OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies.

BACKGROUND: Aberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. Here, we addressed species-specific splicing mechanisms triggered by the OTC c.386G>A mutation, relatively frequent in humans, leading to Ornithine TransCarbamylase Deficiency (OTCD) in patients and spfash mice, and its differential susceptibility to RNA therapeutics based on engineered U1snRNA. METHODS: Creation and co-expression of engineered U1snRNAs with human and mouse minigenes, either wild-type or harbouring different nucleotide changes, in human (HepG2) and mouse (Hepa1-6) hepatoma cells followed by analysis of splicing pattern. RNA pulldown studies to evaluate binding of specific splicing factors. RESULTS: Comparative nucleotide analysis suggested a role for the intronic +10-11 nucleotides, and pull-down assays showed that they confer preferential binding to the TIA1 splicing factor in the mouse context, where TIA1 overexpression further increases correct splicing. Consistently, the splicing profile of the human minigene with mouse +10-11 nucleotides overlapped that of mouse minigene, and restored responsiveness to TIA1 overexpression and to compensatory U1snRNA. Swapping the human +10-11 nucleotides into the mouse context had opposite effects. Moreover, the interplay between the authentic and the adjacent cryptic 5'ss in the human OTC dictates pathogenic mechanisms of several OTCD-causing 5'ss mutations, and only the c.386+5G>A change, abrogating the cryptic 5'ss, was rescuable by engineered U1snRNA. CONCLUSIONS: Subtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1snRNAs, which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans.

Distinguishing among complex evolutionary models using unphased whole-genome data through random forest approximate Bayesian computation.

Inferring past demographic histories is crucial in population genetics, and the amount of complete genomes now available should in principle facilitate this inference. In practice, however, the available inferential methods suffer from severe limitations. Although hundreds complete genomes can be simultaneously analysed, complex demographic processes can easily exceed computational constraints, and the procedures to evaluate the reliability of the estimates contribute to increase the computational effort. Here we present an approximate Bayesian computation framework based on the random forest algorithm (ABC-RF), to infer complex past population processes using complete genomes. To this aim, we propose to summarize the data by the full genomic distribution of the four mutually exclusive categories of segregating sites (FDSS), a statistic fast to compute from unphased genome data and that does not require the ancestral state of alleles to be known. We constructed an efficient ABC pipeline and tested how accurately it allows one to recognize the true model among models of increasing complexity, using simulated data and taking into account different sampling strategies in terms of number of individuals analysed, number and size of the genetic loci considered. We also compared the FDSS with the unfolded and folded site frequency spectrum (SFS), and for these statistics we highlighted the experimental conditions maximizing the inferential power of the ABC-RF procedure. We finally analysed real data sets, testing models on the dispersal of anatomically modern humans out of Africa and exploring the evolutionary relationships of the three species of Orangutan inhabiting Borneo and Sumatra.

The genetic structure and adaptation of Andean highlanders and Amazonians are influenced by the interplay between geography and culture.

Western South America was one of the worldwide cradles of civilization. The well-known Inca Empire was the tip of the iceberg of an evolutionary process that started 11,000 to 14,000 years ago. Genetic data from 18 Peruvian populations reveal the following: 1) The between-population homogenization of the central southern Andes and its differentiation with respect to Amazonian populations of similar latitudes do not extend northward. Instead, longitudinal gene flow between the northern coast of Peru, Andes, and Amazonia accompanied cultural and socioeconomic interactions revealed by archeology. This pattern recapitulates the environmental and cultural differentiation between the fertile north, where altitudes are lower, and the arid south, where the Andes are higher, acting as a genetic barrier between the sharply different environments of the Andes and Amazonia. 2) The genetic homogenization between the populations of the arid Andes is not only due to migrations during the Inca Empire or the subsequent colonial period. It started at least during the earlier expansion of the Wari Empire (600 to 1,000 years before present). 3) This demographic history allowed for cases of positive natural selection in the high and arid Andes vs. the low Amazon tropical forest: in the Andes, a putative enhancer in HAND2-AS1 (heart and neural crest derivatives expressed 2 antisense RNA1, a noncoding gene related to cardiovascular function) and rs269868-C/Ser1067 in DUOX2 (dual oxidase 2, related to thyroid function and innate immunity) genes and, in the Amazon, the gene encoding for the CD45 protein, essential for antigen recognition by T and B lymphocytes in viral-host interaction.

Climate shaped how Neolithic farmers and European hunter-gatherers interacted after a major slowdown from 6,100 BCE to 4,500 BCE.

The Neolithic transition in Europe was driven by the rapid dispersal of Near Eastern farmers who, over a period of 3,500 years, brought food production to the furthest corners of the continent. However, this wave of expansion was far from homogeneous, and climatic factors may have driven a marked slowdown observed at higher latitudes. Here, we test this hypothesis by assembling a large database of archaeological dates of first arrival of farming to quantify the expansion dynamics. We identify four axes of expansion and observe a slowdown along three axes when crossing the same climatic threshold. This threshold reflects the quality of the growing season, suggesting that Near Eastern crops might have struggled under more challenging climatic conditions. This same threshold also predicts the mixing of farmers and hunter-gatherers as estimated from ancient DNA, suggesting that unreliable yields in these regions might have favoured the contact between the two groups.

A genetic perspective on Longobard-Era migrations.

From the first century AD, Europe has been interested by population movements, commonly known as Barbarian migrations. Among these processes, the one involving the Longobard culture interested a vast region, but its dynamics and demographic impact remains largely unknown. Here we report 87 new complete mitochondrial sequences coming from nine early-medieval cemeteries located along the area interested by the Longobard migration (Czech Republic, Hungary and Italy). From the same areas, we sampled necropoleis characterized by cultural markers associated with the Longobard culture (LC) and coeval burials where no such markers were found, or with a chronology slightly preceding the presumed arrival of the Longobards in that region (NLC). Population genetics analysis and demographic modeling highlighted a similarity between LC individuals, as reflected by the sharing of quite rare haplogroups and by the degree of genetic resemblance between Hungarian and Italian LC necropoleis estimated via a Bayesian approach, ABC. The demographic model receiving the strongest statistical support also postulates a contact between LC and NLC communities, thus indicating a complex dynamics of admixture in medieval Europe.

The female ancestor's tale: Long-term matrilineal continuity in a nonisolated region of Tuscany.

OBJECTIVES: With the advent of ancient DNA analyses, it has been possible to disentangle the contribution of ancient populations to the genetic pool of the modern inhabitants of many regions. Reconstructing the maternal ancestry has often highlighted genetic continuity over several millennia, but almost always in isolated areas. Here we analyze North-western Tuscany, a region that was a corridor of exchanges between Central Italy and the Western Mediterranean coast. MATERIALS AND METHODS: We newly obtained mitochondrial HVRI sequences from 28 individuals, and after gathering published data, we collected genetic information for 119 individuals from the region. Those span five periods during the last 5,000 years: Prehistory, Etruscan age, Roman age, Renaissance, and Present-day. We used serial coalescent simulations in an approximate Bayesian computation framework to test for continuity between the mentioned groups. RESULTS: Our analyses always favor continuity over discontinuity for all groups considered, with the Etruscans being part of the genealogy. Moreover, the posterior distributions of the parameters support very small female effective population sizes. CONCLUSIONS: The observed signals of long-term genetic continuity and isolation are in contrast with the history of the region, conquered several times (Etruscans, Romans, Lombards, and French). While the Etruscans appear as a local population, intermediate between the prehistoric and the other samples, we suggest that the other conquerors-arriving from far-had a consistent social or sex bias, hence only marginally affecting the maternal lineages. At the same time, our results show that long-term genealogical continuity is not necessarily linked to geographical isolation.

Genome diversity in the Neolithic Globular Amphorae culture and the spread of Indo-European languages.

It is unclear whether Indo-European languages in Europe spread from the Pontic steppes in the late Neolithic, or from Anatolia in the Early Neolithic. Under the former hypothesis, people of the Globular Amphorae culture (GAC) would be descended from Eastern ancestors, likely representing the Yamnaya culture. However, nuclear (six individuals typed for 597 573 SNPs) and mitochondrial (11 complete sequences) DNA from the GAC appear closer to those of earlier Neolithic groups than to the DNA of all other populations related to the Pontic steppe migration. Explicit comparisons of alternative demographic models via approximate Bayesian computation confirmed this pattern. These results are not in contrast to Late Neolithic gene flow from the Pontic steppes into Central Europe. However, they add nuance to this model, showing that the eastern affinities of the GAC in the archaeological record reflect cultural influences from other groups from the East, rather than the movement of people.

Analysis of the R1b-DF27 haplogroup shows that a large fraction of Iberian Y-chromosome lineages originated recently in situ.

Haplogroup R1b-M269 comprises most Western European Y chromosomes; of its main branches, R1b-DF27 is by far the least known, and it appears to be highly prevalent only in Iberia. We have genotyped 1072 R1b-DF27 chromosomes for six additional SNPs and 17 Y-STRs in population samples from Spain, Portugal and France in order to further characterize this lineage and, in particular, to ascertain the time and place where it originated, as well as its subsequent dynamics. We found that R1b-DF27 is present in frequencies ~40% in Iberian populations and up to 70% in Basques, but it drops quickly to 6-20% in France. Overall, the age of R1b-DF27 is estimated at ~4,200 years ago, at the transition between the Neolithic and the Bronze Age, when the Y chromosome landscape of W Europe was thoroughly remodeled. In spite of its high frequency in Basques, Y-STR internal diversity of R1b-DF27 is lower there, and results in more recent age estimates; NE Iberia is the most likely place of origin of DF27. Subhaplogroup frequencies within R1b-DF27 are geographically structured, and show domains that are reminiscent of the pre-Roman Celtic/Iberian division, or of the medieval Christian kingdoms.

Complete mitochondrial sequences from Mesolithic Sardinia.

Little is known about the genetic prehistory of Sardinia because of the scarcity of pre-Neolithic human remains. From a genetic perspective, modern Sardinians are known as genetic outliers in Europe, showing unusually high levels of internal diversity and a close relationship to early European Neolithic farmers. However, how far this peculiar genetic structure extends and how it originated was to date impossible to test. Here we present the first and oldest complete mitochondrial sequences from Sardinia, dated back to 10,000 yBP. These two individuals, while confirming a Mesolithic occupation of the island, belong to rare mtDNA lineages, which have never been found before in Mesolithic samples and that are currently present at low frequencies not only in Sardinia, but in the whole Europe. Preliminary Approximate Bayesian Computations, restricted by biased reference samples for Mesolithic Sardinia (the two typed samples) and Neolithic Europe (limited to central and north European sequences), suggest that the first inhabitants of the island have had a small or negligible contribution to the present-day Sardinian population, which mainly derives its genetic diversity from continental migration into the island by Neolithic times.

Effect of migration patterns on maternal genetic structure: a case of Tai-Kadai migration from China to Thailand.

The migration of the Tai-Kadai speaking people from southern China to northern Thailand over the past hundreds of years has revealed numerous patterns that have likely been influenced by routes, purposes and periods of time. To study the effects of different migration patterns on Tai-Kadai maternal genetic structure, mitochondrial DNA hypervariable region I sequences from the Yong and the Lue people having well-documented histories in northern Thailand were analyzed. Although the Yong and Lue people were historically close relatives who shared Xishuangbanna Dai ancestors, significant genetic differences have been observed among them. The Yong people who have been known to practice mass migration have exhibited a closer genetic affinity to their Dai ancestors than have the Lue people. Genetic heterogeneity and a sudden reduced effective population size within the Lue group is likely a direct result of the circumstances of the founder effect.

The Uromodulin Gene Locus Shows Evidence of Pathogen Adaptation through Human Evolution.

Common variants in the UMOD gene encoding uromodulin, associated with risk of hypertension and CKD in the general population, increase UMOD expression and urinary excretion of uromodulin, causing salt-sensitive hypertension and renal lesions. To determine the effect of selective pressure on variant frequency, we investigated the allelic frequency of the lead UMOD variant rs4293393 in 156 human populations, in eight ancient human genomes, and in primate genomes. The T allele of rs4293393, associated with CKD risk, has high frequency in most modern populations and was the one detected in primate genomes. In contrast, we identified only the derived, C allele in Denisovan and Neanderthal genomes. The distribution of the UMOD ancestral allele did not follow the ancestral susceptibility model observed for variants associated with salt-sensitive hypertension. Instead, the global frequencies of the UMOD alleles significantly correlated with pathogen diversity (bacteria, helminths) and prevalence of antibiotic-resistant urinary tract infections (UTIs). The inverse correlation found between urinary levels of uromodulin and markers of UTIs in the general population substantiates the link between UMOD variants and protection against UTIs. These data strongly suggest that the UMOD ancestral allele, driving higher urinary excretion of uromodulin, has been kept at a high frequency because of its protective effect against UTIs.

Early modern human dispersal from Africa: genomic evidence for multiple waves of migration.

BACKGROUND: Anthropological and genetic data agree in indicating the African continent as the main place of origin for anatomically modern humans. However, it is unclear whether early modern humans left Africa through a single, major process, dispersing simultaneously over Asia and Europe, or in two main waves, first through the Arab Peninsula into southern Asia and Oceania, and later through a northern route crossing the Levant. RESULTS: Here, we show that accurate genomic estimates of the divergence times between European and African populations are more recent than those between Australo-Melanesia and Africa and incompatible with the effects of a single dispersal. This difference cannot possibly be accounted for by the effects of either hybridization with archaic human forms in Australo-Melanesia or back migration from Europe into Africa. Furthermore, in several populations of Asia we found evidence for relatively recent genetic admixture events, which could have obscured the signatures of the earliest processes. CONCLUSIONS: We conclude that the hypothesis of a single major human dispersal from Africa appears hardly compatible with the observed historical and geographical patterns of genome diversity and that Australo-Melanesian populations seem still to retain a genomic signature of a more ancient divergence from Africa.

Across language families: Genome diversity mirrors linguistic variation within Europe.

OBJECTIVES: The notion that patterns of linguistic and biological variation may cast light on each other and on population histories dates back to Darwin's times; yet, turning this intuition into a proper research program has met with serious methodological difficulties, especially affecting language comparisons. This article takes advantage of two new tools of comparative linguistics: a refined list of Indo-European cognate words, and a novel method of language comparison estimating linguistic diversity from a universal inventory of grammatical polymorphisms, and hence enabling comparison even across different families. We corroborated the method and used it to compare patterns of linguistic and genomic variation in Europe. MATERIALS AND METHODS: Two sets of linguistic distances, lexical and syntactic, were inferred from these data and compared with measures of geographic and genomic distance through a series of matrix correlation tests. Linguistic and genomic trees were also estimated and compared. A method (Treemix) was used to infer migration episodes after the main population splits. RESULTS: We observed significant correlations between genomic and linguistic diversity, the latter inferred from data on both Indo-European and non-Indo-European languages. Contrary to previous observations, on the European scale, language proved a better predictor of genomic differences than geography. Inferred episodes of genetic admixture following the main population splits found convincing correlates also in the linguistic realm. DISCUSSION: These results pave the ground for previously unfeasible cross-disciplinary analyses at the worldwide scale, encompassing populations of distant language families.

Genealogical relationships between early medieval and modern inhabitants of Piedmont.

In the period between 400 to 800 AD, also known as the period of the Barbarian invasions, intense migration is documented in the historical record of Europe. However, little is known about the demographic impact of these historical movements, potentially ranging from negligible to substantial. As a pilot study in a broader project on Medieval Europe, we sampled 102 specimens from 5 burial sites in Northwestern Italy, archaeologically classified as belonging to Lombards or Longobards, a Germanic people ruling over a vast section of the Italian peninsula from 568 to 774. We successfully amplified and typed the mitochondrial hypervariable region I (HVR-I) of 28 individuals. Comparisons of genetic diversity with other ancient populations and haplotype networks did not suggest that these samples are heterogeneous, and hence allowed us to jointly compare them with three isolated contemporary populations, and with a modern sample of a large city, representing a control for the effects of recent immigration. We then generated by serial coalescent simulations 16 millions of genealogies, contrasting a model of genealogical continuity with one in which the contemporary samples are genealogically independent from the medieval sample. Analyses by Approximate Bayesian Computation showed that the latter model fits the data in most cases, with one exception, Trino Vercellese, in which the evidence was compatible with persistence up to the present time of genetic features observed among this early medieval population. We conclude that it is possible, in general, to detect evidence of genealogical ties between medieval and specific modern populations. However, only seldom did mitochondrial DNA data allow us to reject with confidence either model tested, which indicates that broader analyses, based on larger assemblages of samples and genetic markers, are needed to understand in detail the effects of medieval migration.

Genetic evidence does not support an Etruscan origin in Anatolia.

The debate on the origins of Etruscans, documented in central Italy between the eighth century BC and the first century AD, dates back to antiquity. Herodotus described them as a group of immigrants from Lydia, in Western Anatolia, whereas for Dionysius of Halicarnassus they were an indigenous population. Dionysius' view is shared by most modern archeologists, but the observation of similarities between the (modern) mitochondrial DNAs (mtDNAs) of Turks and Tuscans was interpreted as supporting an Anatolian origin of the Etruscans. However, ancient DNA evidence shows that only some isolates, and not the bulk of the modern Tuscan population, are genetically related to the Etruscans. In this study, we tested alternative models of Etruscan origins by Approximate Bayesian Computation methods, comparing levels of genetic diversity in the mtDNAs of modern and ancient populations with those obtained by millions of computer simulations. The results show that the observed genetic similarities between modern Tuscans and Anatolians cannot be attributed to an immigration wave from the East leading to the onset of the Etruscan culture in Italy. Genetic links between Tuscany and Anatolia do exist, but date back to a remote stage of prehistory, possibly but not necessarily to the spread of farmers during the Neolithic period.

Origins and evolution of the Etruscans' mtDNA.

The Etruscan culture is documented in Etruria, Central Italy, from the 8(th) to the 1(st) century BC. For more than 2,000 years there has been disagreement on the Etruscans' biological origins, whether local or in Anatolia. Genetic affinities with both Tuscan and Anatolian populations have been reported, but so far all attempts have failed to fit the Etruscans' and modern populations in the same genealogy. We extracted and typed the hypervariable region of mitochondrial DNA of 14 individuals buried in two Etruscan necropoleis, analyzing them along with other Etruscan and Medieval samples, and 4,910 contemporary individuals from the Mediterranean basin. Comparing ancient (30 Etruscans, 27 Medieval individuals) and modern DNA sequences (370 Tuscans), with the results of millions of computer simulations, we show that the Etruscans can be considered ancestral, with a high degree of confidence, to the current inhabitants of Casentino and Volterra, but not to the general contemporary population of the former Etruscan homeland. By further considering two Anatolian samples (35 and 123 individuals) we could estimate that the genetic links between Tuscany and Anatolia date back to at least 5,000 years ago, strongly suggesting that the Etruscan culture developed locally, and not as an immediate consequence of immigration from the Eastern Mediterranean shores.

No evidence of Neandertal admixture in the mitochondrial genomes of early European modern humans and contemporary Europeans.

Neandertals, the archaic human form documented in Eurasia until 29,000 years ago, share no mitochondrial haplotype with modern Europeans. Whether this means that the two groups were reproductively isolated is controversial, and indeed nuclear data have been interpreted as suggesting that they admixed. We explored the range of demographic parameters that may have generated the observed mitochondrial diversity, simulating 3.0 million genealogies under six models differing as for the relationships among contemporary Europeans, Neandertals, and Upper Palaeolithic European early modern humans (EEMH), who coexisted with Neandertals for millennia. We compared by Approximate Bayesian Computations the simulation results with mitochondrial diversity in 7 Neandertals, 3 EEMH, and 150 opportunely chosen modern Europeans. A model of genealogical continuity between EEMH and contemporary Europeans, with no Neandertal contribution, received overwhelming support from the analyses. The maximum degree of Neandertal admixture, under the model of gene flow supported by nuclear data, was estimated at 1.5%, but this model proved 20-32 times less likely than a model without any gene flow. Nuclear and mitochondrial evidence might be reconciled if smaller population sizes led to faster lineage sorting for mitochondrial DNA, and Neandertals shared a longer period of common ancestry with the non-African's than with the African's ancestors.