. The mammalian skin provides a remarkable case study on the occurrence of adaptive morphological innovations. Skin sebaceous glands (SGs), for instance, emerged in the ancestor of mammals serving pivotal roles, such as lubrication, waterproofing, immunity, and thermoregulation, through the secretion of sebum, a complex mixture of various neutral lipids such as triacylglycerol, free fatty acids, wax esters, cholesterol, and squalene. Remarkably, SGs are absent in a few mammalian lineages, including the iconic Cetacea. We investigated the evolution of the key molecular components responsible for skin sebum production: Dgat2l6, Awat1, Awat2, Elovl3, Mogat3, and Fabp9. We show that all analyzed genes have been rendered nonfunctional in Cetacea species (toothed and baleen whales). Transcriptomic analysis, including a novel skin transcriptome from blue whale, supports gene inactivation. The conserved mutational pattern found in most analyzed genes, indicates that pseudogenization events took place prior to the diversification of modern Cetacea lineages. Genome and skin transcriptome analysis of the common hippopotamus highlighted the convergent loss of a subset of sebum-producing genes, notably Awat1 and Mogat3. Partial loss profiles were also detected in non-Cetacea aquatic mammals, such as the Florida manatee, and in terrestrial mammals displaying specialized skin phenotypes such as the African elephant, white rhinoceros and pig. Our findings reveal a unique landscape of "gene vestiges" in the Cetacea sebum-producing compartment, with limited gene loss observed in other mammalian lineages: suggestive of specific adaptations or specializations of skin lipids.

Essential genetic findings in neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) represent a growing medical challenge in modern societies. Ever-increasing sophisticated diagnostic tools have been continuously revealing a remarkably complex architecture that embraces genetic mutations of distinct types (chromosomal rearrangements, copy number variants, small indels, and nucleotide substitutions) with distinct frequencies in the population (common, rare, de novo). Such a network of interacting players creates difficulties in establishing rigorous genotype-phenotype correlations. Furthermore, individual lifestyles may also contribute to the severity of the symptoms fueling a large spectrum of gene-environment interactions that have a key role on the relationships between genotypes and phenotypes.Herein, a review of the genetic discoveries related to NDDs is presented with the aim to provide useful general information for the medical community.

Convergent inactivation of the skin-specific C-C motif chemokine ligand 27 in mammalian evolution.

The appearance of mammalian-specific skin features was a key evolutionary event contributing for the elaboration of physiological processes such as thermoregulation, adequate hydration, locomotion, and inflammation. Skin inflammatory and autoimmune processes engage a population of skin-infiltrating T cells expressing a specific C-C chemokine receptor (CCR10) which interacts with an epidermal CC chemokine, the skin-specific C-C motif chemokine ligand 27 (CCL27). CCL27 is selectively produced in the skin by keratinocytes, particularly upon inflammation, mediating the adhesion and homing of skin-infiltrating T cells. Here, we examined the evolution and coding condition of Ccl27 in 112 placental mammalian species. Our findings reveal that a number of open reading frame inactivation events such as insertions, deletions, and start and stop codon mutations independently occurred in Cetacea, Pholidota, Sirenia, Chiroptera, and Rodentia, totalizing 18 species. The diverse habitat settings and lifestyles of Ccl27-eroded lineages probably implied distinct evolutionary triggers rendering this gene unessential. For example, in Cetacea, the rapid renewal of skin layers minimizes the need for an elaborate inflammatory mechanism, mirrored by the absence of epidermal scabs. Our findings suggest that the convergent and independent loss of Ccl27 in mammalian evolution concurred with unique adaptive roads for skin physiology.

Retention of fatty acyl desaturase 1 (fads1) in Elopomorpha and Cyclostomata provides novel insights into the evolution of long-chain polyunsaturated fatty acid biosynthesis in vertebrates.

BACKGROUND: Provision of long-chain polyunsaturated fatty acids (LC-PUFA) in vertebrates occurs through the diet or via endogenous production from C18 precursors through consecutive elongations and desaturations. It has been postulated that the abundance of LC-PUFA in the marine environment has remarkably modulated the gene complement and function of Fads in marine teleosts. In vertebrates two fatty acyl desaturases, namely Fads1 and Fads2, encode ∆5 and ∆6 desaturases, respectively. To fully clarify the evolutionary history of LC-PUFA biosynthesis in vertebrates, we investigated the gene repertoire and function of Fads from species placed at key evolutionary nodes. RESULTS: We demonstrate that functional Fads1Δ5 and Fads2∆6 arose from a tandem gene duplication in the ancestor of vertebrates, since they are present in the Arctic lamprey. Additionally, we show that a similar condition was retained in ray-finned fish such as the Senegal bichir and spotted gar, with the identification of fads1 genes in these lineages. Functional characterisation of the isolated desaturases reveals the first case of a Fads1 enzyme with ∆5 desaturase activity in the Teleostei lineage, the Elopomorpha. In contrast, in Osteoglossomorpha genomes, while no fads1 was identified, two separate fads2 duplicates with ∆6 and ∆5 desaturase activities respectively were uncovered. CONCLUSIONS: We conclude that, while the essential genetic components involved LC-PUFA biosynthesis evolved in the vertebrate ancestor, the full completion of the LC-PUFA biosynthesis pathway arose uniquely in gnathostomes.

Cetacea are natural knockouts for IL20.

The Cetacea infraorder comprises a very unique group within the mammalian lineage. While sharing common ancestors with terrestrial mammals, their exclusive dependence on aquatic environments makes them attractive models to explore the landscape of molecular shifts in radical habitat transitions. Among their diverse anatomical and physiological solutions, we find detectable genetic remodeling of the immune system. In agreement, here we show that the gene sequence of interleukin-20 (IL20) displays unambiguous signs of inactivation with several disruptive mutations, including stop codons, insertions, and a conserved trans-species mutation abolishing a canonical splice site, in nine analyzed cetacean genomes. Considering the suggested role of IL20 in skin immunity processes, including inflammation, epithelization, and remodeling, we propose that gene inactivation follows specific adaptations of cetacean skin to the aquatic environment, in frame with the less-is-more hypothesis.

Evolutionary Exploitation of Vertebrate Peroxisome Proliferator-Activated Receptor γ by Organotins.

Globally persistent man-made chemicals display ever-growing ecosystemic consequences, a hallmark of the Anthropocene epoch. In this context, the assessment of how lineage-specific gene repertoires influence organism sensitivity toward endocrine disruptors is a central question in toxicology. A striking example highlights the role of a group of compounds known as obesogens. In mammals, most examples involve the modulation of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). To address the structural and biological determinants of PPARγ exploitation by a model obesogen, tributyltin (TBT), in chordates, we employed comparative genomics, transactivation and ligand binding assays, homology modeling, and site-directed-mutagenesis. We show that the emergence of multiple PPARs (α, ß and γ) in vertebrate ancestry coincides with the acquisition of TBT agonist affinity, as can be deduced from the conserved transactivation and binding affinity of the chondrichthyan and mammalian PPARγ. The amphioxus single-copy PPAR is irresponsive to TBT; as well as the investigated teleosts, this is a probable consequence of a specific mutational remodeling of the ligand binding pocket. Our findings endorse the modulatory ability of man-made chemicals and suggest an evolutionarily diverse setting, with impacts for environmental risk assessment.

Unusual loss of chymosin in mammalian lineages parallels neo-natal immune transfer strategies.

Gene duplication and loss are powerful drivers of evolutionary change. The role of loss in phenotypic diversification is notably illustrated by the variable enzymatic repertoire involved in vertebrate protein digestion. Among these we find the pepsin family of aspartic proteinases, including chymosin (Cmy). Previous studies demonstrated that Cmy, a neo-natal digestive pepsin, is inactivated in some primates, including humans. This pseudogenization event was hypothesized to result from the acquisition of maternal immune immunoglobulin G (IgG) transfer. By investigating 94 mammalian subgenomes we reveal an unprecedented level of Cmy erosion in placental mammals, with numerous independent events of gene loss taking place in Primates, Dermoptera, Rodentia, Cetacea and Perissodactyla. Our findings strongly suggest that the recurrent inactivation of Cmy correlates with the evolution of the passive transfer of IgG and uncovers a noteworthy case of evolutionary cross-talk between the digestive and the immune system, modulated by gene loss.

Diversity and history of the long-chain acyl-CoA synthetase (Acsl) gene family in vertebrates.

BACKGROUND: Fatty acids, a considerable fraction of lipid molecules, participate in fundamental physiological processes. They undergo activation into their corresponding CoA esters for oxidation or esterification into complex lipids (e.g. triglycerides, phospholipids and cholesterol esters), a process that is carried out by acyl-CoA synthases (ACS). Here we analyze the evolution of the gene family encoding for the long-chain acyl-CoA synthetases (Acsl) in vertebrates. RESULTS: By means of phylogenetics and comparative genomics we show that genome duplications (2R) generated the diversity of Acsl genes in extant vertebrate lineages. In the vertebrate ancestor two separate genes originated the current Acsl1/5/6 and the Acsl3/4 gene families, and the extra gene duplicates in teleosts are a consequence of the teleost specific third round of genome duplication (3R). Moreover, the diversity of Acsl family members is broader than anticipated. Our strategy uncovered a novel uncharacterized Acsl-like gene found in teleosts, spotted gar, coelacanth and possibly lamprey, which we designate Acsl2. The detailed analysis of the Acsl2 teleost gene locus strongly supports the conclusion that it corresponds to a retained 2R paralogue, lost in tetrapods. CONCLUSIONS: We provide here the first evolutionary analysis of the Acsl gene family in vertebrates, showing the specific contribution of 2R/3R to the diversity of this gene family. We find also that the division of ACSL enzymes into two groups predates at least the emergence of deuterostomes. Our study indicates that genome duplications significantly contributed to the elaboration of fatty acid activation metabolism in vertebrates.

A partial duplication of an X-linked gene exclusive of a primate lineage (Macaca).

Gene duplication plays a significant role in evolution. Paralogous gene copies may be lost due to the successive accumulation of deleterious mutations or remain active in the genome. In this work, a partial duplication of an X-linked region in the Macaca genus is identified and explored. Genomic comparisons reveal that the duplication encompasses the genes encoding ornithine transcarbamylase (OTC) and retinitis pigmentosa GTPase regulator (RPGR), spanning over 0.1 Mb on the chromosome 9 of Macaca. According to our analyses, the duplicated region of chromosome 9 involves partial coding sequences of both OTC and RPGR genes. Analyses of the selective pressures did not reveal significant differences in the ratio between nonsynonymous and synonymous mutations (w<1), suggesting that no selective pressures were acting in the evolutionary process. Reports for a biological role regarding some partial duplications exist in the literature, therefore, although being rare events, partial duplications of functionally important genes are worthy of study so that their impact can be explored.

Human carbamoyl phosphate synthetase I (CPSI): insights on the structural role of the unknown function domains.

Carbamoyl phosphate synthetase (CPS) is an ancient protein. In mammals it intervenes in the urea cycle. This enzyme is organized into six domains, three of which have no established role in the mammalian enzyme. Taking advantage of the high degree of conservation between the human and the Escherichia coli homologue a comparative study was carried out in order to infer about the biological role of these less characterized domains. We show that among the residues involved in the maintenance of quaternary structure of the E. coli enzyme, several are highly conserved between human and bacterial enzyme and match the homologous positions of the "unknown function" domains in human enzyme, suggesting they are involved in the structural stability of the human enzyme as they are in bacteria.

A Robust Assay to Monitor Ataxin-3 Amyloid Fibril Assembly.

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a glutamine repeat in the protein ataxin-3, which is deposited as intracellular aggregates in affected brain regions. Despite the controversial role of ataxin-3 amyloid structures in SCA3 pathology, the identification of molecules with the capacity to prevent aberrant self-assembly and stabilize functional conformation(s) of ataxin-3 is a key to the development of therapeutic solutions. Amyloid-specific kinetic assays are routinely used to measure rates of protein self-assembly in vitro and are employed during screening for fibrillation inhibitors. The high tendency of ataxin-3 to assemble into oligomeric structures implies that minor changes in experimental conditions can modify ataxin-3 amyloid assembly kinetics. Here, we determine the self-association rates of ataxin-3 and present a detailed study of the aggregation of normal and pathogenic ataxin-3, highlighting the experimental conditions that should be considered when implementing and validating ataxin-3 amyloid progress curves in different settings and in the presence of ataxin-3 interactors. This assay provides a unique and robust platform to screen for modulators of the first steps of ataxin-3 aggregation-a starting point for further studies with cell and animal models of SCA3.

Complex interactions between p.His558Arg and linked variants in the sodium voltage-gated channel alpha subunit 5 (Na V 1.5).

Common genetic polymorphisms may modify the phenotypic outcome when co-occurring with a disease-causing variant, and therefore understanding their modulating role in health and disease is of great importance. The polymorphic p.His558Arg variant of the sodium voltage-gated channel alpha subunit 5 (Na V 1.5) encoded by the SCN5A gene is a case in point, as several studies have shown it can modify the clinical phenotype in a number of cardiac diseases. To evaluate the genetic backgrounds associated with this modulating effect, we reanalysed previous electrophysiological findings regarding the p.His558Arg variant and further assessed its patterns of genetic diversity in human populations. The Na V 1.5 p.His558Arg variant was found to be in linkage disequilibrium with six other polymorphic variants that previously were also associated with cardiac traits in GWAS analyses. On account of this, incongruent reports that Arg558 allele can compensate, aggravate or have no effect on Na V 1.5, likely might have arose due to a role of p.His558Arg depending on the additional linked variants. Altogether, these results indicate a major influence of the epistatic interactions between SCN5A variants, revealing also that phenotypic severity may depend on the polymorphic background associated to each individual genome.

The repertoire of the elongation of very long-chain fatty acids (Elovl) protein family is conserved in tambaqui (Colossoma macropomum): Gene expression profiles offer insights into the sexual differentiation process.

Elongation of very long-chain fatty acids (Elovl) proteins are critical players in the regulation of the length of a fatty acid. At present, eight members of the Elovl family (Elovl1-8), displaying a characteristic fatty acid substrate specificity, have been identified in vertebrates, including teleost fish. In general, Elovl1, Elovl3, Elovl6 and Elovl7 exhibit a substrate preference for saturated and monounsaturated fatty acids, while Elovl2, Elovl4, Elovl5 and Elovl8 use polyunsaturated fatty acids (PUFA) as substrates. PUFA elongases have received considerable attention in aquatic animals due to their involvement in the conversion of C18 PUFAs to long-chain polyunsaturated fatty acids (LC-PUFA). Here, we identified the full repertoire of elovl genes in the tambaqui Colossoma macropomum genome. A detailed phylogenetic and synteny analysis suggests a conservation of these genes among teleosts. Furthermore, based on RNAseq gene expression data, we discovered a gender bias expression of elovl genes during sex differentiation of tambaqui, toward future males. Our findings suggest a role of Elovl enzymes and fatty acid metabolism in tambaqui sexual differentiation.

Genetic Variability of the Functional Domains of Chromodomains Helicase DNA-Binding (CHD) Proteins.

In the past few years, there has been an increasing neuroscientific interest in understanding the function of mammalian chromodomains helicase DNA-binding (CHD) proteins due to their association with severe developmental syndromes. Mammalian CHDs include nine members (CHD1 to CHD9), grouped into subfamilies according to the presence of specific functional domains, generally highly conserved in evolutionary terms. Mutations affecting these domains hold great potential to disrupt protein function, leading to meaningful pathogenic scenarios, such as embryonic defects incompatible with life. Here, we analysed the evolution of CHD proteins by performing a comparative study of the functional domains of CHD proteins between orthologous and paralogous protein sequences. Our findings show that the highest degree of inter-species conservation was observed at Group II (CHD3, CHD4, and CHD5) and that most of the pathological variations documented in humans involve amino acid residues that are conserved not only between species but also between paralogs. The parallel analysis of both orthologous and paralogous proteins, in cases where gene duplications have occurred, provided extra information showing patterns of flexibility as well as interchangeability between amino acid positions. This added complexity needs to be considered when the impact of novel mutations is assessed in terms of evolutionary conservation.

SLC35A2-CDG: Novel variant and review.

SLC35A2 encodes the X-linked transporter that carries uridine diphosphate (UDP)-galactose from the cytosol to the lumen of the Golgi apparatus and the endoplasmic reticulum. Pathogenic variants have been associated to a congenital disorder of glycosylation (CDG) with epileptic encephalopathy as a predominant feature. Among the sixty five patients described so far, a strong gender bias is observed as only seven patients are males. This work is a review and reports a SLC35A2-CDG in a male without epilepsy and with growth deficiency associated with decreased serum IGF1, minor neurological involvement, minor facial dysmorphism, and camptodactyly of fingers and toes. Sequence analysis revealed a hemizygosity for a novel de novo variant: c.233A > G (p.Lys78Arg) in SLC35A2. Further analysis of SLC35A2 sequence by comparing both orthologous and paralogous positions, revealed that not only the variant found in this study, but also most of the reported mutated positions are conserved in SLC35A2 orthologous, and many even in the paralogous SLC35A1 and SLC35A3. This is strong evidence that replacements at these positions will have a critical pathological effect and may also explain the gender bias observed among SLC35A2-CDG patients.